key: cord-0291239-01i0bbn2
authors: Moncunill, G.; Aguilar, R.; Ribes, M.; Ortega, N.; Rubio, R.; Salmeron, G.; Molina, M. J.; Vidal, M.; Barrios, D.; Mitchell, R. A.; Jimenez, A.; Castellana, C.; Hernandez-Luis, P.; Rodo, P.; Mendez, S.; Llupia, A.; Puyol, L.; Rodrigo Melero, N.; Carolis, C.; Mayor, A.; Izquierdo, L.; Varela, P.; Trilla, A.; Vilella, A.; Barroso, S.; Angulo, A.; Engel, P.; Tortajada, M.; Garcia-Basteiro, A. L.; Dobano, C.
title: Determinants of early antibody responses to COVID-19 mRNA vaccines in exposed and naive healthcare workers
date: 2021-09-12
journal: nan
DOI: 10.1101/2021.09.08.21263232
sha: 7834e6ffd40cd7ea7aa6e1d0d83c026be490c709
doc_id: 291239
cord_uid: 01i0bbn2

Background Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naive adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. Methods We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. Findings Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects. A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with lower neutralization and antibody levels. Among fully vaccinated, 6.3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. Interpretation Our data support administering a single-dose in pre-exposed healthy individuals. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year.

The unprecedented fast development of highly efficacious COVID-19 vaccines has changed 93 the fate of the SARS-CoV-2 pandemic [1] . The COVID-19 vaccines from Pfizer/BioNTech 94

We measured IgA, IgG and IgM antibody levels (median fluorescence intensity, MFI) to 179 different SARS-CoV-2 antigens using previously developed assays based on the quantitative 180 suspension array technology Luminex (Supplementary Information) [37, 41] For feasibility reasons, we selected 165 samples from the study visit M12 with a balanced 194 representation of BNT162b2 and mRNA-1273 vaccinees and non-vaccinated participants 195 (previously exposed and naive individuals). We already had pre-vaccination neutralization 196 data from 33 of the selected 165 individuals [37] . Plasma neutralizing capacity was assessed 197 as the percentage of inhibition of RBD binding to ACE2 receptor and was measured through 198 a flow cytometric-based assay that correlates with a validated pseudovirus neutralization 199 assay [37] . Briefly, a murine stable cell line expressing the ACE2 receptor was incubated 200 with RBD-mFc fusion protein, composed of RBD fused to the Fc region of murine IgG1, 201 previously exposed to the different plasma samples at a 1:400 dilution. Cells were stained 202 with anti-mouse IgG-PE, washed, and analyzed by flow cytometry using standard 203

procedures. Study samples were tested alongside 30 negative pre-pandemic controls, in 204 duplicates. 205

For feasibility, a subset of 58 M12 samples from BNT162b2 and mRNA-1273 vaccinated 207 participants (48 naive and 10 exposed), were randomly selected for the avidity assay. 208

Antibody avidity was determined as the percentage of IgA and IgG levels against RBD, S 209 and S2 antigens measured incubating samples with a chaotropic agent (urea 4M, 30 min at 210 room temperature) over the IgA and IgG levels measured in the same samples without 211 chaotropic agent. Antibody levels with and without chaotropic agents were measured in 212 plasma samples (dilution 1:5000) using the Luminex assay described above. 213 214

MFIs were log 10 -transformed. In vaccinated participants MFIs for S-related antigen IgGs 216 correspond to the 1:5000 dilution, except in plots where we compare pre and post 217 vaccination levels, in which the 1:500 dilution was used. Any other MFIs correspond to the 218 dilution 1:500, and for seropositivity calculations, only 1:500 dilution was used. 219

Assay positivity cutoffs specific for each isotype and analyte were calculated as 10 to the 220 mean plus 3 standard deviations (SD) of log 10 -transformed MFI of 128 pre-pandemic 221 controls. Results were defined as undetermined when the MFI levels for a given isotype-222 analyte were between the positivity threshold and an upper limit defined as 10 to the mean 223 plus 4.5 SD of the log 10 -transformed MFIs of 128 pre-pandemic samples, and no other 224 isotype-antigen combination was above the positivity cutoff, and the participant did not have 225 any previous evidence of seropositivity or rRT-PCR positivity. 226

Analysis of antibody levels after the second dose included only data from samples collected 227 12-19 days after vaccination, while for the first dose we included data from all samples 228 collected 7 or more days after vaccination, as no previous visit window was established. 229 Groups were compared using the Wilcoxon Sum Rank test for continuous non-parametric 230 variables and with the Wilcoxon Signed-Sum Rank Test for paired continuous data. 231

Correlations between continuous variables were analyzed using linear regression models 232 and Spearman's rank test. Locally estimated scatterplot smoothing (LOESS) plots were used 233 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 to visualize trends in antibody levels over days post vaccination, days post-symptom onset 234 (PSO) or post rRT-PCR diagnosis. 235

Univariable and multivariable linear regression models were fitted to assess factors 236 associated with antibody responses to SARS-CoV-2 RBD and S antigens and their 237 neutralization capacity (%) after vaccination among exposed and naive individuals, and 238 overall. Models having both exposed and naive participants included the following 239 independent variables: sex, age, days since first dose administration, days since second 240 dose administration, smoking habits, chronic medication, presence of baseline illness (heart 241 and liver disease, diabetes, chronic respiratory and renal disease, cancers and autoimmune 242 and other immunological disorders), antibody levels (log 10 MFI) to endemic common cold 243 human coronaviruses (HCoV: 229E, NL63, OC43, and HKU1) at M6, vaccine type, and 244

presence of systemic or local AEs (systemic AEs included fever, arthralgia, fatigue, chills, 245 muscle pain and headache, while local AEs included pain, erythema and/or swelling at the 246 injection site or swollen glands near the injection site) after 1 st or 2 nd vaccine dose. In 247 addition, the predictor variable "presence of any cough, 248 dyspnea and other respiratory symptoms, anosmia or ageusia, sore throat, fever, rhinorrhea, 249 headache, chills and digestive symptoms)" was included in models having only exposed 250 participants. Predictor variables that had a P-value of 0.2 in the univariable models were 251 selected for stepwise multivariable models performed with the function stepAIC (R package 252 MASS). The betas obtained in each model for each of the predictor variables were 253 transformed into a percentage of antibody increase for easier interpretation. For continuous 254 log 10 -transformed variables (log-log model) the beta transformed value (%) was calculated 255 with the formula ((10^(beta*log 10 (1.1)))-1)*100. This represents the effect (in percentage) on 256

IgG levels of a 10% increase in the corresponding predictor variable. For categorical 257 predictor variables (log-linear models), the beta transformed value (%) was calculated with 258 the formula ((10^beta)-1)*100. This gives the difference (in percentage) in IgG levels 259 between the reference and the study group. A P-value of ≤ 0.05 was considered statistically 260 significant and 95% confidence intervals (CI) were calculated for all estimates. We 261 . 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. (which was not certified by peer review) preprint (Table 1 ). Most of the study participants were females (73%) and had a mean 277 age of 42.7 (SD 11.65) years. Around 20% had underlying comorbidities and 22% were 278 under chronic medication (Table 1) . Thirty-two per cent of all participants and 22% of those 279 vaccinated had previously been infected by SARS-CoV-2 according to rRT-PCR or serology 280 data (Table 1) . Seventy-three per cent of the participants had AEs to vaccination, systemic in 281 28% after one dose and 68% after two doses (Table 1) . Among the 159 participants fully 282 vaccinated with two doses, 10 (6.3%) vaccine breakthroughs were detected by rRT-PCR 283 after 15 days post-second dose with a median of 144.5 days (49-189 days) post-vaccination. 284

Among the 53 individuals non-vaccinated at M12, 4 (7.5%) had a SARS-CoV-2 infection in 285 the same period of time. 286 287

After 7 to 72 days post one dose of the BNT162b2 or the mRNA-1273 vaccines, 92.2% 289 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;  (59/64) participants were seropositive, and seropositivity increased to 95.9% (47/49) when 290 excluding samples from less than 10 days post-vaccination. IgA and IgG levels against all S 291 antigens tested (RBD, S full length and S2) increased in most of the participants after one 292 dose, albeit at very heterogeneous levels (Fig.1a) . After 2 doses of the BNT162b2 or the 293 mRNA-1273 vaccines (12-19 days post-vaccination), all participants were seropositive with 294 the exception of a participant receiving the BNT162b2 who had renal insufficiency and was 295 under corticoids and immunomodulatory cytokine treatments (Fig. 1b) . IgA, IgG and IgM 296 levels ( Fig. 1b) and neutralization capacity (Fig. 2) increased in all individuals after the two 297 doses compared to pre-vaccination but at varying levels. 298

IgG antibodies produced after two doses in all seropositive individuals recognized the S full 299 length from the Alpha, the Beta and the Gamma VoCs (Fig. S1 ). However, the odds of being 300

IgM seronegative were 4.7 times higher for the Gamma variant, 3.8 times higher for the Beta 301 variant and 2.5 times higher for the Alpha variant than for the wild-type. 302 303

Previously SARS-CoV-2 infected individuals produced higher IgA, IgG and IgM levels 305 against the S antigens RBD, S full length, and S2 after 1 (5.53 median fold-change increase 306

for IgG, all antigens pooled) and 2 doses (1.36 median fold-change increase for IgG, all 307 antigens pooled) of the vaccine than naive participants (Fig. 3) . Kinetics after vaccination 308 ( Fig. S2 ) also show that vaccinated people who were previously exposed mounted higher 309 antibody levels than naive individuals. Differences were larger after a 1 st dose than after 2 310 doses ( Fig. 3-Fig. S2 ). Indeed, in previously infected individuals, antibody levels after the 2 nd 311 dose were similar to levels after the 1 st dose with the exception of IgG against S2 that were 312 lower after the 2 nd dose (Fig. 3) , while for unexposed individuals, antibody levels clearly 313 reached their maximum after the second dose. Differences in antibody levels between pre-314 exposed and unexposed individuals were similar for the S proteins of the Alpha, Beta and 315

Gamma VoCs (Fig. S2) . 316 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; Antibody neutralization capacity after 2 vaccine doses was higher in pre-exposed than naive 317 individuals (Fig. 4a) . Similarly, the avidity of IgA and IgG in pre-exposed individuals after the 318 2 nd dose was higher compared to unexposed individuals (Fig. 4b) . The plasma neutralization 319 capacity positively and strongly correlated with IgG levels (for IgG RBD and S: rho=0.81-320 0.76 in naive and 0.83-0.84 in pre-exposed, p<0.001; Fig. S3 ) and moderately with IgA 321 levels, particularly for RBD and in previously exposed participants (for IgA RBD: rho=0.45 322 p=0.002 in naïve and rho=0.61 in pre-exposed p<0.001; Fig. S3 ). 323 324

When comparing responses between HCW who had the infection more than 11 months vs 326 less than 11 months before vaccination, the first ones induced higher levels of IgA and IgG 327 ( Fig. S4 ). However, when using different cutoff values for the time passed between infection 328 and vaccination, there were no differences in antibody levels induced by the vaccines. 329

Previously exposed participants who had symptoms during infection produced higher IgA 330 and IgG levels against RBD and S2 after 2 vaccine doses than asymptomatic participants 331 ( Fig. S5a ). Symptomatic individuals also had higher IgA and IgG levels against S full length 332 VoCs ( Fig. S5b ) and had higher plasma neutralization capacity (Fig. S5c ). In contrast, an 333 inverse tendency was observed for IgM ( Fig. S5a,b) . 334

Pre-vaccination IgG and IgA levels in exposed participants positively and moderately 335 correlated with antibody levels post-1 st and 2 nd dose (Fig S6a-b) . Pre-vaccination IgM levels 336 also correlated with post-vaccination levels but to a lesser extent. Antibody levels elicited 337 after one dose positively correlated with the antibody levels elicited after the 2 nd dose among 338 previously exposed (Fig. S6c) . 339 340 mRNA-1273 vaccine elicits higher antibody responses than BNT162b2 341

Two doses of the mRNA-1273 vaccine elicited higher IgA and IgG levels against RBD, S full 342 length and the S2 subunit, and of higher neutralizing capacity and avidity, than two doses of 343 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;  the BNT162b2 vaccine (Fig. 5) . Similarly, IgA and IgG levels against the S full length protein 344 of the tested VoCs were higher after mRNA-1273 than BNT162b2 vaccination (Fig.5) . 345 346 AEs after vaccination are associated with induction of higher antibody levels 347

Having had systemic AEs after 1 st dose was associated with higher levels of IgA and IgG 348 against RBD and IgG against the S protein from the wild-type and the VoCs compared to 349 having no or only local AEs (Fig. S7a) . Similarly, having had systemic AEs after the 2 nd dose 350 was associated with higher IgA, IgG and IgM levels to almost all S antigens than not having 351 or only local AEs (Fig. S7b) . Systemic AEs were also positively associated with higher 352 neutralization capacity and avidity after the 2 nd dose ( Fig. S7c-d) . 353

In univariable models, previously exposed HCW had 839% (260-2347, 95%CI; P-356 value<0.001) higher IgG S levels than naive HCW after a single-dose of the vaccines. 357

BNT162b2 vaccination was associated with 78% lower IgG S levels (38-93, 95%CI; P-358 value=0.005) compared to mRNA-1273, whereas having had systemic AEs in contrast to 359 local AEs or no AEs and days since 1 st dose were significantly and positively associated with 360 350% (56-1202, 95% CI; P-value=0.006) and 10% (2-19, 95% CI; P-value=0.13) higher IgG 361 S levels, respectively. 362

In a stepwise multivariable model, these variables were retained but only previous exposure 363 to SARS-CoV-2 and systemic AEs after vaccination were statistically significant (Table 2) . In 364 addition, smoking was associated with significantly less IgG S levels (63%, 6-85, 95% CI; P-365 value=0.038). 366 367

In univariable models, we found that males had higher IgG levels against S full length protein 369 than females, and that IgG levels decreased by age in unexposed vaccinated participants, 370 but not in exposed participants or when analyzing all participants together (Table S1) . 371 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;  Comorbidities and receiving the BNT162b2 vaccine instead of the mRNA-1273 vaccine were 372 associated with lower IgG levels (Table S1 ) and plasma neutralizing capacity (Table S2) . 373

Having been previously exposed, having had systemic AEs compared to local AEs or no 374

AEs after the 1 st dose (for all and pre-exposed HCW) or the 2 nd dose (for all and naïve HCW) 375 and days since the 1 st dose, were associated with higher IgG levels (Table S1 ). Curiously, 376

IgG levels against the N protein of the HCoV HKU were negatively associated with post 377 vaccination IgG levels against S full length in pre-exposed participants (Table S1 ). Being a 378 smoker was also associated with lower plasma neutralizing capacity, while systemic AEs 379 after the 2 nd dose were associated with higher neutralizing capacity (Table S2) . 380

In stepwise multivariable models, age and sex were not significantly associated with IgG 381 levels against S protein (Table 2) . Previous SARS-CoV-2 exposure was associated with 382 38% (13-69%, 95% CI) higher IgG levels to S, whereas BNT162b2 vaccination was 383 associated with 43% (31-54%, 95% CI) less IgG-S levels than mRNA-1273 vaccination, 384 regardless of exposure. In addition, in all participants and in the unexposed ones, having 385 had systemic AEs compared to local or no AEs after the 2 nd dose was associated with 23-386 28% higher IgG-S levels. In the pre-exposed HCW, being a smoker or having underlying 387 comorbidities were independently associated with 35% (3-57%, 95% CI) and 55% (33-70%, 388 95% CI) less IgG-S levels, whereas there was a trend towards higher IgG-S levels when the 389 HCW had a symptomatic infection compared to an asymptomatic infection. 390

Being smoker, having comorbidities, and receiving the BNT162b2 vaccine instead of the 391 mRNA-1273 vaccine were also associated with 43%, 45% and 30% lower plasma 392 neutralizing capacity, respectively (Table 3) . Having had systemic AEs compared to local or 393 no AEs after the 2 nd dose was associated with 60.54% higher neutralizing capacity. SARS-394

CoV-2 exposure was also associated with 30% higher plasma neutralizing capacity though 395 the statistical significance was borderline (P-value=0.051). 396 397 IgG levels to S antigens induced by natural infection are maintained for up to a year 398 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; Antibody kinetics since the onset of symptoms along 6 time-points for 102 exposed non-399 vaccinated individuals are shown in Fig. 6 . At study month 12, 53 of the 414 HCW who 400 visited had not received any vaccine dose yet, 36 of whom had been previously infected by 401 SARS-CoV-2. IgM levels rapidly fell below the seropositivity thresholds. Similarly, IgA 402 against full length N and its C-term region and IgG against N C-term decayed over time 403 below the seropositivity thresholds. On the contrary, IgG and IgA levels against any of the S 404 antigens tested (RBD, S or S2) remained positive over time for most of the participants for 405 up to 1 year of follow-up, with IgG at higher levels than IgA. There were 31 exposed 406 individuals with more than 300 days post-infection who had not been vaccinated. IgM, IgA 407 and IgG seropositivity was 12.9%, 64.5% and 90.3%, respectively, for any of the antigens 408 affecting it, such as previous SARS-CoV-2 infection, is essential to understanding immunity 415 elicited by vaccination and its heterogeneity in the general population, which can be used to 416 improve the design of vaccination policies and guide personalized recommendations. We 417 analyzed IgA, IgG and IgM responses to the COVID-19 mRNA vaccines mRNA-1273 and 418

BNT162b2 in a well-characterized cohort of HCW with detailed demographic and clinical 419 information, accurate history of SARS-CoV-2 exposure, and antibody responses since the 420 beginning of the pandemic. Our results show that COVID-19 mRNA vaccines induce robust 421 antibody responses to S antigens in most of the HCW but mRNA-1273 elicited higher 422 antibody levels and quality than BNT162b2. Independently of the vaccine received, antibody 423 responses were higher in previously SARS-CoV-2 exposed individuals, particularly if they 424 had a symptomatic infection, and a 2 nd dose of the vaccine in pre-exposed individuals did not 425 increase their antibody levels, supporting the strategy of a single-dose vaccination for 426 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;  previously infected individuals to achieve a higher vaccination coverage and in more 427 populations. However, our data also highlights the need for more personalized strategies as 428 antibody responses may be diminished in asymptomatic, smokers and individuals with 429 chronic diseases. 430 431 Higher IgG responses induced by mRNA-1273 than BNT162b2 have also recently been 432 reported by others [47, 48] , but to our knowledge, we are the first to report higher neutralizing 433 capability and higher antibody avidity. The type of vaccine was not randomly administrated, 434 but HCW were not allowed to choose the vaccine brand since this depended on vaccine 435 availability and did not follow a pre-established pattern. In addition, results were adjusted by 436 relevant confounders such as previous SARS-CoV-2 exposure. Albeit very high antibody 437 levels are induced by both vaccines, differences may be relevant for those individuals 438 responding more poorly to vaccination or naive individuals. Although both vaccines use the 439 same technology, they differ in the amount of mRNA per dose (100 μ g vs 30 μ g) [2,3] and 440

formulation, but also in the schedule of the 2 nd dose: 4 weeks after 1 st dose for mRNA-1273 441 vs. 3 weeks for BNT162b2, which could be related to the differences observed here. A delay 442 in the 2 nd dose of the COVID-19 vaccine AstraZeneca (ChAdOx1-SARS-COV-2) showed 443 improved immunogenicity and protection [49, 50] . This suggests that there may be room for 444 optimization in the dose quantity and schedules. 445 446 Upon vaccination, exposed participants had higher levels of IgG and IgA against S antigens 447 than naive participants after one dose of the vaccine and, after two vaccine doses, naive 448 individuals still had lower IgG and IgA levels against S antigens and of lower neutralizing 449 capacity and avidity than exposed individuals. As mentioned, the 2 nd dose seemed to not be 450 beneficial in expanding the antibody response further, similarly to what has been reported by 451 others [7, 8, 11] . Nevertheless, antibody responses were very heterogeneous, even among 452 previously exposed individuals. We found that HCW who had an asymptomatic infection 453 tended to ahve less IgG levels after vaccination than symptomatic HCW, and that SARS-454 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;

CoV-2 antibodies before vaccination positively correlated with post-vaccination levels. 455

Smokers and individuals with underlying comorbidities had considerably lower antibody 456 levels and lower plasma neutralizing capacity. Therefore, a 2 nd dose should be considered 457 for exposed individuals who were asymptomatic, are smokers or people with comorbidities, 458 especially the immunosuppressed, or could be administered depending on previous antibody 459 titers, although this approach depends on the identification of correlates of protection and 460 would only be feasible in high-income countries. 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;  for more than 6 months [52] [53] [54] [55] recommendations to wait up to 6-month post-infection to get 483 vaccinated were issued in some countries, including Spain [23] . Nevertheless, at HCB, all 484 HCW were recommended to get the vaccine although naïve individuals were prioritized. 485

Here, we show maintenance of IgG responses up to a year post-infection. After more than 487 300 days (up to 383 days) following infection among unvaccinated HCW, 90% were still 488 seropositive for IgG against any of the S antigens, demonstrating persistence of immunity to 489 natural exposure. This suggests maintenance of a certain level of protection irrespective of 490 the additional role of memory T cell responses [52, 54] . Based on our data, it is difficult to 491 recommend how long previously exposed individuals could wait to get vaccinated, although 492 receiving at least a dose of a mRNA vaccine if previously exposed clearly increases antibody 493 levels and neutralizing capacity regardless of the time since infection. 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. (which was not certified by peer review) preprint Neutralising capacity against Delta (B.1.617.2) and other variants of concern following 622 . 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. (which was not certified by peer review) preprint Immunogenicity and crossreactivity of antibodies to the nucleocapsid protein of 678 . 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 this version posted September 12, 2021. ; SARS-CoV-2: utility and limitations in seroprevalence and immunity studies. 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. 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 this version posted September 12, 2021. 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 this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint 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 this version posted September 12, 2021. ; . 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. glycoprotein (S), the S protein and its subunit S2 at pre-and post-vaccination after 1 dose 754 . 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 this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint (N=44) (A) and 2 doses (N=253) (B). All plasma samples were analyzed at 1:500 dilution. 755

Pre-vaccination samples were collected at study month 6 for those who were already 756 vaccinated at month 9, and at month 9 for those vaccinated at month 12. Post-vaccination 757 samples analyzed are those collected >10 days after the 1 st dose (A) and 2 weeks after the 758 2 nd dose (B). Paired samples are joined by grey lines. The center line of boxes depicts the 759 median of MFIs; the lower and upper hinges correspond to the first and third quartiles; the 760 distance between the first and third quartiles corresponds to the interquartile range (IQR); 761 whiskers extend from the hinge to the highest or lowest value within 1.5 × IQR of the 762 respective hinge. Wilcoxon signed-rank test was used to assess statistically significant 763 differences in antibody levels between pre-and post-vaccination. and upper hinges correspond to the first and third quartiles; the distance between the first 774 and third quartiles corresponds to the interquartile range (IQR); whiskers extend from the 775 hinge to the highest or lowest value within 1.5 × IQR of the respective hinge. Wilcoxon 776 signed-rank test was used to assess statistically significant differences between pre-and 777 post-vaccination neutralization. Pre-vaccination levels correspond to visit M6. Pre-778 vaccination samples were analyzed at a 1:50 dilution while post vaccination were at 1:400. 779

We standardized the post-vaccination results to make them comparable by dividing them by 780 8. 781 782 783 . 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. IgA, IgG and IgM levels (log 10 MFI) against the receptor-binding domain (RBD) of the SARS-787

CoV-2 Spike glycoprotein (S), the S protein and its subunit S2 after 1 dose (N=64, 20 naive 788 and 44 pre-exposed) and 2 doses (N=263, 211 naive and 52 pre-exposed). Post-vaccination 789 samples analyzed were those collected >7 days after the 1 st dose and 2 weeks after the 2 nd 790 dose. The center line of boxes depicts the median of MFIs; the lower and upper hinges 791 correspond to the first and third quartiles; the distance between the first and third quartiles 792 corresponds to the interquartile range (IQR); whiskers extend from the hinge to the highest 793 or lowest value within 1.5 × IQR of the respective hinge. Wilcoxon rank test was used to 794 assess statistically significant differences in antibody levels between naive and pre-exposed 795 participants for a same dosage, and between 1 st and 2 nd dose into each group. We selected 796 all dilutions at 1:500 to make levels comparable. 797 798 799 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;

mRNA vaccines in naive and pre-exposed participants. A) Antibody neutralizing 802 capacity, as a percentage of RBD-ACE2 binding inhibition by plasma samples assayed at 803 1:400 dilution (N=92, 47 naive and 45 pre-exposed). B) Antibody avidity, as % of IgA and 804

IgG levels against RBD, S and S2 antigens measured incubating samples with a chaotropic 805 agent over the IgA and IgG levels measured in the same samples without chaotropic agent, 806 all at 1:5000 dilution (N=58, 48 naive and 10 pre-exposed). The center line of boxes depicts 807 the median of MFIs; the lower and upper hinges correspond to the first and third quartiles; 808 the distance between the first and third quartiles corresponds to the interquartile range 809 (IQR); whiskers extend from the hinge to the highest or lowest value within 1.5 × IQR of the 810 respective hinge. Wilcoxon rank test was used to assess statistically significant differences 811 in antibody neutralization and avidity between naive and pre-exposed participants. 812 813 814 . 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. mRNA-1273 among naive and pre-exposed participants (N = 263, 205 BNT162b2 / 56 818 mRNA-1273, 211 naïve, 52 exposed). Plasma samples were analyzed at 1:5000 dilution for 819

IgG and 1:500 for IgA/IgM. B) Plasma neutralization capacity elicited by BNT162b2 and 820 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint mRNA-1273 among naive and pre-exposed participants (N=92, 45 BNT162b2r/47 mRNA-821 1273, 47 naive, 45 exposed). Plasma dilution used was 1:400. C) Antibody avidity elicited by 822

BNT162b2 vs mRNA-1273 among naive and pre-exposed participants (N=58, 36 BNT162b2 823 and 22 mRNA-1273, 48 naive, 10 pre-exposed). Plasma dilution used was 1:5000 for IgG 824 and 1:500 for IgA. Red and green dots correspond to naive and pre-exposed participants, 825

respectively is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 controls and test samples (prediluted 1:50 in 96 round-bottom well plates), were added to a 874 384-well plate using Assist Plus Integra device with 12 channels Voyager pipette (final test 875 sample dilution of 1:500). To quantify IgM and IgA responses, test samples and controls 876

were pre-treated with anti-Human IgG (Gullsorb) at 1:10 dilution, to avoid IgG interferences. 877

Technical blanks consisting of Luminex Buffer (PBS with 1% BSA) and microspheres without 878 samples were added in 4 wells to detect and adjust for non-specific microsphere signals. 879

Plates were incubated for 1 h at room temperature in agitation (Titramax 1000) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Supplementary figures 903 904 905 Figure S1 . Antibody levels and seropositivity against the S antigen from the Wuhan 906 and the Alpha, Beta and Gamma variants of concern. The graphs show IgA, IgG and IgM 907 levels (median fluorescence intensity, MFI) against the Spike protein of Alpha, Beta and 908

Gamma variants A) before and after two vaccine doses in a subset of individuals with 909 available pre-vaccination data (N=13, 12 mRNA-1273 and 1 BNT162b2) at M9 (1:500 910 dilution for pre and 1:5000 for post) and B) after two vaccine doses in all participants 911 (N=328). The center line of boxes depicts the median of MFIs; the lower and upper hinges 912 correspond to the first and third quartiles; the distance between the first and third quartiles 913 corresponds to the interquartile range (IQR); whiskers extend from the hinge to the highest 914 or lowest value within 1.5 × IQR of the respective hinge. In A, paired samples are joined by 915 grey lines and Wilcoxon signed-rank test was used to assess statistically significant 916 differences in antibody levels between pre-and post-vaccination. In B, dashed lines 917 represent the positivity threshold, and numbers and percentages indicate the number and 918

proportion of seropositive participants for each isotype and S protein. 919 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101 /2021 doi: medRxiv preprint 920 921 Figure S2 . Kinetics of antibody levels against the S antigens since 1 st vaccination 922 dose in naive and pre-exposed participants after 1 st and 2 nd doses. Levels (median 923 fluorescence intensity, MFI) of IgA, IgG and IgM against each antigen (the Receptor Binding 924

Domain (RBD), full S protein, its subregion S2 and full S protein of the variants of concern) 925 measured in 426 samples from 368 participants. The red and green solid lines represent the 926 fitted curve calculated using the LOESS (locally estimated scatterplot smoothing) method. 927

Shaded areas represent 95% confidence intervals. Red and Green shaded areas 928 correspond to naive and pre-exposed participants, respectively. Filled and empty points 929 correspond to samples collected after 1 and 2 doses, respectively. Samples were analyzed 930 at the 1:5000 dilution. 931 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint 932 933 Figure S3 . Correlations between plasma neutralization activity and antibody levels 934 after 2 vaccine doses in naive and exposed participants. Scatter plots showing the 935 correlation between plasma neutralization capacity (as a percentage of RBD-ACE2 binding 936 inhibition) and IgA, IgG and IgM levels (median fluorescence intensity, MFI) against each 937 antigen (the Receptor Binding Domain (RBD), full S protein, its subregion S2 and full S 938 proteins of the variants of concern). P-values and rho correlation coefficients were computed 939 through Spearman's rank correlation tests. Lines represent the fitted curve calculated using 940 the linear model method. Red and green dots and lines correspond to naive (N=47) and pre-941 exposed (N= 45) participants, respectively. A dilution of 1:400 was used for the 942 neutralization assay, 1:500 for IgA and IgM levels, and 1:5000 for IgG levels. 943 944 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint 945 946 Figure S4 . Comparison of antibody levels to S antigens after two COVID-19 mRNA 947 vaccine doses between participants infected more vs less than 11 months prior to 948 vaccination. Plots show IgA, IgG and IgM levels (log 10 MFI) against the receptor-binding 949 domain (RBD) of the SARS-CoV-2 Spike glycoprotein (S), the full S protein, its subunit S2 950 and full S proteins for the variants of concern in participants infected more (N=6) or less 951 (N=34) than 11 months prior to vaccination. The center line of boxes depicts the median of 952

MFIs; the lower and upper hinges correspond to the first and third quartiles; the distance 953 between the first and third quartiles corresponds to the interquartile range (IQR); whiskers 954 extend from the hinge to the highest or lowest value within 1.5 × IQR of the respective hinge. 955

Wilcoxon rank test was used to assess statistically significant differences in antibody levels 956 between groups. 957 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint 958 959 Figure S5 . Comparison of antibody levels to S antigens and plasma neutralization 960 capacity after two COVID-19 mRNA vaccine doses between symptomatic and 961 asymptomatic pre-exposed participants. Antibody levels to spike (S) antigens from 962

Wuhan strain (A) and to S proteins from the variants of concern (B) (total N= 51, 25 963 asymptomatic and 26 symptomatic). Plasma dilutions tested were 1:500 for IgA/M and 964 1:5000 for IgG. C) Plasma neutralizing capacity after two vaccine doses (N=45) in previously 965 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263232 doi: medRxiv preprint infected symptomatic (N=25) and asymptomatic (N=20) participants (dilution tested was 966 1:400). 967 968 969 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ;

970 Figure S6 . Correlation of antibody levels before and after COVID-19 mRNA vaccination 971 and between doses in pre-exposed participants. A) Correlations between pre-vaccination 972 levels at M6 and >7 days post dose 1 (BNT162b2, N=27). Sample dilution analyzed was 973 . 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. (which was not certified by peer review) preprint N=13) (D). Red and green dots correspond to naive and pre-exposed participants, 990

respectively. 991 . 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 12, 2021. ; 1 . 5 0 < 0 . 0 0 1 n a n a n a n a n a n a n a n a n a n a n a n a n a n a n a n a 3 0 . 2 7 -1 0 . 5 9 8 9 . 7 9 0 . 1 6 5 D a y s s i n c e i n f e c t i o n n a n a n a n a n a n a n a n a -0 . 1 4 -3 . 8 7 3 . 7 3 0 . 9 3 9 a Beta transformed values to percentage to facilitate interpretation of the effect from variables. HCW: health care workers.

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It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review) 

B) Correlations between pre-vaccination (M6) and post-vaccination antibody levels 974 after 2 doses (BNT162b2 and mRNA-1273 pooled, N=49)