key: cord-1041172-dekbeuh5 authors: Wang JIong, J.; Young, B. E.; Li, D.; Seppo, A. E.; Zhou, Q.; Wiltse, A.; Nowak-wegrzyn, A.; Murphy, K.; Widrick, K.; Diaz, N.; CruzVasquez, J.; Jarvinen, K. M.; Zand, M. S. title: Broad Cross-reactive IgA and IgG Against Human Coronaviruses in Milk Induced by COVID-19 Vaccination and Infection date: 2022-03-14 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2022.03.13.22272281 sha: 9bd6d30ca1f77b6627433ed43db84aadf9a50f80 doc_id: 1041172 cord_uid: dekbeuh5 It is currently unclear if SARS-CoV-2 infection or mRNA vaccination can also induce IgG and IgA against common human coronaviruses (HCoVs) in lactating parents. Here we prospectively analyzed human milk (HM) and blood samples from lactating parents to measure the temporal patterns of anti-SARS-CoV-2 specific and anti-HCoV cross-reactive IgA and IgG responses. Two cohorts were analyzed: a vaccination cohort (n=30) who received mRNA-based vaccines for COVID-19 (mRNA-1273 or BNT162b2), and an infection cohort (n=45) with COVID-19 disease. Longitudinal HM and fingerstick blood samples were collected pre- and post-vaccination or, for infected subjects, at 5 time-points 14 - 28 days after confirmed diagnosis. The anti-spike(S) and anti-nucleocapsid(N) IgA and IgG antibody levels against SARS-CoV-2 and HCoVs were measured by multiplex immunoassay (mPlex-CoV). We found that vaccination significantly increased the anti-S IgA and IgG levels in HM. In contrast, while IgG levels increased after a second vaccine dose, blood and HM IgA started to decrease. Moreover, HM and blood anti-S IgG levels were significantly correlated, but anti-S IgA levels were not. SARS2 acute infection elicited anti-S IgG and IgA that showed much higher correlations between HM and blood compared to vaccination. Vaccination and infection were able to significantly increase the broadly cross-reactive IgG recognizing HCoVs in HM and blood than the IgA antibodies in HM and blood. In addition, the broader cross-reactivity of IgG in HM versus blood indicates that COVID-19 vaccination and infection might provide passive immunity through HM for the breastfed infants not only against SARS-CoV-2 but also against common cold coronaviruses. The severe acute respiratory syndrome virus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19) pandemic, causing more than 5 million deaths worldwide as of January 2022 (1) . SARS-CoV-2 belongs to the zoonotic coronaviridae family (2) . The human coronaviruses include SARS-CoV-1 (SARS1), Middle Eastern Respiratory Syndrome (MERS), and the endemic β -(OC43, HKU1) and α-human cold coronaviruses (229E, NL63) (3), which are responsible for 30% of mild upper respiratory infections (4, 5) . The major immunodominant epitopes for SARS-CoV-2 are located on the homotrimeric spike (S) glycoprotein (2) , which shares sequence homologies with the β -HCoVs. The S-protein N-terminal S1 subunit has a receptor-binding domain (RBD), mediating viral binding via high-affinity interactions with host cell angiotensin-converting enzyme 2 (ACE2). In contrast, the S2 subunit is responsible for virus-cell membrane fusion (6) , and displays more sequence homology between HCoV strains than the S1 subunit (7, 8). Individuals with symptomatic COVID-19 infection exhibit increases in an extensive range of anti-SARS-CoV-2 antibodies (6, 9, 10) . The patterns of seroconversion in most individuals are similar to those of secondary immune responses, and rapid and robust antibody response was correlated with disease severity (6) . Our previous clinical study suggested that pre-existing OC43-reactive antibodies are involved in the early humoral response to SARS-CoV-2 (11) . Other groups have reported that pre-existing cross-reactive antibodies or memory B cell immunity might be protective against COVID-19 (12, 13, 14) . Human milk (HM) provides protection against various infectious diseases, including respiratory illnesses in infants (15) . This protection is in part due to the passive immunity transferred via maternal immunoglobulins (16) . Anti-SARS-CoV-2 secretory IgA is present in HM during and after acute infection (17, 18) , and those antibodies neutralize SARS-CoV-2 in vitro (19, 20) . Currently, COVID-19 mRNA vaccination is the most effective way to prevent SARS-CoV-2 infection and transmission (21) . Studies have also shown that mRNA vaccination can elicit high titers of IgA and IgG antibodies in HM (22, 23) , which have neutralizing activity against SARS-CoV-2 (20) . The majority of evidence suggests that transmission of SARS-CoV-2 is overwhelmingly airborne, with minimal evidence of transmission via HM (24, 25) . Thus, passive transfer of maternal antibodies in HM may provide neonatal protection from SARS-CoV-2 infection and mitigate severe disease. Antibody cross-reactivity across different viral strains is an important source of pre-existing immunity to emerging viral variants (26, 27, 10) . Similarly, vaccination against a new viral strain may "back-boost" cross-reactive immunity to previously cir- culating strains (28, 29) . This appears to be the case for SARS-CoV-2. It has been reported that pre-pandemic HM samples exhibited low-level cross-reactivity to the RBD subunit of SARS-CoV-2 (18) . However, it is unclear whether SARS-CoV-2 infection or vaccination can produce antibodies against common HCoVs in HM or blood, and the characteristics of any such elicited cross-reactive antibodies. This study quantitatively assessed anti-S and anti-N HM IgA and IgG antibodies against SARS-CoV-2, SARS-CoV-1, and four other common HCoVs (OC43, HKU1, NL63, 229E) following COVID-19 infection and vaccination. We first estimated anti-SARS-CoV-2 IgA and IgG antibody responses against whole S protein (SARS-CoV-2-S), S1, S2, and RBD subunits elicited by mRNA vaccination. The HM and blood IgA and IgG antibodies significantly increased by 18 days after the first dose of SARS-CoV-2 mRNA vaccine (Vac1) compared with prevaccination (P<0.0001) (Figure 2 ), consistent with previous reports for HM (30, 31, 22, 20) and our recent findings of blood IgG responses in healthy adults (32) . Except for one participant, who had higher milk anti-S and anti-N IgG levels before vaccination consistent with previous SARS-CoV-2 exposure, HM anti-S levels uniformly increased 10 (IgA) and 100 fold (IgG) post-vaccination. However, milk and blood anti-S IgA levels 3 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Antibody concentration (ng/ml) 4 IgA IgG **** **** **** **** **** **** **** *** **** **** **** **** **** *** **** **** **** ** * *** ** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** **** ** **** **** **** Antibody concentration (ng/ml) Vac1 Vac2 S1 S2 RBD S1 S2 RBD S1 S2 RBD S1 S2 RBD IgA IgG S1 S2 RBD S1 S2 RBD All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.22272281 doi: medRxiv preprint remained constant or decreased after the second vaccine dose (Vac2). In contrast, HM and blood IgG levels continued to increase up to 187 days after Vac2, compared with levels after Vac1 (P<0.0001). In addition, the anti-S2 subunit-specific IgG levels did not increase as high as IgG against the full S protein, S1, and RBD sub-regions. As expected, we did not detect N-specific antibody responses following vaccination (Figure 2 A) . To understand the relationship between the vaccine-induced SARS-CoV-2 reactive IgA and IgG responses in blood versus HM, we performed a correlation analysis (Figure 2B) . Significant positive correlations exist between HM and blood IgA and IgG antibodies against full-spike protein, S1, S2, and RBD subunits (Pearson's r in the range 0.22-0.46, Figure 2 B ). Elevated IgG antibody levels after Vac2 showed a higher correlation between milk and blood compared to Vac1, but r values decreased after Vac2. Overall, milk IgA levels did not correlate with blood IgG (P>0.05). In contrast, milk IgG had the highest correlation with blood IgG (mean r= 0.36, P<0.05). This suggests a common source for milk and serum IgG, but a different source for milk IgA, consistent with our previous work (33) . It is noteworthy that anti-SARS-CoV-2-S, S2 and RBD IgA concentrations in milk did not correlate with concentrations in blood after Vac1. This lack of correlation strongly suggests that IgA in milk originates from a pool of mucosal B-cells and is distinct from serum IgA. Figure 3A , as well as antibodies that recognized S1, S2, and RBD-subunits of spike proteins. These results demonstrate that SARS-CoV-2 infection can significantly increase anti-S IgA and IgG levels in most subjects compared with the naive healthy pre-vaccination cohort; only one subject's milk IgG remained undetectable over the 28 days. In the Con- Compared to anti-S IgA, IgG concentrations did not exhibit such robust changes from day 0-28. Given that we collected our first sample (Day0) at an average 8 days post diagnosis, we likely missed the initial surge in IgA antibodies, which has been shown to occur earlier than the increase in milk IgG (20) . This pattern is also consistent with the trajectory of HM IgA and IgG after vaccination and other reports (34, 35, 31) . To our knowledge, this is the first study to report milk anti-N IgA and IgG levels in (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Days from Enrollment Correlation Coefficient (r) 6 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 8 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Additional correlation analysis was performed between the antibodies in HM versus blood, and IgG versus IgA. HM IgG was highly correlated to blood IgG levels after vaccination, whereas infection caused a higher correlation between HM and blood and IgG with IgA, especially in the early phase of COVID-19 ( Figure 5) . Additionally, anti-β -HCoV antibodies are more correlated to each other than to anti-α -HCoV antibodies, and anti-SARS2 S2 subunit antibodies are highly correlated to all β -HCoV S antibodies. This is the first study to document that in human milk (HM) both COVID-19 mRNA vaccination and SARS-CoV-2 (SARS2) infection induced broadly cross-reactive IgG and IgA 9 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.22272281 doi: medRxiv preprint antibodies against human common cold coronaviruses (HCoVs), as estimated by multiplex immunoassay (mPlex-CoV) (32) . In general, we found that the acute infection elicited a broader cross-reactive antibody response against HCoV spike proteins compared to the two dose mRNA vaccinations. Furthermore, our results demonstrate a high correlation between broadly cross reactive anti-S IgG in HM and peripheral blood broad post-infection and vaccination, with no milk-blood correlation for anti-S IgA. Interestingly, HM cross-reactive IgG demonstrated broader and higher magnitude crossreactivity to S proteins tested, including OC43 and HKU1 β -HCoVs, 229E and NL63 α-HCoVs. These result highlight the question of the origin of such broadly cross-reactive anti-coronavirus S protein IgG and IgA. Mucosal IgA is produced by plasma cells in the lamina propria and are transported across epithelial cells by the polymeric immunoglobulin receptor (pIgR) (36) . Milk IgA is produced by mammary gland B cells that have migrated from the mother's intestine via the "enteromammary link" (37) , as shown in animal studies (38, 39, 40, 41) . This is controlled by the mucosal vascular addressin, MadCAM-1, which interacts with the gut homing receptor α4β 7 integrin (42) and mucosal-associated CCR10 (43) . Furthermore, in a rabbit model, either oral or inhaled RSV resulted in RSV-specific IgA production in milk, bronchial and enteral secretions, whereas systemic immunization did not (44) . Human studies have shown that oral immunization in women results in an increase in plasma cells specific to non-pathogenic E. coli strains in milk, but not in saliva or blood (45) . This compartmentalization of mucosal IgA secreting B cells may explain the lack of correlation between HM and blood IgA anti-HCoVs post-infection or vaccination. In contrast to IgA, we found a high correlation between the anti-S HCoV binding profiles in IgG. This may be due to the dichotomous source of HM IgG: In HIV, for example, it has been demonstrated that specific IgG secreting cells can be found in HM, which predominate over IgA-secreting cells and have a mucosal homing profile similar to gut-associated lymphoid tissue B cells (46) . Moreover, specific anti-HIV IgGs isolated from HM and plasma have similar neutralization potencies, despite lower HM concentrations, suggesting that the neutralization responses in HM are mainly due to plasma-derived IgG (47) . Therefore, Neutralizing capacity of HM IgG may be a function of the ratio between specific anti-HIV IgG produced by mucosal versus peripheral blood IgG secreting cells, which may vary between mothers (48). The source for the broadly cross-reactive anti-S IgG antibodies found in HM is not precisely known. HM IgG antibodies are produced by B cells, which originate in the gut-or bronchus associated lymphoid tissue and home to HM, or circulate in plasma during respiratory infections (47) . Broad cross-reactivity with seasonal human coronaviruses may indicate that that this IgG production is boosted in the airways via mucosal associated memory B cells within lymphoid tissue at the site of previous exposure to seasonal coronaviruses. This would be a novel mechanism providing broadly crossreactive antibodies. Irrespective of their origin, these anti-SARS-CoV-2 IgA and IgG antibodies potentially serve to provide broadly protective antibodies to the infant. It is well known that the S2 subdomain of spike protein shares more homology with other HCoVs than the S1 subdomain, including β -(SARS-CoV-1, OC43, and HKU1), and α-(229E and NL63) HCoVs (2, 3, 5) . Studies have also shown that SARS-CoV-2 infection can enhance preexisting HCoV immunity through anti-S2 IgG and memory B cell formation (14) . During infection, a rapid increase in S2-reactive IgG levels highly 10 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.22272281 doi: medRxiv preprint correlates with a rapid increase SARS-CoV-2 IgG and, counterintuitively, the severity of COVID-19 disease (11) . Thus, it remains unclear, whether a predominance of anti-S2 IgG in milk contributes substantially to passive infant protective immunity to SARS-CoV-2 exposure in infants. In this study, we found a high correlation between the IgG antibodies against SARS-CoV-2 with β -HCoVs IgG in milk and blood ( Figure 5 Infected cohort. Forty-six lactating parents with COVID-19 infection were recruited nationally between July 2020 -April 2021. To be eligible, participants needed to have an RT-PCR COVID-19 diagnosis within the previous 14 days and be providing HM to an infant ≤6 months of age. The eligible subjects aged ≥18 years collected milk samples at home on days 0, 3, 7, 10, and 28 as described below. Sample Collection See Figure1 for details. HM samples: All participants collected milk and fingerstick capillary blood samples at home as previously described (20) . 5-10 mL HM samples were stored in the home -20 0 C freezer until transport to the lab, packaged on ice via overnight mail. Milk samples were spun at 10,000g for 10 minutes at 4 degrees to remove the skim milk in the lab, and were aliquoted and stored at -80 0 C until analysis. Fingerstick blood samples: All capillary blood samples were collected with volumet-11 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In the infection cohort, the IgG antibodies in fingerstick blood samples were evaluated by mPLEX-CoV assay. All samples were tested at the same time in duplicate, and significant results were calculated with generalized linear mixed-effects models (**** P < 0.0001, *** P< 0.001, ** P<0.01, * P<0.05). KJ and MZ contributed to funding acquisition for this project. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. **** **** **** **** In the infection cohort, the anti-N IgG antibodies against SARS2, SARS1, and other common coronaviruses (HCoVs) in the milk and fingerstick blood samples had evaluated by mPLEX-CoV assay. All samples were tested at the same time in duplicate, and significant results were calculated with generalized linear mixed-effects models (**** P < 0.0001, *** P< 0.001, ** P<0.01, * P<0.05). 2 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. HCoV-NL63 NL63 NC_005831.2 a Spike (S) proteins of human coronaviruses are recombinant proteins expression by the baculovirus system with the trimerization domain at the C-terminal with His-tag. b Nucleocapsid proteins of human coronaviruses are recombinant proteins with C-terminal His-tag . 3 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 14, 2022. ; https://doi.org/10.1101/2022.03.13.22272281 doi: medRxiv preprint WHO. 2021. 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In the infection cohort, the IgG antibodies in fingerstick blood samples had evaluated by mPLEX-CoV assay. All samples were tested at the same time in duplicate, and significant results were calculated with generalized linear mixed-effects models **** *** **** **** **** *** *** 2