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/ PMCID: PMC8438590PMID: 34656289


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

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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.


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:


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Vaccine-associated enhanced disease: Case definition and guidelines for data collection, analysis, and presentation of immunization safety data

Authors: Flor M. Munoz,a,⁎Jakob P. Cramer,bCornelia L. Dekker,cMatthew Z. Dudley,dBarney S. Graham,eMarc Gurwith,fBarbara Law,gStanley Perlman,hFernando P. Polack,iJonathan M. Spergel,jEva Van Braeckel,kBrian J. Ward,lArnaud M. Didierlaurent,mPaul Henri Lambert,m and for the Brighton Collaboration Vaccine-associated Enhanced Disease Working Group


This is a Brighton Collaboration Case Definition of the term “Vaccine Associated Enhanced Disease” to be utilized in the evaluation of adverse events following immunization. The Case Definition was developed by a group of experts convened by the Coalition for Epidemic Preparedness Innovations (CEPI) in the context of active development of vaccines for SARS-CoV-2 vaccines and other emerging pathogens. The case definition format of the Brighton Collaboration was followed to develop a consensus definition and defined levels of certainty, after an exhaustive review of the literature and expert consultation. The document underwent peer review by the Brighton Collaboration Network and by selected Expert Reviewers prior to submission.Keywords: Adverse event, Immunization, Guidelines, Case definition, Vaccine, Enhanced disease, Respiratory, Systemic diseaseGo to:

1. Preamble

Vaccine-associated enhanced diseases (VAED) are modified presentations of clinical infections affecting individuals exposed to a wild-type pathogen after having received a prior vaccination for the same pathogen [1]. Vaccine-associated enhanced respiratory (VAERD) disease refers to disease with predominant involvement of the lower respiratory tract. Classic examples of VAED are atypical measles and enhanced respiratory syncytial virus (RSV) occurring after administration of inactivated vaccine for these pathogens. In this situation, severe disease has been documented resulting from infection in individuals primed with non-protective immune responses against the respective wild-type viruses [2][3][4][5][6]. Given that these enhanced responses are triggered by failed attempts to control the infecting virus, VAED typically presents with symptoms related to the target organ of the infection pathogen. In order to recognize vaccine associated disease enhancement, it is therefore necessary to have a clear understanding of the clinical presentation and usual course of the natural disease.

Disease enhancement independent of vaccine priming has also been described for pathogens causing sequential infections with different cross-reactive but not cross-protective serotypes, including dengue and pandemic influenza [7][8][9][10][11][12].

In late 2019, a novel severe respiratory illness emerged in Wuhan, China [13]. The causative agent, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was promptly identified, and determined to be closely related to SARS and the Middle East Respiratory Syndrome (MERS) coronaviruses, which had caused geographically localized outbreaks in 2002–2004 and from 2012 onwards, respectively. SARS-CoV-2 progressed to a global pandemic with substantial consequences due to its high infectivity and transmissibility, and its ability to cause both a severe respiratory illness, and a systemic disease with fatal consequences for vulnerable populations. The natural history of coronavirus infectious disease caused by SARS-CoV-2 (COVID-19), is yet to be fully described. However, a case fatality rate that ranges from 0.5% to nearly 20% depending on age and other risk factors, and the understanding that SARS-CoV-2 is now a well-adapted human pathogen that will continue to cause disease in susceptible populations, makes the development of an effective vaccine a global priority.

The potential for vaccination against SARS-CoV-2 to be associated with disease enhancement is of theoretical concern, given similar observations with other respiratory viruses in general, and in animal models of highly pathogenic coronaviruses in particular [14]. Importantly, VAED has not been seen following SARS or MERS vaccines given to humans, albeit the number of people who received these experimental vaccines remains very small. At this time, the pathogenesis, host responses and immunity to SARS-CoV-2 are still being evaluated and are not fully understood. SARS-CoV-2 infection is associated with a spectrum of disease that varies from asymptomatic infection to severe lung disease with acute respiratory distress syndrome (ARDS) and a fatal multiorgan disease with inflammatory, cardiovascular, hematologic and coagulation dysregulation [15][16][17]. Post-infectious, possibly immune-mediated systemic disease has also been described, particularly the multisystemic inflammatory syndrome in children (MIS-C) and adults (MIS-A) of unclear pathogenesis at this time [18][19][20][21].

Given the broad spectrum of disease associated with SARS-CoV-2, clinical assessment of both systemic VAED and lung-specific VAERD will be challenging during the pre-licensure evaluation of candidate vaccines and after the implementation of widespread vaccination for COVID-19. The broad spectrum of natural disease manifestations in different populations and age groups makes it very difficult, if not impossible, to determine how severe COVID-19 infection would have been in the absence of vaccination in the individual case. Someone who might have been completely asymptomatic without prior vaccination but who develops mild respiratory symptoms in association with prior vaccination could logically be considered a case of VAERD. However, this end of the spectrum of possible VAERD would have very little clinical significance for this individual person. At the population level however, even a small shift in the spectrum of disease towards greater severity could have major clinical and societal impact. Furthermore, given that severe illness is more feasible to detect and characterize, the case definitions discussed herein focus on the more severe presentations of VAED/VAERD.

There is no uniformly accepted definition of VAED or VAERD. Frequently used related terms include “vaccine-mediated enhanced disease (VMED)”, “enhanced respiratory disease (ERD)”, “vaccine-induced enhancement of infection”, “disease enhancement”, “immune enhancement”, and “antibody-dependent enhancement (ADE)”. This is potentially confusing as the mechanisms for disease enhancement likely vary, and data comparability across trials or surveillance systems can be problematic when the systems do not utilize a consistent case definition and do not collect comparable data. However, the assessment of this potential adverse event following immunization is particularly important for SARS-CoV-2, given the urgent global need for safe and effective vaccines. While this case definition was developed for the identification of potential cases of VAED/VAERD in the context of SARS-CoV-2 vaccine development, it is not exclusive for COVID-19 vaccines and may be applied in the evaluation of possible VAED/VAERD after any vaccine.

1.1. Methods for the development of the case definition

The Brighton Collaboration VAED working group was formed in March 2020 and included members with expertise in basic science, virology, animal models, immunology, vaccinology, vaccine safety, clinical care, clinical research, public health, regulatory sciences and ethics.

To guide the decision-making for the case definition and guidelines, a series of literature searches were performed using PubMed. The search terms and results of the searches are described in detail in [99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128].

To address the current state of knowledge and knowledge gaps for the assessment of VAED in the context of the assessment of vaccines for SARS-CoV-2, a Consensus Conference of Experts, including the authors of this case definition, was convened on March 12–13, 2020. The topics of discussion and conclusions of this meeting are published [22]. The group of experts in this Consensus Meeting concluded that the demonstration of some disease enhancement with any candidate vaccine after viral challenge in animal models should not necessarily represent a “no-go” signal for deciding whether to progress into early trials in clinical development of a COVID-19 vaccine. However, continuous monitoring of this risk during clinical trials in an epidemic context will be needed. Each observed effect should be discussed by the vaccine developers with the respective regulatory agencies who will ultimately define the actual requirements for clinical studies.

The working group determined that describing the known pathophysiologic pathways leading to VAED/VAERD was necessary to highlight the different potential mechanisms of disease, because an established clinical, laboratory or histopathologic definition of VAED or VAERD is not available. The case definition focuses on the identification of possible and probable cases of VAED/VAERD after any vaccination based on clinical presentation and frequency of clinical outcomes of concern, and provides suggested clinical and laboratory evaluation tools. However, while describing options for evaluation of possible cases when planning clinical trials or safety surveillance, a specific biomarker or histopathologic finding of VAED does not exist, and therefore, the case definition is not prescriptive regarding the specific tests to perform, nor when and who should be conducting or interpreting such testing. Furthermore, with the current state of knowledge, the “gold standard” evaluation to diagnose a definitive case of VAED/VAERD may be pathogen- and vaccine-specific and cannot be defined until more clinical and research data become available, in consultation with experts.

1.2. Defining pathophysiologic pathways leading to VAED/VAERD

Previous experiences of specific vaccine enhanced diseases serve as examples of how various pathophysiologic mechanisms can lead to VAED or VAERD. The mechanisms are outlined below.

1.2.1. Immune complex mediated enhanced disease (RSV, measles, pandemic influenza)

Shortly after the successful inactivation of polioviruses with formaldehyde, and the success of that vaccine for the control of epidemic polio, other pediatric pathogens were targeted for vaccine development using similar methods. In the mid-1960s, a formalin-inactivated vaccine against RSV was administered to infants and young children in four studies in the United States [23][24][25][26]. Children were subsequently exposed to wild-type virus in the community, and those immunized children who were seronegative for RSV before vaccination experienced an enhanced and atypical presentation of RSV disease, with fever, wheezing, and bronchopneumonia. These children were more frequently hospitalized and two children vaccinated in infancy, died as a consequence of the RSV infection [23]. In contrast, enhanced respiratory disease (ERD) was not observed in infants who were seropositive for the virus at the time of administration of the formalin-inactivated RSV vaccine [23].

The potential reason for the enhanced disease associated with the formalin-inactivated vaccine was that a non-protective antibody response of low affinity for the RSV fusion (F) protective antigen was generated [27][28][29]. This low-avidity, non-protective response elicited by formalin-inactivated vaccines has been linked to deficient Toll-like receptor (TLR) activation at the time of immunization [27][28][29][30][31]. Subsequent RSV infection leads to immune complex formation and complement activation with pulmonary injury, exacerbation of bronchopneumonia with a Th2 biased CD4 T-cell response (a distinctive phenotype of the disease) [32][33][34][35][36] and abundant mucus production [37][38]. Another potential contributing factor to non-protective antibody responses elicited by formalin-inactivated vaccine may have been the administration of RSV F in its post-fusion conformation, which is less stable and results in antibodies with lower neutralizing capacity than the pre-fusion conformation [30].

Interestingly, affinity maturation during an earlier exposure to RSV explains why children who were seropositive for the virus before inoculation never developed ERD. Preexisting acquisition of high-avidity antibodies against wild-type RSV likely outcompeted low-affinity B cell clones elicited by the formalin-inactivated RSV vaccine thus eliminating the low affinity non-protective B cells. This same paradigm explains why no children experienced ERD twice. After experiencing RSV disease enhancement post-wild-type infection, new antibodies with high affinity for the virus in these individuals established a healthy response against subsequent reinfections.

A formalin-inactivated vaccine against measles virus was licensed in the United States in 1963 simultaneously with the first live-attenuated measles vaccine [39][40]. Although most people were initially protected by the formalin-inactivated vaccine, the relatively low-avidity antibodies elicited by this vaccine failed to protect at lower titers and led to a severe form of illness known as atypical measles, in immunized individuals exposed to wild-type virus [41]. Children with atypical measles presented with high fever, a petechial rash in the extremities, and bibasilar pneumonia [42]. In this case, low-avidity antibodies elicited by the vaccine failed to neutralize virus that bound to the CD150 high-affinity receptor in exposed individuals, and promoted immune complex-mediated illness at sites of measles virus infection, mainly the skin and lungs [43][44][45][46]. Importantly, atypical measles occurred many years after exposure to the formalin-inactivated vaccine.

After observing the first cases of atypical measles, formalin-inactivated vaccine recipients not yet exposed to wild-type measles were inoculated with the licensed live-attenuated vaccine, with the intent of generating protective antibodies to prevent further atypical cases. These individuals developed an erythematous nodule at the subcutaneous injection site, characterized histopathologically by measles virus-specific immune complexes [47][48]. Some failed to mount a corrective immune response following the live-attenuated vaccination, presumably due to neutralization of the vaccine-strain virus by pre-existing antibodies. In most however, the live attenuated vaccine successfully induced a high affinity IgG response that could outcompete the potentially pathogenic antibodies and provide long-term protection.

1.2.2. Cellular immunity in enhanced respiratory disease and atypical measles.

Atypical measles and ERD are also characterized by a Th2 polarization of their immune response. Although mice are not an ideal small animal model for either RSV or measles virus infection, an early evaluation of ERD pathogenesis by Graham et al., showed increased production of interleukin 4 (IL-4) in lungs of affected BALB/c mice [35]. Subsequent depletion of CD4+  T lymphocytes and co-depletion of IL-4 and IL-10 ameliorated ERD lung pathology, suggesting that the disease was due, at least in part, to an exacerbated Th2 response [29][36]. These findings were expanded by reports of high levels of IL-5 and IL-13, increased numbers of eosinophils, and CD4+  T lymphocytes in mice with ERD [32][33]. In recent years, a critical role for Th2 bias has been described for enhanced disease components, including airway hyperreactivity and mucus hypersecretion [32][33]. Formaldehyde inactivation of RSV may also have contributed to Th2 polarization during ERD by generating carbonyl groups on viral antigens [48].

In addition, other T lymphocyte populations may have contributed to ERD pathogenesis and its phenotype. Marked suppression of T regulatory cells (T-reg) may have exacerbated the Th2 bias in formalin-inactivated RSV vaccine recipients; [49] absence of RSV-specific cytotoxic T lymphocytes response after immunization was permissive for viral replication in the lungs and contributed to a Th2 bias in the anamnestic CD4+  T lymphocyte response during wild-type infection; [50][51] and eosinophils, though probably not a critical factor in disease pathogenesis [52], may be a useful biomarker of undesirable responses in animal models of disease.

1.2.3. Antibody-mediated enhanced disease (Dengue)

Dengue viruses (DENV) belong to the genus Flavivirus, with four serologically and genetically distinct serotypes, which differ by 30–35% amino acid identity. The symptoms of dengue infections range from asymptomatic in about two-thirds to mild flu-like symptoms to dengue fever. Dengue hemorrhagic fever/dengue shock syndrome is the most severe form of dengue disease and is characterized by vascular leakage, hemorrhagic manifestations, thrombocytopenia, and hypotensive shock. People exposed to their first DENV infection develop memory B cells and long-lived plasma cells that produce antibodies that can either be cross-reactive or specific to the serotype of infection. Secondary DENV infections induce cross-neutralizing antibodies and protective immunity. However, priming with one DENV serotype can sometimes increase the risk of severe dengue upon secondary infection with a different DENV serotype. The mechanism for increased disease severity is thought to be associated with antibody-dependent enhancement (ADE) [53][54]. It has been postulated that low levels of antibodies or non-neutralizing antibodies induced by a previous DENV infection bind to the new serotype of DENV and facilitate viral entry into Fcγ receptor (FcγR)-bearing cells, leading to higher viremia and immune activation. Strong evidence for this ADE mechanism after natural infection comes from a pediatric cohort study in Nicaragua, where an increased risk for viremia and severe disease (7.64-fold higher, 95% confidence interval (CI): 2.19–18.28) was observed in children with preexisting DENV- antibody levels of 1:21–1:80 compared to DENV-naïve or those with high (>1:1280) antibody titers [55].

There is concern that dengue vaccines can induce similar ADE. If a vaccine produces antibodies with poor neutralizing activity that bind heterotypic virions without achieving neutralization, the opsonized viral particle may have an increased ability to infect Fcγ-R-bearing cells (i.e., facilitated entry). If a vaccine does not induce enough neutralizing antibodies against one or more serotypes, VAED may develop upon exposure to these serotypes. In a phase 3 clinical trial, children who received vaccine (Dengvaxia™: Sanofi Pasteur) had an increased risk of hospitalization due to dengue compared to the placebo group in year 3 after vaccination [9][56]. While the mechanism of ADE is not proven, prior exposure to dengue (positive serostatus) is thought to play a critical role, and those that are dengue-naïve seem to have higher risk of severe disease after vaccination when subsequently infected with another serotype of dengue virus [57]. Nevertheless, the data for ADE are controversial as most of the evidence is anecdotal or based on animal models.

In in vitro and animal models, a peak enhancement titer (i.e., a specific concentration of antibodies that most efficiently enhances DENV infection) has been observed. By contrast, higher antibody concentrations effectively neutralize virions, whereas lower concentrations can enhance infection. However, neutralizing assays vary from laboratory to laboratory and standardization of such bioassays across laboratories can be challenging. ADE is also postulated for other arboviruses including Zika and Japanese Encephalitis Virus [58][59][60].

1.2.4. Cytokine activation/storm and enhanced disease (SARS, MERS, SARS-CoV-2)

Exuberant cytokine activation is considered an important component in severe disease caused by SARS-CoV, MERS-CoV or SARS-CoV-2. The precise mechanism of this immunopathologic response still remains unclear, since active cytokine/chemokine production may be an appropriate response to uncontrolled virus replication as opposed to a truly excessive response. In any case, prolonged cytokine responses in patients with SARS, characterized by expression of mainly INF-gamma [61][62], are correlated with worse outcomes. In these patients, lymphopenia was commonly observed, which likely reflected effects of elevated levels of cytokines and endogenous corticosteroids. In one study, prolonged levels of type I interferon (INF) and other cytokines were observed in SARS patients who did poorly, while these levels were generally lower in patients who had better clinical responses, coincident with the development of protective antibody responses [63]. However, in other studies, antibody responses were higher in patients with worse outcomes, raising the possibility that the antibody response actually contributed to more severe disease. Macrophages are considered an important source of pro-inflammatory cytokines, but these cells are not productively infected with SARS-CoV [64][65]. Both macrophages and dendritic cells are abortively infected with SARS-CoV. While the cells do not support productive infection, in acute respiratory distress syndrome (ARDS) they were shown to produce pro-inflammatory cytokines such as TNF, IL-1beta, IL-6, IL-8, CCL2 and CCL7 [66][67][68]. Additional insight into the role of excessive cytokine activation in SARS comes from mouse studies. Mice infected with mouse-adapted SARS-CoV develop a lethal pneumonia, characterized by rapid virus replication and peak virus titers within 16–24 h. However, 100% of mice survive if type I IFN signaling is blocked either by genetic deletion or treatment with antibody that blocks INF signaling [69].

Similar mechanisms appear to occur in MERS patients, although less is known because there have been only approximately 2500 cases since MERS was first identified in 2012. As in SARS, MERS patients tend to have (delayed) elevated levels of pro-inflammatory cytokines such as IL-1beta, IL-6 and IL-8 [70]. MERS-CoV, unlike SARS-CoV, actively inhibits the induction of an early IFN-I response, allowing for enhanced virus replication [71]. Also unlike SARS-CoV, MERS-CoV is sensitive to IFN-I therapy [71]. MERS-CoV was shown to productively infect macrophages and dendritic cells, with delayed induction of IFN-I and other cytokines [72]. Thus both SARS-CoV and MERS-CoV induce the expression of pro-inflammatory molecules, even though their ability to replicate in myeloid cells differs substantially.

Although SARS-CoV-2 has been in human populations for only a few months, several studies have suggested that excessive cytokine activation contributes to pathogenesis. Molecules, such as IL-1, IL-6, TNF, IL-8 are upregulated in patients with more severe disease, raising the possibility that some of them may contribute to poor outcomes [73]. IL blocking antibodies are being used clinically in both controlled and uncontrolled clinical trials.

ADE has only been convincingly demonstrated in coronavirus infections in cats that were previously seropositive from infection or vaccination and immunized with S protein expressing vectors followed by challenge with feline infectious peritonitis virus [73]. However, VAERD has occurred after immunization with SARS inactivated vaccines or alphavirus vectors expressing the nucleocapsid protein. In many instances, inflammatory infiltrates exhibit a Th2 rather than a Th1 phenotype and are characterized by increased numbers of eosinophils [74][75][76][77]. In another study, macaques were immunized with vaccinia virus expressing the SARS protein or passively immunized with plasma from macaques immunized with the same vector. They were then challenged with SARS-CoV. While the animals remained asymptomatic, the nature of the inflammatory infiltrates changed, most prominently from M2- to M1-type macrophages, with increased expression of pro-inflammatory cytokines. Of note, this modification in the immune response did not result in a change in clinical disease [78].

1.2.5. Vaccine-induced enhancement of acquisition of infection

The first large placebo controlled trial (STEP trial) of an Ad5-HIV vaccine candidate was terminated early when a planned interim analysis demonstrated a significantly higher rate of HIV infection in male vaccinees who had been Ad5 seropositive at baseline vs. placebo (5.1% versus 2.2% per year) and/or were uncircumcised (5.2% versus 1.4% per year) [79]. A longer term follow-up analysis (after unblinding) supported the initial finding of enhanced acquisition of HIV infection in the vaccinees compared to the placebo group [80]. Although the difference in the rate of HIV infection was relatively small, it was statistically significant (Hazard ratio of 1.40 (95% CI, 1.03–1.92; p = 0.03)). In an expanded analysis of data, 49 of the 914 male vaccine recipients became HIV infected (annual incidence 4.6%, 95% CI 3.4 to 6.1) and 33 of the 922 male placebo recipients became HIV infected (annual incidence 3.1%, 95% CI 2.1 to 4.3). Potential explanations of this increased susceptibility to HIV infection in the vaccinees included the lack of an HIV-Env antigen in the vaccine, with the possibility that the HIV immune response induced by the vaccine potentially induced attachment of HIV to cellular surface, but without killing or neutralizing the virus, thus enabling viral entry into the cell. While the cause of the apparent vaccine-induced increased susceptibility to acquisition of HIV infection was never fully explained, the conclusion that the vaccine enhanced acquisition of HIV infection remained firm. The clinical data in those vaccinees who were infected were also suggestive of disease enhancement, or at least reduction of the time from acquisition of infection to onset of disease manifestations.

Although no other vaccine has been definitively linked to enhanced acquisition of infection, it is likely that this is not an outcome unique to HIV infection. Enhanced disease associated with inactivated measles or RSV vaccines has become accepted has having occurred, but enhanced acquisition of infection was not widely reported for either the formalin-inactivated RSV and inactivated measles vaccines. However, this may not have been noted due to the high infection rate for measles or RSV in the study populations at the time. Additionally, the four formalin-inactivated RSV vaccine trials were not placebo-controlled [23][24][25][26]. A control vaccine (Parainfluenza 3 vaccine candidate – PIV3) or historical controls were used. Nevertheless, in two of the RSV vaccine trials [23], the attack rates, particularly in infants less than 12 months of age, were higher than in the PIV3 control group. In one study, 23 of 31 (74%) of RSV vaccinated infants later developed RSV infection compared to 21 of 40 (53%) RSV infections in the infants receiving PIV3 (unadjusted chi-square 5.1505; p-value is 0.023) [23]. In a second study, 13 of 43 (30%) RSV vaccinated infants later developed RSV infection compared to 5 of 46 (11%) RSV infections in the infants receiving PIV3 (unadjusted chi-square 5.1645; p-value is 0.023) [24].

In the case of the inactivated measles vaccine, the inactivated measles vaccine was evaluated in trials which compared inactivated vaccine plus at least one dose of live measles vaccine to a live measles vaccine alone control group [81]. Thus, although enhancement of clinical measles was noted in the inactivated measles vaccine group, the number of breakthrough cases of measles was too small to detect a difference in incidence.

Thus, while the occurrence of enhanced acquisition of HIV infection, induced by an Ad5-HIV vaccine candidate became generally acknowledged as a consequence of immunization with that particular vaccine, enhanced acquisition of infection has not been perceived as having occurred with other vaccines. Although the difference in the numbers of HIV infection was relatively small in the STEP trial, the denominators were large and the conclusion that the Ad5-HIV vaccine caused enhancement of acquisition is now generally accepted. For the inactivated RSV vaccine, the trials were relatively small, but the rates of breakthrough RSV infections were much higher and also resulted in statistically significant increases in infection rates in the RSV vaccine groups. This apparent enhanced acquisition of infection may have been overlooked because the severity of enhanced disease occurring in the vaccinees overshadowed the higher risk of RSV infection associated with the vaccine and because the background rates of RSV infection were high in both RSV and PIV3 control vaccines recipients.

1.3. How do existing animal models inform assessment of VAED in humans?

Although animal models have been developed for most respiratory viruses, they rarely reproduce the full spectrum of the corresponding human disease. Therefore, the assessment of the risk of VAED in animal models is imperfect and limited. However, animal models can still provide useful information on potential pathogenic mechanisms and identify markers of potential risks that can be considered for inclusion in clinical trials.

Some lessons may be retained from the previous VAED experience. Today, preclinical RSV vaccine studies that indicate the induction of weak neutralizing and strong non-neutralizing responses with Th2 types of T-cell responses suggesting a risk of VAED may lead to changes in vaccine design strategies. First the selection of antigen(s) is critical to ensure an appropriate balance of neutralizing vs non-neutralizing antibody production. Second, some candidate subunit RSV vaccines are now formulated with Th1-driving adjuvants, which will diminish a prominent Th2 response and an eosinophilic reaction after exposure to the wild virus. Third, preference can be given to RSV vaccines that can induce long-lasting and powerfully neutralizing antibody responses and affinity maturation in order to avoid a gradual waning of antibody levels.

When planning phase 1/2 clinical trials of new candidate vaccines against acute respiratory infections, it is useful to analyze markers of innate and acquired immunity, and observations made in animal models may be informative. Regarding innate immunity, a detailed assessment of monocytes and NK-cell phenotypic markers and various circulating cytokines (e.g. IFN-alpha, IL-10) in the first 24 h post-immunization has been shown to correlate with long term features of subsequent antibody responses [82]. T-cell responses also need to be monitored, including a cytokine profile analysis of both CD4 and CD8 T-cell responses, with multiple Th1 and Th2 markers. The absence of Th2 markers such as IL-4, IL-5 or IL-13, in the presence of consistent IFN-gamma responses may indicate a lower risk of some forms of VAED. Animal models have identified antibody response patterns that are associated with low risk of VAED including a high ratio of neutralizing vs. antigen-binding antibodies anti-receptor binding domain (RBD) antibodies of high affinity (nanomolar range), and antibody kinetics showing sustained IgG responses over time. One may also consider the passive transfer of serum (containing different antibody levels) from immunized Phase 1 trial participants into suitable animal models, prior to viral challenge, to assess the risk of enhanced disease after infection.

1.4. Knowledge gaps in current understanding of potential VAED in the context of SARS-CoV-2

1.4.1. Mechanisms

Various distinct pathways may lead to VAED. SARS-CoV-2 is a novel pathogen facing no specific immunity in populations of all ages and it presents a considerable degree of variability in its clinical manifestations. In addition, its mechanisms of pathogenesis are still unclear. Therefore, understanding how aberrant immune responses may alter a process that is not yet well characterized and that itself presents a considerable range of clinical manifestations is difficult. However, VAED always involves a memory response primed by vaccination and, in the experiences best characterized until now, targets the same organs as wild-type infections. The availability of clinical data and samples from patients with wild-type infections (not vaccinated) is therefore critical to make any assessments and decisions regarding the presence of disease enhancement when analyzing a vaccine candidate.

1.4.2. Animal models

There are a few critical details that should be considered when testing vaccine candidates for the risk of VAED in animal models, regardless of the species selected: [1] the need for a negative wild-type infection control group is paramount. A vaccine may seem safe in the absence of a baseline wild-type infection control, particularly when the VAED positive control presents an exaggerated phenotype; [2] the importance of methodically clearing all control inoculations and challenges from cellular debris, which may enhance reactogenicity in animal models and bias observations; [3] while acknowledging the urgency of the ongoing pandemic, it may be important to wait a considerable period of time between immunization and challenge, since early challenge might prevent investigators from seeing effects that would become obvious later. For this purpose, challenging after antibody titers fall to low – possibly non-protective – levels may be most informative; [4] carefully selecting reproducible models of VAED as positive controls in the evaluation. Some animal models may not exhibit the same manifestations reproducibly and a negative test in the absence of proper positive controls may be deceptive; and [5] prioritize models with clinical manifestations of illness over those exhibiting only pathological changes. Otherwise, results may be distorted by emphasizing pathological differences of uncertain clinical relevance.

1.4.3. Vaccine platforms

Numerous vaccines are under evaluation for SARS-CoV-2 and other emerging pathogens [83]. These include well established vaccine constructs used in existing licensed vaccines (protein subunit, inactivated, virus-like particle, and replicating viral vectored vaccines), and new technologies (nucleic acid, DNA or mRNA-based vaccines) that allow for the rapid development of vaccine candidates [84]. Certain vaccines may be more appropriate for specific populations, such as the elderly, children and pregnant women, compared with healthy adults. The safety, immunogenicity and efficacy of SARS-CoV-2 vaccines must be carefully evaluated prior to their use in the general population, particularly given concerns for disease enhancement and the global need for effective vaccines to control the COVID-19 pandemic.

1.4.4. Adjuvants

Adjuvants have been used in vaccines and given to billions of individuals. Based on this experience and in the context of vaccine development against other pandemic viruses responsible for acute respiratory viral infections, adjuvants may help to: [1] enhance the level and durability of protective humoral response and broaden its epitope-related specificity [2] induce skewed response towards more functional immune responses, including cellular response and [3] generate antigen-sparing approaches able to deliver more vaccine doses in the context of an ongoing pandemic.

Adjuvants have the capacity to increase immune responses through the activation of innate immunity, conditioning the level and quality of antibodies and T-cell responses specific to the vaccine antigen [85]. In addition to antibodies, the most potent adjuvants such as emulsions or those containing saponins and Toll-like receptor ligands have been shown to induce robust and long lasting polyfunctional CD4 + T-cell responses, with a predominant IL-2, IFN-g and TNF response, but remarkably little Th2-associated cytokines [86][87]. CD8 T-cell responses are also generally not increased by adjuvanted recombinant vaccines.

In the context of disease enhancement, the use of appropriate adjuvants in subunit vaccines may therefore be a possible avenue to manage the potential risk of VAED, in particular those that induce a more potent innate response. However, both pre-existing immunity and the type of antigen influence the impact of the adjuvant on the immune response, and therefore each antigen/adjuvant combination needs to be specifically evaluated. The safety profile of adjuvanted vaccines will also depend on the adjuvant’s mode of action. In early clinical trials of candidate vaccines, it will be important to assess the impact of adjuvant not only on the magnitude, but more critically, on the quality of the immune response, such as antibody functionality and T-cell profiling. In this regard, non-human primates rather than mice have been shown to best reflect the behavior of adjuvants observed in humans and therefore constitutes a good predictive model for formulation selection [88].

1.5. Diagnosis and differential diagnosis of VAED/VAERD

No single or combination of specific confirmatory tests is available to diagnose VAED. As the clinical manifestations of VAED lies within the spectrum of natural disease – occurring more frequently and/or severely in vaccinated individuals – it is also difficult to separate vaccine failure (also called breakthrough disease) from VAED in vaccinated individuals. All cases of vaccine failure should be investigated for VAED. Vaccine failure is defined as the occurrence of the specific vaccine-preventable disease in a person who is appropriately and fully vaccinated, taking into consideration the incubation period and the normal delay for the protection to be acquired as a result of immunization [89]. Assessment of single or multiorgan dysfunction, atypical immune and inflammatory responses, viral identification and quantification, and histopathology may aid in the diagnosis and classification of the extent and severity of disease occurring after vaccination. However, definitive case ascertainment of VAED/VAERD might not be possible, and ascertainment of occurrence of VAED/VAERD might only be feasible in the context of large randomized controlled clinical trials or during post-licensure safety surveillance.

1.6. Disease severity assessment and classification

A classification or a standardized method for the assessment of disease severity are not available for VAED/VAERD. In the case of dengue infection, where the occurrence of antibody-mediated disease enhancement upon reinfection is a well described phenomenon, the existing clinical classification characterizes the more severe manifestations of disease. Similarly, existing clinical disease characterizations and severity of illness scores may be utilized to identify and classify cases of severe or enhanced disease occurring after vaccination.

2. Rationale for selected decisions about the case definition of vaed/vaerd as an adverse event following immunization

In general, VAED is a modified and/or severe presentation of an infectious disease affecting individuals exposed to the wild-type pathogen after having received vaccine designed to prevent infection.

An accepted case definition of VAED does not yet exist. Similarly, harmonized, specific guidance from regulatory bodies regarding the assessment of clinical trial subjects for VAED, is not yet available. A consensus definition is necessary not only in the context of vaccine clinical trials to allow for comparability among different vaccines and clinical studies, but also for the assessment of safety after vaccine licensure and implementation.

2.1. Case definition

2.1.1. Vaccine-associated enhanced disease (VAED)

  • 1.Is an illness that occurs in persons who receive a vaccine and who are subsequently infected with the pathogen that the vaccine is meant to protect against. This definition assumes previously antigen-naïve vaccine recipients, which can be assessed by determining seronegative status prior to vaccination, when feasible. The need for documentation of seronegativity prior to vaccination, which can be done retrospectively, is particularly relevant in Phase II-III clinical trials. In the context of such trials, the working group acknowledged the difficulty in distinguishing between vaccine failure and VAED. Thus, all cases of vaccine failure should be evaluated for VAED.
  • 2.VAED may present as severe disease or modified/unusual clinical manifestations of a known disease presentation. The illness presumably is more severe or has characteristics that distinguish it from illness that might occur in unvaccinated individuals.
  • 3.VAED may involve one or multiple organ systems.
  • 4.VAED may also present as an increased incidence of disease in vaccinees compared with controls or known background rates.

2.1.2. Vaccine-associated enhanced respiratory disease (VAERD)

  • 5.Refers to the predominant lower respiratory tract presentation of VAED. The mechanisms of pathogenesis might be specific to the lower respiratory tract or part of a systemic process.

2.1.3. Approach for identification of cases of VAED/VAERD

In the context of vaccine clinical trials, the routine collection of adverse events (AE), serious adverse events (SAE), and adverse events of special interest (AESI) is an existing mechanism to evaluate the occurrence of illnesses and outcomes that are serious, including those that are new, require medical care, result in disability, are life threating or result in hospitalization or death. Similarly, AEs are evaluated for severity, using existing tools, such as severity grading scales and toxicity tables for clinical and laboratory outcomes that are adapted to various populations including adults, children and pregnant women [DAIDS Toxicity tables]. The working group concurs that these methods of assessment of events occurring after vaccination are appropriate to identify triggers that point towards potential cases of VAED/VAERD. Potential cases may be initially identified though clinical characteristics alone (Table 1 ), or complemented with laboratory evaluation (Table 2 ).

Table 1

Factors to consider in the assessment of the clinical presentation of VAED and VAERD.

A• Recognizing VAED in an individual patient is particularly challenging.VAED might be identified first as a vaccine failure. The clinical presentation may be variable within a spectrum of disease that ranges from mild to severe, life threatening, with or without long term sequelae, to fatal.
B• Identification of VAED requires the recognition of a clinical presentation that is different, atypical, modified or more severe in comparison to the natural or known (typical) disease presentation, or that occurs at a higher frequency from the control group or expected background rates in the specific target population.• No clinical presentation is pathognomonic for VAED.
C• Identification requires that the clinical syndrome is new or distinct from the typical presentation or from other known diseases, similar or associated disorders, or that such clinical syndrome occurs at higher frequency from the control group or expected background rates. For example, acute respiratory distress syndrome (ARDS) is a distinct entity characterized by rapid and progressive inflammatory changes in the lung parenchyma, resulting in respiratory failure. Diagnosis is based on clinical characteristics and documentation of hypoxemia using accepted standardized definitions (eg. Berlin classification of ARDS). ARDS may occur as a result of a variety of insults that cause inflammation, alveolar cell injury, surfactant dysfunction, and other vascular and hematologic abnormalities, including SARS-CoV-2 infection. ARDS may be a form of clinical presentation of VAED or VAERD.
D• Assessment of the type and frequency of the clinical presentations by developing a clinical profile of cases would be helpful to aid in the more efficient identification of cases through the development of algorithms [90].
E• Grading of clinical manifestations of disease based on severity using a standardized and/or validated severity of illness score to evaluate all cases is recommended. Several tools are utilized in clinical practice for the assessment of disease severity in adults and children. Commonly used and practical scoring tools for adults are shown in Appendix B, and for children in Appendix C. Appropriate tools for the assessment of severity in various settings (eg. community vs. hospitalized cases) should be selected and used consistently [91].• Whenever feasible, the same clinical scoring tool should be used across related studies and validated.

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Table 2

Assessment for VAED in the context of vaccine development: relevant clinical and laboratory diagnostic parameters.

Organ systemClinical parametersLaboratory parameters
Respiratory system● Cough● Tachypnea● Dyspnea● Lower respiratory tract disease● Respiratory failure● Pulmonary hemorrhage● Radiographic abnormalities● Oxygen requirement● Hypoxemia● PaO2● PaO2/FiO2 ratio● Aa gradient
Cardiovascular system● Tachycardia● Hypotension/ Hypertension● Acute cardiac injury● Vasculitis/ Vasculopathy● Myocarditis● Heart failure● Cardiogenic shock● Abnormal ECG● Abnormal Echocardiogram● Troponin● B-Natriuretic Peptide (BNP)
Hematopoietic and Immune system● Coagulopathy● Disseminated intravascular coagulation● Bleeding/ Thrombotic events● Leukopenia, lymphopenia● Thrombocytopenia● B and T cell function assays● Altered coagulation parameters (PT, PTT, D-Dimer, INR)
Inflammatory markers● Pro-inflammatory state● Elevated inflammatory markers (CRP, procalcitonin)● Elevated Ferritin, LDH● Elevated cytokines
Renal system● Renal dysfunction● Acute kidney injury● Renal replacement therapy● Decreased urine output● Serum creatinine● Glomerular filtration rate
Gastrointestinal and hepatic system● Emesis/Diarrhea● Abdominal pain● Hematochezia/Melena● Hepatitis● Liver dysfunction● Acute liver failure● Electrolyte abnormalities● Elevation of liver enzymes● Elevated bilirubin
Central Nervous System● Altered mental status● Convulsions/seizures● Cranial nerve involvement● Unconsciousness● Elevated intracranial pressure● Abnormal CSF parameters
Other● Fatigue● Myalgia/myositis/myonecrosis● Arthralgia/arthritis● Multiorgan failure● Death● Viral load (PCR Ct value)● Antibody titers● Histopathology

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2.1.4. Identification of VAED/VAERD in clinical trials

Identifying cases of VAED/VAERD might be impossible when assessing individual patients, however, in clinical studies, a control group is helpful to compare the frequency of cases and the severity of illness in vaccinees vs. controls, including the occurrence of specific events of concern such as hospitalization and mortality. A comparison group of unvaccinated subjects or active comparator control group is particularly important when background rates of the outcome of interest are not available in the target population. If a control group is not available, comparisons should be made to the expected background rate of the event of interest when it occurs after natural disease in an unvaccinated population. When available, background rates of specific clinical manifestations and outcomes should be used to compare frequencies. Participation of an epidemiologist and statistician is recommended in clinical trial design. Given that the background rate of VAED is unknown, study sample size calculations in early phases of vaccine evaluation should not be based on the occurrence of VAED/VAERD. However, in large studies and in post-implementation phases, reliable surveillance systems should be in place for the timely detection of potential cases, using estimates of defined specific outcomes based on expected background rates or control group rates. All cases of vaccine failure should be evaluated for the possibility of VAED/VAERD, but not all cases of VAED/VAERD will represent vaccine failures. When feasible, a thorough evaluation for alternative etiologies should be conducted, and an adjudication committee, or a safety monitoring committee, or independent expert consultation should be convened to evaluate potential cases.

2.2. Factors to consider in the ascertainment of a case of VAED/VAERD and levels of Diagnostic certainty are described in Table 3

Table 3

Factors to consider in the ascertainment of a case of VAED/VAERD and Levels of Diagnostic Certainty.

Background ratesBackground rates of specific relevant conditions and outcomes, including hospitalization and mortality should be used when available. Backgrounds rates appropriate to the study population and contemporary to the vaccine evaluation should be used. This information might be unavailable or difficult to obtain. Alternatively, assessment of the frequency of events in a control group of unvaccinated individuals is necessary to ascertain the occurrence of events suggestive of VAED or VAERD. Whenever feasible, it is also important to distinguish VAED or VAERD from vaccine failure (as previously defined).
Age and genderThe expected severity of outcomes by age group must be described. This is an important factor to consider given that a different clinical presentation from what is expected for a specific age group could be considered VAED or VAERD. When pertinent, gender differences should be considered.
Time of onset after vaccination and after infectionVAED or VAERD may occur at any time after vaccination. The timing of occurrence of clinical manifestations of VAED or VAERD after vaccination will be dependent on the mechanism or pathophysiologic pathway leading to disease enhancement after natural infection. VAED or VAERD may present within 2–4 weeks of natural infection, if the expected initial antibody responses are inadequate; or may present at a later time (>1 month or longer) after natural infection if antibody waning is noted or if the mechanism is not exclusively antibody mediated.
Duration of follow upThe working group recommends that prolonged follow up is established, at least one year after vaccination or more, depending on the epidemiology of the disease, followed by population-based surveillance in the post-licensure period. In addition to taking into consideration what is realistic in the context of a clinical trial, it is important to consider the circulation of the target pathogen.In the case of endemic, ongoing active circulation, there is a possibility of exposure at any time after vaccination, which requires close follow up immediately after vaccination and potentially, for a prolonged period, depending on the risk of exposure. When pathogens exhibit a seasonal circulation, exposure can be identified through seasonal surveillance and may include follow up for a period of at least one, and preferably two, or more seasons, depending on the pathogen. In cases of sporadic circulation, the exposure periods may be unknown, and the follow up period may be prolonged.
Clinical course and progression of symptomsThe following outcomes would be concerning for VAED or VAERD in a person with confirmed infection:a.Death. This would be particularly concerning if death occurs in person without other risk factors for mortality (note phase I-II trials with selected healthy population) or if it occurs at higher rates than expected.b.Hospitalization, including hospitalization above expected rates.c.Worsening or clinical deterioration over time, particularly, although not exclusively, if differing from the anticipated natural course of the disease.d.Prolonged clinical course compared to natural disease.e.Complications of acute disease, new morbidities or new diagnoses subsequent to natural infection post-vaccination (for example higher rate of MIS-C or MIS-A)
Control for confounders and comorbiditiesIn the context of evaluating a case for VAED, it will be important to rule out other infections, comorbidities, drug effects, toxicities, etc. If no alternative explanation for the frequency or severity of illness is identified, including vaccine failure, VAED or VAERD may be considered.
Influence of treatment or response to treatment on fulfilment of case definitionTreatment of VAED and VAERD is for the most part, supportive and focused on managing the specific organ dysfunction resulting from the disease. The use of specific antiviral therapy when available, and of immunomodulatory treatments, should be documented. However, the working group considers that treatment or response to treatment is unlikely to be relevant for this case definition as ascertainment is based on comparative clinical severity of illness at presentation.
Type of vaccineVaccines vary based on the antigen utilized and the addition of adjuvants. At this time, there is insufficient data to determine a priori if any of these platforms is less or more likely to be associated with VAED/VAERD. The working group agrees that it is not possible to know the potential risk for VAED/VAERD of an individual vaccine given various mechanisms leading to disease enhancement, and the different affinity for specific receptors. The use of convalescent sera or monoclonal antibodies might inform potential antibody mediated effect vs. cell mediated mechanisms.
Vaccine enhancement vs. vaccine failureIn the event of low/poor vaccine efficacy, infection will occur in vaccinated subjects, with breakthrough disease associated with viral replication. When assessing the safety of a vaccine, there is a need to distinguish between a case where an immune response is not induced from a case where an aberrant non-protective immune response is induced. A thorough assessment of immune responses along with protection from serious disease outcomes is necessary to distinguish enhancement from break-through infection.
Geographic and population specific variability in vaccine responsesOther factors including geographic and genetic factors, and individual or population factors such as nutritional status, co-infections, and the effect of co-administration of medications and non-medical products, might also play a role in enhanced disease after vaccination.

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2.3. Diagnostic tests for the assessment of VAED

In addition to clinical parameters and clinical severity of illness grading, the diagnosis of VAED/VAERD should be supported by laboratory, radiographic, and pathology findings, as pertinent. A minimum set of recommended tests to be applied in the assessment of a possible case of VAED based on our current knowledge, is described here and in Table 4 .

Table 4

Suggested laboratory evaluation for the assessment of VAED/VAERD.

ParameterLaboratory findings suggestive of VAED/VAERD
Evidence inadequate or unbalanced neutralizing antibody responses• Low or inappropriate total binding (IgG, IgM, IgA) antibody titers• Low neutralizing antibody titers• Low ratio of neutralizing to binding antibody• Low absolute affinity of IgG antibody to receptor binding domain (RBD)• Lack of acquisition or loss of affinity of IgG to RBD• Increased viral load
Evidence of inadequate or inappropriately biased cellular immune responses• Lymphopenia or lymphocytosis• High CD4 lymphocyte subset• Low CD8 lymphocyte subset• Th2 (IL-4, IL-5, IL-13) CD4 T cell predominant response over Th1 (INFg, TNF) responses (testing in vitro stimulation with viral peptides or proteins, ELISPOT, or intracellular cytokine staining assays).• Low virus-specific cytotoxic T-cells (CTL)
Evidence of exuberant inflammatory responses• Elevated IL-1, IL-6, IL-8• Increased pro-inflammatory chemo/cytokines: INF-g, type 1-INF, TNF, CCL2, CCL7• Reduced expression of type I interferons (eg. IFN-α, INF-b)• Elevated C-reactive protein, Ferritin, Lactate dehydrogenase (LDH), D-dimers
Evidence of immunopathology in target organs involved, by histopathology• Present or elevated tissue eosinophils in tissue• Elevated pro-inflammatory Th2 cytokines in tissue (IL4, IL5, IL10, IL13)• C4d tissue deposition (evidence for complement activation through immune complex deposition)• C1q assessments of immune complexes in fluids• Low C3 levels as evidence complement consumption

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2.3.1. Viral identification and quantitation

Confirmation of viral infection by detection and quantitation of virus in specific sites is recommended. These include blood, the upper and lower respiratory tracts, tissue, and other pertinent sterile sites. Characterization of the virus should be performed when feasible (e.g., wild-type vs. vaccine virus, sequencing, emergence of mutations, etc.) Viral quantitation findings should be compared to the extent of observed clinical disease and assessed for consistency.

2.3.2. Immune responses

Evaluation of the immune response after vaccination and at the time of infection could inform the ascertainment of VAED. Whenever feasible, the immune responses should be compared to the expected immune response after natural infection or vaccination. Assessment of neutralizing and total antibody against specific epitopes/targets (for SARS-CoV-2, S and NP) as well as T-cell responses is recommended. Further studies of antibody neutralization, affinity and other stimulation and proliferation assays could be helpful to characterize the immune response.

2.3.3. Antibodies

Several characteristics can assist in exploring the potential risk of VAED in vaccine candidates during early clinical trials. After immunization, the kinetics of the response (sustained vs. peak-valley), neutralization titers, the ratio of neutralizing to S-binding antibodies, and both the absolute affinity for IgG against the RBD compared to that observed in IgG after wild-type infection and the progressive acquisition of affinity for RBD over time, may inform about the quality of antibodies elicited by the immunogens. After infection, the best information to define the potential for VAED when exploring antibody-mediated injury is, when possible, obtaining a biopsy of affected tissues or surrogate materials (e.g., from a small skin biopsy in atypical measles to a nasopharyngeal aspirate in respiratory diseases) that allows detection of C4d deposition as evidence for complement activation through immune complex deposition, C1q assessments of immune complexes in fluids, and C3 levels to explore complement consumption. All these determinations are particularly useful when matched against control samples from subjects experiencing wild-type disease.

2.3.4. Cell mediated immunity

Cell mediated immunity may be assessed by measurement of cell counts to determine the presence of lymphopenia or lymphocytosis, and quantification of specific cell subtypes, such as CD4 and CD8 T-cells. Functional assays will provide information about a change from a Th1 to a Th2 CD4 T-cell response. These assays will measure Th1 (IFN gamma, TNF) vs. Th2 (IL-4, IL-5, IL-13) patterns of response after in vitro stimulation with viral peptides or proteins, in ELISPOT or intracellular cytokine staining assays.

2.3.5. Serum cytokines and other markers

Cytokines are molecules which are secreted by a multitude of cells and effect other cells. They are divided into broad categories: 36 different types of interleukins; 17 types of interferons; 48 chemokines, and 17 members of TNF family at the current time, and more being identified on a regular basis. These cytokines affect growth, maturation, differentiation, regulation and chemotaxis of cells. Cytokines may be biomarkers of VAED and part of the mechanistic process.

Cytokines can also be used as marker of viral disease process and worsening infection. For example, in a severe dengue virus infection, a cytokine storm develops with increased levels of IL-6, IL-8, IFN-α and IFN-γ [92]. Therefore, measuring these cytokines might indicate a worsening of infection. These cytokines or a subset (IL-6 and IL-8) could also be elevated if a VAED-dependent cytokine storm is developing. In ADE seen after viral infections or vaccines, decreased antiviral activity with reduced expression of IFN-α [93] or evidence of worsening virus infection with high titers or increased pro-inflammatory cytokines may be seen. Skewing to Th2 cytokines (IL-4, IL-5, IL-13) and associated eosinophilia may occur as seen in RSV-associated VAED [31]. In the murine model of RSV, TNF-α and IFN-γ are necessary to induce this cytokine storm as other possible biomarkers [94]. Currently, no cytokine/chemokine ‘signature’ associated with VAED has been defined and variability would be expected with different mechanisms of VAED.

2.3.6. Inflammatory responses

A basic assessment of host immune responses after infection should include the evaluation of total white blood cell count and subpopulations (e.g. lymphocyte count, lymphocyte subtypes such as CD8 or CD4), and measurement of inflammatory markers such as C-reactive protein (CRP), Ferritin, Lactate dehydrogenase (LDH), D-dimers, and other specific cytokines (e.g., IL-1, IL-6).

2.3.7. Histopathology (if available)

If available, tissue obtained by biopsy from affected organs or autopsy should be evaluated for evidence of immunopathology.

2.3.8. Radiographic findings

Atypical or more severe involvement of the lower respiratory tract would be anticipated in patients with VAERD. Chest computed tomography (CT) has a high sensitivity for diagnosis of lower respiratory tract disease involvement, including for COVID-19 [95][96][97][98]. A standardized reporting system has been proposed for patients with suspected COVID-19 infection by means of the “CO-RADS classification”, integrating CT findings with clinical symptoms and duration of disease (, to:

3. Case definition of vaccine associated enhanced disease (VAED)

The case definition of VAED is described in Table 5 .

Table 5

Case definition and Levels of Certainty of Vaccine Associated Enhanced Disease.

LEVEL 1 of Diagnostic Certainty (Definitive case)
The working group considers that a Definitive Case (LOC 1) of VAED cannot be ascertained with current knowledge of the mechanisms of pathogenesis of VAED.

LEVEL 2 of Diagnostic Certainty (Probable)
Rationale for level 2: Ascertainment is based on confirmed infection, with known (2A, higher level of certainty) or without previously known (2B, lower certainty) serostatus, clinical and epidemiologic criteria, and available histopathology.
LEVEL 2A. A probable case of VAED is defined by the occurrence of disease in a previously seronegative vaccinated individual with:
Laboratory confirmed infection with the pathogen targeted by the vaccine
Clinical findings of disease involving one or more organ systems (a case of VAERD if the lung is the primarily affected organ)
Severe disease as evaluated by a clinical severity index/score (systemic in VAED or specific to the lungs in VAERD)
Increased frequency of severe outcomes (including severe disease, hospitalization and mortality) when compared to a non-vaccinated population (control group or background rates)
Evidence of immunopathology in target organs involved by histopathology, when available, including any of the following:• Present or elevated tissue eosinophils in tissue• Elevated pro-inflammatory Th2 cytokines in tissue (IL4, IL5, IL10, IL13)• C4d tissue deposition (evidence for complement activation through immune complex deposition)• C1q assessments of immune complexes in fluids• Low C3 levels as evidence complement consumptionAND
No identified alternative etiology
LEVEL 2B. A probable case of VAED is defined by the occurrence of disease in a vaccinated individual with no prior history of infection and unknown serostatus, with:
Laboratory confirmed infection with the pathogen targeted by the vaccine
Clinical findings of disease involving one or more organ systems (a case of VAERD if the lung is the primarily affected organ)
Severe disease as evaluated by a clinical severity index/score (systemic in VAED or specific to the lungs in VAERD)
Increased frequency of severe outcomes (including severe disease, hospitalization and mortality) when compared to a non-vaccinated population (control group or background rates)
Evidence of immunopathology in target organs involved by histopathology, if available, including any of the following:• Present or elevated tissue eosinophils in tissue• Elevated pro-inflammatory Th2 cytokines in tissue (IL4, IL5, IL10, IL13)• C4d tissue deposition (evidence for complement activation through immune complex deposition)• C1q assessments of immune complexes in fluids• Low C3 levels as evidence complement consumptionAND
No identified alternative etiology

LEVEL 3 of Diagnostic Certainty (Possible)
Rationale for level 3: Ascertainment is based on confirmed or suspected infection, known (3A higher level of certainty) or unknown (3B lower level of certainty) serostatus, clinical and epidemiologic criteria, but no histopathology findings.
LEVEL 3A. A possible case of VAED is defined by the occurrence of disease in a previously seronegative vaccinated individual with:
Laboratory confirmed infection with the pathogen targeted by the vaccine
Clinical findings of disease involving one or more organ systems (a case of VAERD if the lung is the primarily affected organ)
Severe disease as evaluated by a clinical severity index/score (systemic in VAED or specific to the lungs in VAERD)
Increased frequency of severe outcomes (including severe disease, hospitalization and mortality) when compared to a non-vaccinated population (control group or background rates)
No identified alternative etiology
LEVEL 3B. A possible case of VAED is defined by the occurrence of disease in vaccinated individual with no prior history of infection and unknown serostatus, with:
Laboratory confirmed infection with the pathogen targeted by the vaccine
Clinical findings of disease involving one or more organ systems (a case of VAERD if the lung is the primarily affected organ)
Severe disease as evaluated by a clinical severity index/score (systemic in VAED or specific to the lungs in VAERD)
Increased frequency of severe outcomes (including severe disease, hospitalization and mortality) when compared to a non-vaccinated population (control group or background rates)
No identified alternative etiology

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Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: F. M. Munoz is a consultant for the Coalition for Epidemic Preparedness Innovations (CEPI) for the development of Brighton Collaboration Case Definitions for the Safety Platform for Emergency vACcines (SPEAC) Project.

J. Cramer is a member of the Coalition for Epidemic Preparedness Innovations (CEPI).

C. Dekker is a consultant for the Coalition for Epidemic Preparedness Innovations (CEPI) via Brighton Collaboration for the Safety Platform for Emergency vACcines (SPEAC) Project and a consultant for Medicago Inc.

M. Dudley is a consultant for the Coalition for Epidemic Preparedness Innovations (CEPI) via Brighton Collaboration, for the Safety Platform for Emergency vACcines (SPEAC) Project.

B. Graham is inventor on pending patent applications related to coronavirus vaccines and monoclonal antibodies.

M. Gurwith is a consultant for the Coalition for Epidemic Preparedness Innovations (CEPI) via Brighton Collaboration for the Safety Platform for Emergency vACcines (SPEAC) Project; and is Chief Medical Officer for Verndari, Inc., which is developing vaccines for influenza and Covid-19.

S. Perlman is a member of the ACIP COVID-19 Vaccine Work Group.

F. Polack is an investigator in the Pfizer’s COVID-19 vaccine trial.

B. Ward is Chief Medical Officer for Medicago Inc., which is developing vaccines for influenza and Covid-19.

PH Lambert is a consultant for the Coalition for Epidemic Preparedness Innovations (CEPI) for the development of Case Definitions in the Brighton Collaboration format and for DSMB members training. He is also member of several vaccine DSMBs.

The following authors have no conflict of interests to disclose: J. Spergel, B. Law, E. Van Braeckel, A. Didierlaurent.Go to:


The authors are grateful for the support and helpful comments provided by the Brighton Collaboration Reference group who provided peer review, the expert reviewers of the final draft of the manuscript, Kathy Edwards, Kanta Subbarao and Neil Halsey, as well as members of the Safety Platform for Emergency Vaccines and other experts consulted as part of the process, including Stephen Evans, Steve Black, Svein R. Andersen, Miriam Sturkenboom, Wan-Ting Huang, and Robert Chen.

We acknowledge the financial support provided by the Coalition for Epidemic Preparedness Innovations (CEPI) for our work under a service order entitled Safety Platform for Emergency vACcines (SPEAC) Project with the Brighton Collaboration, a program of the Task Force for Global Health, Decatur, GA.


The findings, opinions and assertions contained in this consensus document are those of the individual scientific professional members of the working group. They do not necessarily represent the official positions of each participant’s organization (e.g., government, university, or corporation). Specifically, the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of their respective institutions.Go to:


Appendix ASupplementary data to this article can be found online at to:

Appendix A. Supplementary material

The following are the Supplementary data to this article:Supplementary data 1:Click here to view.(22K, docx)

Supplementary data 2:Click here to view.(277K, docx)

Supplementary data 3:Click here to view.(615K, docx)

Supplementary data 4:Click here to view.(31K, docx)Go to:


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COVID-19 vaccine surveillance report Week 3

Latest UK Data Shows Covid Infection RATE Among the Triple Jabbed (Boosted) Is HIGHER And RISING FASTER Than The Unvaccinated Across ALMOST EVERY Age Group

20 January 2022

Executive summary
Four coronavirus (COVID-19) vaccines have now been approved for use in the UK. Rigorous
clinical trials have been undertaken to understand the immune response, safety profile and
efficacy of these vaccines as part of the regulatory process. Ongoing monitoring of the vaccines
as they are rolled out in the population is important to continually ensure that clinical and public health guidance on the vaccination program is built upon the best available evidence. UK Health Security Agency (UKHSA), formerly Public Health England (PHE), works closely with the Medicines and Healthcare Regulatory Agency (MHRA), NHS England, and other government, devolved administration and academic partners to monitor the COVID-19 vaccination program. Details of the vaccine surveillance strategy are set on the page COVID-19: vaccine surveillance strategy (1). As with all vaccines, the safety of COVID-19 vaccines is continuously being monitored by the MHRA. They conclude that overall, the benefits of COVID-19 vaccines outweigh any potential risks (2).

Vaccine effectiveness

Several studies of vaccine effectiveness have been conducted in the UK against different
COVID-19 variants. Vaccine effectiveness against symptomatic disease with the Omicron
variant is substantially lower than against the Delta variant, with rapid waning. However,
protection against hospitalisation remains high, particularly after 3 doses.

Population impact

The impact of the vaccination program on the population is assessed by taking into account
vaccine coverage, evidence on vaccine effectiveness and the latest COVID-19 disease surveillance indicators. Vaccine coverage tells us about the proportion of the population that have received 1, 2 and 3 doses of COVID-19 vaccines. By 16 January 2022, the overall vaccine uptake in England for dose 1 was 68.9% and for dose 2 was 63.6%. Overall vaccine uptake in England in people with at least 3 doses was 48.4%. In line with the program rollout, coverage is highest in the oldest age groups.

We present data on COVID-19 cases, hospitalizations and deaths by vaccination status. These
raw data should not be used to estimate vaccine effectiveness as the data does not take into account inherent biases present such as differences in risk, behavior and testing in the
vaccinated and unvaccinated populations. Vaccine effectiveness is measured in other ways as
detailed in the ‘Vaccine Effectiveness’ section. Based on antibody testing of blood donors, 98.7% of the adult population now have antibodies to COVID-19 from either infection or vaccination compared to 24.1% that have antibodies from infection alone.

COVID-19 vaccine surveillance report – week 3- 4

Vaccine effectiveness

Large clinical trials have been undertaken for each of the COVID-19 vaccines approved in the
UK which found that they are highly efficacious at preventing symptomatic disease in the
populations that were studied. The clinical trials have been designed to be able to assess the
efficacy of the vaccine against laboratory confirmed symptomatic disease with a relatively short follow up period so that effective vaccines can be introduced as rapidly as possible. Post implementation real world vaccine effectiveness studies are needed to understand vaccine effectiveness against different outcomes (such as severe disease and onwards transmission), effectiveness in different subgroups of the population and against different variants as well as to understand the duration of protection. Vaccine effectiveness is estimated by comparing rates of disease in vaccinated individuals to rates in unvaccinated individuals.

Below we outline the latest real-world evidence on vaccine effectiveness from studies in UK
populations. Where available we focus on data related to the Omicron variant which is currently dominant in the UK. The findings are also summarized in Table 2.

Effectiveness against symptomatic disease

Vaccine effectiveness against symptomatic COVID-19 has been assessed in England based on
community testing data linked to vaccination data from the National Immunisation Management
System (NIMS), cohort studies such as the COVID Infection Survey and GP electronic health
record data. After 2 doses of AstraZeneca vaccine, vaccine effectiveness against the Omicron
variant starts at 45 to 50% then drops to almost no effect from 20 weeks after the second dose.
With 2 doses of Pfizer or Moderna effectiveness dropped from around 65 to 70% down to
around 10% by 20 weeks after the 2nd dose. 2 to 4 weeks after a booster dose of either the
Pfizer or Moderna vaccine, effectiveness ranges from around 65 to 75%, dropping to 55 to 65%
at 5 to 9 weeks and 45 to 50% from 10+ weeks after the booster. Vaccine effectiveness
estimates for the booster dose are very similar, irrespective of the primary course received (3).
Vaccine effectiveness is generally slightly higher in younger compared to older age groups.

SEE FULL REPORT by selecting link below:

VAERS Summary for COVID-19 Vaccines through 11/05/2021

Official Reported Vaccine Adverse Events in the FDA Data Base

For a Complete Report of All Vaccine Reported Adverse Events Through November 5th, 2021 Click Link Below: