At least 58% of U.S. population has natural antibodies from previous Covid infection, CDC says

Authors: Spencer Kimball PUBLISHED TUE, APR 26 2022 CNBC

KEY POINTS

  • Three out of every 5 people in the U.S. now have antibodies from a previous Covid-19 infection, according to a new CDC analysis.
  • The proportion is even higher among children, demonstrating how widespread the virus was during the winter omicron surge.
  • CDC officials told reporters on a call Tuesday that the study did not measure whether people with prior infections had high enough antibody levels to protect against reinfection and severe illness.
  • However, CDC Director Dr. Rochelle Walensky said health officials believe there is a lot of protection against the virus in communities from vaccination, boosting and infection taken together.

Three out of every 5 people in the U.S. now have antibodies from a previous Covid-19 infection with the proportion even higher among children, demonstrating how widespread the virus was during the winter omicron surge, according to data from the Centers for Disease Control and Prevention.

The proportion of people with natural Covid antibodies increased substantially from about 34% of the population in December to about 58% in February during the unprecedent wave of infection driven by the highly contagious omicron variant. The CDC’s analysis didn’t factor in people who had antibodies from vaccination.

The CDC published the data in its Morbidity and Mortality Weekly Report on Tuesday.

The increase in antibody prevalence was most pronounced among children, indicating a high rate of infection among kids during the winter omicron wave. About 75% of children and teenagers now have antibodies from past Covid infections, up from about 45% in December.

The high rate of infection among children is likely due to lower vaccination rates than adults. Only 28% of children 5- to 11-years-old and 59% of teens 12- to 17-years-old were fully vaccinated as of April. Children under 5-years-old are not yet eligible for vaccination.

About 33% of people ages 65 and older, the group with the highest vaccination rate, had antibodies from infection. Roughly 64% of adults ages 18 to 49 and 50% of people 50 to 64 had the antibodies.

The CDC analyzed about 74,000 blood samples every month from September through January from a national commercial lab network. The sample size decreased to about 46,000 in February. The CDC tested the samples for a specific type of antibody that is produced in response to Covid infection, not from vaccination.

CDC officials told reporters on a call Tuesday that the study did not measure whether people with prior infections had high enough antibody levels to protect against reinfection and severe illness. However, CDC Director Dr. Rochelle Walensky said health officials believe there is a lot of protection in communities across the country from vaccination, boosting and infection taken together, while cautioning that vaccination is the safest strategy to protect yourself against the virus.

“Those who have detectable antibody from prior infection, we still continue to encourage them to get vaccinated,” Walensky told reporters during the call. “We don’t know when that infection was. We don’t know whether that protection has waned. We don’t know as much about that level of protection than we do about the protection we get from both vaccines and boosters.”

Scientists in Qatar affiliated with Cornell University found that natural infection provides about 73% protection against hospitalization if a person is reinfected with BA.2. However, three doses of Pfizer’s vaccine provided much higher protection against hospitalization at 98%. The study, published in March, has not undergone peer-review.

About 66% of the U.S. population is fully vaccinated and 77% have received at least one dose, according to data from the CDC.

Infections and hospitalizations have dropped more than 90% from the peak of the omicron wave in January when infections in the U.S. soared to an average of more than 800,000 a day. New cases are rising again due to the BA.2 subvariant. Another subvariant, BA.2.12.1, is now gaining ground in the U.S., representing about 29% of new infections, according to CDC data. Walensky said the public health agency believes BA.2.12.1 spreads about 25% faster than BA.2. However, she said the CDC does not expect to see more severe disease from BA.2.12.1though studies are ongoing.

More than 98% of the U.S. population lives in areas where they do not need to wear masks indoors under CDC guidance due to low Covid community levels, which takes into account both infections and hospitalizations. A U.S. district judge last week struck down the CDC’s mask mandate for public transportation, though the Justice Department has filed an appeal. Walensky said the CDC continues to recommend that people wear masks on public transportation.

Consequences of COVID-19 for the Pancreas

Authors: Urszula Abramczyk,1,*Maciej Nowaczyński,2Adam Słomczyński,2Piotr Wojnicz,2Piotr Zatyka,2 and Aleksandra Kuzan1 Int J Mol Sci. 2022 Jan; 23(2): 864.Published online 2022 Jan 13. doi: 10.3390/ijms23020864

Abstract

Although coronavirus disease 2019 (COVID-19)-related major health consequences involve the lungs, a growing body of evidence indicates that COVID-19 is not inert to the pancreas either. This review presents a summary of the molecular mechanisms involved in the development of pancreatic dysfunction during the course of COVID-19, the comparison of the effects of non-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on pancreatic function, and a summary of how drugs used in COVID-19 treatment may affect this organ. It appears that diabetes is not only a condition that predisposes a patient to suffer from more severe COVID-19, but it may also develop as a consequence of infection with this virus. Some SARS-CoV-2 inpatients experience acute pancreatitis due to direct infection of the tissue with the virus or due to systemic multiple organ dysfunction syndrome (MODS) accompanied by elevated levels of amylase and lipase. There are also reports that reveal a relationship between the development and treatment of pancreatic cancer and SARS-CoV-2 infection. It has been postulated that evaluation of pancreatic function should be increased in post-COVID-19 patients, both adults and children.

1. Effects of Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome-Related Coronavirus (MERS-CoV) on the Pancreas

Coronaviruses are enveloped, single- and positive-stranded RNA viruses that infect birds and mammals. In humans, coronaviruses cause respiratory tract infection, usually the common cold, but they can also cause severe respiratory illness including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), caused by severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), respectively [1]. Coronaviruses tend to cause epidemics and even pandemics. The first coronavirus pandemic was the SARS outbreak in 2002–2003 [2]. With the experience gained during the SARS pandemic, it was possible to more quickly identify subsequent outbreaks of the MERS epidemic in 2012 [3]. The pathomechanism of both viruses is very similar—they even both use transmembrane protease serine 2 (TMPRSS2), except SARS-CoV uses angiotensin-converting enzyme 2 (ACE2) as its receptor, whereas MERS uses dipeptidyl peptidase-4 (DPP4) [4,5]. Moreover, there is a difference in terms of the severity and frequency of symptoms, which was observed in MERS patients as more frequent hospitalization in the intensive care unit (ICU) compared to SARS patients [2] (Table 1). Diabetes was one of the significant and independent predictors for developing severe SARS-CoV and MERS-CoV [6,7,8]. In MERS, no viral antigen was detected in any tissue other than pneumocytes [7], despite multiple organ dysfunction syndrome in critically ill patients. In SARS-CoV, the presence of the virus was detected not only in respiratory epithelial cells, but also in small intestinal and colonic epithelial cells, in which it also revealed replication features [9]. It is known that the ACE2 receptor is also present in tissues such as the heart, kidney, and pancreas [8,9]. According to some authors, the presence of the receptor is sufficient for tissue entry and pathogenic activity, although other researchers do not support this thesis [9,10]. Yang et al. were some of the first researchers who hypothesized that SARS coronavirus enters islets using ACE2 as its receptor and damages islets causing acute diabetes [8]. Yang’s study revealed that SARS-CoV had a much higher affinity for pancreatic islet cells than for pancreatic exocrine cells, which was consistent with the hyperglycemia observed in some patients and rarely reported acute pancreatitis (AP) [8]. Furthermore, insulin-dependent diabetes mellitus (IDDM) and high fasting blood glucose values were observed in some inpatients [8]. A 3-year follow-up revealed that both abnormalities were transient, which may be indicative of only temporary damage to the pancreatic islets [8]. However, another reason (different from that given by Young et al.) for high fasting blood glucose value in patients may result from increased stress hormones release. Cortisol, catecholamines, growth hormone, and glucagon, which are released during infection, fever, and trauma, can lead to hyperglycemia to the same degree as SARS-CoV can [11]. No information was found in the literature about a direct impact of the MERS virus on the pancreas or on glycemia during or after infection. This may be due to an insufficiently detailed analysis of the available data during previous studies that oscillated primarily, for laboratory tests, between complete blood count (CBC), lactate dehydrogenase (LDH), urea, and creatinine analysis. A summary of SARS-CoV, MERS, and SARS-CoV-2 is shown in Table 1.Table 1. The summary of characteristics of SARS and MERS coronaviruses. Dipeptidyl peptidase-4 (DPP4), transmembrane protease serine 2 (TMPRSS2), hospitalization in the intensive care unit (ICU), and cathepsin L (CTSL).

Table

In 2019, a new coronavirus named SARS-CoV-2 was identified, causing COVID-19. This virus has many characteristics that are analogous to SARS-CoV, for example, ACE2 is also used as its receptor [12]. Patients with diabetes are among those with the most severe forms of COVID-19 and related mortality; insights from recent experience can guide future management [17], particularly for the consequences on the pancreas. As the COVID-19 pandemic has been ongoing for nearly two years, this study aims to collect data concerning the impact of SARS-CoV-2 on the pancreas and analyze them to estimate the future health consequences of COVID-19 in populations.

2. Pancreatic Damage during Diabetes Mellitus and COVID-19

Pancreas tissue damage may cause to the lack of control over normal blood glucose levels in the body. Type 1 diabetes (T1D) is caused by insulin deficiency due to βcell dysfunction of immunologic or idiopathic cause. In contrast, β pancreatic cells in type 2 diabetes (T2D) become depleted over time due to compensatory insulin secretion caused by insulin resistance. There is also type 3 diabetes (T3D), which is described as diabetes associated with the development of Alzheimer’s disease [18]. It should not be confused with type 3c (pancreatogenic) diabetes, which relates to the exocrine and digestive functions of the pancreas. The issue concerning the impairing effect of hyperglycemia (glucotoxicity) on the secretory function of the islets of Langerhans has also been increasingly raised. In addition to endocrine dysfunction, some diabetic patients may also develop moderate exocrine pancreatic insufficiency (EPI), in which pancreatic enzyme secretion is impaired. EPI can be observed in almost all patients with type 3c (pancreatogenic) diabetes (secondary to pancreatic pathology), whereas the prevalence of this dysfunction in patients with T1D or T2D is 40% and 27%, respectively [19].With the ongoing SARS-CoV-2 pandemic, patients with reduced normal pancreatic function are at high risk for COVID-19 requiring hospitalization. In particular, elevated blood glucose levels in patient with and without diabetes makes them at high risk of mortality [20]. Hyperglycemia impairs the immune response (e.g., by reducing the activity of macrophages and polymorphonuclear leukocytes), which in addition influences the excessive cytokine response, and thus has a strong proinflammatory effect.The receptors for ACE2, which are also present in the pancreas, are a target of SARS-CoV-2 in the body, which may result in acute failure of both the islets of Langerhans and exocrine cells [15]. Infection-induced, transient β cell dysfunction may cause an uncontrolled hyperglycemic state, especially in patients whose pancreas is already affected by diabetes mellitus. Persistent hyperglycemia usually predisposes to severe COVID-19 and to viral infection complicated by secondary infections. The aforementioned risk can be found in T1D, T2D, and gestational diabetes mellitus (GDM). In T2D patients, the much more frequent coexistence of other risk factors such as atherosclerosis, hypertension, and obesity should be taken into consideration, which usually implies a worse prognosis for the course of COVID-19 [21,22]. In GDM, SARS-CoV-2 infection not only increases the risk of more severe course of the disease in a patient, but may also result in diabetic fetopathy or, in more advanced pregnancies, increase the risk of future pathologies involving glucose metabolism (such as T2D) in a child [23].

3. Pancreatic Damage in Patients without Pre-Existing Diabetes Infected with SARS-CoV-2

It has been postulated that, either by direct invasion of pancreatic cells by the virus or by indirect mechanisms described below, SARS-CoV-2 has a destructive effect on the pancreas and can lead to insulin deficiency and development of T1D [24].If the hypothesis that SARS-CoV-2 infection causes hyperglycemia is true, increased statistics of new T1D cases should be observed. Indeed, there are publications that describe such a phenomenon. For instance, Unsworth et al. and Kamrath et al. describe an increase in new-onset T1D in children during the COVID-19 pandemic [16,25]. Although pancreatic β cell damage induced transient hyperglycemia in SARS-CoV, it is still unclear whether β cell damage is transient or permanent in SARS-CoV-2 [22]. This information appears to be of great importance because COVID-19 in children is frequently considered “harmless”. Therefore, it is reasonable to sensitize parents to the fact that the consequences of COVID-19 may be potentially dangerous for their children.Below you will find the proposed molecular mechanisms that may participate in pancreatic damage that causes carbohydrate metabolism disorders.

4. Etiology Associated with ACE2, TMPRSS2, and Na+/H+ Exchanger

As previously mentioned, SARS-CoV infection of host cells is facilitated by ACE2, but also by the transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL) [13].ACE2 is an enzyme that is expressed to varying degrees in most cells of the human body [14,26,27]. This enzyme catalyzes the conversion of angiotensin II to angiotensin 1–7, taking part in the maintenance of body homeostasis by influencing the regulation of blood pressure and water–electrolyte balance through the renin–angiotensin–aldosterone (RAA) system [28]. Moreover, ACE2/angiotensin (1–7) stimulates insulin secretion, reduces insulin resistance, and increases pancreatic βcell survival [27,28].In addition to the key role it plays in maintaining body homeostasis, ACE2 is now also the best-studied target for SARS-CoV-2 S glycoprotein, enabling infection of host cells [27,29]. ACE2 in the pancreas is expressed mainly within the pericytes of pancreatic microvessels and to a lesser extent on the surface of the islets of Langerhans, including pancreatic β cells [30]. SARS-CoV-2 shows 10–20 times more activity against ACE2 than SARS-CoV, which significantly increases the infectivity of SARS-CoV-2 [31,32]. Furthermore, studies indicate that SARS-CoV may also downregulate ACE2 expression in cells. This causes an imbalance between ACE and ACE2, consequently leading to blood pressure disorders and systemic inflammation [27,33,34]. Due to the 79% genetic similarity between SARS-CoV and SARS-CoV-2 [35], it is speculated that ACE2 expression may also be downregulated during SARS-CoV-2 infection, causing i.a. MODS observed in COVID-19 [27].During cell infection by SARS-CoV-2, in addition to the role played by ACE2, it is also appropriate to consider the significant pathogenic role of TMPRSS2 that is necessary for the preparation of S glycoprotein by its cleavage, thereby enabling fusion of the virus with the host cell [36,37]. The S1 and S2 domains can be distinguished in the SARS-CoV-2 S glycoprotein. The S1 domain is involved in binding to the ACE2 receptor and then TMPRSS2 intersects with the S protein, including at the boundary of the S1 and S2 domains and within the S2 domain, which enables the virus–cell fusion [38,39]. According to studies, TMPRSS2 expression is significantly increased in obese patients, which may contribute to the poorer prognosis that is observed during COVID-19 in this patient group [40]. Moreover, obese patients are frequently already burdened with problems such as insulin resistance at baseline, while the presence of ACE2 and TMPRSS2 within the pancreas as a binding site for SARS-CoV-2 may exacerbate insulin resistance causing problems in terms of diabetes management in COVID-19 patients.There are also other mechanisms by which COVID-19 may affect the development of hyperglycemia. It is reported that the virus may also affect the glucose regulation through the Na+/H+ exchanger and lactate pathways. The mechanism is that angiotensin II, which accumulates during infection, contributes to insulin resistance and—by activating the Na+/H+ exchanger in the pancreas—it leads to hypoxia and extracellular acidification, which, through the accumulation of calcium and sodium ions in the cells and the production of reactive oxygen species, damages pancreatic tissue [41]. Simultaneously, the concentration of lactate increases, which in COVID-19 infection is intensively released, among other things, from adipose tissue, and then monocarboxylate transporters transport lactate and H+ ion inward in the cell, which increases Na+/H+ exchanger activation, further disrupting pancreatic homeostasis [41].

5. The Etiology Associated with a Systemic Proinflammatory Environment, Immune System Aggression, and Production of Novel Autoantigens

A broad spectrum of proinflammatory cytokines, such as IL-2, IL-6, IL-7, IL-8, interferon-γ, and Tumor Necrosis Factor α (TNF-α), is released during, in particular severe, COVID-19 infection [42,43,44]. Based on current studies, it is reasonable to suspect that these cytokines are released in response to the binding of the virus to ACE2 receptors that are also located in the pancreas [9,42]. The cause of pancreatic damage during COVID-19 is the cytokine storm that plays a key role in this case, because in both acute pancreatitis (AP) and severe COVID-19, elevated levels of the aforementioned interleukins are associated with the severity of these both disease entities. Particular attention should be paid to IL-6, because it is suspected to play a key role in the pathogenesis of AP as well as acute respiratory distress syndrome (ARDS) that is the most common and most severe clinical manifestation of COVID-19. In COVID-19-induced ARDS, IL-6 levels are correlated with disease-related mortality [45,46,47]. At the same time, high IL-6 levels correlate with an increased risk of developing severe pancreatitis [48,49].The production of neutralizing antibodies is also an important response of the body in the course of COVID-19 [50,51,52]. It has been observed that early seroconversion and very high antibody titers occur in patients with severe SARS-CoV-2 infection [53,54]. The available literature details a mechanism called antibody-dependent enhancement (ADE), which is associated with a pathological response of the immune system [53]. ADE exploits the existence of FcRS receptors located on various cells of the immune system, for example, macrophages and B lymphocytes [53]. This relationship may lead to a likely bypass of the classical viral infection pathway by ACE2, and virus–antibody complexes may stimulate macrophages to overproduce cytokines including significant IL-6 [53,55].Molecular mimicry may be also one of potential causes of pancreatic cell damage [56]. There are similarities in the protein structure of the virus and β-pancreatic cells, which may induce cross-reactivity and lead to autoimmunity [56]. Furthermore, viral infection may also lead to increased cytokine secretion by surrounding dendritic cells and activation of naive T cells in genetically predisposed individuals [56].

6. Pancreatitis in COVID-19

Although the impact of the discussed coronavirus-induced disease on exocrine function is not fully understood, available literature is not able to unambiguously determine whether the tissue damage leading to AP occurs as a result of direct SARS-CoV-2 infection [57] or as a result of systemic MODS with increased levels of amylase and lipase [42]. Liu et al.’s study involving 121 COVID-19 patients with a mean age of 57 years and a variable course of infection proved above-normal levels of amylase and lipase in 1–2% of patients with moderate COVID-19 infection and in 17% of patients with severe COVID-19 infection. This may support the hypothesis that SARS-CoV-2-induced disease has a destructive effect not only on the endocrine portion of this gland, but also on the exocrine one [15].However, elevated levels of pancreatic enzymes in question do not have to mean the destruction of pancreatic cells—after all, such a situation may occur during kidney failure or diarrhea in the course of COVID-19. Furthermore, there remains the question of the effect of drugs administered during SARS-CoV-2 infection on changes in pancreatic function [42], discussed further in this article.According to the International Association of Pancreatology (IAP) and the American Pancreatic Association (APA), the diagnosis of AP is based on meeting two out of three of the following criteria: clinical (epigastric pain), laboratory (serum amylase or lipase > 3 × upper limit of normal), and/or imaging criteria (computed tomography, magnetic resonance imaging, ultrasound) [58]. Pancreatic lipase is considered as a potential marker of SARS-CoV-2 severity with concomitant AP. In Hemant Goyal et al.’s study, as many as 11.7% out of 756 COVID-19 patients had hyperlipidemia and they were three times more likely to have severe COVID-19 [59]. Those with higher lipase levels—17% out of 83 patients—required hospitalization [60]. However, it is difficult to distinguish whether these patients required hospitalization for severe systemic COVID-19 infection or for pancreatitis in the course of COVID-19 infection.AP in the course of COVID-19 was analyzed in different age groups; however, some studies only involve children [61]. Compared to pancreatic islet cells, cells of the exocrine pancreatic ducts are more abundant in ACE2 and TMPRSS2 that are necessary for the virus to penetrate the cell [62]. Infection of these cells may be one of the causes of AP [63]. Infections, both bacterial and viral, are one of the causes of AP. The definitive mechanism of how viral infections affect pancreatic cells is not known; however, a study by Maria K Smatti et al. found that there is infection of pancreatic islet cells and replication of the virus within them, ultimately resulting in autoimmune reactions that eventually affect both diabetes and AP in a negative way [64]. For non-SARS-CoV-2 patients, the etiology of AP is known and confirmed in most cases, although 69% of those undergoing infection do not have definite etiology of AP while meeting the AP-Atlanta criteria for diagnosis [65].Hegyi et al. show the mechanism of MODS formation during COVID-19 infection and AP [66]. This is lipotoxicity, involving an interstitial increase in pancreatic lipase levels, which leads to the breakdown of triacylglycerols contained in adipose tissue cells and the release of unsaturated fatty acids. These in turn exert a toxic effect on mitochondria causing the release of cytokines, which results in a cytokine storm.There is also a hypothesis, which claims that AP can develop because of blood circulatory centralization resulting from uncontrolled cytokine storm created by SARS-CoV-2 infection [67]. There exist reports that say that pancreatic ischemia may be the cause of different degrees of acute pancreatitis [68,69]. This statement can be supported by the reports that state that pancreatic blood reperfusion inhibits the development of AP and accelerate pancreas recovery [70].Another mechanism of developing AP during COVID-19 may be a coagulation cascade activation caused by active inflammatory process due to SARS-CoV-2 infection [71]. The ongoing inflammatory process causes not only hemostasis imbalance for blood clotting, but it also leads to intensification of coagulation by removing epithelial cell protein C receptor (EPCR) from epithelial by the means of inflammatory mediators and thrombin [71]. This means that both processes intensify each other. Simultaneously, it was proved that COVID-19 predisposes patients to venous thromboembolism resulting from excessive inflammation, platelet activation, and endothelial dysfunction [72]. It is also important to notice that AP is inherently connected with a coagulation cascade activation, increased fibrinolysis and, hence, higher level of D-dimers [73]. Acute pancreatitis severity may depend on hemostasis imbalance; local coagulation results in mild AP whereas, in more severe AP cases, the imbalance may lead to development of disseminated intravascular coagulation (DIC) [74]. These observations have been supported by the results of experimental studies showing that the inhibition of coagulation reduces the development of AP [75,76,77] and exhibits therapeutic effect in this disease [78,79]. Additionally it is worth noticing that infection-related hyperglycemia has powerful inflammation-promoting effects on the organism (especially when organism is under stress), thus increasing the number of inflammatory mediators [74]. Unfortunately, it is impossible to decide which process is dominant in causing AP in COVID-19 patients: local inflammation caused by SARS-CoV-2 or systemic hemostasis imbalance.Clinical reports on low molecular weight heparin (LMWH) treatment in AP seem to emphasize a more significant role of hemostasis imbalance in causing AP [74,80,81]. Heparin is extremely significant in the treatment of COVID19 patients due to its properties, mainly its similarity to heparan sulphate, which appears in a respiratory tract, its interactions with SARS-CoV-2 S protein, leading to viral adhesion inhibiting to the cell membrane [82], and its anti-inflammatory effects. Thanks to these properties, heparin may not only show its therapeutic effect as the anticoagulant, but also its protective role in acute pancreatitis or respiratory inflammations [83,84,85].

7. Drugs Used against SARS-CoV-2 Infection (Glucocorticoids, Lopinavir, Ritonavir, Remedesivir, Interferon-β1 (IFN-β1), and Azithromycin) Induce Pancreatic β Cell Damage

Statistical analyses revealed a significantly higher incidence of AP with the concomitant systemic use of glucocorticosteroids (GCS) [86]. In one study analyzing the development of drug-induced AP, dexamethasone, was classified as type IB—there was one case report in which administration of this drug-induced AP occurred; however, other causes of pancreatitis such as alcohol consumption could not be excluded [87]. Other GCS such as hydrocortisone, prednisone, and prednisolone were used in patients with mild to moderate AP; however, they cannot be classified into any group because they are frequently used together with other drugs that cause AP [86,87]. However, it has been determined that GCS independently increase the risk of AP, and patients with residual AP risk factors during GCS treatment should be more monitored for the development of AP [23]. Javier A. Cienfuegos et al. additionally observed that one of mechanisms of AP formation in COVID-19 patients may be GCS administered at the time of admission to the ICU with severe respiratory failure [88]. Because GCS were used in severe COVID-19 cases, it is difficult to say what true reason for AP was—either a severe course of COVID-19 or GCS application or both.GCS are used in the treatment of many diseases due to their immunosuppressive and anti-inflammatory nature. They induce diabetes in previously healthy patients as well as significantly exacerbate diabetes in diabetic patients [89,90]. Diabetes develops in these patients likely due to pancreatic β cell dysfunction, decreased insulin secretion, and increased insulin resistance in other tissues, which may depend on the timing and the dose of GCS used [89,91]. Long-acting or intermediate-acting insulin alone or combined with short-acting insulin should be used during the treatment [90]. At the same time, no advantage was found over the use of oral hypoglycemics [92]. Certainly, patients after long-term GCS therapy will need further observation for diabetes.Lopinavir/ritonavir was classified in the previously mentioned study as a type IV drug—medications reported with little information [87]. Both drugs are included in the group of antiretrovirals that act as protease inhibitors, and they are primarily used for HIV infection. Although Lopinavir is an active drug, it is not used alone. There have been reports about the occurrence of AP during the use of protease inhibitors in question, which is also described in the Summary of Product Characteristics (SmPC) of products approved by Committee for Medicinal Products for Human Use (CHMP). It has been proved that the use of lopinavir/ritonavir causes hyperglycemia [93,94].Remdesivir is an adenosine analogue with antiviral activity. There are single reports about the occurrence of pancreatitis as a result of the use of the aforementioned medication [95,96]. At the same time, it should be noted that other nucleoside-derivative drugs may cause pancreatitis [97].The current state of knowledge does not clearly indicate the therapeutic benefit of interferon-β in the treatment of COVID-19 patients [98,99]. To date, only single cases suggesting induction of pancreatitis by interferon-β have been reported. Based on this, Badalov et al. classified interferon into type III [87].There are few reports about the development of AP due to the use of azithromycin [100]. In the previously mentioned study by Badalov et al., two macrolide antibiotics were classified as type II and III. Unfortunately, there are no direct data concerning azithromycin. Interestingly, there were cases of patients with concomitant symptoms of AP and viral pneumonia caused by SARS-CoV-2 who were treated with azithromycin, which resulted in complete resolution of symptoms for both conditions [96,101]. Based on available data, the risk of azithromycin-induced AP is low.There is no clear evidence that azithromycin affects blood glucose levels in humans. However, it is known for its prokinetic effects, which may be helpful in patients who suffer from diabetic gastroparesis [102]). The incidence of hypo- and hyperglycemic episodes was not proved to be significant for azithromycin [103]; however, the risk of dysglycemia is emphasized [94]. In the SmPC, where azithromycin is the main ingredient, it is not possible to establish a causal relationship between the occurrence of pancreatitis and taking medications (Zithromax) based on the available data. In contrast, glycemic disturbances were not indicated as side effects (Zithromax) [104].Hydroxychloroquine has been extensively promoted for COVID-19 due to its anti-inflammatory and antiviral action; yet, the use of this agent in diabetes deserves particular attention for its documented hypoglycemic action, and its benefit on COVID-19 is controversial, although there is large usage [105].Table 2 shows a comparison of the side effects of medications in question.Table 2. Side effects of medications used in SARS-CoV-2 infection in the area of pancreatic effects and hyperglycemia.

Table

8. COVID-19, Pancreas, and Glycation

In T2D diabetics, oxidative stress leading to pancreatic damage may be stimulated by, among other things, the intense glycation that accompanies hyperglycemia [24]. Glycation is a non-enzymatic process involving reducing sugar and amino groups of proteins, which contributes to the formation of advanced glycation end products (AGEs). These products have significantly altered biochemical properties relative to the substrates, including proteins that have altered conformation, increased rigidity, resistance to proteolysis, etc. [106,107].Part of the pathomechanism involved in facilitating coronavirus infection in diabetics may be due to glycation of ACE2 and SARS-CoV-2 spike protein [108,109].An interesting hypothesis is that COVID-19 has a worse prognosis in patients with intense glycation, and thus high tissue AGE content. Glycated hemoglobin (HbA1c) is a commonly used diagnostic tool that estimates intensity of glycation. The parameter is not only a marker of long-term persistent hyperglycemia, but an active participant in immune processes, as HbA1c levels are associated with NK cell activity [110].Zhang et al.’s retrospective cohort study concerning COVID-19 patients revealed that glycated hemoglobin correlates negatively with saturation (SaO2) and positively with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and fibrinogen (Fbg). It was concluded that determination of HbA1c levels may be helpful in assessing inflammation, hypercoagulability, and prognosis of COVID-19 patients [111].According to the meta-analysis by Chen et al. (2020), Hba1c levels were slightly higher in patients with severe COVID-19 compared to patients with mild COVID-19; however, this correlation was not statistically significant. However, it is of great importance to note that only two studies analyzing HbA1c in COVID-19 patients were included in this analysis because only these studies were available in May 2020 [112].Glycation plays its physiological effects not only directly by changing the properties of various proteins, but also indirectly through various receptors. RAGE is the most common receptor for AGEs. Binding of RAGE to its ligands activates a proinflammatory response primarily by mitogen-activated protein kinase (MAPK) and nuclear factor κβ (NFκβ) pathways. This interaction was proved to be significant in the pathogenesis of cancer, diabetes mellitus, and other inflammatory disorders [113]. RAGE was found to be expressed in the pancreas, and S100P-derived RAGE antagonistic peptide (RAP) reduces pancreatic tumor growth and metastasis [113]. The implications of this fact may also apply to the etiology and treatment of COVID-19. It has been postulated that targeting RAGE by various antagonists of this receptor may inhibit damage to various organs including the pancreas [114].

9. COVID-19 vs. Pancreatic Cancer

Immunosuppression as a treatment effect, elevated cytokine levels, altered expression of receptors for SARS-CoV-2, and a prothrombotic state in patients with various types of cancer may exacerbate the effects of COVID-19 [115].Focusing on pancreatic cancer, it can be observed that the pathomechanism of both diseases—COVID-19 and tumorigenesis in the pancreas—overlap in several molecular mechanisms. As mentioned above, SARS-CoV-2 infection of host cells is facilitated by ACE-2, TMPRSS2, and CTSL. Cathepsin L is upregulated in a wide variety of cancers, including pancreatic adenocarcinoma [13]. TMPRSS2 upregulation in pancreatic cancers is moderate, whereas ACE-2 is overexpressed in some cancers, including pancreatic carcinomas [115]. Interestingly, ACE2 upregulation seems to be associated with favorable survival in pancreatic cancer [116], and it is known that SARS-CoV-2 reduces ACE2 expression [22]. Furthermore, the above-mentioned RAGE may also participate in both pancreatic cancer development and SARS-CoV-2 infection. RAGE facilitates neutrophil extracellular trap (NET) formation in pancreatic cancer [117]. In conclusion, pancreatic cancer predisposes to an increased risk of COVID-19 and its more severe course, and coronavirus infection may contribute to pancreatic cancer.It also seems important how the COVID-19 epidemic has affected the treatment of patients with pancreatic cancer of SARS-CoV-2-independent etiology. According to the study by Pergolini et al., care of patients with pancreatic cancer can be disrupted or delayed, particularly in the context of treatment selection, postoperative course, and outpatient care [118].A separate issue is how patients after pancreatoduodenectomy respond to SARS-CoV-2 infection. A case series reported by Bacalbasa reveal that patients who develop SARS-CoV-2 infection postoperatively require re-admission in the ICU and a longer hospital stay; however, these infections are not fatal [119]. Although the analysis was performed on single cases, it is concluded that these results are an argument to perform elective oncological surgeries [119].There are also reports that chemotherapy in pancreatic cancer patients who become ill between treatment series can be successfully completed after a complete cure of the infection [120]. Guidelines for, e.g., prioritization and treatment regimens regarding pancreatic cancer treatment in the era of the pandemic, are developed and described, for example, by Catanese et al. or Jones et al. [121,122].

10. Conclusions

Evidence shows that SARS-CoV-2 infection contributes to damage within the pancreas. The mechanisms that are involved in this include but are not limited to direct cytopathic effect of SARS-CoV-2 replication and systemic and local inflammatory response [123]. At the current state of knowledge, it is certain that the virus attacks the endocrine portion of the pancreas as well as, to a much lesser extent, the exocrine portion. It has been shown that a bidirectional relationship between COVID-19 and diabetes exists; indeed, diabetes is associated with COVID-19 severity and mortality but, at the same time, patients with COVID-19 have shown new onset of diabetes [124]. SARS-CoV-2 virus infection not only directly affects glycemic levels, but also exacerbates already existing hyperglycemia through its negative impact on the functional competence of the islets of Langerhans. It cannot be excluded that the real cause of exocrine dysfunction of this gland is the negative effect of the drugs used for treatment of the infection. As the pandemic progresses, special attention should be given to the evaluation of chronic and acute pancreatic diseases, including pancreatic cancer, so that faster diagnosis enables faster implementation of treatment.

Author Contributions

Conceptualization, A.K.; investigation, U.A., M.N., A.S., P.W., P.Z. and A.K.; resources, U.A., M.N., A.S., P.W., P.Z. and A.K.; writing—original draft preparation, U.A., M.N., A.S., P.W., P.Z. and A.K.; visualization, U.A.; supervision, A.K. All authors have read and agreed to the published version of the manuscript.

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SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans

Authors: Jackson S. TurnerWooseob KimElizaveta KalaidinaCharles W. GossAdriana M. RauseoAaron J. SchmitzLena HansenAlem HaileMichael K. KlebertIskra PusicJane A. O’HalloranRachel M. Presti & Ali H. Ellebedy 

Nature volume 595, pages421–425 (2021)

Abstract

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1,2,3,4,5,6,7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28,9,10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11,12,13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

Main

Reinfections by seasonal coronaviruses occur 6 to 12 months after the previous infection, indicating that protective immunity against these viruses may be short-lived14,15. Early reports documenting rapidly declining antibody titres in the first few months after infection in individuals who had recovered from COVID-19 suggested that protective immunity against SARS-CoV-2 might be similarly transient11,12,13. It was also suggested that infection with SARS-CoV-2 could fail to elicit a functional germinal centre response, which would interfere with the generation of long-lived plasma cells3,4,5,7,16. More recent reports analysing samples that were collected approximately 4 to 6 months after infection indicate that SARS-CoV-2 antibody titres decline more slowly than in the initial months after infection8,17,18,19,20,21. Durable serum antibody titres are maintained by long-lived plasma cells—non-replicating, antigen-specific plasma cells that are detected in the bone marrow long after the clearance of the antigen1,2,3,4,5,6,7. We sought to determine whether they were detectable in convalescent individuals approximately 7 months after SARS-CoV-2 infection.

Biphasic decay of anti-S antibody titres

Blood samples were collected approximately 1 month after the onset of symptoms from 77 individuals who were convalescing from COVID-19 (49% female, 51% male, median age 49 years), the majority of whom had experienced mild illness (7.8% hospitalized, Extended Data Tables 12). Follow-up blood samples were collected three times at approximately three-month intervals. Twelve convalescent participants received either the BNT162b2 (Pfizer) or the mRNA-1273 (Moderna) SARS-CoV-2 vaccine between the last two time points; these post-vaccination samples were not included in our analyses. In addition, bone marrow aspirates were collected from 18 of the convalescent individuals at 7 to 8 months after infection and from 11 healthy volunteers with no history of SARS-CoV-2 infection or vaccination. Follow-up bone marrow aspirates were collected from 5 of the 18 convalescent individuals and from 1 additional convalescent donor approximately 11 months after infection (Fig. 1a, Extended Data Tables 34). We first performed a longitudinal analysis of circulating anti-SARS-CoV-2 serum antibodies. Whereas anti-SARS-CoV-2 spike protein (S) IgG antibodies were undetectable in blood from control individuals, 74 out of the 77 convalescent individuals had detectable serum titres approximately 1 month after the onset of symptoms. Between 1 and 4 months after symptom onset, overall anti-S IgG titres decreased from a mean loge-transformed half-maximal dilution of 6.3 to 5.7 (mean difference 0.59 ± 0.06, P < 0.001). However, in the interval between 4 and 11 months after symptom onset, the rate of decline slowed, and mean titres decreased from 5.7 to 5.3 (mean difference 0.44 ± 0.10, P < 0.001; Fig. 1a). In contrast to the anti-S antibody titres, IgG titres against the 2019–2020 inactivated seasonal influenza virus vaccine were detected in all control individuals and individuals who were convalescing from COVID-19, and declined much more gradually, if at all over the course of the study, with mean titres decreasing from 8.0 to 7.9 (mean difference 0.16 ± 0.06, P = 0.042) and 7.9 to 7.8 (mean difference 0.02 ± 0.08, P = 0.997) across the 1-to-4-month and 4-to-11-month intervals after symptom onset, respectively (Fig. 1b).

figure 1
Fig. 1: SARS-CoV-2 infection elicits durable serum anti-S antibody titres.

Induction of S-binding long-lived BMPCs

The relatively rapid early decline in the levels of anti-S IgG, followed by a slower decrease, is consistent with a transition from serum antibodies being secreted by short-lived plasmablasts to secretion by a smaller but more persistent population of long-lived plasma cells generated later in the immune response. The majority of this latter population resides in the bone marrow1,2,3,4,5,6. To investigate whether individuals who had recovered from COVID-19 developed a virus-specific long-lived BMPC compartment, we examined bone marrow aspirates obtained approximately 7 and 11 months after infection for anti-SARS-CoV-2 S-specific BMPCs. We magnetically enriched BMPCs from the aspirates and then quantified the frequencies of those secreting IgG and IgA directed against the 2019–2020 influenza virus vaccine, the tetanus–diphtheria vaccine and SARS-CoV-2 S by enzyme-linked immunosorbent spot assay (ELISpot) (Fig. 2a). Frequencies of influenza- and tetanus–diphtheria-vaccine-specific BMPCs were comparable between control individuals and convalescent individuals. IgG- and IgA-secreting S-specific BMPCs were detected in 15 and 9 of the 19 convalescent individuals, respectively, but not in any of the 11 control individuals (Fig. 2b). Notably, none of the control individuals or convalescent individuals had detectable S-specific antibody-secreting cells in the blood at the time of bone marrow sampling, indicating that the detected BMPCs represent bone-marrow-resident cells and not contamination from circulating plasmablasts. Frequencies of anti-S IgG BMPCs were stable among the 5 convalescent individuals who were sampled a second time approximately 4 months later, and frequencies of anti-S IgA BMPCs were stable in 4 of these 5 individuals but had decreased to below the limit of detection in one individual (Fig. 2c). Consistent with their stable BMPC frequencies, anti-S IgG titres in the 5 convalescent individuals remained consistent between 7 and 11 months after symptom onset. IgG titres measured against the receptor-binding domain (RBD) of the S protein—a primary target of neutralizing antibodies—were detected in 4 of the 5 convalescent individuals and were also stable between 7 and 11 months after symptom onset (Fig. 2d). Frequencies of anti-S IgG BMPCs showed a modest but significant correlation with circulating anti-S IgG titres at 7–8 months after the onset of symptoms in convalescent individuals, consistent with the long-term maintenance of antibody levels by these cells (r = 0.48, P = 0.046). In accordance with previous reports22,23,24, frequencies of influenza-vaccine-specific IgG BMPCs and antibody titres exhibited a strong and significant correlation (r = 0.67, P < 0.001; Fig. 2e). Nine of the aspirates from control individuals and 12 of the 18 aspirates that were collected 7 months after symptom onset from convalescent individuals yielded a sufficient number of BMPCs for additional analysis by flow cytometry. We stained these samples intracellularly with fluorescently labelled S and influenza virus haemagglutinin (HA) probes to identify and characterize antigen-specific BMPCs. As controls, we also intracellularly stained peripheral blood mononuclear cells (PBMCs) from healthy volunteers one week after vaccination against SARS-CoV-2 or seasonal influenza virus (Fig. 3a, Extended Data Fig. 1a–c). Consistent with the ELISpot data, low frequencies of S-binding BMPCs were detected in 10 of the 12 samples from convalescent individuals, but not in any of the 9 control samples (Fig. 3b). Although both recently generated circulating plasmablasts and S- and HA-binding BMPCs expressed BLIMP-1, the BMPCs were differentiated by their lack of expression of Ki-67—indicating a quiescent state—as well as by higher levels of CD38 (Fig. 3c).

figure 2
Fig. 2: SARS-CoV-2 infection elicits S-binding long-lived BMPCs.
figure 3
Fig. 3: SARS-CoV-2 S-binding BMPCs are quiescent and distinct from circulating plasmablasts.

Robust S-binding memory B cell response

Memory B cells form the second arm of humoral immune memory. After re-exposure to an antigen, memory B cells rapidly expand and differentiate into antibody-secreting plasmablasts. We examined the frequency of SARS-CoV-2-specific circulating memory B cells in individuals who were convalescing from COVID-19 and in healthy control individuals. We stained PBMCs with fluorescently labelled S probes and determined the frequency of S-binding memory B cells among isotype-switched IgDloCD20+ memory B cells by flow cytometry. For comparison, we co-stained the cells with fluorescently labelled influenza virus HA probes (Fig. 4a, Extended Data Fig. 1d). S-binding memory B cells were identified in convalescent individuals in the first sample that was collected approximately one month after the onset of symptoms, with comparable frequencies to influenza HA-binding memory B cells (Fig. 4b). S-binding memory B cells were maintained for at least 7 months after symptom onset and were present at significantly higher frequencies relative to healthy controls—comparable to the frequencies of influenza HA-binding memory B cells that were identified in both groups (Fig. 4c).

figure 4
Fig. 4: SARS-CoV-2 infection elicits a robust memory B cell response.

Discussion

This study sought to determine whether infection with SARS-CoV-2 induces antigen-specific long-lived BMPCs in humans. We detected SARS-CoV-2 S-specific BMPCs in bone marrow aspirates from 15 out of 19 convalescent individuals, and in none from the 11 control participants. The frequencies of anti-S IgG BMPCs modestly correlated with serum IgG titres at 7–8 months after infection. Phenotypic analysis by flow cytometry showed that S-binding BMPCs were quiescent, and their frequencies were largely consistent in 5 paired aspirates collected at 7 and 11 months after symptom onset. Notably, we detected no S-binding cells among plasmablasts in blood samples collected at the same time as the bone marrow aspirates by ELISpot or flow cytometry in any of the convalescent or control samples. Together, these data indicate that mild SARS-CoV-2 infection induces a long-lived BMPC response. In addition, we showed that S-binding memory B cells in the blood of individuals who had recovered from COVID-19 were present at similar frequencies to those directed against influenza virus HA. Overall, our results are consistent with SARS-CoV-2 infection eliciting a canonical T-cell-dependent B cell response, in which an early transient burst of extrafollicular plasmablasts generates a wave of serum antibodies that decline relatively quickly. This is followed by more stably maintained levels of serum antibodies that are supported by long-lived BMPCs.

Although this overall trend captures the serum antibody dynamics of the majority of participants, we observed that in three participants, anti-S serum antibody titres increased between 4 and 7 months after the onset of symptoms, after having initially declined between 1 and 4 months. This could be stochastic noise, could represent increased net binding affinity as early plasmablast-derived antibodies are replaced by those from affinity-matured BMPCs, or could represent increases in antibody concentration from re-encounter with the virus (although none of the participants in our cohort tested positive a second time). Although anti-S IgG titres in the convalescent cohort were relatively stable in the interval between 4 and 11 months after symptom onset, they did measurably decrease, in contrast to anti-influenza virus vaccine titres. It is possible that this decline reflects a final waning of early plasmablast-derived antibodies. It is also possible that the lack of decline in influenza titres was due to boosting through exposure to influenza antigens. Our data suggest that SARS-CoV-2 infection induces a germinal centre response in humans because long-lived BMPCs are thought to be predominantly germinal-centre-derived7. This is consistent with a recent study that reported increased levels of somatic hypermutation in memory B cells that target the RBD of SARS-CoV-2 S in convalescent individuals at 6 months compared to 1 month after infection20.

To our knowledge, the current study provides the first direct evidence for the induction of antigen-specific BMPCs after a viral infection in humans. However, we do acknowledge several limitations. Although we detected anti-S IgG antibodies in serum at least 7 months after infection in all 19 of the convalescent donors from whom we obtained bone marrow aspirates, we failed to detect S-specific BMPCs in 4 donors. Serum anti-S antibody titres in those four donors were low, suggesting that S-specific BMPCs may potentially be present at very low frequencies that are below the limit of detection of the assay. Another limitation is that we do not know the fraction of the S-binding BMPCs detected in our study that encodes neutralizing antibodies. SARS-CoV-2 S protein is the main target of neutralizing antibodies17,25,26,27,28,29,30 and a correlation between serum anti-S IgG binding and neutralization titres has been documented17,31. Further studies will be required to determine the epitopes that are targeted by BMPCs and memory B cells, as well as their clonal relatedness. Finally, although our data document a robust induction of long-lived BMPCs after infection with SARS-CoV-2, it is critical to note that our convalescent individuals mostly experienced mild infections. Our data are consistent with a report showing that individuals who recovered rapidly from symptomatic SARS-CoV-2 infection generated a robust humoral immune response32. It is possible that more-severe SARS-CoV-2 infections could lead to a different outcome with respect to long-lived BMPC frequencies, owing to dysregulated humoral immune responses. This, however, has not been the case in survivors of the 2014 Ebola virus outbreak in West Africa, in whom severe viral infection induced long-lasting antigen-specific serum IgG antibodies33.

Long-lived BMPCs provide the host with a persistent source of preformed protective antibodies and are therefore needed to maintain durable immune protection. However, the longevity of serum anti-S IgG antibodies is not the only determinant of how durable immune-mediated protection will be. Isotype-switched memory B cells can rapidly differentiate into antibody-secreting cells after re-exposure to a pathogen, offering a second line of defence34. Encouragingly, the frequency of S-binding circulating memory B cells at 7 months after infection was similar to that of B cells directed against contemporary influenza HA antigens. Overall, our data provide strong evidence that SARS-CoV-2 infection in humans robustly establishes the two arms of humoral immune memory: long-lived BMPCs and memory B cells. These findings provide an immunogenicity benchmark for SARS-CoV-2 vaccines and a foundation for assessing the durability of primary humoral immune responses that are induced in humans after viral infections.

Methods

Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded during outcome assessment.

Sample collection, preparation and storage

All studies were approved by the Institutional Review Board of Washington University in St Louis. Written consent was obtained from all participants. Seventy-seven participants who had recovered from SARS-CoV-2 infection and eleven control individuals without a history of SARS-CoV-2 infection were enrolled (Extended Data Tables 14). Blood samples were collected in EDTA tubes and PBMCs were enriched by density gradient centrifugation over Ficoll 1077 (GE) or Lymphopure (BioLegend). The remaining red blood cells were lysed with ammonium chloride lysis buffer, and cells were immediately used or cryopreserved in 10% dimethyl sulfoxide in fetal bovine serum (FBS). Bone marrow aspirates of approximately 30 ml were collected in EDTA tubes from the iliac crest of 18 individuals who had recovered from COVID-19 and the control individuals. Bone marrow mononuclear cells were enriched by density gradient centrifugation over Ficoll 1077, and the remaining red blood cells were lysed with ammonium chloride buffer (Lonza) and washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA. Bone marrow plasma cells were enriched from bone marrow mononuclear cells using the CD138 Positive Selection Kit II (Stemcell) and immediately used for ELISpot or cryopreserved in 10% dimethyl sulfoxide in FBS.

Antigens

Recombinant soluble spike protein (S) and its receptor-binding domain (RBD) derived from SARS-CoV-2 were expressed as previously described35. In brief, mammalian cell codon-optimized nucleotide sequences coding for the soluble version of S (GenBank: MN908947.3, amino acids (aa) 1–1,213) including a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag cloned into the mammalian expression vector pCAGGS. The S protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations were introduced (K986P and V987P, wild-type numbering). The RBD, along with the signal peptide (aa 1–14) plus a hexahistidine tag were cloned into the mammalian expression vector pCAGGS. Recombinant proteins were produced in Expi293F cells (Thermo Fisher Scientific) by transfection with purified DNA using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Supernatants from transfected cells were collected 3 (for S) or 4 (for RBD) days after transfection, and recombinant proteins were purified using Ni-NTA agarose (Thermo Fisher Scientific), then buffer-exchanged into PBS and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). For flow cytometry staining, recombinant S was labelled with Alexa Fluor 647- or DyLight 488-NHS ester (Thermo Fisher Scientific); excess Alexa Fluor 647 and DyLight 488 were removed using 7-kDa and 40-kDa Zeba desalting columns, respectively (Pierce). Recombinant HA from A/Michigan/45/2015 (aa 18–529, Immune Technology) was labelled with DyLight 405-NHS ester (Thermo Fisher Scientific); excess DyLight 405 was removed using 7-kDa Zeba desalting columns. Recombinant HA from A/Brisbane/02/2018 (aa 18–529) and B/Colorado/06/2017 (aa 18–546) (both Immune Technology) were biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher Scientific); excess biotin was removed using 7-kDa Zeba desalting columns.

ELISpot

Plates were coated with Flucelvax Quadrivalent 2019/2020 seasonal influenza virus vaccine (Sequiris), tetanus–diphtheria vaccine (Grifols), recombinant S or anti-human Ig. Direct ex vivo ELISpot was performed to determine the number of total, vaccine-binding or recombinant S-binding IgG- and IgA-secreting cells present in BMPC and PBMC samples using IgG/IgA double-colour ELISpot Kits (Cellular Technology) according to the manufacturer’s instructions. ELISpot plates were analysed using an ELISpot counter (Cellular Technology).

ELISA

Assays were performed in 96-well plates (MaxiSorp, Thermo Fisher Scientific) coated with 100 μl of Flucelvax 2019/2020 or recombinant S in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 10% FBS and 0.05% Tween-20 in PBS. Serum or plasma were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG–HRP (Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween-20 in PBS, and then washed 3 times with PBS before the addition of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression (GraphPad Prism v.8). The limit of detection was defined as 1:30.

Statistics

Spearman’s correlation coefficients were estimated to assess the relationship between 7-month anti-S and anti-influenza virus vaccine IgG titres and the frequencies of BMPCs secreting IgG specific for S and for influenza virus vaccine, respectively. Means and pairwise differences of antibody titres at each time point were estimated using a linear mixed model analysis with a first-order autoregressive covariance structure. Time since symptom onset was treated as a categorical fixed effect for the 4 different sample time points spaced approximately 3 months apart. P values were adjusted for multiple comparisons using Tukey’s method. All analyses were conducted using SAS v.9.4 (SAS Institute) and Prism v.8.4 (GraphPad), and P values of less than 0.05 were considered significant.

Flow cytometry

Staining for flow cytometry analysis was performed using cryo-preserved magnetically enriched BMPCs and cryo-preserved PBMCs. For BMPC staining, cells were stained for 30 min on ice with CD45-A532 (HI30, Thermo Fisher Scientific, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD19-PE (HIB19, 1:200), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD71-PE-Cy7 (CY1G4, 1:400), CD20-APC-Fire750 (2H7, 1:400), CD3-APC-Fire810 (SK7, 1:50) and Zombie Aqua (all BioLegend) diluted in Brilliant Stain buffer (BD Horizon). Cells were washed twice with 2% FBS and 2 mM EDTA in PBS (P2), fixed for 1 h using the True Nuclear permeabilization kit (BioLegend), washed twice with perm/wash buffer, stained for 1h with DyLight 405-conjugated recombinant HA from A/Michigan/45/2015, DyLight 488- and Alexa 647-conjugated S, Ki-67-BV711 (Ki-67, 1:200, BioLegend) and BLIMP-1-A700 (646702, 1:50, R&D), washed twice with perm/wash buffer, and resuspended in P2. For memory B cell staining, PBMCs were stained for 30 min on ice with biotinylated recombinant HAs diluted in P2, washed twice, then stained for 30 min on ice with Alexa 647-conjugated S, IgA-FITC (M24A, Millipore, 1:500), IgG-BV480 (goat polyclonal, Jackson ImmunoResearch, 1:100), IgD-SB702 (IA6-2, Thermo Fisher Scientific, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific Blue (2H7, 1:400), CD4-BV570 (OKT4, 1:50), CD24-BV605 (ML5, 1:100), streptavidin-BV650, CD19-BV750 (HIB19, 1:100), CD71-PE (CY1G4, 1:400), CXCR5-PE-Dazzle 594 (J252D4, 1:50), CD27-PE-Cy7 (O323, 1:200), IgM-APC-Fire750 (MHM-88, 1:100), CD3-APC-Fire810 (SK7, 1:50) and Zombie NIR (all BioLegend) diluted in Brilliant Stain buffer (BD Horizon), and washed twice with P2. Cells were acquired on an Aurora using SpectroFlo v.2.2 (Cytek). Flow cytometry data were analysed using FlowJo v.10 (Treestar). In each experiment, PBMCs were included from convalescent individuals and control individuals.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Relevant data are available from the corresponding author upon reasonable request.

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

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

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

Abstract

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

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

Introduction

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

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

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

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

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

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

Structure and pathogenesis of SARS-CoV-2

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

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

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

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

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Open in a separate windowFigure 1.

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

Innate immune response

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

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

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

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

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

Adaptive immune response

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

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

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

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

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

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

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

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

The devil in disguise: What happens when antibodies go bad

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

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Figure 2.

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

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

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

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

Molecular mechanism of ADE

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

ADE via enhanced infection

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

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

ADE via enhanced immune activation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

ADE as a possible threat to vaccine efficacy

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conclusions

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

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

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

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

Acknowledgments

The figures were created with BioRender.comGo to:

Appendix

Abbreviations

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

Footnotes

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

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

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

ORCID iDs

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

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

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COVID-19 and autoimmune diseases

Authors: Yu Liu,aAmr H. Sawalha,b and Qianjin Lua,cAuthor information Copyright and License information Disclaimer This article has been cited by other articles in PMC.

Abstract

Purpose of review

The aim of this study was to evaluate the relationship between infection with SARS-CoV-2 and autoimmunity.

Recent findings

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome (SARS) associated coronavirus 2 (SARS-CoV-2). Although most of the infected individuals are asymptomatic, a proportion of patients with COVID-19 develop severe disease with multiple organ injuries. Evidence suggests that some medications used to treat autoimmune rheumatologic diseases might have therapeutic effect in patients with severe COVID-19 infections, drawing attention to the relationship between COVID-19 and autoimmune diseases. COVID-19 shares similarities with autoimmune diseases in clinical manifestations, immune responses and pathogenic mechanisms. Robust immune reactions participate in the pathogenesis of both disease conditions. Autoantibodies as a hallmark of autoimmune diseases can also be detected in COVID-19 patients. Moreover, some patients have been reported to develop autoimmune diseases, such as Guillain–Barré syndrome or systemic lupus erythematosus, after COVID-19 infection. It is speculated that SARS-CoV-2 can disturb self-tolerance and trigger autoimmune responses through cross-reactivity with host cells. The infection risk and prognosis of COVID-19 in patients with autoimmune diseases remains controversial, but patient adherence to medication regimens to prevent autoimmune disease flares is strongly recommended.

Summary

We present a review of the association between COVID-19 and autoimmune diseases, focusing on similarities in immune responses, cross-reactivity of SARS-CoV-2, the development of autoimmune diseases in COVID-19 patients and the risk of COVID-19 infection in patients with preexisting autoimmune conditions.

INTRODUCTION

Since December 2019, a novel infection named coronavirus disease 2019 (COVID-19) broke out in Wuhan, China, and has been sweeping across the globe. COVID-19 was officially declared a pandemic by WHO on 11 March 2020 [1]. The disease is caused by a newly identified strain of severe acute respiratory syndrome (SARS) associated coronavirus, which was named SARS-CoV-2 after SARS-CoV that caused the epidemic of SARS in 2002 [2].

SARS-CoV-2 belongs to the coronavirus family, which are enveloped viruses with a spherical morphology and a single-stranded RNA (ssRNA) genome [3]. The spike glycoproteins (S protein) cross through the peplos of the virus and form a crown-like surface [4]. Through the receptor binding domain (RBD) located in the S1 subunit of the S protein, the virus can ligate to the host cell receptor angiotensin-converting enzyme 2 (ACE2) and invade into the cell [57].

In many cases, hosts infected by SARS-CoV-2 present with flu-like symptoms, such as fever, fatigue and dry cough. Headache, myalgia, sore throat, nausea and diarrhoea can also be seen in patients with COVID-19 [8,9]. Shortness of breath and hypoxemia occur in severe cases. In critical cases, the disease progresses rapidly and patients can develop septic shock and multiorgan dysfunction [10]. As such, COVID-19 can be a systemic disease affecting multiple organ systems, including the skin, kidneys, respiratory system, cardiovascular system, digestive system, nervous system and haematological system [11]. The dysregulated immune response and increased pro-inflammatory cytokines induced by SARS-CoV-2 contribute to the disease pathogenesis and organ damage, which brought attention to immune-regulatory therapy in the treatment of COVID-19 [12]. Medications used to treat autoimmune diseases are widely used in critical cases of COVID-19 [13]. Further, some autoantibodies can be detected in patients with COVID-19 [14]. These observations suggest that examining pathways known to contribute to the pathogenesis of autoimmunity might provide clues to better understand and treat COVID-19. 

For More Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880581/

Trained Innate Immunity, Epigenetics, and Covid-19

Authors: Alberto Mantovani, M.D., and Mihai G. Netea, M.D.

Innate immunity is mediated by different cell types and cell-associated or fluid-phase pattern-recognition molecules and plays a key role in tissue repair and resistance against pathogens.1 Exposure to selected vaccines, such as bacille Calmette–Guérin (BCG) or microbial components, can increase the baseline tone of innate immunity and trigger pathogen-agnostic antimicrobial resistance (known as trained innate immunity). Such training is directly relevant to resistance against infectious diseases, including Covid-19. A recent study by de Laval et al.2 pinpoints a driver of durable innate immune memory conferred by myeloid cells (monocytes, macrophages, and neutrophils).

Myeloid cells are central players in innate immunity: they produce effector molecules and contribute to the activation, orientation, and regulation of adaptive immune responses. Diversity and plasticity are fundamental properties of myeloid cells, particularly macrophages. To some extent, these properties are imprinted through ontogenetic origin (embryonal vs. adult bone marrow), but they are also influenced by environmental cues in the tissue. Moreover, in response to microbial molecules, metabolic products, or cytokines, macrophages increase effector function (“activation”), are primed for short-term responses (“priming”), or become unresponsive (“tolerance”). Microbial components can also cause long-term imprinting (“training”) of innate immunity and myeloid-cell function (Figure 1).3 (This type of imprinting is distinct from genomic imprinting whereby methyl groups are added to DNA in or near specific genes.)

For More Information: https://www.nejm.org/doi/10.1056/NEJMcibr2011679

Good news: Mild COVID-19 induces lasting antibody protection

People who have had mild illness develop antibodyproducing cells that can last lifetime

Authors: by Tamara Bhandari•May 24, 2021

Months after recovering from mild cases of COVID-19, people still have immune cells in their body pumping out antibodies against the virus that causes COVID-19, according to a study from researchers at Washington University School of Medicine in St. Louis. Such cells could persist for a lifetime, churning out antibodies all the while.

The findings, published May 24 in the journal Nature, suggest that mild cases of COVID-19 leave those infected with lasting antibody protection and that repeated bouts of illness are likely to be uncommon.

“Last fall, there were reports that antibodies wane quickly after infection with the virus that causes COVID-19, and mainstream media interpreted that to mean that immunity was not long-lived,” said senior author Ali Ellebedy, PhD, an associate professor of pathology & immunology, of medicine and of molecular microbiology. “But that’s a misinterpretation of the data. It’s normal for antibody levels to go down after acute infection, but they don’t go down to zero; they plateau. Here, we found antibody-producing cells in people 11 months after first symptoms. These cells will live and produce antibodies for the rest of people’s lives. That’s strong evidence for long-lasting immunity.”

For More Information: https://medicine.wustl.edu/news/good-news-mild-covid-19-induces-lasting-antibody-protection/

COVID-19 Science Update released: June 4, 2021 Edition 92

Authors: From the Office of the Chief Medical Officer, CDC COVID-19 Response, and the CDC Library, Atlanta GA. Intended for use by public health professionals responding to the COVID-19 pandemic.

PEER-REVIEWED

Safety, immunogenicity, and efficacy of the BNT162b2 COVID-19 vaccine in adolescents.external icon Frenck et al. NEJM (May 27, 2021).

Key findings:

  • Vaccine efficacy was 100% (95% CI 75.3%-100%) in 12- to 15-year-olds.
    • There were no cases in the vaccinated group compared with 16 cases among the placebo group, 7 or more days after dose 2.
  • Compared with baseline, geometric mean neutralizing antibody titers were 118.3-fold higher 1 month after dose 2.
  • Vaccine reactions were mainly transient, mild to moderate, and similar to a comparator group of 16–25-year-olds.
    • Injection-site pain was reported by 79% to 86%, fatigue was reported by 60% to 66%, and headache was reported by 55% to 65% of participants (Figure).

Methods: A randomized, placebo-controlled, observer-blinded trial of Pfizer/BioNTech BNT162b2 in 2,260 adolescents 12–15 years old (1,129 received placebo). Efficacy of the vaccine was assessed based on confirmed SARS-CoV-2 infection with onset 7 or more days after dose 2. Reactogenicity events (assessed for 7 days after each dose) and unsolicited adverse events compared with 16–25 age group (n = 3,610). SARS-CoV-2 serum neutralization assays were performed. LimitationsRacial and ethnic diversity of participants 12-15 years does not reflect the general US population; short (1 month) post-vaccination safety evaluation.

Implications: Vaccination of adolescents with BNT162b2 was safe and effective. Vaccinating adolescents will broaden community protection, and it will likely facilitate reintegration into society and resumption of in-person learning.

Figure:Graphs showing systemic events with 7 days after dose 1 or dose 2 of vaccine or placeboresize iconView Larger

Note: Adapted from Frenck et al. Systemic events reported within 7 days after receiving dose 1 (top) or dose 2 (bottom) of vaccine or placebo. 1 participant in the 12-to-15-year-old group had a fever with a temperature >40°C after dose 1. From the New England Journal of Medicine, Frenck et al., Safety, immunogenicity, and efficacy of the BNT162b2 COVID-19 vaccine in adolescents. May 27, 2021, online ahead of print. Copyright © 2021 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

Occurrence of severe COVID-19 in vaccinated transplant patientsexternal icon. Caillard et al. Kidney International. (May 21, 2021).

Key findings:

  • 55 solid organ transplant recipients developed COVID-19 after receiving 2 doses of mRNA vaccine.
    • Symptoms began a median of 22 days after the second vaccine dose (Figure).
    • 15 cases required hospitalization; of these, 6 were admitted to an intensive care unit, and 3 died.
  • Of 25 patients with post-vaccination serology, 24 were antibody negative; 1 was antibody positive but had low titers.

For More Information: https://www.cdc.gov/library/covid19/06042021_covidupdate.html

Pros and Cons of Adenovirus-Based SARS-CoV-2 Vaccines

Authors: Eric J. Kremer1,∗

Main Text

Most of us might be surprised by the rudimentary scientific rationale prevalent in the field of vaccine research just 50 years ago. For over a century after Louis Pasteur’s vaccine against rabies, approaches usually consisted of inactivating a virus, injecting it, and seeing if it protected the host. Unlike today, interactions between vaccinologists and immunologists to improve vaccine efficacy were marginal.

With the rise of molecular biology, vaccine designs became more nuanced and the use of viral vectors emerged. An example is the evolution and checkered history of vaccines based on adenoviruses (Ads). Live Ad types 4 (Ad4) and 7 (Ad7) have been used in North American military recruits since the 1950s to prevent severe respiratory illness.1 Similarly, dogs in western countries are vaccinated with an attenuated canine Ad type 2 (CAV-2) to prevent infection of the more virulent CAV-1.

Many of the first replication-defective Ad “vectors” in the early 1980s were vaccines. The original Ad vaccine design was relatively simple: delete a region of the viral genome that the virus needs to propagate, provide these functions via transcomplementing cells (e.g., Frank Graham’s 293 cells) so that one could grow the vaccine, and then insert into the virus genome an expression cassette encoding the targeted epitopes.

Fast forward to 2020. The SARS-CoV-2 pandemic may be headed toward historic proportions—although still far from the 1918 Spanish flu (50 million deaths) and AIDS (35 million deaths)—inflicting havoc on families, communities, and economies and overwhelming health care facilities. Clearly, we need a vaccine. Are Ad-based vaccines targeting the SARS-CoV-2 spike and capsid proteins our best bet? After almost 70 years of working with Ads, their biochemical properties are well characterized: Ads are simple to make (in ∼2 weeks a graduate student could generate enough of a novel Ad vaccine to treat a thousand mice and dozens of monkeys), easy to purify to high titer, genetically stable, easily stockpiled, relatively inexpensive, and can be delivered via aerosol, oral, intradermal, and intramuscular routes. The aerosol route is particularly relevant when targeting a respiratory virus because inducing protective immune responses that home to the tissue where infections will occur is strategically important. It is also worth noting that Ad-based vaccines tend to induce B cell and T cell responses.

Hundreds of millions of euros, dollars, and yen have been invested in advancing Ad-based vaccines. These advances include production and purification methods, genetic incorporation of epitopes into the capsid so that mononuclear phagocytes present these antigens via major histocompatibility complex (MHC) class I and II pathways, cloaking the capsid with polymers/shields or using Ad types with a lower level of seroprevalence to prevent neutralization by antibodies (NAbs) to common types found in many individuals, retargeting the vector to professional antigen-presenting cells, using helper-dependent vectors (so that the vector-infected cell only expresses the target epitopes and not Ad antigens), and single-cycle replication of vaccines to produce massive amounts of antigens. Each tweak, alone or in combination with others, has improved vaccine efficacy in preclinical trials.

As SARS-CoV-2 became a pandemic, it is astonishing that, in the case of the Ad-based vaccine frontrunners, little has changed from the basic design of 40 years ago. Some used the well-trodden path of an Ad5-based vaccine, while others switched to human (e.g., Ad26) or simian (monkey and gorilla) Ads that have low seroprevalence in Europe and North America (but not necessarily in Africa or Asia).2 Conceptually, Ad type switching to avoid NAbs is at least 30 years old. The advent of simian Ad vaccines was not developed following a rigorous testing of all of the >200 different Ad types but was most likely the result of intellectual property issues and the ability to produce simian Ads in good manufacturing practice (GMP)-compliant cells. One presumes that subsequent rounds of Ad-based coronavirus disease 2019 (COVID-19) vaccine candidates will be more sophisticated.

Should we go “all in” on an Ad-based vaccine against SARS-CoV-2? The first issue is safety. There are few drugs or biologicals that do not have side effects or cause adverse reactions. Weighing the advantages versus disadvantages during the current pandemic can be idiosyncratic, and the strength of the reasoning varies by population, culture, religious beliefs, and bizarrely (for those of us outside the USA) even political affiliation. Current criteria limit the window to identify adverse reactions to 2 months. In addition to swelling and pain at the injection site, common to some vaccines, Ad-based vaccine adverse effects include fever, pneumonia, diarrhea, transient neutropenia and lymphopenia, fatigue, labored breathing, headaches, liver damage, and fasting hyperglycaemia. Rare but grave adverse reactions include neuropathies such as Bell’s palsy, Guillain-Barré syndrome, gait disturbance, and transverse myelitis, an inflammatory condition in the spinal cord.

For More Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546260/

COVID-19 survivors may possess wide-ranging resistance to the disease

Authors: Rajee Suri rajee.suri@emory.edu

Recovered COVID-19 patients retain broad and effective longer-term immunity to the disease, suggests a recent Emory University study, which is the most comprehensive of its kind so far. The findings have implications for expanding understanding about human immune memory as well as future vaccine development for coronaviruses.

The longitudinal study, published recently on Cell Reports Medicine, looked at 254 patients with mostly mild to moderate symptoms of SARS-CoV-2 infection over a period for more than eight months (250 days) and found that their immune response to the virus remained durable and strong.

Emory Vaccine Center director Rafi Ahmed, PhD, and a lead author on the paper, says the findings are reassuring, especially given early reports during the pandemic that protective neutralizing antibodies did not last in COVID-19 patients.

“The study serves as a framework to define and predict long-lived immunity to SARS-CoV-2 after natural infection. We also saw indications in this phase that natural immunity could continue to persist,” Ahmed says. The research team will continue to evaluate this cohort over the next few years.

Researchers found that not only did the immune response increase with disease severity, but also with each decade of age regardless of disease severity, suggesting that there are additional unknown factors influencing age-related differences in COVID-19 responses. 

In following the patients for months, researchers got a more nuanced view of how the immune system responds to COVID-19 infection. The picture that emerges indicates that the body’s defense shield not only produces an array of neutralizing antibodies but activates certain T and B cells to establish immune memory, offering more sustained defenses against reinfection.

“We saw that antibody responses, especially IgG antibodies, were not only durable in the vast majority of patients but decayed at a slower rate than previously estimated, which suggests that patients are generating longer-lived plasma cells that can neutralize the SARS-CoV-2 spike protein.”

For More Information: https://news.emory.edu/stories/2021/07/covid_survivors_resistance/index.html