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PLOS ONE logoLink to PLOS ONE
. 2021 Aug 27;16(8):e0256482. doi: 10.1371/journal.pone.0256482

Original antigenic sin responses to Betacoronavirus spike proteins are observed in a mouse model, but are not apparent in children following SARS-CoV-2 infection

Stacey A Lapp 1,2, Venkata Viswanadh Edara 1,2,3, Austin Lu 1,2, Lilin Lai 1,2,3, Laila Hussaini 1,2, Ann Chahroudi 1,2, Larry J Anderson 1,2, Mehul S Suthar 1,2,3, Evan J Anderson 1,2,4, Christina A Rostad 1,2,*
Editor: Victor C Huber5
PMCID: PMC8396729  PMID: 34449792

Abstract

Background

The effects of pre-existing endemic human coronavirus (HCoV) immunity on SARS-CoV-2 serologic and clinical responses are incompletely understood.

Objectives

We sought to determine the effects of prior exposure to HCoV Betacoronavirus HKU1 spike protein on serologic responses to SARS-CoV-2 spike protein after intramuscular administration in mice. We also sought to understand the baseline seroprevalence of HKU1 spike antibodies in healthy children and to measure their correlation with SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome (MIS-C).

Methods

Groups of 5 mice were injected intramuscularly with two doses of alum-adjuvanted HKU1 spike followed by SARS-CoV-2 spike; or the reciprocal regimen of SARS-Cov-2 spike followed by HKU1 spike. Sera collected 21 days following each injection was analyzed for IgG antibodies to HKU1 spike, SARS-CoV-2 spike, and SARS-CoV-2 neutralization. Sera from children hospitalized with acute COVID-19, MIS-C or healthy controls (n = 14 per group) were analyzed for these same antibodies.

Results

Mice primed with SARS-CoV-2 spike and boosted with HKU1 spike developed high titers of SARS-CoV-2 binding and neutralizing antibodies; however, mice primed with HKU1 spike and boosted with SARS-CoV-2 spike were unable to mount neutralizing antibodies to SARS-CoV-2. HKU1 spike antibodies were detected in all children with acute COVID-19, MIS-C, and healthy controls. Although children with MIS-C had significantly higher HKU1 spike titers than healthy children (GMT 37239 vs. 7551, P = 0.012), these titers correlated positively with both SARS-CoV-2 binding (r = 0.7577, P<0.001) and neutralizing (r = 0.6201, P = 0.001) antibodies.

Conclusions

Prior murine exposure to HKU1 spike protein completely impeded the development of neutralizing antibodies to SARS-CoV-2, consistent with original antigenic sin. In contrast, the presence of HKU1 spike IgG antibodies in children with acute COVID-19 or MIS-C was not associated with diminished neutralizing antibody responses to SARS-CoV-2.

Introduction

As the coronavirus disease 2019 (COVID-19) pandemic continues and the first vaccinations are administered, our understanding of the immune responses to SARS-CoV-2 continues to evolve. A prevailing question has been what role pre-existing immunity to endemic human coronaviruses (HCoVs) plays in the serologic responses to SARS-CoV-2. The Betacoronaviruses, HKU1 and OC43, and Alphacoronaviruses, 229E and NL63, cause seasonal respiratory illnesses in both adults and children worldwide. Seroprevalence data indicate that infection with HCoVs occurs during early childhood [1], and the majority of adults are seropositive with antibody titers that wane over time [2, 3]. Cross-reactive antibodies are elicited within genera, but less so between Alphacoronaviruses and Betacoronaviruses [4]. Cross-reactive antibodies are predominantly directed against non-neutralizing antigens, including the S2 subunit of the spike protein and nucleocapsid proteins. Because children have more frequent exposures to HCoVs, some have hypothesized that pre-existing cross-reactive immunity to HCoVs may in part explain the reduced COVID-19 disease severity observed in children [57].

Although SARS-CoV-2 is a Betacoronavirus, it shares only 33% amino acid identity with the HCoV Betacoronaviruses within the spike protein, which is the predominant immunogen and neutralizing antigen of coronaviruses. Thus, although some pre-pandemic sera do have SARS-CoV-2 spike-reactive antibodies, these antibodies are poorly neutralizing [8]. The widespread seroprevalence of SARS-CoV-2 binding, non-neutralizing antibodies has led to concern that pre-existing immunity from prior HCoV exposures may contribute to aberrant serologic responses to the antigenically similar SARS-CoV-2, as in original antigenic sin (OAS). OAS refers to the preferential induction of antibodies directed against an original, priming antigen rather than a structurally similar boosting antigen. The mechanism of OAS is thought to be attributable to the initial development and differentiation of memory B cells directed against the original antigen of exposure. Upon secondary exposure to a structurally similar antigen, these memory B cells undergo clonal expansion to preferentially produce antibodies directed against the original priming antigen. The phenomenon of OAS has been well described with several other viruses including dengue and influenza, and has led to concerns about its impact on disease severity and vaccine development [9].

In this study, our objectives included determining if prior exposure to HKU1 spike protein in a mouse model impacted serologic responses to SARS-CoV-2 upon spike protein challenge. We further sought to determine the baseline seroprevalence of HKU1 spike antibodies in healthy children and to assess if the presence or titer of HKU1 antibody affected the response to SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute COVID-19 or multisystem inflammatory syndrome (MIS-C).

Experimental results

Murine results

Two groups of five BALB/c mice were injected intramuscularly (IM) with 10 μg each of alum-adjuvanted full-length spike protein antigens according to the schedule shown in Fig 1. One group received a prime (d0) and boost (d21) with SARS-CoV-2 full-length spike protein, followed by a prime (d42) and boost (d63) with HCoV HKU1 full-length spike protein. The second group received a reciprocal immunization regimen of a prime and boost with HKU1 spike followed by SARS-CoV-2 spike prime and boost. Submandibular bleed samples were collected at 3 weeks following each injection, and a terminal bleed was performed 3 weeks following the last injection.

Fig 1. Schematic of intramuscular spike protein administrations in groups of five BALB/c mice.

Fig 1

Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.

We measured IgG antibody titers to SARS-CoV-2 spike and HKU1 spike antigens in the two groups of mice by ELISA. We also measured the neutralizing antibody titers of these plasma samples using a Focus Reduction Neutralization Test (FRNT) with an infectious clone of SARS-CoV-2 virus (Wuhan strain, A.1 based on PANGO lineage) as previously described [10, 11] (Fig 2 and S1 Dataset). As expected, the SARS-CoV-2 spike-primed mice developed SARS-CoV-2 spike IgG antibodies that significantly increased in titer after the first injection (1914 vs. 85, P = 0.012) and peaked at 21 days after the SARS-CoV-2 spike boost (139316 vs 85, P<0.001, Fig 2A). The SARS-CoV-2 spike IgG antibody titers did not further increase after subsequent administration of HKU1 spike. The HKU1 spike-primed mice also generated cross-reactive anti-SARS-CoV-2 spike IgG antibodies that reached statistical significance on day 21 post-HKU1 spike boost (2594 vs. 85, P = 0.018, Fig 2B). Subsequent injection with SARS-CoV-2 spike incrementally boosted SARS-CoV-2 spike IgG titers, which increased significantly after the second boost (51523 vs. 2594, P = 0.031). These findings suggested that heterologous boosting drove affinity maturation against related epitopes on the boosting SARS-CoV-2 spike antigen.

Fig 2. Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies.

Fig 2

SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). *P<0.05; **P<0.01; ***P<0.005; ****P<0.001.

Analogously, SARS-CoV-2 spike-primed mice developed cross-reactive HKU1 spike binding IgG antibodies, but these did not significantly increase above baseline following either the first (137 vs. 85, P = 0.990) or second injection (2851 vs. 85, P = 0.116, Fig 2C). Subsequent immunization with HKU1 spike protein increased the HKU1 spike IgG titers significantly over baseline (10351 vs. 85, P = 0.038) which were maintained after HKU1 spike boost (39719 vs. 10351, P = 0.750, Fig 2C). As expected, the HKU1 spike-primed mice generated HKU1 spike-binding IgG titers that increased significantly after the initial HKU1 injection (1197 vs. 85, P = 0.001) and peaked following HKU1 boost (106167 vs. 85, P<0.001, Fig 2D). Subsequent injection with SARS-CoV-2 spike protein did not further increase HKU1 spike IgG titers. The plateau in antibody titers observed following repeated boosting is a known phenomenon whereby antibody feedback limits further B cell expansion by masking immunodominant epitopes [12].

We then performed SARS-CoV-2 neutralizing FRNT antibody assays with these samples using an infectious clone of SARS-CoV-2. Only the SARS-CoV-2 spike primed mice generated neutralizing antibodies to SARS-CoV-2 (Fig 2E). Neutralizing titers significantly increased compared to baseline after the boost immunization of SARS-CoV-2 spike (180 vs. 31, P = 0.006) and further increased following the second HKU1 spike boost (520 vs. 180, P = 0.050). These results suggested that HKU1 spike boosted neutralizing antibodies to SARS-CoV-2, although ongoing affinity maturation to SARS-CoV-2 spike in the germinal centers may have also contributed to these findings. In contrast, the HKU1 spike/SARS-CoV-2 prime-boosted mice did not generate neutralizing antibodies to SARS-CoV-2 following any administration of antigen (Fig 2F). Thus, recent prior exposure to the endemic HKU1 coronavirus adjuvanted spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies in mice upon SARS-CoV-2 spike protein administration, despite the presence of SARS-CoV-2 binding antibodies. These data indicate that the SARS-CoV-2 spike boost immunization predominantly boosted cross-reactive antibodies to the original priming (HKU1) antigen at shared, non-neutralizing epitopes.

Results in children

Because our mouse data suggested that prior exposure to endemic coronaviruses may blunt serologic responses to SARS-CoV-2 by the phenomenon of original antigenic sin, we next measured the baseline seroprevalence of IgG antibodies to both SARS-CoV-2 and HKU1 spike proteins in healthy children (n = 14) using pre-pandemic sera. The healthy, asymptomatic children were recruited from the community to participate in a phlebotomy study between 2016–2018, and they were comprised of 10 females (71%), median age 8 years (IQR 2.3–11.8 years), 8 (57%) Black, 4 (29%) White, 2 (14%) other race, and 14 (100%) non-Hispanic ethnicity. The geometric mean antibody titers from healthy children were then compared to children hospitalized at Children’s Healthcare of Atlanta with acute COVID-19 (n = 14) or MIS-C (n = 14) (Table 1 and Fig 3) using one-way analysis of variance (ANOVA) of log-transformed titers.

Table 1. Clinical and demographic characteristics of patient cohort of children hospitalized with acute COVID-19 or MIS-C at Children’s Healthcare of Atlanta.

COVID-19 (n = 14) MIS-C (n = 14) P-value
n n
Age, years, mean (SD) 10.9 (7.6) 14 9.1 (3.9) 14 0.436
Gender, female, n (%) 8 (57%) 14 7 (50%) 14 0.379
Race, n (%) 0.006
 Black 4 (29%) 14 12 (86%) 14
 White 9 (64%) 14 1 (7%) 14
 Declined 1 (7%) 14 14
Ethnicity, n (%) 0.065
 Hispanic 5 (36%) 14 1 (7%) 14
 Non-Hispanic 9 (64%) 14 13 (93%) 14
Disease Severity, n (%) 0.002
 Mild/moderate 10 (71%) 14 2 (14%) 14
 Severe 4 (29%) 14 12 (86%) 14
Labs, mean (SD)
 WBC max, x103cells/μL 11.7 (5.8) 13 11.9 (6.2) 14 0.932
 ALC min, cells/μL 2262.9 (1703.4) 13 1055.1 (673.1) 14 0.021
 Platelets min, x103cells/μL 262.2 (106.0) 13 153.8 (84.4) 14 0.007
 ESR max, mm/hr 34.9 (36.2) 7 53.6 (22.8) 10 0.209
 Sodium min, mmol/L 137.3 (2.1) 13 133.1 (4.6) 14 0.006
 Creatinine max, mg/dL 0.6 (0.2) 13 1.1 (0.7) 14 0.02
 ALT max, U/L 148.5 (396.6) 13 71.9 (48.5) 14 0.479
 BNP max, pg/mL 21.5 (16.1) 5 1774.1 (1260.3) 13 0.008
 Troponin max, ng/mL 0.015 (0.0) 4 1.2 (1.7) 10 0.193
 Ferritin max, ng/mL 217.5 (134.2) 10 1385.4 (1278.5) 14 0.009
 CRP max, mg/dL 6.8 (7.5) 12 19.5 (10.4) 14 0.002
Imaging
CXR, n (%)
 Infiltrates 3 (33%) 9 10 (83%) 12 0.02
 Pleural effusions 3 (33%) 9 9 (75%) 12 0.056
Echocardiogram, n (%)
 Depressed function 0 (0%) 4 7 (50%) 14 0.07
 Coronary artery dilation 0 (0%) 4 2 (14%) 14 0.423
Treatment, n (%)
 Remdesivir 4 (29%) 14 1 (7%) 14 0.139
 IVIG 0 (0%) 14 13 (93%) 14 <0.001
 Steroids 5 (36%) 14 12 (86%) 14 0.007
 Antiplatelet 0 (0%) 14 13 (93%) 14 <0.001
Outcomes
 Days of hospitalization, mean (SD) 6.1 (7.8) 14 9.4 (6) 14 0.221
 ICU admission, n (%) 5 (36%) 14 12 (86%) 14 0.007
 Days of ICU, mean (SD) 2.6 (5.6) 14 5.8 (5) 14 0.123
 Low-flow O2, n (%) 5 (36%) 14 11 (79%) 14 0.022
 Mechanical ventilation, n (%) 0 (0%) 14 2 (14%) 14 0.142
 Vasopressors, n (%) 0 (0%) 14 10 (71%) 14 <0.001
 Death, n (%) 0 (0%) 14 0 (0%) 14 ---

Max, maximum value obtained during the hospitalization; Min, minimum value obtained during the hospitalization; WBC, white blood cell count; ALC, absolute lymphocyte count; ESR, erythrocyte sedimentation rate; ALT, alanine aminotransferase; BNP, brain natriuretic peptide; CRP, C-reactive protein; CXR, chest radiograph; IVIG, intravenous immunoglobulin; ICU, intensive care unit; O2, oxygen.

Fig 3. HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C.

Fig 3

SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. Each dot represents a unique patient, and data represent the geometric mean titers of *P<0.05; **P<0.01; ***P<0.005; ****P<0.001.

As expected, we found significantly higher SARS-CoV-2 spike IgG titers in children with acute COVID-19 (7727 vs. 1019, P<0.001) and MIS-C (46989 vs. 1019, P<0.001) compared to healthy controls (Fig 3A). Moreover, children with MIS-C had significantly higher SARS-CoV-2 spike IgG titers compared to those with acute COVID-19 (Fig 3A, P<0.001), as we have previously shown [13]. All healthy controls had detectable antibodies to HKU1 spike with a wide range of titers (GMT 7551, 95% CI 3317 to 17190, Fig 3B). We found no difference in HKU1 spike IgG titers between healthy controls and children with COVID-19 (7551 vs. 9376, P = 0.917), but children with MIS-C had significantly higher HKU1 spike IgG titers compared with healthy children (37239 vs. 7551, P = 0.012) and those with acute COVID-19 (37239 vs. 9376, P = 0.043, respectively).

We then tested the ability of plasma from the groups of children to neutralize an infectious clone of the SARS-CoV-2 virus using the FRNT assay. Plasma from healthy controls failed to neutralize SARS-CoV-2. In contrast, plasma from children with acute COVID-19 (33 vs. 10 (lower LOD), P = 0.002) and MIS-C (94 vs. 10, P<0.001) had significantly higher SARS-CoV-2 neutralization titers compared to healthy children (Fig 3C). The variability in neutralizing antibody titers observed in children with acute COVID-19 was likely attributable to the timing of sample collection post-symptom onset (POS) (median 6 days, IQR 4–11 days). In adults, the average time to detection of neutralizing antibodies against SARS-CoV-2 is 14.3 days POS with a wide range (range 3–59 days) [14], and these factors are likely reflected in our results. Children with MIS-C, which typically occurs 2–6 weeks post-COVID-19, had significantly higher neutralizing titers compared to children with acute COVID-19 (P = 0.005). In summary, although HKU1 spike antibodies were prevalent in healthy children with acute COVID-19 and MIS-C were able to mount strong neutralizing antibody responses to SARS-CoV-2.

We next performed multiple linear regression analyses and determined the Spearman correlations between the serologic assays among children with acute COVID-19 and MIS-C. We found that HKU1 spike IgG binding antibodies correlated strongly with SARS-CoV-2 spike binding antibody titers in children with acute COVID-19 and MIS-C (r = 0.7577, P<0.001) (Fig 4A). HKU1 spike IgG antibodies also correlated positively with SARS-CoV-2 neutralizing antibodies (r = 0.6201, P = 0.001) (Fig 4B), although the correlation was not as strong as that of SARS-CoV-2 spike IgG with neutralizing antibodies (r = 0.7599, P<0.001) (Fig 4C). Thus, our clinical data demonstrated that HKU1 spike antibodies are prevalent in healthy children, that they are higher in titer in children with MIS-C, and that they correlate with both SARS-CoV-2 spike binding and neutralizing antibodies in acute COVID-19 and MIS-C.

Fig 4. HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C.

Fig 4

Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) are shown.

Discussion

In this study, we found that priming mice with an endemic coronavirus HKU1 spike protein impeded the development of neutralizing antibodies to SARS-CoV-2 upon challenge with SARS-CoV-2 spike, consistent with original antigenic sin (OAS). Boosting mice intramuscularly with the heterologous coronavirus adjuvanted spike protein appeared to preferentially boost antibodies directed at the priming spike protein at shared, non-neutralizing epitopes. The phenomenon of OAS has been described in humans and animals with influenza [15, 16], dengue [17, 18], and human rhinovirus [19]. The pathophysiology of OAS is thought to be attributable to sequential exposure to similar, but non-identical antigens which preferentially elicits antibodies to the original antigen due to immunologic memory B cells [9]. In our study, long-lived plasma cells may have also contributed to the antibody responses observed following early boosting (3 weeks post-prime). Our data provides evidence that OAS plays a role in the murine serologic response to the spike protein of coronaviruses upon sequential exposure.

In contrast, analysis of clinical specimens from children with COVID-19 and MIS-C suggests that the serologic responses to SARS-CoV-2 in children may be amoral. Healthy pediatric controls had a wide range of pre-existing, binding antibody titers to HCoV HKU1 spike, reflecting a high baseline seroprevalence in children. Nevertheless, children with acute COVID-19 and MIS-C were able to effectively mount a neutralizing antibody response that correlated positively with both SARS-CoV-2 and HKU1 spike IgG antibodies. Thus, there was no direct evidence of OAS observed in our pediatric cohort. These results are consistent with previously published data, which indicate that SARS-CoV-2 infection elicits antibodies that cross-react with shared epitopes of endemic coronavirus spike proteins, predominantly within the conserved S2 subunit [20]. The discrepancy we observed with the experimental model of adjuvanted antigen administration in mice may be in part because the human immune system is capable of overcoming OAS, dependent upon factors relative to the original and subsequent antigen exposures (e.g. timing, duration, and magnitude).

To date, the literature describing the role of pre-existing HCoV immunity on SARS-CoV-2 serologic and clinical responses has been conflicting. Ng, et al. found that SARS-CoV-2 spike antibodies were prevalent in pre-pandemic sera, and that these were primarily IgG antibodies directed against the S2 subunit [8]. Interestingly, these cross-reactive antibodies possessed some neutralizing activity against SARS-CoV-2 pseudotyped viruses. This data conflicts with the pre-print findings of Anderson, et al. which demonstrated that endemic HCoV antibodies are boosted by SARS-CoV-2 infection, but are not associated with neutralization or protection against SARS-CoV-2 infections or hospitalizations [21]. However, Sagar, et al. found that although recent endemic coronavirus infections did not impact susceptibility to SARS-CoV-2 infections or hospitalizations, they were associated with significant improvements in patient outcomes including mortality [22]. Thus, the role of pre-existing antibodies to endemic coronaviruses in SARS-CoV-2 immune responses and outcomes is incompletely understood.

Limitations of this study include the small number of mice in each group, and the limited number of available clinical samples. Importantly, we also lacked pre- and post-COVID-19 sera in a single patient cohort to definitively answer the question of the effects of pre-existing HCoV antibodies on SARS-CoV-2 acquisition and clinical outcomes. Population-based studies may provide greater insights into subtler effects of pre-existing HCoV cross-reactive immunity on SARS-CoV-2 infection. We only analyzed serologic immunity to one endemic coronavirus (HKU1 in the Betacoronavirus genus), and differences could exist among Alphacoronaviruses (229E and NL63) or with the other endemic Betacoronavirus (OC43). While HKU1 infection is less prevalent than NL63 and OC43, it shares more homology in the spike protein with SARS-CoV-2 compared to the other HCoVs [23, 24], so we chose to evaluate it for this reason in these experiments. Pre-existing immunity to other SARS-CoV-2 antigens such as the nucleocapsid protein may contribute to clinical response, but we did not evaluate these antibodies in this study. The converse question of whether SARS-CoV-2 antibodies could blunt serologic responses to endemic coronaviruses or to emerging SARS-CoV-2 variants remains to be determined.

Conclusions

Prior exposure to endemic coronaviruses may blunt serologic responses to SARS-CoV-2 in mice by impeding the development of neutralizing antibodies, consistent with original antigenic sin. In comparison, our data in children suggest an amoral immune response. Future studies are needed to determine the effects of pre-existing immunity to endemic coronaviruses on clinical outcomes in children with acute COVID-19 and MIS-C.

Experimental methods

Mouse experiments

Six to 8-week-old female BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME) and housed in pathogen-free conditions with 5 mice per cage. Animals were allowed to acclimate to the environment for 2 weeks prior to experimentation. All animal experiments were conducted according to approved protocols by the Emory University Institutional Animal Care and Use Committee (PROTO202000026). Treatment regimens described below were randomly assigned by cage, and investigators were not blinded to the group assignments or results.

One cage of Balb/c mice (n = 5) was primed and boosted at 21 days IM with 10 μg SARS-CoV-2 nucleocapsid protein (SinoBiological, 40588-V08B) in 50 μl with alum (Alhydrogel adjuvant 2%, Invivogen). The purpose of this was to generate polyclonal antiserum to the nucleocapsid protein early during the COVID-19 pandemic, which was not a part of the present study. These mice were immunized 3 weeks later analogously with 10 μg alum-adjuvanted HCoV-HKU1 S1+S2 ECD-His (SinoBiological, 40606-V08B) IM, followed by an identical boost 21 days later. Three weeks later the same mice were immunized with 10 μg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later. Plasma was collected by submandibular bleeding on day 21 following each administration. A terminal bleed via cardiac puncture was conducted 21 days after the final boost.

A second cage of mice (n = 5) was immunized with 10 μg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His followed by a boost 21 days later. The mice were immunized 3 weeks later with 10 μg alum-adjuvanted HCoV-HKU1 S1+S2 ECD-His, followed by a boost 21 days later. Blood was collected as described above. Serum samples from each group were pooled and all serologic analyses were performed on pooled samples in duplicate. A small convenience sample size was chosen for this initial pilot study in mice. All animals (n = 10) were included in the final analyses, with no exclusions.

Human subjects

Children and adolescents, 0 to 21 years of age, hospitalized at Children’s Healthcare of Atlanta (CHOA) with confirmed or suspected COVID-19 or MIS-C were enrolled into an IRB-approved specimen collection protocol (Emory University IRB protocols STUDY00022371 and STUDY00000723) following written or verbal informed consent and assent as appropriate for age as previously described [13]. The Emory University IRB approved obtaining verbal consent and assent under specific circumstances, including to limit staff exposure to COVID-19. If verbal consent and assent were obtained, this was documented on the approved ICF form by the staff member who obtained consent. Children were classified as having MIS-C if they met the Centers for Disease Control and Prevention case definition [25]. They were classified as having acute COVID-19 if they were hospitalized with symptomatic disease and had SARS-CoV-2 detected by nasopharyngeal (NP) real-time polymerase chain reaction (RT-PCR). Prospective blood samples were collected from patients at the time of enrollment, and residual samples leftover from clinical labs were also obtained from the clinical laboratory. Plasma from healthy pediatric controls was collected through a separate IRB-approved protocol (STUDY0008846) following written informed consent and assent, as appropriate for age. Partial non-HKU1 serologic data from a subset of these children (4 with acute COVID-19 and 7 with MIS-C) were included in a prior publication [13]. Of the 126 planned analyses (3 groups, 14 patients per group, 3 types of serologic assays), there was insufficient volume to complete 5 assays. Data is available in the Supporting information.

Serology

Recombinant SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) and HCoV-HKU1 S1+S2 ECD-His (SinoBiological, 40606-V08B) were coated onto Nunc MaxiSorp plates at a concentration of 0.5 μg/mL in 100 μL phosphate-buffered saline (PBS) at 4°C overnight. Plates were blocked for two hours at room temperature in PBS/1% BSA/0.05% Tween-20 (ELISA buffer). Serum or plasma samples were heated to 56°C for 30 min, aliquoted, and stored at -20°C before use. Samples were serially diluted 1:3 in dilution buffer (PBS/1% BSA/0.05% Tween-20) starting at a dilution of 1:100. Coated plates were washed 4 times with 300 μl PBS/0.05% Tween-20 before adding 100 μL of each dilution and incubated for 90 minutes at room temperature. Plates were washed 4 times with PBS/0.05% Tween-20, and 100 μL of horseradish peroxidase-conjugated anti-Fc IgG antibody (Jackson ImmunoResearch Laboratories, 109-035-098), diluted 1:5,000 in ELISA buffer, was added and incubated for 60 minutes at room temperature. Plates were washed 4 times with PBS/0.05% Tween-20, followed by one additional wash with 1X PBS. Development was performed using 0.4 mg/mL o-phenylenediamine substrate (Sigma) in 0.05 M phosphate-citrate buffer pH 5.0, supplemented with 0.012% hydrogen peroxide before use. Reactions were incubated for 5 minutes then stopped with 1 M HCl and absorbance was measured at 490 nm.

Focus reduction neutralization assays

FRNT assays were performed as previously described [11]. Briefly, samples were diluted at 3-fold in 8 serial dilutions using DMEM (VWR, #45000–304) in duplicates with an initial dilution of 1:10 in a total volume of 60 μl. Serially diluted samples were incubated with an equal volume of an infectious clone of SARS-CoV-2 (Wuhan strain, A.1 from PANGO lineage) (100–200 foci per well) at 37°C for 1 hour in a round-bottomed 96-well culture plate. The antibody-virus mixture was then added to Vero cells and incubated at 37°C for 1 hour. Post-incubation, the antibody-virus mixture was removed and 100 μl of prewarmed 0.85% methylcellulose (Sigma-Aldrich, #M0512-250G) overlay was added to each well. Plates were incubated at 37°C for 24 hours. After 24 hours, methylcellulose overlay was removed, and cells were washed three times with PBS. Cells were then fixed with 2% paraformaldehyde in PBS (Electron Microscopy Sciences) for 30 minutes. Following fixation, plates were washed twice with PBS and 100 μl of permeabilization buffer (0.1% BSA [VWR, #0332], Saponin [Sigma, 47036-250G-F] in PBS), was added to the fixed Vero cells for 20 minutes. Cells were incubated with an anti-SARS-CoV spike primary antibody directly conjugated to biotin (CR3022-biotin) for 1 hour at room temperature. Next, the cells were washed three times in PBS and avidin-HRP was added for 1 hour at room temperature followed by three washes in PBS. Foci were visualized using TrueBlue HRP substrate (KPL, # 5510–0050) and imaged on an ELISPOT reader (CTL).

Statistical analysis

Statistical comparisons were made with GraphPad Prism (v9.0). Antibody geometric mean titers (GMTs) of replicates were determined and log-transformed titers were compared using one-way analysis of variance (ANOVA) with Tukey’s post-hoc comparison. Linear regression was performed on log-transformed antibody titers, and the Spearman’s correlation coefficients (r) were calculated. P values ≤0.05 were considered statistically significant. Raw data with statistical comparisons and 95% confidence intervals are shown in the S1 Dataset.

Supporting information

S1 Dataset. Antibody titers in murine and human samples.

(XLSX)

S1 File. The ARRIVE essential 10: Compliance questionnaire.

(PDF)

Acknowledgments

We thank clinical research coordinators Beena Desai, Kerry Dibernardo, Felicia Glover, Vikash Patel, Maureen Richardson, Amber Samuel, and clinical research nurses Lisa Macoy and Kathy Stephens for their assistance enrolling patients and collecting specimens. We thank Nadine Rouphael and the Hope Clinic laboratory, Theda Gibson, Hui-Mien Hsiao, Wensheng Li, and the Emory Vaccine Research Center laboratory for their assistance processing specimens. We thank the Children’s Healthcare of Atlanta research laboratory for their assistance in collecting residual specimens. We thank the professional staff of Emory Division of Animal Resources for their assistance with animal studies. Lastly, we thank the clinical study participants and their families for generously donating their time and samples to further our understanding of COVID-19 and MIS-C in children.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was funded by a Center for Childhood Infections and Vaccines (CCIV) pilot award from Children’s Healthcare of Atlanta and Emory University School of Medicine (to C.A.R.) and a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (to A.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Victor C Huber

12 May 2021

PONE-D-21-10490

Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

PLOS ONE

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[Partial non-HKU1 serologic data from a subset of these children (4 with acute COVID-19 and 7 with MIS-C) were included in a prior publication: https://pediatrics.aappublications.org/content/pediatrics/146/6/e2020018242.full.pdf]

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Reviewer #1: Lapp and colleagues consider the important question of whether pre-existing immunity to endemic human coronaviruses impacts the Ab response to SARS-CoV-2 infection. Specifically, they investigate whether previous B cell responses to the spike (S) protein of the endemic betacoronavirus HKU1 modulate Ab production against the antigenically-related S protein of SARS-CoV-2. The authors postulate that the response might reflect original antigenic sin (OAS) and feature preferential induction of Abs against the original priming antigen, the HKU1 S protein, diminishing Ab production against the SARS-CoV-2 S protein. The study is in two parts: (i) a mouse model study using IM immunization with the HKU1 and SARS-CoV-2 S proteins to test for OAS Ab responses, and (ii) analysis of serum Abs to HKU1 and SARS-CoV-2 S proteins in pre-pandemic children and hospitalized children with acute COVID-19 or MIS-C. The authors conclude from the mouse study that OAS impedes the Ab response to SARS-CoV-2 S after priming with HKU1 S, but that OAS does not appear to be a factor in the Ab responses of children to SARS-CoV-2 infection.

The study is clearly described and well justified. However, I have concerns about conclusions drawn from the data, as described under the following points.

1. In the mouse studies, IgG levels generated in response to SARS-CoV-2 S did not further increase after the HKU1 S boost (Fig. 2A). The result was the same in the reciprocal regimen: priming with HKU1 S and boosting with SARS-CoV-2 S (Fig. 2D). In these experiments, the boost did not elicit a response primarily against the priming antigen. Why was there no OAS response in these experiments, since other experiments (Figs 2B and C) demonstrate cross-reactivity between the S of HKU1 and SARS-CoV-2? In fact, Figs 2B and C indicate that the boost increased titers to the boosting antigen rather than to the priming antigen. Could the protein administered in the boost drive affinity maturation against related epitopes on the boosting antigen?

2. Fig. 2E: The authors conclude that HKU1 S boosted neutralizing Abs to SARS-CoV-2. Why is this “OAS” boost not evident in Fig. 2A? Could the result in Fig. 2E reflect an ongoing germinal center response (ongoing affinity maturation) to the priming SARS-CoV-2 S? Note that 10 micrograms given IM with alum is a relatively large dose with a depot adjuvant that could potentially maintain germinal center reactions for weeks/months. Could the same results be obtained without the HKU1 boosts?

3. Fig. 2F: The authors state that the HKU1 S immunization completely impeded development of SARS-CoV-2 neutralizing Abs on SARS-CoV-2 S immunization (line 138) and suggest that the SARS-CoV-2 S predominantly boosted cross-reactive Abs to the priming HKU1 S at shared non-neutralizing epitopes (line 140). This is hard to reconcile with the suggestion that HKU1 S boosted neutralizing Abs to SARS-CoV-2 (line 135), presumably by boosting Abs to shared neutralizing epitopes. Furthermore, why is boosting of any Abs to the priming HKU1 S by SARS-CoV-2 S not evident in Fig. 2D?

4. The analysis performed on sera from healthy children (pre-pandemic) and children with acute COVID-19 and MIS-C does not provide any insight into OAS effects on the Ab response to SARS-CoV-2 S. Multiple studies have demonstrated that SARS-CoV-2 infection generates Abs to the conserved S2 of the SARS-CoV-2 S that cross-react with seasonal betacoronavirus S proteins, as well as neutralizing Abs to novel S1 epitopes. Hence, the described correlations (Fig. 4) are expected and do not exclude an anti-S response that has OAS features and occurs early in the response.

Reviewer #2: The authors present a study to investigate the effects of original antigenic sin on the development of the adaptive immune response to SARS-CoV-2. Overall, the methodology and analysis are satisfactory. I have concerns regarding the interpretation of the results and the extent to which the data support the conclusions drawn.

1) The authors point out a discrepancy in their results in the mouse model and the results from analyzing sera from children. Specifically, results were consistent with OAS in the mouse model but not in the serological study. In the mouse study, the time between the last HKU exposure and first SARS-CoV-2 exposure was approximately 3 weeks. In general, imprinting effects are attributed to the recall of memory B-cells, but this short amount of time between exposures could mean that long-lived plasma cells might be playing an important role in clearing antigen before memory recall could occur. Can the authors address the extent to which the observed effects might be due to long-lived plasma cells as opposed to memory?

2) For the serological study, the authors show that children are able to mount a neutralizing antibody response to SARS-CoV-2 despite having high titers to HKU. However, looking at Figure 3C, although the group with acute infection does have higher mean neutralization titer than the control group, it appears as though most of the points have nearly undetectable or undetectable titer. Was the time between symptom onset and subject enrollment relatively consistent across the acutely-infected group, and could that explain the observed variance in neutralizing titer? Is it possible that not all acutely infected individuals had a previous HKU infection? In general, I find it hard to evaluate how the serological results are/are not consistent with OAS given the uncertainty of prior HKU exposure and the lack of specific information on the timing of patient sampling.

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PLoS One. 2021 Aug 27;16(8):e0256482. doi: 10.1371/journal.pone.0256482.r002

Author response to Decision Letter 0


8 Jul 2021

We thank the reviewers for their time and feedback to provide us with the opportunity to strengthen our manuscript. Please find below our point-by-point responses.

Review Comments to the Author

Reviewer #1: Lapp and colleagues consider the important question of whether pre-existing immunity to endemic human coronaviruses impacts the Ab response to SARS-CoV-2 infection. Specifically, they investigate whether previous B cell responses to the spike (S) protein of the endemic betacoronavirus HKU1 modulate Ab production against the antigenically-related S protein of SARS-CoV-2. The authors postulate that the response might reflect original antigenic sin (OAS) and feature preferential induction of Abs against the original priming antigen, the HKU1 S protein, diminishing Ab production against the SARS-CoV-2 S protein. The study is in two parts: (i) a mouse model study using IM immunization with the HKU1 and SARS-CoV-2 S proteins to test for OAS Ab responses, and (ii) analysis of serum Abs to HKU1 and SARS-CoV-2 S proteins in pre-pandemic children and hospitalized children with acute COVID-19 or MIS-C. The authors conclude from the mouse study that OAS impedes the Ab response to SARS-CoV-2 S after priming with HKU1 S, but that OAS does not appear to be a factor in the Ab responses of children to SARS-CoV-2 infection. The study is clearly described and well justified. However, I have concerns about conclusions drawn from the data, as described under the following points.

1. In the mouse studies, IgG levels generated in response to SARS-CoV-2 S did not further increase after the HKU1 S boost (Fig. 2A). The result was the same in the reciprocal regimen: priming with HKU1 S and boosting with SARS-CoV-2 S (Fig. 2D). In these experiments, the boost did not elicit a response primarily against the priming antigen. Why was there no OAS response in these experiments, since other experiments (Figs 2B and C) demonstrate cross-reactivity between the S of HKU1 and SARS-CoV-2? In fact, Figs 2B and C indicate that the boost increased titers to the boosting antigen rather than to the priming antigen. Could the protein administered in the boost drive affinity maturation against related epitopes on the boosting antigen?

The authors thank the reviewer for this interesting question. Antibody and B cell responses following repeated vaccination/administration peak and plateau, such that further boosting does not further increase antibody titers. The mechanism for this has been attributed to “antibody feedback,” such that high titers of antibodies mask the immunodominant epitopes and limit further B cell expansion. Repeated boosting is thought to diversify the antibody responses, as subdominant responses expand. This was recently described for human immune responses to malaria vaccine (McNamara HA, et al. Cell. July 2020). We have added the text in the results section to describe this (Line 130): “The plateau in antibody titers observed following repeated boosting is a known phenomenon whereby antibody feedback limits further B cell expansion by masking immunodominant epitopes.”

Our findings do suggest that heterologous boosting drives affinity maturation against related epitopes on the boosting antigen, as seen in Figures 2B and 2C. But the fact that these antibodies lack neutralization activity indicates that they are predominantly cross-reactive antibodies directed against the original priming strain at shared, but non-neutralizing epitopes. We have modified the text to clarify this point on Line 120: “These findings suggested that heterologous boosting drove affinity maturation against related epitopes on the boosting SARS-CoV-2 spike antigen.” And Line 142: “… recent prior exposure to the endemic HKU1 coronavirus adjuvanted spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies in mice upon SARS-CoV-2 spike protein administration, despite the presence of SARS-CoV-2 binding antibodies. These data indicate that the SARS-CoV-2 spike boost immunization predominantly boosted cross-reactive antibodies to the original priming (HKU1) antigen at shared, non-neutralizing epitopes.”

2. Fig. 2E: The authors conclude that HKU1 S boosted neutralizing Abs to SARS-CoV-2. Why is this “OAS” boost not evident in Fig. 2A?

We thank the reviewer for this question. The magnitude of binding antibodies have peaked and plateaued following repeated boosting in Fig. 2A, as described above. Further boosting allows for diversification of antibody response as non-dominant epitopes are exposed.

Could the result in Fig. 2E reflect an ongoing germinal center response (ongoing affinity maturation) to the priming SARS-CoV-2 S? Note that 10 micrograms given IM with alum is a relatively large dose with a depot adjuvant that could potentially maintain germinal center reactions for weeks/months. Could the same results be obtained without the HKU1 boosts?

This is an interesting point, and we cannot exclude that ongoing affinity maturation to SARS-CoV-2 spike in the germinal center is contributing to the increases in IgG titers following HKU1 boost. We have therefore modified the text accordingly on Line 135: “Neutralizing titers significantly increased compared to baseline after the boost immunization of SARS-CoV-2 spike (180 vs. 31, P=0.006) and further increased following the second HKU1 spike boost (520 vs. 180, P=0.050). These results suggested that HKU1 spike boosted neutralizing antibodies to SARS-CoV-2, although ongoing affinity maturation to SARS-CoV-2 spike in the germinal centers may have also contributed to these findings.”

3. Fig. 2F: The authors state that the HKU1 S immunization completely impeded development of SARS-CoV-2 neutralizing Abs on SARS-CoV-2 S immunization (line 138) and suggest that the SARS-CoV-2 S predominantly boosted cross-reactive Abs to the priming HKU1 S at shared non-neutralizing epitopes (line 140). This is hard to reconcile with the suggestion that HKU1 S boosted neutralizing Abs to SARS-CoV-2 (line 135), presumably by boosting Abs to shared neutralizing epitopes.

Yes, to state these findings another way:

- HKU1 S prime immunization completely impeded the development of neutralizing antibodies to SARS-CoV-2 upon SARS-CoV-2 S boost immunization because the boosted antibodies were directed toward the priming (HKU1) strain at non-neutralizing epitopes for SARS-CoV-2.

- By this same rationale, HKU1 S boost administered after SARS-CoV-2 prime elicited antibodies directed toward the priming (SARS-CoV-2) strain at neutralizing epitopes for SARS-CoV-2.

- In both cases, the boost elicited antibodies directed against cross-reactive epitopes on the priming antigen. The antibodies were only able to neutralize SARS-CoV-2 if the prime was SARS-CoV-2 spike.

Furthermore, why is boosting of any Abs to the priming HKU1 S by SARS-CoV-2 S not evident in Fig. 2D?

In figure 2D, the antibodies have peaked and plateaued, as described above (McNamara HA, et al. Cell. July 2020) due to antibody feedback. Further boosting is not expected to further increase antibody titers.

4. The analysis performed on sera from healthy children (pre-pandemic) and children with acute COVID-19 and MIS-C does not provide any insight into OAS effects on the Ab response to SARS-CoV-2 S. Multiple studies have demonstrated that SARS-CoV-2 infection generates Abs to the conserved S2 of the SARS-CoV-2 S that cross-react with seasonal betacoronavirus S proteins, as well as neutralizing Abs to novel S1 epitopes. Hence, the described correlations (Fig. 4) are expected and do not exclude an anti-S response that has OAS features and occurs early in the response.

We acknowledge that the human data does not directly prove or disprove the presence of OAS in children with COVID-19 or MIS-C, and we have discussed this in the limitations paragraph. We do think that the human data provides context for interpreting the animal studies, as we think it is helpful to show that HKU1 antibodies are prevalent in healthy children. Further, if OAS plays a role in human serologic responses, children with COVID-19 or MIS-C were nevertheless able to mount SARS-CoV-2 neutralizing antibodies, which correlated strongly with HKU1 spike IgG. We have modified the text to address the reviewer’s comments on Line 284: “These results are consistent with previously published data, which indicate that SARS-CoV-2 infection elicits antibodies that cross-react with shared epitopes of endemic coronavirus spike proteins, predominantly within the conserved S2 subunit (Ladner, Cell Reports Medicine, Jan 2021).”

Reviewer #2: The authors present a study to investigate the effects of original antigenic sin on the development of the adaptive immune response to SARS-CoV-2. Overall, the methodology and analysis are satisfactory. I have concerns regarding the interpretation of the results and the extent to which the data support the conclusions drawn.

1) The authors point out a discrepancy in their results in the mouse model and the results from analyzing sera from children. Specifically, results were consistent with OAS in the mouse model but not in the serological study. In the mouse study, the time between the last HKU exposure and first SARS-CoV-2 exposure was approximately 3 weeks. In general, imprinting effects are attributed to the recall of memory B-cells, but this short amount of time between exposures could mean that long-lived plasma cells might be playing an important role in clearing antigen before memory recall could occur. Can the authors address the extent to which the observed effects might be due to long-lived plasma cells as opposed to memory?

We thank the reviewer for this interesting question. We did not determine the extent to which long-lived plasma cells vs. memory B cells were contributing to OAS responses observed in the mice. Long-lived plasma cells can survive weeks to months following a murine exposure, so they likely did contribute to the responses we observed, and perhaps more so because of the early boost at 3 weeks post-prime. We have modified the discussion to take this into consideration in Line 274: “In our study, long-lived plasma cells may have also contributed to the antibody responses observed following early boosting regimen (3 weeks post-prime).”

2) For the serological study, the authors show that children are able to mount a neutralizing antibody response to SARS-CoV-2 despite having high titers to HKU. However, looking at Figure 3C, although the group with acute infection does have higher mean neutralization titer than the control group, it appears as though most of the points have nearly undetectable or undetectable titer. Was the time between symptom onset and subject enrollment relatively consistent across the acutely-infected group, and could that explain the observed variance in neutralizing titer? Is it possible that not all acutely infected individuals had a previous HKU infection? In general, I find it hard to evaluate how the serological results are/are not consistent with OAS given the uncertainty of prior HKU exposure and the lack of specific information on the timing of patient sampling.

The timing of sample collection post-onset of symptoms (POS) in the patients with COVID-19 was median 6 days (IQR 4-11 days). At this early time point POS, it is not surprising that some patients had not yet developed neutralizing antibodies. In adults, the average time to detection of neutralizing antibodies against SARS-CoV-2 is 14.3 days POS with a wide range (range 3-59 days) (Seow, Nature Microbiology, October 2020). In our cohort, it is likely that the variability in timing POS of sample collection, in addition to the intrinsic variability in timing of seroconversion, contributed to the observed variability in neutralizing titer. It is possible that not all acutely infected individuals had a previous HKU1 infection, although all had detectable HKU1 IgG antibodies. Nevertheless, we concur that our lack of pre-infection samples to ascertain prior HKU1 exposure is a limitation of the study.

We have included text in the manuscript to communicate this information on Line 230: “The variability in neutralizing antibody titers observed in children with acute COVID-19 was likely attributable to the timing of sample collection post-symptom onset (POS) (median 6 days, IQR 4-11 days). In adults, the average time to detection of neutralizing antibodies against SARS-CoV-2 is 14.3 days POS with a wide range (range 3-59 days) (Seow, Nature Microbiology, Oct 2020), and these factors are likely reflected in our results. Children with MIS-C, which typically occurs 2-6 weeks post-COVID-19, had significantly higher neutralizing titers compared to children with acute COVID-19 (P=0.005).” and Line 304: “Importantly, we also lacked pre- and post-COVID-19 sera in a single patient cohort to definitively answer the question of the effects of pre-existing HCoV antibodies on SARS-CoV-2 acquisition and clinical outcomes.”

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Decision Letter 1

Victor C Huber

9 Aug 2021

Original antigenic sin responses to Betacoronavirus spike proteins are observed in a mouse model, but are not apparent in children following SARS-CoV-2 infection

PONE-D-21-10490R1

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Acceptance letter

Victor C Huber

20 Aug 2021

PONE-D-21-10490R1

Original antigenic sin responses to Betacoronavirus spike proteins are observed in a mouse model, but are not apparent in children following SARS-CoV-2 infection

Dear Dr. Rostad:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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    S1 Dataset. Antibody titers in murine and human samples.

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    Submitted filename: 06_25_2021 Reviewer Responses PLOS One.docx

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