Does SARS-CoV-2 Possess “Allergen-Like” Epitopes?

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting manuscript addressing the role of viral “allergen-like” epitopes which induce the synthesis of IgE to evade the immune system and generate a chronic infection. In response, the immune system produces IgG4 antibodies to neutralize the negative effects of IgE. Thus, repeated vaccination leading to an allergen-like antigen might not be adequate if it results in tolerance rather than effective immunity. The manuscript addresses a clinically relevant issue, however, some immunological effects of COVID-19 infection have not be considered and should be discussed. As historically described in other viral infections (EBV, CMV, HSV), also COVID-19 infection has been associated with the development of autoimmune phenomena, as recently reported (J Microbiol Immunol Infect. 2021 Jun;54(3):531-533. doi: 10.1016/j.jmii.2020.08.006; doi: 10.1111/cts.12908.). Of clinical relevance, antinuclear antibodies with nucleolar immunofluorescence pattern, has been associated with a negative prognostic significance, as demonstrated (doi: 10.1111/cts.13026.). The role of COVID-19 as autoimmune-response trigger in genetically predisposed subjects, should be recalled and discussed as well as the potential impact of genetic factors to explain the different clinical profiles associated with COVID-19 infections.

Author Response

We thank the reviewers for their excellent suggestions and criticisms, which contributed to improving the quality of our work. We have highlighted in green the information added because of the reviewers' comments and in yellow the new information we added to improve the understanding of our hypothesis. We hope we have satisfactorily addressed your excellent comments. We hope that our revisions and responses satisfactorily address the reviewers’ concerns. Please let us know if any additional modifications are required. We sincerely appreciate your consideration and look forward to your response.

Reviewer 1: This is an interesting manuscript addressing the role of viral “allergen-like” epitopes which induce the synthesis of IgE to evade the immune system and generate a chronic infection. In response, the immune system produces IgG4 antibodies to neutralize the negative effects of IgE. Thus, repeated vaccination leading to an allergen-like antigen might not be adequate if it results in tolerance rather than effective immunity. The manuscript addresses a clinically relevant issue, however, some immunological effects of COVID-19 infection have not be considered and should be discussed. As historically described in other viral infections (EBV, CMV, HSV), also COVID-19 infection has been associated with the development of autoimmune phenomena, as recently reported (J Microbiol Immunol Infect. 2021 Jun;54(3):531-533. doi: 10.1016/j.jmii.2020.08.006; doi: 10.1111/cts.12908.). Of clinical relevance, antinuclear antibodies with nucleolar immunofluorescence pattern, has been associated with a negative prognostic significance, as demonstrated (doi: 10.1111/cts.13026.). The role of COVID-19 as autoimmune-response trigger in genetically predisposed subjects, should be recalled and discussed as well as the potential impact of genetic factors to explain the different clinical profiles associated with COVID-19 infections.

R: We thank the Reviewer for this important suggestion. We have added the following important contributions:

In addition to high IgE and IgG4 levels, several studies have reported the presence of autoanti-bodies, such as antiphospholipid antibodies, antinuclear antibodies (ANAs), and anti-cytoplasmic neutrophil antibodies (ANCAs). Fifteen out of thirty-three patients (45%) had at least one auto-antibody test positive, including eleven with ANAs (33%), eight with anti-cardiolipin antibodies (immunoglobulin (Ig)G and/or IgM; 24%), and three with anti-β2-glycoprotein antibodies (IgG and/or IgM; 9%). ANCA reactivity was not found in any patient. The prognosis for patients who tested positive for auto-antibodies was significantly worse than that of other patients: only 1 out of 18 patients (5.5%) who did not have auto-antibodies died (P = 0.03), whereas 6 out of 15 patients (40%) with auto-antibodies passed away as a result of COVID-19 complications [101-103].

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors address an interesting and relatively unexplored aspect of COVID-19, namely the significance of anti-SARS-CoV-2 antibodies belonging to IgE and IgG4 isotypes. They propose that SARS-CoV-2 proteins possess allergen-like epitopes, which induce the production of IgE, whereby the virus evades an immune response, potentially leading to chronic infection. They discuss this in the context of literature data regarding IgE antibodies against some other viruses, such as HIV-1 and RSV. They also address an important issue of IgG4 antibody induction in COVID-19 patients and especially in subjects undergoing repeated vaccinations against SARS-CoV-2. They suggest that the induction of IgG4 during infection might be an attempt of the immune system to counteract the adverse effects of IgE. On the other hand, the specific IgG4 induced after repeated vaccinations, by lacking the ability to trigger Fc receptor or complement activation, and by possessing reduced virus-neutralizing activity, may hamper the effective antiviral response and promote ‘immune tolerance’ to the virus (a better term is probably immune evasion). This may be the reason behind the increased frequency of SARS-CoV-2 infections after multiple vaccinations.

While proposed scenarios may be possible, the authors make quite many assumptions that have little experimental support. They should try to gather more data to support the importance of IgE/IgG4 in COVID-19 pathogenesis. My suggestions are listed below.

1. Natural SARS-CoV-2 infection induces relatively low levels of virus-specific IgE and IgG4 (refs 45, 67 etc), which are likely to be outcompeted by other, more abundant virus-specific antibody isotypes for antigen binding. So, specific IgE/IgG4 might have little effect on the immune response against the virus, which would question the idea of induction of IgG4 to counteract the adverse effects of IgE in acute infection (lines 243-246). Please comment.
2. Are there any experimental data on the epitope specificity of IgE or IgG4 antibodies against SARS-CoV-2? Also, in section 6, the authors mention a number of tools to search for ‘allergen-like’ epitopes of vaccine antigens. But have they tried themselves to use these tools to predict such epitopes in SARS-CoV-2? Such data would strengthen their hypothesis.
3. Is there any experimental evidence that virus-specific IgE or IgG4 interfere with virus-neutralizing or ADCC/ADCP-inducing antibodies? For example, in ref. 146 (which is the only cited reference dealing with function of IgG4 in COVID-19), IgG4 depletion does not affect virus-neutralization activity of convalscent plasma. But this argues against IgG4 competing with neutralizing antibodies.
4. Elevated total IgE and to a lesser extent total IgG4 are associated with negative prognosis in COVID-19 (ref. 51, 41, 85). However, elevated IgE apparently does not worsen COVID-19 in Th2-associated allergic asthma. How does this fit with the proposed adverse role of IgE in COVID-19?
5. Tissue damage in severe COVID-19 is driven by macrophage activation, which might be counteracted by Th2 cytokines. If we consider IgE as a marker of Th2 response, wouldn’t IgE induction reflect an attempt of the immune system to reduce tissue damage? Also, can IgE or IgG4 affect antibody-dependent enhancement of cytokine production by SARS-CoV-2-infected cells?
6. The authors suggest that induction of IgE and/or IgG4 may lead to chronic SARS-CoV-2 infection (e.g. lines 34, 118). However, such chronic infections have so far been documented only in immunocompromised subjects suffering e.g. from agammaglobulinemia. Has a chronic SARS-CoV-2 infection been described in immunocompetent subjects? Is there any relationship between anti-viral IgE/IgG4 and development of long COVID, if we consider virus persistence as one of its putative mechanisms?
7. Lines 223-233. No matter how these epitopes were inserted in the S protein sequence, there are no data that they are targets of IgE and/or IgG4. So the relevance of this reference and the entire paragraph is unclear.
8. Lines 235-245. The proposal that S protein possesses a galectin-3-like fold (ref. 103) is interesting, although not proven experimentally. However, the references 105 and 106 do not support the idea of that S protein binds IgE via this galectin fold, because ref. 106 does not deal with IgE or galectin-3, while ref. 105 does not deal with SARS-CoV-2. Please find more relevant ones or rephrase the sentence. Further, it is unclear how this can lead to augmented IgG4 synthesis.

 

Minor comments

Line 50. Fab fragment exchange will not affect affinity of individual antigen-bindings sites of IgG4. Therefore, the resulting antibody will have reduced avidity, not affinity.

Line 132. The term ‘negative predictor’ is unclear. Better wording would be ‘a predictor of negative clinical outcome’ or similar.

Line 135. It is unclear, which outcome is predicted by low eosinophils (the word ‘adverse’ or ‘negative’ is probably missing).

Line 183. It is hard to imagine how elevated IgG4 can be a consequence of death. Perhaps a consequence of severe disease leading to death? Please find a better wording.

References 34 and 47 are basically the same, perhaps #47 can be removed.

 

Author Response

Reviewer 2:

The authors address an interesting and relatively unexplored aspect of COVID-19, namely the significance of anti-SARS-CoV-2 antibodies belonging to IgE and IgG4 isotypes. They propose that SARS-CoV-2 proteins possess allergen-like epitopes, which induce the production of IgE, whereby the virus evades an immune response, potentially leading to chronic infection. They discuss this in the context of literature data regarding IgE antibodies against some other viruses, such as HIV-1 and RSV. They also address an important issue of IgG4 antibody induction in COVID-19 patients and especially in subjects undergoing repeated vaccinations against SARS-CoV-2. They suggest that the induction of IgG4 during infection might be an attempt of the immune system to counteract the adverse effects of IgE. On the other hand, the specific IgG4 induced after repeated vaccinations, by lacking the ability to trigger Fc receptor or complement activation, and by possessing reduced virus-neutralizing activity, may hamper the effective antiviral response and promote ‘immune tolerance’ to the virus (a better term is probably immune evasion). This may be the reason behind the increased frequency of SARS-CoV-2 infections after multiple vaccinations.

R: In the version you reviewed, we envisaged 2 possible hypotheses explaining the elevated presence of IgG4 antibodies: On lines 181-191, we wrote: “Interestingly, other studies discovered that lethal COVID-19 was associated with high IgG4 levels [41,85-87].  It is hypothesized that elevated IgG4 levels were a consequence, rather than a cause of death.  The etiology of death (among other factors) may be attributable to a severe IgE-mediated allergic reaction directed against the N and spike proteins. It is conceivable that the immune system generated the production of IgG4s in an attempt to minimize the cytokine storm. Indeed, it has been reported that IgG4 competes with specific IgE for allergen binding as a "blocking antibody," which prevents mast cell and basophil degranulation [88,89].  Another possibility is that in severe COVID-19 pneumonia, SARS-CoV-2 induces IgG4 synthesis in an IgE-independent manner, inhibiting IgG3 binding to its Fc receptor, thus impairing viral phagocytosis [90].”

In the current version, we have eliminated the first hypothesis. This is because it is not possible to explain that elevated IgG4 levels in acute infection could minimize the negative effects of IgE. In other words, it takes time (several weeks) to develop an IgG4 response. During allergen-specific immunotherapy (AIT), there is an initial increase in IgE levels, which then decrease, followed by a sustained increase in specific IgG4 levels. Indeed, clinical improvement has been associated with increases in IgG4 levels after prolonged exposure to allergens. We therefore retain only the second hypothesis for further consideration.

While proposed scenarios may be possible, the authors make quite many assumptions that have little experimental support. They should try to gather more data to support the importance of IgE/IgG4 in COVID-19 pathogenesis. My suggestions are listed below.

1. Natural SARS-CoV-2 infection induces relatively low levels of virus-specific IgE and IgG4 (refs 45, 67 etc), which are likely to be outcompeted by other, more abundant virus-specific antibody isotypes for antigen binding. So, specific IgE/IgG4 might have little effect on the immune response against the virus, which would question the idea of induction of IgG4 to counteract the adverse effects of IgE in acute infection (lines 243-246). Please comment.

R:  We thank the reviewer for this important insight. We agree that IgG4 could not counteract the adverse effects of IgE in acute infection. That is why we reformulated our hypothesis, as explained above. Furthermore, we added important information showing that in severe COVID-19, IgG4 levels were high, while in mild disease, they remained low (lines 198-209 in the revised version):

“… specifically, it was reported that high serum IgG4 concentrations (>700 mg/dL) were associated with a considerably higher 30-day death rate and were significantly correlated with IL-6 levels [97].

Another study found that patients who passed away between 8-14 and 15-21 days also had higher levels of anti-RBD IgG4 compared to those who recovered (P < 0.05).  This indicates that certain patients in critical condition developed an IgG4 to RBD antibody response during the initial weeks after the appearance of symptoms. The investigation also revealed that over 50% of the blood samples from deceased patients exhibited positive IgG4 antibody tests. Conversely, the majority of recovered patients exhibited negative IgG4 antibody test results within the same period [98]. On the other hand, in mild COVID-19 cases, strong IgG1 and IgG3 antibody responses targeting the RBD predominated, while IgG4 levels remained low [66,67].

 

 

1. Are there any experimental data on the epitope specificity of IgE or IgG4 antibodies against SARS-CoV-2? Also, in section 6, the authors mention a number of tools to search for ‘allergen-like’ epitopes of vaccine antigens. But have they tried themselves to use these tools to predict such epitopes in SARS-CoV-2? Such data would strengthen their hypothesis.

R: We thank the reviewer for this important observation. We searched databases such as PUBMED and Google Scholar and found that some studies have indeed investigated the presence of allergen-like epitopes in SARS-CoV-2. It is important to clarify that these researchers looked for the presence of "allergen-like" epitopes, trying to explain why asthma does not represent a risk factor for COVID-19 in several published cohorts. To our knowledge, our work is the first to propose that these epitopes could be used by SARS-CoV-2 to evade the immune system by inducing immunological tolerance and achieving persistence within the host. We have described these findings in lines 304-315. In addition, we will initiate new work using the proposed tools to find other epitopes in SARS-CoV-2 that could induce IgE responses.

An in-silico study investigated potentially reactive T cell epitopes between allergens and SARS-CoV-2. Regardless of the bioinformatic method used, allergen sources including Aspergillus fumigatus, Phleum pratense, and Dermatophagoides species were particularly noteworthy because they correlated with several cross-reactive peptides. A major candidate was found to be the fungus Aspergillus fumigatus [104]. Notably, an amino acid region in the HIV-1 gp41 heptad repeat 2 (HR2) protein also resembles the allergen domain in the Aspergillus fumigatus Asp f1 protein [120]. This fungus employs multiple sophisticated strategies to evade the human immune system, particularly in immunocompromised individuals [121-124]. Intriguingly, Aspergillus fumigatus can indeed induce a Th2 shift after chronic exposure. Such a shift is associated with the production of cytokines such as IL-4, IL-5, and IL-13, contributing to disease progression [125-128]. IL-4 and IL-13 suppress protective Th1 responses, creating a permissive environment for fungal colonization [125,129].   

1. Is there any experimental evidence that virus-specific IgE or IgG4 interfere with virus-neutralizing or ADCC/ADCP-inducing antibodies? For example, in ref. 146 (which is the only cited reference dealing with the function of IgG4 in COVID-19), IgG4 depletion does not affect virus-neutralization activity of convalescent plasma. But this argues against IgG4 competing with neutralizing antibodies.

R: Thank you for this relevant question. Yes, there is experimental evidence. Selva et al. demonstrated that elevated IgG4 is negatively correlated with FcγR engagement

- K. J. Selva, P. Ramanathan, E. R. Haycroft, A. Reynaldi, D. Cromer, C. W. Tan, L. F. Wang, B. D. Wines, P. M. Hogarth, L. E. Downie, S. K. Davis, R. A. Purcell, H. E. Kent, J. A. Juno, A. K. Wheatley, M. P. Davenport, S. J. Kent, A. W. Chung, Preexisting immunity restricts mucosal antibody recognition of SARS-CoV-2 and Fc profiles during breakthrough infections. JCI Insight 8, e172470 (2023).

Furthermore, Irrgang et al. demonstrated that IgG4 antibodies impair ADCC/ADCP.  They wrote:

“In our study, antibody-mediated phagocytic activity and complement deposition were reduced in sera after the third immunization, in parallel to higher proportions of anti-spike IgG4 antibodies”.

“IgG2 and IgG4 are considered to have a lower potential to mediate FcγR-dependent secondary effector function (20). Therefore, we performed an ADCP assay with the monocytic THP-1 cell line (30) (Fig. 3GOpens in image viewer). Using fluorescently labeled microbeads loaded with spike protein as targets and equal amounts of our recombinant monoclonal anti-RBD antibodies, we confirmed that IgG3 and IgG1 are more potent in inducing phagocytosis than IgG4 and IgG2 (fig. S7). Using FcγRIIA-, FcγRIIB-, or FcγRIII-expressing reporter cells (31), engagement of IgG2 and IgG4 results in reduced activation of the FcγRIIA, which was reported to be a key mediator of ADCP (30, 32) (fig. S7). Consistent with this, sera taken after the third vaccination and normalized to the amount of anti-spike antibodies yielded significantly lower phagocytic scores than sera from the same donors after two immunizations (Fig. 3HOpens in image viewer). Furthermore, ADCD on spike-coated microbeads was also significantly reduced after incubation with sera taken after the third vaccination (Fig. 3IOpens in image viewer). Together, these data show that spike protein–reactive IgG2 and IgG4 exhibit reduced Fc-mediated effector functions.

https://www.science.org/doi/10.1126/sciimmunol.ade2798

 

Besides ref 146, other works have demonstrated that IgG4s are not neutralizing antibodies

• Suthar, M.S.; Zimmerman, M.G.; Kauffman, R.C.; Mantus, G.; Linderman, S.L.; Hudson, W.H.; Vanderheiden, A.; Nyhoff, L.; Davis, C.W.; Adekunle, O. Rapid generation of neutralizing antibody responses in COVID-19 patients. Cell Reports Medicine 2020, 1.
• Whitcombe, A.L.; McGregor, R.; Craigie, A.; James, A.; Charlewood, R.; Lorenz, N.; Dickson, J.M.; Sheen, C.R.; Koch, B.; Fox‐Lewis, S. Comprehensive analysis of SARS‐CoV‐2 antibody dynamics in New Zealand. Clinical & translational immunology 2021, 10, e1261
• Kalkeri, Raj, et al. "P-1972. SARS-CoV-2 Vaccine–Induced IgG4 Response Negatively Correlates with Neutralizing Antibodies and Fc Effector Functions." Open Forum Infectious Diseases. Vol. 12. No. Suppl 1. 2025.

 

We also wish to clarify that the competition between IgG4 and IgG3 does not occur at the level of the Fab (variable) region of the antibody but rather at the Fc region. We have included a new figure in the text to explain this:

Figure 2. (A) The IgG3 antibody typically binds to spike protein via its variable region. The corresponding receptor on macrophages and other immune cells recognizes the constant region (Fc) of this antibody. This process, known as "opsonization," identifies foreign pathogens so that phagocytes can destroy them. (B) The presence of SARS-CoV-2 has been observed to trigger the production of IL-6, which in turn prompts a shift in the typical phenotype of B cells, resulting in the production of IgG4 antibodies (illustrated in red). The Fc region of the IgG4 antibody interacts with the Fc region of IgG3, hindering its ability to bind to its receptor on macrophages. As a result, IgG3 effector functions are inhibited. Source: [100].

1. Elevated total IgE and, to a lesser extent, total IgG4 are associated with a negative prognosis in COVID-19 (ref. 51, 41, 85). However, elevated IgE apparently does not worsen COVID-19 in Th2-associated allergic asthma. How does this fit with the proposed adverse role of IgE in COVID-19?

R: This is an important question. We have added a text explaining why elevated IgE levels do not worsen COVID-19 in allergic asthma.  SARS-CoV-2 and other viruses induce an “allergic-like” response, but it is not the classic allergic response. Please see the details in the next sentences.

A large-scale study utilizing the Optum database identified over a million patients with allergic conditions and over two million without. The analysis showed that allergic conditions were associated with an increased risk of receiving a COVID-19 diagnosis, but reduced mortality after infection [109]. From the immunological perspective, the differential risk of severe COVID-19 in individuals with non-allergic versus allergic asthma can be attributed to the distinct immune pathways underlying these conditions [108,110,111]. As mentioned above, in allergic people with elevated blood eosinophil counts, COVID-19 was less severe [51]. Conversely, low eosinophil counts were considered to be a negative disease predictor. In people who died from COVID-19, such a decrease was frequently seen [52]. Eosinophils have been found to contain and produce molecules with antiviral activity, including RNases and reactive nitrogen species. Furthermore, eosinophils have the capacity to participate in adaptive immunity, functioning as antigen-presenting cells. The eosinophil antiviral response has been demonstrated against various respiratory viruses in vitro and in vivo, including RSV and influenza [112-116].

These viruses induce an “allergic-like” response that counteracts antiviral defenses, potentially facilitating viral persistence. This suggests that certain viral epitopes could trigger IgE production, not to cause typical allergic symptoms in the traditional sense, but rather as a part of a viral strategy to subvert the host´s defense mechanism. For example, infection with Ascaris lumbricoides (a large parasitic roundworm) has been shown to induce high total IgE levels.  It should be noted that the majority of these antibodies are not specific [117], as a result of polyclonal B cell activation [118]. This nonspecific IgE binds to its receptors on mast cells and eosinophils, preventing their degranulation and reducing the effector response against larvae [119]. Although helminths and viruses are not phylogenetically related, they have likely evolved similar immune evasion mechanisms through convergent evolution.

 

1. Tissue damage in severe COVID-19 is driven by macrophage activation, which might be counteracted by Th2 cytokines. If we consider IgE as a marker of Th2 response, wouldn’t IgE induction reflect an attempt of the immune system to reduce tissue damage? Also, can IgE or IgG4 affect antibody-dependent enhancement of cytokine production by SARS-CoV-2-infected cells?

R: Thank you for these important questions. Unfortunately, there is little information about the role of IgE and IgG4 in CoVID-19. We know that in severe cases elevated levels of IgE and IgG4 were detected. That is why we have included relevant information on HIV and RSV infections that could explain these elevated levels. In fact, we thought the same thing at the beginning, i.e. the immune system produces IgG4 to try to minimize the damage caused by IgE. However, we have modified our hypothesis after studying how HIV and RSV induce IgE to disable antiviral responses. They generate a shift from Th1 to Th2 to promote tolerance by increasing Th2 cytokine levels. Such a shift causes an inhibition of the interferon synthesis, which is essential for antiviral responses.

Moreover, Th2 cytokines may counteract macrophage activation in normal conditions. However, the cytokine storm is an uncontrolled, hyper-reactive response in severe COVID-19. Although IL-10 is frequently thought of as an anti-inflammatory cytokine, this is only true at low concentrations; at higher concentrations, it has pro-inflammatory actions:

• Lauw, F.N.; Pajkrt, D.; Hack, C.E.; Kurimoto, M.; van Deventer, S.J.; van der Poll, T. Proinflammatory effects of IL-10 during human endotoxemia. J. Immunol. 2000, 165, 2783–2789.
• Nagata, K.; Nishiyama, C. IL-10 in mast cell-mediated immune responses: Anti-inflammatory and proinflammatory roles. Int. J. Mol. Sci. 2021, 22, 4972.
• Saxton, R.A.; Tsutsumi, N.; Su, L.L.; Abhiraman, G.C.; Mohan, K.; Henneberg, L.T.; Aduri, N.G.; Gati, C.; Garcia, K.C. Structure-based decoupling of the pro-and anti-inflammatory functions of interleukin-10. Science 2021, 371, eabc8433.

The same occurs with IL-4, which is normally considered an anti-inflammatory cytokine. At moderate levels, it might counterbalance inflammation and reduce damage. However, at high levels, it could exacerbate tissue fibrosis, airway pathology, or persistent inflammation via eosinophils and mast cells:

• Milner, Joshua D., et al. "Sustained IL-4 exposure leads to a novel pathway for hemophagocytosis, inflammation, and tissue macrophage accumulation." Blood, The Journal of the American Society of Hematology14 (2010): 2476-2483.
• Dang, Buyun, et al. "The glycolysis/HIF-1α axis defines the inflammatory role of IL-4-primed macrophages." Cell Reports5 (2023).
• Hua, Xiaoyang, et al. "IL-4 amplifies the pro-inflammatory effect of adenosine in human mast cells by changing expression levels of adenosine receptors." PloS one9 (2011): e24947.

 

1. The authors suggest that induction of IgE and/or IgG4 may lead to chronic SARS-CoV-2 infection (e.g. lines 34, 118). However, such chronic infections have so far been documented only in immunocompromised subjects suffering e.g. from agammaglobulinemia. Has a chronic SARS-CoV-2 infection been described in immunocompetent subjects? Is there any relationship between anti-viral IgE/IgG4 and development of long COVID, if we consider virus persistence as one of its putative mechanisms?

R: Thank you very much for these relevant questions. While chronic SARS-CoV-2 infections have been well documented in immunocompromised individuals, there is emerging evidence suggesting that viral persistence may also occur in immunocompetent individuals, though it is less frequently reported. Some studies have detected SARS-CoV-2 RNA or proteins for months after acute infection in immunocompetent hosts, including in tissues such as the gut, brain, and lungs.

• Natarajan, Aravind, et al. "Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA suggest prolonged gastrointestinal infection." Med6 (2022): 371-387.
• Leitao, Isabela de Carvalho, et al. "Prolonged SARS-CoV-2 positivity in immunocompetent patients: virus isolation, genomic integrity, and transmission risk." Microbiology spectrum3 (2021): e00855-21.
• Swank, Zoe, et al. "Persistent circulating severe acute respiratory syndrome coronavirus 2 spike is associated with post-acute coronavirus disease 2019 sequelae." Clinical Infectious Diseases3 (2023): e487-e490.
• Stein, Sydney R., et al. "SARS-CoV-2 infection and persistence in the human body and brain at autopsy." Nature7941 (2022): 758-763.
• Tejerina F., Catalan P., Rodriguez-Grande C., Adan J., Rodriguez-Gonzalez C., Muñoz P., Aldamiz T., Diez C., Perez L., Fanciulli C., et al. Post-COVID-19 syndrome. SARS-CoV-2 RNA detection in plasma, stool, and urine in patients with persistent symptoms after COVID-19. BMC Infect. Dis. 2022;22 doi: 10.1186/S12879-022-07153-4.

 

We have added such information in lines 335-341:

During the acute phase of COVID-19, evasion of the immune system would enable rapid viral replication. In the long term, this could lead to the establishment of a chronic infection. While chronic SARS-CoV-2 infections have been well documented in immunocompromised individuals, there is emerging evidence suggesting that viral persistence may also occur in immunocompetent individuals, though it is less frequently reported. Some studies have detected SARS-CoV-2 RNA or proteins for months after acute infection in immunocompetent hosts, including in tissues such as the gut, brain, and lungs [139-143].

 

 

Regarding the relationship between anti-viral IgE/IgG4 and development of long COVID, we found some works:

• Giménez-Orenga, Karen, et al. "HERV-W ENV antigenemia and correlation of increased anti-SARS-CoV-2 immunoglobulin levels with post-COVID-19 symptoms." Frontiers in Immunology13 (2022): 1020064.
• Körner, Robert Walter, et al. "Atopy and elevation of IgE, IgG3, and IgG4 may be risk factors for post COVID-19 condition in children and adolescents." Children10 (2023): 1598.

 

1. Lines 223-233. No matter how these epitopes were inserted in the S protein sequence, there are no data that they are targets of IgE and/or IgG4. So the relevance of this reference and the entire paragraph is unclear.

R:  We agree with the reviewer that the validity of this reference is unclear. We sent an email to the authors asking for more information on why their work was withdrawn, but they did not reply. So we have deleted such reference.

1. Lines 235-245. The proposal that S protein possesses a galectin-3-like fold (ref. 103) is interesting, although not proven experimentally. However, the references 105 and 106 do not support the idea of that S protein binds IgE via this galectin fold, because ref. 106 does not deal with IgE or galectin-3, while ref. 105 does not deal with SARS-CoV-2. Please find more relevant ones or rephrase the sentence. Further, it is unclear how this can lead to augmented IgG4 synthesis.

R: Thank you for this observation. We have deleted unrelated references and modified the text accordingly:

In respect of the Spike protein, a galectin-3 (Gal-3)---like fold has been identified within the N-terminal domain (NTD) of SARS-CoV-2 [131]. Indeed, Gal-3 was originally identified as an IgE-binding protein [132]. The “galectin-fold” of the S1-NTD region of spike protein has a structural homology almost identical to that of human galectin-3.  This supports our hypothesis that signaling pathways promoted by binding to antigenic sites on the NTD would likely correlate with signaling pathways and pathologies caused by human galectin-3 (gal-3) [133]. Further significant experimental support is through research published in 2022.  The authors sought to test the hypothesis that the S1-NTD is specifically responsible for a pattern of cytokines and innate immune mechanisms that match those of Gal-3.  The authors used microtiter wells to immobilize S components and confirmed that the S1-NTD produced a near-identical cytokine pattern to that of Gal-3 through activation of human monocytes.  In contrast, ACE2-binding S1-CTD/RBD failed to trigger the same response. Researchers postulated that the spike protein's S1-NTD would thereafter function similarly to EC-Gal-3, activating innate immune cells including monocytes and macrophages that invade virally infected lesion sites. While it is currently uncertain whether such a mechanism has a role in the cytokine storm frequently observed in severe COVID-19 disease, the theory makes sense and needs more research [133]. Gal-3-induced IgE may also contribute to post-acute COVID-19 syndrome in children, adolescents, and adults [134,135].

Minor comments

Line 50. Fab fragment exchange will not affect affinity of individual antigen-bindings sites of IgG4. Therefore, the resulting antibody will have reduced avidity, not affinity.

R: Thank you for this important error. We have corrected it accordingly.

Line 132. The term ‘negative predictor’ is unclear. Better wording would be ‘a predictor of negative clinical outcome’ or similar.

R: Thank you for the suggestion. We have modified the text accordingly.

Line 135. It is unclear, which outcome is predicted by low eosinophils (the word ‘adverse’ or ‘negative’ is probably missing).

R: Thank you for this observation. We have changed the original sentence and written instead:

Low eosinophil counts are considered a potential indicator of disease progression.

Line 183. It is hard to imagine how elevated IgG4 can be a consequence of death. Perhaps a consequence of severe disease leading to death? Please find a better wording.

R: Thank you for this observation. We agree with the reviewer. As mentioned above, we deleted the first hypothesis due to its improbability.

References 34 and 47 are basically the same, perhaps #47 can be removed.

47. Thanks for pointing out this mistake. We have removed ref. 47.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review describes a very unique hypothesis, but there are some problems.

1. Clarifying the role of IgE in COVID-19

The biggest problem is whether IgE is involved in the pathology of COVID-19. The main symptoms of COVID-19 are cough, sore throat, joint pain, and fever, which are like influenza. These symptoms are not consistent with an IgE-dependent allergic reaction. On the other hand, runny nose and dermatitis seen in some patients are like allergic symptoms involving IgE. When runny nose symptoms are common in the later stages of infection, IgE may be involved. Therefore, it is necessary to clearly describe which symptoms of COVID-19 are consistent with the IgE hypothesis.

 

2. Clear distinction between facts and hypotheses

Another important challenge is to clearly distinguish between facts and hypotheses. For example, IgG4 induced by corona vaccination does not correlate with IgG4-related diseases. Also, an increase in IgG4 does not affect the efficacy of the vaccine. The role of IgG4 is only speculated from the results of in vitro experiments. Furthermore, since IgG4 does not exist in rodents, it is difficult to create gene-deficient animals, and its physiological significance has not been fully elucidated. These facts should be clearly described, and the predicted role should be discussed. Some expressions are likely to be cited in the anti-vaccination movement, so please be careful when writing.

 

3. Standardization of Description

This review was written by multiple authors and requires consistency in the descriptions. For example, there are numerous errors in spelling and abbreviations. Terminology should be standardized and consistent.

Comments on the Quality of English Language

This review was written by multiple authors and requires consistency in the descriptions. For example, there are numerous errors in spelling and abbreviations. Terminology should be standardized and consistent.

Author Response

Reviewer 3:

This review describes a very unique hypothesis, but there are some problems.

1. Clarifying the role of IgE in COVID-19

The biggest problem is whether IgE is involved in the pathology of COVID-19. The main symptoms of COVID-19 are cough, sore throat, joint pain, and fever, which are like influenza. These symptoms are not consistent with an IgE-dependent allergic reaction. On the other hand, runny nose and dermatitis seen in some patients are like allergic symptoms involving IgE. When runny nose symptoms are common in the later stages of infection, IgE may be involved. Therefore, it is necessary to clearly describe which symptoms of COVID-19 are consistent with the IgE hypothesis.

R: Thank you for this important observation. In point 4, Reviewer 2 made a similar comment. We believe that the following information can explain this apparent paradox:

These viruses induce an “allergic-like” response that counteracts antiviral defenses, potentially facilitating viral persistence. This suggests that certain viral epitopes could trigger IgE production, not to cause typical allergic symptoms in the traditional sense, but rather as a part of a viral strategy to subvert the host´s defense mechanism. For example, Ascaris lumbricoides infection has been shown to induce high total IgE levels, though it should be noted that the majority of these antibodies are not specific [114], as a result of polyclonal B cell activation [115]. This nonspecific IgE binds to its receptors on mast cells and eosinophils, preventing their degranulation and reducing the effector response against larvae [116]. Although helminths and viruses are not phylogenetically related, it is likely that they have evolved similar immune evasion mechanisms through convergent evolution.

 

 

 

 

1. Clear distinction between facts and hypotheses

Another important challenge is to clearly distinguish between facts and hypotheses. For example, IgG4 induced by corona vaccination does not correlate with IgG4-related diseases. Also, an increase in IgG4 does not affect the efficacy of the vaccine. The role of IgG4 is only speculated from the results of in vitro experiments. Furthermore, since IgG4 does not exist in rodents, it is difficult to create gene-deficient animals, and its physiological significance has not been fully elucidated. These facts should be clearly described, and the predicted role should be discussed. Some expressions are likely to be cited in the anti-vaccination movement, so please be careful when writing.

R: We thank reviewer for this important observation. We have removed references related to IgG4-related diseases.  Regarding the affirmation that an increase in IgG4 does not affect the efficacy of the vaccine, that is correct with the first three doses. However, in the revised version, we added important information:

Recent work demonstrated the appearance of tolerance-induction and non-inflammatory SARS-CoV-2 spike-specific IgG4 antibodies after COVID-19 booster vaccinations. The study also discovered that individuals who received mRNA for both the initial and booster doses had higher serum IL-10 levels following each vaccination treatment [39]. This aligns with research showing that prolonged immunization with SARS-CoV-2 RBD boosters (more than four doses) generated IL-10 responses that ultimately caused humoral and cellular immunological tolerance in mice [180]. Furthermore, the study discovered that extended administration of RBD booster vaccinations significantly elevated the levels of Programmed Cell Death Protein 1 (PD-1) and the Lymphocyte Activation gene 3 (LAG-3), accompanied by a considerable decline in memory CD8⁺ T cells. PD-1 and LAG-3 activation is commonly associated with T-cell exhaustion [180]. This is especially significant because it has been demonstrated that memory CD8⁺ T cell responses are crucial for a successful defense against new SARS-CoV-2 variants.  These may significantly compromise humoral immunity due to the accumulation of neutralization escape mutations [181-183].

Akhtar et al (ref.39) concluded: “Future vaccination strategies need to be designed using thorough research data, requiring larger studies that encompass diverse populations across different settings. This is crucial to prevent immunological tolerance and the induction of IgG4 subclasses after administering booster COVID-19 vaccinations using various regimens.”

 

We acknowledge that this IgG4-induced tolerance could not be detrimental for young and healthy individuals. However, due to immunological senescence, elderly individuals may not benefit from repeated boosting. Older individuals are particularly vulnerable to severe infection, probably reflecting age-related changes in the immune system, which can also compromise vaccine efficacy.

• https://www.nature.com/articles/s41541-024-00874-4

We also acknowledge the reviewer´s concern regarding the misinterpretation of our hypothesis. Our goal is not to discourage vaccination but to understand the full spectrum of immune responses, which is essential for optimizing future vaccine designs. Indeed, we have found evidence for the presence of “allergen-like” epitopes in SARS-CoV-2. This should encourage virologists and vaccine companies to remove such epitopes to avoid the proposed mechanism of immune tolerance. In this way, the Th2 responses should be redirected to an effective Th1 response. 

 

 

1. Standardization of Description

This review was written by multiple authors and requires consistency in the descriptions. For example, there are numerous errors in spelling and abbreviations. Terminology should be standardized and consistent.

1. Thank you for this suggestion. We have made the necessary corrections.

 

Comments on the Quality of English Language

This review was written by multiple authors and requires consistency in the descriptions. For example, there are numerous errors in spelling and abbreviations. Terminology should be standardized and consistent.

1. Thank you very much for this observation. One of the coauthors is a native English speaker, so he did a final proofreading of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their detailed responses.

Author Response

Dear Reviewer: We sincerely appreciate your time and effort in reviewing our work. Your comments and suggestions have significantly improved our work.

Reviewer 3 Report

Comments and Suggestions for Authors

Unfortunately, the revised manuscript still fails to adequately address the reviewers' comments.

Regarding the Omicron variant, the symptoms of COVID-19 closely resemble those of influenza. If the IgE-like epitopes of the Spike protein, which the authors focus on, play a crucial role in viral infection and disease onset, the authors should explicitly specify which COVID-19 symptoms are thought to be linked to IgE. Additionally, if there are similarities between parasitic infections and SARS-CoV-2, the specific overlapping symptoms should be clearly identified.

The manuscript should explicitly state that all vaccines, not just mRNA vaccines, effectively prevent severe COVID-19. Furthermore, the authors should clarify what additional measures could enhance vaccine efficacy and explain why they are focusing on IgE epitopes as a potential approach.

The increase in IgG4 and IgE levels following viral infection or vaccination may not be of physiological significance. Therefore, the introduction should clearly justify why these two immunoglobulins were chosen as the focus of this study.

There are multiple issues related to terminology and consistency. For example, while immunoglobulin E (IgE) is introduced in line 129, it is already mentioned from line 62 onward. In line 123, RSV appears abruptly without prior introduction, and its full name, respiratory syncytial virus, should be provided. Conversely, abbreviations such as FAE, ADCC, and ADCP appear only once and should be spelled out instead. Numerous similar errors remain throughout the manuscript. A professional English language editor is strongly recommended to improve clarity and readability.

Comments on the Quality of English Language

The use of an English proofreader is strongly recommended.

Author Response

We thank the reviewer for his/her insightful comments and the opportunity to clarify our hypothesis regarding the role of IgE-like epitopes in SARS-CoV-2 infection and their potential overlap with symptoms observed in other conditions, such as influenza and parasitic infections. 

Author Response File: Author Response.pdf