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.