The potential for antibody-dependent enhancement of SARS-CoV-2 infection: Translational implications for vaccine development

Authors:  Jiong Wang andMartin S. Zand Published online by Cambridge University Press: 13 April 2020

Journal of Clinical and Translational Science>Volume 5 Issue 1

Abstract

There is an urgent need for vaccines to the 2019 coronavirus (COVID19; SARS-CoV-2). Vaccine development may not be straightforward, due to antibody-dependent enhancement (ADE). Antibodies against viral surface proteins can, in some cases, increase infection severity by ADE. This phenomenon occurs in SARS-CoV-1, MERS, HIV, Zika, and dengue virus infection and vaccination. Lack of high-affinity anti-SARS-CoV-2 IgG in children may explain the decreased severity of infection in these groups. Here, we discuss the evidence for ADE in the context of SARS-CoV-2 infection and how to address this potential translational barrier to vaccine development, convalescent plasma, and targeted monoclonal antibody therapies.

Introduction

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 coronavirus (CoV), is currently an immense global health threat. There are currently no effective treatments available, sparking a global rush to develop vaccines, small molecule inhibitors, plasma therapies, and to test a variety of existing compounds for antiviral activity. Estimates are that 14–20% of infected patients develop severe illness requiring hospitalization [Reference Wu and McGoogan1,Reference Bi2]. Approximately ∼5% of those infected develop acute respiratory distress syndrome (ARDS), with high mortality. SARS-CoV-2 infection appears to occur at similar rates across age groups, although the severity of disease is less for those <20 years of age [Reference Bi2,3]. Interestingly, younger individuals are also known to lack or have a lower incidence of high-affinity anti-CoV IgG. This is relevant as certain antibodies can potentiate, rather than protect against, coronavirus infection through antibody-dependent enhancement (ADE), wherein normal mechanisms of antigen–antibody complex clearance fail and instead provide an alternative route for host cell infection [Reference Tetro4].

These observations have serious implications for the development strategy of vaccines that induce anti-SARS-CoV-2 IgG antibodies. Rapid translational vaccine development should include checks for ADE at multiple stages in vaccine development across translational stages. Here we discuss the data underlying our concerns and suggest strategies for assessing this risk during vaccine development and deployment.

Epidemiology of COVID-19

Initial data regarding the epidemiology of SARS-CoV-2 show that individuals ≤20 years of age accounted for <3% of all confirmed cases [Reference Wu and McGoogan1,3]. Multiple reports have also indicated that individuals ≤20 years old have milder symptoms, a lower hospitalization risk, and lower case fatality rates [Reference Wu and McGoogan1,Reference Bi2]. However, recent work by the Shenzhen Center for Disease Control, following 1286 close contacts of 391 index cases over a 28-day period, demonstrated that SARS-CoV-2 infection rates among close contacts ≤20 years old were equivalent to those found in older cohorts [Reference Bi2]. Importantly, this younger cohort was often asymptomatic (<50% presenting with fever) and had less severe infection even when symptomatic. Similar patterns have been observed for the SARS-CoV-1 [Reference Chan-Yeung and Xu5] coronavirus from 2003, with a low incidence of symptomatic infection and fewer severe cases. The inverse relationship of age and asymptomatic coronavirus infection with less pathogenic human strains (e.g. 229 E, NL63, OC43) has been known for over a decade [Reference van der Zalm6].

An important difference between children and adults is the presence of IgG antibodies directed at common circulating human coronavirus strains. Children lack anti-CoV IgG prior to 6 years of age, but then begin to develop antibodies against the common circulating strains in humans (229 E, NL63, OC43, HKU1). Anti-CoV IgG increases with age, with high titers ∼75% of those >6 years old [Reference Zhou7]. Importantly, the anti-CoV IgG repertoire in children may consist of predominately low-affinity IgG, which will mature to high-affinity anti-CoV IgG only after repeated infections.

Antibody-Dependent Enhancement in CoV Infections

Coronaviruses make use of ADE as an alternative mode of viral fusion with target cells (Fig. 1A) [Reference Wang8,Reference Wan9]. Both SARS virus S (spike) proteins contain a binding domain for the the angiotensin-converting enzyme 2 (ACE2) protein [Reference Yan10]. Antibodies targeting the receptor binding sites can prevent S-protein:ACE2 binding and potentially viral fusion [Reference Tian11]. However, anti-S protein IgG complexed with virus will facilitate virus-IgG uptake via the Fc family of receptors [Reference Wang8,Reference Wan9]. This can lead to subsequent viral fusion and infection in macrophages, B cells, monocytes, increasing sources of viral production, and decreasing viral clearance. Binding of complement to antigen–antibody complexes formed by IgG1 and IgG3 may also facilitate ADE via complement receptors. Normally, a mechanism for viral clearance and antigen presentation, phagocytosis now potentiates viral infection. This mechanism is well known for SARS-CoV-1, respiratory syncytial (RSV), HIV, and dengue virus (DENV) [Reference Wan9].

Fig. 1.Antibody-dependent enhancement (ADE). (A) Mechanism – normal viral fusion occurs with binding of the coronavirus spike protein to its receptor, the angiotensin-converting enzyme 2 protein (ACE2). This induces a conformational change in the S protein, exposing a membrane fusion domain, resulting in viral fusion and mRNA release into the cell. With ADE, antibody binding to the S-protein both facilitates cell binding via the FcRγ and induces a conformational change in the spike protein exposing the fusion domain. A similar process can occur if the IgG binds complement, with the C3b:IgG:virus complex being taken up via the C3b receptor. (B) The ADE-associated spike glycoprotein peptide sequences S579-603 from SARS [12] are also conserved in SARS-CoV-2 strains (bold) and the closely related bat CoV strain (RaTG). There is less sequence homology in MERS and the common human CoV strains (HKU1, OC43, 229 E). Sequence homologies analyzed with the Clustal Omega method using Unipro UGENE v33.0 software.

The protein sequences responsible for ADE have been identified on the S protein (Fig. 1B) [Reference Wang12]. Importantly, sera from SARS-CoV-1 patients contain a mixture of IgG antibodies that both inhibit infection and cause ADE [Reference Jaume13,Reference Ho14]. Similarly, vaccination with recombinant S protein in animal models can elicit both neutralizing and ADE-inducing IgG antibodies [Reference Wang12]. Even the presence of neutralizing antibodies can cause severe disease, including cytokine storm, similar to that seen in DENV [Reference Rothman15].

Preexisting anti-coronavirus IgG antibodies that cross-react with SARS-CoV-2, including those against common and less pathogenic coronavirus strains, may increase the risk of ADE and the severity of COVID-19 disease [Reference Wan9]. This is a well-described phenomenon for DENV, where antibodies against one strain are a risk factor for severe disease during infection with another DENV strain [Reference Dejnirattisai16]. Thus, high-affinity anti-CoV IgG may be most effective not only in neutralizing CoV binding during infection but also increase the risk for ADE. This is not a hypothetical concern. Clinical trials for DENV and RSV vaccines were halted when vaccinated subjects were found to have increased disease severity after viral infection [Reference Halstead17,Reference Kim18].

Anti-CoV IgG Levels and Infection Severity

Paradoxically, these findings suggest that lower levels of anti-SARS-CoV-2 IgG antibodies might, in some cases, explain decreased severity of COVID-19 in subjects ≤20 years of age. Their relative lack of high-affinity, cross-reactive, anti-SARS-CoV antibodies, with the associated absence of ADE, may contribute to lower viral loads as fewer host cells become infected and produce virus. Second, the development of high-affinity, class-switched IgG antibodies can occur during the immune response around days 7–14 and can increase after multiple rounds of antigenic exposure with serial vaccination (e.g. priming and boosting) or recurrent infection. Emergence of such antibodies during a primary infection, or after prior vaccination or infection, may increase the risk of ADE [Reference Wan9]. Importantly, we currently lack data on how the balance of neutralizing versus ADE-inducing IgG to SARS-CoV-2 may differ in children and adults. Finally, ADE has been linked to the development of cytokine storm syndrome, which occurs in the most severe cases of MERS, SARS, and COVID-19 infection [Reference Tetro4,Reference Jaume13]. Thus, absence of high-affinity anti-SARS-CoV-2 IgG could potentially mitigate infection severity and explain the milder disease in children and younger adults.

This hypothesis comes with a number of questions and caveats. It is important to note that a lower severity of disease in children will also be influenced by other immune-related factors, including the relative immaturity of macrophages and monocytes in infants, lower levels of Th1, Th2, and Th17 CD4 memory T cells in adolescents and young adults, and lower memory B cell repertoire diversity in younger individuals [Reference Simon, Hollander and McMichael19]. In addition, we currently lack data on whether IgG antibodies against spike proteins from less pathogenic human strains are prevalent in the 6–20-year-old age group and on the balance of neutralizing versus ADE-inducing IgG before, during, and after SARS-CoV-2 infection. Further work will be needed to assess this hypothesis.

Implications for Vaccine Development

The above data suggest that development of a SARS-CoV-2 vaccine will require careful design and testing to assure efficacy and safety. There are several vaccine types currently being pursued including mRNA, DNA, recombinant protein, virus-like particle, and live-attenuated or killed virus. With the potential exception of live, attenuated virus vaccines, the general goal is to induce adaptive immune response resulting in high-affinity IgG against S or N viral capsid proteins. However, unless care is taken to modify the protein sequences to remove or inactivate regions highly associated with ADE, if this is even possible, we may produce vaccines that enhance, rather than protect against, severe SARS-CoV-2 infection. This could be particularly problematic in children, with their reduced risk of severe infection.

Given these issues, we suggest several translational considerations for vaccine development and clinical trials. It may be useful to group these by translational stage and category (Table 1). First, the immunodominance of various antibody subsets should be assessed carefully. This should include mapping epitope targets on SARS-CoV-2 protein sites, especially those known to induce ADE, with the goal of altering the vaccine antigens to minimize ADE. Next, clinical trials will need to be designed to specifically look for ADE in vaccine recipients who are subsequently infected. These should include pre- and post-vaccination measurement of anti-SARS-CoV-2-reactive IgG and the fraction of such antibodies directed against ADE-associated epitopes. Additional in vitro and in vivo testing of vaccine recipient sera for ability to induce ADE should be performed. Considering the incidence of milder disease in many younger individuals, and the potential for increasing the risk of ADE in vaccinated children subsequently infected with SARS-CoV-2, initial clinical trials should carefully consider whether to include children.

Table 1. Translational considerations for SARS-CoV-2 vaccine development

Importantly, long-term follow-up of vaccinated cohorts will be essential to assess vaccine efficacy and the risk of ADE. We will likely need to track the proportion of protective versus ADE-inducing antibodies generated by each vaccine type. Vaccine-specific variations in ADE could occur for many reasons, including differences in vaccine adjuvant, vaccine protein glycosylation, and prior exposure to other CoV strains. Multiplex methods developed for influenza can be quickly adapted to this use [Reference Wang, Wiltse and Zand20], especially to assess the balance between vaccine-induced protection from infection versus increased risk of severe disease with subsequent infection despite vaccination. Further clinical studies will be needed to assess this risk in both vaccinated and infected individuals. Such data may become even more critical as SARS-CoV-2 virus mutates or becomes seasonal.

Similar issues may emerge with the use of convalescent plasma to treat SARS-CoV-2 infection [Reference Casadevall and Anne Pirofski21], especially with hyperimmune plasma from vaccinated individuals, and with targeted monoclonal antibodies. The hypothesis is that such plasma or antibodies may improve infection morbidity and mortality, if given early enough in the course of COVID-19 illness [Reference Casadevall and Anne Pirofski21]. However, the presence of neutralizing antibodies has also been associated with a worse prognosis in some individuals with SARS-CoV-1 [Reference Ho14]. Similar phenomena could also occur with targeted monoclonal antibody therapies. It may be prudent to assess for ADE-inducing antibodies in convalescent plasma and therapeutic monoclonal antibodies prepared for clinical administration. Given the paucity of data on this issue, further work will be needed to rigorously determine whether this is indeed a significant issue.

The current urgency for COVID-19 therapies has brought together the scientific community to find treatments. In our rush to develop vaccines and antibody-based therapies, we should be mindful of what we have learned about ADE from SARS-CoV-1, HIV, and dengue virus research. A rapid but careful approach to vaccine, convalescent plasma, and targeted monoclonal antibody therapies for COVID-19 treatment seems warranted until we have more data on the risks of ADE.

Acknowledgments

This work was supported by the National Institutes of Health Institute of Allergy, Immunology and Infectious Diseases grant R21 AI138500, and the University of Rochester Clinical and Translational Science Award UL1 TR002001 from the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. None of the above funders had any role in the decision to publish or preparation of the manuscript.

Disclosures

The authors have no competing interests to declare.


References

1 Wu, Z, McGoogan, JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72,000 cases from the Chinese Center for Disease Control and Prevention. JAMA 2020. doi: 10.1001/jama.2020.2648.CrossRefGoogle Scholar

2 Bi, Q, et al. Epidemiology and transmission of COVID-19 in Shenzhen China: analysis of 391 cases and 1,286 of their close contacts. medRxiv. 2020; p. 2020.03.03.20028423. doi: 10.1101/2020.03.03.20028423.Google Scholar

3 COVID-19 Response Team C. Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) – United States. MMWR Morbidity Mortality Weekly Report 2020; 69. doi: 10.15585/mmwr.mm6912e2.CrossRefGoogle Scholar

4Tetro, JA. Is COVID-19 receiving ADE from other coronaviruses? Microbes and Infection 2020. doi: 10.1016/j.micinf.2020.02.006.CrossRefGoogle ScholarPubMed

5Chan-Yeung, M, Xu, RH. SARS: epidemiology. Respirology 2003; 8 Suppl: S9–S14. doi: 10.1046/j.1440-1843.2003.00518.x.CrossRefGoogle ScholarPubMed

6 van der Zalm, MM, et al. Respiratory pathogens in children with and without respiratory symptoms. The Journal of Pediatrics 2009; 154(3): 396–400.e1. doi: 10.1016/j.jpeds.2008.08.036.CrossRefGoogle ScholarPubMed

7 Zhou, W, et al. First infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood. BMC Infectious Diseases 2013; 13(1): 433. doi: 10.1186/1471-2334-13-433.CrossRefGoogle ScholarPubMed

8 Wang, SF, et al. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochemical Biophysical Research Communications 2014; 451(2): 208–214. doi: 10.1016/j.bbrc.2014.07.090.CrossRefGoogle ScholarPubMed

9 Wan, Y, et al. Molecular mechanism for antibody-dependent enhancement of coronavirus entry. Journal of Virology 2020; 94(5). doi: 10.1128/JVI.02015-19.Google ScholarPubMed

10 Yan, R, et al. Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2. Science 2020; doi: 10.1126/science.abb2762.CrossRefGoogle ScholarPubMed

11 Tian, X, et al. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerging Microbes & Infections 2020; 9(1): 382–385. doi: 10.1080/22221751.2020.1729069.CrossRefGoogle ScholarPubMed

12 Wang, Q, et al. Immunodominant SARS coronavirus epitopes in humans elirefd both enhancing and neutralizing effects on infection in non-human primates. ACS Infectious Diseases 2016; 2(5): 361–376. doi: 10.1021/acsinfecdis.6b00006.CrossRefGoogle ScholarPubMed

13 Jaume, M, et al. Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcgammaR pathway. Journal of Virology 2011; 85(20): 10582–10597. doi: 10.1128/JVI.00671-11.CrossRefGoogle Scholar

14 Ho, MS, et al. Neutralizing antibody response and SARS severity. Emerging Infectious Diseases 2005; 11(11): 1730–1737. doi: 10.3201/eid1111.040659.CrossRefGoogle ScholarPubMed

15 Rothman, AL. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nature Reviews Immunology 2011; 11(8): 532–543. doi: 10.1038/nri3014.CrossRefGoogle ScholarPubMed

16 Dejnirattisai, W, et al. Cross-reacting antibodies enhance dengue virus infection in humans. Science 2010; 328(5979): 745–748. doi: 10.1126/science.1185181.CrossRefGoogle ScholarPubMed

17 Halstead, SB. Dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age. Vaccine 2017; 35(47): 6355–6358. doi: 10.1016/j.vaccine.2017.09.089.CrossRefGoogle ScholarPubMed

18 Kim, HW, et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. American Journal of Epidemiology 1969; 89(4): 422–434. doi: 10.1093/oxfordjournals.aje.a120955.CrossRefGoogle ScholarPubMed

19 Simon, AK, Hollander, GA, McMichael, A. Evolution of the immune system in humans from infancy to old age. Proceedings of the Royal Society B: Biological Sciences 2015; 282(1821): 20143085. doi: 10.1098/rspb.2014.3085.CrossRefGoogle ScholarPubMed

20 Wang, J, Wiltse, A, Zand, MS. A complex dance: measuring the multidimensional worlds of influenza virus evolution and anti-influenza immune responses. Pathogens 2019; 8(4). doi: 10.3390/pathogens8040238.CrossRefGoogle ScholarPubMed

21 Casadevall, A, Anne Pirofski, L. The convalescent sera option for containing COVID-19. The Journal of Clinical Investigation 2020; 130(4). doi: 10.1172/JCI138003.CrossRefGoogle ScholarPubMed

Worse Than the Disease? Reviewing Some Possible Unintended Consequences of the mRNA Vaccines Against COVID-19

Authors: Stephanie Seneff1 and Greg Nigh Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge MA, 02139, USA, E- Naturopathic Oncology, Immersion Health, Portland, OR 97214,

ABSTRACT

Operation Warp Speed brought to market in the United States two mRNA vaccines, produced by Pfizer and Moderna. Interim data suggested high efficacy for both of these vaccines, which helped legitimize Emergency Use Authorization (EUA) by the FDA. However, the exceptionally rapid movement of these vaccines through controlled trials and into mass deployment raises multiple safety concerns. In this review we first describe the technology underlying these vaccines in detail. We then review both components of and the intended biological response to these vaccines, including production of the spike protein itself, and their potential relationship to a wide range of both acute and long-term induced pathologies, such as blood disorders, neurodegenerative diseases and autoimmune diseases. Among these potential induced pathologies, we discuss the relevance of prion-protein-related amino acid sequences within the spike protein. We also present a brief review of studies supporting the potential for spike protein “shedding”, transmission of the protein from a vaccinated to an unvaccinated person, resulting in symptoms induced in the latter. We finish by addressing a common point of debate, namely, whether or not these vaccines could modify the DNA of those receiving the vaccination. While there are no studies demonstrating definitively that this is happening, we provide a plausible scenario, supported by previously established pathways for transformation and transport of genetic material, whereby injected mRNA could ultimately be incorporated into germ cell DNA for transgenerational transmission. We conclude with our recommendations regarding surveillance that will help to clarify the long-term effects of these experimental drugs and allow us to better assess the true risk/benefit ratio of these novel technologies. Keywords: antibody dependent enhancement, autoimmune diseases, gene editing, lipid nanoparticles, messenger RNA, prion diseases, reverse transcription, SARS-CoV-2 vaccines Introduction Unprecedented. This word has defined so much about 2020 and the pandemic related to SARSCoV-2. In addition to an unprecedented disease and its global response, COVID-19 also initiated an unprecedented process of vaccine research, production, testing, and public distribution (Shaw, International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 38 2021). The sense of urgency around combatting the virus led to the creation, in March 2020, of Operation Warp Speed (OWS), then-President Donald Trump’s program to bring a vaccine against COVID-19 to market as quickly as possible (Jacobs and Armstrong, 2020). OWS established a few more unprecedented aspects of COVID-19. First, it brought the US Department of Defense into direct collaboration with US health departments with respect to vaccine distribution (Bonsell, 2021). Second, the National Institutes of Health (NIH) collaborated with the biotechnology company Moderna in bringing an unprecedented type of vaccine against infectious disease to market, one utilizing a technology based on messenger RNA (mRNA) (National Institutes of Health, 2020). The confluence of these unprecedented events has rapidly brought to public awareness the promise and potential of mRNA vaccines as a new weapon against infectious diseases into the future. At the same time, events without precedent are, by definition, without a history and context against which to fully assess risks, hoped-for benefits, safety, and long-term viability as a positive contribution to public health. Unprecedented In this paper we will be briefly reviewing one particular aspect of these unprecedented events, Many aspects of Covid-19 and subsequent namely the development and deployment of vaccine development are unprecedented for a mRNA vaccines against the targeted class of vaccine deployed for use in the general infectious diseases under the umbrella of “SARS- population. Some of these includes the CoV-2.” We believe many of the issues we raise following. here will be applicable to any future mRNA 1. First to use PEG (polyethylene glycol) in an vaccine that might be produced against other injection (see text) infectious agents, or in applications related to 2. First to use mRNA vaccine technology cancer and genetic diseases, while others seem against an infectious agent 3. First time Moderna has brought any product specifically relevant to mRNA vaccines currently to market being implemented against the subclass of corona 4. First to have public health officials telling viruses. While the promises of this technology those receiving the vaccination to expect an have been widely heralded, the objectively adverse reaction assessed risks and safety concerns have received 5. First to be implemented publicly with nothing more than preliminary efficacy data far less detailed attention. It is our intention to (see text) review several highly concerning molecular 6. First vaccine to make no clear claims about aspects of infectious disease-related mRNA reducing infections, transmissibility, or technology, and to correlate these with both deaths documented and potential pathological effects. 7. First coronavirus vaccine ever attempted in humans 8. First injection of genetically modified Vaccine Development polynucleotides in the general population Development of mRNA vaccines against infectious disease is unprecedented in many ways. In a 2018 publication sponsored by the Bill and Melinda Gates Foundation, vaccines were divided into three categories: Simple, Complex, and Unprecedented (Young et al., 2018). Simple and Complex vaccines represented standard and modified applications of existing vaccine technologies. Unprecedented represents a category of International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 39 vaccine against a disease for which there has never before been a suitable vaccine. Vaccines against HIV and malaria are examples. As their analysis indicates, depicted in Figure 1, unprecedented vaccines are expected to take 12.5 years to develop. Even more ominously, they have a 5% estimated chance of making it through Phase II trials (assessing efficacy) and, of that 5%, a 40% chance of making it through Phase III trials (assessing population benefit). In other words, an unprecedented vaccine was predicted to have a 2% probability of success at the stage of a Phase III clinical trial. As the authors bluntly put it, there is a “low probability of success, especially for unprecedented vaccines.” (Young et al., 2018) Figure 1. Launching innovative vaccines is costly and time-consuming, with a low probability of success, especially for unprecedented vaccines (adapted from Young et al, 2018). With that in mind, two years later we have an unprecedented vaccine with reports of 90-95% efficacy (Baden et al. 2020). In fact, these reports of efficacy are the primary motivation behind public support of vaccination adoption (U.S. Department of Health and Human Services, 2020). This defies not only predictions, but also expectations. The British Medical Journal (BMJ) may be the only prominent conventional medical publication that has given a platform to voices calling attention to concerns around the efficacy of the COVID-19 vaccines. There are indeed reasons to believe that estimations of efficacy are in need of re-evaluation. Peter Doshi, an associate editor of the BMJ, has published two important analyses (Doshi 2021a, 2021b) of the raw data released to the FDA by the vaccine makers, data that are the basis for the claim of high efficacy. Unfortunately, these were published to the BMJ’s blog and not in its peerreviewed content. Doshi, though, has published a study regarding vaccine efficacy and the questionable utility of vaccine trial endpoints in BMJ’s peer reviewed content (Doshi 2020). A central aspect of Doshi’s critique of the preliminary efficacy data is the exclusion of over 3400 “suspected COVID-19 cases” that were not included in the interim analysis of the Pfizer vaccine data submitted to the FDA. Further, a low-but-non-trivial percent of individuals in both Moderna International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 40 and Pfizer trials were deemed to be SARS-CoV-1-positive at baseline despite prior infection being grounds for exclusion. For these and other reasons the interim efficacy estimate of around 95% for both vaccines is suspect. A more recent analysis looked specifically at the issue of relative vs. absolute risk reduction. While the high estimates of risk reduction are based upon relative risks, the absolute risk reduction is a more appropriate metric for a member of the general public to determine whether a vaccination provides a meaningful risk reduction personally. In that analysis, utilizing data supplied by the vaccine makers to the FDA, the Moderna vaccine at the time of interim analysis demonstrated an absolute risk reduction of 1.1% (p= 0.004), while the Pfizer vaccine absolute risk reduction was 0.7% (p<0.000) (Brown 2021). Others have brought up important additional questions regarding COVID-19 vaccine development, questions with direct relevance to the mRNA vaccines reviewed here. For example, Haidere, et. al. (2021) identify four “critical questions” related to development of these vaccines, questions that are germane to both their safety and their efficacy: • Will Vaccines Stimulate the Immune Response? • Will Vaccines Provide Sustainable Immune Endurance? • How Will SARS-CoV-2 Mutate? • Are We Prepared for Vaccine Backfires? Lack of standard and extended preclinical and clinical trials of the two implemented mRNA vaccines leaves each of these questions to be answered over time. It is now only through observation of pertinent physiological and epidemiological data generated by widescale delivery of the vaccines to the general public that these questions will be resolved. And this is only possible if there is free access to unbiased reporting of outcomes — something that seems unlikely given the widespread censorship of vaccine-related information because of the perceived need to declare success at all cost. The two mRNA vaccines that have made it through phase 3 trials and are now being delivered to the general population are the Moderna vaccine and the Pfizer-BioNTech vaccine. The vaccines have much in common. Both are based on mRNA encoding the spike protein of the SARS-CoV-2 virus. Both demonstrated a relative efficacy rate of 94-95%. Preliminary indications are that antibodies are still present after three months. Both recommend two doses spaced by three or four weeks, and recently there are reports of annual booster injections being necessary (Mahose, 2021). Both are delivered through muscle injection, and both require deep-freeze storage to keep the RNA from breaking down. This is because, unlike double-stranded DNA which is very stable, singlestrand RNA products are apt to be damaged or rendered powerless at warm temperatures and must be kept extremely cold to retain their potential efficacy (Pushparajah et al., 2021). It is claimed by the manufacturers that the Pfizer vaccine requires storage at -94 degrees Fahrenheit (-70 degrees Celsius), which makes it very challenging to transport it and keep it cold during the interim before it is finally administered. The Moderna vaccine can be stored for 6 months at -4 degrees Fahrenheit (- 20 degrees Celsius), and it can be stored safely in the refrigerator for 30 days following thawing (Zimmer et al., 2021). International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 41 Two other vaccines that are now being administered under emergency use are the Johnson & Johnson vaccine and the AstraZeneca vaccine. Both are based on a vector DNA technology that is very different from the technology used in the mRNA vaccines. While these vaccines were also rushed to market with insufficient evaluation, they are not the subject of this paper so we will just describe briefly how they are developed. These vaccines are based on a defective version of an adenovirus, a double-stranded DNA virus that causes the common cold. The adenovirus has been genetically modified in two ways, such that it cannot replicate due to critical missing genes, and its genome has been augmented with the DNA code for the SARS-CoV-2 spike protein. AstraZeneca’s production involves an immortalized human cell line called Human Embryonic Kidney (HEK) 293, which is grown in culture along with the defective viruses (Dicks et al., 2012). The HEK cell line was genetically modified back in the 1970s by augmenting its DNA with segments from an adenovirus that supply the missing genes needed for replication of the defective virus (Louis et al., 1997). Johnson & Johnson uses a similar technique based on a fetal retinal cell line. Because the manufacture of these vaccines requires genetically modified human tumor cell lines, there is the potential for human DNA contamination as well as many other potential contaminants. The media has generated a great deal of excitement about this revolutionary technology, but there are also concerns that we may not be realizing the complexity of the body’s potential for reactions to foreign mRNA and other ingredients in these vaccines that go far beyond the simple goal of tricking the body into producing antibodies to the spike protein. In the remainder of this paper, we will first describe in more detail the technology behind mRNA vaccines. We devote several sections to specific aspects of the mRNA vaccines that concern us with regard to potential for both predictable and unpredictable negative consequences. We conclude with a plea to governments and the pharmaceutical industry to consider exercising greater caution in the current undertaking to vaccinate as many people as possible against SARS-CoV-2. Technology of mRNA Vaccines In the early phase of nucleotide-based gene therapy development, there was considerably more effort invested in gene delivery through DNA plasmids rather than through mRNA technology. Two major obstacles for mRNA are its transient nature due to its susceptibility to breakdown by RNAses, as well as its known power to invoke a strong immune response, which interferes with its transcription into protein. Plasmid DNA has been shown to persist in muscle up to six months, whereas mRNA almost certainly disappears much sooner. For vaccine applications, it was originally thought that the immunogenic nature of RNA could work to an advantage, as the mRNA could double as an adjuvant for the vaccine, eliminating the arguments in favor of a toxic additive like aluminum. However, the immune response results not only in an inflammatory response but also the rapid clearance of the RNA and suppression of transcription. So this idea turned out not to be practical. There was an extensive period of time over which various ideas were explored to try to keep the mRNA from breaking down before it could produce protein. A major advance was the realization that substituting methyl-pseudouridine for all the uridine nucleotides would stabilize RNA against degradation, allowing it to survive long enough to produce adequate amounts of protein antigen International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 42 needed for immunogenetics (Liu, 2019). This form of mRNA delivered in the vaccine is never seen in nature, and therefore has the potential for unknown consequences. The Pfizer-BioNTech and Moderna mRNA vaccines are based on very similar technologies, where a lipid nanoparticle encloses an RNA sequence coding for the full-length SARS-CoV-2 spike protein. In the manufacturing process, the first step is to assemble a DNA molecule encoding the spike protein. This process has now been commoditized, so it’s relatively straightforward to obtain a DNA molecule from a specification of the sequence of nucleotides (Corbett et al., 2020). Following a cell-free in vitro transcription from DNA, utilizing an enzymatic reaction catalyzed by RNA polymerase, the single-stranded RNA is stabilized through specific nucleoside modifications, and highly purified. The company Moderna, in Cambridge, MA, is one of the developers of deployed mRNA vaccines for SARS-CoV-2. Moderna executives have a grand vision of extending the technology for many applications where the body can be directed to produce therapeutic proteins not just for antibody production but also to treat genetic diseases and cancer, among others. They are developing a generic platform where DNA is the storage element, messenger RNA is the “software” and the proteins that the RNA codes for represent diverse application domains. The vision is grandiose and the theoretical potential applications are vast (Moderna, 2020). The technology is impressive, but manipulation of the code of life could lead to completely unanticipated negative effects, potentially long term or even permanent. SARS-CoV-2 is a member of the class of positive-strand RNA viruses, which means that they code directly for the proteins that the RNA encodes, rather than requiring a copy to an antisense strand prior to translation into protein. The virus consists primarily of the single-strand RNA molecule packaged up inside a protein coat, consisting of the virus’s structural proteins, most notably the spike protein, which facilitates both viral binding to a receptor (in the case of SARS-CoV-2 this is the ACE2 receptor) and virus fusion with the host cell membrane. The SARS-CoV-2 spike protein is the primary target for neutralizing antibodies. It is a class I fusion glycoprotein, and it is analogous to haemagglutinin produced by influenza viruses and the fusion glycoprotein produced by syncytial viruses, as well as gp160 produced by human immunodeficiency virus (HIV) (Corbett et al., 2020). The mRNA vaccines are the culmination of years of research in exploring the possibility of using RNA encapsulated in a lipid particle as a messenger. The host cell’s existing biological machinery is co-opted to facilitate the natural production of protein from the mRNA. The field has blossomed in part because of the ease with which specific oligonucleotide DNA sequences can be synthesized in the laboratory without the direct involvement of living organisms. This technology has become commoditized and can be done at large-scale, with relatively low cost. Enzymatic conversion of DNA to RNA is also straightforward, and it is feasible to isolate essentially pure single-strand RNA from the reaction soup (Kosuri and Church, 2014). 1. Considerations in mRNA Selection and Modification While the process is simple in principle, the manufacturers of mRNA vaccines do face some considerable technical challenges. The first, as we’ve discussed, is that extracellular mRNA itself can induce an immune response which would result in its rapid clearance before it is even taken up by International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 43 cells. So, the mRNA needs to be encased in a nanoparticle that will keep it hidden from the immune system. The second issue is getting the cells to take up the nanoparticles. This can be solved in part by incorporating phospholipids into the nanoparticle to take advantage of natural pathways of lipid particle endocytosis. The third problem is to activate the machinery that is involved in translating RNA into protein. In the case of SARS-CoV-2, the protein that is produced is the spike protein. Following spike protein synthesis, antigen-presenting cells need to present the spike protein to T cells, which will ultimately produce protective memory antibodies (Moderna, 2020). This step is not particularly straightforward, because the nanoparticles are mostly taken up by muscle cells, which, being immobile, are not necessarily equipped to launch an immune response. As we will see, the likely scenario is that the spike protein is synthesized by muscle cells and then handed over to macrophages acting as antigen-presenting cells, which then launch the standard B-cell-based antibody-generating cascade response. The mRNA that is enclosed in the vaccines undergoes several modification steps following its synthesis from a DNA template. Some of these steps involve preparing it to look exactly like a human mRNA sequence appropriately modified to support ribosomal translation into protein. Other modifications have the goal of protecting it from breakdown, so that sufficient protein can be produced to elicit an antibody response. Unmodified mRNA induces an immune response that leads to high serum levels of interferon-α (IF- α), which is considered an undesirable response. However, researchers have found that replacing all of the uridines in the mRNA with N-methyl-pseudouridine enhances stability of the molecule while reducing its immunogenicity (Karikó et al. 2008; Corbett et al., 2020). This step is part of the preparation of the mRNA in the vaccines, but, in addition, a 7- methylguanosine “cap” is added to the 5’ end of the molecule and a poly-adenine (poly-A) tail, consisting of 100 or more adenine nucleotides, is added to the 3’ end. The cap and tail are essential in maintaining the stability of the mRNA within the cytosol and promoting translation into protein (Schlake et al., 2012; Gallie, 1991). Normally, the spike protein flips very easily from a pre-fusion configuration to a post-fusion configuration. The spike protein that is in these vaccines has been tweaked to encourage it to favor a stable configuration in its prefusion state, as this state provokes a stronger immune response (Jackson et al., 2020). This was done via a “genetic mutation,” by replacing a critical two-residue segment with two proline residues at positions 986 and 987, at the top of the central helix of the S2 subunit (Wrapp et al., 2020). Proline is a highly inflexible amino acid, so it interferes with the transition to the fusion state. This modification provides antibodies much better access to the critical site that supports fusion and subsequent cellular uptake. But might this also mean that the genetically modified version of the spike protein produced by the human host cell following instructions from the vaccine mRNA lingers in the plasma membrane bound to ACE2 receptors because of impaired fusion capabilities? What might be the consequence of this? We don’t know. Researchers in China published a report in Nature in August 2020 in which they presented data on several experimental mRNA vaccines where the mRNA coded for various fragments and proteins in the SARS-CoV-2 virus. They tested three distinct vaccine formulations for their ability to induce an appropriate immune response in mice. The three structural proteins, S (spike), M and E are minimal requirements to assemble a “virus-like particle” (VLP). Their hypothesis was that providing M and E as well as the S spike protein in the mRNA code would permit the assembly of VLPs that might International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 44 elicit an improved immune response, because they more closely resemble the natural virus than S protein exposed on the surface of cells that have taken up only the S protein mRNA from the vaccine nanoparticles. They were also hoping that critical fragments of the spike protein would be sufficient to induce immunity, rather than the entire spike protein, if viral-like particles could be produced through augmentation with M and E (Lu et al., 2020). They confirmed experimentally that a vaccine containing the complete genes for all three proteins elicited a robust immune response that lasted for at least eight weeks following the second dose of the vaccine. Its performance was far superior to that of a vaccine containing only the spike protein. Disappointingly, a vaccine that contained only critical components of the spike protein, augmented with the other two envelope proteins, elicited practically no response. Moderna researchers have conducted similar studies with similar results. They concluded that the spike protein alone was clearly inferior to a formulation containing RNA encoding all three envelope proteins, and they hypothesized that this was due to the fact that all three proteins were needed to allow the cell to release intact virus-like particles, rather than to just post the spike protein in the plasma membrane. The spike protein alone failed to initiate a T cell response in animal studies, whereas the formulation with all three proteins did (Corbett et al., 2020). The two emergency-approved vaccines only contain mRNA code for spike protein (without E or M), and there must have been a good reason for this decision, despite its observed poor performance. It is possible that more sophisticated design of the lipid nanoparticle (see below) resulted in the ability to have the lipids serve as an adjuvant (similar to aluminum that is commonly added to traditional vaccines) while still protecting the RNA from degradation. Another curious modification in the RNA code is that the developers have enriched the sequence in cytosines and guanines (Cs and Gs) at the expense of adenines and uracils (As and Us). They have been careful to replace only the third position in the codon in this way, and only when it does not alter the amino acid map (Hubert, 2020). It has been demonstrated experimentally that GC-rich mRNA sequences are expressed (translated into protein) up to 100-fold more efficiently than GCpoor sequences (Kudla et al., 2006). So this appears to be another modification to further assure synthesis of abundant copies of the spike protein. We do not know the unintended consequences of this maneuver. Intracellular pathogens, including viruses, tend to have low GC content compared to the host cell’s genome (Rocha and Danchin, 2020). So, this modification may have been motivated in part by the desire to enhance the effectiveness of the deception that the protein is a human protein. All of these various modifications to the RNA are designed to make it resist breakdown, appear more like a human messenger RNA protein-coding sequence, and efficiently translate into antigenic protein. 2. Lipid Nanoparticle Construction Lipid nanoparticles (LNPs), also known as liposomes, can encapsulate RNA molecules, protecting them from enzymatic degradation by ribonucleases, and thus they form an essential ingredient of a successful delivery method (Wadhwa et al., 2020; Xu et al., 2020). These artificial constructs closely resemble exosomes. Exosomes are extracellullar vesicles secreted by cells and taken up by their International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 45 neighbors, and they also often embed DNA or RNA. Thus, these nanoparticles can take advantage of natural endocytosis processes that normally internalize extracellular exosomes into endosomes. As the endosome acidifies to become a lysosome, the mRNA is released into the cytoplasm, and this is where translation into protein takes place. Liposomes have actually been found to be more successful at enhancing antigen presentation and maturation of dendritic cells, when compared to fusion proteins that encapsulate virus-based vaccines (Norling et al., 2019). The lipid nanoparticles (LNPs) in these vaccines are composed of ionizable cationic lipids, phospholipids, cholesterol and polyethyleine glycol (PEG). Together, this mixture assembles into a stable lipid bilayer around the mRNA molecule. The phospholipids in these experimental vaccines consist of a phosphatidylcholine headgroup connected to two saturated alkyl tails through a glycerol linker. The lipid used in these vaccines, named 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), has 18 repeat carbon units. The relatively long chain tends to form a gel phase rather than a fluid phase. Molecules with shorter chains (such as a 12-carbon chain) tend to stay in a fluid phase instead. Gel phase liposomes utilizing DSPC have been found to have superior performance in protecting RNA from degradation because the longer alkyl chains are much more constrained in their movements within the lipid domain. They also appear to be more efficient as an adjuvant, increasing the release of the cytockines tumor necrosis factor- α (TNF- α), interleukin (IL)-6 and IL1β from exposed cells (Norling et al., 2019). However, their ability to induce an inflammatory response may be the cause of the many symptoms people are experiencing, such as pain, swelling, fever and sleepiness. A study published in bioRxiv verified experimentally that these ionizable cationic lipids in lipid nanoparticles induce a strong inflammatory response in mice (Ndeupen et al., 2021). The current mRNA vaccines are delivered through intramuscular injection. Muscles contain a large network of blood vessels where immune cells can be recruited to the injection site (Zeng et al., 2020). Muscle cells generally can enhance an immune reaction once immune cells infiltrate, in response to an adjuvant (Marino et al., 2011). Careful analysis of the response to an mRNA vaccine, administered to mice, revealed that antigen is expressed initially within muscle cells and then transferred to antigen-presenting cells, suggesting “cross-priming” as the primary path for initiating a CD8 T cell response (Lazzaro et al., 2015). One can speculate that muscle cells make use of an immune response that is normally used to deal with misfolded human proteins. Such proteins induce upregulation of major histocompatibility complex (MHC) class II proteins, which then bind to the misfolded proteins and transport them intact to the plasma membrane (Jiang et al., 2013). The MHC-bound surface protein then induces an inflammatory response and subsequent infiltration of antigen-presenting cells (e.g., dendritic cells and macrophages) into the muscle tissue, which then take up the displayed proteins and carry them into the lymph system to present them to T cells. These T cells can then finally launch the cascade that ultimately produces memory antibodies specific to the protein. Muscle cells do express MHC class II proteins (Cifuentes-Diaz et al., 1992). As contrasted with class I, class II MHC proteins specialize in transporting intact proteins to the surface as opposed to small peptide sequences derived from the partial breakdown of the proteins (Jiang et al., 2013). An in vitro study on non-human primates demonstrated that radiolabeled mRNA moved from the injection site into the draining lymph node and remained there for at least 28 hours. Antigen International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 46 presenting cells (APCs) in both the muscle tissue as well as the draining lymph nodes were shown to contain radiolabeled mRNA (Lindsay et al., 2019). Classical APCs include dendritic cells, macrophages, Langerhans cells (in the skin) and B cells. Many of the side effects associated with these vaccines involve pain and inflammation at the injection site, as would be expected given the rapid infiltration of immune cells. Lymphadenopathy is an inflammatory state in the lymph system associated with swollen lymph nodes. Swollen lymph nodes in the arm pit (axillary lymphadenopathy) is a feature of metastatic breast cancer. A paper published in 2021 described four cases of women who developed axillary lymphadenopathy following a SARS-CoV-2 vaccine (Mehta et al., 2021). The authors urged caution in misinterpreting this condition as an indicator requiring biopsy follow-up for possible breast cancer. This symptom corroborates tracer studies showing that the mRNA vaccine is predominantly taken up by APCs that then presumably synthesize the antigen (spike protein) from the mRNA and migrate into the lymph system, displaying spike protein on their membranes. A list of the most common adverse effects reported by the FDA that were experienced during the Pfizer-BioNTech clinical trials include “injection site pain, fatigue, headache, muscle pain, chills, joint pain, fever, injection site swelling, injection site redness, nausea, malaise, and lymphadenopathy.” (US Food and Drug Administration, 2021). We turn now to individual molecular and organ system concerns that arise with these mRNA vaccines. Adjuvants, Polyethylene Glycol, and Anaphylaxis Adjuvants are vaccine additives intended to “elicit distinctive immunological profiles with regard to the direction, duration, and strength of immune responses” from the vaccines to which they are added (Liang et al., 2020). Alum or other aluminum compounds are most commonly utilized in traditional vaccines, and they elicit a wide range of systemic immune activation pathways as well as stromal cell activation at the site of the injection (Lambrecht et al., 2009; Danielsson & Eriksson, 2021). An aluminum-based adjuvant was determined not to be optimal for a coronavirus vaccine, so other solutions were sought (Liang et. al., 2020). A solution presented itself in the form of the widely used pharmaceutical ingredient polyethylene glycol, or PEG. A limiting factor in the use of nucleic-acidbased vaccines is the tendency for the nucleic acids to be quickly degraded by nuclease enzymes (Ho et al., 2021). Regarding the RNAse enzymes targeting injected mRNA, these enzymes are widely distributed both intracellularly (primarily within the lysosomes) (Fujiwara et al., 2017) and extracellularly (Lu et al., 2018). To overcome this limitation, both mRNA vaccines currently deployed against COVID-19 utilize lipid-based nanoparticles as delivery vehicles. The mRNA cargo is placed inside a shell composed of synthetic lipids and cholesterol, along with PEG to stabilize the mRNA molecule against degradation. The vaccine produced by Pfizer/BioNTech creates nanoparticles from 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide, or ALC-0159, commonly abbreviated simply as PEG (World Health Organization, 2021, January 14). The Moderna vaccine contains another PEG variant, SM102, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol2000 (World Health Organization, International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 47 2021, January 19). For convenience we will abbreviate both PEG-modified lipids as PEG, and refer to the vaccines as PEGylated according to standard nomenclature. The lipid shell plays a triple role. First, it protects the genetic material from degradation prior to cellular uptake. Second, the lipid shell, which also contains cholesterol, facilitates cellular uptake through fusion with the lipid membrane of the cell and subsequent endocytosis of the lipid particle, invoking naturally existing processes. And finally, it acts as an adjuvant (Ho et al., 2021). It is in this latter role as immune stimulant that most concerns have been raised regarding the widespread use of PEG in an injection therapy. In an article published in May 2019, prior to large clinical trials involving these PEGylated vaccines, Mohamed et. al. (2019) described a number of concerning findings regarding PEG and the immunological activation it had been shown to produce, which includes humoral, cell-mediated, and complement-based activation. They note that, paradoxically, large injection doses of PEG cause no apparent allergic reaction. Small doses, though, can lead to dramatic pathological immune activation. Vaccines employing PEGylation utilize micromolar amounts of these lipids, constituting this potentially immunogenic low-dose exposure. In animal studies it has been shown that complement activation is responsible for both anaphylaxis and cardiovascular collapse, and injected PEG activates multiple complement pathways in humans as well. The authors of one study conclude by noting that “This cascade of secondary mediators substantially amplifies effector immune responses and may induce anaphylaxis in sensitive individuals. Indeed, recent studies in pigs have demonstrated that systemic complement activation (e.g., induced following intravenous injection of PEGylated liposomes) can underlie cardiac anaphylaxis where C5a played a causal role.” (Hamad et al., 2008) It is also important to note that anaphylactoid shock in pigs occurred not with first injected exposure, but following second injected exposure (Kozma et al., 2019). The presence of antibodies against PEG is widespread in the population (Zhou et al., 2020). Yang and Lai (2015) found that around 42% of blood samples surveyed contained anti-PEG antibodies, and they warn that these could have important consequences for any PEG-based therapeutics introduced. Hong et. al. (2020) found anti-PEG antibodies with a prevalence up to 72% in populations with no prior exposure to PEG-based medical therapy. Lila et. al. (2018) note that the “existence of such anti-PEG antibodies has been intimately correlated with an impairment of therapeutic efficacy in tandem with the development of severe adverse effects in several clinical settings employing PEGylated-based therapeutics.” Anaphylaxis to vaccines has previously been assumed to be rare based on the frequency of such events reported to VAERS, a database established by the Centers for Disease Control and Prevention in 1990 for reporting of adverse events related to vaccines (Centers for Disease Control and Prevention, 1990; Su et al., 2019). While rare, anaphylaxis can be life-threatening, so it is important to monitor for the possibility in the short period following vaccination (McNeil et al., 2016). Sellaturay et. al., after reviewing 5 cases of anaphylaxis they link to PEG exposure, one near-fatal and involving cardiac arrest, write, “PEG is a high-risk ’hidden’ allergen, usually unsuspected and can cause frequent allergic reactions due to inadvertent re-exposure. Allergy investigation carries the risk International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 48 of anaphylaxis and should be undertaken only in specialist drug allergy centres.” (Sellaturay et al., 2020). In fact it has already been demonstrated that pre-existing antibodies to PEG are linked to more common and more severe reactions upon re-exposure (Ganson et al., 2016). Is anaphylaxis upon exposure to PEG happening with a frequency relevant to public health? Numerous studies have now documented the phenomenon (Lee et al., 2015; Povsic et al., 2016; Wylon et al., 2016). Anaphylactic reactions to the mRNA vaccines are widely reported in the media (Kelso, 2021) and, as noted above, have been frequently reported in the VAERS database (690 reports of anaphylaxis following SARS-CoV-2 vaccines up to January 29, 2021). There are also some initial case studies published in the peer-reviewed literature (Garvey & Nasser, 2020; CDC COVID19 Response Team, 2021, January 15). Anaphylaxis reactions to vaccines prior to these COVID-19 vaccines were generally reported at rates less than 2 cases per million vaccinations (McNeil et al., 2016), while the current rate with the COVID-19 vaccinations was reported by the CDC to be more than 11 cases per million (CDC COVID-19 Response Team, 2021, January 29). However, a published prospective study on 64,900 medical employees, where their reactions to their first mRNA vaccination were carefully monitored, found that 2.1% of the subjects reported acute allergic reactions. A more extreme reaction involving anaphylaxis occurred at a rate of 247 per million vaccinations (Blumenthal et al., 2021). This is more than 21 times as many as were initially reported by the CDC. The second injection exposure is likely to cause even larger numbers of anaphylactic reactions. mRNA Vaccines, Spike Proteins, and Antibody-Dependent Enhancement (ADE) ADE is an immunological phenomenon first described in 1964 (Hawkes et al., 1964). In that publication Hawkes described a set of experiments in which cultures of flavivirus were incubated with avian sera containing high titers of antibodies against those viruses. The unexpected finding was that, with increasingly high dilutions of the antibody-containing sera, cell infectivity was enhanced. Lack of an explanation for how this could happen is likely responsible for its being largely ignored for almost 20 years (Morens et al., 1994). Multiple pathways have been proposed through which antibodies both directly and indirectly participate in the neutralization of infections (Lu et al., 2018b). ADE is a special case of what can happen when low, non-neutralizing levels of either specific or cross-reactive antibodies against a virus are present at the time of infection. These antibodies might be present due to prior exposure to the virus, exposure to a related virus, or due to prior vaccination against the virus. Upon reinfection, antibodies in insufficient numbers to neutralize the virus nevertheless bind to the virus. These antibodies then dock at the Fc receptor on cell surfaces, facilitating viral entry into the cell and subsequently enhancing the infectivity of the virus (Wan et. al., 2020). ADE is believed to underlie the more severe dengue fever often observed in those with previous exposure (Beltramello et al., 2010), and might also play a role in more severe disease among those previously vaccinated against the disease (Shukla et al., 2020). ADE is also believed to play a role in Ebola (Takada et al., 2003), zika virus infection (Bardina et al., 2017), and other flavivirus infections (Campos et al., 2020). International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 49 In an extended correspondence published in Nature Biotechnology, Eroshenko et. al. offer a comprehensive review of evidence suggesting that ADE could become manifest with any vaccinations employed against SARS-CoV-2. Importantly, they note that ADE has been observed with coronavirus vaccines tested in both in vitro and in vivo models (Eroshenko et al., 2020). Others have warned about the same possibility with SARS-CoV-2 vaccines. A theory for how ADE might occur in the case of a SARS-CoV-2 vaccine suggests that non-neutralizing antibodies form immune complexes with viral antigens to provoke excessive secretion of pro-inflammatory cytokines, and, in the extreme case, a cytokine storm causing widespread local tissue damage (Lee et al., 2020). One extensive review of ADE potentially associated with SARS-CoV-2 vaccines noted, “At present, there are no known clinical findings, immunological assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells or intrinsic host responses” (Arvin et al. 2020; Liu et al., 2019). We will return to this point again below. Preexisting immunoglobulin G (IgG) antibodies, induced by prior vaccination, contribute to severe pulmonary damage by SARS-CoV in macaques (Liu et al., 2019). Peron and Nakaya (2020) provide evidence suggesting that the much more diverse range of prior exposures to coronaviruses experienced by the elderly might predispose them to ADE upon exposure to SARS-CoV-2. A concerning pre-print article reported that plasma from 76% of patients who had recovered from severe COVID-19 disease, when added to cultures of SARS-CoV-2 and susceptible cells, exhibited enhanced ability for SARS-CoV-2 viral infection of Raji cells (Wu et al., 2020). The authors note that “the antibody titers [against the spike protein] were higher in elderly patients of COVID-19, and stronger antibody response was associated with delayed viral clearance and increased disease severity in patients. Hence it is reasonable to speculate that S protein-specific antibodies may contribute to disease severity during SARS-CoV-2 infection.” (Wu et al., 2020) It has been reported that all three US vaccine manufacturers – Moderna, Pfizer, and Johnson & Johnson – are working to develop booster shots (Zaman 2021).With tens of millions of young adults and even children now with vaccine-induced coronavirus spike protein antibodies, there exists the possibility of triggering ADE related to either future SARS-CoV-2 infection or booster injection among this younger population. Time will tell. The mRNA vaccines ultimately deliver the highly antigenic spike protein to antigen-presenting cells. As such, monoclonal antibodies against the spike protein are the expected outcome of the currently deployed mRNA vaccines. Human spike protein monoclonal antibodies were found to produce high levels of cross-reactive antibodies against endogenous human proteins (Vojdani et. al., 2021; reviewed in more detail below). Given evidence only partially reviewed here, there is sufficient reason to suspect that antibodies to the spike protein will contribute to ADE provoked by prior SARS-CoV-2 infection or vaccination, which may manifest as either acute or chronic autoimmune and inflammatory conditions. We have noted above that it is not possible to distinguish an ADE manifestation of disease from a true, non-ADE viral infection. In this light it is important to recognize that, when diseases and deaths occur shortly after vaccination with an mRNA vaccine, it can never be definitively determined, even with a full investigation, that the vaccine reaction was not a proximal cause. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 50 Pathogenic Priming, Multisystem Inflammatory Disease, and Autoimmunity Pathogenic priming is a concept that is similar in outcome to ADE, but different in the underlying mechanism. We discuss it here as a unique mechanism through which the mRNA vaccines could provoke associated pathologies. In April 2020 an important paper was published regarding the potential for self-reactive antibodies to be generated following exposure to the spike protein and other antigenic epitopes spread over the length of SARS-CoV-2. Lyons-Weiler (2020) coined the phrase “pathogen priming” because he believed the more commonly used “immune enhancement” fails to capture the severity of the condition and its consequences. In his in silico analysis, Lyons-Weiler compared all antigenic SARSCoV-2 protein epitopes flagged in the SVMTriP database (http://sysbio.unl.edu/SVMTriP/) and searched the p-BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for homology between those epitopes and endogenous human proteins. Of the 37 SARS-CoV-2 proteins analyzed, 29 had antigenic regions. All but one of these 29 had homology with human proteins (putative selfantigens) and were predicted to be autoreactogenic. The largest number of homologies were associated with the spike (S) protein and the NS3 protein, both having 6 homologous human proteins. A functional analysis of the endogenous human proteins homologous with viral proteins found that over 1/3 of them are associated with the adaptive immune system. The author speculates that prior virus exposure or prior vaccination, either of which could initiate antibody production that targets these endogenous proteins, may be playing a role in the development of more severe disease in the elderly in particular. In this case the pre-existing antibodies act to suppress the adaptive immune system and lead to more severe disease. Another group (Ehrenfeld et. al., 2020), in a paper predominantly about the wide range of autoimmune diseases found in association with a prior SARS-CoV-2 infection, also investigated how the spike protein could trigger such a range of diseases. They report, in Table 1 of that reference, strings of heptapeptides within the human proteome that overlap with the spike protein generated by SARS-CoV-2. They identified 26 heptapeptides found in humans and in the spike protein. It is interesting to note that 2 of the 26 overlapping heptapeptides were found to be sequential, a strikingly long string of identical peptides to be found in common between endogenous human proteins and the spike protein. Commenting on the overlapping peptides they had discovered and the potential for this to drive many types of autoimmunity simultaneously, they comment, “The clinical scenario that emerges is upsetting.” Indeed, it is. In May of 2020 another important paper in this regard was published by Vojdani and Kharrazian (2020). The authors used both mouse and rabbit monoclonal antibodies against the 2003 SARS spike protein to test for reactivity against not only the spike protein of SARS-CoV-2, but also against several endogenous human proteins. They discovered that there was a high level of binding not only with the SARS-CoV-2 spike protein, but against a wide range of endogenous proteins. “[W]e found that the strongest reactions were with transglutaminase 3 (tTG3), transglutaminase 2 (tTG2), ENA, myelin basic protein (MBP), mitochondria, nuclear antigen (NA), α-myosin, thyroid peroxidase (TPO), collagen, claudin 5+6, and S100B.” (Vojdani and Kharrazian, 2020). International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 51 These important findings need to be emphasized. Antibodies with a high binding affinity to SARSCoV-2 spike and other proteins also have a high binding affinity with tTG (associated with Celiac Disease), TPO (Hashimoto’s thyroiditis), myelin basic protein (multiple sclerosis), and several endogenous proteins. Unlike the autoimmune process associated with pathogen priming, these autoimmune diseases typically take years to manifest symptomatically. The autoantibodies generated by the spike protein predicted by Lyons-Weiler (2020) and described above were confirmed with an in vitro study published more recently. In this follow-on paper, Vojdani et. al., (2021) looked again at the issue of cross-reactivity of antibodies, this time using human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike protein rather than mouse and rabbit mAbs. Their results confirmed and extended their prior findings. “At a cutoff of 0.32 OD [optical density], SARS-CoV-2 membrane protein antibody reacted with 18 out of the 55 tested antigens.” These 18 endogenous antigens encompass reactivity to tissue in liver, mitochondria, the nervous and digestive system, the pancreas, and elsewhere in the body. In a report on multisystem inflammatory syndrome in children (MIS-C), Carter et. al. (2020) studied 23 cases. Seventeen of 23 (68%) patients had serological evidence of prior SARS-CoV-2 infection. Of the three antibodies assessed in the patient population (nucleocapsid, RBD, and spike), IgG spike protein antibody optical density (which quantifies antibody concentrations against a standardized curve (Wikipedia, 2021)), was highest (see Figure 1d in Carter et al., 2020). MIS-C is now commonly speculated to be an example of immune priming by prior exposure to SARS-CoV-2 or to other coronaviruses. Buonsenso et. al. (2020) reviewed multiple immunologic similarities between MIS-C and disease related to prior β-hemolytic Group A streptococcal infection (GAS). The authors write, “We can speculate that children’s multiple exposition to SARS-CoV-2 with parents with COVID-19 can work as a priming of the immune system, as happens with GAS infection and, in genetically predisposed children, lead to [MIS-C] development. Another hypothesis is that previous infections with other coronaviruses, much more frequent in the pediatric population, may have primed the child immune system to SARS-CoV-2 virus.” In June 2019 Galeotti and Bayry (2020) reviewed the occurrence of both autoimmune and inflammatory diseases in patients with COVID-19. They focus their analysis on MIS-C. After reviewing several previously published reports of a temporal link between COVID-19 and onset of MIS-C and describing a number of possible mechanistic connections between the two, the authors noted that no causal link had been established. In a somewhat prescient recommendation, they wrote, “A fine analysis of homology between various antigens of SARS-CoV-2 and self-antigens, by use of in silico approaches and validation in experimental models, should be considered in order to confirm this hypothesis.” It is precisely this type of in silico analysis carried out by Lyons-Weiler (2020) and by Ehrenfeld et. al. (2020) described in the opening paragraphs of this section which found the tight homology between viral antigens and self-antigens. While this may not definitively confirm the causal link hypothesized by Galeotti and Bayry, it is strong supporting evidence. Autoimmunity is becoming much more widely recognized as a sequela of COVID-19. There are multiple reports of previously healthy individuals who developed diseases such as idiopathic thrombocytopenic purpura, Guillain-Barré syndrome and autoimmune haemolytic anaemia (Galeotti and Bayry, 2020). There are three independent case reports of systemic lupus erythemosus (SLE) International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 52 with cutaneous manifestations following symptomatic COVID-19. In one case a 39-year-old male had SLE onset two months following outpatient treatment for COVID-19 (Zamani et.al., 2021). Another striking case of rapidly progressing and fatal SLE with cutaneous manifestations is described by Slimani et.al. (2021). Autoantibodies are very commonly found in COVID-19 patients, including antibodies found in blood (Vlachoyiannopoulos et. al., 2020) and cerebrospinal fluid (CFS) (Franke et. al., 2021). Though SARS-CoV-2 is not found in the CSF, it is theorized that the autoantibodies created in response to SARS-CoV-2 exposure may lead to at least some portion of the neurological complications documented in COVID-19 patients. One important Letter to the Editor submitted to the journal Arthritis & Rheumatology by Bertin et. al. (2020) noted the high prevalence and strong association (p=0.009) of autoantibodies against cardiolipin in COVID-19 patients with severe disease. Zuo et. al. (2020) found anti-phospholipid autoantibodies in 52% of hospitalized COVID-19 patients and speculated that these antibodies contribute to the high incidence of coagulopathies in these patients. Schiaffino et. al. (2020) reported that serum from a high percentage of hospitalized COVID-19 patients contained autoantibodies reactive to the plasma membrane of hepatocytes and gastric cells. One patient with Guillain-Barre Syndrome was found to have antibody reactivity in cerebrospinal fluid (CFS), leading the authors to suggest that cross-reactivity with proteins in the CFS could lead to neurological complications seen in some COVID-19 patients. In a more recent review, Gao et. al. (2021) noted high levels of autoantibodies in COVID-19 patients across multiple studies. They conclude, “[O]ne of the potential side effects of giving a mass vaccine could be an mergence [sic] of autoimmune diseases especially in individuals who are genetically prone for autoimmunity.” A recent publication compiles a great deal of evidence that autoantibodies against a broad range of receptors and tissue can be found in individuals who have had previous SARS-CoV-2 infection. “All 31 former COVID-19 patients had between 2 and 7 different GPCR-fAABs [G-protein coupled receptor functional autoantibodies] that acted as receptor agonists.” (Wallukat et. al. 2021) The diversity of GPCR-fAABs identified, encompassing both agonist and antagonist activity on target receptors, strongly correlated with a range of post-COVID-19 symptoms, including tachycardia, bradycardia, alopecia, attention deficit, PoTS, neuropathies, and others. The same study, referencing the autoantibodies predicted by Lyons-Weiler (2020) mentioned above, notes with obvious grave concern: “The Sars-CoV-2 spike protein is a potential epitopic target for biomimicry-induced autoimmunological processes [25]. Therefore, we feel it will be extremely important to investigate whether GPCR-fAABs will also become detectable after immunisation by vaccination against the virus.” We have reviewed the evidence here that the spike protein of SARS-CoV-2 has extensive sequence homology with multiple endogenous human proteins and could prime the immune system toward development of both auto-inflammatory and autoimmune disease. This is particularly concerning given that the protein has been redesigned with two extra proline residues to potentially impede its clearance from the circulation through membrane fusion. These diseases could present acutely and International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 53 over relatively short timespans such as with MIS-C or could potentially not manifest for months or years following exposure to the spike protein, whether via natural infection or via vaccination. Many who test positive for COVID-19 express no symptoms. The number of asymptomatic, PCRpositive cases varies widely between studies, from a low of 1.6% to a high of 56.5% (Gao et. al., 2020). Those who are insensitive to COVID-19 probably have a very strong innate immune system. The healthy mucosal barrier’s neutrophils and macrophages rapidly clear the viruses, often without the need for any antibodies to be produced by the adaptive system. However, the vaccine intentionally completely bypasses the mucosal immune system, both through its injection past the natural mucosal barriers and its artificial configuration as an RNA-containing nanoparticle. As noted in Carsetti (2020), those with a strong innate immune response almost universally experience either asymptomatic infection or only mild COVID-19 disease presentation. Nevertheless, they might face chronic autoimmune disease, as described previously, as a consequence of excessive antibody production in response to the vaccine, which was not necessary in the first place. The Spleen, Platelets and Thrombocytopenia Dr. Gregory Michael, an obstetrician in Miami Beach, died of a cerebral hemorrhage 16 days after receiving the first dose of the Pfizer/BioNTech COVID-19 vaccine. Within three days of the vaccine, he developed idiopathic thrombocytopenic purpura (ITP), an autoimmune disorder in which the immune cells attack and destroy the platelets. His platelet count dropped precipitously, and this caused an inability to stop internal bleeding, leading to the stroke, as described in an article in the New York Times (Grady and Mazzei, 2021). The New York Times followed up with a second article that discussed several other cases of ITP following SARS-CoV-2 vaccination (Grady, 2021), and several other incidences of precipitous drop of platelets and thrombocytopenia following SARSCoV-2 vaccination have been reported in the Vaccine Adverse Event Reporting System (VAERS). 1. Biodistribution of mRNA Vaccines Several studies on mRNA-based vaccines have confirmed independently that the spleen is a major center of activity for the immune response. A study on an mRNA-based influenza virus vaccine is extremely relevant for answering the question of the biodistribution of the mRNA in the vaccine. This vaccine, like the SARS-CoV-2 vaccines, was designed as lipid nanoparticles with modified RNA coding for hemagglutinin (the equivalent surface fusion protein to the spike protein in corona viruses), and was administered through muscular injection. The concentration of mRNA was tracked over time in various tissue samples, and the maximum concentration observed at each site was recorded. Not surprisingly, the concentration was highest in the muscle at the injection site (5,680 ng/mL). This level decreased slowly over time, reaching half the original value at 18.8 hours following injection. The next highest level was observed in the proximal lymph node, peaking at 2,120 ng/mL and not dropping to half this value until 25.4 hours later. Among organs, the highest levels by far were found in the spleen (86.69 ng/mL) and liver (47.2 ng/mL). Elsewhere in the body the concentration was at 100- to 1,000-fold lower levels. In particular, distal lymph nodes only had a peak concentration of 8 ng/mL. They concluded that the mRNA distributes from the injection site to the liver and spleen via the lymphatic system, ultimately reaching the general circulation. This likely happens through its transport inside macrophages and other immune cells that take it up at the International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 54 muscular injection site. Disturbingly, it also reaches into the brain, although at much lower levels (Bahl et al., 2017). The European Medicines Agency assessment report for the Moderna vaccine also noted that mRNA could be detected in the brain following intramuscular administration at about 2% of the level found in the plasma (European Medicines Agency, 2021). In another experiment conducted to track the biodistribution pathway of RNA vaccines, a rabies RNA vaccine was administered intramuscularly to rats in a single dose. The vaccine included a code for an immunogenic rabies protein as well as the code for RNA polymerase and was formulated as an oil-in-water nanoemulsion. Thus, it is not entirely representative of the SARS-CoV-2 mRNA vaccines. Nevertheless, its intramuscular administration and its dependence on RNA uptake by immune cells likely means that it would migrate through the tissues in a similar pathway as the SARS-CoV-2 vaccine. The authors observed an enlargement of the draining lymph nodes, and tissue studies revealed that the rabies RNA appeared initially at the injection site and in the draining lymph nodes within one day, and was also found in blood, lungs, spleen and liver (Stokes et al., 2020). These results are consistent with the above study on influenza mRNA vaccines. Finally, a study comparing luciferase-expressing mRNA nanoparticles with luciferase-expressing mRNA dendritic cells as an alternative approach to vaccination revealed that the luciferase signal reached a broader range of lymphoid sites with the nanoparticle delivery mechanism. More importantly, the luciferase signal was concentrated in the spleen for the nanoparticles compared to dominance in the lungs for the dendritic cells (Firdessa-Fite and Creuso, 2020). 2. Immune Thrombocytopenia Immune thrombocytopenia (ITP) has emerged as an important complication of COVID-19 (Bhattacharjee and Banerjee, 2020). In many cases, it emerges after full recovery from the disease, i.e, after the virus has been cleared, suggesting it is an autoimmune phenomenon. A likely pathway by which ITP could occur following vaccination is through the migration of immune cells carrying a cargo of mRNA nanoparticles via the lymph system into the spleen. These immune cells would produce spike protein according to the code in the nanoparticles, and the spike protein would induce B cell generation of IgG antibodies to it. ITP appears initially as petechiae or purpura on the skin, and/or bleeding from mucosal surfaces. It has a high risk of fatality through haemorrhaging and stroke. ITP is characterized by both increased platelet destruction and reduced platelet production, and autoantibodies play a pivotal role (Sun and Shan, 2019). Platelets are coated by anti-platelet antibodies and immune complexes, and this induces their clearance by phagocytes. Particularly under conditions of impaired autophagy, the resulting signaling cascade can also result in suppression of production of megakaryocytes in the bone marrow, which are the precursor cells for platelet production (Sun and Shan, 2019). A case study of a patient diagnosed with COVID-19 is revealing because he developed sudden onset thrombocytopenia a couple of days after he had been released from the hospital based on a negative COVID-19 nucleic acid test. Following this development, it was verified that the patient had a reduced number of platelet-producing megakaryocytes, while autoimmune antibodies were negative, suggesting a problem with platelet production rather than platelet destruction (Chen et al., 2020). International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 55 Autophagy is essential for clearing damaged proteins, organelles, and bacterial and viral pathogens. Alterations in autophagy pathways are emerging as a hallmark of the pathogenesis of many respiratory viruses, including influenza virus, MERS-CoV, SARS-CoV and, importantly, SARS-CoV2 (Limanaqi et al., 2020). Autophagy is surely critical in the clearance of spike protein produced by immune cells programmed to produce it through the mRNA vaccines. One can speculate that impaired autophagy prevents clearance of the spike protein produced by macrophages from the vaccine mRNA. As we will show later, platelets possess autophagic proteins and use autophagy to clear viruses. Impaired autophagy is a characteristic feature of ITP, and it may be key to the autoimmune attack on the platelets (Wang et al., 2019). 3. A Critical Role for the Spleen The spleen is the largest secondary lymphoid organ in humans and it contains as much as 1/3 of the body’s platelet supplies. The spleen is the primary site for platelet destruction during ITP, as it controls the antibody response against platelets. The two main autoantibodies associated with ITP are against immunoglobulin G (IgG) and the glycoprotein (GP) IIb/IIIa complex on platelets (Aslam et al., 2016). The spleen plays a central role in the clearance of foreign antigens and the synthesis of IgG by B cells. Upon exposure to an antigen, such as the spike protein, neutrophils in the marginal zone of the spleen acquire the ability to interact with B cells, inducing antibody production (Puga et al., 2011). This is likely crucial for successful vaccination outcome. The pseudouridine modification of mRNA is important for assuring RNA survival long enough for it to reach the spleen. In an experiment on injection of mRNA nanoparticles into mice, both the delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after injection, at significantly higher levels than when non-modified RNA was used (Karikó et al., 2008). A sophisticated platelet-neutrophil cross-communication mechanism in the spleen can lead to thrombocytopenia, mediated by a pathological response called NETosis. Platelet-TLR7 (toll-like receptor 7) recognizes influenza particles in circulation and leads to their engulfment and endocytosis by the platelets. After engulfing the viruses, the platelets stimulate neutrophils to release their DNA within Neutrophil Extracellular Traps (NETs) (Koupenova et al., 2019), and the DNA, in excessive amounts, launches a prothrombotic cascade. 4. Lessons from Influenza The influenza virus, like the corona virus, is a single-strand RNA virus. Thrombocytopenia is a common complication of influenza infection, and its severity predicts clinical outcomes in critically ill patients (Jansen et al., 2020). Platelets contain abundant glycoproteins in their membranes which act as receptors and support adhesion to the endothelial wall. Autoantibodies against platelet glycoproteins are found in the majority of patients with autoimmune thrombocytopenia (Lipp et al., 1998). The influenza virus binds to cells via glycoproteins, and it releases an enzyme called neuraminidase that can break down the glycosaminoglycans bound to the glycoproteins and release them. This action likely exposes the platelet glycoproteins to B cells, inducing autoantibody production. Neuraminidase expressed by the pathogen Streptococcus pneumoniae has been shown to desialylate platelets, leading to platelet hyperactivity (Kullaya et al., 2018). International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 56 Platelets appear to play an important role in viral clearance. Within one minute after platelets were incubated together with influenza viruses, the viruses had already attached to the platelets. Subsequent internalization, possibly by phagocytosis, peaked at 30 minutes (Jansen et al., 2020). The SARS-CoV-2 spike protein binds sialic acid, which means it could attach to glycoproteins in the platelet membranes (Baker et al., 2020). There is a structural similarity between the S1 spike protein in SARS CoV and neuraminidase expressed by the influenza virus, which might mean that the spike protein possesses neuraminidase activity (Zhang et al., 2004). Several viruses express neuraminidase, and it generally acts enzymatically to catabolize the glycans in glycoproteins through desialylation. Thus, it seems plausible that a dangerous cascade leading to ITP could ensue following mRNA vaccination, even with no live virus present, particularly in the context of impaired autophagy. Immune cells in the arm muscle take up the RNA particles and circulate within the lymph system, accumulating in the spleen. There, the immune cells produce abundant spike protein, which binds to the platelet glycoproteins and desialylates them. Platelet interaction with neutrophils causes NETosis and the launch of an inflammatory cascade. The exposed glycoproteins become targets for autoimmune antibodies that then attack and remove the platelets, leading to a rapid drop in platelet counts, and a life-threatening event. Activation of Latent Herpes Zoster An observational study conducted at Tel Aviv Medical Center and the Carmel Medical Center in Haifa, Israel, found a significantly increased rate of herpes zoster following the Pfizer vaccination (Furer 2021). This observational study monitored patients with pre-existing autoimmune inflammatory rheumatic diseases (AIIRD). Among the 491 patients with AIIRD over the study period, 6 (1.2%) were diagnosed with herpes zoster as a first-ever diagnosis between 2 days and 2 weeks after either the first or second vaccination. In the control group of 99 patients there were no herpes zoster cases identified. The CDC’s VAERS database, queried on April 19, 2021, contains 278 reports of herpes zoster following either the Moderna or Pfizer vaccinations. Given the documented underreporting to VAERS (Lazarus et al. 2010), and given the associational nature of VAERS reports, it is not possible to prove any causal link between the vaccinations and the zoster reports. However, we believe the occurrence of zoster is another important ‘signal’ in VAERS. This increased risk to shingles, if valid, may have important broader implications. Multiple studies have shown that patients with either primary or acquired immune deficiency are more susceptible to severe herpes zoster infection (Ansari et al., 2020). This suggests that the mRNA vaccines may be suppressing the innate immune response. There is cross-talk between TNF- α and type I interferon in autoimmune disease, wherein each suppresses the other (Palucka et al., 2005). Type I interferon inhibits varicella-zoster virus replication (Ku et al., 2016). TNF- α is sharply upregulated in an inflammatory response, which is induced by the lipid nanoparticles in the vaccine. Its upregulation is also associated with the chronic inflammatory state of rheumatoid arthritis (Matsuno et al., 2002). Exuberant TNF-α expression following vaccination may be interfering with the dendritic cell INF-α response that keeps latent herpes zoster in check. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 57 Spike Protein Toxicity The picture is now emerging that SARS-CoV-2 has serious effects on the vasculature in multiple organs, including the brain vasculature. As mentioned earlier, the spike protein facilitates entry of the virus into a host cell by binding to ACE2 in the plasma membrane. ACE2 is a type I integral membrane protein that cleaves angiotensin II into angiotensin(1-7), thus clearing angiotensin II and lowering blood pressure. In a series of papers, Yuichiro Suzuki in collaboration with other authors presented a strong argument that the spike protein by itself can cause a signaling response in the vasculature with potentially widespread consequences (Suzuki, 2020; Suzuki et al., 2020; Suzuki et al., 2021; Suzuki and Gychka, 2021). These authors observed that, in severe cases of COVID-19, SARSCoV-2 causes significant morphological changes to the pulmonary vasculature. Post-mortem analysis of the lungs of patients who died from COVID-19 revealed histological features showing vascular wall thickening, mainly due to hypertrophy of the tunica media. Enlarged smooth muscle cells had become rounded, with swollen nuclei and cytoplasmic vacuoles (Suzuki et al., 2020). Furthermore, they showed that exposure of cultured human pulmonary artery smooth muscle cells to the SARSCoV-2 spike protein S1 subunit was sufficient to promote cell signaling without the rest of the virus components. Follow-on papers (Suzuki et al., 2021, Suzuki and Gychka, 2021) showed that the spike protein S1 subunit suppresses ACE2, causing a condition resembling pulmonary arterial hypertension (PAH), a severe lung disease with very high mortality. Their model is depicted here in Figure 2. Ominously, Suzuki and Gychka (2021) wrote: “Thus, these in vivo studies demonstrated that the spike protein of SARS-CoV-1 (without the rest of the virus) reduces the ACE2 expression, increases the level of angiotensin II, and exacerbates the lung injury.” The “in vivo studies” they referred to here (Kuba et al., 2005) had shown that SARS coronavirus-induced lung injury was primarily due to inhibition of ACE2 by the SARS-CoV spike protein, causing a large increase in angiotensin-II. Suzuki et al. (2021) went on to demonstrate experimentally that the S1 component of the SARS-CoV-2 virus, at a low concentration of 130 pM, activated the MEK/ERK/MAPK signaling pathway to promote cell growth. They speculated that these effects would not be restricted to the lung vasculature. The signaling cascade triggered in the heart vasculature would cause coronary artery disease, and activation in the brain could lead to stroke. Systemic hypertension would also be predicted. They hypothesized that this ability of the spike protein to promote pulmonary arterial hypertension could predispose patients who recover Figure 2: A simple model for a process by which the spike protein produced through the mRNA vaccines could induce a pathological response distinct from the desirable induction of antibodies to suppress viral entry. Redrawn with permission from Suzuki and Gychka, 2021. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 58 from SARS-CoV-2 to later develop right ventricular heart failure. Furthermore, they suggested that a similar effect could happen in response to the mRNA vaccines, and they warned of potential longterm consequences to both children and adults who received COVID-19 vaccines based on the spike protein (Suzuki and Gychka, 2021). An interesting study by Lei et. al. (2021) found that pseudovirus — spheres decorated with the SARS-CoV-2 S1 protein but lacking any viral DNA in their core — caused inflammation and damage in both the arteries and lungs of mice exposed intratracheally. They then exposed healthy human endothelial cells to the same pseudovirus particles. Binding of these particles to endothelial ACE2 receptors led to mitochondrial damage and fragmentation in those endothelial cells, leading to the characteristic pathological changes in the associated tissue. This study makes it clear that spike protein alone, unassociated with the rest of the viral genome, is sufficient to cause the endothelial damage associated with COVID-19. The implications for vaccines intended to cause cells to manufacture the spike protein are clear and are an obvious cause for concern. Neurological symptoms associated with COVID-19, such as headache, nausea and dizziness, encephalitis and fatal brain blood clots are all indicators of damaging viral effects on the brain. Buzhdygan et al. (2020) proposed that primary human brain microvascular endothelial cells could cause these symptoms. ACE2 is ubiquitously expressed in the endothelial cells in the brain capillaries. ACE2 expression is upregulated in the brain vasculature in association with dementia and hypertension, both of which are risk factors for bad outcomes from COVID-19. In an in vitro study of the blood-brain barrier, the S1 component of the spike protein promoted loss of barrier integrity, suggesting that the spike protein acting alone triggers a pro-inflammatory response in brain endothelial cells, which could explain the neurological consequences of the disease (Buzhdygan et al., 2020). The implications of this observation are disturbing because the mRNA vaccines induce synthesis of the spike protein, which could theoretically act in a similar way to harm the brain. The spike protein generated endogenously by the vaccine could also negatively impact the male testes, as the ACE2 receptor is highly expressed in Leydig cells in the testes (Verma et al., 2020). Several studies have now shown that the coronavirus spike protein is able to gain access to cells in the testes via the ACE2 receptor, and disrupt male reproduction (Navarra et al., 2020; Wang and Xu, 2020). A paper involving postmortem examination of testicles of six male COVID-19 patients found microscopic evidence of spike protein in interstitial cells in the testes of patients with damaged testicles (Achua et al., 2021). A Possible Link to Prion Diseases and Neurodegeneration Prion diseases are a collection of neurodegenerative diseases that are induced through the misfolding of important bodily proteins, which form toxic oligomers that eventually precipitate out as fibrils causing widespread damage to neurons. Stanley Prusiner first coined the name `prion’ to describe these misfolded proteins (Prusiner, 1982). The best-known prion disease is MADCOW disease (bovine spongiform encephalopathy), which became an epidemic in European cattle beginning in the 1980s. The CDC web site on prion diseases states that “prion diseases are usually rapidly progressive and always fatal.” (Centers for Disease Control and Prevention, 2018). It is now believed that many neurodegenerative diseases, including Alzheimer’s, Parkinson’s disease, and International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 59 amyotrophic lateral sclerosis (ALS) may be prion diseases, and researchers have identified specific proteinaceous infectious particles linked to these diseases (Weickenmeier et al., 2019). Furthermore, researchers have identified a signature motif linked to susceptibility to misfolding into toxic oligomers, called the glycine zipper motif. It is characterized by a pattern of two glycine residues spaced by three intervening amino acids, represented as GxxxG. The bovine prion linked to MADCOW has a spectacular sequence of ten GxxxGs in a row (see uniprot.org/uniprot/P10279). More generally, the GxxxG motif is a common feature of transmembrane proteins, and the glycines play an essential role in cross-linking α-helices in the protein (Mueller et al., 2014). Prion proteins become toxic when the α-helices misfold as β-sheets, and the protein is then impaired in its ability to enter the membrane (Prusiner, 1982). Glycines within the glycine zipper transmembrane motifs in the amyloid-β precursor protein (APP) play a central role in the misfolding of amyloid- β linked to Alzheimer’s disease (Decock et al., 2016). APP contains a total of four GxxxG motifs. When considering that the SARS-CoV-2 spike protein is a transmembrane protein, and that it contains five GxxxG motifs in its sequence (see uniprot.org/uniprot/P0DTC2), it becomes extremely plausible that it could behave as a prion. One of the GxxxG sequences is present within its membrane fusion domain. Recall that the mRNA vaccines are designed with an altered sequence that replaces two adjacent amino acids in the fusion domain with a pair of prolines. This is done intentionally in order to force the protein to remain in its open state and make it harder for it to fuse with the membrane. This seems to us like a dangerous step towards misfolding potentially leading to prion disease. A paper published by J. Bart Classen (2021) proposed that the spike protein in the mRNA vaccines could cause prion-like diseases, in part through its ability to bind to many known proteins and induce their misfolding into potential prions. Idrees and Kumar (2021) have proposed that the spike protein’s S1 component is prone to act as a functional amyloid and form toxic aggregates. These authors wrote that S1 has the ability “to form amyloid and toxic aggregates that can act as seeds to aggregate many of the misfolded brain proteins and can ultimately lead to neurodegeneration.” According to Tetz and Tetz (2020), the form of the spike protein in SARS-CoV-2 has prion regions that are not present in the spike proteins for other coronaviruses. While this was reported in a nonpeer-reviewed article, the authors had published a previous paper in 2018 identifying prion-like regions in multiple eukaryotic viruses, so they have considerable expertise in this area (Tetz and Tetz, 2018). A final point here relates to information about the Pfizer vaccine in particular. The European Medicines Agency (EMA) Public Assessment Report is a document submitted to gain approval to market the vaccine in Europe. It describes in detail a review of the manufacturing process as well as a wide range of associated testing data. One concerning revelation is the presence of “fragmented species” of RNA in the injection solution. These are RNA fragments resulting from early termination of the process of transcription from the DNA template. These fragments, if translated by the cell following injection, would generate incomplete spike proteins, again resulting in altered and unpredictable three-dimensional structure and a physiological impact that is at best neutral and at worst detrimental to cellular functioning. There were considerably more of these fragmented International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 60 forms of RNA found in the commercially manufactured products than in the products used in clinical trials. The latter were produced via a much more tightly controlled manufacturing process. Pfizer claims the RNA fragments “likely… will not result in expressed proteins” due to their assumed rapid degradation within the cell. No data was presented to rule out protein expression, though, leaving the reviewers to comment, “These [fragmented RNA] forms are poorly characterised, and the limited data provided for protein expression do not fully address the uncertainties relating to the risk of translating proteins/peptides other than the intended spike protein” (EMA 2020). To our knowledge no data has been forthcoming since that time. While we are not asserting that non-spike proteins generated from fragmented RNA would be misfolded or otherwise pathological, we believe they would at least contribute to the cellular stress that promotes prion-associated conformational changes in the spike protein that is present. 1. Lessons from Parkinson’s Disease Parkinson’s disease is a neurodegenerative disease associated with Lewy body deposits in the brain, and the main protein found in these Lewy bodies is α-synuclein. That protein, α-Synuclein, is certainly prion-like insofar as under certain conditions it aggregates into toxic soluble oligomers and fibrils (Lema Tomé et al., 2013). Research has shown that misfolded α-synuclein can form first in the gut and then travel from there to the brain along the vagus nerve, probably in the form of exosomes released from dying cells where the misfolded protein originated (Kakarla et al., 2020; Steiner et al., 2011). The cellular conditions that promote misfolding include both an acidic pH and high expression of inflammatory cytokines. It is clear that the vagus nerve is critical for transmission of misfolded proteins to the brain, because severance of the vagus nerve protects from Parkinson’s. Vagus nerve atrophy in association with Parkinson’s disease provides further evidence of the involvement of the vagus nerve in transport of misfolded α-synuclein oligomers from the gut to the brain (Walter et al., 2018). Another pathway is through the olfactory nerve, and a loss of a sense of smell is an early sign of Parkinson’s disease. Ominously, diminution or loss of the sense of smell is also a common symptom of SARS-CoV-2 infection. There are many parallels between α-synuclein and the spike protein, suggesting the possibility of prion-like disease following vaccination. We have already shown that the mRNA in the vaccine ends up in high concentrations in the liver and spleen, two organs that are well connected to the vagus nerve. The cationic lipids in the vaccine create an acidic pH conducive to misfolding, and they also induce a strong inflammatory response, another predisposing condition. Germinal centers are structures within the spleen and other secondary lymphoid organs where follicular dendritic cells present antigens to B cells, which in turn perfect their antibody response. Researchers have shown that mRNA vaccines, in contrast with recombinant protein vaccines, elicit a robust development of neutralizing antibodies at these germinal centers in the spleen (Lederer et al., 2020). However, this also means that mRNA vaccines induce an ideal situation for prion formation from the spike protein, and its transport via exosomes along the vagus nerve to the brain. Studies have shown that prion spread from one animal to another first appears in the lymphoid tissues, particularly the spleen. Differentiated follicular dendritic cells are central to the process, as they accumulate misfolded prion proteins (Al-Dybiat et al., 2019). An inflammatory response International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 61 upregulates synthesis of α-synuclein in these dendritic cells, increasing the risk of prion formation. Prions that accumulate in the cytoplasm are packaged up into lipid bodies that are released as exosomes (Liu et al., 2017). These exosomes eventually travel to the brain, causing disease. 2. Vaccine Shedding There has been considerable chatter on the Internet about the possibility of vaccinated people causing disease in unvaccinated people in close proximity. While this may seem hard to believe, there is a plausible process by which it could occur through the release of exosomes from dendritic cells in the spleen containing misfolded spike proteins, in complex with other prion reconformed proteins. These exosomes can travel to distant places. It is not impossible to imagine that they are being released from the lungs and inhaled by a nearby person. Extracellular vesicles, including exosomes, have been detected in sputum, mucus, epithelial lining fluid, and bronchoalveolar lavage fluid in association with respiratory diseases (Lucchetti et al., 2021). A Phase 1/2/3 study undertaken by BioNTech on the Pfizer mRNA vaccine implied in their study protocol that they anticipated the possibility of secondary exposure to the vaccine (BioNTech, 2020). The protocol included the requirement that “exposure during pregnancy” should be reported by the study participants. They then gave examples of “environmental exposure during pregnancy” which included exposure “to the study intervention by inhalation or skin contact.” They even suggested two levels of indirect exposure: “A male family member or healthcare provider who has been exposed to the study intervention by inhalation or skin contact then exposes his female partner prior to or around the time of conception.” Emergence of Novel Variants of SARS-CoV-2 An interesting hypothesis has been proposed in a paper published in Nature, which described a case of serious COVID-19 disease in a cancer patient who was taking immune-suppressing cancer chemotherapy drugs (Kemp et al., 2021). The patient survived for 101 days after admission to the hospital, finally succumbing in the battle against the virus. The patient constantly shed viruses over the entire 101 days, and therefore he was moved to a negative-pressure high air-change infectious disease isolation room, to prevent contagious spread. During the course of the hospital stay, the patient was treated with Remdesivir and subsequently with two rounds of antibody-containing plasma taken from individuals who had recovered from COVID-19 (convalescent plasma). It was only after the plasma treatments that the virus began to rapidly mutate, and a dominant new strain eventually emerged, verified from samples taken from the nose and throat of the patient. An immune-compromised patient offers little support from cytotoxic T cells to clear the virus. An in vitro experiment demonstrated that this mutant strain had reduced sensitivity to multiple units of convalescent plasma taken from several recovered patients. The authors proposed that the administered antibodies had actually accelerated the mutation rate in the virus, because the patient was unable to fully clear the virus due to their weak immune response. This allowed a “survival of the fittest” program to set in, ultimately populating the patient’s body with a novel antibody-resistant strain. Prolonged viral replication in this patient led to “viral immune escape,” and similar resistant International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 62 strains could potentially spread very quickly within an exposed population (Kemp et al., 2021). Indeed, a similar process might plausibly be at work to produce the highly contagious new strains that are now appearing in the United Kingdom, South Africa and Brazil. There are at least two concerns that we have regarding this experiment, in relation to the mRNA vaccines. The first is that, via continued infection of immune-compromised patients, we can expect continued emergence of more novel strains that are resistant to the antibodies induced by the vaccine, such that the vaccine may quickly become obsolete, and there may well be demands for the population to undergo another mass vaccination campaign. Already a published study by researchers from Pfizer has shown that vaccine effectiveness is reduced for many of these variant strains. The vaccine was only 2/3 as effective against the South African strain as against the original strain (Liu et al., 2021). The second more ominous consideration is to ponder what will happen with an immunecompromised patient following vaccination. It is conceivable that they will respond to the vaccine by producing antibodies, but those antibodies will be unable to contain the disease following exposure to COVID-19 due to impaired function of cytotoxic T cells. This scenario is not much different from the administration of convalescent plasma to immune-compromised patients, and so it might engender the evolution of antibody-resistant strains in the same way, only on a much grander scale. This possibility will surely be used to argue for repeated rounds of vaccines every few months, with increasing numbers of viral variants coded into the vaccines. This is an arms race that we will probably lose. Potential for Permanent Incorporation of Spike Protein Gene into human DNA It has been claimed that mRNA-based vaccines are safer than DNA-vectored vaccines that work by incorporating the genetic code for the target antigenic protein into a DNA virus, because the RNA cannot become inadvertently incorporated into the human genome. However, it is not at all clear that this is true. The classic model of DNA → RNA → protein is now known to be false. It is now indisputable that there is a large class of viruses called retroviruses that carry genes that reverse transcribe RNA back into complementary DNA (cDNA). In 1975, Howard Temin, Renato Dulbecco, and David Baltimore shared the Nobel Prize in Physiology or Medicine in 1975 for their discovery of reverse transcriptase and its synthesis by retroviruses (such as human immunodeficiency virus (HIV)) to derive DNA from RNA (Temin and Mizutani, 1970, Baltimore, 1970). Much later, it was discovered that reverse transcriptase is not unique to retroviruses. More than a third of the human genome is devoted to mysterious mobile DNA elements called SINEs and LINEs (short and long interspersed nuclear elements, respectively). LINEs provide reverse transcriptase capabilities to convert RNA into DNA, and SINEs provide support for integrating the DNA into the genome. Thus, these elements provide the tools needed to convert RNA into DNA and incorporate it into the genome so as to maintain the new gene through future generations (Weiner, 2002). SINEs and LINEs are members of a larger class of genetic elements called retrotransposons. Retrotransposons can copy and paste their DNA to a new site in the genome via an RNA International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 63 intermediate, while possibly introducing genetic alterations in the process (Pray, 2008). Retrotransposons, also known as “jumping genes,” were first identified by the geneticist Barbara McClintock of Cold Spring Harbor Laboratory in New York, over 50 years ago (McClintock, 1965). Much later, in 1983, she was recognized with a Nobel prize for this work. Remarkably, retrotransposons seem to be able to expand their domain from generation to generation. LINEs and SINEs collaborate to invade new genomic sites through translation of their DNA to RNA and back to a fresh copy of DNA, which is then inserted at an AT-rich region of the genome. These LINEs and SINEs had long been considered to be “junk” DNA, an absurd idea that has now been dispelled, as awareness of their critical functions has grown. In particular, it has now become clear that they can also import RNA from an exogenous source into a mammalian host’s DNA. Retroviral-like repeat elements found in the mouse genome called intracisternal A particles (IAPs) have been shown to be capable of incorporating viral RNA into the mouse genome. Recombination between an exogenous nonretroviral RNA virus and an IAP retrotansposon resulted in reverse transcription of the viral RNA and integration into the host’s genome (Geuking et al., 2009). Furthermore, as we shall see later, the mRNA in the new SARS-CoV-2 vaccines could also get passed on from generation to generation, with the help of LINEs expressed in sperm, via nonintegrated cDNA encapsulated in plasmids. The implications of this predictable phenomenon are unclear, but potentially far-reaching. 1. Exogenous and Endogenous Retroviruses There is also a concern that the RNA in the mRNA vaccines could be transferred into the human genome with assistance from retroviruses. Retroviruses are a class of viruses that maintain their genomic information in the form of RNA, but that possess the enzymes needed to reverse transcribe their RNA into DNA and insert it into a host genome. They then rely on existing natural tools from the host to produce copies of the virus through translation of DNA back into RNA and to produce the proteins that the viral RNA codes for and assemble them into a fresh viral particle (Lesbats et al., 2016). Human endogenous retroviruses (HERVs) are benign sections in the DNA of humans that closely resemble retroviruses, and that are believed to have become permanent sequences in the human genome through a process of integration from what was originally an exogenous retrovirus. Endogenous retroviruses are abundant in all jawed vertebrates and are estimated to occupy 5-8% of the human genome. The protein syncytin, which has become essential for placental fusion with the uterine wall and for the fusion step between the sperm and the egg at fertilization, is a good example of an endogenous retroviral protein. Syncytin is the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W (Mi et al., 2000). During gestation, the fetus expresses high levels of another endogenous retrovirus, HERV-R, and it appears to protect the fetus from immune attack from the mother (Luganini and Gribaudo, 2020). Endogenous retroviral elements closely resemble retrotransposons. Their reverse transcriptase, when expressed, has the theoretical capability to convert spike protein RNA from the mRNA vaccines into DNA. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 64 2. Permanent DNA integration of Exogenous Retrovirus Genes Humans are colonized by a large collection of exogenous retroviruses that in many cases cause no harm to the host, and may even be symbiotic (Luganini and Gribaudo, 2020). Exogenous viruses can be converted to endogenous viruses (permanently incorporated into host DNA) in the laboratory, as demonstrated by Rudolf Jaenisch (Jaenisch, 1976), who infected preimplantation mouse embryos with the Moloney murine leukemia virus (M-MuLV). The mice generated from these infected embryos developed leukemia, and the viral DNA was integrated into their germ line and transmitted to their offspring. Besides the incorporation of viral DNA into the host genome, it was also shown as early as 1980 that DNA plasmids could be microinjected into the nuclei of mouse embryos to produce transgenic mice that breed true (Gordon et al., 1980). The plasmid DNA was incorporated into the nuclear genome of the mice through existing natural processes, thus preserving the newly acquired genetic information in the offspring’s genome. This discovery has been the basis for many genetic engineering experiments on transgenic mice engineered to express newly acquired human genes since then (Bouabe and Okkenhaug, 2013). 3. LINE-1 is Widely Expressed LINEs alone make up over 20% of the human genome. The most common LINE is LINE-1, which encodes a reverse transcriptase that regulates fundamental biological processes. LINE-1 is expressed in many cell types, but at especially high levels in sperm. Sperm cells can be used as vectors of both exogenous DNA and exogenous RNA molecules through sperm-mediated gene transfer assays. Sperm can reverse transcribe exogenous RNA directly into cDNA and can deliver plasmids packaging up this cDNA to the fertilized egg. These plasmids are able to propagate themselves within the developing embryo and to populate many tissues in the fetus. In fact, they survive into adulthood as extrachromosomal structures and are capable of being passed on to progeny. These plasmids are transcriptionally competent, meaning that they can be used to synthesize proteins encoded by the DNA they contain (Pittoggi et al., 2006). In addition to sperm, embryos also express reverse transcriptase prior to implantation, and its inhibition causes developmental arrest. LINE-1 is also expressed by cancer cells, and RNA interference-mediated silencing of human LINE-1 induces differentiation in many cancer cell lines. Reverse-transcriptase machinery is implicated in the genesis of new genetic information, both in cancer cells and in germ cells. Many tumor tissues have been found to express high levels of LINE1, and to contain many extrachromosomal plasmids in their nucleus. Malignant gliomas are the primary tumors of the central nervous system. It has been shown experimentally that these tumors release exosomes containing DNA, RNA and proteins, that end up in the general circulation (Vaidya and Sugaya, 2020). LINE-1 is also highly expressed in immune cells in several autoimmune diseases such as systemic lupus erythematosus, Sjögrens and psoriasis (Zhang et al., 2020). 4. Integrating Spike Protein Gene into Human Genome Remarkably, it has been demonstrated that neurons from the brain of Alzheimer’s patients harbor multiple variants of the gene for amyloid precursor protein APP, incorporated into the genome, which are created through a process called somatic gene recombination (SGR) (Kaeser et al., 2020). SGR requires gene transcription, DNA strand-breaks, and reverse transcriptase activity, all of which International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 65 may be promoted by well-known Alzheimer’s disease risk factors. The DNA coding for APP is reverse transcribed into RNA and then transcribed back into DNA and incorporated into the genome at a strand break site. Since RNA is more susceptible to mutations, the DNA in these mosaic copies contains many mutant variants of the gene, so the cell becomes a mosaic, capable of producing multiple variants of APP. Neurons from Alzheimer’s patients contained as many as 500 million base pairs of excess DNA in their chromosomes (Bushman et al., 2015). Researchers from MIT and Harvard published a disturbing paper in 2021, where they provided strong evidence that the SARS-CoV-2 RNA can be reverse transcribed into DNA and integrated into human DNA (Zhang et al., 2021). They were led to investigate this idea after having observed that many patients continue to test positive for COVID-19 after the virus has already been cleared from their body. The authors found chimeric transcripts that contained viral DNA sequences fused to cellular DNA sequences in patients who had recovered from COVID-19. Since COVID-19 often induces a cytokine storm in severe cases, they confirmed the possibility of enhanced reverse transcriptase activity through an in vitro study using cytokine-containing conditioned media in cell cultures. They found a 2-3-fold upregulation of endogenous LINE-1 expression in response to cytokines. The exogenous RNA from the virus incorporated into human DNA could produce fragments of viral proteins indefinitely after the infection has been cleared, and this yields a falsepositive on a PCR test. 5. Bovine Viral Diarrhea: A Disturbing Model Bovine Viral Diarrhea (BVD) is an infectious viral disease that affects cattle throughout the world. It is a member of the class of pestiviruses, which are small, spherical, single-stranded, enveloped RNA viruses. The disease is associated with gastrointestinal, respiratory and reproductive diseases. A unique characteristic of BVD is that the virus can cross the placenta of an infected pregnant dam. This can result in the birth of a calf which carries intra-cellular viral particles which it mistakes as `self.’ Its immune system refuses to recognize the virus as a foreign invasion, and, as a result, the calf sheds the virus in large quantities throughout its life, potentially infecting the entire herd. It has become a widespread practice to identify such carrier calves and cull them from the herd in an attempt to curtail infection (Khodakaram-Tafti & Farjanikish, 2017). It seems plausible that a dangerous situation may arise in the future where a woman receives an mRNA vaccine for SARS-CoV-2 and then conceives a child shortly thereafter. The sperm would be free to take up RNA-embedded liposomes from the vaccine and convert them to DNA using LINE-1. They would then produce plasmids containing the code for the spike protein which would be taken up by the fertilized egg through the process described above. The infant that is born is then potentially unable to mount antibodies to the spike protein because their immune system considers it to be `self.’ Should that infant get infected with SARS-CoV-2 at any time in its lifespan, its immune system would not mount a defense against the virus, and the virus would presumably be free to multiply in the infant’s body without restraint. The infant would logically become a superspreader in such a situation. Admittedly, this is speculation at this time, but there is evidence from what we know about retrotransposons, sperm, fertilization, the immune system and viruses, that such a scenario cannot be ruled out. It has already been demonstrated in mouse experiments that the genetic elements in DNA vector vaccines, which are essentially plasmids, can integrate into the host International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 66 genome (Wang et al., 2004). In fact, such a process has been suggested as a basis for Lamarckian evolution defined as the inheritance of acquired traits (Steele, 1980). The realization that what was formerly called “junk DNA” is not junk, is just one of the results coming out of the new philosophical paradigm in human language, biology and genetics that is based on fractal genomics (Pellionisz, 2012) — a paradigm that Pellionisz has linked to the involvement of “true narrative representations” (TNRs; Oller, 2010), realized as “iterations of a fractal template” in the highly repetitive processes of normal development of the many branching structures of the human body. These processes are numerous in the lungs, kidneys, veins and arteries, and most importantly in the brain. The mRNA vaccines are an experimental gene therapy with the potential to incorporate the code for the SARS-CoV-2 spike protein into human DNA. This DNA code could instruct the synthesis of large numbers of copies of proteinaceous infectious particles, and this has the potential to insert multiple false signals into the unfolding narrative, resulting in unpredictable outcomes. Conclusion Experimental mRNA vaccines have been heralded as having the potential for great benefits, but they also harbor the possibility of potentially tragic and even catastrophic unforeseen consequences. The mRNA vaccines against SARS-CoV-2 have been implemented with great fanfare, but there are many aspects of their widespread utilization that merit concern. We have reviewed some, but not all, of those concerns here, and we want to emphasize that these concerns are potentially serious and might not be evident for years or even transgenerationally. In order to adequately rule out the adverse potentialities described in this paper, we recommend, at a minimum, that the following research and surveillance practices be adopted: • A national effort to collect detailed data on adverse events associated with the mRNA vaccines with abundant funding allocation, tracked well beyond the first couple of weeks after vaccination. • Repeated autoantibody testing of the vaccine-recipient population. The autoantibodies tested could be standardized and should be based upon previously documented antibodies and autoantibodies potentially elicited by the spike protein. These include autoantibodies against phospholipids, collagen, actin, thyroperoxidase (TPO), myelin basic protein, tissue transglutaminase, and perhaps others. • Immunological profiling related to cytokine balance and related biological effects. Tests should include, at a minimum, IL-6, INF-α, D-dimer, fibrinogen, and C-reactive protein. • Studies comparing populations who were vaccinated with the mRNA vaccines and those who were not to confirm the expected decreased infection rate and milder symptoms of the vaccinated group, while at the same time comparing the rates of various autoimmune diseases and prion diseases in the same two populations. • Studies to assess whether it is possible for an unvaccinated person to acquire vaccine-specific forms of the spike proteins from a vaccinated person in close proximity. • In vitro studies to assess whether the mRNA nanoparticles can be taken up by sperm and converted into cDNA plasmids. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 67 • Animal studies to determine whether vaccination shortly before conception can result in offspring carrying spike-protein-encoding plasmids in their tissues, possibly integrated into their genome. • In vitro studies aimed to better understand the toxicity of the spike protein to the brain, heart, testes, etc. Public policy around mass vaccination has generally proceeded on the assumption that the risk/benefit ratio for the novel mRNA vaccines is a “slam dunk.” With the massive vaccination campaign well under way in response to the declared international emergency of COVID-19, we have rushed into vaccine experiments on a world-wide scale. At the very least, we should take advantage of the data that are available from these experiments to learn more about this new and previously untested technology. And, in the future, we urge governments to proceed with more caution in the face of new biotechnologies. Finally, as an obvious but tragically ignored suggestion, the government should also be encouraging the population to take safe and affordable steps to boost their immune systems naturally, such as getting out in the sunlight to raise vitamin D levels (Ali, 2020), and eating mainly organic whole foods rather than chemical-laden processed foods (Rico-Campà et al., 2019). Also, eating foods that are good sources of vitamin A, vitamin C and vitamin K2 should be encouraged, as deficiencies in these vitamins are linked to bad outcomes from COVID-19 (Goddek, 2020; Sarohan, 2020).

Acknowledgements

This research was funded in part by Quanta Computers, Inc., Taiwan, under the auspices of the Qmulus project. Competing interests The authors have no competing interests or conflicts to declare.

References

Achua, J. K., Chu, K. Y., Ibrahim, E., Khodamoradi, K., Delma, K. S., Ramsamy, R. … Arora, H. (2021). Histopathology and Ultrastructural Findings of Fatal COVID-19 Infections on Testis. The World Journal of Men’s Health 39(1): 65-74. https://doi.org/10.5534/wjmh.200170.

Al-Dybiat, I., Moudjou, M., Martin, D., Reine, F., Herzog, L., Truchet, S., … Sibille, P. (2019) Prion Strain-dependent Tropism is Maintained between Spleen and Granuloma and Relies on Lymphofollicular Structures. Scientific Reports 9: 14656. https://doi.org/10.1038/s41598-019-51084-1.

Ali, N. (2020). Role of Vitamin D in Preventing of COVID-19 Infection, Progression and Severity. Journal of Infection and Public Health 13(10): 1373-1380. https://doi.org/10.1016/j.jiph.2020.06.021.

Ansari, B. Rosen, L. B., Lisco, A., Gilden, D., Holland, S. M., Zerbe, C. S., … Cohen, J. I. (2020). Primary and Acquired Immunodeficiencies Associated with Severe Varicella-Zoster Virus Infections. Clinical Infectious Diseases August 28 [Epub ahead of print]. https://doi.org/10.1093/cid/ciaa1274.

Arvin, A. M., Fink, K. Schmid, M. A., Cathcart, A., Spreafico, R., Havenar-Daughton, C. … Virgin, H. W. (2020). A Perspective on Potential Antibody-Dependent Enhancement of SARS-CoV-2. Nature 584(7821): 353-363. https://doi.org/10.1038/s41586-020-2538-8.

Aslam, R., Kapur, R., egel, G. B., Guo, L., Zufferey, A., Ni, H. & Semple, J. W. (2016). The Spleen Dictates Platelet International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 68 Destruction, Anti-platelet Antibody Production, and Lymphocyte Distribution Patterns in a Murine Model of Immune Thrombocytopenia. Experimental Hematology 44(10): 924-930. https://doi.org/10.1016/j.exphem.2016.07.004.

Baden, L. R., El Sahly, H. M., Essink, B.,Kotloff, K., Frey, S., Novak, R. … Zaks, T. (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. The New England Journal of Medicine 384: 403-416. https://doi.org/10.1056/NEJMoa2035389.

Bahl, K., Senn, J. J., Yuzhakov, O., Bulychev, A., Brito, L. A., Hassett, K. J. … Ciaramella, G. (2017). Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Molecular Therapy 25(6): 1316-1327. http://dx.doi.org/10.1016/j.ymthe.2017.03.035.

Baker, A. N., Richards,S.-J., Guy, C. S., Congdon, T. R., Hasan, M., Zwetsloot, A. J., … Gibson, M. I. (2020). The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device. ACS Central Science 6(11): 2046-2052. https://doi.org/10.1021/acscentsci.0c00855.

Baltimore, D. (1970). Viral RNA-dependent DNA Polymerase: RNA-dependent DNA Polymerase in visions of RNA Tumor Viruses. Nature 226(5252): 1209-1211. https://doi.org/10.1038/2261209a0.

Bardina, S. V., Bunduc, P., Tripathi, S., Duehr, J., Frere, J. J., Brown, J. A. … Lim, J. K. (2017). Enhancement of Zika Virus Pathogenesis by Preexisting Antiflavivirus Immunity. Science 356(6334): 175-180. https://doi.org/10.1126/science.aal4365.

Beltramello, M., Williams, K. L., Simmons, C. P., Macagno, A., Simonelli, L., Ha Quyen, N. T. … Sallusto, F. (2010). The Human Immune Response to Dengue Virus is Dominated by Highly Cross-Reactive Antibodies Endowed with Neutralizing and Enhancing Activity. Cell Host Microbe 8(3): 271-83. https://doi.org/10.1016/j.chom.2010.08.007.

Bertin, D., Brodovitch, A., Beziane, A., Hug, S., Bouamri, A., Mege, J. L. … Bardin, N. (2020). Anticardiolipin IgG Autoantibody Level Is an Independent Risk Factor for COVID‐19 Severity. Arthritis & Rheumatology, 72(11), 1953-1955. https://doi.org/10.1002/art.41409.

Bhattacharjee, S. & Banerjee, M. (2020). Immune Thrombocytopenia Secondary to COVID-19: a Systematic Review SN Comprehensive Clinical Medicine 2: 2048-2058. https://doi.org/10.1007/s42399-020-00521-8.

BioNTech (2020). A Phase 1/2/3, Placebo-Controlled, Randomized, Observer-Blind, Dose-Finding Study to Evaluate the Safety, Tolerability, Immunogenicity, and Efficacy of Sars-CoV-2 RNA Vaccine Candidates against COVID-19 in Healthy Individuals. PF-07302048 (BNT162 RNA-Based COVID-19 Vaccines) Protocol C4591001. November. https://media.tghn.org/medialibrary/ 2020/11/C4591001_Clinical_Protocol_Nov2020_Pfizer_BioNTech.pdf.

Blumenthal, K. G., Robinson, L. B., Camargo, C. Jr., Shenoy, E. S., Banerji, A., Landman, A. B., Wickner, P. (2021) Acute Allergic Reactions to mRNA COVID-19 Vaccines. Journal of the American Medical Association 325(15):1562-1565. https://doi.org/10.1001/jama.2021.3976.

Bonsell, D. (2021, January 10). Largest Multi-Site Distribution Complex in Defense Department Delivers for Operation Warp. Defense Logistics Agency. Retrieved January 27, 2021, from https://www.dla.mil/ AboutDLA/News/NewsArticleView/Article/2467282/largest-warehouse-in-defensedepartment-delivers-for-operation-warp-speed/

Bouabe, H. & Okkenhaug, K. (2013). Gene Targeting in Mice: a Review. Methods in Molecular Biology 2013; 1064: 315- 336. https://doi.org/10.1007/978-1-62703-601-6_23.

Brown, R. B. (2021) Outcome Reporting Bias in COVID-19 mRNA Vaccine Clinical Trials. Medicina (Kaunas) 57(3): 199. https://www.doi.org/10.3390/medicina57030199.

Buonsenso, D., Riitano, F., & Valentini, P. (2020). Pediatric Inflammatory Multisystem Syndrome Temporally Related with SARS-CoV-2: Immunological Similarities with Acute Rheumatic Fever and Toxic Shock Syndrome. Frontiers in Pediatrics 8: 574. https://doi.org/10.3389/fped.2020.00574.

Bushman, D. M., Kaeser, G. E., Siddoway, B., Westra, J. W., Rivera, R. R., Rehen, S. K. … Chun, J. (2015). Genomic Mosaicism with Increased Amyloid Precursor Protein (APP) Gene Copy Number in Single Neurons from Sporadic Alzheimers Disease Brains. eLife 4: e05116. https://doi.org/10. 7554/eLife.05116.

Buzhdygana, T. P., DeOrec, B. J., Baldwin-Leclair, A., Bullock, T. A., McGary, H. M. … Ramirez, S. H. (2020). The International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 69 SARS-CoV-2 Spike Protein Alters Barrier Function in 2D Static and 3D Microfluidic in-Vitro Models of the Human Blood-Brain Barrier. Neurobiology of Disease 146: 105131. https://doi.org/10.1016/j.

nbd.2020.105131. CDC COVID-19 Response Team; Food and Drug Administration (2021, January 15). Allergic Reactions Including Anaphylaxis After Receipt of the First Dose of Pfizer-BioNTech COVID-19 Vaccine—United States, December 14–23, 2020. Morbidity and Mortality Weekly Report 70(2): 46. https://www.cdc.gov/mmwr/volumes/70/wr/mm7002e1.htm.

CDC COVID-19 Response Team; Food and Drug Administration (2021, January 29). Allergic Reactions Including Anaphylaxis After Receipt of the First Dose of Moderna COVID-19 Vaccine-United States. December 21, 2020 — January 10, 2021. MMWR. Morbidity and Mortality Weekly Report 70(4): 125-129. https://www.cdc.gov/mmwr/volumes/70/wr/mm7004e1.htm.

Campos, J. Slon, L., Mongkolsapaya, J., & Screaton, G. R. (2018). The Immune Response Against Flaviviruses. Nature immunology 19(11): 1189-1198. https://doi.org/10.1038/s41590-018-0210-3.

Carsetti, R., Zaffina, S., Piano Mortari, E., Terreri, S., Corrente, F., Capponi, C., … & Locatelli, F. (2020). Different Innate and Adaptive Immune Responses to SARS-CoV-2 Infection of Asymptomatic, Mild, and Severe Cases. Frontiers in immunology, 11, 3365. https://www.frontiersin.org /articles/10.3389/fimmu.2020.610300/full

Carter, M. J., Fish, M., Jennings, A., Doores, K. J., Wellman, P., Seow, J., … Shankar-Hari, M. (2020). Peripheral Immunophenotypes in Children with Multisystem Inflammatory Syndrome Associated with SARS-CoV-2 Infection. Nature Medicine, 26(11), 1701-1707. https://doi.org/10.1038/s41591-020-1054-6. Centers for Disease Control and Prevention. COVID Data Tracker. https://covid.cdc.gov/covid-datatracker/#vaccinations. Accessed 2/6/21.

Centers for Disease Control and Prevention, Prion Diseases. October 9, 2018. https://www.cdc.gov/prions/index.html.

Centers for Disease Control and Prevention (1990). Vaccine Adverse Events Reporting System [database]. Retrieved February 11, 2021 from https://vaers.hhs.gov/about.html

Chen, W., Yang, B., Li, Z., Wang, P., Chen, Y. & Zhou, H. (2020). Sudden Severe Thrombocytopenia in a Patient in the Recovery Stage of COVID-19. Lancet Haematology 7(8): e624. https://doi.org/10.1016 /S2352-3026(20)30175-7.

Cifuentes-Diaz, C., Delaporte, C., Dautréaux,B., Charron, D. & Fardeau, M. (1992) Class II MHC Antigens in Normal Human Skeletal Muscle. Muscle Nerve 15(3): 295-302. https://doi.org/10.1002/ mus.880150307.

Classen, J. B. (2021). Review of COVID-19 Vaccines and the Risk of Chronic Adverse Events Including Neurological Degeneration. Journal of Medical-Clinical Research and Reviews 5(4): 1-7. https://foundationforhealthresearch.org/review-of-covid-19-vaccines-and-the-risk-of-chronic-adverse-events/.

Corbett, K. S., Edwards, D.K., Leist, S. R., Abiona, O. M., Boyoglu-Barnum, S., Gillespie, R. A. … Graham, B. S. (2020) SARS-CoV-2 mRNA Vaccine Design Enabled by Prototype Pathogen Preparedness. Nature 586(7830): 567-571. https://doi.org/10.1038/s41586-020-2622-

0. Danielsson, R. & Eriksson, H. (2021, January 7). Aluminium Adjuvants in Vaccines — A Way to Modulate the Immune Response. Seminars in Cell & Developmental Biology. (Epub ahead of print) https://doi.org/10.1016/j.semcdb.2020.12.008.

Decock, M, Stanga, S., Octave, J.-N., Dewachter, I., Smith, S. O., Constantinescu, S. N., and Kienlen-Campard, P. (2016). Glycines from the APP GXXXG/GXXXA Trans- membrane Motifs Promote Formation of Pathogenic A Oligomers in Cells. Frontiers in Aging Neuroscience 8: 107. https://doi.org/ 10.3389/fnagi.2016.00107.

Dicks, M. D. J., Spencer, A. J., Edwards, N. J., Wadell, G., Bojang, %K., Gilbert, S.C., … Cottingham, M. G. (2012). A Novel Chimpanzee Adenovirus Vector with Low Human Seroprevalence: Improved Systems for Vector Derivation and Comparative Immunogenicity. PLoS ONE 7(7): e40385. https://doi.org/10.1371/journal.pone.0040385.

Doshi, P. (2020). Will COVID-19 Vaccines Save Lives? Current Trials Aren’t Designed to Tell Us. BMJ 371: m4037. https://doi.org/10.1136/bmj.m4037. Doshi, P. (2021a).

Peter Doshi: Pfizer and Moderna’s “95% effective” vaccines—we need more details and the raw data. BMJ blog. Accessed 02/20/2021. https://blogs.bmj.com/bmj/2021/01/04/peter-doshi-pfizer-and-modernas-95- effective-vaccines-we-need-more-details-and-the-raw-data/ International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 70

Doshi, P. (2021b). Clarification: Pfizer and Moderna’s “95% effective” Vaccines — We Need More Details and the Raw Data. BMJ blog. Accessed 02/20/21. https://blogs.bmj.com/bmj/ 2021/02/05/clarification-pfizer-and-modernas95-effective-vaccines-we-need-more-details-and-the-raw-data/

Ehrenfeld, M., Tincani, A., Andreoli, L., Cattalini, M., Greenbaum, A., Kanduc, D. … Shoenfeld, Y. (2020). COVID-19 and Autoimmunity. Autoimmunity Reviews 19(8): 102597. https://www.ncbi.nlm.nih. gov/pmc/articles/PMC7289100/

EMA Public Assessment Report on Pfizer-BioNTech Vaccine. (2020). Accessed 5/2/21. https://www.documentcloud.org/documents/20516010-ema-assessment-report-12-21- 2020#document/p35/a2023027

Eroshenko, N., Gill, T., Keaveney, M. K., Church, G. M., Trevejo, J. M. & Rajaniemi, H. (2020). Implications of Antibody-dependent Enhancement of Infection for SARS-CoV-2 Countermeasures. Nature Biotechnology 38(7): 789-791. https://doi.org/10.1038/s41587-020-0577-1.

European Medicines Agency. Committee for Medicinal Products for Human Use (CHMP) Assessment report. COVID19 Vaccine Moderna. Common name: COVID-19 mRNA Vaccine (nucleoside-modified) Procedure. No. EMEA/H/C/005791/0000. March 11 2021. p. 47. https://www.ema.europa. eu/en/documents/assessmentreport/covid-19-vaccine-moderna-epar-public-assessment-report_en.pdf

Firdessa-Fite, R. & Creusot, R. J. (2020). Nanoparticles versus Dendritic Cells as Vehicles to Deliver mRNA Encoding Multiple Epitopes for Immunotherapy. Molecular Therapy: Methods & Clinical Development 16: 50-62. https://doi.org/10.1016/j.omtm.2019.10.015.

Franke, C., Ferse, C., Kreye, J., Reincke, S. M., Sanchez-Sendin, E., Rocco, A., … & Pruess, H. (2021). High Frequency of Cerebrospinal Fluid Autoantibodies in COVID-19 Patients with Neurological Symptoms. Brain, Behavior, and Immunity 93: 415-419. https://doi.org/10.1016/j.bbi.2020.12.022.

Fujiwara, Y., Wada, K. & Kabuta, T. (2017). Lysosomal Degradation of Intracellular Nucleic Acids — Multiple Autophagic Pathways. The Journal of Biochemistry 161(2): 145-154. https://doi.org/10.1093/jb/mvw085.

Furer, V., Zisman, D., Kibari, A., Rimar, D., Paran, Y., & Elkayam, O. (2021). Herpes zoster Following BNT162b2 mRNA Covid-19 Vaccination in Patients with Autoimmune Inflammatory Rheumatic Diseases: a Case Series. Rheumatology keab345. April 12 [Epub ahead of print] https://doi.org/ 10.1093/rheumatology/keab345.

Galeotti, C., & Bayry, J. (2020). Autoimmune and Inflammatory Diseases Following COVID-19. Nature Reviews Rheumatology, 16(8), 413-414. https://doi.org/10.1038/s41584-020-0448-7.

Gallie, D. R., (1991) The Cap and Poly(A) Tail Function Synergistically to Regulate mRNA Translational Efficiency. Genes & Development 5: 2108–2116. https://doi.org/10.1101/gad.5.11.2108.

Ganson, N. J., Povsic, T. J., Sullenger, B. A., Alexander, J. H., Zelenkofske, S. L., … Hershfield, M. S. (2016). Pre-existing Anti–Polyethylene Glycol Antibody Linked to First-Exposure Allergic Reactions to Pegnivacogin, A PEGylated RNA Aptamer. Journal of Allergy and Clinical Immunology 137(5): 1610-1613. https://doi.org/10.1016/j.jaci.2015.10.034.

Garvey, L. H., & Nasser, S. (2020, December 17) Allergic Reactions to the First COVID-19 Vaccine: is Polyethylene Glycol (PEG) the Culprit? British Journal of Anaesthesia. Epub ahead of print. https://doi.org/10.1016/j.bja.2020.12.020.

Gao, Z., Xu, Y., Sun, C., Wang, X., Guo, Y., Qiu, S., & Ma, K. (2020). A systematic review of asymptomatic infections with COVID-19. Journal of Microbiology, Immunology and Infection 54(1): 12-16. https://www.sciencedirect.com/science/article/pii/S1684118220301134.

Gao, Z. W., Zhang, H. Z., Liu, C., & Dong, K. (2021). Autoantibodies in COVID-19: Frequency and Function. Autoimmune Reviews 20(3): 102754. https://doi.org/10.1016/j.autrev.2021.102754.

Geuking, M. B., Weber, J., Dewannieux, M., Gorelik, E., Heidmann, T., Hengartner, H., … Hangartner, L. (2009). Recombination of Retrotransposon and Exogenous RNA Virus Results in Nonretroviral cDNA Integration. Science 323(5912): 393-6. https://doi.org/10.1126/science.1167375.

Goddek, S. (2020). Vitamin D3 and K2 and Their Potential Contribution to Reducing the COVID-19 Mortality Rate. International Journal of Infectious Diseases 99: 286-290. https://doi.org/10.1016/ j.ijid.2020.07.080. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 71

Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A. & Ruddle, F.H. (1980). Genetic Transformation of Mouse Embryos by Microinjection of Purified DNA. Proceedings of the National Academy of Sciences USA.77: 7380-84. https://doi.org/10.1073/pnas.77.12.7380.

Grady, D. & Mazzei, P. (2021). Doctor’s Death After COVID Vaccine Is Being Investigated. New York Times Jan. 12. https://www.nytimes.com/2021/01/12/health/covid-vaccine-death.html.

Grady, D. (2021). A Few Covid Vaccine Recipients Developed a Rare Blood Disorder. New York Times Feb. 8. https://www.nytimes.com/2021/02/08/health/immune-thrombocytopenia-covid-vaccine-blood.html.

Haidere, M. F., Ratan, Z. A., Nowroz, S., Zaman, S. B., Jung, Y. J., Hosseinzadeh, H., & Cho, J. Y. (2021). COVID-19 Vaccine: Critical Questions with Complicated Answers. Biomolecules & therapeutics, 29(1), 1. https://doi.org/10.4062/biomolther.2020.178.

Hamad, I., Hunter, A. C., Szebeni, J. & Moghimi, S. M. (2008). Poly (Ethylene Glycol)s Generate Complement Activation Products in Human Serum through Increased Alternative Pathway Turnover and a MASP-2-Dependent Process. Molecular immunology 46(2): 225-232. https://doi.org/10.1016/j .molimm.2008.08.276.

Hawkes, R. A. (1964). Enhancement of the Infectivity of Arboviruses by Specific Antisera Produced in Domestic Fowls. Australian Journal of Experimental Biology and Medical Science 42(4): 465-482. https://doi.org/10.1038/icb.1964.44.

Ho, W., Gao, M.,Li, F., Li, J., Zhang, X.-Q. & Xu, X. (2021, January 18). Next‐Generation Vaccines: Nanoparticle‐ Mediated DNA and mRNA Delivery. Advanced Healthcare Materials 10(8): e2001812. https://doi.org/10.1002/adhm.202001812.

Hong, L., Wang, Z., Wei, X., Shi, J. & Li, C. (2020). Antibodies Against Polyethylene Glycol in Human Blood: A Literature Review. Journal of Pharmacological and Toxicological Methods 102: 106678. https://doi.org/10.1016/j.vascn.2020.106678.

Hubert, B. Reverse Engineering the source code of the BioNTech/Pfizer SARS-CoV-2 Vaccine. Dec. 25, 2020. https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/ Idrees

D, Kumar V. SARS-CoV-2 spike protein interactions with amyloidogenic proteins: Potential clues to Neurodegeneration. Biochemical and Biophysical Re- search Com- munications. 2021; 554: 94-98. https://www.doi.org/10.1016/j.bbrc.2021.03.100.

Jackson, L. A., Anderson, E. J., Rouphael, N. G., Roberts, P. C., Makhene, M., Coler, R. N. … Beigel, J. H. (2020). An mRNA Vaccine against SARS-CoV-2 Preliminary Report. The New England Journal of Medicine 383: 1920-31. https://doi.org/10.1056/NEJMoa2022483.

Jacobs, J. & Armstrong, D. (2020, April 29) Trump’s `Operation Warp Speed’ Aims to Rush Coronavirus Vaccine Bloomberg. Retreived February 11 from https://www.bloomberg.com/news/articles/2020-04-29/trump-soperation-warp-speed-aims-to-rush-coronavirus-vaccine.

Jaenisch R. (1976). Germ Line Integration and Mendelian Transmission of the Exogenous Moloney Leukemia Virus. Proceedings of the National Academy of Sciences of the United States of America 73: 1260-1264. https://doi.org/10.1073/pnas.73.4.1260.

Jansen, A. J. G., Spaan, T., Low, H. Z., Di Iorio D., van den Brand, J., Malte Tieke, M., … van der Vries, E. (2020). Influenza-Induced Thrombocytopenia is Dependent on the Subtype and Sialoglycan Receptor and Increases with Virus Pathogenicity. Blood Advances 4(13): 2967-2978. https://doi.org/10. 1182/bloodadvances.2020001640.

Jiang, Y., Arase, N., Kohyama, M., Hirayasu, K., Suenaga, T., Jin, H., … Hisashi Arase , H. (2013) Transport of Misfolded Endoplasmic Reticulum Proteins to the Cell Surface by MHC Class II Molecules. International Immunology 25(4): 235-246. https://doi.org/10.1093/intimm/dxs155

Kaeser, G. E. & Chun, J. (2020). Mosaic Somatic Gene Recombination as a Potentially Unifying Hypothesis for Alzheimers Disease. Frontiers in Genetics 11: 390. https://doi.org/10.3389/ fgene.2020.00390.

Kakarla, R., Hur, J., Kim, Y. J., Kim, J., and Chwae, Y.-J. (2020). Apoptotic Cell- derived Exosomes: Messages from Dying Cells. Experimental & Molecular Medicine 52: 16 https://www.doi.org/1 0.1038/s12276-019-0362-8.

Karikó, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S. & Weissman, D. (2008). Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 72 Biological Stability. Molecular Therapy 16(11): 1833-1840. https://doi.org/ 10.1038/mt.2008.200.

Kelso, J. M. (2021) Anaphylactic Reactions to Novel mRNA SARS-CoV-2/COVID-19 Vaccines. Vaccine 39(6): 865– 867. https://doi.org/10.1016/j.vaccine.2020.12.084.

Kemp, S. A., Collier, D. A. Datir, R. P., Ferreira, I. A. T. M. Gayed, S., Jahun, A. … Gupta, R. K. (2021) SARS-CoV-2 Evolution during Treatment of Chronic Infection. Nature 2021 Apr;592(7853):277-282. https://doi.org/10.1038/s41586-021-03291-y.

Khodakaram-Tafti, A. & Farjanikish, G. H. (2017) Persistent Bovine Viral Diarrhea Virus (BVDV) Infection in Cattle Herds. Iranian Journal of Veterinary Research, Shiraz University 18(3): 154-163. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674437/.

Kosuri, S. & Church, G. M., Large-Scale de Novo DNA Synthesis: Technologies and Applications. Nature Methods 2014; 11 (5): 499–507. https://doi.org/10.1038/nmeth.2918.

Koupenova, M., Corkrey, H. A., Vitseva, O., Manni, G., Pang, C. J., Clancy, L. … Freedman, J. E. (2019). The Role of Platelets in Mediating a Response to Human Influenza Infection. Nature Communications 2019;10: 1780. https://doi.org/10.1038/s41467-019-09607-x.

Kozma, G. T., Mészáros, T., Vashegyi, I., Fülöp, T., Örfi, E., Dézsi, L., … Szebeni, J. (2019). Pseudo-anaphylaxis to Polyethylene Glycol (PEG)-Coated Liposomes: Roles of Anti-PEG IgM and Complement Activation in a Porcine Model of Human Infusion Reactions. ACS Nano 13(8): 9315-9324. https://doi.org/10.1021/acsnano.9b03942.

Ku, C.-C., Chang, Y.-H., Chien, Y., & Lee, T.-L. (2016). Type I Interferon Inhibits Varicella-zoster Virus Replication by Interfering with the Dynamic Interaction between Mediator and IE62 within Replication Compartments. Cell & Bioscience 6: 21. https://doi.org/10.1186/s13578-016-0086-6.

Kuba, K., Imai, Y., Rao, S., Gao, H., Guo, F., Guan, B. … Penninger, J. M. (2005). A Crucial Role of Angiotensin Converting Enzyme 2 (ACE2) in SARS Coronavirus-Induced Lung Injury. Natural Medicine 11: 875-879. https://doi.org/10.1038/nm1267.

Kudla, G., Lipinski, L., Caffin, F., Helwak, A., Zylicz, M. (2006) High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells. PlOS Biology 4(6): e180. https://doi.org/10.1371/journal.pbio.0040180.

Kullaya, V., de Jonge, M. E., Langereis, J. D., van der Gaast-de Jongh, C. E., Büll, C., Adema, G. J. … van der Ven, A. J. (2018). Desialylation of Platelets by Pneumococcal Neuraminidase A Induces ADP-Dependent Platelet Hyperreactivity. Infection and Immunity 86(10): e00213-18. https://doi.org/10.1128/IAI.00213-18.

Lambrecht, B. N., Kool, M., Willart, M. A. M. & Hammad, H. (2009). Mechanism of Action of Clinically Approved Adjuvants. Current opinion in immunology 21.1 (2009): 23-29. https://doi.org/10.1016/j.coi.2009.01.004.

Lazzaro, S., Giovani, C., Mangiavacchi, S., Magini,D., Maione, D., Baudner, B., … Buonsanti, C. (2015). CD8 T-cell Priming upon mRNA Vaccination is Restricted to Bone-marrow-derived Antigen-presenting Cells and May Involve Antigen Transfer from Myocytes. Immunology 146: 312-326. https://doi.org/10.1111/imm.12505.

Michael Klompas, Steve Bernstein, and Harvard Pilgrim Health Care, Inc. 2010. “Electronic Support for Public Health– Vaccine Adverse Event Reporting System (ESP-VAERS).” Rockville, MD: Harvard Pilgrim Health Care, Inc. https://healthit.ahrq.gov/sites/default/files/docs/publication/r18hs017045-lazarus-final-report-2011.pdf.

Lederer, K., Castaño, D., Gómez Atria, D., Oguin T. H., III, Wang, S., Manzoni, T. B., … (2020).SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation.Immunity 53: 1281-1295. https://doi.org/10.1016/j. immuni.2020.11.009.

Lee, S. H., Cha, J. M., Lee, J. I., Joo, K. R., Shin, H. P., Baek, I. H. … Cho, J. L. (2015). Anaphylactic Shock Caused by Ingestion of Polyethylene Glycol. Intestinal research 13(1): 90-94. https://doi.org/10.5217/ ir.2015.13.1.90.

Lee, W. S., Wheatley, A. K., Kent, S. J. & DeKosky, B. J. (2020). Antibody-Dependent Enhancement and SARS-CoV-2 Vaccines and Therapies. Nature Microbiology 5(10): 1185-1191. https://doi.org/ 10.1038/s41564-020-00789-5.

Lema Tomé, C. M., Tyson, T., Rey, N. L., Grathwohl, S., Britschgi, M. and Brundin, P. (2013). Inflammation and αSynuclein Prion-like Behavior in Parkinson’s Disease – Is There a Link? Molecular Neurobiology 47: 561-574. https://www.doi.org/10.1007/s12035-012-8267-8.

Lesbats, P., Engelman, A. N. & Cherepanov, P. (2016). Retroviral DNA Integration. Chemical Reviews 2016 116(20): International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 73 12730012757. https://doi.org/10.1021/acs.chemrev.6b00125.

Liang, J., Zhu, H., Wang, X., Jing, B., Li, Z., Xia, X. … Sun, B. (2020). Adjuvants for Coronavirus Vaccines. Frontiers in Immunology 11: 2896. https://doi.org/10.3389/fimmu.2020.589833.

Lila, A. S., Shimizu, A. T. & Ishida, T. (2018). PEGylation and Anti-PEG Antibodies. Engineering of Biomaterials for Drug Delivery Systems. Woodhead Publishing 51-68. https://doi.org/10.1016/B978-0-08-101750-0.00003-9.

Limanaqi, F., Letizia Busceti, C., Biagioni, F., Lazzeri, G., Forte, M., Schiavon, S. … Fornai, F. (2020). Cell Clearing Systems as Targets of Polyphenols in Viral Infections: Potential Implications for COVID-19 Pathogenesis. Antioxidants 9: 1105. https://doi.org/10.3390/antiox9111105.

Lindsay, K. E., Bhosle, S. M., Zurla, C., Beyersdorf, J., Rogers, K. A., Vanover D. & Xiao, P. (2019). Visualization of Early Events in mRNA Vaccine Delivery in Non-Human Primates via PET–CT and Near-Infrared Imaging. Nature Biomedical Engineering 3: 371-380. https://doi.org/10.1038/s41551-019-0378-3.

Lipp, E., von Felten, A., Sax, H., Mller, D. & Berchtold, P. (1998). Antibodies Against Platelet Glycoproteins and Antiphospholipid Antibodies in Autoimmune Thrombocytopenia. European Journal of Haematology 60(5): 283-8. https://doi.org/10.1111/j.1600-0609.1998.tb01041.x.

Liu, L., Wei, Q., Lin, Q., Fang, J., Wang, H., Kwok, H., … Chen, Z. (2019). Anti–spike IgG Causes Severe Acute Lung Injury by Skewing Macrophage Responses During Acute SARS-CoV Infection. JCI Insight 4(4): e123158. https://doi.org/10.1172/jci.insight.123158.

Liu, M. A. (2019). A Comparison of Plasmid DNA and mRNA as Vaccine Technologies. Vaccines (Basel) 7(2): 37. https://doi.org/10.3390/ vaccines7020037.

Liu, S., Hossinger, A., Gbbels, S., and Ina M. Vorberga, I. M. (2017). Prions on the Run: How Extracellular Vesicles Serve as Delivery Vehicles for Self-templating Protein Aggregates. Prion 11(2): 98-112. https://www.doi.org/10.1080/19336896.2017.1306162.

Liu, Y., Liu, J., Xia, H., Zhang, X., Fontes-Garfias, C. R., Swanson, K. A. … Shi, P.-Y. (2021). Neutralizing Activity of BNT162b2-Elicited Serum. N Engl J Med 384: 1466-1468. https://doi.org/10.1056/ NEJMc2102017.

Louis, N., Evelegh, C., Graham, F. L. (1997) Cloning and Sequencing of the Cellular-Viral Junctions from the Human Adenovirus Type 5 Transformed 293 Cell Line. Virology 233: 423-429. https://doi.org/10.1006/viro.1997.8597.

Lu, J., Lu, G., Tan, S., Xia, J., Xiong, H., Yu, X. … Lin, J. (2020). A COVID-19 mRNA Vaccine Encoding SARS-CoV-2 Virus-like Particles Induces a Strong Antiviral-like Immune Response in Mice. Cell Research 30: 936-939. https://doi.org/10.1038/s41422-020-00392-7.

Lu, L., Li, J., Moussaoui, M. & Boix, E. (2018). Immune Modulation by Human Secreted RNases at the Extracellular Space. Frontiers in Immunology 9: 1012. https://doi.org/10.3389/fimmu.2018.01012.

Lu, L. L., Suscovich, T. J., Fortune, S. M. & Alter G. (2018b). Beyond Binding: Antibody Effector Functions in Infectious Diseases. Nature Reviews Immunology18(1): 46-61. https://doi.org/10.1038/ nri.2017.106.

Lucchetti, D., Santini, G., Perelli, L., Ricciardi-Tenore, C., Colella, F., Mores, N., … Montuschi, P. (2021). Detection and Characterization of Extracellular Vesicles in Exhaled Breath Condensate and Sputum of COPD and Severe Asthma Patients. European Respiratory Journal Apr 1; 2003024. [Epub ahead of print]. https://www.doi.org/10.1183/13993003.03024-2020.

Luganini, A. & Gribaudo, G. (2020). Retroviruses of the Human Virobiota: The Recycling of Viral Genes and the Resulting Advantages for Human Hosts During Evolution. Frontiers in Microbiology 11: 1140. https://doi.org/10.3389/fmicb.2020.01140.

Lyons-Weiler, J. (2020). Pathogenic Priming Likely Contributes to Serious and Critical Illness and Mortality in COVID19 via Autoimmunity. Journal of Translational Autoimmunity 3: 100051. https://www.sciencedirect.com/science/article/pii/S2589909020300186.

Mahose, E. (2021) Covid-19: Booster Dose will be Needed in Autumn to Avoid Winter Surge, Says Government Adviser. BMJ 372: n664. https://doi.org/10.1136/bmj.n664.

Marino, M., Scuderi, F., Provenzano, C. & Bartoccioni, E. (2011) Skeletal Muscle Cells: from Local Inflammatory Response to Active Immunity. Gene Therapy 18: 109-116. https://doi.org/10.1038/ gt.2010.124. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 74

Matsuno, H., Yudoh, K., Katayama, R., Nakazawa, F., Uzuki, M., Sawai, T., … Kimura, T. (2002). The Role of TNF-α iin the Pathogenesis of Inflammation and Joint Destruction in Rheumatoid Arthritis (RA): a Study Using a Human RA/SCID Mouse Chimera. Rheumatology (Oxford) 41(3): 329-37. https://doi.org/10.1093/rheumatology/41.3.329.

McClintock, B. (1965). Components of Action of the Regulators Spm and Ac. Carnegie Institution of Washington Year Book 64: 527-536. http://repository.cshl.edu/id/eprint/34634/.

McNeil, M. M., Weintraub, E. S., Duffy, J., Sukumaran, L., Jacobsen, S. J., Klein, N. P. … DeStefano, F. (2016). Risk of Anaphylaxis after Vaccination in Children and Adults. The Journal of Allergy and Clinical Immunology 137(3): 868-78. https://doi.org/10.1016/j.jaci.2015.07.048.

Mehta, N., Sales, R. M., Babagbemi, K., Levy, A. D., McGrath, A. L., Drotman, M. & Dodelzon. K. (2021). Unilateral axillary Adenopathy in the setting of COVID-19 vaccine. Clinical Imaging 75: 12-15. https://doi.org/10.1016/j.clinimag.2021.01.016.

Mi, S., Lee, X., Li, X., Veldman, G. M., Finnerty, H., Racie, L. … McCoy, J. M. (2000). Syncytin is a Captive Retroviral Envelope Protein Involved in Human Placental Morphogenesis. Nature 403(6771): 785-9. https://doi.org/10.1038/35001608.

Moderna. mRNA Platform: Enabling Drug Discovery & Development. 2020. https://www.modernatx.com/mrnatechnology/mrna-platform-enabling-drug-discovery-development

Mohamed, M., Lila, A. S., Shimizu, T., Alaaeldin, E., Hussein, A., Sarhan, H. A., Szebeni, J. & Ishida, T. (2019).PEGylated Liposomes: Immunological Responses. Science and Technology of Advanced Materials 20(1): 710-724. https://doi.org/10.1080/14686996.2019.1627174.

Morens, D. M. (1994). Antibody-dependent Enhancement of Infection and the Pathogenesis of Viral Disease. Clinical Infectious Diseases 19(3): 500-512, https://doi.org/10.1093/clinids/19.3.500.

Mueller, B. K., Subramaniam, S., and Senes, A. (2014). A Frequent, GxxxG-mediated, Transmembrane Association Motif is Optimized for the Formation of Interhelical Cα-H Hydrogen Bonds. PNAS E888-E895. Proceedings of the Natural Academy of Sciences USA 111(10): E888-95. https://doi.org/10.1073/pnas.1319944111. National Institutes of Health (December 11, 2020).

NIH-Moderna COVID-19 Vaccine Shows Promising Interim. Results. NIH Record Vol. LXXII, No. 25. Retrieved January 27, 2021 from https://nihrecord.nih.gov/2020/12/11/nih-moderna-covid-19-vaccine-shows-promising-interim-results

Navarra, A., Albani, E., Castellano, S., Arruzzolo L., & Levi-Setti P. E. (2020). Coronavirus Disease-19 Infection: Implications on Male Fertility and Reproduction. Frontiers in Physiology 11: 574761. https://www.doi.org/10.3389/fphys.2020.574761.

Ndeupen, S., Qin, Z., Jacobsen, S., Estanbouli, H., Bouteau, A., & Igyártó, B.Z. (2021) The mRNA-LNP Platform’s Lipid Nanoparticle Component Used in Preclinical Vaccine Studies is Highly Inflammatory. bioRxiv 2021.03.04.430128. https://doi.org/10.1101/2021.03.04.430128.

Norling, K., Bernasconi, V., Hernández, V. A., Parveen, N., Edwards, K., Lycke, N. Y. … Bally. M. (2019). Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro. ACS Infectious Diseases 5(11): 1867-1878. https://doi.org/10.1021/acsinfecdis.9b00189.

Oller, J. W., Jr. (2010). The Antithesis of Entropy: Biosemiotic Communication from Genetics to Human Language with Special Emphasis on the Immune Systems. Entropy 12: 631-705. https://www.doi.org/10.3390/e12040631.

Palucka, A. K., Blanck, J. P., Bennett, L., Pascual, V,, Banchereau, J. (2005) Cross-regulation of TNF and IFN-α in Autoimmune Diseases. Proceedings of the National Academy of Sciences USA 102: 3372-3377. https://doi.org/10.1073/pnas.0408506102.

Pellionisz, A. J. (2012). The Decade of Fractogene: From Discovery to Utility – Proofs of Concept Open Genome-Based Clinical Applications. International Journal of Systemics, Cybernetics and Informatics 12-02: 17-28. http://www.junkdna.com/pellionisz_decade_of_fractogene.pdf.

Peron, J. P. S. & Nakaya, H. (2020). Susceptibility of the Elderly to SARS-CoV-2 Infection: ACE-2 Overexpression, Shedding, and Antibody-dependent Enhancement (ADE). Clinics (Sao Paulo) 75: e1912. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 75 https://doi.org/10.6061/clinics/2020/e1912.

Pittoggi, C., Beraldi, R., Sciamanna, I., Barberi, L., Giordano, R., Magnano, A. R.& Spadafora C (2006). Generation of Biologically Active Retro-genes upon Interaction of Mouse Spermatozoa with Exogenous DNA. Molecular Reproduction and Development 73(10): 1239-46. https://doi.org/10.1002/mrd.20550.

Povsic, T. J., Lawrence, M. G., Lincoff, A. M., Mehran, R., Rusconi, C. P. … REGULATE-PCI Investigators. (2016). Pre-existing Anti-PEG Antibodies are Associated with Severe Immediate Allergic Reactions to Pegnivacogin, a PEGylated Aptamer. Journal of Allergy and Clinical Immunology 138(6): 1712-1715. https://doi.org/10.1016/j.jaci.2016.04.058. Pray, L. (2008) T

ransposons, or Jumping Genes: Not Junk DNA? Nature Education 1(1): 32. https://www.nature.com /scitable/topicpage/transposons-or-jumping-genes-not-junk-dna-1211/.

Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie Science 216(4542): 136-44. https://www.doi.org/10.1126/science.6801762.

Puga, I., Cols, M., Barra, C. M., He, B., Cassis, L., Gentile, M. … Cerutti, A. (2011). B Cell-helper Neutrophils Stimulate the Diversification and Production of Immunoglobulin in the Marginal Zone of the Spleen. Natural Immunology 13(2): 170-80. https://doi.org/10.1038/ni.2194.

Pushparajah, D., Jimeneza, S., Wong, S., Alattas, H., Nafissi, N. & Slavcev, R. A. (2021) Advances in Gene-Based Vaccine Platforms to Address the COVID-19 Pandemic. Advanced Drug Delivery Reviews 170: 113-141. https://doi.org/10.1016/j.addr.2021.01.003.

Rico-Campà, A., Martínez-González, M. A., Alvarez-Alvarez, I., de Deus Mendonça, R., de la Fuente-Arrillaga, C., Gómez-Donoso, C. & Bes-Rastrollo, M. (2019). Association Between Consumption of Ultra-Processed Foods and All Cause Mortality: SUN Prospective Cohort Study. Journal of Infection and Public Health 13(10): 1373-1380. https://pubmed.ncbi.nlm.nih.gov/31142450/

Rocha, E. P. C., & Danchin, A. (2002). Base composition bias might result from competition for metabolic resources. Trends in Genetics, 18(6), 291–294. https://doi.org/10.1016/S0168-9525(02)02690-2

Sarohan, A. R. (2020). COVID-19: Endogenous Retinoic Acid Theory and Retinoic Acid Depletion Syndrome. Medical Hypotheses 144: 110250. https://www.doi.org/10.1016/j.mehy.2020.110250.

Schiaffino, M. T., Di Natale, M., García-Martínez, E., Navarro, J., Muñoz-Blanco, J. L., Demelo-Rodríguez, P., & Sánchez-Mateos, P. (2020). Immunoserologic Detection and Diagnostic Relevance of Cross-reactive Autoantibodies in Coronavirus Disease 2019 Patients. The Journal of Infectious Diseases, 222(9), 1439-1443. https://doi.org/10.1093/infdis/jiaa485.

Schlake, T., Thess, A., Fotin-Mleczek, M. & Kallen, K.-J. (2012). Developing mRNA-vaccine technologies, RNA Biology 9: 1319–1330. https://doi.org/10.4161/rna.22269.

Sellaturay, P., Nasser, S., & Ewan, P. (2020). Polyethylene Glycol (PEG)-Induced Anaphylactic Reaction During Bowel Preparation. ACG Case Reports Journal 2(4) 216-217. https://doi.org/10.14309/crj.2015.63.

Sellaturay, P., Nasser, S., & Ewan, P. (2020). Polyethylene Glycol–Induced Systemic Allergic Reactions (Anaphylaxis). The Journal of Allergy and Clinical Immunology: In Practice 9(2): 670-675. https://doi.org/10.1016/j.jaip.2020.09.029.

Shaw, C.A. (2021). The Age of COVID-19: Fear, Loathing, and the New Normal. International Journal of Vaccine Theory, Practice, and Research 1: 98-142. https://ijvtpr.com/index.php/IJVTPR/article/ view/11.

Shukla, R., Ramasamy, V., Shanmugam, R. K., Ahuja, R. & Khanna, N. (2020). Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine. Frontiers in Cellular and Infection Microbiology 10: 572681. https://doi.org/10.3389/fcimb.2020.572681.

Slimani, Y., Abbassi, R., El Fatoiki, F. Z., Barrou, L., & Chiheb, S. (2021). Systemic Lupus Erythematosus and Varicella‐ Like Rash Following COVID‐19 in a Previously Healthy Patient. Journal of Medical Virology 93(2): 1184-1187. https://doi.org/10.1002/jmv.26513.

Steele, E. J., Gorczynski, R. M., Lindley, R. A., Liu, Y., Temple, R., Tokoro, G., … Wickramasinghe, , N. C. (2019). Lamarck and Panspermia – On the Efficient Spread of Living Systems Throughout the Cosmos. Progress in Biophysics and Molecular Biology 149: 10-32. https://doi.org/10.1016/j. pbiomolbio.2019.08.010.

International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 76

Steiner, J. A., Angot, E., and Brundin, P. (2011). A Deadly Spread: Cellular Mechanisms of α-Synuclein Transfer. Cell Death and Differentiation 18: 1425-1433. https://www.doi.org/10.1038/cdd.2011.53.

Stokes, A., Pion, J., Binazon, O., Laffont, B., Bigras, M., Dubois, G. … Rodriguez L.-A. (2020). Nonclinical Safety Assessment of Repeated Administration and Biodistribution of a Novel Rabies Self-amplifying mRNA Vaccine in Rats. Regulatory Toxicology and Pharmacology 113: 104648. https://doi.org/10.1016/j.yrtph.2020.104648.

Su, J. R., Moro, P. L., Ng, C. S., Lewis, P. W., Said, M. A., & Cano, M.V. (2019). Anaphylaxis after vaccination reported to theVaccine Adverse Event Reporting System, 1990-2016.Journal of Allergy and Clinical Immunology 143(4): 1465-1473. https://doi.org/10.1016/j.jaci.2018.12.1003.

Sun, R.-J. & Shan, N.-N. (2019). Megakaryocytic Dysfunction in Immune Thrombocytopenia is Linked to Autophagy Cancer Cell International 19: 59. https://doi.org/10.1186/s12935-019-0779-0.

Suzuki, Y. J. & Gychka, S. G. (2021). SARS-CoV-2 Spike Protein Elicits Cell Signaling in Human Host Cells: Implications for Possible Consequences of COVID-19 Vaccines. Vaccines 9: 36. https://doi.org/10.3390/vaccines9010036.

Suzuki, Y. J. (2020). The Viral Protein Fragment Theory of COVID-19 Pathogenesis. Medical Hypotheses 144: 110267. https://doi.org/10.1016/j.mehy.2020.110267.

Suzuki, Y. J., Nikolaienko, S. I., Dibrova, V. A., Dibrova, Y. V., Vasylyk, V. M., Novikov, M. Y. … Gychka, S. G. (2021). SARS-CoV-2 Spike Protein-Mediated Cell Signaling in Lung Vascular Cells. Vascular Pharmacology 137: 106823. https://doi.org/10.1016/j.vph.2020.106823.

Suzuki, Y.J., Nikolaienko, S.I., Dibrova, V.A., Dibrova, Y.V., Vasylyk, V.M., Novikov, M.Y. … Gychka, S.G. (2020). SARS-CoV-2 Spike Protein-Mediated Cell Signaling in Lung Vascular Cells. Vascular Pharmacology 137: 106823. https://www.doi.org/10.1016/j.vph.2020.106823.

Takada, A., Feldmann, H., Ksiazek, T. G. & Kawaoka, Y. (2003). Antibody-Dependent Enhancement of Ebola Virus Infection. Virology 77(13): 7539-7544. https://doi.org/10.1128/JVI.77.13.7539-7544.2003.

Temin, H. M. and Mizutani, S. (1970). RNA-dependent DNA polymerase in virions of Rous Sarcoma Virus. Nature 226: 1211–3. https://www.doi.org/10.1038/2261211a0.

Tetz, G. and Tetz, V. (2020). SARS-CoV-2 Prion-Like Domains in Spike Proteins Enable Higher Affinity to ACE2. Preprints 2020030422. https://www.doi.org/10.20944/preprints202003.0422.v1. Tetz, G. and Tetz,V (2018). Prion-like Domains in Eukaryotic Viruses. Scientific Reports 8: 8931. https://doi.org/10.1038/s41598-018-27256-w.

U.S. Department of Health and Human Services, Food and Drug Administration. Center for Biologics Evaluation and Research. (2020, June) Development and Licensure of Vaccines to Prevent COVID-19 Guidance for Industry. Retrieved February 11, 2021 from https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/development-and-licensure-vaccines-prevent-covid-19. US Food and Drug Administration (2021).

Pfizer-BioNTech COVID-19 Vaccine EUA Fact Sheet for Healthcare Providers Administering Vaccine (Vaccination Providers). https://www.fda.gov/media/144413

Verma, S., Saksena, S. & Sadri-Ardekani, H. (2020). ACE2 Receptor Expression in Testes: Implications in Coronavirus Disease 2019 Pathogenesis. Biology of Reproduction 103(3): 449-451. https://doi.org/10.1093/biolre/ioaa080.

Vlachoyiannopoulos, P. G., Magira, E., Alexopoulos, H., Jahaj, E., Theophilopoulou, K., Kotanidou, A., & Tzioufas, A. G. (2020). Autoantibodies Related to Systemic Autoimmune Rheumatic Diseases in Severely Ill Patients with COVID-19. Annals of the Rheumatic Diseases 79(12): 1661-1663. http://dx.doi.org/10.1136/annrheumdis-2020- 218009.

Vaidya, M. and Sugaya, K (2020). DNA Associated with Circulating Exosomes as a Biomarker for Glioma. Genes 11: 1276. https://www.doi.org/10.3390/genes11111276.

Vojdani, A., & Kharrazian, D. (2020). Potential Antigenic Cross-Reactivity Between SARS-CoV-2 and Human Tissue with a Possible Link to an Increase in Autoimmune Diseases. Clinical Immunology (Orlando, Fla.) 217: 108480. https://doi.org/10.1016/j.clim.2020.108480.

Vojdani, A., Vojdani, E., & Kharrazian, D. (2021). Reaction of Human Monoclonal Antibodies to SARS-CoV-2 Proteins International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 77 with Tissue Antigens: Implications for Autoimmune Diseases. Frontiers in Immunology 11: 3679. https://doi.org/10.3389/fimmu.2020.617089.

Wadhwa, A., Aljabbari, A., Lokras, A., Foged, C. & Thakur, A. (2020). Opportunities and Challenges in the Delivery of mRNA-based Vaccines. Pharmaceutics 12(2): 102. https://doi.org/10.3390/ pharmaceutics12020102.

Wallukat, G., Hohberger, B., Wenzel, K.,Fürst, J.,Schulze-Rothe, S., Wallukat, A. … Müller, J. (2021). Functional Autoantibodies against G-protein Coupled Receptors in Patients with Persistent Post-COVID-19 Symptoms. Journal of Translational Autoimmunity 4: 100100. https://doi.org/10.1016/j .jtauto.2021.100100.

Walter, U., Tsiberidou, P., Kersten, M., Storch, A., and Lohle, M. (2018). Atrophy of the Vagus Nerve in Parkinsons Disease Revealed by High-resolution Ultrasonography. Frontiers in Neurology 9:805. https://www.doi.org/10.3389/fneur.2018.00805.

Wan, Y., Shang, J., Sun, S., Tai, W., Chen, J., Geng, Q., … & Li, F. (2020). Molecular Mechanism for AntibodyDependent Enhancement of Coronavirus Entry. Journal of virology, 94(5). https://doi.org/10.1128/JVI.02015- 19.

Wang, C.-Y., Ma, S., Bi, S.-J., Su,L., Huang, S.-Y. … Peng, J. (2019). Enhancing Autophagy Protects Platelets in Immune Thrombocytopenia Patients. Ann Transl Med 7(7): 134. https://doi.org/10.21037/atm.2019.03.04.

Wang, Z., Troilo, P. J., Wang, X., Griffiths, T.G. II, Pacchione, S. J., Barnum, A. B., … Ledwith, B. J. (2004). Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Therapy 11: 711-721. https://doi.org/10.1038/sj.gt.3302213.

Wang, Z.& Xu, X. (2020). ScRNA-seq Profiling of Human Testes Reveals the Presence of the ACE2 Receptor, a Target for SARS-CoV-2 Infection in Spermatogonia, Leydig and Sertoli Cells. Cells 9: 920. https://doi.org/10.3390/cells9040920.

Weickenmeier, J., Jucker, M., Goriely, A., and Kuhl, E. (2019). A Physics-based Model Explains the Prion-like Features of Neurodegeneration in Alzheimer’s Disease, Parkinson’s Disease, and Amyotrophic Lateral Sclerosis. Journal of the Mechanics and Physics of Solids 124: 264-281. https://doi.org/10.1016/j.jmps.2018.10.013.

Weiner, A. M. (2002). SINEs and LINEs: the Art of Biting the Hand that Feeds You. Current Opinions in Cell Biology 14(3): 343-50. https://doi.org/10.1016/s0955-0674(02)00338-1. Wikipedia contributors. (2021, February 13). ELISA. Retrieved February 16, 2021, from Wikipedia, The Free Encyclopedia. https://en.wikipedia.org/w/index.php?title=ELISA&oldid=1006455262.

World Health Organization (2021, January 19). mRNA-1273 Vaccine (Moderna) Against COVID-19 Background Document: Draft Prepared by the Strategic Advisory Group of Experts (SAGE) on Immunization Working Group on COVID-19 vaccines. No. WHO/2019-nCoV/vaccines/mRNA-1273/2021.1. https://policycommons.net/artifacts/1424630/mrna-1273-vaccine-moderna-against-covid-19-backgrounddocument/ World Health Organization. (2021, January 14).

Background document on mRNA vaccine BNT162b2 (PfizerBioNTech) against COVID-19. License: CC BY-NC-SA 3.0 IGO. https://apps.who.int/iris/handle/10665/338671.

Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C.-L., Abiona, O. … Graham, B. S. (2020). Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation. Science 2020; 367: 1260-3. https://doi.org/10.1126/science.abb2507.

Wu, F., Yan, R., Liu, M., Liu, Z., Wang, Y., Luan, D., … Huang, J. (2020). Antibody-Dependent Enhancement (ADE) of SARS-CoV-2 Infection in Recovered COVID-19 Patients: Studies Based on Cellular and Structural Biology Analysis. medRxiv preprint. https://doi.org/10.1101/2020 .10.08.20209114.

Wylon, K. Sabine Dölle, S., & Margitta Worm, M. (2016). Polyethylene Glycol as a Cause of Anaphylaxis. Allergy, Asthma & Clinical Immunology 12(1): 1-3. https://doi.org/10.1186/s13223-016-0172-7.

Xu, S., Yang, K., Li, R. & Zhang, L. (2020) mRNA Vaccine Era — Mechanisms, Drug Platform and Clinical Prospection. International Journal of Molecular Science 21(18): 6582. https://doi.org/10.3390 /ijms21186582.

Yang, Q. & Lai, S. K. (2015). Anti‐PEG Immunity: Emergence, Characteristics, and Unaddressed Questions. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 7(5): 655-677. International Journal of Vaccine Theory, Practice, and Research 2(1), May 10, 2021 Page | 78 https://doi.org/10.1002/wnan.1339.

Young, R., Bekele, T., Gunn, A., Chapman, N., Chowdhary, V., Corrigan, K., … Yamey, G. (2018). Developing New Health Technologies for Neglected Diseases: A Pipeline Portfolio Review and Cost Model. Gates Open Res 2:23. https://doi.org/10.12688/gatesopenres.12817.2.

Zaman, M. (2021). COVID Vaccine Booster Shots Are Coming — Here’s What to Know. https://www.msn.com/enus/health/medical/covid-vaccine-booster-shots-are-coming-here-s-what-to-know/ar-BB1foY4s. Accessed 5/1/2021.

Zamani, B., Moeini Taba, S.-M. & Shayestehpour, M. (2021). Systemic Lupus Erythematosus Manifestation Following COVID-19: A Case Report. Journal of Medical Case Reports 15(1): 1-4. https://doi.org/10.1186/s13256-020- 02582-8.

Zeng, C., Zhang, C, Walker, P. G. & Dong, Y. (2020). Formulation and Delivery Technologies for mRNA Vaccines. Current Topics in Microbiology and Immunology June 2. [Epub ahead of print]. https://doi.org/10.1007/82_2020_217.

Zhang, L., Richards, A., Barrasa, M, I., Hughes, S. H., Young, R. A. & Jaenisch, R. (2021). Reverse-transcribed SARSCoV-2 RNA can Integrate into the Genome of Cultured Human Cells and can be Expressed in Patient-derived Tissues. Proceedings of the National Academy of Sciences 118(21): e2105968118. https://doi.org/10.1073/pnas.2105968118.

Zhang, X. W. & Yap, Y. L. (2004). The 3D Structure Analysis of SARS-CoV S1 Protein Reveals a Link to Influenza Virus Neuraminidase and Implications for Drug and Antibody Discovery. Theochemistry 681(1): 137-141. https://doi.org/10.1016/j.theochem.2004.04.065.

Zhou, Z.-H., Stone, C. A., Jr., Jakubovic, B., Phillips, E. J., Sussman, G., Park, J.-M. … Kozlowski, S. (2020). Anti-PEG IgE in Anaphylaxis Associated with Polyethylene Glycol. The Journal of Allergy and Clinical Immunology in Practice ;9(4): 1731-1733.e3. https://doi.org/10.1016/j.jaip.2020.11.011.

Zimmer, C., Corum, J., Wee, S.-L. Coronavirus Vaccine Tracker. New York Times. Updated Jan. 28, 2021. https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html.

Zuo, Y., Estes, S. K., Ali, R. A., Gandhi, A. A., Yalavarthi, S., Shi, H., … & Knight, J. S. (2020). Prothrombotic Autoantibodies in Serum from Patients Hospitalized with COVID-19. Science Translational Medicine, 12(570): eabd3876. https://doi.org/10.1126/scitranslmed.abd3876.

Legal Disclaimer

The information on the website and in the IJVTPR is not intended as a diagnosis, recommended treatment, prevention, or cure for any human condition or medical procedure that may be referred to in any way. Users and readers who may be parents, guardians, caregivers, clinicians, or relatives of persons impacted by any of the morbid conditions, procedures, or protocols that may be referred to, must use their own judgment concerning specific applications. The contributing authors, editors, and persons associated in any capacity with the website and/or with the journal disclaim any liability or responsibility to any person or entity for any harm, financial loss, physical injury, or other penalty that may stem from any use or application in any context of information, conclusions, research findings, opinions, errors, or any statements found on the website or in the IJVTPR. The material presented is freely offered to all users who may take an interest in examining it, but how they may choose to apply any part of it, is the sole responsibility of the viewer/user. If material is quoted or reprinted, users are asked to give credit to the source/author and to conform to the non-commercial, no derivatives, requirements of the Creative Commons License 4.0 NC ND.

Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies

Nature Microbiology volume 5, pages1185–1191 (2020)

Abstract

Antibody-based drugs and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being expedited through preclinical and clinical development. Data from the study of SARS-CoV and other respiratory viruses suggest that anti-SARS-CoV-2 antibodies could exacerbate COVID-19 through antibody-dependent enhancement (ADE). Previous respiratory syncytial virus and dengue virus vaccine studies revealed human clinical safety risks related to ADE, resulting in failed vaccine trials. Here, we describe key ADE mechanisms and discuss mitigation strategies for SARS-CoV-2 vaccines and therapies in development. We also outline recently published data to evaluate the risks and opportunities for antibody-based protection against SARS-CoV-2.

Main

The emergence and rapid global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has resulted in substantial global morbidity and mortality along with widespread social and economic disruption. SARS-CoV-2 is a betacoronavirus closely related to SARS-CoV (with ~80% sequence identity), which caused the SARS outbreak in 2002. Its next closest human coronavirus relative is Middle East respiratory syndrome-related coronavirus (MERS-CoV; ~54% sequence identity), which caused Middle East respiratory syndrome in 2012 (refs. 1,2). SARS-CoV-2 is also genetically related to other endemic human coronaviruses that cause milder infections: HCoV-HKU1 (~52% sequence identity), HCoV-OC43 (~51%), HCoV-NL63 (~49%) and HCoV-229E (~48%)1. SARS-CoV-2 is even more closely related to coronaviruses identified in horseshoe bats, suggesting that horseshoe bats are the primary animal reservoir with a possible intermediate transmission event in pangolins3.

Cellular entry of SARS-CoV-2 is mediated by the binding of the viral spike (S) protein to its cellular receptor, angiotensin-converting enzyme 2 (ACE2)4,5. Other host entry factors have been identified, including neuropilin-1 (refs. 6,7) and TMPRSS2, a transmembrane serine protease involved in S protein maturation4. The SARS-CoV-2 S protein consists of the S1 subunit, which contains the receptor binding domain (RBD), and the S2 subunit, which mediates membrane fusion for viral entry8. A major goal of vaccine and therapeutic development is to generate antibodies that prevent the entry of SARS-CoV-2 into cells by blocking either ACE2–RBD binding interactions or S-mediated membrane fusion.

One potential hurdle for antibody-based vaccines and therapeutics is the risk of exacerbating COVID-19 severity via antibody-dependent enhancement (ADE). ADE can increase the severity of multiple viral infections, including other respiratory viruses such as respiratory syncytial virus (RSV)9,10 and measles11,12. ADE in respiratory infections is included in a broader category named enhanced respiratory disease (ERD), which also includes non-antibody-based mechanisms such as cytokine cascades and cell-mediated immunopathology (Box 1). ADE caused by enhanced viral replication has been observed for other viruses that infect macrophages, including dengue virus13,14 and feline infectious peritonitis virus (FIPV)15. Furthermore, ADE and ERD has been reported for SARS-CoV and MERS-CoV both in vitro and in vivo. The extent to which ADE contributes to COVID-19 immunopathology is being actively investigated.

In this Perspective, we discuss the possible mechanisms of ADE in SARS-CoV-2 and outline several risk mitigation principles for vaccines and therapeutics. We also highlight which types of studies are likely to reveal the relevance of ADE in COVID-19 disease pathology and examine how the emerging data might influence clinical interventions.

Box 1 ADE and ERD

ERD

ERD describes severe clinical presentations of respiratory viral infections associated with medical interventions (especially vaccines). Similar clinical presentations can occur as a result of natural infections, and so ERD is detected during preclinical and clinical trials by comparing the distribution of disease severities between the intervention and placebo study arms. ERD can be associated with a broad range of molecular mechanisms, including FcR-dependent antibody activity and complement activation (that is, ADE), but also to other antibody-independent mechanisms such as tissue cell death, cytokine release and/or local immune cell activation.

ADE

ADE can be broadly categorized into two different types based on the molecular mechanisms involved:

ADE via enhanced infection. Higher infection rates of target cells occur in an antibody-dependent manner mediated by Fc–FcR interactions. ADE via enhanced infection is commonly measured using in vitro assays detecting the antibody-dependent infection of cells expressing FcγRIIa, such as monocytes and macrophages. The link between in vitro ADE assay results and clinical relevance is often implied, rather than directly observed. Dengue virus represents the best documented example of clinical ADE via enhanced infection.

ADE via enhanced immune activation. Enhanced disease and immunopathology are caused by excessive Fc-mediated effector functions and immune complex formation in an antibody-dependent manner. The antibodies associated with enhanced disease are often non-neutralizing. ADE of this type is usually examined in vivo by detecting exacerbated disease symptoms, including immunopathology and inflammatory markers, and is most clearly associated with respiratory viral infections. RSV and measles are well-documented examples of ADE caused by enhanced immune activation.

ERD and ADE (of the second type described above) are often identified by clinical data, including symptom prevalence and disease severity, rather than by the specific molecular mechanisms that drive severe disease. The presence of complex feedback loops between different arms of the immune system makes it very difficult (although not impossible) to conclusively determine molecular mechanisms of ADE and ERD in human and animal studies, even if the clinical data supporting ADE and ERD are quite clear. Many different measurements and assays are used to track ADE and ERD, which can vary based on the specific virus, preclinical and/or clinical protocols, biological samples collected and in vitro techniques used.

Respiratory ADE is a specific subset of ERD.Show more

Mechanisms of ADE

ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology (Fig. 1, Box 1). Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection. ADE can be measured in several ways, including in vitro assays (which are most common for the first mechanism involving FcγRIIa-mediated enhancement of infection in phagocytes), immunopathology or lung pathology. ADE via FcγRIIa-mediated endocytosis into phagocytic cells can be observed in vitro and has been extensively studied for macrophage-tropic viruses, including dengue virus in humans16 and FIPV in cats15. In this mechanism, non-neutralizing antibodies bind to the viral surface and traffic virions directly to macrophages, which then internalize the virions and become productively infected. Since many antibodies against different dengue serotypes are cross-reactive but non-neutralizing, secondary infections with heterologous strains can result in increased viral replication and more severe disease, leading to major safety risks as reported in a recent dengue vaccine trial13,14. In other vaccine studies, cats immunized against the FIPV S protein or passively infused with anti-FIPV antibodies had lower survival rates when challenged with FIPV compared to control groups17. Non-neutralizing antibodies, or antibodies at sub-neutralizing levels, enhanced entry into alveolar and peritoneal macrophages18, which were thought to disseminate infection and worsen disease outcome19.

figure 1
Fig. 1: Two main ADE mechanisms in viral disease.

In the second described ADE mechanism that is best exemplified by respiratory pathogens, Fc-mediated antibody effector functions can enhance respiratory disease by initiating a powerful immune cascade that results in observable lung pathology20,21. Fc-mediated activation of local and circulating innate immune cells such as monocytes, macrophages, neutrophils, dendritic cells and natural killer cells can lead to dysregulated immune activation despite their potential effectiveness at clearing virus-infected cells and debris. For non-macrophage tropic respiratory viruses such as RSV and measles, non-neutralizing antibodies have been shown to induce ADE and ERD by forming immune complexes that deposit into airway tissues and activate cytokine and complement pathways, resulting in inflammation, airway obstruction and, in severe cases, leading to acute respiratory distress syndrome10,11,22,23. These prior observations of ADE with RSV and measles have many similarities to known COVID-19 clinical presentations. For example, over-activation of the complement cascade has been shown to contribute to inflammatory lung injury in COVID-19 and SARS24,25. Two recent studies found that S- and RBD-specific immunoglobulin G (IgG) antibodies in patients with COVID-19 have lower levels of fucosylation within their Fc domains26,27—a phenotype linked to higher affinity for FcγRIIIa, an activating Fc receptor (FcR) that mediates antibody-dependent cellular cytotoxicity. While this higher affinity can be beneficial in some cases via more vigorous FcγRIIIa-mediated effector functions28,29, non-neutralizing IgG antibodies against dengue virus that were afucosylated were associated with more severe disease outcomes30. Larsen et al. further show that S-specific IgG in patients with both COVID-19 and acute respiratory distress syndrome had lower levels of fucosylation compared to patients who had asymptomatic or mild infections26. Whether the lower levels of fucosylation of SARS-CoV-2-specific antibodies directly contributed to COVID-19 immunopathology remains to be determined.

Importantly, SARS-CoV-2 has not been shown to productively infect macrophages31,32. Thus, available data suggest that the most probable ADE mechanism relevant to COVID-19 pathology is the formation of antibody–antigen immune complexes that leads to excessive activation of the immune cascade in lung tissue (Fig. 1).

Evidence of ADE in coronavirus infections in vitro

While ADE has been well documented in vitro for a number of viruses, including human immunodeficiency virus (HIV)33,34, Ebola35,36, influenza37 and flaviviruses38, the relevance of in vitro ADE for human coronaviruses remains less clear. Several studies have shown increased uptake of SARS-CoV and MERS-CoV virions into FcR-expressing monocytes or macrophages in vitro32,39,40,41,42. Yip et al. found enhanced uptake of SARS-CoV and S-expressing pseudoviruses into monocyte-derived macrophages mediated by FcγRIIa and anti-S serum antibodies32. Similarly, Wan et al. showed that a neutralizing monoclonal antibody (mAb) against the RBD of MERS-CoV increased the uptake of virions into macrophages and various cell lines transfected with FcγRIIa39. However, the fact that antigen-specific antibodies drive phagocytic uptake is unsurprising, as monocytes and macrophages can mediate antibody-dependent phagocytosis via FcγRIIa for viral clearance, including for influenza43. Importantly, macrophages in infected mice contributed to antibody-mediated clearance of SARS-CoV44. While MERS-CoV has been found to productively infect macrophages45, SARS-CoV infection of macrophages is abortive and does not alter the pro-inflammatory cytokine gene expression profile after antibody-dependent uptake41,42. Findings to date argue against macrophages as productive hosts of SARS-CoV-2 infection31,32.

ADE in human coronavirus infections

No definitive role for ADE in human coronavirus diseases has been established. Concerns were first raised for ADE in patients with SARS when seroconversion and neutralizing antibody responses were found to correlate with clinical severity and mortality46. A similar finding in patients with COVID-19 was reported, with higher antibody titres against SARS-CoV-2 being associated with more severe disease47. One simple hypothesis is that greater antibody titres in severe COVID-19 cases result from higher and more prolonged antigen exposure due to higher viral loads48,49. However, a recent study showed that viral shedding in the upper respiratory tract was indistinguishable between patients with asymptomatic and symptomatic COVID-19 (ref. 50). Symptomatic patients showed higher anti-SARS-CoV-2 antibody titres and cleared the virus from the upper respiratory tract more quickly, contradicting a simpler hypothesis that antibody titres are simply caused by higher viral loads. Other studies showed that anti-SARS-CoV-2 T-cell responses could be found at high levels in mild and asymptomatic infections51,52. Taken together, the data suggest that strong T-cell responses can be found in patients with a broad range of clinical presentations, whereas strong antibody titres are more closely linked to severe COVID-19. One important caveat is that viral shedding was measured in the upper respiratory tract rather than in the lower respiratory tract50. The lower respiratory tract is likely more important for severe COVID-19 lung pathology, and it is unclear how closely SARS-CoV-2 viral shedding in the upper and lower respiratory tracts correlate throughout the disease course.

Beyond the host response to new SARS-CoV-2 infections, the potential of pre-existing antibodies against other human coronavirus strains to mediate ADE in patients with COVID-19 is another possible concern53. Antibodies elicited by coronavirus strains endemic in human populations (such as HKU1, OC43, NL63 and 229E) could theoretically mediate ADE by facilitating cross-reactive recognition of SARS-CoV-2 in the absence of viral neutralization. Preliminary data show that antibodies from SARS-CoV-2-naïve donors who had high reactivity to seasonal human coronavirus strains were found to have low levels of cross-reactivity against the nucleocapsid and S2 subunit of SARS-CoV-2 (ref. 54). Whether such cross-reactive antibodies can contribute to clinical ADE of SARS-COV-2 remains to be addressed.

Risk of ERD for SARS-CoV-2 vaccines

Safety concerns for SARS-CoV-2 vaccines were initially fuelled by mouse studies that showed enhanced immunopathology, or ERD, in animals vaccinated with SARS-CoV following viral challenge55,56,57,58. The observed immunopathology was associated with Th2-cell-biased responses55 and was largely against the nucleocapsid protein56,58. Importantly, immunopathology was not observed in challenged mice following the passive transfer of nucleocapsid-specific immune serum56, confirming that the enhanced disease could not be replicated using the serum volumes transferred. Similar studies using inactivated whole-virus or viral-vector-based vaccines for SARS-CoV or MERS-CoV resulted in immunopathology following viral challenge59,60,61, which were linked to Th2-cytokine-biased responses55 and/or excessive lung eosinophilic infiltration57. Rational adjuvant selection ensures that Th1-cell-biased responses can markedly reduce these vaccine-associated ERD risks. Candidate SARS-CoV vaccines formulated with either alum, CpG or Advax (a delta inulin-based adjuvant) found that while the Th2-biased responses associated with alum drove lung eosinophilic immunopathology in mice, protection without immunopathology and a more balanced Th1/Th2 response were induced by Advax62. Hashem et al. showed that mice vaccinated with an adenovirus 5 viral vector expressing MERS-CoV S1 exhibited pulmonary pathology following viral challenge, despite conferring protection. Importantly, the inclusion of CD40L as a molecular adjuvant boosted Th1 responses and prevented the vaccine-related immunopathology63.

Should it occur, ERD caused by human vaccines will first be observed in larger phase II and/or phase III efficacy trials that have sufficient infection events for statistical comparisons between the immunized and placebo control study arms. Safety profiles of COVID-19 vaccines should be closely monitored in real time during human efficacy trials, especially for vaccine modalities that may have a higher theoretical potential to cause immunopathology (such as inactivated whole-virus formulations or viral vectors)64,65.

Risk of ADE for SARS-CoV-2 vaccines

Evidence for vaccine-induced ADE in animal models of SARS-CoV is conflicting, and raises potential safety concerns. Liu et al. found that while macaques immunized with a modified vaccinia Ankara viral vector expressing the SARS-CoV S protein had reduced viral replication after challenge, anti-S IgG also enhanced pulmonary infiltration of inflammatory macrophages and resulted in more severe lung injury compared to unvaccinated animals66. They further showed that the presence of anti-S IgG prior to viral clearance skewed the wound-healing response of macrophages into a pro-inflammatory response. In another study, Wang et al. immunized macaques with four B-cell peptide epitopes of the SARS-CoV S protein and demonstrated that while three peptides elicited antibodies that protected macaques from viral challenge, one of the peptide vaccines induced antibodies that enhanced infection in vitro and resulted in more severe lung pathology in vivo67.

In contrast, to determine whether low titres of neutralizing antibodies could enhance infection in vivo, Luo et al. challenged rhesus macaques with SARS-CoV nine weeks post-immunization with an inactivated vaccine, when neutralizing antibody titres had waned below protective levels68. While most immunized macaques became infected following viral challenge, they had lower viral titres compared to placebo controls and did not show higher levels of lung pathology. Similarly, Qin et al. showed that an inactivated SARS-CoV vaccine protected cynomolgus macaques from viral challenge and did not result in enhanced lung immunopathology, even in macaques with low neutralizing antibody titres69. A study in hamsters demonstrated that despite enhanced in vitro viral entry into B cells via FcγRII, animals vaccinated with the recombinant SARS-CoV S protein were protected and did not show enhanced lung pathology following viral challenge70.

SARS-CoV immunization studies in animal models have thus produced results that vary greatly in terms of protective efficacy, immunopathology and potential ADE, depending on the vaccine strategy employed. Despite this, vaccines that elicit neutralizing antibodies against the S protein reliably protect animals from SARS-CoV challenge without evidence of enhancement of infection or disease71,72,73. These data suggest that human immunization strategies for SARS-CoV-2 that elicit high neutralizing antibody titres have a high chance of success with minimal risk of ADE. For example, subunit vaccines that can elicit S-specific neutralizing antibodies should present lower ADE risks (especially against S stabilized in the prefusion conformation, to reduce the presentation of non-neutralizing epitopes8). These modern immunogen design approaches should reduce potential immunopathology associated with non-neutralizing antibodies.

Vaccines with a high theoretical risk of inducing pathologic ADE or ERD include inactivated viral vaccines, which may contain non-neutralizing antigen targets and/or the S protein in non-neutralizing conformations, providing a multitude of non-protective targets for antibodies that could drive additional inflammation via the well-described mechanisms observed for other respiratory pathogens. However, it is encouraging that a recent assessment of an inactivated SARS-CoV-2 vaccine elicited strong neutralizing antibodies in mice, rats and rhesus macaques, and provided dose-dependent protection without evidence of enhanced pathology in rhesus macaques74. Going forward, increased vaccine studies in the Syrian hamster model may provide critical preclinical data, as the Syrian hamster appears to replicate human COVID-19 immunopathology more closely than rhesus macaque models75.

ADE and recombinant antibody interventions

The discovery of mAbs against the SARS-CoV-2 S protein is progressing rapidly. Recent advances in B-cell screening and antibody discovery have enabled the rapid isolation of potent SARS-CoV-2 neutralizing antibodies from convalescent human donors76,77 and immunized animal models78, and through re-engineering previously identified SARS-CoV antibodies79. Many more potently neutralizing antibodies will be identified in the coming weeks and months, and several human clinical trials are ongoing in July 2020. Human trials will comprise both prophylactic and therapeutic uses, both for single mAbs and cocktails. Some human clinical trials are also incorporating FcR knockout mutations to further reduce ADE risks80. Preclinical data suggest a low risk of ADE for potently neutralizing mAbs at doses substantially above the threshold for neutralization, which protected mice and Syrian hamsters against SARS-CoV-2 challenge without enhancement of infection or disease81,82. ADE risks could increase in the time period where mAb concentrations have waned below a threshold for protection (which is analogous to the historical mother–infant data that provided important clinical evidence for ADE in dengue83). The sub-protective concentration range will likely occur several weeks or months following mAb administration, when much of the initial drug dose has cleared the body. Notably, Syrian hamsters given low doses of an RBD-specific neutralizing mAb prior to challenge with SARS-CoV-2 showed a trend for greater weight loss than control animals82, though differences were not statistically significant and the low-dose animals had lower viral loads in the lung compared to control animals. Non-neutralizing mAbs against SARS-CoV-2 could also be administered before or after infection in a hamster model to determine whether non-neutralizing antibodies enhance disease. Passive transfer of mAbs at various time points after infection (for example, in the presence of high viral loads during peak infection) could also address the question of whether immune complex formation and deposition results in the enhancement of disease and lung immunopathology. If ADE of neutralizing or non-neutralizing mAbs is a concern, the Fc portion of these antibodies could be engineered with mutations that abrogate FcR binding80. Animal studies can help to inform whether Fc-mediated effector functions are crucial in preventing, treating or worsening SARS-CoV-2 infection, in a similar way to previous studies of influenza A and B infection in mice84,85 and simian-HIV infection in macaques86,87. An important caveat for testing human mAbs in animal models is that human antibody Fc regions may not interact with animal FcRs in the same way as human FcRs88. Whenever possible, antibodies used for preclinical ADE studies will require species-matched Fc regions to appropriately model Fc effector function.

ADE and convalescent plasma interventions

Convalescent plasma (CP) therapy has been used to treat patients with severe disease during many viral outbreaks in the absence of effective antiviral therapeutics. It can offer a rapid solution for therapies until molecularly defined drug products can be discovered, evaluated and produced at scale. While there is a theoretical risk that CP antibodies could enhance disease via ADE, case reports in SARS-CoV and MERS-CoV outbreaks showed that CP therapy was safe and was associated with improved clinical outcomes89,90. One of the largest studies during the SARS outbreak reported the treatment of 80 patients with SARS in Hong Kong91. While there was no placebo control group, no CP-associated adverse effects were detected and there was a higher discharge rate among patients treated earlier in infection. Several small studies of individuals with severe COVID-19 disease and a study of 5,000 patients with COVID-19 have shown that CP therapy appears safe and may improve disease outcomes92,93,94,95,96, although the benefits appear to be mild97. However, it is difficult to determine whether CP therapy contributed to recovery as most studies to date were uncontrolled and many patients were also treated with other drugs, including antivirals and corticosteroids. The potential benefits of CP therapy in patients with severe COVID-19 is also unclear, as patients with severe disease may have already developed high antibody titres against SARS-CoV-2 (refs. 47,98). CP has been suggested for prophylactic use in high-risk populations, including people with underlying risk factors, frontline healthcare workers and people with exposure to confirmed COVID-19 cases99. CP for prophylactic use may pose an even lower ADE risk compared to its therapeutic use, as there is a lower antigenic load associated with early viral transmission compared to established respiratory infection. As we highlighted above with recombinant mAbs, and as shown in historical dengue virus mother–infant data, the theoretical risk of ADE in CP prophylaxis is highest in the weeks following transfusion, when antibody serum neutralization titres fall to sub-protective levels. ADE risks in CP studies will be more difficult to quantify than in recombinant mAb studies because the precise CP composition varies widely across treated patients and treatment protocols, especially in CP studies that are performed as one-to-one patient–recipient protocols without plasma pooling.

To mitigate potential ADE risks in CP therapy and prophylaxis, plasma donors could be pre-screened for high neutralization titres. Anti-S or anti-RBD antibodies could also be purified from donated CP to enrich for neutralizing antibodies and to avoid the risks of ADE caused by non-neutralizing antibodies against other SARS-CoV-2 antigens. Passive infusion studies in animal models are helping to clarify CP risks in a well-controlled environment, both for prophylactic and therapeutic use. Key animal studies (especially in Syrian hamsters, and ideally with hamster-derived CP for matched antibody Fc regions) and human clinical safety and efficacy results for CP are now emerging contemporaneously. These preclinical and clinical data will be helpful to deconvolute the risk profiles for ADE versus other known severe adverse events that can occur with human CP, including transfusion-related acute lung injury96,100.

Conclusion

ADE has been observed in SARS, MERS and other human respiratory virus infections including RSV and measles, which suggests a real risk of ADE for SARS-CoV-2 vaccines and antibody-based interventions. However, clinical data has not yet fully established a role for ADE in human COVID-19 pathology. Steps to reduce the risks of ADE from immunotherapies include the induction or delivery of high doses of potent neutralizing antibodies, rather than lower concentrations of non-neutralizing antibodies that would be more likely to cause ADE.

Going forwards, it will be crucial to evaluate animal and clinical datasets for signs of ADE, and to balance ADE-related safety risks against intervention efficacy if clinical ADE is observed. Ongoing animal and human clinical studies will provide important insights into the mechanisms of ADE in COVID-19. Such evidence is sorely needed to ensure product safety in the large-scale medical interventions that are likely required to reduce the global burden of COVID-19.

References

  1. Zhou, Y. et al. Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2. Cell Discov. 6, 14 (2020).CAS PubMed PubMed Central Google Scholar 
  2. Lu, R. et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565–574 (2020).CAS PubMed PubMed Central Google Scholar 
  3. Lam, T. T. et al. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins. Nature 583, 282–285 (2020).CAS PubMed Google Scholar 
  4. Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280 (2020).CAS PubMed PubMed Central Google Scholar 
  5. Yan, R. et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444–1448 (2020).CAS PubMed PubMed Central Google Scholar 
  6. Daly, J. L. et al. Neuropilin-1 is a host factor for SARS-CoV-2 infection. Preprint at https://www.biorxiv.org/content/10.1101/2020.06.05.134114v1 (2020).
  7. Cantuti-Castelvetri, L. et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system. Preprint at https://www.biorxiv.org/content/10.1101/2020.06.07.137802v1 (2020).
  8. Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260–1263 (2020).CAS PubMed PubMed Central Google Scholar 
  9. Kim, H. W. et al. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am. J. Epidemiol. 89, 422–434 (1969).CAS PubMed Google Scholar 
  10. Graham, B. S. Vaccines against respiratory syncytial virus: the time has finally come. Vaccine 34, 3535–3541 (2016).PubMed PubMed Central Google Scholar 
  11. Nader, P. R., Horwitz, M. S. & Rousseau, J. Atypical exanthem following exposure to natural measles: eleven cases in children previously inoculated with killed vaccine. J. Pediatr. 72, 22–28 (1968).Google Scholar 
  12. Polack, F. P. Atypical measles and enhanced respiratory syncytial virus disease (ERD) made simple. Pediatr. Res. 62, 111–115 (2007).PubMed Google Scholar 
  13. Dejnirattisai, W. et al. Cross-reacting antibodies enhance dengue virus infection in humans. Science 328, 745–748 (2010).CAS PubMed Google Scholar 
  14. Sridhar, S. et al. Effect of dengue serostatus on dengue vaccine safety and efficacy. N. Engl. J. Med. 379, 327–340 (2018).PubMed Google Scholar 
  15. Hohdatsu, T. et al. Antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line U937 by serum of cats experimentally or naturally infected with feline coronavirus. J. Vet. Med. Sci. 60, 49–55 (1998).CAS PubMed Google Scholar 
  16. Halstead, S. B. & O’Rourke, E. J. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146, 201–217 (1977).CAS PubMed PubMed Central Google Scholar 
  17. Vennema, H. et al. Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization. J. Virol. 64, 1407–1409 (1990).CAS PubMed PubMed Central Google Scholar 
  18. Hohdatsu, T., Nakamura, M., Ishizuka, Y., Yamada, H. & Koyama, H. A study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies. Arch. Virol. 120, 207–217 (1991).CAS PubMed PubMed Central Google Scholar 
  19. Weiss, R. C. & Scott, F. W. Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever. Comp. Immunol. Microbiol. Infect. Dis. 4, 175–189 (1981).CAS PubMed PubMed Central Google Scholar 
  20. Ye, Z. W. et al. Antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin head region of pandemic H1N1 influenza virus play detrimental roles in H1N1-infected mice. Front. Immunol. 8, 317 (2017).PubMed PubMed Central Google Scholar 
  21. Winarski, K. L. et al. Antibody-dependent enhancement of influenza disease promoted by increase in hemagglutinin stem flexibility and virus fusion kinetics. Proc. Natl Acad. Sci. USA 116, 15194–15199 (2019).CAS PubMed Google Scholar 
  22. Polack, F. P. et al. A role for immune complexes in enhanced respiratory syncytial virus disease. J. Exp. Med. 196, 859–865 (2002).CAS PubMed PubMed Central Google Scholar 
  23. Polack, F. P., Hoffman, S. J., Crujeiras, G. & Griffin, D. E. A role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles. Nat. Med. 9, 1209–1213 (2003).CAS PubMed Google Scholar 
  24. Gao, T. et al. Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation. Preprint at https://www.medrxiv.org/content/10.1101/2020.03.29.20041962v3 (2020).
  25. Gralinski, L. E. et al. Complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis. mBio 9, e01753-18 (2018).PubMed PubMed Central Google Scholar 
  26. Larsen, M. D. et al. Afucosylated immunoglobulin G responses are a hallmark of enveloped virus infections and show an exacerbated phenotype in COVID-19. Preprint at https://www.biorxiv.org/content/10.1101/2020.05.18.099507v1 (2020).
  27. Chakraborty, S. et al. Symptomatic SARS-CoV-2 infections display specific IgG Fc structures. Preprint at https://www.medrxiv.org/content/10.1101/2020.05.15.20103341v1 (2020).
  28. Hiatt, A. et al. Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy. Proc. Natl Acad. Sci. USA 111, 5992–5997 (2014).CAS PubMed Google Scholar 
  29. Zeitlin, L. et al. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc. Natl Acad. Sci. USA 108, 20690–20694 (2011).CAS PubMed Google Scholar 
  30. Wang, T. T. et al. IgG antibodies to dengue enhanced for FcγRIIIA binding determine disease severity. Science 355, 395–398 (2017).CAS PubMed PubMed Central Google Scholar 
  31. Hui, K. P. Y. et al. Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures. Lancet Respir. Med. 8, 687–695 (2020).CAS PubMed PubMed Central Google Scholar 
  32. Yip, M. S. et al. Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus. Virol. J. 11, 82 (2014).PubMed PubMed Central Google Scholar 
  33. Robinson, W. E. Jr, Montefiori, D. C. & Mitchell, W. M. Antibody-dependent enhancement of human immunodeficiency virus type 1 infection. Lancet 1, 790–794 (1988).PubMed Google Scholar 
  34. Robinson, W. E. Jr et al. Antibody-dependent enhancement of human immunodeficiency virus type 1 (HIV-1) infection in vitro by serum from HIV-1-infected and passively immunized chimpanzees. Proc. Natl Acad. Sci. USA 86, 4710–4714 (1989).PubMed Google Scholar 
  35. Takada, A., Watanabe, S., Okazaki, K., Kida, H. & Kawaoka, Y. Infectivity-enhancing antibodies to Ebola virus glycoprotein. J. Virol. 75, 2324–2330 (2001).CAS PubMed PubMed Central Google Scholar 
  36. Takada, A., Feldmann, H., Ksiazek, T. G. & Kawaoka, Y. Antibody-dependent enhancement of Ebola virus infection. J. Virol. 77, 7539–7544 (2003).CAS PubMed PubMed Central Google Scholar 
  37. Ochiai, H. et al. Infection enhancement of influenza A NWS virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody. J. Med. Virol. 36, 217–221 (1992).CAS PubMed Google Scholar 
  38. Sariol, C. A., Nogueira, M. L. & Vasilakis, N. A tale of two viruses: does heterologous flavivirus immunity enhance Zika disease? Trends Microbiol. 26, 186–190 (2018).CAS PubMed Google Scholar 
  39. Wan, Y. et al. Molecular mechanism for antibody-dependent enhancement of coronavirus entry. J. Virol. 94, e02015-19 (2020).PubMed PubMed Central Google Scholar 
  40. Jaume, M. et al. Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway. J. Virol. 85, 10582–10597 (2011).CAS PubMed PubMed Central Google Scholar 
  41. Cheung, C. Y. et al. Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis. J. Virol. 79, 7819–7826 (2005).CAS PubMed PubMed Central Google Scholar 
  42. Yip, M. S. et al. Antibody-dependent enhancement of SARS coronavirus infection and its role in the pathogenesis of SARS. Hong Kong Med. J. 22, 25–31 (2016).CAS PubMed Google Scholar 
  43. Ana-Sosa-Batiz, F. et al. Influenza-specific antibody-dependent phagocytosis. PLoS ONE 11, e0154461 (2016).PubMed PubMed Central Google Scholar 
  44. Yasui, F. et al. Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus. Virology 454–455, 157–168 (2014).PubMed Google Scholar 
  45. Zhou, J. et al. Active replication of Middle East respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis. J. Infect. Dis. 209, 1331–1342 (2014).CAS PubMed Google Scholar 
  46. Ho, M. S. et al. Neutralizing antibody response and SARS severity. Emerg. Infect. Dis. 11, 1730–1737 (2005).CAS PubMed PubMed Central Google Scholar 
  47. Zhao, J. et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin. Infect. Dishttps://doi.org/10.1093/cid/ciaa344 (2020).
  48. Liu, Y. et al. Viral dynamics in mild and severe cases of COVID-19. Lancet Infect. Dis. 20, 656–657 (2020).CAS PubMed PubMed Central Google Scholar 
  49. Zheng, S. et al. Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January–March 2020: retrospective cohort study. BMJ 369, m1443 (2020).PubMed PubMed Central Google Scholar 
  50. Long, Q. X. et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat. Med. 26, 1200–1204 (2020).CAS PubMed Google Scholar 
  51. Sekine, T. et al. Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell https://doi.org/10.1016/j.cell.2020.08.017 (2020).
  52. Mathew, D., Giles, J. R., Baxter, A. E., Oldridge, D. A. & Greenplate, A. R. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science https://doi.org/10.1126/science.abc8511 (2020).
  53. Tetro, J. A. Is COVID-19 receiving ADE from other coronaviruses? Microbes Infect. 22, 72–73 (2020).CAS PubMed PubMed Central Google Scholar 
  54. Khan, S. et al. Analysis of serologic cross-reactivity between common human coronaviruses and SARS-CoV-2 using coronavirus antigen microarray. Preprint at https://www.biorxiv.org/content/10.1101/2020.03.24.006544v1 (2020).
  55. Tseng, C. T. et al. Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus. PLoS ONE 7, e35421 (2012).CAS PubMed PubMed Central Google Scholar 
  56. Deming, D. et al. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants. PLoS Med. 3, e525 (2006).PubMed PubMed Central Google Scholar 
  57. Bolles, M. et al. A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge. J. Virol. 85, 12201–12215 (2011).CAS PubMed PubMed Central Google Scholar 
  58. Yasui, F. et al. Prior immunization with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV. J. Immunol. 181, 6337–6348 (2008).CAS PubMed Google Scholar 
  59. Agrawal, A. S. et al. Immunization with inactivated Middle East respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus. Hum. Vaccin. Immunother. 12, 2351–2356 (2016).PubMed PubMed Central Google Scholar 
  60. Weingartl, H. et al. Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets. J. Virol. 78, 12672–12676 (2004).CAS PubMed PubMed Central Google Scholar 
  61. Czub, M., Weingartl, H., Czub, S., He, R. & Cao, J. Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets. Vaccine 23, 2273–2279 (2005).CAS PubMed PubMed Central Google Scholar 
  62. Honda-Okubo, Y. et al. Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology. J. Virol. 89, 2995–3007 (2015).CAS PubMed Google Scholar 
  63. Hashem, A. M. et al. A highly immunogenic, protective, and safe adenovirus-based vaccine expressing Middle East respiratory syndrome coronavirus S1-CD40L fusion protein in a transgenic human dipeptidyl peptidase 4 mouse model. J. Infect. Dis. 220, 1558–1567 (2019).CAS PubMed PubMed Central Google Scholar 
  64. London, A. J. & Kimmelman, J. Against pandemic research exceptionalism. Science 368, 476–477 (2020).CAS PubMed Google Scholar 
  65. Lurie, N., Saville, M., Hatchett, R. & Halton, J. Developing Covid-19 vaccines at pandemic speed. N. Engl. J. Med. 382, 1969–1973 (2020).CAS PubMed Google Scholar 
  66. Liu, L. et al. Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight 4, e123158 (2019).PubMed Central Google Scholar 
  67. Wang, Q. et al. Immunodominant SARS coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates. ACS Infect. Dis. 2, 361–376 (2016).CAS PubMed PubMed Central Google Scholar 
  68. Luo, F. et al. Evaluation of antibody-dependent enhancement of SARS-CoV infection in rhesus macaques immunized with an inactivated SARS-CoV vaccine. Virol. Sin. 33, 201–204 (2018).PubMed PubMed Central Google Scholar 
  69. Qin, E. et al. Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine. Vaccine 24, 1028–1034 (2006).CAS PubMed Google Scholar 
  70. Kam, Y. W. et al. Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro. Vaccine 25, 729–740 (2007).CAS PubMed Google Scholar 
  71. Yang, Z. Y. et al. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428, 561–564 (2004).CAS PubMed PubMed Central Google Scholar 
  72. Bukreyev, A. et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 363, 2122–2127 (2004).CAS PubMed PubMed Central Google Scholar 
  73. Bisht, H. et al. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc. Natl Acad. Sci. USA 101, 6641–6646 (2004).CAS PubMed Google Scholar 
  74. Gao, Q. et al. Rapid development of an inactivated vaccine candidate for SARS-CoV-2. Science 369, 77–81 (2020).CAS PubMed Google Scholar 
  75. Chan, J. F. et al. Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin. Infect. Dishttps://doi.org/10.1093/cid/ciaa325 (2020).
  76. Ju, B. et al. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115–119 (2020).CAS PubMed Google Scholar 
  77. Brouwer, P. J. M. et al. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science 369, 643–650 (2020).CAS PubMed Google Scholar 
  78. Hansen, J. et al. Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. Science 369, 1010–1014 (2020).CAS PubMed Google Scholar 
  79. Yuan, M. et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630–633 (2020).CAS PubMed PubMed Central Google Scholar 
  80. Shi, R. et al. A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature 584, 120–124 (2020).CAS PubMed Google Scholar 
  81. Cao, Y. et al. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients’ B cells. Cell 182, 73–84 (2020).CAS PubMed PubMed Central Google Scholar 
  82. Rogers, T. F. et al. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science 369, 956–963 (2020).CAS PubMed Google Scholar 
  83. Halstead, S. B. Neutralization and antibody-dependent enhancement of dengue viruses. Adv. Virus. Res. 60, 421–467 (2003).CAS PubMed Google Scholar 
  84. DiLillo, D. J., Palese, P., Wilson, P. C. & Ravetch, J. V. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J. Clin. Invest. 126, 605–610 (2016).PubMed PubMed Central Google Scholar 
  85. Liu, Y. et al. Cross-lineage protection by human antibodies binding the influenza B hemagglutinin. Nat. Commun. 10, 324 (2019).PubMed PubMed Central Google Scholar 
  86. Hessell, A. J. et al. Fc receptor but not complement binding is important in antibody protection against HIV. Nature 449, 101–104 (2007).CAS PubMed Google Scholar 
  87. Parsons, M. S. et al. Fc-dependent functions are redundant to efficacy of anti-HIV antibody PGT121 in macaques. J. Clin. Invest. 129, 182–191 (2019).PubMed Google Scholar 
  88. Crowley, A. R. & Ackerman, M. E. Mind the gap: how interspecies variability in IgG and its receptors may complicate comparisons of human and non-human primate effector function. Front. Immunol. 10, 69 (2019).Google Scholar 
  89. Mair-Jenkins, J. et al. The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis. J. Infect. Dis. 211, 80–90 (2015).CAS Google Scholar 
  90. Ko, J. H. et al. Challenges of convalescent plasma infusion therapy in Middle East respiratory coronavirus infection: a single centre experience. Antivir. Ther. 23, 617–622 (2018).CAS PubMed Google Scholar 
  91. Cheng, Y. et al. Use of convalescent plasma therapy in SARS patients in Hong Kong. EurJ. Clin. Microbiol. Infect. Dis. 24, 44–46 (2005).CAS Google Scholar 
  92. Shen, C. et al. Treatment of 5 critically ill patients with COVID-19 with convalescent plasma. JAMA 323, 1582–1589 (2020).CAS PubMed PubMed Central Google Scholar 
  93. Duan, K. et al. Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc. Natl Acad. Sci. USA 117, 9490–9496 (2020).CAS Google Scholar 
  94. Ahn, J. Y. et al. Use of convalescent plasma therapy in two COVID-19 patients with acute respiratory distress syndrome in Korea. J. Korean Med. Sci. 35, e149 (2020).CAS PubMed PubMed Central Google Scholar 
  95. Zhang, B. et al. Treatment with convalescent plasma for critically ill patients with SARS-CoV-2 infection. Chest 158, e9–e13 (2020).CAS PubMed PubMed Central Google Scholar 
  96. Joyner, M. J. et al. Early safety indicators of COVID-19 convalescent plasma in 5,000 patients. J. Clin. Investhttps://doi.org/10.1172/JCI140200 (2020).
  97. Li, L. et al. Effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening COVID-19: a randomized clinical trial. JAMA 324, 460–470 (2020).CAS Google Scholar 
  98. Gharbharan, A. et al. Convalescent plasma for COVID-19. A randomized clinical trial. Preprint at https://www.medrxiv.org/content/10.1101/2020.07.01.20139857v1 (2020).
  99. Casadevall, A. & Pirofski, L. A. The convalescent sera option for containing COVID-19. J. Clin. Invest. 130, 1545–1548 (2020).CAS Google Scholar 
  100. Pandey, S. & Vyas, G. N. Adverse effects of plasma transfusion. Transfusion 52 (Suppl. 1), 65S–79S (2012).CAS PubMed PubMed Central Google Scholar 

A review: Antibody-dependent enhancement in COVID-19: The not so friendly side of antibodies

Authors: Gabriela Athziri Sánchez-Zuno,1,†Mónica Guadalupe Matuz-Flores,1,†Guillermo González-Estevez,1Ferdinando Nicoletti,2Francisco Javier Turrubiates-Hernández,1Katia Mangano,2 and José Francisco Muñoz-Valle1

Int J Immunopathol Pharmacol. 2021 Jan-Dec; 35: 20587384211050199.Published online 2021 Oct 10. doi: 10.1177/20587384211050199PMCID: PMC8512237PMID: 34632844

Abstract

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents an unprecedented global public health emergency with economic and social consequences. One of the main concerns in the development of vaccines is the antibody-dependent enhancement phenomenon, better known as ADE. In this review, we provide an overview of SARS-CoV-2 infection as well as the immune response generated by the host. On the bases of this principle, we also describe what is known about the ADE phenomenon in various viral infections and its possible role as a limiting factor in the development of new vaccines and therapeutic strategies.

Keywords: COVID-19, SARS-CoV-2, ADE, vaccine, antibody-dependent enhancement

Introduction

The first cases of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were identified in December 2019 in China. The spread of this disease occurred rapidly throughout the world mainly due to its forms of transmission, being the most important the contact with respiratory fluids (exposure to respiratory droplets carrying infectious viruses). 1

Following the outbreak in China, a trend of increasing cases grew exponentially as it was observed. As a consequence of this rapid spread, the World Health Organization (WHO) declared COVID-19 as an international public health emergency, and in March 2020, it was declared a pandemic. As a result of this unexpected progression, health authorities around the world entered into a state of alert facing the need to implement unprecedented sanitation and isolation protocols. 2

The state of pandemic has caused a great impact on both the economic and public health level around the world, because of social distancing, border closures, and the performance of essential activities only. According to the WHO, in the week of April 12th to April 20th, 2021, more than 140 million cases and more than 3 million deaths have already been reported worldwide, new cases continued to rise globally, in the past week to over 5.2 million new reported cases. Possible reasons for this increase include the continued spread of more transmissible variants of concern (VOCs). The countries such as India, the United States of America, Brazil, Turkey, and France reported the highest number of cases. 2

Regarding the health implications of this disease, it has been described that the majority of patients infected by COVID-19 have symptoms of a common cold such as fever, cough, fatigue, headache, and muscle pain as well as diarrhea. In some cases, severe shortness of breath can also occur. 3 Although most patients have a favorable prognosis, in some cases this may not be the scenario. A poor prognosis has been associated with the presence of some chronic diseases and comorbidities including hypertension, diabetes, coronary heart disease, and obesity. In the case of diabetes, patients are more susceptible to developing the so-called “cytokine storm” that leads to a rapid deterioration of COVID-19.4,5

Another important aspect regarding the pathogenesis of COVID-19 is the occurrence of the phenomenon called antibody-dependent enhancement (ADE). This mechanism involves endocytosis of virus–antibody immune complexes into cells through interaction of the antibody Fc region with cellular Fc receptors (FcRs). In this event, pre-existing non-neutralizing or sub-neutralizing antibodies to viral surface proteins that were generated during a previous infection can promote the subsequent entry of viruses into the cell and therefore intensify the inflammatory process during a secondary infection with any antigenic-related virus.68 The occurrence of ADE may represent one of the greatest challenges for scientists working on the development of a safe vaccine against COVID-19.

For the aforementioned, in this review, we provide an overview of SARS-CoV-2 infection as well as the immune response generated by the host. On the bases of this principle, we also describe what is known about the ADE phenomenon in various viral infections and its role as a limiting factor in the development of new vaccines and therapeutic strategies.

Structure and pathogenesis of SARS-CoV-2

The Coronaviruses (CoVs) are viruses that show morphological similarity to a solar corona appearance under an electron microscope due to the presence of “spike” glycoproteins. These CoVs belong to the large family Coronaviridae, which consists of two subfamilies: Orthocoronavirinae and Torovirinae. The Orthocoronavirinae subfamily is classified into four genera: alpha coronaviruses, beta coronaviruses, gamma coronaviruses, and delta coronaviruses. Among these, the beta genus is the one that has been described as capable of causing severe illness and even death among infected individuals.9,10

The genome of this beta-CoV has been classified as a single-stranded ribonucleic acid (RNA) virus consisting of 26–32 kbp and contains 7–10 open reading frames (ORF). Two-thirds of the genome encodes the replicase-transcriptase proteins, and a third part encodes the four structural proteins: spike (S), envelope, membrane, and nucleoprotein. The S-glycoproteins on the surface of CoVs comprise the receptor-binding domain(s) and contribute for host cell binding, host–viral cell membrane fusion, and virus internalization while the M-glycoprotein plays a role in the virion envelope formation and assembly.912 Therefore, the entry of the coronavirus into susceptible cells is a complex process that requires receptor binding and proteolytic processing of protein S to promote virus–cell fusion. As anticipated above, SARS-CoV-2 is acquired by exposure to respiratory fluids of infected individuals and less through contact with fomites. 13

SARS CoV interacts directly with angiotensin-converting enzyme 2 (ACE2) to enter target cells. At the onset of the infection, SARS-CoV-2 targets mainly host cells that express ACE2, including bronchial cells, airway epithelial cells, alveolar epithelial cells, macrophages in the lung, and vascular endothelial cells. 14

After the recognition and binding of the SARS-CoV-2 S-glycoprotein with ACE2 in the host cells, the S-protein is cleaved by transmembrane protease serine 2 (TMPRSS2) to reveal the S2 domain necessary for the fusion of the viral membrane–host cell and the entry of the virus. Once the viral content is released into host cells, the viral RNA that enters, begins its replication, production, and release of new viral particles (Figure 1(a)).14,15

An external file that holds a picture, illustration, etc.
Object name is 10.1177_20587384211050199-fig1.jpg

Open in a separate windowFigure 1.

The Immune Response and Immunopathology of COVID-19. (a) The entry of SARS-CoV-2 into cells is mediated by the binding of TMPRSS2 and S-glycoprotein with the ACE2 acting as a receptor that facilitates viral binding to the membrane of the host cells. The virus enters by endocytosis and releases its RNA, replicates and creates new virions that cause a rapid progression of the infection. (b) Bronchial epithelial cells, type I and type II alveolar pneumocytes, and capillary endothelial cells become infected and a response occurs that leads to recruitment of macrophages, monocytes, neutrophils, and cytokine production in response to virus entry. (c) Sub-epithelial dendritic cells recognize the virus antigen and present them to CD4 + T cells that induce the differentiation of B cells into plasma cells that promote the production of virus-specific antibodies. Neutralizing antibodies can interact with phagocytes and NK cells and enhance antibody-mediated clearance of SARS-CoV. (d) A dysfunctional immune response leads to excessive cell infiltration, cytokine storm, inflammation, apoptosis, and multi-organ damage. Ab, antibody; ACE2, angiotensin-converting enzyme 2; FcγR, Fcγ receptor; IL, interleukin; MHC, major histocompatibility complex; TCR, T-cell receptor; TMPRSS2, transmembrane protease serine 2; TNF-a, tumor necrosis factor.

Innate immune response

After SARS-CoV-2 enters the host cells, it is recognized by pattern recognition receptors (PRRs) such as Toll-like receptor-7 (TLR7) and TLR8, which are expressed by epithelial cells that activate the local immune response, recruiting macrophages and monocytes that respond to infection (Figure 1(b)).

Once SARS-CoV-2 binds to PRRs, the recruited adapter proteins activate transcription factors. This includes interferon regulatory factor (IRF) and Nuclear factor κB (NF-κB), that lead to the production of antiviral type I interferon (IFN), and cytokines that induce an alarm signal in neighboring cells to attract other cells of innate immunity including polymorphonuclear cells, natural killer cells (NKs), dendritic cells, and monocytes.16,17 One of the signature features of this disease in patients with worst prognosis is the high serum levels of cytokines such as IL-1β, IL-6, TNF, IL1RA, and IL-8. These cytokines have an important role in the exacerbation of the inflammatory process and lead to the recruitment of other immune cells such as neutrophils and T cells. Among infiltrated innate cells, neutrophils can promote the destruction of viruses, but they can also worsen disease progression by inducing severe lung lesions.18,19

Types I and III IFNs are considered to be crucial in the antiviral response, and SARS-CoV-2 has been shown to be sensitive to pretreatment with IFN-I and III in vitro assays.20,21 The IFN timing and location are a key factor for an effective response against the virus. A study of the Middle East respiratory syndrome (MERS) in mice demonstrated that blockade of IFN signaling leads to a delayed virus clearance with increased neutrophil infiltration and alteration in T cell response. Conversely, 1 day of IFN-I administration protected mice from lethal infection, meanwhile, delayed IFN treatment failed to inhibit the replication of the virus. 22

One of the most important questions that arises in relation to innate immunity is how the SARS-CoV-2 evades the immune response. In a recent study, Kaneko et al., propose that the evasion of the antiviral aspects of innate immunity and the inflammatory process as a consequence of the virus can probably result in an alteration of the environment that leads to the attenuation of immunity of CD8 + T cells. In addition, there is an absence of germinal centers with reduction of B cells; therefore, it gives rise to a memory with a short duration and to B cells without high affinity. So far, it is still a very difficult question to answer. 23 However, it has been shown that patients with COVID-19 with worst prognosis showed poor IFN-I signals compared to patients with a favorable prognosis. 24

Additionally, various evasion mechanisms have been described for CoVs, with viral factors that antagonize pathways from PRR detection, cytokine secretion, and IFN signal induction. CoVs are able to evade PRRs by protecting the double strand RNA (dsRNA) with membrane-bound compartments formed during viral replication. Furthermore, SARS-CoV-2 is protected with guanosine and methylated by nonstructural proteins. They resemble host mRNA to promote translation, prevent degradation, and avoid detection of RIG-I-like receptors (RLRs).2527

Adaptive immune response

The main mechanisms for decreasing viral replication, limit virus spread, and inflammation include the production of various pro-inflammatory cytokines, the activation of CD4 + and CD8 + T cells.28,29

The mechanism for the presentation of viral peptides occurs once the virus is inside respiratory cells. They are presented through the major histocompatibility complex (MHC) class I for cytotoxic CD8+ T cells which are essential to mediate elimination of cells infected by the virus. Additionally, the virus and its viral particles can be presented in the context of MHC class II by means of antigen-presenting cells, including dendritic cells and macrophages. They are in charge of presenting viral proteins to CD4+ T cells that provide the signals necessary for the induction of B cells and differentiation of plasma cells producing virus-specific neutralizing antibodies (Figure 1(c)).28,29

However, in patients with COVID-19, a low count of lymphocytes, CD4+ T cells, CD8+ T cells, B cells, and NK cells has been shown. Likewise, severe cases have presented lower levels of these cells compared to mild cases. 30 Secretion of type I IFNs dramatically increases the response of CD8 + T cells against viruses, but SARS-CoV-2 has been shown to possess nonstructural proteins that induce a decreased response to type I interferon (IFN) in infected cells. Therefore, the decrease in type I IFNs by different non-structural proteins of SARS-CoV-2 could explain the marked absence of CD8+ T cell response in COVID-19 patients.3133

Kaneko et al., evaluated subsets of CD4+ T cells in lymph nodes and the spleen and observed that TH1 cells increase steadily at the beginning and end in lymph nodes and the spleen, also, a constant decrease in TH2 cells was described. Furthermore, FOXP3 + T reg cells make up a large part of the CD4+ T cell population at the end of disease. 23 Furthermore, it was shown that patients with significant decreases in T cell counts, especially CD8+ T cells, have elevated levels of IL-6, IL-10, IL-2, and IFN-γ in the peripheral blood. 34

Elevated cytokine secretion promotes cell infiltration inflammatory by establishing an aberrant inflammatory feedback loop that can cause damage to the lung. It can also cause damage through the secretion of proteases and reactive oxygen species (ROS) with subsequent alveolar damage and desquamation of alveolar cells. This results in inefficient gas exchange in the lung, which is reflected in low oxygen levels in patients. 35

Overall, impaired acquired immune responses and uncontrolled innate inflammatory responses to SARS-CoV-2 can cause cytokine storms that are associated with COVID-19 severity states and can lead to migration to different organs, causing multi-organ damage (Figure 1(d)). 36

Antibody responses in COVID-19 patients occur in conjunction with CD4+ T cell responses that induce B cells to differentiate into plasma cells and subsequently produce antibodies. In patients with SARS-CoV infection, the main target of neutralizing antibodies is the virus S glycoprotein, particularly with its receptor-binding domain (RBD), which is responsible for the binding of the virus to the ACE2 in host cells. 37 Neutralizing antibody responses to protein S possibly begin to develop in week two, and in most patients, antibody titers are detected by the third week.38,39

A recent study conducted by Ni et al., 2020 40 showed the presence of specific IgM and IgG antibodies for the structural proteins N (nuclear) and S-RBD in serum of recently negative COVID-19 patients compared to healthy donors. The IgG anti-SARS-CoV-2 was also higher in titers than IgM in follow-up patients compared to healthy donors. This indicates that patients with COVID-19 have IgG- and IgM-mediated responses to SARS-CoV-2 proteins, especially N and S-RBD. It also proposes that previously infected patients could maintain their IgG levels for at least 2 weeks after receiving a negative COVID-19 test result. 40Go to:

The devil in disguise: What happens when antibodies go bad

All viruses initiate infection by adhering to host cells through the interaction between viral proteins and receptor/coreceptor molecules on target cells (Figure 2(a)) As mentioned above, the host’s humoral response is responsible for generating specific antibodies to surface proteins that inhibit this step of the infection cycle, resulting in virus neutralization. Conversely, in some cases, these antibodies may paradoxically favor the infection process as part of a phenomenon better known as antibody-dependent enhancement (ADE). 41

An external file that holds a picture, illustration, etc.
Object name is 10.1177_20587384211050199-fig2.jpg

Figure 2.

ADE phenomenon. (a) The conventional mechanism of infection by SARS-CoV 2 consists of the binding of its S-protein to the cellular receptor ACE2. After the union of the SARS-CoV-2 virus to the receptor, a conformational change occurs in the S-protein necessary for the fusion of the viral envelope with the cell membrane for subsequent endocytosis. Subsequently, SARS-CoV-2 releases its genetic material into the host cell. The RNA of the viral genome is then translated into proteins necessary for the subsequent assembly of viriomes in the ER and Golgi. These visions are then transported through vesicles outside the cell by exocytosis. The ADE phenomenon can be classified as two different mechanisms: ADE through enhanced infection and ADE through enhanced immune activation. (b) In ADE through increased infection, antibodies of a non-neutralizing or sub-neutralizing nature cause viral infection through FcγRIIa-mediated endocytosis, resulting in a more severe disease phenotype. (c) In ADE via enhanced immune activation, non-neutralizing antibodies can form immune complexes with viral antigens inside airway tissues, resulting in the secretion of pro-inflammatory cytokines, immune cell recruitment, and activation of the complement cascade within lung tissue. ADE, antibody-dependent enhancement; ACE2, angiotensin-converting enzyme 2; CR, compliment receptor; ER, endoplasmic reticulum; FcγRIIa, Fc γ receptor IIa; IFN-a, interferon a; IL, interleukin; IRF, interferon regulatory factors; iNOS, inducible nitric oxide synthase; PGE2, prostaglandin E2, RNA, ribonucleic acid; TNF-a, tumor necrosis factor.

Regarding the mechanism of ADE, it has been described that it involves endocytosis of virus–antibody immune complexes into cells through interaction of the antibody Fc region with cellular Fc receptors (FcRs). It is well known that the FcγRI (CD64) binds with high affinity to IgG monomerically while FcγRII (CD32) and FcγRIII (CD16) do so with low affinity and are activated by immune complexes. 42 In this regard, it is postulated that myeloid cells that express FcRs such as monocytes and macrophages, dendritic cells, and certain granulocytes can promote ADE through phagocytic uptake of the immune complexes. Although ADE is principally mediated by IgG antibodies, IgM along with complement, and IgA antibodies have also been described as capable of ADE 43

The phenomenon of ADE is an event that occurs in some viruses, where pre-existing non-neutralizing or sub-neutralizing antibodies to viral surface proteins that were generated during a previous infection can promote the subsequent entry of viruses into the cell and therefore intensify the inflammatory process during a secondary infection with any antigenic-related virus.6,8

ADE was first described in 1964 by Hawkes, who demonstrated increased infectivity of various arboviruses such as Japanese encephalitis virus, West Nile virus, Murray Valley encephalitis virus, and Murray Valley virus and Getah virus under in vitro conditions. 6 Prior to that, there were also previous reports positing pre-existing non-neutralizing antibodies as responsible for increased infection with various human and animal viruses, including dengue virus (DENV), Zika virus (ZIKV), Ebola virus, human immunodeficiency virus (HIV), Aleutian mink disease parvovirus, Coxsackie B virus, equine infectious anemia virus, feline infectious peritonitis virus, simian hemorrhagic fever virus, caprine arthritis virus, respiratory syndrome virus, and reproductive disease and African swine fever virus. To date, ADE has also been demonstrated with models using monoclonal antibodies and in vitro models of polyclonal sera using cells expressing the Fc receptor, including K562 and U937 cell lines, as well as primary human monocytes, macrophages, and dendritic cells. 44

Molecular mechanism of ADE

In order to clearly understand ADE, it has been broadly categorized into two different mechanisms; when the specific antibody enhances viral entry into host monocytes/macrophages and granulocytes or when it promotes viral infection in cells through interaction with FcR and/or complement receptor. Although these mechanisms are not mutually exclusive, their classification was proposed in order to understand the biological process involved at the molecular level.8,45

ADE via enhanced infection

As mentioned earlier, FcRs are mostly expressed by immune cells and are receptors directed to Fc portion of an antibody. In ADE, via enhanced infection, non-neutralizing or sub-neutralizing antibodies bind to the viral surface and traffic virions directly to macrophages, this complex is internalized by Fc-receptor-bearing cells, including monocytes/macrophages and dendritic cells and subsequently leads to the phosphorylation of Syk and PI3K that triggers signaling for FcγR-mediated phagocytosis. Alternatively, activating FcγR can concentrate immune complexes on the surface of the cell. The virion can then bind to its receptor to enter the cell via receptor-mediated endocytosis. These processes culminate in an increased virus load and disease severity (Figure 2(b)).8,44,46

It is also worth mentioning that this mechanism can be abrogated in the absence of the Fc receptor. The activation of Fc receptors triggers signaling molecules that also induce IFN-stimulated gene (ISG) expression, independent of type-I IFN. Because ISGs have powerful antiviral effects, viruses must develop tools to suppress these antiviral responses in target cells for ADE to occur. For example, in DENV infection, the ADE phenomenon requires the binding of DENV to the leukocyte immunoglobulin receptor B1 (LILRB1). As a result, LILRB1 signaling can inhibit the pathway that induces ISG expression.47,48

ADE via enhanced immune activation

The second, recently described and less studied mechanism, through which ADE can occur, is well represented by pathogens that cause respiratory infections. In these conditions, Fc-mediated antibody effector functions are capable of enhancing respiratory disease by initiating a strong immune cascade that results in severe lung pathology (Figure 2(c)). 45

This mechanism can also be induced when virus–antibody C1q complexes promote fusion between the viral capsule and the cell membrane by deposition of C1q and its receptor. This complex binds to the C1q receptor in cells and initiates the intracellular signaling pathway. The classical complement pathway is then initiated, leading to the activation of C3, whose fragment can be covalently linked to the bound antibodies or the surface of the virus particles then favors the binding of the virus and its receptor, as well as the subsequent endocytosis.10,43

Interestingly, another mechanism for the ADE phenomenon that has been rather described in the multisystemic inflammatory syndrome in children is that mediated by mast cells; these cells are capable of degranulating both IgE and IgG antibodies bound to Fc receptors. 49

In this sense, a model of multisystemic inflammatory syndrome in children has been proposed in babies with maternally transferred antibodies against SARS-CoV-2 in which the activation and degranulation of mast cells with SARS-CoV-2 antibodies bound to the Fc receptor lead to an increase in histamine levels. In this model, the binding of the SARS-CoV-2 nucleocapsid to the PTGS2 promoter results in prostaglandin E2 (PGE2) which may be driving overactive mast cells as an alternative mechanism that drives increased histamine levels in older children and adults. 49

The best known so far (but also misunderstood) ADE phenomenon: ADE in DENV infection

The DENV is a mosquito-borne virus of the Flaviviridae family (with four serotypes identified DENV1-4) capable of causing classic dengue (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) showing tropism for monocytes, macrophages, and dendritic cells.42,48,50

There exists no cross-antibody protection for the four serotypes, which means the antibodies induced by each serotype cannot work on others. In the case of a secondary infection, if infected by the virus of same serotype, the antibodies produced in previous infections are capable of effectively neutralizing the virus. On the contrary, these antibodies will not only neutralize viruses, but may also even facilitate viral entry through Fc portions of antibodies and will increase viral load in vivo. 8

According to this hypothesis of ADE, the antibodies produced in a DENV infection can recognize and bind to a different serotype of DENV than that of the primary infection but are not able to neutralize it. Instead, these antibodies facilitate the entry of non-neutralized virus–antibody complexes (immune complexes), primarily through FcγR into phagocytic mononuclear cells (MPCs). 48

The DENV represents the best documented example of clinical ADE via enhanced infection. After ligation of FcR, DENV activates IL-10 production at an early phase of infection. The suppressor activity of IL-10 during ADE infection induces Th2 bias and inhibits the JAK-STAT signaling pathway through the suppressor cytokine signaling system (SOCS). ADE also results in a higher rate of virus internalization by increasing the number of fusions per cell.44,45,51

Since many antibodies to different dengue serotypes are cross-reactive, secondary infections with heterologous strains can lead to increased viral replication and more severe disease. Typically, both DHF and DSS occur in this setting, presenting more severe forms of symptoms, such as thrombocytopenia, fever, and hemorrhagic manifestations. It has also been shown that the presence of these cross-reactivated non-neutralizing antibodies can predispose to more severe disease and even the development of DHF and DSS.45,52

SARS CoV-2 and ADE, what is known and what remains to be known?

Despite all reports generated in recent months in response to the pandemic, there is still no detailed information regarding the mechanism of the ADE that occurs in SARS-CoV-2 infection. One of the best accepted hypotheses so far is that in the SARS-CoV-2 infection, pre-existing CoV-specific antibodies are capable of promoting viral entry into FcR-expressing cells. ADE is mediated by the binding of FcRs, mainly CD32 expressed in different immune cells, including monocytes, macrophages, and B cells. The infection of CD32+ cells is a key step in the development of the COVID-19 and its progression from mild to severe form.53,54

A potential hypothesis states that circulating non-neutralizing antibodies, instead of helping to eliminate circulating SARS-CoV-2, can then bind to viral particles and thus contribute to the worsening of COVID-19 by promoting its Fc-mediated internalization by pulmonary epithelial cells and infiltrating monocytes, as it has been observed in previously mentioned diseases such as SARS-CoV-1. 55

One particularity about this mechanism is that ADE of SARS-CoV does not use endosomal/lysosomal pathway as used by ACE2 during normal virus transport into the cell, but instead it has been described as a possible mechanism for viral entry where non-neutralizing antibodies recognizing the RBD of the S-protein of the coronavirus bind to the Fc receptor and allow virus entry. The non-neutralizing antibodies–Fc receptor complex mimics the cell surface virus receptor and favors virus entry pathways into IgG Fc receptor-expressing cells.6,52

This phenomenon could also explain the observed impairment of immune regulation such as apoptosis of immune cells leading to the development of T-cell lymphopenia, an inflammatory cascade, as well as a storm of cytokines.8,54

An important difference between the ADE phenomenon previously described for DENV and SARS-CoV is that there is no evidence that ADE facilitates the spread of SARS-CoV in infected hosts. Therefore, ADE in this disease would be best described as “ADE of viral entry” which does not necessarily result in a productive viral infection, meaning that ADE of viral entry in vitro does not predict ADE of infection and ADE of disease. 56

Antibodies are capable of promoting virus attachment and entry into the immune cell, where they start to replicate without production of viable virions. This pseudo infection may be due to the inability of macrophages to express the serine proteases necessary for virion activation. For their part, immune complexes (virus–antibody) can promote an infectious process after being internalized through the FcRs. Furthermore, pulmonary epithelial cells have been reported to express high levels of FcγRIIa. The virus introduced into the endosome through this pathway will likely involve TLR3, TLR7, and TLR8 capable of recognizing RNA. SARS-CoV infection by ADE in macrophages leads to elevated production of TNF and IL-6. It was also observed in a murine SARS-CoV model that ADE is associated with a decrease in the levels of the anti-inflammatory cytokines IL-10 and TGFβ and increased levels of the pro-inflammatory chemokines CCL2 and CCL3.7,53,54

ADE in the case of SARS-CoV-2 can occur due to the priming caused by other CoVs, leading to development of non-neutralizing or poorly neutralizing antibodies. It is known that antibodies to the S-proteins of SARS-CoV and SARS-CoV-2—and, to a much lesser extent, MERS-CoV—can cross-react, and both high-potency neutralizing antibodies that also mediate antibody-dependent cytotoxicity and antibody-dependent cellular phagocytosis, as well as non-neutralizing antibodies, can be elicited against conserved S-epitopes. Despite the above, the limited spread of SARS-CoV and MERS-CoV means that it is not feasible that antibodies with cross-reactivity due to another coronavirus infection are the responsible element for the development of ADE, but rather those that were generated during a first infection or after passive immunization.8,57

The ADE hypothesis is further supported by the results of a study on viral kinetics and antibody responses in patients with COVID-19 where it was found that stronger antibody response was associated with delayed viral clearance and increased disease severity. Patients with an elevated IgG response showed only 9% of virus shedding on day 7 after IgG developed. In the case of weak IgG patients, 57% shed the virus. Furthermore, an association was found between a more severe disease phenotype and earlier IgG response, concurrently with IgM and higher IgG antibody titers. 58

The hypotheses regarding ADE are however conflictive and somehow even contradictory. As stated by Jaume et al., it was observed in an in vitro analysis that ADE infection promoted viral gene transcription and the production of viral gene protein synthesis and intermediate species, which can be then recognized by immune sensors and potentiate an immune response. Therefore, proposing a possible participation of immune-mediated enhanced disease during SARS pathogenesis suggests very little clinical significance for this mechanism. In this same study, it was observed in a different cell line, (Raji cells, derived from a Burkitt’s lymphoma patient) that ADE infected cells did not support replication of SARS-CoV-1, ultimately ending in an abortive viral cycle without the detectable release of progeny virus. 59

In addition to the above, recent reports indicate that the percentage of patients with COVID-19 that develop cross-reactive antibodies is significant. In a study by Shrock et al., a serological profile of patients with and without previous COVID-19 infection was performed. In this study, it was found that the studied patients presented cross-reactive antibody titers, and it is suggested that this may have various effects on the disease, from a less severe prognosis when they were able to neutralize the virus to a serious infection when ADE is developed. 60

Another important aspect that needs to be studied further is the relationship between ADE-epitopes. This was previously reported for DENV and ZIKV.61,62 In the case of SARS-CoV-2, this association was reported for the first time in the article by Zhou et al., where monoclonal cells were isolated from memory B cells, later a group of non-overlapping receptor-binding domain was identified. (RBD) epitopes that were directly associated with ADE and favored the entry of the virus into Raji cells via an Fcg receptor-dependent mechanism. 56 This is of utmost importance especially when considering the design of vaccines, which, as mentioned later, must be capable of triggering a strong neutralizing response, which is why the epitopes to which they will be directed must be carefully selected.

Finally, it is also important to take into account that detailed research is lacking to elucidate the possible mechanism of ADE in SARS-CoV-2 infection, mainly due to the fact that the studies carried out at present have been carried out in viral infections (such as DENV) with differences in their pathological mechanisms as well as in animal models (such as the feline infectious peritonitis virus [FIPV]) where mechanisms of pathogenesis in the human host differ among viruses, therefore difficult to translate the mechanisms of infection. 57

ADE as a possible threat to vaccine efficacy

All vaccines have the objective of generating a response from the host against an antigen that is not capable of causing a disease but of provoking a response against that antigen that will be effective in subsequent encounters with it. As we have been discussing, the mechanism of ADE makes vaccine development particularly difficult due to similarity to a natural infection. Vaccines against one specific serotype produce cross-reactive non-neutralizing antibodies against other serotypes, predisposing the enhanced illness in secondary heterotypic infection. 52

The immune mechanisms of this phenomenon involve from ADE of infection to the formation of immune complexes by antibodies, although accompanied by various coordinated cellular responses, such as Th2 T-cell skewing. 63 Another important point to consider is that not only sub-neutralizing or non-neutralizing antibodies are associated with the development of ADE; according to the study by Liu et al., 55 anti-spike IgG (S-IgG), in productively infected lungs, causes severe ALI by skewing inflammation-resolving response.

To avoid the development of ADE, the strategy used in the development of current vaccines was to target the immunodominant epitope, in this case, that corresponds to the S-protein. The S1 subunit presents two highly immunogenic domains, the N-terminal domain (NTD) and the RBD, which are the major targets of polyclonal and monoclonal neutralizing antibodies.64,65 Because the S-proteins of SARS-CoV-2 are accessible and play an essential role in the entry of the virus into the host cell, and therefore the mechanism of infection, they are considered to be prime antibody targets. 66

Understanding the structure of SARS-CoV-2 epitopes, particularly within S, provides essential information for the development of vaccines that favors the production of neutralizing antibodies rather than antibodies that could exacerbate the severity of ADE infection. 60 In general, RNA viruses are known to be highly susceptible to random mutations due to the lack of exonuclease proofreading activity of virus-encoded RNA-dependent RNA polymerases (RdRp) 67 with some exceptions such as Nidovirales order (to which the Coronavirus genus belongs). In SARS-CoV, an exonuclease activity with proofreading function has been described for the nsp14 (ExoN), and a homologue nsp14 protein is found in the SARS-CoV-2 as well. 68

The high error rate and subsequent rapid evolution of virus populations, which could lead to the accumulation of amino acid mutations, could affect the virus’ transmissibility, its cellular tropism, and even its pathogenicity. 69

Although several vaccines have gained (emergence) regulatory approval and are being distributed worldwide, we cannot ignore the possibility that the evolution of the virus, based on natural selection, can directly affect the S-protein to which these vaccines are directed, and therefore the newly mutated virus can escape antibody-mediated protection induced by previous infection or vaccination. 70

Amino acid sequences of SARS-CoV-2 are available from NCBI GenBank and by the Global Initiative on Sharing All Influenza Data (GISAID). The first complete genome sequence of SARS-CoV-2 was released on NCBI GenBank (NC 045512.2). 67 According to these reported sequences, the linear genome of the SARS-CoV-2 virus is 29,903 bases long and houses 25 genes. 71 To date, 4150 mutations have been identified in the S-gene of SARS-CoV-2 isolated from humans, resulting in 1246 changes in amino acids, including 187 RBD substitutions compared to the reference genome. 72

The main variants identified that seem to have high relevance in the immunogenicity of the virus are D614G, N501Y, and E484K mutations of the RBD.7376

The D614G mutation in protein S represents a change from nucleotide A to G at position 23,403 in the first Wuhan reference strain. The D614G change is commonly detected along with three other mutations: a C to T change in the UTR 5 ‘ (position 241 relative to the Wuhan reference sequence), a silent mutation from C to T at position 3037, and a C-to-T mutation at position 14,408 that results in an amino acid change in the RdRp P323L. This, comprised the four aforementioned mutations, represented the dominant global form as of May (78% of a total of 12,194 sequences). 73 The D614G mutation has been reported as capable of improving the replication capacity of SARS-CoV-2 in the upper respiratory tract through increased virion infectivity, this was demonstrated in the human lung cell line Calu-3 and the primary tissues of the human upper respiratory tract. 73

It was also observed that patients infected with the G614 variant of the virus developed higher levels of viral RNA in nasopharyngeal smears than those with the D614 virus but did not develop a more severe disease. This suggests that despite affecting the replication capacity of the virus, this mutation did not influence the severity of the infection. 77

The N501Y variant was identified in the UK as VUI-2020/01 or lineage B.1.1.7. This lineage is composed of 14 defining mutations in protein S. This variant has a mutation in the RBD of the peak protein at position 501, where the amino acid asparagine (N) has been replaced by tyrosine (Y). The N501Y mutation is one of the six key contact residues within the RBD. 78

This change in different fundamental residues in the binding site could affect the fusion of the host cells–virus and, therefore, the infectivity of the virus. 79 As of December 28, 2020, this variant accounted for approximately 28% of cases of SARS-CoV-2 infection in England. 74

The E484K mutation in the S-protein of the virus has been identified in the South African (B.1.351) and Brazilian (B.1.1.28) variants and has been reported to be an escape mutation from the immune response. 80

This variant consists of a change in codon 484 in that of the RBD where a negatively charged amino acid (E, glutamic acid) is substituted with a positively charged amino acid (K, lysine). 81

Due to the location of this mutation, like the other variants, it has been directly associated with changes in the mechanism of infection of the virus and even on the efficacy of the immune response of the organism or that induced by a vaccine to the virus. 80 Studies have also shown that the presence of this variant directly affects the average binding of convalescent sera (>10 times) reducing the neutralization activity of some individuals. 75

Recently, the BNT162b2 nucleoside modified RNA vaccine encoding the full-length SARS-CoV-2 protein (S) was reported to be effective in inducing neutralizing geometric mean titers of antibodies against SARS-CoV-2 virus constructs containing key peak mutations of the newly emerging UK (UK) and South African (SA) variants: N501Y from the UK and South Africa; Deletion 69/70 + N501Y + D614G from the UK; and E484K + N501Y + D614G de SA, thus suggesting that the efficacy of this vaccine is not significantly affected by these variants. 82

Recently, the delta variant (B.1.617.2) was described, which is characterized by mutations in the peak protein P681R, T19R, D614G, L452R, T478K, Δ157-158, and D950N, first detected in India in December 2020. 83 According to what is believed, these mutations directly affect key antigenic regions of RBD. This variant also appears to cause mutations at sites that trigger an increase in viral replication and therefore an increase in viral load. 84 This variant and its rapid transmission capacity represent an imminent threat to the population and a concern about the effectiveness of vaccines. In this sense, in the study by Lopez-Bernal et al., it was reported that the effectiveness after a dose of vaccine (BNT162b2 or ChAdOx1 nCoV-19) was lower among people with the delta variant (30.7%) than among those with the alpha variant (48.7%). With the BNT162b2 vaccine, the effectiveness of two doses was 93.7% among people with the alpha variant and 88.0% among people with the delta variant. With the ChAdOx1 nCoV-19 vaccine, the two-dose efficacy was 74.5% among people with the alpha variant and 67.0% among people with the delta variant. 84

In addition to this, there are reports regarding the kinetics of natural immunity in patients who had COVID-19. In a study, 85 the humoral response was evaluated in a total of 76 patients (IgM and IgG antibodies that recognized the nucleocapsid protein or the RBD of the S-protein). In these patients 1 year after infection, approximately 90% of recovered patients still had detectable SARS-CoV-2-specific IgG antibodies recognizing N and RBD-S. However, when evaluating the neutralizing capacity, it was only detected in ∼43% of patients. 85

In addition to concerns regarding natural immunity, there are also reports about the duration of the humoral immune response in response to a vaccine. In a study in health personnel vaccinated with BNT162b2, it was observed that the antibody response was greater in seropositive participants compared to seronegative participants. In both seropositive and seronegative subjects, a significant decrease in antibodies was observed at 3 months compared to maximum response. 86 Similar results were found by our work group in the study by Morales-Nuñez et al., where it was observed that after the second dose with this same vaccine, individuals developed antibodies with high neutralizing capacity. 87 In a study by Pegu et al., 2021, the efficacy of the immune response generated by the mRNA-1273 vaccine was evaluated, in this work the impact of the variants B.1.1.7 (Alpha), B.1.351 (Beta), P.1 was also evaluated (Gamma), B.1.429 (Epsilon), B.1.526 (Iota) for SARS-CoV-2, and B.1.617.2 (Delta) on binding, neutralization, and ACE2-competing antibodies elicited by this vaccine for 7 months. The results of this study turned out to be interesting because all included individuals responded to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273 vaccine. 88

The imminent risk that may be triggered by a vaccine-mediated antibody response is that the mechanism of ADE occurs and places vaccinated individuals at greater risk of a more severe disease phenotype compared to unvaccinated individuals. Closely monitoring of these mutations is essential for the scientists in charge of the design and development of vaccines to make the necessary modifications that go hand in hand with the high mutation rate of SARS-CoV-2. 63

The light at the end of the tunnel; an inhibitor as a possible therapeutic alternative

As described above in the presence of cross-reactive antibodies (responsible for the ADE phenomenon), the entry of the virus is promoted in monocytes/macrophages through the FcR. Once inside the cell, the viruses are replicated and released in large quantities after escaping the immune response. The exacerbated activation of macrophages and mass liberation of cytokines support a hypothesis that states that the so-called cytokine storm is the secondary event of the activation of macrophages, mainly mediated by the ADE phenomenon, reason why its specific blockade will provide therapeutic potentials for patients suffering from severe COVID-19. 89

In this context, it has been stated that the mammalian Target of Rapamycin (mTOR) is one of the main signaling pathways involved in the exacerbated immune response triggered by SARS-CoV2. 90 mTOR is a serine-threonine kinase family protein, a key regulator in protein synthesis, and cellular metabolism that forms two major complexes, mTORC1 with Raptor and mTORC2 with Rictor and plays a pivotal role in cell proliferation and cellular metabolism; therefore, inhibition of mTOR has shown to suppress virus growth and replication. 91

In this regard, in a recent study, a specific set of biological pathways was described in the primary human pulmonary epithelium of SARS-COV-2 infection, among them the mTOR signaling pathway was identified. 92 It has also been stated that the mTOR pathway plays an important role in B‐cell development; mTORC1 controls BCL6 expression and controlling the fate of B cells in the germinal center reaction, therefore contributing in an essential way to the development of ADE by favoring the production of cross-reactive or sub-neutralizing antibodies. 89

These findings propose that selective inhibition of mTOR by an inhibitory agent, such as rapamycin, could have detrimental effects over memory B cell activation and therefore beneficial effects over the characteristic immune response of COVID-19 92

The mechanism of action of rapamycin consists of its ability to bind to the FK506 Immunophilin-binding protein (FKBP12A) and to inhibit the activity of mTORC1 as well as to interrupt the interaction between Raptor and mTOR. The inhibition of mTORC1 by rapamycin then leads to autophagy of infected cells and inhibition of translation of SARS‐CoV‐2 viral polymerase and structural proteins. 90

Overall, it is suggested that the antiviral action of rapamycin, together with its immunomodulatory potential that reduces the excessive production of pro-inflammatory cytokines, would justify clinical studies in patients with COVID-19.90,91

Conclusions

The outbreak and rapid spread of SARS-CoV-2 are a health threat with unprecedented consequences throughout the world. Considering the great economic and health burden of the COVID-19 pandemic, any means to improve the condition of patients, accelerate their recovery, and reduce the risk of deterioration and death would be considered of significant clinical and economic importance. With respect to the immune response generated by the host, the specific neutralizing antibodies generated against the virus are considered essential in the control of virus infections in various ways. However, in some cases, the presence of specific antibodies can be beneficial for the virus. This activity known as antibody-dependent enhancement (ADE) of virus infection enhances virus entry and in some cases virus replication into host cells through interaction with Fc and/or complement receptors. It has been also reported in data from previous CoV research studies that ADE may play a role in the virus’s pathology.

Even though several vaccines have been approved from regulatory bodies under emergency conditions and are distributed worldwide, we cannot rule out the possibility that the evolution of the virus can directly affect its targets, and therefore, the newly mutated virus can escape antibody-mediated protection induced by previous infection or vaccination.

If the vaccines are not capable of generating neutralizing antibodies against the possible mutagenic variants to mount a response, the result may lead to the generation of sub-neutralizing antibodies that will even be capable of facilitating uptake by macrophages that express FcR, with the subsequent stimulation of macrophages and production of pro-inflammatory cytokines.

One advantage of the current pandemics is the unprecedented availability of scientific and technological means to face COVID-19, on these bases, careful design and testing of vaccines will be necessary to evaluate which viral mutations can escape from antibodies-mediated neutralization as well as which one significantly affects the efficacy of the currently approved vaccines.Go to:

Acknowledgments

The figures were created with BioRender.comGo to:

Appendix

Abbreviations

ACE2Angiotensin-converting enzyme 2ADEAntibody-dependent enhancementCoVsCoronavirusesCOVID-19Coronavirus-19DENVDengue virusDFDengue feverDHFDengue hemorrhagic feverDSSDengue shock syndromeEREndoplasmic reticulumFcRsFc receptorsGISAIDGlobal Initiative on Sharing All Influenza DataHIVHuman immunodeficiency virusIDIntradermalIFNInterferonILInterleukinIMIntramusculariNOSInducible nitric oxide synthaseIRFInterferon regulatory factorISGIFN stimulated geneLILRB1Leukocyte immunoglobulin-like receptor B1MERSMiddle East respiratory syndromeMHCMajor histocompatibility complexMPCsMononuclear phagocytic cellsmTORMammalian Target of RapamycinNF-KBNuclear factor kBNKsNatural killer cellsNTDN-terminal domainORFOpen reading framesPGE2Prostaglandin E2PRRsPattern recognition receptorsRBDReceptor-binding domainRdRpRNA-dependent RNA polymeraseRLRRIG-I-like receptorsRNARibonucleic acidROSReactive oxygen speciesRSVRespiratory Syncytial VirusSOCSSuppressor of cytokine signallingThT helper cellTLRToll-like receptorTMPRRS2Serine protease transmembrane type 2TNF-αTumor necrosis factorWHOWorld Health OrganizationZIKVZika virusGo to:

Footnotes

Author contributions: Gabriela Athziri Sánchez-Zuno, Mónica Guadalupe Matuz-Flores, and José Francisco Muñoz-Valle conceived, drafted, and finalized the manuscript.José Francisco Muñoz-Valle, Francisco Javier Turrubiates-Hernández, and Guillermo González-Estevez critically reviewed the draft of the manuscript and approved the final version.All the authors contributed significantly and agreed to the published version of the manuscript.

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by the National Council of Science and Technology (CONACYT Ciencia Básica grant number A1-S-8774) and the Universidad de Guadalajara through Fortalecimiento de la Investigación y el Posgrado 2020.Go to:

ORCID iDs

Francisco Javier Turrubiates-Hernández https://orcid.org/0000-0001-9637-168X

José Francisco Muñoz-Valle https://orcid.org/0000-0002-2272-9260Go to:

References

1. National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral Diseases (2020) Scientific Brief: SARS-CoV-2 Transmission. In CDC COVID-19 Science Briefs Atlanta (GA): Centers for Disease Control and Prevention (US). [Google Scholar]

2. Coronavirus Disease (COVID-19) Situation reports. [online] Available at: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports (accessed April 2021).

3. Baloch S, Baloch MA, Zheng T, et al. (2020) The Coronavirus Disease 2019 (COVID-19) pandemic. Tohoku J Exp Med 250: 271–278. doi:10.1620/tjem.250.271. [PubMed] [CrossRef] [Google Scholar]

4. Zhou Y, Chi J, Lv W, et al. (2021) Obesity and diabetes as high‐risk factors for severe coronavirus disease 2019 (Covid‐19). Diabetes Metab Res Rev 37(2): e3377. doi:10.1002/dmrr.3377. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

5. Mehta P, McAuley DF, Brown M, et al. (2020) COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet 395: 1033–1034. doi:10.1016/S0140-6736(20)30628-0. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

6. Karthik K, Senthilkumar TMA, Udhayavel S, et al. (2020) Role of antibody-dependent enhancement (ADE) in the virulence of SARS-CoV-2 and its mitigation strategies for the development of vaccines and immunotherapies to counter COVID-19. Hum Vaccines Immunother 16(12): 3055–3060. doi:10.1080/21645515.2020.1796425. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

7. Peron JPS, Nakaya H. (2020) Susceptibility of the elderly to SARS-CoV-2 infection: ACE-2 overexpression, shedding, and antibody-dependent enhancement (ADE). Clinics 75, e1912. doi:10.6061/clinics/2020/e1912. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

8. Wen J, Cheng Y, Ling R, et al. (2020) Antibody-dependent enhancement of coronavirus. Int J Infect Dis 100: 483–489. doi:10.1016/j.ijid.2020.09.015. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

9. Ujike M, Taguchi F. (2015) Incorporation of spike and membrane glycoproteins into coronavirus virions. Viruses 7: 1700–1725. doi:10.3390/v7041700. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

10. Comas-Garcia M. (2019) Packaging of genomic RNA in positive-sense single-stranded RNA viruses: a complex story. Viruses 11: 253. doi:10.3390/v11030253. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

11. Kirchdoerfer RN, Cottrell CA, Wang N, et al. (2016) Pre-fusion structure of a human coronavirus spike protein. Nature 531; 118–121. doi:10.1038/nature17200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

12. Song W, Gui M, Wang X, et al. (2018) Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLOS Pathog 14: e1007236. doi:10.1371/journal.ppat.1007236. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

13. National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral Diseases (2020) Science brief: SARS-CoV-2 and surface (Fomite) transmission for indoor community environments. In CDC COVID-19 Science Briefs. Atlanta (GA): Centers for Disease Control and Prevention (US). [Google Scholar]

14. Li W, Moore MJ, Vasilieva N, et al. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426: 450–454. doi:10.1038/nature02145. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

15. HCA Lung Biological Network. Sungnak W, Huang N, Bécavin C, et al. (2020) SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat Med 26: 681–687. doi:10.1038/s41591-020-0868-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

16. Fung TS, Liu DX. (2019) Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 73: 529–557. doi:10.1146/annurev-micro-020518-115759. [PubMed] [CrossRef] [Google Scholar]

17. Hur S. (2019) Double-stranded RNA sensors and modulators in innate immunity. Annu Rev Immunol 37: 349–375. doi:10.1146/annurev-immunol-042718-041356. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

18. Young RE, Thompson RD, Larbi KY, et al. (2004) Neutrophil Elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo. J Immunol 172: 4493–4502. doi:10.4049/jimmunol.172.7.4493. [PubMed] [CrossRef] [Google Scholar]

19. Liu S, Su X, Pan P, et al. (2016) Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Sci Rep 6: 37252. doi:10.1038/srep37252. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

20. Lokugamage KG, Hage A, de Vries M, et al. (2020) Type I Interferon Susceptibility Distinguishes SARS-CoV-2 from SARS-CoV. J Virol 94(23): e01410–e01420. [PMC free article] [PubMed] [Google Scholar]

21. Stanifer ML, Kee C, Cortese M, et al. (2020) Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells. Cell Rep 32(1): 107863. [PMC free article] [PubMed] [Google Scholar]

22. Channappanavar R, Fehr AR, Zheng J, et al. (2019) IFN-I response timing relative to virus replication determines MERS coronavirus infection outcomes. J Clin Invest 129: 3625–3639. doi:10.1172/JCI126363. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

23. Kaneko N, Kuo H-H, Boucau J, et al. (2020) Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19. Cell 183: 143–157.e13. doi:10.1016/j.cell.2020.08.025. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

24. Hadjadj J, Yatim N, Barnabei L, et al. (2020) Impaired type I interferon activity and exacerbated inflammatory responses in severe covid-19 patients. Science 369(6504): 718–724. [PMC free article] [PubMed] [Google Scholar]

25. Knoops K, Kikkert M, Van den Worm SHE, et al. (2008) SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6: e226. doi:10.1371/journal.pbio.00602

26. [PMC free article] [PubMed] [CrossRef] [Google Scholar]26. Chen Y, Cai H, Pan J, et al. (2009) Functional screen reveals SARS coronavirus nonstructural Protein Nsp14 as a Novel Cap N7 methyltransferase. Proc Natl Acad Sci Unit States Am 106: 3484–3489. doi:10.1073/pnas.0808790106. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

27. Bouvet M, Debarnot C, Imbert I, et al. (2010) Vitro reconstitution of SARS-coronavirus MRNA cap methylation. PLoS Pathog 6: e1000863. doi:10.1371/journal.ppat.1000863. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

28. Jansen JM, Gerlach T, Elbahesh H, et al. (2009) Influenza virus-specific CD4+ and CD8+ T cell-mediated immunity induced by infection and vaccination. J Clin Virol 119: 44–52. doi:10.1016/j.jcv.2019.08.009. [PubMed] [CrossRef] [Google Scholar]

29. Long Q-X, Liu B-Z, Deng H-J, et al. (2020) Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med 26: 845–848. doi:10.1038/s41591-020-0897-1. [PubMed] [CrossRef] [Google Scholar]

30. Wang F, Nie J, Wang H, et al. (2020) Characteristics of peripheral lymphocyte subset alteration in COVID-19 pneumonia. J Infect Dis 221: 1762–1769. doi:10.1093/infdis/jiaa150. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

31. Fung S-Y, Yuen K-S, Ye Z-W, et al. (2020) A tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses. Emerg Microb Infect 9: 558–570, doi:10.1080/22221751.2020.1736644. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

32. Welsh RM, Bahl K, Marshall HD, et al. (2012) Type 1 interferons and antiviral CD8 T-cell responses. PLoS Pathog 8: e1002352. doi:10.1371/journal.ppat.1002352. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

33. Sallard E, Lescure F-X, Yazdanpanah Y, et al. (2020) Type 1 interferons as a potential treatment against COVID-19. Antivir Res 178: 104791. doi:10.1016/j.antiviral.2020.104791. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

34. Liu J, Li S, Liu J, et al. (2020) Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients. EBioMedicine 55: 102763. doi:10.1016/j.ebiom.2020.102763. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

35. Qin C, Zhou L, Hu Z, et al. (2020) Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China. Clin Infect Dis 71: 762–768. doi:10.1093/cid/ciaa248. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

36. Hu B, Huang S, Yin L. (2021) The cytokine storm and COVID‐19. J Med Virol 93(1): 250–256. doi:10.1002/jmv.26232. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

37. Zhu Z, Chakraborti S, He Y, et al. (2007) Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. Proc Natl Acad Sci Unit States Am 104: 12123–12128. doi:10.1073/pnas.0701000104. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

38. Temperton NJ, Chan PK, Simmons G, et al. (2007) Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes. Emerg Infect Dis 11: 411–416. doi:10.3201/eid1103.040906. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

39. Yuchun N, Guangwen W, Xuanling S, et al. (2004) Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection. J Infect Dis 190: 1119–1126. doi:10.1086/423286. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

40. Ni L, Ye F, Cheng M-L, et al. (2020) Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity 52: 971–977.e3. doi:10.1016/j.immuni.2020.04.023. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

41. Takada A, Kawaoka Y. (2003) Antibody-dependent enhancement of viral infection: molecular mechanisms andin vivo implications. Rev Med Virol 13: 387–398. doi:10.1002/rmv.405. [PubMed] [CrossRef] [Google Scholar]

42. Cloutier M, Nandi M, Ihsan AU, et al. (2020) ADE and hyperinflammation in SARS-CoV2 infection- comparison with dengue hemorrhagic fever and feline infectious peritonitis. Cytokine 136: 155256. doi:10.1016/j.cyto.2020.155256. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

43. Kulkarni R. (2020) Antibody-dependent enhancement of viral infections. In: Bramhachari PV. (ed) Dynamics of Immune Activation in Viral Diseases. Singapore: Springer Singapore. pp. 9–41. ISBN 9789811510441. [Google Scholar]

44. Khandia R, Munjal A, Dhama K, et al. (2018) Modulation of Dengue/Zika virus pathogenicity by antibody-dependent enhancement and strategies to protect against enhancement in zika virus infection. Front Immunol 9: 597. doi:10.3389/fimmu.2018.00597. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

45. Lee WS, Wheatley AK, Kent SJ, et al. (2020) Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nat Microbiol 5: 1185–1191. doi:10.1038/s41564-020-00789-5. [PubMed] [CrossRef] [Google Scholar]

46. Gan ES, Ting DHR, Chan KR. (2017) The mechanistic role of antibodies to dengue virus in protection and disease pathogenesis. Expert Rev Anti Infect Ther 15: 111–119. doi:10.1080/14787210.2017.1254550. [PubMed] [CrossRef] [Google Scholar]

47. Wan Y, Shang J, Sun S, et al. (2020) Molecular mechanism for antibody-dependent enhancement of coronavirus entry. J Virol 94: e02015–e02019. doi:10.1128/JVI.02015-19. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

48. Langerak T, Mumtaz N, Tolk VI, et al. (2019) The possible role of cross-reactive dengue virus antibodies in zika virus pathogenesis. PLOS Pathog 15: e1007640. doi:10.1371/journal.ppat.1007640. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

49. Ricke DO, et al. (2021) Two different antibody-dependent enhancement (ADE) risks for SARS-CoV-2 antibodies. Front Immunol 12: 640093. doi:10.3389/fimmu.2021.640093. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

50. Pang X, Zhang R, Cheng G. (2017) Progress towards understanding the pathogenesis of dengue hemorrhagic fever. Virol Sin 32: 16–22. doi:10.1007/s12250-016-3855-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

51. Halstead SB, Mahalingam S, Marovich MA, et al. (2010) Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis 10: 712–722. doi:10.1016/S1473-3099(10)70166-3. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

52. Ulrich H, Pillat MM, Tárnok A, et al. (2020) Dengue fever, COVID ‐19 (SARS‐CoV ‐2), and antibody‐dependent enhancement (ADE): a perspective. Cytometry 97: 662–667. doi:10.1002/cyto.a.24047. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

53. Iwasaki A, Yang Y. (2020) The potential danger of suboptimal antibody responses in COVID-19. Nat Rev Immunol 20: 339–341. doi:10.1038/s41577-020-0321-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

54. Zaichuk TA, Nechipurenko YD, Adzhubey AA, et al. (2020) The challenges of vaccine development against betacoronaviruses: antibody dependent enhancement and sendai virus as a possible vaccine vector. Mol Biol 54(6): 922–938. doi:10.1134/S0026893320060151. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

55. Liu L, Wei Q, Lin Q, et al. (2019) Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during Acute SARS-CoV infection. JCI Insight 4: e123158. doi:10.1172/jci.insight.123158. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

56. Zhou Y, Liu Z, Li S, et al. (2021) Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD. Cell Rep 34: 108699. doi:10.1016/j.celrep.2021.108699. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

57. Arvin AM, Fink K, Schmid MA, et al. (2020) A perspective on potential antibody-dependent enhancement of SARS-CoV-2. Nature 584: 353–363. doi:10.1038/s41586-020-2538-8. [PubMed] [CrossRef] [Google Scholar]

58. Fierz W, Walz B. (2020) Antibody dependent enhancement due to original antigenic sin and the development of SARS. Front Immunol 11: 1120. doi:10.3389/fimmu.2020.01120. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

59. Jaume M, Yip MS, Cheung CY, et al. (2011) Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a PH- and cysteine protease-independent Fc R pathway. J Virol 85: 10582–10597. doi:10.1128/JVI.00671-11. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

60. Shrock E, Fujimura E, Kula T, et al. (2020) Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Science 370(6520): eabd4250. doi:10.1126/science.abd4250. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

61. Shukla R, Ramasamy V, Shanmugam RK, et al. (2020) Antibody-dependent enhancement: a challenge for developing a safe dengue vaccine. Front Cell Infect Microbiol 10: 572681. doi:10.3389/fcimb.2020.572681. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

62. Cui G, Si L, Wang Y, et al. (2021) Antibody‐dependent enhancement (ADE) of dengue virus: identification of the key amino acid that is vital in denv vaccine research. J Gene Med 23(2): e3297. doi:10.1002/jgm.3297. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

63. Cardozo T, Veazey R. (2021) Informed consent disclosure to vaccine trial subjects of risk of COVID‐19 vaccines worsening clinical disease. Int J Clin Pract 75(3): e13795. doi:10.1111/ijcp.13795. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

64. Andreano E, Piccini G, Licastro D, et al. (2020) SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma. bioRxiv, in press. [PMC free article] [PubMed] [Google Scholar]

65. Letko M, Marzi A, Munster V. (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol 5: 562–569. doi:10.1038/s41564-020-0688-y. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

66. Weisblum Y, Schmidt F, Zhang F, et al. (2020) Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. eLife 9: e61312. doi:10.7554/eLife.61312. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

67. Chen J, Wang R, Wang M, et al. (2020) Mutations strengthened SARS-CoV-2 infectivity. J Mol Biol 432: 5212–5226. doi:10.1016/j.jmb.2020.07.009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

68. Pachetti M, Marini B, Benedetti F, et al. (2020) Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med 18: 179. doi:10.1186/s12967-020-02344-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

69. Giovanetti M, Benedetti F, Campisi G, et al. (2021) Evolution patterns of SARS-CoV-2: snapshot on its genome variants. Biochem Biophys Res Commun 538: 88–91. doi:10.1016/j.bbrc.2020.10.102. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

70. Jangra S, Ye C, Rathnasinghe R, et al. (2021) The E484K mutation in the SARS-CoV-2 spike protein reduces but does not abolish neutralizing activity of human convalescent and post-vaccination sera. medRxiv, in press. [Google Scholar]

71. Nagy Á, Pongor S, Győrffy B. (2021) Different mutations in SARS-CoV-2 associate with severe and mild outcome. Int J Antimicrob Agents 57: 106272. doi:10.1016/j.ijantimicag.2020.106272. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

72. Liu Z, VanBlargan LA, Bloyet L-M, et al. (2021) Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. Cell Host Microbe 29: 477–488.e4. doi:10.1016/j.chom.2021.01.014. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

73. Korber B, Fischer WM, Gnanakaran S, et al. (2020) Tracking changes in SARS-CoV-2 spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell 182: 812–827.e19. doi:10.1016/j.cell.2020.06.043. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

74. Davies NG, Abbott S, Barnard RC, et al. (2020) Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science 372(6538): eabg3055. [PMC free article] [PubMed] [Google Scholar]

75. Greaney AJ, Loes AN, Crawford KHD, et al. (2021) Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies. Cell Host Microbe 29(3): 463–476.e6. [PMC free article] [PubMed] [Google Scholar]

76. National Institute of Infectious Diseases , JAPAN (2021) Brief report: new variant strain of SARS-CoV-2 identified in travelers from Brazil. January 12, 2021. [Online] Available at: https://www.niid.go.jp/niid/en/2019-ncov-e/10108-covid19-33-en.html (accessed September 2021).

77. Plante JA, Liu Y, Liu J, et al. (2021) Spike mutation D614G alters SARS-CoV-2 fitness. Nature 592: 116–121. doi:10.1038/s41586-020-2895-3. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

78. Erol A. (2021) Are the emerging SARS-COV-2 mutations friend or foe? Immunol Lett 230: 63–64. doi:10.1016/j.imlet.2020.12.014. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

79. Ali F, Kasry A, Amin M. (2021) The new SARS-CoV-2 strain shows a stronger binding affinity to ACE2 due to N501Y mutant. Med. Drug Discov 10: 100086. doi:10.1016/j.medidd.2021.100086. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

80. Wise J. (2021) Covid-19: the E484K mutation and the risks it poses. BMJ 372: n359. doi:10.1136/bmj.n359. [PubMed] [CrossRef] [Google Scholar]

81. da Silva Francisco R, Jr, Benites LF, Lamarca AP, et al. (2021) Pervasive transmission of E484K and emergence of VUI-NP13L with evidence of SARS-CoV-2 Co-infection events by two different lineages in Rio Grande Do Sul, Brazil. Virus Res 296: 198345. doi:10.1016/j.virusres.2021.198345. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

82. Xie X, Liu Y, Liu J, et al. (2021) Neutralization of SARS-CoV-2 Spike 69/70 Deletion, E484K, and N501Y Variants by BNT162b2 Vaccine-Elicited Sera. Nat Med 27(4): 620–621. [PubMed] [Google Scholar]

83. European Centre for Disease Prevention and Control (2021) Emergence of SARS-CoV-2 B.1.617 variants in India and situation in the EU/EEA– 11 May 2021. Stockholm: ECDC [Google Scholar]

84. Lopez Bernal J, Andrews N, Gower C, et al. (2021) Effectiveness of Covid-19 vaccines against the B.1.617.2 (Delta) variant. N Engl J Med 385: 585–594. doi:10.1056/NEJMoa2108891. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

85. Xiang T, Liang B, Fang Y, et al. (2021) Declining levels of neutralizing antibodies against SARS-CoV-2 in convalescent COVID-19 patients one year post symptom onset. Front Immunol 12: 708523. doi:10.3389/fimmu.2021.708523. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

86. Favresse J, Bayart J-L, Mullier F, et al. (2021) Antibody titres decline 3-month post-vaccination with BNT162b2. Emerg Microb Infect 10: 1495–1498. doi:10.1080/22221751.2021.1953403. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

87. Morales-Núñez JJ, Muñoz-Valle JF, Meza-López C, et al. (2021) Neutralizing antibodies titers and side effects in response to BNT162b2 vaccine in healthcare workers with and without prior SARS-CoV-2 infection. Vaccines 9: 742. doi:10.3390/vaccines9070742. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

88. Pegu A, O’Connell S, Schmidt SD, et al. (2021) Durability of MRNA-1273 vaccine–induced antibodies against SARS-CoV-2 variants. Science 2021: eabj4176. doi:10.1126/science.abj4176. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

89. Zheng Y, Li R, Liu S. (2020) Immunoregulation with MTOR inhibitors to prevent COVID‐19 severity: a novel intervention strategy beyond vaccines and specific antiviral medicines. J Med Virol 92: 1495–1500. doi:10.1002/jmv.26009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

90. Karam BS, Morris RS, Bramante CT, et al. (2021) MTOR inhibition in COVID‐19: a commentary and review of efficacy in RNA viruses. J Med Virol 93: 1843–1846. doi:10.1002/jmv.26728. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

91. Ramaiah MJ. (2020) MTOR inhibition and P53 activation, MicroRNAs: the possible therapy against pandemic COVID-19. Gene Rep 20: 100765. doi:10.1016/j.genrep.2020.100765. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

92. Fagone P, Ciurleo R, Lombardo SD, et al. (2020) Transcriptional landscape of SARS-CoV-2 infection dismantles pathogenic pathways activated by the virus, proposes unique sex-specific differences and predicts tailored therapeutic strategies. Autoimmun Rev 19: 102571. doi:10.1016/j.autrev. 2020. 102571. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

Antibody dependent enhancement: Unavoidable problems in vaccine development

Authors: Lele XuZhiqian MaYang LiZhaoxia Pang, and Shuqi Xiao*

Adv Immunol. 2021; 151: 99–133.Published online 2021 Sep 4. doi: 10.1016/bs.ai.2021.08.003 PMCID: PMC8438590PMID: 34656289

Abstract

In some cases, antibodies can enhance virus entry and replication in cells. This phenomenon is called antibody-dependent infection enhancement (ADE). ADE not only promotes the virus to be recognized by the target cell and enters the target cell, but also affects the signal transmission in the target cell. Early formalin-inactivated virus vaccines such as aluminum adjuvants (RSV and measles) have been shown to induce ADE. Although there is no direct evidence that there is ADE in COVID-19, this potential risk is a huge challenge for prevention and vaccine development. This article focuses on the virus-induced ADE phenomenon and its molecular mechanism. It also summarizes various attempts in vaccine research and development to eliminate the ADE phenomenon, and proposes to avoid ADE in vaccine development from the perspective of antigens and adjuvants.

Keywords: Coronavirus, Antibody-dependent infection enhancement, Vaccine.

1. Introduction

In 1967 Hawkes first confirmed that IgG in serum can induce ADE (Lafferty, 1967). Many viruses have been proven to have ADE effects, such as Arthropod borne viruses (ABV), Dengue virus (DENV), Respiratory syncytial virus (RSV), Human Immunodeficiency virus (HIV), Feline infectious perionitis virus (FIPV), Coronaviruses (CoV). For many viruses (including DENV, HIV) that pose a major threat to human health, the presence of ADE is considered to be a major obstacle to vaccine development.

Fc receptor (FcR)-mediated ADE is the most common form of ADE, which was first discovered by Halstead (1977). At first, it was thought that the antigen antibody immune complex formed by virus and specific antibody combined with the host cell with the help of FcR on the cell surface, which was more conducive to the entry of virus, and increased the infection amount and infection rate of virus, finally led to the increase of infection and replication of virus. By phagocytosing immune complexes, the cells expressing FcR on the surface, such as monocytes, macrophages, dendritic cells, and certain granulocytes, can produce ADE. This kind of ADE is mainly mediated by IgG antibody, but IgM, IgE and IgA antibody have also shown the ability of ADE (Janoff, Wahl, Kelly, & Smith, 1995Shi et al., 2018Takada, Ebihara, Feldmann, Geisbert, & Kawaoka, 2007).

Complement mediated ADE(C-ADE) refers to the combination of virus and antibody to form an immune complex, which activates the complement and combines with the complement to form a complex, and then enters the cell through the complement receptor on the cell surface. After the complement inactivation, the ability of the serum to mediate the enhancement of viral infection decreased. However, the enhanced effect of virus infection returned to normal after the addition of excessive complement which indicated that C-ADE needed the participation of complement and antibody. The antibody of CR3 can block ADE infection of West Nile virus on cells expressing FcR, while the antibody of FcR receptor cannot block ADE infection, indicating that ADE infection can also be mediated by complement alone (Gordon, 1983).

With the research on ADE, the concept of intrinsic antibody-dependent enhancement(iADE) was proposed (Chareonsirisuthigul, Kalayanarooj, & Ubol, 2007Halstead, Mahalingam, Marovich, Ubol, & Mosser, 2010). Combining Fc and FcR also changes intracellular signaling pathways, causing them to shift from antiviral mode to viral promotion mode. This process is called iADE. In other words, ADE changes the innate immune response in cells and inhibits the antiviral response in cells, thus enhancing the virus infection. iADE infection can change some molecular signal transduction pathways in the process of immune cell response, especially the changes in the expression of IFN-β and IL-10, the changes in the phosphorylation levels of key molecules in signal transduction (NF-κB, STAT and IRF, etc.) (Patro et al., 2019Taylor et al., 2015Tsai et al., 2014Ubol, Phuklia, & Kalayanarooj, 2010). iADE could be the key to tackling the dangers of ADE in the future.

The mechanism or interaction of ADE is not fully understood. It is necessary to understand the upstream and downstream molecular signal events of ADE. In the development of vaccines for a variety of viral diseases, ADE needs to be overcome.

2. ADE of representative virus

2.1. Coronavirus

The mechanism of MERS ADE was mediated by neutralizing MAb targeting the coronavirus S protein RBD (Wan et al., 2020). ADE of MERS-CoV followed the same entry pathway as that of DPP4-dependent MERS-CoV. RBD specific neutralizing monoclonal antibody mediates coronavirus entry into ADE by functionally mimicking viral receptors. If other parts of the targeted neutralizing antibody do not trigger the conformational change of the spike, they are unlikely to mediate ADE. Similar findings have been found in SARS-CoV. The monoclonal antibody specific to SARS-CoV RBD (S230) binds to ACE2 in SARS-CoV RBD, triggering the conformational change of SARS-CoV. S230 can block the connection with DPP4 or ACE2 through the competitive mechanism, respectively. The antibody attributes membrane fusion by mimicking the action of the receptor (Walls et al., 2019). Therefore, SARS-CoV is likely to have ADE mechanism similar to MERS-CoV.

In the acidic pH environment of the endosome leads to the aggravation of antibody mediated infection, which is the opposite of SARS-CoV infection mediated by ACE2 receptor. Virus particles that infect cells via ADE pathway may still be trapped in acid compartment, and inhibition of internal acidification will prevent its degradation. It was also found that the sera induced by ADE did not contain anti-Spike IgG2a antibody, while the two neutralized/non-enhanced sera did (Jaume et al., 2011). IgG2a is a marker of Th1 type response, and Th2 type is more prone to induce ADE. Whether this phenomenon is related to the preference for Th1 type response and whether the deficiency of IgG2a has a causal relationship with the occurrence of ADE infection needs further research.

Daniel found two VHH (variable domain of the heavy-chain of heavy chain antibody) targeting SARS-CoV and MERS-CoV RBD, which neutralizes SARS-CoV and MERS-CoV and interferes with receptor binding (Wrapp et al., 2020). The possible mechanisms of neutralization are the blocking of the receptor binding interface and the capture of the up-conformation of RBDs, acting as receptor simulators that trigger the premature transition from the prefusion-to-post fusion conformation. The author did not discuss the possibility of this antibody ADE, but according to Wan’s results mentioned above (Wan et al., 2020), it is likely to have the phenomenon of ADE. It should be noted that the VHH directed by SARS-CoV RBD reacts with SARS-CoV-2 RBD, and can block receptor binding. SARS-CoV-2 and SARS-CoV have 79.6% sequence identity (Zhou et al., 2020). They use the same receptor ACE2 and cause similar acute respiratory syndrome. There is a cross reaction between SARS-CoV-2 and SARS-CoV S protein antibody, but cross neutralization reaction is rare (Lv et al., 2020).The key epitope of ADE induced by SARS-CoV S protein has been identified (LYQDANC), which is highly similar in SARS-CoV-2 region (Wang & Zand, 2020Wang et al., 2016). 48 kinds of SARS-CoV-2 antibodies were isolated by Zhou, 11 (23%) of which significantly increased the level of SARS-CoV-2 ADE in vitro, and 9 of them were RBD binding antibodies. It was further confirmed that the epitope of RBD was related to SARS-CoV-2 ADE in vitro (Zhou et al., 2021). Recent studies have found that antibodies that can promote SARS-CoV-2 infection can bind to specific sites of N-terminal domain(NTD), which leads to the conformational change of RBD and makes it easier to bind to ACE2 (Liu et al., 2021).

Observations in patients with SARS-CoV-2 were similar to those with SARS during the 2003 epidemic (Cheung et al., 2005Tetro, 2020). It is speculated whether individuals with severe disease may have been exposed to one or more coronavirons and are experiencing an ADE due to antigen epitope heterogeneity (Tetro, 2020). ADE has been found in SARS-CoV, and is believed to be one of the reasons for such a high mortality rate (Ho et al., 2005). High concentration of anti SARS-CoV antiserum neutralized the infection of SARS-CoV, while highly diluted antiserum significantly increased the infection of SARS-CoV and induced a higher level of apoptosis (Wang et al., 2014). Rhesus monkeys immunized with S-glycoprotein of full-length SARS-CoV can cause serious acute lung injury (ALI) when they attack the virus (Chen et al., 2005Liu et al., 2019). All these suggest that the development of SARS-COV vaccine requires special attention to ADE. The SARS-CoV S protein trimer-immunized hamster serum mediated the ADE of SARS-CoV, but the viral load and lung lesions did not increase in the vaccinated animals. These data indicate that the enhancement of virus entry into cells in vitro cannot fully predict the adverse effects in vivo (Arvin, Fink, Schmid, Cathcart, & Virgin, 2020Jaume et al., 2011). Therefore, more in-depth research is needed to correlate in vitro laboratory test results with clinical results, so as to provide a reference for the SARS-CoV-2 ADE study.

At present, the clinical experience is not enough to prove the existence or non-existent of ADE in SARS-CoV-2 (Arvin et al., 2020). Without the support of data to believe that ADE will definitely hinder the development of SARS-CoV-2 vaccine. However, the experience of dengue fever vaccine and RSV vaccine reminds us that if there is a risk of ADE in the COVID-19 vaccine, special attention should be paid to the safety of any candidate SARS-CoV-2 vaccine (Coish & MacNeil, 2020Sharma, 2020). The good news is that immunity to SARS-CoV-2 RBD or inactivated SARS-CoV-2 vaccine can cause a strong neutralizing antibody response in rodents, and antiserum does not mediate ADE (Gao et al., 2020Quinlan et al., 2020). Even though some antibodies can enhance virus infection in cell experiments, they have not been shown to enhance virus infection in vitro experiments by mice and monkeys (Li, Edwards, Manne, Martinez, & Saunders, 2021).

2.2. Flaviviruses

Perhaps the most famous example of ADE infection is the virus-specific antibody enhanced DENV (Izmirly, Alturki, Alturki, Connors, & Haddad, 2020). The main representative viruses include Moray Valley encephalitis virus (MVEV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and DENV(Schweitzer, Chapman, & Iwen, 2009Weaver & Barrett, 2004).

Dengvaxia (CYD-TDV) is first licensed vaccine. This vaccine is only approved in dengue-endemic countries and only dengue seropositive people. CYD-TDV has shown a powerful effect in preventing serious diseases. But it increases the risk of severe dengue fever in seronegative patients. The potential risk of ADE is undoubtedly the difficulty of dengue fever vaccine development (Izmirly et al., 2020Sridhar et al., 2018). DENV has four different serotypes (DENV1-4), ADE is easily caused between different serotypes (Heinz & Stiasny, 2012). Global epidemiological studies show that the vast majority of DHF/ DSS cases occur after the secondary infection of DENV. In addition, it has been proposed that ADE is also triggered by sub-neutralization concentrations DENV antibodies. From the perspective of mechanism, antibody mediated DENV entry into cells expressing FcR, such as monocytes, leads to increased viral replication, which triggers the release of inflammatory and vasoactive mediators, thus aggravating the severity of the disease. The critical time for DHF/DSS is approximately 2 months after degrade below a protective level of maternal dengue 2 neutralizing antibodies, maternally derived DENV-reactive IgG is a determinant of the viral burden in vivo (Chau et al., 2008Chau et al., 2009Kliks, Nimmanitya, Nisalak, & Burke, 1988). ADE can also be reproduced in vitro by primary human monocytes, macrophages, and mature DC, as well as cell lines expressing human and mouse Fc tissues (e.g., U937, K562, THP-1, and P388D1) in the presence of subneutralizing concentrations of antibodies. In addition, the expression of FcγRs can also promote ADE in non Fc cell lines (Goncalvez, Engle, Claire, Purcell, & Lai, 2007Chareonsirisuthigul et al., 2007Halstead, Chow, & Marchette, 1973Littaua, Kurane, & Ennis, 1990Morens, Larsen, & Halstead, 1987).

DENV could be divided into the compact and expanded mature DENV, the fully immature DENV, and the partially mature DENV. Some of these DENV particles are completely infectious, while others need to be infectious by binding with other molecules (Lok, 2016). The prM is a precursor protein of a membrane protein (M), which is present on the surface of immature DENV. The prM forms a heterodimer with the E protein, blocks the fusion peptide of the E protein, and prevents membrane fusion of the virus and the host cell membrane. In the process of virus maturation, prM is cleaved into M protein by furin protease under the induction of low pH, which rearranges E protein, and makes immature virus particles that are not infectious become mature virus and become infectious. Antibodies to prM mediate the binding of immature virus to target cells, thereby enhancing the infectivity of the virus. Using JEV Pr instead of DENV Pr will not change the neutralization ability of anti PrM antibody, and will not enhance the infection of K562 cells with Fc receptor, so as to avoid the ADE of anti prM antibody (Wang et al., 2017). Recent studies have found that the DENV gene for ADE is located at the fifth, sixth, seventh, and sixteenth amino acids of pr4, which can reduce the occurrence of ADE by replacing key amino acids (Cui et al., 2021).

Renner confirmed two neutralizing antibodies of DENV2, 2C8 can induce ADE, while 3H5 does not. Fc region of 3H5 (IgG1 subtype) was switched to Fc region of 2C8 (IgG2a subtype), it did not change this feature, which indicated that the difference was not caused by different IgG subtypes. 2C8: DENV can strongly bind to FcγR, but 3H5: DENV cannot bind to FcγR receptor. The binding of 3H5 antibody may lead to the deformation of virus surface, and the organization of the whole immunoglobulins will be too crowded to allow FcγR to bind. On the contrary, The upright conformation of 2c8 will completely expose the Fc region of the whole immunoglobulin, so that it can combine with FcγR to induce ADE (Morrone & Lok, 2019). 3H5 showed a strong binding force at neutral pH and low pH, while 2C8 showed a significant reduction in binding force at low pH. Due to the low pH value of the late endosomes, 2C8 may dissociate from the surface of the virus, leading to infection, which partly explains the mechanism of ADE in mature dengue granules (Lok, 2016Morrone & Lok, 2019).

It should also be noted that DENV and ZIKV belong to the Flaviviridae genus in the virus classification, and there is a large amount of antigen overlap between them. DENV and ZIKV infection can induce cross-reactive antibody responses (Rathore & St John, 2020). The antibodies of one virus may promote the development of the other virus. Two immunodominant epitopes, the precursor membrane protein (prME) and the envelope (E) protein, can be recognized by cross-reactive antibodies. These antibodies not only neutralize the ills, but also promote viral replication and disease severity through Fc receptor-mediated myeloid cell infection. Serum from patients infected with DENV, as well as DENV-specific human monoclonal antibodies, bind to ZIKV and promote its infection of cells carrying Fc receptors. The broadly neutralizing anti-DENV E protein linear fusion ring epitope (FLE) are not neutralize to ZIKV, but can increase ZIKV infection (Dejnirattisai et al., 2016Paul et al., 2016). Cross-reactive flavivirus antibodies may also cause other harmful effects of ZIKV infection by promoting placental transmission via FcRn-mediated endocytosis (Hermanns et al., 2018Langerak et al., 2019). In the case of ADE, the co-infection of DENV and ZIKV will cause more damage to the host. The initial virus (ZIKV) will produce a higher viral load and a second peak, while the second virus (DENV) will produce a larger peak viral load and an earlier peak. ADE may have a greater impact on the second virus during co-infection (Tang et al., 2020). Antibodies produced against ZIKV or DENV could enhanced the entry of ROCV, SLEV, WNV and ILHV into K562 cells, which may lead to a risk of serious infection (Oliveira et al., 2019). The serum of WNV antibody-positive persons can significantly enhance ZIKV infection in vitro. Considering the prevalence of WNV infection in the United States, this may have a great potential risk. Further research found that the flavivirus E antibody is related to this cross ADE (Garg et al., 2021).

2.3. Porcine reproductive and respiratory syndrome virus (PRRSV)

PRRSV is an arteritis virus with a capsule (Karl-Klaus, Nico, et al., 1993). It is one of the most serious diseases faced by the global pig industry and causes huge losses to the pig industry every year (Li et al., 2021Wang, Yu, Cai, Zhou, & Zimmerman, 2020).

ADE is present in PRRSV infection both in vivo and in vitro. In vitro, PRRSV infection was added with a certain concentration of PRRSV antibodies for 1 hour, and then the mixture was used to infect porcine alveolar macrophages, which was significantly higher than the control group by tens of times (Cancel-Tirado, Evans, & Yoon, 2004). In vivo studies have found that the mean level and duration of vireaemia in pigs treated with PRRSV-specific IgG subneutralization were greater than in control pigs injected with normal serum globulin (Yoon, Wu, Zimmerman, Hill, & Platt, 1996). This suggests that when maternal PRRSV-specific antibody, exposure to wild-type or vaccine PRRSV-induced antibody level decreases, ADE of PRRSV may occur, resulting in increased disease severity and possibly increased susceptibility of pigs to PRRSV infection. FcγR is the main way to mediate PRRSV ADE. FcγRI, FcγRIIb and FcγRIII are all related to ADE (Gu et al., 2015Qiao et al., 2011Shi et al., 2019). After infection with the PRRSV-antibody complex, the transcription level of FcγRI slightly increased, while the transcription level of FcγRIIb (inhibitor receptor) increased significantly. In addition to the receptor of IgG, the receptor FCɛRI of IgE also affects PRRSV infection. FcɛRI may be related to the antigen presenting process and regulation of the inflammatory response, PRRSV reproduction and the regulation of antiviral response during PRRSV infection (Shi et al., 2018).

Cancel-Tirado used a variety of monoclonal antibodies against M/N/GP3/CP5, and found that some monoclonal antibodies not only failed to inhibit virus infection, but also promoted virus replication (Cancel-Tirado et al., 2004). GP5 exists the main neutralizing epitope of the virus. There are two B-cell antigenic sites A and B on the GP5 protein. A epitope is the decoy epitope, and the antibody induced by A epitope has no neutralizing activity. Non-neutralizing antigen site A can be recognized by non-neutralizing antibody, and cannot be recognized by monoclonal antibody ISU25-c1 and pig neutralizing serum antibody. The A epitope is highly variable in the strains and has an immune advantage. Antibodies at this epitope appear early in PRRSV infection. Therefore, the epitope of GP5 antibody that can induce ADE is presumed to be epitope A. The antibody against N protein is the earliest and most common antibody after infection, but the N protein is not exposed on the virus surface. The antibody against N protein ISU15A can promote PRRSV infection. This process should not be an antibody against N that promotes virus adsorption and entry into cells. It is speculated that it may affect the replication of PRRSV in cells. Carmen a. Sautter’s study showed that immunized with the inactivated PRRSV vaccine and then attacked the homologous virulent virus, the vaccine-mediated enhancement of clinical symptoms was observed, but the results showed that it was not neutralizing antibody or other antibody mediated enhanced infection, increased the release of virus or the production of cytokines (Sautter, Trus, Nauwynck, & Summerfield, 2019). However, the heat-inactivated serum used in the test may affect the participation of complement, and the test is the use of monocyte Derived Macrophages. The immune complex enhanced infection may target other cells, such as FcR on monocytes and dendritic cells.

3. The molecular mechanism that produces ADE

3.1. FcγR-mediated ADE

FcγR, also known as IgG Fc receptors for immunoglobulin G, is an important cell surface molecule that is widely expressed on the surface of most immune cells and can specifically bind to the Fc region of immunoglobulin. At present, the identified human FcγR can be divided into three subclasses: FcγRI (CD64), FcγRI (CD32), FcγRIII (CD16). In addition, specific FcRIV was found in mice (Nimmerjahn, Bruhns, Horiuchi, & Ravetch, 2005Taylor et al., 2015). As a bridge between humoral immunity and cellular immunity, FcγR mediates the interaction between immune complex and immune cells through specific binding with Fc region of immunoglobulin, so as to participate in the activation and regulation of various immune effects (de Taeye, Rispens, & Vidarsson, 2019). FcγR can also mediate the interaction between immune cells. The immune response mediated by FcγR includes the elimination of immune complex, the regulation of antibody production, the lysis of tumor cells, antigen processing and presentation, and the regulation of T cell proliferation and differentiation. FcγR plays a role in resisting the invasion of foreign microorganisms by triggering various immune functions of immune cells.

The connection between FcγR and viral immune complex can control ADE through internalization, and the result is the balance of activation and inhibition of FcγR. FcγRI, FcγRII and FcγRIII can all mediate the occurrence of ADE, but their action modes and intensity are different. In the cells expressing FcR, the production of cytokines mediated by FcγR depends on the proportion of activated and inhibited FcγR.

FcγRI is a high affinity receptor and the only FcR binding to IgG monomer. It consists of extracellular regions of three Ig like domains, a transmembrane domain and an intracellular domain (Hanson & Barb, 2015). It is mainly expressed on the surfaces of macrophages, monocytes, polymorphonuclear cells and dendritic cells. FcγRI is mainly involved in the process of antigen intake, processing and presentation in the early stage of immune response.

It has been proved that FcγRI can mediate the ADE of DENV, HIV, PRRSV and other viruses. The extracellular domain of FcγRI is sufficient to internalize the immune complex of infectious dengue virus through a mechanism that does not involve classical ITAM dependent signaling (Schlesinger & Chapman, 1999). Rodrigo found that enhanced immune complex infectivity mediated by FcγRIA was greatest when the receptor was associated with a γ-chain in its native form and that abrogation of γ-chain ITAM signaling capacity by Tyr-to-Phe mutation reduced but did not entirely eliminate this function (Rodrigo, Jin, Blackley, Rose, & Schlesinger, 2006). FcγRIA may have at least two internalization mechanisms of viral immune complexes. The first is γ-chain signal dependence. Aggregates of infectious viral immune complexes of sufficient size trigger the classical phagocytosis entry pathway. The second is a somewhat less efficient entry mechanism that did not require γ-chain activation and relied simply on concentrating partially neutralized virions onto the cell for entry by a parallel endocytosis mechanism (Rodrigo et al., 2006). The study found six different alternatively spliced of porcine FcγRI, and different FcγRI alternatively spliced have different effects on PRRSV ADE. The membrane pCD64-T1 enhances the replication of PRRSV in the form of promoting the endocrine of the PRRSV-antibody complex. Soluble pCD64-T3 exhibits inflammation-enhancing effects. It is speculated that pCD64-T3 can enhance the activation of TLR in ADE mode, and up-regulate the transcription of inflammatory factors such as TNF-α, IL-1β and IFN-β, and negatively regulate PRRSV ADE. Porcine FcγRI plays a dual regulatory role in PRRSV infection and PRRSV inflammation through alternatively spliced mechanism (Shi et al., 2019). Different spliceosomes lead to different viral infection results, which also increase the complexity of PRRSV ADE.

FcγRII is a low-affinity receptor and requires high-affinity binding through IgG immune complexes. Among them, FcγRIIA and FcγRIIC are activated receptors, while FcγRIIB is an inhibitory receptor (Guilliams, Bruhns, Saeys, Hammad, & Lambrecht, 2014). The activated receptor participates in intracellular signal transduction through immunoreceptor tyrosine-based activation motif (ITAM), for example, the combination of the antibody-virus complex of Ebola virus with the cell surface Fc eagerly RIIa will trigger the phosphorylation of Src family PTK and activate the subsequent signal transduction pathway, thereby leading to increased viral uptake through phagocytosis and/or macropinocytosis (Furuyama et al., 2016). The inhibited receptor contains the suppressed motif based on the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. FcγRIIb mainly exerts its negative regulatory function through a series of cascade reactions generated after phosphorylation of its intracellular ITIM motif.

FcγRIIA is more effective than FcyRIA in enhancing the infectivity of dengue virus immune complexes. FcγRIIA is better equipped than FcγRIA to utilize alternative signaling pathways and entry mechanisms made available by relocation to such sites where weakly bound immune complexes might be more easily transferred to favorable entry pathways. After the immune complex is connected to FcyRIIa, the internalized FcyRIIa-immune complex enters the endosomal compartment, where the low affinity of FcyRIIa promotes the release of the immune complex. The attachment or dissociation of the FcγRIIa-immune complex enables innate immunity from antiviral to immunosuppression, induces the expression of IL-6, TNF and IL-10, and suppresses the expression of type I IFN (Rodrigo et al., 2006).

In vitro studies conducted in the monocyte cell lines U937 and K562 showed that FcγRII-mediated entry occurred through clathrin-coated vesicles, while FcγRI-mediated entry was not related to clathrin. In addition, since FcγRII translocates into lipid rafts after the immune complex is bound, entry through FcγRII is affected by membrane cholesterol levels (Carro, Piccini, & Damonte, 2018).

The activation of FcγR signaling can be blocked by the negative signaling of the inhibitory receptor FcγRIIb on the same cell surface. The destruction of tyrosine residues in ITIM or the removal of the poFcγRIIb cytoplasmic domain eliminates the ability of poFcγRII to mediate PRRSV-infected ADE (Wan, Chen, Li, Pang, & Bao, 2019). FcγRIIa promotes DENV-infected ADE, while FcγRIIb restricts it. By switching between these two isoforms containing the motifs of ITAM and ITIM, it was found that the intracellular part of FcγRII is the main determinant of ADE infection.

FcγRIII can not only significantly inhibit the levels of IFN-α and TNF-α mRNA, but also significantly increase the levels of IL-10 and viral mRNA during PRRSV viral infection. These clarify a mechanism of PRRSV-ADE regulated by poFcγRIII, which may be to increase virus internalization in its host cells and reduce antiviral cytokine levels to promote virus entry and replication (Gu et al., 2015Zhang et al., 2016). FcγRIIIA promotes aggravation of dengue fever. The IgG1 subclass antibodies produced by DHF/DSS patients have a high affinity for the non-fucosylated Fc receptor FcγRIIIA, and the combination of the two induces platelet reduction, which is an important risk factor for thrombocytopenia syndrome, leads to the ADE effect of the disease (Wang, Sewatanon, et al., 2017).

3.2. Complement-mediated ADE

Complement dependent ADE is quite different from FcγR-dependent ADE, Complement can synergize with FCγR or cause ADE alone. High levels of CR2 and CD4 are required to enhance HIV-1 infection in T lymphocyt-like cell lines in the presence of subneutralizing levels of HIV-specific antibodies (Robinson, Montefiori, & Mitchell, 1990). In the absence of antibodies, complement CR3 and CR1 can promote HIV infection. Using antibodies to block CR1 or CR3 functions can completely inhibit complement-mediated enhancement (Thieblemont, 2010). Another independent pathway of complement mediated ADE in HIV-1 infection has been shown to require the complement component C1q. The IgG antibody binds closely to the viral epitope, so that the C1q binds to the Fc part of the antibody, and the immune complex binds to the C1q receptor on the cell surface (Prohászka et al., 1997). Similarly, C1q mediated ADE can enhance the infection of Ebola virus in non-monocytes by promoting the binding or endocytosis of the virus to the target cells (Takada, Feldmann, Ksiazek, & Kawaoka, 2003). Unlike FcR mediated intracellular signaling to promote ADE, C1q mediated ADE is only caused by the increase of virus particles attached to the cell surface (Byrne & Talarico, 2021Furuyama, Nanbo, Maruyama, Marzi, & Takada, 2020). Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. This may be related to the increased uptake of human parvovirus B19 on myocardial endothelial cells by complement factor C1q and its receptor CD93 mediated ADE. Despite its strong host tropism for erythroid progenitor cells, in the presence of specific antibodies to human parvovirus B19, complement dependent ADE increased the uptake of viral endothelial cells by 4000 times. ADE results in the accumulation of B19V DNA in the nucleus of infected cells, but it has no significant effect on gene expression and genome replication. The enhancement did not depend on the interaction between the virus antibody complex and FcR (von Kietzell et al., 2014).

3.3. Molecular signaling events in ADE

In addition to promoting the entry of viruses, ADE also affects the immune response of cells, inhibiting the antiviral activity of cells and thus promoting infection. The connection or dissociation of FcγR-immune complexes changed the innate response of antiviral and immunosuppression through unknown pathways, induced IL-6, TNF and IL-10, and inhibited the expression of type I IFN (Izmirly et al., 2020Patro et al., 2019).

When immune complex entering the target cell can activates the negative regulator, dihydroxyacetone kinase (DAK) and autophagy-related 5 autophagy-related 12 (Atg5-Atg12), and then disrupts the RIG-I/MDA-5 signal cascade, down-regulating the downstream molecular signal (Ubol & Halstead, 2010Ubol et al., 2010). Meanwhile, the connection between antibody complex and FcR downregulated TLRs gene expression, strongly stimulated the negative regulators of TIR-domain-containing adapter-inducing interferon-β (TRIF) and TNF receptor-associated factor 6 (TRAF-6), Sterile-alpha and Armadillo motif-containing protein (SARM), TRAF family member-associated NF-κB activator (TANK), negative regulators of the NF-κB pathway. Up-regulation of SARM and TANK results in down-regulation of TLR signaling molecules (Modhiran, Kalayanarooj, & Ubol, 2010Taylor et al., 2015). But TLRs express upstream events and how SARM and TANK are activated is not entirely clear. These events lead to inhibition of innate responses mediated through the TLR signaling pathway and increased viral production. Both MyD88-dependent and non-dependent signaling molecules are downregulated during DENV-ADE infection (Kulkarni, 2020Modhiran et al., 2010).

The connection between FcγRIIa and immune complex induced the activation of PI3K/PKB mediated by spleen tyrosine kinase (Syk). Activation of PI3K/PKB phosphorylates and inactivates glycogen synthase kinase (GSK)-3b, resulting in the observed high levels of IL-10. IL-10 can induce the cytokine response of Th2 and inhibit the production of proinflammatory cytokines, such as IFN-γ, IL-12, TNF-α and nitric oxide free radicals (Kuczera, Assolini, Tomiotto-Pellissier, Pavanelli, & Silveira, 2018). PRRSV ADE can up-regulate the production of IL10 through FcγRI and FcγRIII to promote the replication of PRRSV (Zhang et al., 2020).

IL-10 can effectively activate the SOCS system and inhibit Janus kinase/signal transduction and transcriptional activator (JAK/STAT) signal transduction pathways (Sukathida Ubol et al., 2010). The secreted IL10 cytokine binds to the IL10 receptor 1(IL10R1) on the membrane surface, and IL10R1 dimerizes with IL10R2 to play its downstream role. IL10R2 recruited the cytoplasmic protein Jak1, Phosphorylated STAT3 forms homodimers that are then transferred to the nucleus to promote transcriptional regulation of the target genes, thereby inhibiting iNOS gene expression and the production of nitric oxide free radicals (Kulkarni, 2020Taylor et al., 2015). (As shown in Fig. 1 )

Fig 1

Fig. 1

Host innate immune responses during ADE. (i) Dengue fever immune complex can enter target cells through FcR to activate negative regulators, dihydroxyacetone kinase (DAK) and autophagy-related 5 autophagy-related 12(Atg5-Atg12), thereby destroying the RIG-I / MDA-5 signaling pathway. Including beta interferon promoter stimulator 1 (IPS-1), inducible I-kappa B predominate kinase (IKKi), tumor necrosis factor receptor-associated factor 3(TRAF-3), and TANK-binding kinase 1 (TBK-I), etc Eventually inhibit type I IFN production. (ii) The expression of TLR-3, -4, -7 and TLR signal molecules was down regulated when the immune complex entered the target cells, resulting in inhibition of NF-κB activity and negative regulation of TLR signal transduction activity. (iii) The binding of leukocyte immunoglobulin like receptor B1 (LILRB1) with DENV virus inhibited the activation of Syk and eliminated the expression of ISG. Activated Syk upregulates IL10 via PI 3K/PKB, CREB signaling pathwayIL-10 stimulates the expression of SOCS3 and inactivates Janus kinase JAK-STAT signal, thus inhibiting the production of proinflammatory cytokines.IL-10 also inhibits the expression of nitric oxide synthase (iNOS) by inhibiting the activities of STAT-1 and IRF-1, thus inhibiting the production of nitric oxide (NO).

NO inhibits late viral protein synthesis and DNA synthesis, for example viral cysteine proteases are inactivated by NO-dependent s-nitrosoylation, NO inhibits PRRSV infection by cGMP-PKG dependent signaling (Saura et al., 1999Zhang, Duan, Li, Zhao, & Xiao, 2016). The PBMC of DHF patients showed higher NS1 and lower NO serum level in acute fever period (Carvalho et al., 2014). The effect of NO is contradictory. NO is related to the severity of viral haemorrhagic fever. The increase of NO level in blood vessels will lead to the increase of vascular permeability and the damage of homeostasis in vivo. The higher concentration will affect the vascular tension and lead to the shock caused by virus (Thein et al., 2015). NO may play both protective and pathological roles, which depend on the concentration of NO.

The attachment of FcγRIIa to the DENV immune complex and the binding of LILRB1 to the DENV virions can synergistically induce ADE in THP-1 cells. (Cosman et al., 1997Kulkarni, 2020). After Binding to LILRB1 the immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1(SHP-1) to dephosphorylate Syk, which leads to the decrease of ISG expression and the enhancement of DENV replication (Chan et al., 2014Kulkarni, 2020).

In theory, DENV immune complex can use the inhibitory ITIM signal of FcγRIIb to destroy the expression of ISG. PoFcγRIIb mediated ADE pathway of PRRSV infection through recruitment of ship-1. The TBK-1-IRF-3-IFN-β signal transduction pathway was further inhibited to enhance PRRSV infection (Wan et al., 2019). In addition, the ability of poFcγRII to mediate PRRSV ADE can be eliminated by interrupting the tyrosine residues in ITIM or removing the cytoplasmic domain of Fc. The connection between immune complex and FcγRIIb can also weaken the activation of NF-κB triggered by TLR4. But ligation of FcγRIIb inhibits antibody-dependent enhancement of dengue virus infection. Antibodies help to form large viral aggregates, which in turn bind to FcγRIIb to inhibit the phagocytosis of monocytes (Chan et al., 2011). Whether there is ADE phenomenon may be related to the cell type and the concentration of antibodies. In cells with FcR, the production of cytokines mediated by FcγR depends on the proportion of activated and inhibited FcγR. Therefore, the simultaneous stimulation of activated and inhibited FcγR may not induce significant cytokine production.

Antibody-dependent DENV entry upregulates a host of host-dependent genes that support DENV infection. Chan found that some host-dependent factors were induced in primary monocytes under ADE conditions. ADE increases the spliceosome gene, so ADE may modify the splicing map of infected cells to produce an intracellular environment that is more conducive to virus replication and packaging. The higher expression of ribosome genes in ADE may explain the increased virus translation compared with DENV infection alone (Chan et al., 2019). In addition, it also includes DENV RNA binding protein, which requires for effective DENV amplification (Chan et al., 2019Viktorovskaya, Greco, Cristea, & Thompson, 2016); ER-Golgi transport protein, which is necessary for viral transport; mitochondrial complex, which is required for ATP synthesis (Chan et al., 2019Savidis et al., 2016).

3.4. Cellular compartmentalization

Cellular compartmentalization is used as a defense against human pathogens in both specialized and nonspecialized phagocytes. The septal boundary limits the entry of pathogens into the cytoplasm, and the expression of pattern recognition receptors is helpful for the detection and elimination of pathogens (Randow, Macmicking, & James, 2013).

In order to cope with the virus-resistant response triggered by the early activation of Fc, during ADE, the antibody-opsonized DENV was co-connected with the LILRB1 with ITIM to change the cellular compartment. Reduced Syk signal transduction not only leads to ISG-induced inhibition, but also downregulates the acidification of phagocytosis, enabling DENV to escape lysosomal degradation. SHP-1 is thought to inhibit phagocytic acidification by acting on membrane fusion during phagocytic transport. LILRB1 signal directly introduced the phagosome containing DENV into the area with lower acidification degree, thus preventing the rapid lysosomal degradation of DENV.

Similarly, inhibition of phagocytic acidification by lysosomal drugs has led to an increase in antibody-dependent infections, suggesting caution in the use of such drugs in the treatment of dengue (Jaume et al., 2011). Although the decrease or slowing down of phagocytic acidification during ADE may delay virus fusion and membrane removal, this may be a necessary trade-off, ultimately allowing DENV to escape lysosomal degradation, which is conducive to intracellular survival. During the ADE process of ACE2 mediated SARS-CoV, most of the virus particles that enter the cells through the ADE pathway are still trapped in the acid compartment and finally degraded. The inhibition of internal acidification can prevent them from degradation (Jaume et al., 2011Ong et al., 2017).

The differential intracellular rate of fusion or compartmentalization could lead to exposure of the virus to a different repertoire of vesicular receptors, including pathogen recognition receptors, resulting in differential cellular responses (Chan et al., 2019). The differentiation compartmentalization of DENV may affect receptor interaction, viral replication and host response, which bring new research direction for the pathogenesis of virus.

4. Development of vaccine to eliminate ADE

Vaccine induced ADE has been found for a long time. Antibodies with poor affinity induced by formalin inactivated virus vaccines (e.g., RSV and measles) with Al adjuvant induced ADE during the initial infection of infants (Anderson et al., 2013). The development of vaccine to eliminate ADE mainly from two aspects: one is to mask or remove the antigen part that produces ADE, so that the antibody produced does not produce ADE effect; the other is to block the combination of antigen antibody complex and receptor, so as to inhibit Fc receptor or other receptor-mediated ADE.

4.1. Mask or remove the antigen part that produces ADE

Screaton developed a stable ZIKV E protein dimer vaccine that has no precursor membrane proteins and does not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection (Slon-Campos et al., 2019).

The cross reactive antibody of DENV and Zika virus is mainly induced by the DI/ DII region of E protein, especially the highly conserved fusionloop(fl)of DII region, which promotes DENV or ZIKV to enhance infection (Dejnirattisai et al., 2016Priyamvada et al., 2016Screaton, Mongkolsapaya, Yacoub, & Roberts, 2015). Lin showed that the enhanced infection of ij loop mutant of ZIKV E antigen in all four serotypes of DENV was greatly reduced, which indicated that ij loop mutant was a good target of glycan screening strategy, which could shield cross reactive antibody to induce ADE effect against DENV, and also retained the virus neutralization ability. Using ij cyclo-glycan to mask adenovirus vector, and then using DIII of E gene and FLIC of bacteria to express flic-diii fusion protein to enhance immunity, the results showed that the enhanced immunity by tetravalent DENV FLIC-DIII could improve the titer of neutralizing antibody and reduce ADE (Lin et al., 2019).

ZIKV-80E(N-terminal 80% of ZIKV E protein) expressed in yeast can self-assemble into nanoparticles. Nanoparticles without prM retain the antigenic integrity of neutralizing epitopes on E domain III (EDIII). Immunization of BALB/c mice can produce The ZIKV neutralizing antibody of this domain. The most exciting thing is that the antibodies induced by ZIKV-80E NPs did not induce the ADE potential of DENV and ZIKV in vivo (Shukla et al., 2020).

The use of reverse genetics to construct chimeric JEVpr/DENV2 with the Japanese encephalitis virus pr instead of the pr gene showed reduced virulence and good immunogenicity. In addition, anti-JEVpr/DENV2 sera showed extensive cross-reactivity and effective neutralizing activity against all four DENV serotypes and immature DENV2 (ImDENV2), and the ADEV activity of DENV decreased (Wang et al., 2015). Wang replaced DENV pr with JEV pr to construct a novel chimeric protein of JEV pr and DENV2 M peptide (JEVpr/DENV-M). Rabbit anti JEVpr/DENV-m had weak neutralization to both serotype DENV and immature DENV (ImDENV2), which did not change the neutralization ability of anti prM antibody, and did not cause the enhancement of infection of K562 cells with Fc receptor (Wang, Si, et al., 2017).

4.2. Block or interfere with the binding of the antigen-antibody complex to the receptor

Because of its own advantages that monoclonal antibody has become an important direction in the development of new vaccines. In particular, nano antibody does not contain Fc segment of common antibody, thus avoiding complement reaction caused by Fc domains and reducing ADE production. Palivizumab, an antiviral monoclonal antibody against respiratory syncytial virus (RSV) F protein, has significantly reduced RSV hospitalizations in premature infants and infants with chronic lung disease or congenital heart disease. Palizumab is clinically safe that fully demonstrates the development prospect of monoclonal antibody vaccine.

Wan analyzed the difference of ADE of coronavirus in different antibody doses, MERS MAb1 can inhibit virus entry at low concentration (by blocking DPP4 dependent entry pathway), promote virus entry at medium concentration (by enhancing CD32a dependent entry pathway), and inhibit virus entry at high concentration (by blocking DPP4 and CD32a dependent entry pathway), which can also explain why virus ADE only exists at some concentrations, and neither too high nor too low can induce virus entry ADE (Wan et al., 2020).

Widjaja identified eight monoclonal antibodies that bind to non-overlapping epitopes on MERS-S with high affinity and interfere with the three known functions of viral proteins: sialic acid binding, receptor binding, and membrane fusion (Widjaja et al., 2019). At low doses, these antibodies protected mice from the deadly MERS-CoV infection. It is worth noting that there are two mAbs (G2 and G4) do not interfere with receptor binding, but abolish intercellular fusion, which means that they can interfere with spike mediated membrane fusion by blocking the conformational changes required for fusion. The high protective mAb showed only moderate neutralization activity in vitro, indicating that strong neutralization activity was not a prerequisite for protection. This provides us with a huge hint to develop a vaccine to eliminate ADE.

The development of new methods to deliver cross-reactive, neutralizing monoclonal antibodies to the circulatory system could provide rapid, comprehensive and inexpensive protection against related diseases. Flingai using monoclonal antibody DNA encoding antibody (DMAb) encodes for an Fc region-modified with abrogated FcγR binding by way of two leucine-to-alanine (LALA) mutations in the CH2 region and eliminate antibody-dependent enhancement of DENV(Beltramello et al., 2010Flingai et al., 2015). By delivering multiple DENV DMAb plasmids, this increases human IgG levels and the number of targeted serotypes. Injecting the DNA that encodes the antibody produces biologically relevant levels of monoclonal antibodies. The protection provided by DMAb is significantly faster than vaccination protection that takes weeks or months to reach peak effect. Similarly, Elliott uses DMAb technology to encode two monoclonal antibodies with broad cross-protection against influenza A and B (Elliott et al., 2017). It can provide mice with extensive protection against influenza A and B infections.

Zhang made specific antigen peptides, linkers and Fc-III mimetic peptides into Dual-functional Conjugate of Antigenic peptide and Fc-III tag(DCAF) (Zhang et al., 2019). The Fc-III part inhibited the dengue fever ADE process to a certain extent and blocked the antibodies-Virus or antibody-Fc receptor interaction. At the cell level in vitro, DCAF successfully inhibited the ADE effect of dengue fever type 2, indicating that reducing the affinity of Fc region binding to Fc receptor can weaken ADE.

Wang produced anti-idiotypic antibodies against prM antibodies that can cause ADE, and found that they can prevent ADE not only in vitro but also in vivo (Wang et al., 2017). After mice immunized with prM mAb-specific anti-idiotypic antibodies (prM-AID) were infected with DENV-1, interleukin 10 (IL-10) and alanine aminotransferase (ALT) were as low as the negative control level, and the number of platelets increased significantly compared with the control group. It suggesting that anti-idiotypic antibodies may be a new option for treating ADE caused by DENV infection.

Influenza M2 protein extracellular functional area (M2e) has a high degree of conservation which can provide a wide range of protective effects. Antibodies to the influenza M2e protein are well protected by blocking viral ion channel function and mediating ADCC or ADPC action to clear infected cells. Vlieger prepared VHH antibodies against M2e and mouse FcRIV (De Vlieger et al., 2019). Specific antibodies that target only FcRIV may be an advantage because they avoid inhibiting FcRIIb receptor activation in this way. Intranasally administered bi-specific VHH fusion antibodies selectively bind to M2e on infected target cells and activate FcRIV to resist influenza A virus attack. Since antibodies against M2e do not work by neutralizing the virus, this greatly reduces the possibility of ADE. However, the antibody effect of M2e immunization alone is limited, and the development may need to cooperate with neutralization effect for HA to provide a higher protective effect (Wei et al., 2020).

FcγR is highly sensitive to N-linked glycosylation mode of Fc region. Therefore, unique plant N-glycans may affect the characteristics of mAbs produced, including ADE activity (Sun, Chen, & Lai, 2017). E60 (mE60) produced by mammalian cells showed ADE in vivo and in vitro when DENV was infected (Balsitis et al., 2010). E60 produced in plants showed a single predominant expected N-glycoform type with high homogeneity. These E60 glycovariants maintain specific binding to EDII antigen with a kinetics similar to mE60, and showing neutralization activity against a variety of DENV serotypes. Most importantly, E60 produced in plants forwent ADE activity in K562 cells expressing FcγR (Matthew et al., 2016).

5. Effect of adjuvant and inactivation selection on ADE

The choice of adjuvant will also affect the immune effect. The carbonyl group on the vaccine antigen treated with formaldehyde can enhance the T-helper 2 (Th 2) reaction and enhance the respiratory syncytial virus (RSV) disease in mice, which can be partially reversed by the chemical reduction of the carbonyl group (Delgado et al., 2009Moghaddam et al., 2006Ubol & Halstead, 2010).

However, inactivation of RSV by methods other than formalin also makes experimental animals more sensitive to disease. RSV-immunized mice treated with UV-irradiated inactivated purified fusion protein or vaccinia virus RSV replicas developed enhanced diseases after being challenged by wild-type viruses (Delgado et al., 2009Olszewska, Suezer, Sutter, & Openshaw, 2004). Inactivated vaccines have insufficient TLR activation, and adding TLR agonists can prevent ERD. Speculate that this may be related to adjuvant.

Boelen found that unimmunized mice infected with RSV showed a Th1 cell response with mild symptoms; but FI-RSV (formalin inactivated antigen with AL adjuvant added) immunization resulted in aggravated lung histopathology and showed obvious Th2 cellular immune response, although the vaccine is still partially protective, can induce low titer neutralizing antibody response, and virus replication is reduced; Fi-mock (Formalin Inactivated Cell Antigen Negative Control) immunized mice showed increased RSV replication, also manifested by a significant Th2 cell immune response (Boelen et al., 2000). This shows that the Th2 cell immune response induced by the formalin-inactivated vaccine with Al adjuvant seems to be conducive to RSV replication. The combination of Montanide ISA-51 and CpG ODN (oligodeoxynucleotide) has been used in the development of coronavirus vaccine, which can synergistically enhance Th1 immune response (Gupta & Gupta, 2020). Immunization of mice and cynomolgus monkeys with Montanide ISA-51 and CpG emulsified SARS-CoV recombinant N protein can cause a strong Th1 immune response (Liu et al., 2006).

6. Conclusions and future prospects

ADE has been proven in vitro and in animal models for a variety of viruses including DENV, HIV, RSV, SARS-CoV and WNV (Table 1 ).

Table 1

Key characteristics of antibody-dependent enhancement (ADE) infected by different viruses.

VirusMain HostTypes of ADEClinical manifestations of ADEEnhancing epitopes locationImpact of vaccine application
Dengue virusHumans,Primate,Aedes1.FcR mediated virus-antibody immune complexes infect monocytes, macrophages, and dendritic cells
2.Through FcR, LILR-B1 regulates the host’s antiviral response, inhibits the innate response mediated by the TLR signaling pathway, disrupts the RIG-I/MDA-5 signal cascade, and induces IL-10 production
1. Increased susceptibility to other serotypes
2. Increased viral infection and association with severe dengue fever (DHF/DSS)
3. Infants born to dengue fever-immunized mothers, serious diseases that may be infected when maternal antibodies are reduced
prM protein, E protein DII-FL regionVaccine raises the risk of ADE for DENV infection.Sero-negative dengue vaccinators are at increased risk of severe dengue, and WHO recommends vaccination only for sero-positive dengue
SRAS-CoVRhinolophus sinicus,Paguma larvatas,HumansFcR-ADE(mainly mediated by FcγRII)May be related to severe lymphopeniaSpike proteinInfection after immunization may cause severe acute lung injury (ALI)
Influenza virusHumans,Pigs,Birds, FerretsFcR-ADEIncreased risk of a (H1N1) pdm09 diseaseHA,NA1.Trivalent inactivated influenza vaccine (TIV) in 2008-09 increased risk of a (H1N1) pdm09 disease
2. The vaccine may be associated with vaccine associated enhanced respiratory disease (VAERD)
Porcine Reproductive and Respiratory SyndromePigsFcR-ADE (Including FcγRI,FcγRII,FcγRIII, FCɛRI)Promote virus infection and enhance clinical symptoms, Increase the level and duration of viremiaGP5,N ProteinInfected with prrsv after immunization with inactivated vaccine, clinical symptoms increased
Human immunodeficiency virusHumans1.FcR-ADE (Including FcγRI,FcγRII,FcγRIII,FCαR) , FcR promotes virus entry by enhancing adhesion to CD4 receptor
2.CR3, C1q complement mediated ADE
1.ADE and plasma viral load is positive correlation. ADE accelerates immunosuppression and disease progression
2.Enhancing antibodies is beneficial to the emergence of ADE susceptible mutants
N-terminal immune dominant domain of gp41,gp120One of the factors affecting vaccine development,higher rates of infection/risk among vaccinees were observed in RV144 clinical trials, but have not been confirmed to be directly associated with ADE
West nile virusHorses,Humans,Birds1.Fcγ receptor dependent ADE
2.CR3 dependent ADE
Increased viral infectivityDomain I and domain II of the E proteinNo vaccine has been marketed. Plasma samples of human WNV infection during rehabilitation can enhance ZIKV infection in vitro and in vivo
Respiratory syncytial virusHumansFcR mediate the uptake of viruses into monocytes, macrophages, and dendritic cells, leading to enhanced infectionADE infection leads to activation of Th2 response and increased expression of TNF-α and IL-6, resulting in aggravated disease
ADE infection of lung dendritic cells (DCs) can negatively regulate the function of DC cells, resulting in impaired T cell activation
Glycoproteins G and FFormalin inactivated RSV vaccine recipients have increased disease and even led to death

Open in a separate window

The essence of ADE effect is a part of immune regulation. In the next step, we need to clarify the mechanism of ADE and clarify the conditions of ADE occurrence, find out factors that regulate Syk activation and inhibition, the balance between antiviral and inflammatory responses, and upregulation of immunosuppressive IL-10, so as to better block the occurrence of ADE from the perspective of iADE. This will provide ideas and directions for the development of new drugs for infectious diseases such as HIV, DENV, influenza, and even SARS CoV2.

Influenza, SARS-CoV, and PRRSV vaccines all have the potential to cause ADE, or to cause vaccine-related enhanced respiratory disease (VAERD). Although some cannot be directly attributed to ADE, it cannot be denied that ADE is very likely to be one of the potential factors. From the experience of SARS-CoV vaccine development, the development of COIVD-19 vaccines should pay attention to ADE. In the future, it is necessary to identify the antigen epitopes of ADE induced by viruses, effectively avoid them in vaccine design, and further discover effective virus neutralizing epitopes and other non-neutralizing but protective epitopes, which will guide the future design of protective vaccines, therapeutic monoclonal antibodies and nano antibodies.

Activation of the immune system increases the number of activated CD4 (+) T cells, which are more susceptible to infection than inactive T cells (Bukh et al., 2014). Human CMV infection may lead to increased susceptibility to HIV-1 infection. HCMV can preventing antiviral IFN-stimulated gene production by degradation of JAK1, interfering with STAT signaling (Johnson et al., 2018Miller & Boss, 1998Paulus, Krauss, & Nevels, 2006). This is similar to the partially suppressed signal produced by ADE. For the outbreak of COVID-19 worldwide, it is speculated that those individuals with severe illness may have been exposed to one or more previous coronaviruses and are experiencing antibody-dependent enhancement (Tetro, 2020). iADE suppresses the function of the immune system and promotes viral infection or disease. Future research should pay attention to this aspect, that is, whether it will cause the increase of heterologous disease infection when in iADE.

In the experience of RSV vaccine design, especially in the selection of adjuvants in recombinant subunit vaccines, Th1 biased adjuvants should be selected as much as possible, such as poly(I:C) (TLR3),AS04 (TLR4), imiquimod (TLR7/8 ), CpG motifs (TLR9), monophosphoryl lipid A (MPL), and avoid the selection of Th2 biased adjuvants, such as Al adjuvant (Apostolico Jde, Lunardelli, Coirada, Boscardin, & Rosa, 2016Gupta & Gupta, 2020Lindblad, 2004). In the future, the preparation of vaccines also needs to develop new safe and efficient adjuvants to avoid the generation of ADE. Kabir proves that ADE can greatly increase the incidence of new diseases. For multiple serotype infectious diseases such as dengue fever, joint vaccine is more effective. The primary vaccine is a better control tool than the secondary vaccine. In order to suppress secondary infections, secondary vaccinations require high efficiency (Kabir & Tanimoto, 2020). This may be because secondary infections are located further downstream in the dynamic process of infection and require a greater effect to suppress the spread of secondary infections. More rigorous, reasonable and safe immune procedures are also important issues to be solved in future vaccine development.

Acknowledgments

Support for this work was received from the Science Fundation for Distinguished Young Scholars of Shaanxi Province (2021JC-18), the Open Project of the State Key Laboratory of Veterinary Etiological Biology (SKLVEB2020KFKT017), the Youth Innovation Team of Shaanxi Universities, the Science and Technology Extension Project in Northwest A&F University (TGZX2020-24) the Fundamental Research Funds for the Central Universities (2452021154).Go to:

References

  • Anderson L.J., Dormitzer P.R., Nokes D.J., Rappuoli R., Roca A., Graham B.S. Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine. 2013;31(Suppl 2):B209–B215. doi: 10.1016/j.vaccine.2012.11.106. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Apostolico Jde S., Lunardelli V.A., Coirada F.C., Boscardin S.B., Rosa D.S. Adjuvants: Classification, modus operandi, and licensing. Journal of Immunology Research. 2016;2016:1459394. doi: 10.1155/2016/1459394. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Arvin A.M., Fink K., Schmid M.A., Cathcart A., Spreafico R., Havenar-Daughton C. A perspective on potential antibody-dependent enhancement of SARS-CoV-2. Nature. 2020;584(7821):353–363. doi: 10.1038/s41586-020-2538-8. [PubMed] [CrossRef] [Google Scholar]
  • Arvin A.M., Fink K., Schmid M.A., Cathcart A., Virgin H.W. A perspective on potential antibody-dependent enhancement of SARS-CoV-2. Nature. 2020:1–14. [PubMed] [Google Scholar]
  • Balsitis S.J., Williams K.L., Lachica R., Flores D., Kyle J.L., Mehlhop E. Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. PLoS Pathogens. 2010;6(2) doi: 10.1371/journal.ppat.1000790. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Beltramello M., Williams K.L., Simmons C.P., Macagno A., Simonelli L., Quyen N.T. The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host & Microbe. 2010;8(3):271–283. doi: 10.1016/j.chom.2010.08.007. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Boelen A., Andeweg A., Kwakkel J., Lokhorst W., Bestebroer T., Dormans J. Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice. Vaccine. 2000;19(7):982–991. [PubMed] [Google Scholar]
  • Bukh I., Calcedo R., Roy S., Carnathan D.G., Grant R., Qin Q. Increased mucosal CD4 + T cell activation in rhesus macaques following vaccination with an adenoviral vector. Journal of Virology. 2014;88(15):8468–8478. doi: 10.1128/jvi.03850-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Byrne A.B., Talarico L.B. Role of the complement system in antibody-dependent enhancement of flavivirus infections. International Journal of Infectious Diseases. 2021;103:404–411. [PubMed] [Google Scholar]
  • Cancel-Tirado S.M., Evans R.B., Yoon K.J. Monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection. Veterinary Immunology and Immunopathology. 2004;102(3):249–262. doi: 10.1016/j.vetimm.2004.09.017. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Carro A.C., Piccini L.E., Damonte E.B. Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies. PLoS Neglected Tropical Diseases. 2018;12(8) doi: 10.1371/journal.pntd.0006685. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Carvalho D.M., Garcia F.G., Terra A.P., Lopes Tosta A.C., Silva Lde A., Castellano L.R. Elevated dengue virus nonstructural protein 1 serum levels and altered toll-like receptor 4 expression, nitric oxide, and tumor necrosis factor alpha production in dengue hemorrhagic Fever patients. Journal of Tropical Medicine. 2014;2014:901276. doi: 10.1155/2014/901276. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Chan C.Y.Y., Low J.Z.H., Gan E.S., Ong E.Z., Zhang S.L., Tan H.C. Antibody-dependent dengue virus entry modulates cell intrinsic responses for enhanced infection. mSphere. 2019;4(5) doi: 10.1128/mSphere.00528-19. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Chan K.R., Ong E.Z., Tan H.C., Zhang S.L., Zhang Q., Tang K.F. Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue. Proceedings of the National Academy of Sciences of the United States of America. 2014;111(7):2722–2727. doi: 10.1073/pnas.1317454111. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Chan K.R., Zhang S.L.-X., Tan H.C., Chan Y.K., Chow A., Lim A.P.C. Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection. Proceedings of the National Academy of Sciences. 2011 [PMC free article] [PubMed] [Google Scholar]
  • Chareonsirisuthigul T., Kalayanarooj S., Ubol S. Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells. The Journal of General Virology. 2007;88(Pt 2):365–375. doi: 10.1099/vir.0.82537-0. [PubMed] [CrossRef] [Google Scholar]
  • Chau T.N., Hieu N.T., Anders K.L., Wolbers M., Lien le B., Hieu L.T. Dengue virus infections and maternal antibody decay in a prospective birth cohort study of Vietnamese infants. The Journal of Infectious Diseases. 2009;200(12):1893–1900. doi: 10.1086/648407. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Chau T.N., Quyen N.T., Thuy T.T., Tuan N.M., Hoang D.M., Dung N.T. Dengue in Vietnamese infants—results of infection-enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity. The Journal of Infectious Diseases. 2008;198(4):516–524. doi: 10.1086/590117. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Chen Z., Zhang L., Qin C., Ba L., Yi C.E., Zhang F. Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region. Journal of Virology. 2005;79(5):2678–2688. doi: 10.1128/JVI.79.5.2678-2688.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Cheung C.Y., Poon L.L., Ng I.H., Luk W., Sia S.F., Wu M.H. Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: Possible relevance to pathogenesis. Journal of Virology. 2005;79(12):7819–7826. doi: 10.1128/JVI.79.12.7819-7826.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Coish J.M., MacNeil A.J. Out of the frying pan and into the fire? Due diligence warranted for ADE in COVID-19. Microbes and Infection. 2020 doi: 10.1016/j.micinf.2020.06.006. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Cosman D., Fanger N., Borges L., Kubin M., Chin W., Peterson L. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity. 1997;7(2):273. [PubMed] [Google Scholar]
  • Cui G., Si L., Wang Y., Zhou J., Yan H., Jiang L. Antibody-dependent enhancement (ADE) of dengue virus: Identification of the key amino acid that is vital in DENV vaccine research. The Journal of Gene Medicine. 2021;23(2) [PMC free article] [PubMed] [Google Scholar]
  • de Taeye S.W., Rispens T., Vidarsson G. The ligands for human IgG and their effector functions. Antibodies (Basel) 2019;8(2) doi: 10.3390/antib8020030. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • De Vlieger D., Hoffmann K., Van Molle I., Nerinckx W., Van Hoecke L., Ballegeer M. Selective engagement of FcgammaRIV by a M2e-specific single domain antibody construct protects against influenza A virus infection. Frontiers in Immunology. 2019;10:2920. doi: 10.3389/fimmu.2019.02920. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Dejnirattisai W., Supasa P., Wongwiwat W., Rouvinski A., Barba-Spaeth G., Duangchinda T. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nature Immunology. 2016;17(9):1102–1108. doi: 10.1038/ni.3515. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Delgado M.F., Coviello S., Monsalvo A.C., Melendi G.A., Hernandez J.Z., Batalle J.P. Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease. Nature Medicine. 2009;15(1):34–41. doi: 10.1038/nm.1894. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Elliott S.T.C., Kallewaard N.L., Benjamin E., Wachter-Rosati L., McAuliffe J.M., Patel A. DMAb inoculation of synthetic cross reactive antibodies protects against lethal influenza A and B infections. NPJ Vaccines. 2017;2:18. doi: 10.1038/s41541-017-0020-x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Flingai S., Plummer E.M., Patel A., Shresta S., Mendoza J.M., Broderick K.E. Protection against dengue disease by synthetic nucleic acid antibody prophylaxis/immunotherapy. Scientific Reports. 2015;5:12616. doi: 10.1038/srep12616. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Furuyama W., Marzi A., Carmody A.B., Maruyama J., Kuroda M., Miyamoto H. Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection. PLoS Pathogens. 2016;12(12) doi: 10.1371/journal.ppat.1006139. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Furuyama W., Nanbo A., Maruyama J., Marzi A., Takada A. A complement component C1q-mediated mechanism of antibody-dependent enhancement of Ebola virus infection. PLoS Neglected Tropical Diseases. 2020;14(9) doi: 10.1371/journal.pntd.0008602. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Gao Q., Bao L., Mao H., Wang L., Xu K., Yang M. Development of an inactivated vaccine candidate for SARS-CoV-2. Science. 2020;369(6499):77–81. doi: 10.1126/science.abc1932. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Garg H., Yeh R., Watts D.M., Mehmetoglu-Gurbuz T., Resendes R., Parsons B. Enhancement of Zika virus infection by antibodies from West Nile virus seropositive individuals with no history of clinical infection. BMC Immunology. 2021;22(1):5. doi: 10.1186/s12865-020-00389-2. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Goncalvez A.P., Engle R.E., Claire M.S., Purcell R.H., Lai C.-J. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proceedings. National Academy of Sciences. United States of America. 2007;104(22):9422–9427. [PMC free article] [PubMed] [Google Scholar]
  • Gordon M.J.C.J.S.P.S. Complement receptor mediates enhanced flavivirus replication in macrophages. Journal of Experimental Medicine. 1983;158(1):258–263. [PMC free article] [PubMed] [Google Scholar]
  • Gu W., Guo L., Yu H., Niu J., Huang M., Luo X. Involvement of CD16 in antibody-dependent enhancement of porcine reproductive and respiratory syndrome virus infection. The Journal of General Virology. 2015;96(Pt 7):1712–1722. doi: 10.1099/vir.0.000118. [PubMed] [CrossRef] [Google Scholar]
  • Guilliams M., Bruhns P., Saeys Y., Hammad H., Lambrecht B.N. The function of Fcgamma receptors in dendritic cells and macrophages. Nature Reviews. Immunology. 2014;14(2):94–108. doi: 10.1038/nri3582. [PubMed] [CrossRef] [Google Scholar]
  • Gupta T., Gupta S.K. Potential adjuvants for the development of a SARS-CoV-2 vaccine based on experimental results from similar coronaviruses. International Immunopharmacology. 2020;86:106717. doi: 10.1016/j.intimp.2020.106717. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Halstead S.B., O’Rourke E.J., Allison A.C. Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. The Journal of Experimental Medicine. 1977;146:218–229. [PMC free article] [PubMed] [Google Scholar]
  • Halstead S.B., Chow J.S., Marchette N.J. Immunological enhancement of dengue virus replication. Nature: New Biology. 1973;243(122):24–26. [PubMed] [Google Scholar]
  • Halstead S.B., Mahalingam S., Marovich M.A., Ubol S., Mosser D.M. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: Disease regulation by immune complexes. Lancet Infectious Diseases. 2010;10(10):712–722. [PMC free article] [PubMed] [Google Scholar]
  • Hanson Q.M., Barb A.W. A perspective on the structure and receptor binding properties of immunoglobulin G Fc. Biochemistry. 2015;54(19):2931–2942. doi: 10.1021/acs.biochem.5b00299. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Heinz F.X., Stiasny K. Flaviviruses and their antigenic structure. Journal of Clinical Virology the Official Publication of the Pan American Society for Clinical Virology. 2012;55(4):289–295. [PubMed] [Google Scholar]
  • Hermanns K., Gohner C., Kopp A., Schmidt A., Merz W.M., Markert U.R. Zika virus infection in human placental tissue explants is enhanced in the presence of dengue virus antibodies in-vitro. Emerging Microbes & Infections. 2018;7(1):198. doi: 10.1038/s41426-018-0199-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Ho M.-S., Chen W.-J., Chen H.-Y., Lin S.-F., Wang M.-C., Di J. Neutralizing antibody response and SARS severity. Emerging Infectious Diseases. 2005;11(11):1730–1737. [PMC free article] [PubMed] [Google Scholar]
  • Izmirly A.M., Alturki S.O., Alturki S.O., Connors J., Haddad E.K. Challenges in dengue vaccines development: Pre-existing infections and cross-reactivity. Frontiers in Immunology. 2020;11 doi: 10.3389/fimmu.2020.01055. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Janoff E.N., Wahl S.M., Kelly T., Smith P.D. Modulation of human immunodeficiency virus type 1 infection of human monocytes by IgA. Journal of Infectious Diseases. 1995;3:3. [PubMed] [Google Scholar]
  • Jaume M., Yip M.S., Cheung C.Y., Leung H.L., Li P.H., Kien F. Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcgammaR pathway. Journal of Virology. 2011;85(20):10582–10597. doi: 10.1128/JVI.00671-11. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Johnson L., Johnson E.L., Boggavarapu S., Johnson E.S., Lal A.A., Agrawal P. Human cytomegalovirus enhances placental susceptibility and replication of human immunodeficiency virus type 1 (HIV-1), which may facilitate in utero HIV-1 transmission. The Journal of Infectious Diseases. 2018 [PMC free article] [PubMed] [Google Scholar]
  • Kabir K.M.A., Tanimoto J. Cost-efficiency analysis of voluntary vaccination against n-serovar diseases using antibody-dependent enhancement: A game approach. Journal of Theoretical Biology. 2020;503:110379. doi: 10.1016/j.jtbi.2020.110379. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Karl-Klaus C., Nico V.…and Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus, a Member of the Arterivirus Group. Virology. 1993 [PMC free article] [PubMed] [Google Scholar]
  • Kliks S.C., Nimmanitya S., Nisalak A., Burke D.S. Evidence that maternal antibodies are important in the development of dengue hemorrhagic fever in infants. The American Journal of Tropical Medicine and Hygiene. 1988;38(2):411–419. [PubMed] [Google Scholar]
  • Kuczera D., Assolini J.P., Tomiotto-Pellissier F., Pavanelli W.R., Silveira G.F. Highlights for Dengue Immunopathogenesis: Antibody-Dependent Enhancement, Cytokine Storm, and Beyond. Journal of Interferon & Cytokine Research. 2018;38(2):69–80. [PubMed] [Google Scholar]
  • Kulkarni R. Dynamics of Immune Activation in Viral Diseases. 2020. Antibody-Dependent Enhancement of Viral Infections; pp. 9–41. [Google Scholar]
  • Lafferty R.A.H.A.K.J. The Enhancement of Virus Infectivity by Antibody. Virology. 1967;33:250–261. [PubMed] [Google Scholar]
  • Langerak T., Mumtaz N., Tolk V.I., van Gorp E.C.M., Martina B.E., Rockx B. The possible role of cross-reactive dengue virus antibodies in Zika virus pathogenesis. PLoS Pathogens. 2019;15(4) doi: 10.1371/journal.ppat.1007640. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Li D., Edwards R.J., Manne K., Martinez D.R., Saunders K.O. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies. Cell. 2021 [PMC free article] [PubMed] [Google Scholar]
  • Li Y., Xu G., Du X., Xu L., Ma Z., Li Z. Genomic characteristics and pathogenicity of a new recombinant strain of porcine reproductive and respiratory syndrome virus. Archives of Virology. 2021;166(2):389–402. doi: 10.1007/s00705-020-04917-8. [PubMed] [CrossRef] [Google Scholar]
  • Lin H.H., Yang S.P., Tsai M.J., Lin G.C., Wu H.C., Wu S.C. Dengue and Zika Virus Domain III-Flagellin Fusion and Glycan-Masking E Antigen for Prime-Boost Immunization. Theranostics. 2019;9(16):4811–4826. doi: 10.7150/thno.35919. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Lindblad E.B. Aluminium compounds for use in vaccines. Immunology and Cell Biology. 2004;82(5):497–505. doi: 10.1111/j.0818-9641.2004.01286.x. [PubMed] [CrossRef] [Google Scholar]
  • Littaua R.A., Kurane I., Ennis F.A. Human IgG Fc receptor II mediates antibody dependent enhancement of dengue virus infection. Journal of Immunology. 1990;144(8):3183–3186. [PubMed] [Google Scholar]
  • Liu S.J., Leng C.H., Lien S.P., Chi H.Y., Huang C.Y., Lin C.L. Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates. Vaccine. 2006;24(16):3100–3108. doi: 10.1016/j.vaccine.2006.01.058. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Liu Y., Soh W.T., Kishikawa J.I., Hirose M., Nakayama E.E., Li S. An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies. Cell. 2021;184(13) doi: 10.1016/j.cell.2021.05.032. 3452–3466.e3418. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Liu L., Wei Q., Lin Q., Fang J., Wang H., Kwok H. Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight. 2019;4(4) doi: 10.1172/jci.insight.123158. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Lok S.M. The Interplay of Dengue Virus Morphological Diversity and Human Antibodies. Trends in Microbiology. 2016;24(4):284–293. doi: 10.1016/j.tim.2015.12.004. [PubMed] [CrossRef] [Google Scholar]
  • Lv H., Wu N.C., Tsang O.T., Yuan M., Perera R., Leung W.S. Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections. bioRxiv. 2020 doi: 10.1101/2020.03.15.993097. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Matthew D., Jonathan H., Paul A.M., Sun H., Lai H., Ming Y. Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity. Journal of General Virology. 2016;97(12):3280–3290. [PMC free article] [PubMed] [Google Scholar]
  • Miller R., Boss Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway. Journal of Experimental Medicine. 1998 [PMC free article] [PubMed] [Google Scholar]
  • Modhiran N., Kalayanarooj S., Ubol S. Subversion of innate defenses by the interplay between DENV and pre-existing enhancing antibodies: TLRs signaling collapse. PLoS Neglected Tropical Diseases. 2010;4(12) doi: 10.1371/journal.pntd.0000924. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Moghaddam A., Olszewska W., Wang B., Tregoning J.S., Helson R., Sattentau Q.J. A potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines. Nature Medicine. 2006;12(8):905–907. doi: 10.1038/nm1456. [PubMed] [CrossRef] [Google Scholar]
  • Morens D.M., Larsen L.K., Halstead S.B. Study of the distribution of antibody-dependent enhancement determinants on dengue 2 isolates using dengue 2-derived monoclonal antibodies. Journal of Medical Virology. 1987;22(2):163–167. [PubMed] [Google Scholar]
  • Morrone S.R., Lok S.M. Structural perspectives of antibody-dependent enhancement of infection of dengue virus. Current Opinion in Virology. 2019;36:1–8. doi: 10.1016/j.coviro.2019.02.002. [PubMed] [CrossRef] [Google Scholar]
  • Nimmerjahn F., Bruhns P., Horiuchi K., Ravetch J.V. FcγRIV: A Novel FcR with Distinct IgG Subclass Specificity. Immunity. 2005;23(1):41–51. doi: 10.1016/j.immuni.2005.05.010. [PubMed] [CrossRef] [Google Scholar]
  • Oliveira R.A., de Oliveira-Filho E.F., Fernandes A.I., Brito C.A., Marques E.T., Tenorio M.C. Previous dengue or Zika virus exposure can drive to infection enhancement or neutralisation of other flaviviruses. Memórias do Instituto Oswaldo Cruz. 2019;114 doi: 10.1590/0074-02760190098. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Olszewska W., Suezer Y., Sutter G., Openshaw P.J. Protective and disease-enhancing immune responses induced by recombinant modified vaccinia Ankara (MVA) expressing respiratory syncytial virus proteins. Vaccine. 2004;23(2):215–221. doi: 10.1016/j.vaccine.2004.05.015. [PubMed] [CrossRef] [Google Scholar]
  • Ong E.Z., Zhang S.L., Tan H.C., Gan E.S., Chan K.R., Ooi E.E. Dengue virus compartmentalization during antibody-enhanced infection. Scientific Reports. 2017;7:40923. doi: 10.1038/srep40923. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Patro A.R.K., Mohanty S., Prusty B.K., Singh D.K., Gaikwad S., Saswat T. Cytokine signature associated with disease severity in dengue. Viruses. 2019;11(1) doi: 10.3390/v11010034. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Paul L.M., Carlin E.R., Jenkins M.M., Tan A.L., Barcellona C.M., Nicholson C.O. Dengue virus antibodies enhance Zika virus infection. Clinical & Translational Immunology. 2016;5(12) doi: 10.1038/cti.2016.72. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Paulus C., Krauss S., Nevels M. A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(10):3840–3845. doi: 10.1073/pnas.0600007103. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Priyamvada L., Quicke K.M., Hudson W.H., Onlamoon N., Sewatanon J., Edupuganti S. Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(28):7852–7857. doi: 10.1073/pnas.1607931113. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Prohászka Z., Nemes J., Hidvégi T., Tóth F.D., Kerekes K., Erdei A. Two parallel routes of the complement-mediated antibody-dependent enhancement of HIV-1 infection. AIDS. 1997;11(8):949–958. [PubMed] [Google Scholar]
  • Qiao S., Jiang Z., Tian X., Wang R., Xing G., Wan B. Porcine FcgammaRIIb mediates enhancement of porcine reproductive and respiratory syndrome virus (PRRSV) infection. PLoS One. 2011;6(12) doi: 10.1371/journal.pone.0028721. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Quinlan B.D., Mou H., Zhang L., Guo Y., He W., Ojha A. The SARS-CoV-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement. bioRxiv. 2020 doi: 10.1101/2020.04.10.036418. 2020.2004.2010.036418. [CrossRef] [Google Scholar]
  • Randow F., Macmicking J.D., James L.C. Cellular self-defense: How cell-autonomous immunity protects against pathogens. Science. 2013;340(6133):701–706. [PMC free article] [PubMed] [Google Scholar]
  • Rathore A.P.S., St John A.L. Cross-reactive immunity among flaviviruses. Frontiers in Immunology. 2020;11:334. doi: 10.3389/fimmu.2020.00334. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Robinson W.E., Montefiori D.C., Mitchell W.M. Complement-mediated antibody-dependent enhancement of HIV-1 infection requires CD4 and complement receptors. Virology. 1990;175(2):600–604. [PubMed] [Google Scholar]
  • Rodrigo W.W., Jin X., Blackley S.D., Rose R.C., Schlesinger J.J. Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32) Journal of Virology. 2006;80(20):10128–10138. doi: 10.1128/JVI.00792-06. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Saura M., Zaragoza C.A., Quick R., Hohenadl C., Lowenstein J., Lowenstein C. An antiviral mechanism of nitric oxide: Inhibition of a viral protease. Immunity. 1999;10(1):21–28. [PMC free article] [PubMed] [Google Scholar]
  • Sautter C.A., Trus I., Nauwynck H., Summerfield A. No evidence for a role for antibodies during vaccination-induced enhancement of porcine reproductive and respiratory syndrome. Viruses. 2019;11(9) doi: 10.3390/v11090829. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Savidis G., Mcdougall W.M., Meraner P., Perreira J.M., Portmann J.M., Trincucci G. Identification of zika virus and dengue virus dependency factors using functional genomics. Cell Reports. 2016;16(1):232–246. [PubMed] [Google Scholar]
  • Schlesinger J.J., Chapman S.E. Influence of the human high-affinity IgG receptor FcγRI (CD64) on residual infectivity of neutralized dengue virus. Virology. 1999;260(1):84–88. [PubMed] [Google Scholar]
  • Schweitzer B.K., Chapman N.M., Iwen P.C. Overview of the flaviviridae with an emphasis on the japanese encephalitis group viruses. Laboratory Medicine. 2009;40(8):493–499. doi: 10.1309/lm5yws85njpcwesw. [CrossRef] [Google Scholar]
  • Screaton G., Mongkolsapaya J., Yacoub S., Roberts C. New insights into the immunopathology and control of dengue virus infection. Nature Reviews. Immunology. 2015;15(12):745–759. doi: 10.1038/nri3916. [PubMed] [CrossRef] [Google Scholar]
  • Sharma A. It is too soon to attribute ADE to COVID-19. Microbes and Infection. 2020;22(4-5):158. doi: 10.1016/j.micinf.2020.03.005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Shi P., Su Y., Li Y., Zhang L., Lu D., Li R. The alternatively spliced porcine FcgammaRI regulated PRRSV-ADE infection and proinflammatory cytokine production. Developmental and Comparative Immunology. 2019;90:186–198. doi: 10.1016/j.dci.2018.09.019. [PubMed] [CrossRef] [Google Scholar]
  • Shi P., Zhang L., Wang J., Lu D., Li Y., Ren J. Porcine FcepsilonRI mediates porcine reproductive and respiratory syndrome virus multiplication and regulates the inflammatory reaction. Virologica Sinica. 2018;33(3):249–260. doi: 10.1007/s12250-018-0032-3. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Shukla R., Shanmugam R.K., Ramasamy V., Arora U., Batra G., Acklin J.A. Zika virus envelope nanoparticle antibodies protect mice without risk of disease enhancement. eBioMedicine. 2020;54:102738. doi: 10.1016/j.ebiom.2020.102738. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Slon-Campos J.L., Dejnirattisai W., Jagger B.W., Lopez-Camacho C., Wongwiwat W., Durnell L.A. A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection. Nature Immunology. 2019;20(10):1291–1298. doi: 10.1038/s41590-019-0477-z. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Sridhar S., Luedtke A., Langevin E., Zhu M., Bonaparte M., Machabert T. Effect of dengue serostatus on dengue vaccine safety and efficacy. The New England Journal of Medicine. 2018;379(4):327–340. doi: 10.1056/NEJMoa1800820. [PubMed] [CrossRef] [Google Scholar]
  • Sun H., Chen Q., Lai H. Development of antibody therapeutics against flaviviruses. International Journal of Molecular Sciences. 2017;19(1) doi: 10.3390/ijms19010054. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Takada A., Ebihara H., Feldmann H., Geisbert T.W., Kawaoka Y. Epitopes required for antibody-dependent enhancement of Ebola virus infection. The Journal of Infectious Diseases. 2007;196(Suppl 2):S347–S356. doi: 10.1086/520581. [PubMed] [CrossRef] [Google Scholar]
  • Takada A., Feldmann H., Ksiazek T.G., Kawaoka Y. Antibody-dependent enhancement of Ebola virus infection. Journal of Virology. 2003;77(13):7539–7544. doi: 10.1128/jvi.77.13.7539-7544.2003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Tang B., Xiao Y., Sander B., Kulkarni M.A., Radam-Lac Research T., Wu J. Modelling the impact of antibody-dependent enhancement on disease severity of Zika virus and dengue virus sequential and co-infection. Royal Society Open Science. 2020;7(4):191749. doi: 10.1098/rsos.191749. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Taylor A., Foo S.S., Bruzzone R., Vu Dinh L., King N.J.C., Mahalingam S. Fc receptors in antibody-dependent enhancement of viral infections. Immunological Reviews. 2015;268(1):340–364. [PMC free article] [PubMed] [Google Scholar]
  • Tetro J.A. Is COVID-19 receiving ADE from other coronaviruses? Microbes and Infection. 2020;22(2):72–73. doi: 10.1016/j.micinf.2020.02.006. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Thein T.L., Wong J., Leo Y.S., Ooi E.E., Lye D., Yeo T.W. Association between increased vascular nitric oxide bioavailability and progression to dengue hemorrhagic fever in adults. The Journal of Infectious Diseases. 2015;212(5):711–714. doi: 10.1093/infdis/jiv122. [PubMed] [CrossRef] [Google Scholar]
  • Thieblemont N. CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4. Clinical and Experimental Immunology. 2010;92(1):106–113. doi: 10.1111/j.1365-2249.1993.tb05955.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Tsai T.T., Chuang Y.J., Lin Y.S., Chang C.P., Wan S.W., Lin S.H. Antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of IL-10 production in monocytes. PLoS Neglected Tropical Diseases. 2014;8(11) doi: 10.1371/journal.pntd.0003320. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Ubol S., Halstead S.B. How innate immune mechanisms contribute to antibody-enhanced viral infections. Clinical and Vaccine Immunology. 2010;17(12):1829–1835. doi: 10.1128/CVI.00316-10. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Ubol S., Phuklia W., Kalayanarooj S. Mechanisms of immune evasion induced by a complex of dengue virus and preexisting enhancing antibodies. Journal of Infectious Diseases. 2010;201(6):923–935. [PubMed] [Google Scholar]
  • Viktorovskaya O.V., Greco T.M., Cristea I.M., Thompson S.R. Identification of RNA binding proteins associated with dengue virus RNA in infected cells reveals temporally distinct host factor requirements. PLoS Neglected Tropical Diseases. 2016;10(8) doi: 10.1371/journal.pntd.0004921. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • von Kietzell K., Pozzuto T., Heilbronn R., Grossl T., Fechner H., Weger S. Antibody-mediated enhancement of parvovirus B19 uptake into endothelial cells mediated by a receptor for complement factor C1q. Journal of Virology. 2014;88(14):8102–8115. doi: 10.1128/JVI.00649-14. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Walls A.C., Xiong X., Park Y.J., Tortorici M.A., Snijder J., Quispe J. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell. 2019;176(5):1026–1039. doi: 10.1016/j.cell.2018.12.028. e1015. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wan B., Chen X., Li Y., Pang M., Bao D. Porcine FcγRIIb mediated PRRSV ADE infection through inhibiting IFN-β by cytoplasmic inhibitory signal transduction. International Journal of Biological Macromolecules. 2019;138 [PubMed] [Google Scholar]
  • Wan Y., Shang J., Sun S., Tai W., Chen J., Geng Q. Molecular mechanism for antibody-dependent enhancement of coronavirus entry. Journal of Virology. 2020;94(5) doi: 10.1128/JVI.02015-19. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wang T.T., Sewatanon J., Memoli M.J., Wrammert J., Bournazos S., Bhaumik S.K. IgG antibodies to dengue enhanced for Fc gamma RIIIA binding determine disease severity. Science. 2017;355(6323):395–398. [PMC free article] [PubMed] [Google Scholar]
  • Wang Y., Si L.L., Guo X.L., Cui G.H., Fang D.Y., Zhou J.M. Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection. Virus Research. 2017;229:57–64. doi: 10.1016/j.virusres.2016.12.003. [PubMed] [CrossRef] [Google Scholar]
  • Wang Y., Si L., Luo Y., Guo X., Zhou J., Fang D. Replacement of pr gene with Japanese encephalitis virus pr using reverse genetics reduces antibody-dependent enhancement of dengue virus 2 infection. Applied Microbiology and Biotechnology. 2015;99(22):9685–9698. doi: 10.1007/s00253-015-6819-3. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wang S.F., Tseng S.P., Yen C.H., Yang J.Y., Tsao C.H., Shen C.W. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochemical and Biophysical Research Communications. 2014;451(2):208–214. doi: 10.1016/j.bbrc.2014.07.090. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wang J., Zand M.S. The potential for antibody-dependent enhancement of SARS-CoV-2 infection: Translational implications for vaccine development. Journal of Clinical and Translational Science. 2020;5(1):1–4. doi: 10.1017/cts.2020.39. [CrossRef] [Google Scholar]
  • Wang M., Yang F., Huang D., Huang Y., Zhang X., Wang C. Anti-Idiotypic antibodies specific to prM monoantibody prevent antibody dependent enhancement of dengue virus infection. Frontiers in Cellular and Infection Microbiology. 2017;7:157. doi: 10.3389/fcimb.2017.00157. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wang G., Yu Y., Cai X., Zhou E.-M., Zimmerman J.J. Effects of PRRSV infection on the porcine thymus. Trends in Microbiology. 2020;28(3):212–223. [PubMed] [Google Scholar]
  • Wang Q., Zhang L., Kuwahara K., Li L., Liu Z., Li T. Immunodominant SARS coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates. ACS Infectious Diseases. 2016;2(5):361–376. doi: 10.1021/acsinfecdis.6b00006. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Weaver S.C., Barrett A.D. Transmission cycles, host range, evolution and emergence of arboviral disease. Nature Reviews. Microbiology. 2004;2(10):789–801. doi: 10.1038/nrmicro1006. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wei C.J., Crank M.C., Shiver J., Graham B.S., Mascola J.R., Nabel G.J. Next-generation influenza vaccines: Opportunities and challenges. Nature Reviews. Drug Discovery. 2020;19(4):239–252. doi: 10.1038/s41573-019-0056-x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Widjaja I., Wang C., van Haperen R., Gutierrez-Alvarez J., van Dieren B., Okba N.M.A. Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein. Emerging Microbes & Infections. 2019;8(1):516–530. doi: 10.1080/22221751.2019.1597644. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Wrapp D., De Vlieger D., Corbett K.S., Torres G.M., Wang N., Van Breedam W. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies. Cell. 2020;181(5):1004–1015. doi: 10.1016/j.cell.2020.04.031. e1015. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Yoon K.J., Wu L.L., Zimmerman J.J., Hill H.T., Platt K.B. Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs. Viral Immunology. 1996;9(1):51–63. [PubMed] [Google Scholar]
  • Zhang L., Chen J., Wang D., Li N., Qin Y., Du D. Ligation of porcine Fc gamma receptor III inhibits levels of antiviral cytokine in response to PRRSV infection in vitro. Research in Veterinary Science. 2016;105:47–52. doi: 10.1016/j.rvsc.2016.01.009. [PubMed] [CrossRef] [Google Scholar]
  • Zhang A., Duan H., Li N., Zhao L., Xiao S. Heme oxygenase-1 metabolite biliverdin, not iron, inhibits porcine reproductive and respiratory syndrome virus replication. Free Radical Biology & Medicine. 2016;102:149–161. [PubMed] [Google Scholar]
  • Zhang L., Li W., Sun Y., Kong L., Xu P., Xia P. Antibody-mediated porcine reproductive and respiratory syndrome virus infection downregulates the production of interferon-α and tumor necrosis factor-α in porcine alveolar macrophages via Fc gamma receptor I and III. Viruses. 2020;12(2) [PMC free article] [PubMed] [Google Scholar]
  • Zhang L., Shen H., Gong Y., Pang X., Yi M., Guo L. Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) for targeted antibody blocking. Chemical Science. 2019;10(11):3271–3280. doi: 10.1039/c8sc05273e. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Zhou Y., Liu Z., Li S., Xu W., Zhang Q., Silva I.T. Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD. Cell Reports. 2021;34(5):108699. doi: 10.1016/j.celrep.2021.108699. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
  • Zhou P., Yang X.L., Wang X.G., Hu B., Zhang L., Zhang W. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270–273. doi: 10.1038/s41586-020-2012-7. [PMC free article] [PubMed] [CrossRef] [Google Scholar]