Discussion
In this study, we assessed the cross-reactivity of IgG antibodies with SARS-CoV-2 and IBV in poultry farm personnel, pre-pandemic controls, and COVID-19 patients. The poultry farm personnel consisted of vaccine implementers and poultry workers, who had been exposed to IBV vaccines or worked in the poultry houses in which IBV vaccinated chickens are held. Virus neutralization assay was performed on some of the poultry farm personnel sera that had cross-reactive antibodies to SARS-CoV-2 and IBV. Moreover, we investigated the antibody responses against peptides expressed in both SARS-CoV-2 and IBV.
A subset of poultry farm personnel showed elevated levels of IgG specific for all SARS-CoV-2 antigens compared to pre-pandemic controls (Figure 1A-D). This may be the result of prior exposure to IBV via aerosol vaccination, which aims to elicit the development of IBV-specific antibodies in chicken. We found that a significant number of vaccine implementers and one poultry worker developed IBV specific antibodies that cross-react with all four SARS-CoV-2 antigens (S1, RBD, S2, and N) that were analyzed. Due to the partial sequence homology of HCoVs with SARS-CoV-2, exposure to HCoVs may induce cross-reactive immune responses to SARS-CoV-2 proteins. Similarly, Tso et al.28 reported that prior exposure to HCoVs (particularly HCoV-NL63 and HCoV-229E) can also be a source of cross-reactive antibodies against SARS-CoV-2 in pre-COVID-19 pandemic plasma samples. These researchers demonstrated that the SARS-CoV-2 N and S proteins were the predominant sources of the cross-reactivity, which is consistent with the results found in the current study. The sequence homology between SARS-CoV-2 and IBV (4/91, M41, and D274) is 27-29% for S and 30% for N.29 The level of sequence homology between SARS-CoV-2 and other β-CoVs (HCoV-OC43 and HCoV-HKU1) is 32-33% for S and 34% for N, while the homology between SARS-CoV-2 and α-CoVs (HCoV-229E and HCoV-NL63) is 28–30% for S and 28-29% for N.30 Although sequence conservation is lower for more common HCoVs, their high prevalence may lead to widespread antibodies with cross-reactivity to SARS-CoV-2.31 Therefore, IBV exposure has the potential to induce SARS-CoV-2 cross-reactive antibodies.
4/91-, IS/1494/06-, M41-, and D274-specific IgG could be detected in the majority of poultry farm personnel, COVID-19 patients, and pre-pandemic controls. None of the control and COVID-19 individuals in this study are likely to have encountered IBV or IBV vaccines. Therefore, the presence of IBV-specific IgG in these individuals, is indicative of a cross-reactivity that may be associated with exposure to endemic HCoVs. Of note, the IBV-specific IgG levels were significantly lower in COVID-19 patients compared to poultry farm personnel and pre-pandemic controls (Fig 2B-D). In addition, non-hospitalized COVID-19 patients showed significantly higher levels of 4/91-, M41-, and D274-specific IgG than hospitalized patients (Fig S2A, C, D). IgG against IBV could not be measured in the same COVID-19 patients before infection because samples of this timepoint were not available. Therefore, it remains unclear whether SARS-CoV-2 infection resulted in a reduction of IBV-reactive IgG levels in COVID-19 patients or that these levels were already reduced prior to infection. Similarly, we observed reduced IgG levels against rhinovirus A and human herpesvirus 4 in COVID-19 patients (Fig 3J,K). This reduction was most pronounced in hospitalized COVID-19 patients (Fig S3B). These observations are in line with what has been reported in the context of measles virus infection. As suggested by Mina et al.32 measles virus infection is associated with a reduced population immunity against other infections, resulting from a measles-induced immune amnesia. Measles virus can infect by memory T-, B- and plasma-cells33 and measles virus infection is associated with a broad reduction in circulating antibodies against pathogens unrelated to measles.34 Recent findings indicate that SARS-CoV-2 can directly infect T cells in an ACE2-independent manner that is consistent with the previously reported mechanism of SARS-CoV-2-induced lymphopenia.35 Hence, reduced IgG levels against IBV, rhinovirus A, and human herpesvirus 4 observed in hospitalized COVID-19 patients may be because the result of a mechanism similar to measles-induced immune amnesia affecting systemic immune memory. It should be noted that hospitalized COVID-19 patients were older than non-hospitalized patients (Table S3). However, this age difference does not explain the above-mentioned differences in IBV-, rhinovirus A- and human herpesvirus 4-specific IgG, as elderly patients typically do not have significantly reduced antibody levels against common infectious agents.36
Importantly, we noticed a strong correlation between IBV-specific IgG and SARS-CoV-2 S1-, RBD-, S2-, and N-specific IgG in poultry farm personnel compared to the pre-pandemic control group and COVID-19 patients. This finding indicates that these cross-reactive antibodies may be triggered by exposure to IBV in poultry farm personnel. Theoretically, if these cross-reactive antibodies would be the result of HCoV exposure, we would have observed similar correlation results in the pre-pandemic samples. But the present results did not confirm this interpretation.
Several differences were observed between poultry farm personnel and COVID-19 patients in the correlation patterns for IBV- and SARS-CoV-2-specific IgG titers (Figure 5A,B). It can be postulated that IBV-specific antibodies in poultry farm personnel are most likely the result of direct exposure to IBV leading to cross-reactivity. It is obvious that SARS-CoV-2 exposure conduces to SARS-CoV-2 specific antibodies in COVID-19 patients. In these patients, the strong correlation between SARS-CoV-2 peptide-specific IgG and SARS-CoV-2 antigen-specific IgG can be partially explained by the aforementioned exposure to SARS-CoV-2. Concerning pre-pandemic controls, individuals have no IBV and SARS-CoV-2 history, and hence, moderate positive correlations shown in Figure 5C, may be the result of exposure to HCoVs.
COVID-19 patients had higher levels of SARS-CoV-2 S1-, RBD-, S2-, and N-specific IgG than pre-pandemic controls and poultry farm personnel. In addition, we found that SARS-CoV-2 antigen-specific IgG levels in COVID-19 patients correlated with disease severity. Hospitalized COVID-19 patients showed higher levels of S1-, RBD-, S2-, and N-specific IgG compared to non-hospitalized COVID-19 patients. These findings corroborate results published by other research groups.27 Bruni et al.37 reported that non-hospitalized patients had lower S ectodomain-, RBD-, and N-specific IgG titers and blood pro-inflammatory cytokine profiles compared to patients in intensive care units. Similarly, Chen et al.38 demonstrated that severe COVID-19 patients mounted the highest S1-, RBD-, and S2-specific IgG titers compared to moderate, mild, and asymptomatic patients. In addition, Kowitdamrong et al.39 found that the levels of S1-specific IgA and IgG were higher in severe COVID-19 patients. Similar observations have been reported in many other studies.30,40,41 However, the molecular mechanism underlying this association has not yet been elucidated in detail.39 One of the possible explanations is that increased levels of IgG may be related to the high viral loads. Moreover, increased inflammatory signals, antigen presentation, and stimulatory signals for humoral responses may play a role in this process.42
Next, we measured IgG levels against nine SARS-CoV-2 peptides that were defined as highly indicative of SARS-CoV-2 exposure history by Shrock et al.26 in all three groups. We detected elevated levels of antibody responses to SARS-CoV-2 peptides in COVID-19 patients compared to poultry farm personnel and pre-pandemic control samples. IgG levels against SARS-CoV-2 peptides [N (aa 153-176), N (aa 221-244), N (aa 358-381), N (aa 382-405), S (aa 547-570), S (aa 782-805), and S (aa 1138-1161)] of hospitalized COVID-19 patients were significantly higher than non-hospitalized COVID-19 patients. This is in line with the findings published by Shrock et al.26 indicating that hospitalized COVID-19 patients developed stronger and broader antibody responses to SARS-CoV-2 S and N proteins than non-hospitalized patients. The most homologous region among the studied SARS-CoV-2 peptides and IBV is S (aa 807-830) (Figure S8G). This immunodominant Coronavirus peptide domain of spike has been identified as a recognizable region for immune responses. Shrock et al.26 showed antibody responses to SARS-CoV-2 S (aa 807-830) in 79.9% of COVID-19 patients and to the corresponding peptides from HCoV-OC43 and HCoV-229E of 20% in the pre-COVID-19 individuals. On the other hand, Loyal et al.43 achieved remarkable results in the functional role of pre-existing SARS-CoV-2- and HCoV-reactive CD4+ T cells. SARS-CoV-2 S (816-830) region is recognized by CD4+ T cells in 20% of healthy individuals, 50-60% of SARS-CoV-2 convalescents, and 97% of BNT162b2-vaccinated individuals. Another indicative peptide sequence for SARS-CoV-2 recognition is S (aa 1144-1163). In this respect, Shrock et al.26 reported that both SARS-CoV-2 and HCoV-OC43 peptides corresponding to this peptide are recognized much more frequently by COVID-19 patients compared to pre-COVID-19 controls. Notably, we also observed elevated levels of IgG specific for S (aa 1144-1163) in poultry farm personnel (Figure 3I). Cross-reactive responses to S (aa 807-830) and S (aa 1144-1163) were more frequently detected in poultry farm personnel than in pre–COVID-19 era controls. Moreover, some poultry farm personnel had high IgG levels against SARS-CoV-2 N (aa 153-176). It is important to note that, all poultry farm personnel declared that they did not have COVID-19 symptoms. It cannot be excluded that elevated IgG specific for SARS-CoV-2 antigens may be caused by subclinical infection with SARS-CoV-2.
Hospitalized COVID-19 patients showed a modest increase in HIV-1-specific IgG levels compared to non-hospitalized patients. HIV-1 (aa 967-991) shares 28% sequence homology with SARS-CoV-2.29 Slightly increased levels of HIV-1-specific IgG in hospitalized COVID-19 patients may be explained by this sequence homology.
Repeated antigen exposure may stimulate antibody response, and hence the elevated levels of S1-, RBD-, and higher amounts of IBV-specific IgG observed in our study in long-term vaccine implementers support this association. Vaccine implementers with >100 months’ experience showed significantly increased levels of S1-, RBD-, 4/91-, IS/1494/06-, M41-, and D274-specific IgG compared to vaccine implementers with 2-20 months of experience. This suggests that long-term exposure to IBV leads to higher levels of SARS-CoV-2 S1- and RBD-cross-reactive IgG. Kosikova et al.44 reported that repeated influenza exposure imprinted not only increased antibody quantity but also improved quality as shown by higher affinity antibody development.
In the present study, we found cross-reactive antibodies between IBV and SARS-CoV-2, but there was no neutralization in the poultry farm personnel (samples selected based on antibody titers against SARS-CoV-2 and IBV) while this was the case in COVID-19 patients (Figure S7). This could be due to 1) low affinity for the RBD compared to COVID-19 patients, 2) low concentrations of antibody against SARS-CoV-2, or 3) the antibodies do not bind to the part of RBD that is responsible for binding to ACE2. On the other hand, there may be cross-reactive CD4+ and CD8+ T cells that may develop in response to exposure to IBVs and may protect against disease. However, this needs further investigation in future studies.
Although no neutralization was observed in the selected poultry farm personnel as mentioned above, one sample from vaccine implementers made a notable exception. The sample VI17, which showed low IBV-specific IgG titers, was found to have high IgG titers against SARS-CoV-2 antigens. This leads to the neutralization of SARS-CoV-2. A possible explanation may be through subclinical SARS-CoV-2 infection.
Here, we conducted a comprehensive analysis of IgG cross-reactivity between SARS-CoV-2 and IBV. The most conspicuous finding of this study is that a subset of poultry farm personnel, particularly long-time exposed vaccine implementers, showed elevated levels of IgG specific for all IBV and SARS-CoV-2 antigens that were analyzed. Furthermore, there was a strong correlation between IBV-specific IgG and SARS-CoV-2 antigen-specific IgG in these individuals. However, these cross-reactive antibodies did not have neutralizing capacity in the SARS-CoV-2 neutralization assay that we employed. It is important to note that, conducting a similar cohort study may not be possible in the future, because of the extensive SARS-CoV-2 vaccine applications, particularly for farm personnel. In conclusion, our data demonstrate that exposure to IBV may cause SARS-CoV-2-cross-reactive IgG.