key: cord-0792780-0e1126pb
authors: Chan, R. W.; Liu, S.; Cheung, J. Y.; Tsun, J. G.; Chan, K. C.; Chan, K. Y.; Fung, G. P.; Li, A. M.; Lam, H. S.
title: Study on the mucosal and serological immune response to the Novel Coronavirus (SARS-CoV-2) vaccine
date: 2021-06-15
journal: nan
DOI: 10.1101/2021.06.15.21256661
sha: b9d5da5de0d21d3d5519dfb741173f43a0cdf8a4
doc_id: 792780
cord_uid: 0e1126pb

Vaccines that elicit mucosal immune responses against SARS-CoV-2 could potentially be of exceptional importance in providing first line defense at the site of viral entry. The serological antibody response induced by SARS-CoV-2 vaccines have already been well characterized. In order to understand the mucosal immune response profiles of SARS-CoV-2 vaccines, we examined both the mucosal and systemic responses of subjects vaccinated by two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac). Serial nasal epithelial lining fluid (NELF) and peripheral blood samples were collected in ten subjects who had received CoronaVac and thirty-two subjects who had received Comirnaty. We quantified IgA and IgG specific to SARS-CoV-2 S1 protein by ELISA in NELF and plasma samples. The neutralization effect of these two sample types were evaluated by surrogate ACE-SARS-CoV-2 Spike protein ELISA. Only Comirnaty induced nasal SARS-CoV-2 S1 protein-specific (S1-specific) IgA and IgG responses, which were evident as early as on 14 days after the first dose. The NELF samples of 72% of subjects became IgA+IgG+, while in 62.5% of subjects the samples were neutralizing by 7 days after the second dose. In 45% of the subjects their NELF remained neutralizing 50 days after the booster of Comirnaty. In plasma, 91% and 100% Comirnaty subjects possessed S1-specific IgA+IgG+ on 14 days after the first dose and 7 days after booster, respectively. The plasma collected on 7 days after booster was 100% neutralizing. The induction of S1-specific antibody by CoronaVac was IgG dominant, and 70% of the subjects possessed S1-specific IgG by 7 days after booster and were all neutralizing. This study reveals that Comirnaty is able to induce S1-specific IgA and IgG response with neutralizing activity in the nasal mucosa in addition to a consistent systemic response. The clinical implications and the biological mechanism of an additional nasal immune response induced by vaccines such as Comirnaty warrant further investigation.

One Sentence Summary: 1 mRNA vaccine (CoronaVac) elicits mucosal IgA and IgG in the nasal epithelial lining fluid 2 together with ELISA-detected anti-wild-type spike neutralizing antibodies as early as day 14 post 3 vaccination. 4 5 Abstract 6 Vaccines that elicit mucosal immune responses against SARS-CoV-2 could potentially be of 7 exceptional importance in providing first line defense at the site of viral entry. The serological 8 antibody response induced by SARS-CoV-2 vaccines have already been well characterized. In 9 order to understand the mucosal immune response profiles of SARS-CoV-2 vaccines, we 10 examined both the mucosal and systemic responses of subjects vaccinated by two different 11 vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac). Serial nasal 12 epithelial lining fluid (NELF) and peripheral blood samples were collected in ten subjects who 13 had received CoronaVac and thirty-two subjects who had received Comirnaty. We quantified 14 IgA and IgG specific to SARS-CoV-2 S1 protein by ELISA in NELF and plasma samples. The 15 neutralization effect of these two sample types were evaluated by surrogate ACE-SARS-CoV-2 16 Spike protein ELISA. Only Comirnaty induced nasal SARS-CoV-2 S1 protein-specific (S1- 17 specific) IgA and IgG responses, which were evident as early as on 14±2 days after the first 18 dose. The NELF samples of 72% of subjects became IgA+IgG+, while in 62.5% of subjects the 19 samples were neutralizing by 7±2 days after the second dose. In 45% of the subjects their NELF 20 remained neutralizing 50 days after the booster of Comirnaty. In plasma, 91% and 100% 21 Comirnaty subjects possessed S1-specific IgA+IgG+ on 14±2 days after the first dose and 7±2 22 days after booster, respectively. The plasma collected on 7±2 days after booster was 100% 23 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 neutralizing. The induction of S1-specific antibody by CoronaVac was IgG dominant, and 70% 24 of the subjects possessed S1-specific IgG by 7±2 days after booster and were all neutralizing. 25 This study reveals that Comirnaty is able to induce S1-specific IgA and IgG response with 26 neutralizing activity in the nasal mucosa in addition to a consistent systemic response. The 27 clinical implications and the biological mechanism of an additional nasal immune response 28 induced by vaccines such as Comirnaty warrant further investigation. 29 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 INTRODUCTION 30 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes the 31 coronavirus disease 2019 pandemic that has resulted in significant morbidity and a 32 global death toll of over 3 million (1) . the full-length viral spike (S) protein modified by two proline mutations, respectively (2) . The S- 39 protein of SARS-CoV-2 is composed of the subunits S1 and S2, with S1 bearing the receptor- 40 binding domain (RBD) that recognizes host angiotensin-converting enzyme 2 (ACE2) to initiate 41 viral entry (3, 4) and S2 being responsible for membrane fusion (3) . Both vaccines have good 42 safety records with low prevalence of serious adverse events (5) (6) (7) . CoronaVac has been shown 43 to prevent symptomatic COVID-19 in 51% of vaccinated healthcare workers, and an efficacy of 44 100% in preventing severe . Comirnaty is reported to be 95% effective in 45 preventing symptomatic COVID-19 with low incidence of serious adverse events (5) . 46 47 Mechanistic properties of these novel vaccines in conferring immunity to combat COVID-19 are 48 beginning to emerge. It has been shown that by 14 days after the booster dose, recipients of 49 CoronaVac aged 18-50 years had seroconversion rates of 95.6% and 95.7% for S1-RBD IgG and 50 neutralizing antibodies, respectively (9) . In comparison, specific IgG against S1 and RBD of 51 SARS-CoV-2 are detectable in serum at 21 days after the priming dose of Comirnaty, with 100% 52 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 doi: medRxiv preprint 6 seroconversion rate (10) . Due to the differences in the study design, it would be difficult to 53 compare the seroconversion times for both SARS-CoV-2 S1 protein-specific (S1-specific) IgG 54 and SARS-CoV-2 neutralization between the two vaccines. It is also important to note that due to 55 differences in the stages of vaccine development, there is currently more research data available 56 for the mRNA vaccines (e.g., Comirnaty and Moderna) than inactivated viral vaccines (e.g., 57 CoronaVac). 58 59 Comirnaty and Moderna elicit neutralizing antibody (NAb) responses that target the RBD 60 epitopes in the same manner as natural infections (11) . Albeit at much lower titers than IgG 61 levels, the mRNA vaccines also induce IgM and IgA responses against S-protein and RBD in 62 plasma samples (11) . In the sequence of seroconversion, IgM responses are first generated and 63 then class-switch converted to IgA and IgG (12) . As detectable IgM levels after vaccinations are 64 often significantly lower and less sustained when compared to IgA and IgG levels, IgM is 65 suspected to have lesser importance in virus neutralization in vivo (11, 13) . On the other hand, 66 serum RBD IgA from COVID-19 patients has been found to have more potent neutralization 67 potential than paired IgG (14) . SARS-CoV-2 IgA can be sustainably elevated in serum or plasma 68 samples for over 2 months after Comirnaty vaccination (13) . Thus, the importance of systemic 69 SARS-CoV-2 IgA in vaccine-induced immunity against COVID-19 warrants further validation 70 (13, 15) . CoV-2 IgA levels was found to be higher than IgG, with neutralization activity correlating with 75 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 IgA titers, but not IgG (14) . Natural infection induces mucosal antibodies directed against S-76 protein and RBD, and it has been suggested that higher antibody levels correlated with fewer 77 systemic symptoms and reduced viral load (16) . Thus, respiratory mucosal immunity could have 78 unique and specific roles in offering protection against SARS-CoV-2 infection (17) . (14) . SIgA in the upper respiratory tract is 82 from the IgA-secreting plasma cells (18 IgG responses to the S-protein and RBD of SARS-CoV-2, but their capability for viral 90 neutralization are unknown (19) . Determining whether or not current vaccines induce antibody 91 response in the mucosa, and if so, the duration and sustainability of any such response in the 92 context of concurrent data on systemic immunity from plasma samples of vaccinated individuals 93 can provide invaluable information that can help optimize the use of these vaccines in a range of 94 public health strategies and in different community settings. The lack of sampling standardization and validation of the mucosal fluid measurements were the 97 major challenges in the past. Our recent work using nasal strips to collect nasal epithelial lining 98 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 doi: medRxiv preprint 8 fluid (NELF) in SARS-CoV-2 infected children and adults is non-invasive and facilitates the 99 collection of undiluted NELF for SARS-CoV-2 nucleoprotein gene detection (20) . The same 100 method was adopted for the mucosal antibody analysis in the current study. With the possibility 101 that subjects vaccinated with mRNA vaccine could induce salivary IgA against SARS-CoV-2 102 protein, we hypothesize that it might also induce SARS-CoV-2 S-protein specific antibody in 103 other mucosal surfaces. Here, we compared the serological and mucosal immune responses after 104 vaccination with CoronaVac and Comirnaty with a particular focus on S1-specific IgA levels 105 detected and neutralization capacity in NELF and plasma.

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Demographic of the subjects 109 Forty-two subjects were recruited in this study. The median age of all subjects was 41 years old 110 (range 21-74), 40.3% were male. Longitudinal measurements of vaccine induced S1-specific IgG 111 and IgA in serum and NELF at four time points (Figure 1A) , 0 to 2 days before the first dose 112 (Baseline), 14±2 days after the first dose (V+D14), 7±2 days after the booster dose (B+D7) and 113 any time between 14 days after the booster dose and before 3 months after the first dose ( Figure   114 1A) were conducted. The median age was significantly different in subjects from the two vaccine 115 groups, CoronaVac (n=10, median age 59) and Comirnaty (n=32, median age 39.5) (p=0.0004). 116 All subjects declared that they did not have known unprotected exposure to SARS-CoV-2 117 infected subjects. To ensure that the measurements of the change in SARS-CoV-2 specific 118 immunoglobulin levels were not due to active SARS-CoV-2 infection during the study period, all 119 the NELF samples were tested negative for the presence of SARS-CoV-2 nucleoprotein gene. As Table 1 ). 124 125 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted https://doi.org/10.1101 https://doi.org/10. /2021 Comirnaty induced detectable immunoglobulin in NELF 137 Among the ten subjects who have taken CoronaVac, none of them developed detectable NELF 138 SARS-CoV-2 S1-specific IgA and IgG (Figure 2A ) by day 7±2 days after booster, while most 139 subjects who received Comirnaty developed S1-specific antibodies. The elevations in S1-specific 140 IgA and IgG levels detected in NELF along the three time points were significant by two-way 141 ANOVA followed by Tukey's multiple comparisons test ( Figure 2D) . Moreover, S1-specific 142 IgA appeared earlier than IgG in NELF. More subjects developed NELF S1-specific IgA 15/32 143 (46.9%) than IgG 3/32 (9.4%) (Supplementary Figure 1B 149 In the CoronaVac group, plasma S1-specific IgA increased significantly by 14±2 days after the 150 first vaccination dose and 7±2 days after the booster dose ( Figure 2B , green dots), while the 151 significant increase in S1-specific IgG was only detected between baseline and 7±2 days after 152 booster ( Figure 2B , orange dots). On 7±2 days after booster, 7/10 (70%) of the CoronaVac 153 subjects had detectable plasma S1-specific IgG. 154 In the Comirnaty group ( Figure 2E) , 93.8% and 100% of subjects were positive for both plasma 155 S1-specific IgA and IgG by 14±2 days after the first vaccination dose (Supplementary Figure   156 1B, red dots) and 7±2 days after the booster dose (Supplementary Figure 1C , red dots), 157 respectively. The outliers were contributed by three subjects who were IgA-IgG+ (Subject 40), 158 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 doi: medRxiv preprint 12 IgA+IgG-(Subject 11) and IgA-IgG-(Subject 20). Nevertheless, all plasma samples were 159 IgA+IgG+ by 7±2 days after booster. 160 There were no statistically significant correlations between age and the induced NELF and 161 plasma S1-specific IgA and IgG levels in the longitudinal CoronaVac and Comirnaty recipients 162 (Supplementary Figure 2) . However, female subjects who received Comirnaty had higher 163 plasma S1-specific IgG at the 14±2 days timepoint than male subjects (p = 0.007) 164 (Supplementary Figure 2D) .

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The level of S1-specific IgA (green dots) and IgG (red dots) were plotted against the three 180 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 Neutralization potential of NELF and plasma 181 We further tested whether NELF and plasma samples were SARS-CoV-2 neutralizing by using 182 the blocking enzyme-linked immunosorbent assay as a surrogate of the neutralization test. NELF 183 and plasma samples collected at 7±2 days after booster were measured. In the CoronaVac group, 184 as there were no SARS-CoV-2 S1-specific IgA and IgG detected in the NELF, we did not 185 perform NAb measurement for those samples. Whereas 7/10 of the plasma from CoronaVac 186 subjects contained SARS-CoV-2 NAb. In the Comirnaty group, 20/32 NELF samples inhibited 187 the binding of SARS-CoV-2 spike protein to ACE-2 ( Figure 2F ), whereas all plasma samples 188 provided over 80% inhibition to the binding of SARS-CoV-2 spike protein to The correlation between SARS-CoV-2 S1-specific Ig with neutralization antibody level 191 In the CoronaVac group, no significant correlations were found between the plasma IgA and IgG 

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The copyright holder for this preprint this version posted https://doi.org/10.1101 https://doi.org/10. /2021 Neutralizing antibody in NELF is transient 209 Whether this is a transient expression SARS-CoV-2 NAb in NELF is uncertain, as there were 210 five individuals (subjects 9, 33, 34, 37, 41) showing a downward trend of S/C ratio of IgA from 211 14±2 days after the first vaccination to 7±2 days after booster (Supplementary Figure 2A) 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 https://doi.org/10.1101 https://doi.org/10. /2021 18 plasma samples from the younger subjects in the cross-sectional group of CoronaVac recipients 232 had NAb (Figure 4B, pink dots) . Overall, 75% (18/24) (Figure 4C ).

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Our study reveals that both Comirnaty and CoronaVac induces plasma SARS-CoV-2 S1-specific 251 IgA and IgG, and NAb. However, Comirnaty, but not CoronaVac, induced S1-specific IgA and 252 IgG in the nasal mucosa by 7±2 days after booster. Of the Comirnaty recipients 72% produced 253 an NELF antibody response, while 62.5% exhibited neutralizing activities in their NELF 254 samples. The NAb in NELF correlated with the S/C ratio of the S1-specific IgA and IgG 255 detected. The induction of nasal S1-specific Igs and NAb is unique to subjects receiving It has been commonly believed that intramuscular vaccines do not induce mucosal immunity 266 effectively (21) . The mucosal immune response of the upper respiratory tract is partly 267 compartmentalized and usually initiated in nasopharynx-associated lymphoid tissue (NALT) in 268 all age groups and bronchus-associated lymphoid tissue (BALT) in children and adolescents or 269 in adults upon disease induction (18) . These upper respiratory tract associated lymphoid tissues 270 generate IgA-producing mucosal B cells that express the homing receptor, e.g. α4ß1, CCR10, 271 CD62L and LFA-1 (22, 23) . These homing receptors allow the B-cells to traffic efficiently to the 272 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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 https://doi.org/10.1101 /2021 (17), and has anti-inflammatory properties (26) . The delayed NAb response found in CoronaVac subjects compared with those in Comirnaty 315 group was not surprising. It has been reported that seroconversion rates were 47.8% and 95.6% 316 for S1-RBD-specific IgG for CoronaVac at 14 days and 28 days after booster, respectively (9) . 317 Thus, our study design with this interim report at 7 days ± 2 post booster may not have 318 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 CoV-2 is detectable for up to 8 weeks after booster (11) . Furthermore, our study demonstrated 341 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 24 the correlation between plasma IgA levels with the percentage of virus-receptor binding 342 inhibition as reported in a previous study (14) . Together, our findings confirm the reliability of 343 Comirnaty in generating robust humoral immune responses in vaccinated subjects. The current study has the following limitations. First, the smaller sample size and the higher 356 median age of the recipients of CoronaVac in the longitudinal group can be argued to have 357 contributed towards the absence in NELF response and a slower and milder plasma response 358 when compared to Comirnaty. We attempted to recruit the cross-sectional subjects to enrich our 359 data for this important early report, while more subjects would be recruited for a better 360 comparison. Second, we were using a SARS-CoV-2 surrogate virus neutralization test instead of 361 a neutralization assay with live cells and viruses in Biosafety Level 3 settings. Therefore, the 362 NAb measured in this study is a surrogate measure that is solely based on the inhibition of the 363 binding between the SARS-CoV-2 antibody-mediated blockage of ACE2-spike (RBD) protein-364 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 25 protein interaction (30) . The protective effects of the intracellular action of NELF IgA in the 365 Comirnaty recipients or the plasma Ig specific to other SARS-CoV-2 proteins that theoretically 366 should be manufactured in CoronaVac recipients were not considered. Although the surrogate 367 assay used has been validated with the plaque reduction neutralization test (PRNT) utilizing the 368 SARS-CoV-2 virus (31), the current test could underestimate the actual neutralization capacity. 369 Third, we observed tremendous individual variations, for example, some recipients of Comirnaty 370 were found to be IgA-, IgG-or IgA-IgG-for S1 protein. Although Comirnaty induced mucosal 371 immunoglobulins for 72% of vaccinated subjects, there were individuals who, despite having 372 plasma S1-specfic IgA and IgG, did not express NELF S1-specific IgA (n=2) or IgG (n=5) or 373 both (n=4). These variations require a larger sample size to further clarify. Nevertheless, our 374 current study clearly shows qualitative and significant differences in mucosal response between 375 different vaccine technologies. Lastly, the best available assay for the measurement of SARS-376 CoV-2 S1-specific IgA and IgG used in this study was optimized for plasma and serum rather 377 than mucosal lining fluid. We were therefore unable to differentiate between the monomeric and 378 dimeric forms of IgA or identify any secretory component in our subjects' NELF samples. CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 doi: medRxiv preprint 26 2 infection, in addition to its effectiveness in protecting the recipient from hospitalization and 388 severe disease. Though the response may be transient, it is possible that a more rapid elevation in 389 antibodies may occur within the mucosa when the subject is exposed to live viruses, thus 390 conferring protection from infection even before the virus breaches the mucosa. CoronaVac CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 CoronaVac, on the other hand, induced satisfactory systemic humoral response with neutralizing 410 capacity induced by CoronaVac in most individuals, and there is less concern about the potential CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Subject recruitment 431 We released the information of this study through the department website, department social CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 https://doi.org/10. /2021 Strips were cut from sheets of Leukosorb medium (Pall Corporation, BSP0669) using a laser 452 cutter (CMA960, Department of Biomedical Engineering, CUHK) to the dimensions of 4 mm 453 wide and 40mm long with a marking at 12mm as previously described (20, 32 1:100 diluted plasma were added to the assay well and processed as per manufacturer's 473 instructions. After subsequent wash and incubation steps with conjugates or substrates, the 474 . CC-BY-NC-ND 4.0 International license It is made available under a 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 https://doi.org/10.1101 /2021 doi: medRxiv preprint 30 plates were analyzed according to the manufacturer's instructions on the Synergy HTX Multi- 475 Mode Reader. Semi-quantitative readout as a ratio between the sample and the calibrator optical 476 density (OD) values was used. The performance was checked by keeping the optical density of 477 the calibrator within the reference value, and the ratio between the positive and negative controls 478 between 1.6-4.2 and 0-0.7, respectively. 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 https://doi.org/10.1101 https://doi.org/10. /2021 the following cycling conditions: reverse transcription at 50°C for 5 min, inactivation of reverse 498 transcriptase at 95°C for 20 s, 40 cycles of PCR amplification (Denaturing at 95°C for 5 s; 499 Annealing/ Extending at 60°C for 30 s). No template control and positive control using cell 500 lysate from SARS-CoV-2 infected human respiratory cells were included in each run. 501 502 Statistical analysis 503 The demographic variables of subjects were compared between the two vaccinated groups using 504 the Mann Whitney test and Fisher's exact test, as appropriate. For the immunoglobulin profiles, 505 differences between different gender and time points were evaluated using Mann Whitney test 506 and Friedman test followed by Dunn's multiple comparison test, respectively. The correlation of 507 S/C ratio of the specific immunoglobulins with the percentage of signal inhibition in the 508 surrogate neutralization test was examined by Spearman's correlation test. Differences were 509 considered to be statistically significant if p < 0.05. The All statistical tests were performed using 510 Graphpad version 9.1.2 for macOS. Differences were considered statistically significant at p < 511 0.05.

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