key: cord-0297476-7zfrq368 authors: Calcoen, B.; Callebaut, K.; Vandenbulcke, A.; Callewaert, N.; Bossuyt, X.; Van Weyenbergh, J.; Maes, P.; Imbrechts, M.; Vercruysse, T.; Thibaut, H. J.; Zapf, D.; Dieckmann, K.; Vanhoorelbeke, K.; Geukens, N.; De Meyer, S.; Maes, W. title: Real-world monitoring of BNT162b2 vaccine-induced SARS-CoV-2 B and T cell immunity in naive healthcare workers: a prospective single center study date: 2022-01-17 journal: nan DOI: 10.1101/2022.01.17.22269081 sha: 3d9f82bd3813863c1e9dc2f6f6e2cc7722c9cc56 doc_id: 297476 cord_uid: 7zfrq368 Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing COVID-19 pandemic. To prevent the massive COVID-19 burden, several vaccination campaigns were initiated. We performed a single center observational trial to evaluate adaptive immunity in naive healthcare workers upon BNT162b2 vaccination. Methods: Serological analysis was performed through conventional immunoassays. Antibody functionality was analyzed via in vitro neutralization assays. Circulating receptor-binding domain (RBD) specific B cells were assessed via flowcytometry. The induction of SARS-CoV-2 specific T cells was investigated through interferon-{gamma} release assay combined with flowcytometric profiling of activated CD4 and CD8 T cells. Results: Three months after vaccination, all but one of the subjects (N = 31) displayed vaccine-induced neutralizing antibodies. In 10 out of 31 subjects, circulating RBD specific B cells were found of which the rate showed moderate correlation to serological parameters. Specific interferon-{gamma} release was present in all subjects and correlated with the significant upregulation of CD69 on CD4+ and CD8+ T cells and CD40L on CD4+ T cells. Interestingly, no relation was found between B and T cell parameters. In addition, one symptomatic breakthrough infection with the SARS-CoV-2 alpha variant of concern was reported. Conclusion: Three months post vaccination, both humoral and cellular immune responses are detectable in all but one participant. No correlation was found between the magnitude of both B and T cell responses. Back in December 2019, multiple cases with serious pneumonia of unknown origin (later renamed 43 as coronavirus disease 19 or COVID- 19) were described in Wuhan, China (1) . Not long afterwards, 44 the causative pathogen was identified via real-time polymerase chain reaction (RT-PCR) as a novel 45 sarbecovirus (sub-genus of the β-coronaviridae) and named severe acute respiratory syndrome 46 coronavirus 2 (SARS-CoV-2) (2,3). 47 Within a few weeks, SARS-CoV-2 rapidly spread internationally and on 30 January 2020, the 48 world health organization (WHO) declared the outbreak a public health emergency of international 49 concern (4). As a result, many countries introduced strict socio-economic rules (e.g. lockdown, 50 mouth mask) to limit viral spreading and prevent a total collapse of their healthcare system (5). 51 During the pandemic, multiple strategies including the development of a SARS-CoV-2 vaccine 52 were urgently initiated to lower the massive SARS-CoV-2 related burden. Multiple vaccine 53 development strategies were explored, including nucleic acid-based (e.g. BNT162b2 or 54 Comirnaty ® (6,7), mRNA-1732 or Spikevax ® (8,9)), adenovector-based (e.g. Vaxzevria ® (10), 55 COVID19 vaccine Janssen ® (11,12)) and protein-based (Sanofi-GSK ® (13)) vaccines of which 56 several products received conditional authorization by either the European Medical Agency (EMA) 57 or Food and Drug Administration (FDA) at the end of 2020. This resulted in the roll-out of 58 international vaccination campaigns at an unprecedented scale and speed. 59 However, due to the emergence of several SARS-CoV-2 variants of concern (VoC) that already 60 led to new infection waves around the globe (14,15), there is reasonable concern whether the 61 immune response evoked by the currently available vaccines is sufficient to cover these VoC. For 62 example, it is known that several of these SARS-CoV-2 VoC have mutations within their spike (S) 63 protein or receptor-binding domain (RBD) region and therefore could be able to escape 64 functionally active vaccine-induced antibodies that only bind to unique epitopes present on Wuhan 65 strain S or RBD (16). In addition, only a limited number of studies reveal mid-term (i.e. longer 66 than 2 months post mRNA vaccination) sustainability data of vaccine-induced de novo responses 67 to SARS-CoV-2 within healthy subjects (17, 18) . Finally, most vaccine monitoring studies are often 68 solely based on the evaluation of serological responses (19) , although it is known from for example 69 vaccination against smallpox that specific T cells can be detected up to 75 years after vaccination 70 and thus are crucial for long-term immunity (20,21). Indeed, few groups have looked at both 71 vaccine-induced B and T cell immunity, in particular in healthy SARS-CoV-2-naive volunteers 72 (22,23) as most trials focused on either immunocompromised or SARS-CoV-2 convalescent 73 patients. Concerning specific T cell responses upon mRNA-based SARS-CoV-2 vaccination, 74 available data are limited to phase I/II studies (24) of the Comirnaty ® vaccine and studies that either 75 assessed short-term T cell response (i.e. up to one month after booster vaccination) (25,26) or 76 focused on T cell responses upon prime or boost vaccination (27, 28) . Of note, there are several, 77 sometimes contradictory reports concerning both the occurrence rate (29,30) and severity (31,32) 78 of post mRNA vaccination breakthrough infections (BTI) . 79 To address these gaps, we monitored vaccine elicited B and T cell responses in a real-world setting. 80 Hereto, SARS-CoV-2 naive healthcare workers were followed for three months after vaccination 81 with the BNT162b2 SARS-CoV-2 vaccine (Comirnaty ® ) in a prospective single center study. 82 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. sampling was performed three months after tpre (t3m). In case a participant, after receiving two 99 vaccines, presented with COVID-19 symptoms (e.g. dry cough, fever and/or headache) and was 100 positive for SARS-CoV-2 via real-time polymerase chain reaction (RT-PCR), he/she was 101 considered to experience a symptomatic BTI. In this situation, additional sampling was scheduled 102 as soon as possible (tBTI). Sampling details are visualized in Figure 1 . 103 104 105 Figure 1 . Study design and timeline. A total of 32 healthcare workers were enrolled. One day 106 before or on the day of the first BNT162b2 vaccine, a first baseline sampling moment was 107 scheduled (tpre). A second sampling moment (t3m) was planned between 80-100 days after baseline. 108 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. On each moment, serum was collected to determine SARS-CoV-2 serology and neutralization 109 efficacy whereas blood was used both for isolating PBMCs to detect circulating RBD specific B 110 cells and to assess both SARS-CoV-2 specific T cell activity and IFN-γ release. When a participant 111 developed symptoms and was tested positive via RT-PCR after receiving two vaccines, an 112 additional sampling moment was planned (tBTI). At tBTI, a nasopharyngeal swab was collected to 113 execute viral whole genome sequencing. Also, a VSV pseudovirus neutralization assay was 114 additionally performed at this timepoint. Abbreviations: PBMC = peripheral blood mononuclear 115 cells, IFN-γ = interferon γ, RT-PCR = real-time polymerase chain reaction, VSV = vesicular 116 stomatitis virus, VoC = variants of concern. This figure was created using Monoclonal anti-RBD calibrator antibody (Sino Biologicals; cat. Nr. 40150-D004) and serum 148 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The presence of serum anti-N IgG antibodies was determined via the Anti-SARS-CoV-2-NCP 161 (IgG) enzyme immunoassay from EUROIMMUN on an ETI-Max 3000 instrument from DiaSorin. 162 Samples that had a cut-of index greater or equal to 1.1 were considered positive as recommended 163 by the instructions from the manufacturer. 164 Determination of the neutralizing capacity of the vaccine-induced antibodies was generated using 167 the EUROIMMUN SARS-CoV-2 NeutraLISA assay according to the manufacturer's instructions. 168 Samples were diluted 1:5 in sample buffer. A photometric measurement was made on a wavelength 169 of 450 nm together with a reference wavelength of 620 nm. Semiquantitative results were generated 170 by calculating a ratio of the extinction values of the sample over the mean extinction value of the 171 blank (measured in duplicate) and were presented as percentage inhibition. Percentage inhibition 172 values lower than 20 were defined as negative, between 20 and 35 as borderline and higher or equal 173 to 35 as positive. Lot-specific control concentrates (positive and negative) were used as assay 174 references. 175 VSV S-pseudotypes were generated as described previously (35). Briefly, HEK-293T cells 177 (SARS-CoV-2) were transfected with the respective S protein expression plasmids, and one day 178 later infected (MOI = 2) with green fluorescent protein (GFP)-encoding VSVΔG backbone virus 179 (purchased from Kerafast). Two hours later, the medium was replaced by medium containing anti-180 VSV-G antibody (I1-hybridoma, ATCC CRL-2700) to neutralize residual VSV-G input. After 24 181 h incubation at 32 °C, the supernatants were harvested. To quantify neutralizing antibodies 182 (nAbs), serial dilutions of serum samples were incubated for 1 h at 37 °C with an equal volume 183 of S pseudotyped VSV particles and inoculated on Vero E6 cells ( SARS-CoV-2) for 18 h. 184 The percentage of GFP expressing cells was quantified on a Cell Insight CX5/7 High Content 185 Screening platform (Thermo Fischer Scientific) with Thermo Fisher Scientific HCS Studio 186 (v.6.6.0) software. Neutralization IC50 values were determined by normalizing the serum 187 neutralization dilution curve to a virus (100%) and cell control (0%) and fitting in GraphPad Prism 188 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Heparinized whole blood was used for the EUROIMMUN SARS-CoV-2 interferon gamma release 192 assay (IGRA) kit that was executed according to the manufacturer's instructions adapted as 193 described below (Supplementary Figure 1) . Heparinized whole blood was transferred into three 194 different tubes (BLANK, COV2 and STIM) followed by an incubation step for 20-24 h at 37 (±1) 195 °C after inverting 6 times. Following incubation, the tubes were centrifuged at RT for 10 min at 196 700 g. Approximately 200 µL of stimulated heparinized plasma from each tube was pipetted into 197 Eppendorf tubes and centrifuged again at RT for 10 min at 12,000 g. Finally, the supernatant was 198 pipetted into cryovials and stored at -20 °C until measurement via the EUROIMMUN Quant-T-199 Cell ELISA (interferon-γ (IFN-γ) ELISA). Importantly, the pellet containing the cellular fraction 200 was used for additional flowcytometry (see 2.5.2 T cell phenotyping and activity). 201 Specific SARS-CoV-2 induced IFN-γ release was determined via the EUROIMMUN Quant-T-203 Cell ELISA according to the manufacturer's instructions. A photometric measurement was 204 performed at a wavelength of 450 nm with a reference measurement at 620 nm. For each tube, 205 IFN-γ concentrations were determined using a standard curve that was fitted via GraphPad Prism 206 (four parameters model without restrictions). Then, for each subject, the determined IFN-γ 207 concentration from the unstimulated control (BLANK) was subtracted from the determined IFN-γ 208 concentrations of both the stimulation control (STIM) and SARS-CoV-2 stimulated condition 209 (COV2). Lot-specific lyophilized calibrators and controls were used as a standard. 210 Thawed PBMCs were stained with a selective B cell staining panel that is listed in Supplementary 213 Table 1 . After a final washing step, the pellets were resuspended in 300 µL dPBS and immediately 214 acquired on the flowcytometer (FACSVerse device, BD Biosciences, USA). 215 Living B cells were selected from the PBMC pool via a CD3 -/CD19 + /Zombiegating strategy 216 (Supplementary Figure 2A) . Specific B cell reactivity against SARS-CoV-2 was assessed with 217 wild type RBD-biotin and PE-streptavidin. Within each sample, a negative control tube (without 218 RBD-biotin) was included to correct for sample-specific background. For each staining 219 experiment, a sample with documented RBD specific B cells was used as a positive control for 220 quality assessment. 221 The remaining cell pellet from the IGRA tubes (see 2.4 Stimulation of SARS-CoV-2 specific T 223 cells) was immediately resuspended with dPBS in a total volume of 500 µL. A whole blood staining 224 was performed on the reconstituted samples. In summary, T cell staining (Supplementary Table 225 1) was performed in 150 µL of the reconstituted samples. Following incubation for 30 min at 4 °C, 226 3 mL of red blood cell (RBC) lysis buffer (BD FACS TM lysing solution, BD Biosciences, USA) 227 was added for 5 min at RT to allow RBC lysis. After extensive washing, the pellets were 228 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Results 265 All enrolled healthcare workers (N = 32) were Caucasian and exact half of them were women. The 267 age ranged between 25 and 51 years with a mean of 36 ± 7 (95% confidence interval (CI): 33-38) 268 years. During this trial, one subject was excluded for all analyses because she dropped out between 269 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. ; https://doi.org/10.1101/2022.01.17.22269081 doi: medRxiv preprint BNT162b2 vaccine monitoring in naive healthcare workers 9 tpre and t3m (Figure 1) . Anti-N IgG titers were measured at both tpre and t3m to exclude (subclinical) 270 natural SARS-CoV-2 infection. For both timepoints, the anti-N IgG titers remained below the index 271 value with a mean of 0.022 ± 0.038 and 0.100 ± 0.113 at respectively tpre and t3m. 272 response (Figure 2A-B) . Anti-S IgG and IgA titers were moderately but significantly correlated 290 with a Spearman r of 0.5798 (p < 0.001) and a Pearson R² of 0.2692 ( Figure 2C) . 291 Next, anti-RBD IgG titers were determined (Figure 3A ). In line with the above findings, no anti-292 RBD IgG antibodies could be detected at baseline (< 160 BAU/mL), whereas all but one had anti-293 RBD IgG titers three months post-vaccination with a median titer of 827.0 (95% CI: 661.0-1103) 294 with a Q1-Q3 interval of 599.0-1310 BAU/mL. 295 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. Additionally, the capacity of the induced antibodies to neutralize the binding between wild type 305 RBD and human ACE2, was assessed. Overall antibody neutralization capacity was high with a 306 median % inhibition compared to a blank control of 93.91 ± 6.08 (95% CI: 90.45-95.13; Figure 307 3B). A moderate but significant correlation was seen between anti-RBD IgG titers and antibody 308 functionality with a Spearman r of 0.8347 (p < 0.0001) and a Pearson R² of 0.3010 ( Figure 3C) . 309 In parallel to the assessment of serology and functionality of the vaccine-induced antibodies, the 311 precursor rate of circulating RBD specific B cells was examined. For each individual, a PBMC 312 aliquot of tpre and t3m were analyzed within the same experiment to allow the detection of vaccine-313 specific changes. An example of the gating strategy used for the flowcytometric B cell phenotyping 314 is shown in Supplementary Figure 2A . 315 The magnitude of CD3 -/CD19 + B cells was comparable between both timepoints and ranged 316 between 1.93 -14.16 % of gated lymphocytes. Furthermore, a small (0.087 ± 0.068 % of parent, 317 i.e. living B cells) but significant (p < 0.005) population of circulating RBD specific B cells was 318 detected in 10 of the 31 individuals (32.26 %) at three months post vaccination ( Figure 4A) . Within 319 these 10 subjects, the rate of circulating RBD specific B cells showed a significant good correlation 320 with anti-RBD IgG titers (Pearson R² = 0.6314, Spearman r = 0.8571 with p < 0.005, Figure 4B ) 321 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. Next, we also defined the SARS-CoV-2 specific T cell response. Firstly, we assessed the vaccine-341 induced specific IFN-γ release by T cells upon restimulation with SARS-CoV-2 antigens ( Figure 342 5). Three subjects were excluded from these analyses due to technical issues. 343 344 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. CoV-2-specific T cell mediated IFN-γ release detected and even exceeding 10,000-fold for some 357 participants. At both timepoints, there was a clear IFN-γ release observed in the stimulation control 358 condition (data not shown). 359 BNT162b2 vaccination 361 Using the reconstituted cell pellets from the IGRA tubes, we were able to include T cell 362 phenotyping in addition to the SARS-CoV-2 specific IFN-γ release. An example of the applied 363 gating strategy can be retrieved in Supplementary Figure 2B . In accordance with the B cell 364 phenotyping, the magnitudes of CD3 + lymphocytes (i.e. T cells), CD4 -/CD8 + cytotoxic T cells (TC), 365 CD4 + /CD8helper T cells (TH) and CD4 + /CD8 -/CXCR5 hi circulating follicular T helper cells (TCFH) 366 were similar for all three conditions within the IGRA (data not shown). 367 Three months after vaccination, a significant upregulation of the early activation marker CD69 was 368 detected upon SARS-CoV-2 specific restimulation in the TC (p < 0.0001), TH (p < 0.0001) and 369 TCFH (p < 0.005) cell subsets ( Figure 6A) . Furthermore, CD40L was also significantly upregulated 370 in both TH (p < 0.0001) and TCFH (p < 0.005) subsets ( Figure 7A ). For each sample, a condition 371 with mitogenic stimulation was analyzed as well as means of quality control (data not shown). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Additionally, PCA was performed to identify parameters that contribute most to the overall 407 variance found within this cohort (Figure 8) . with PC 2 (Figure 8B) . Combining PC 1 and PC 2 explained approximately 60 % of the overall 420 variance in this cohort ( Figure 8C) . 421 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. ; https://doi.org/10.1101/2022.01.17.22269081 doi: medRxiv preprint BNT162b2 vaccine monitoring in naive healthcare workers Within the timeframe of this study, one subject experienced a RT-PCR confirmed symptomatic 424 BTI. This BTI occurred within the collection period for the t3m moment and was therefore redefined 425 as the tBTI timepoint for this participant. As described in Figure 1 , a nasopharyngeal swab was 426 collected additionally to the blood tubes drawn via venipuncture. 427 This case concerned a woman in her thirties who presented with general malaise, dry cough and 428 dyspnea 46 days after receiving the booster vaccine. The subject was ill for seven days but without 429 the need for hospitalization. Based on the WHO COVID-19 severity score (38), the subject was 430 classified as experiencing a mild SARS-CoV-2 infection. A few days prior to the BTI, this 431 participant reported to have been in close contact with a hospitalized COVID-19 patient. A 432 complete overview of both the humoral and cellular immune parameters at the tBTI timepoint of this 433 subject are listed in Table 1 . 434 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. observed. In order to investigate whether this BTI could be ascribed to any of the currently known 446 VoC, whole viral genome sequencing was performed on the nasopharyngeal swab. Genome 447 sequencing revealed the presence of the B.1.1.7 Pango lineage, which is also known as the alpha 448 VoC. 449 Discussion 450 In this study, we report on SARS-CoV-2 specific B and T cell immunity in healthcare workers in 451 a supraregional hospital in Belgium three months after BNT162b2 vaccination. Serological findings in this cohort were comparable with those described by other groups that 459 studied the serology status post booster BNT162b2 vaccination at different timepoints, i.e. ranging 460 between 2 and 12 weeks post primary vaccination (41) (42) (43) (44) (45) (46) (47) (48) . Of note, many of these studies 461 monitored vaccine induced antibody responses in patients suffering from a myriad of pathologies 462 (41) (42) (43) . In addition, the net effect of vaccination is often difficult to highlight as in many studies 463 subjects with earlier natural SARS-COV-2 infection are not excluded (27, 49 IgA anti-S antibodies just above detection limit, either the mRNA-vaccine encoded spike protein 478 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. antibody functionality on the other hand (Figure 4B-C) . As no signs of ongoing natural infection 497 were found, these data suggest that at least in these participants, the germinal center driven humoral 498 immunity is still being built up three months post mRNA vaccination. these results, we could clearly observe SARS-CoV-2 specific IFN-γ release in all subjects up to 511 three months post vaccination. Moreover, we were able to include T cell phenotyping as secondary 512 read-out to the whole blood IGRA, hereby eliminating the need for PBMCs nor expensive peptide 513 pools covering the most prominent MHC haplotypes in a given population. Using this approach, 514 we were able to show SARS-CoV-2 specific activation of both TC and TH cell subsets which 515 implies that the SARS-CoV-2 specific antigens in this assay were presented on both MHC class I 516 and II proteins respectively. This was also shown in reports from both Braun et al. (65) and Giminez 517 et al. (66) in which they used a related SARS-CoV-2 antigen pool to stimulate T cells and found 518 respectively an upregulation of activated CD4 + and CD8 + T cells. Besides membrane expression 519 of CD69 as general early T cell activation marker, we were able to show upregulation of CD40L 520 both in total TH and in the CXCR5 + TCFH subsets. Both early T cell activation and membrane 521 CD40L expression are correlated with the release of type II interferon, confirming that BNT162b2 522 vaccination induces a TH1 response (25). In addition, as CD40L is one of the key costimulatory 523 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. will most likely lead to a massive and rapid T cell dependent antibody recall response, the latter 526 being exactly the overall goal of vaccination. 527 Next, we addressed whether the humoral and cellular immune parameters measured in this study 528 were associated to each other. From the PCA analysis, two different clusters could be identified 529 based on the loadings plot with PC 2. Interestingly, these clusters included all the parameters 530 determined within B or T cell immunity. This suggests that measuring only one or two parameters 531 from both the humoral and cellular immune response is sufficient to respectively predict 60 to 80 532 percent of the overall variability observed in this cohort ( Figure 8C) . Also, the approximate angle 533 of 90 degrees between both clusters emphasizes the absence of any correlation between each other. 534 Within the three month monitoring time window in this cohort, there was one subject who 535 developed a mild symptomatic BTI (incidence of 3.22 %) although the incidence rate reported in 536 the BNT162b2 phase III trial was very low (< 0.05 %, 8 cases out of 18,198) (67). Of note, one 537 must be careful not to overinterpret this high incidence rate due to the small size of this study. In 538 addition, healthcare workers might be more at risk than the general population, especially during a 539 new infection wave including emergent VoC. Although, it must be mentioned that a recently 540 published report of the Belgian Healthcare Knowledge Centerthat included surveillance data 541 since the start of the vaccination campaign in Belgiummentioned that the national incidence of 542 symptomatic BTI within the general population (of which 71.2% were vaccinated with 543 Comirnaty ® ) was only 0.20 % (68). Antonelli et al. stated that vaccinated people who developed a 544 BTI had mostly milder symptoms and were approximately half less likely to report symptoms of 545 Long COVID-19 Syndrome than infected unvaccinated people (69). This was also the case in our 546 subject as she had a mild infection and recovered completely after seven days. Interestingly, this 547 subject showed a clear vaccine-induced immune response including SARS-CoV-2 specific T cell 548 activity, positive titers of both anti-S and anti-RBD IgG antibodies with a high neutralization 549 efficacy (confirmed in an established pseudovirus assay) and had a small but clearly detectable 550 population of circulating RBD specific B cells. Here, the latter could very well represent the recall 551 response to natural infection in this subject. Whole genome sequencing retrieved that the subject 552 was infected with the alpha variant which was at that time the upcoming VoC within the first phase 553 of the third wave in Belgium. Antibody repertoire profiling of the subject with BTI compared to a 554 pool of age and gender matched controls could yield interesting information, in particular when 555 compared to other vaccine BTI profiles. 556 At last, it must be noted that this trial has several limitations. Firstly, the number of subjects is 557 rather limited and was based on a power analysis for the serological and IFN-γ release read-outs. 558 Hence, this design is less suited to pick up rather unexpected events such as BTI. Larger studies 559 that look at both arms of the adaptive immune response months after vaccination are needed to 560 further finetune our findings and to characterize BTI more reliable. Secondly, only SARS-CoV-2 561 naive individuals were included in this study. The pronounced recall response due to vaccination 562 in convalescent individuals has been described and even led to the discussion whether these 563 individuals should receive one rather than two vaccine doses. Addressing this was beyond our 564 scope as we specifically aimed to study the BNT162b2 vaccine-induced de novo immune response 565 in a real-world setting. Thirdly, the emergence of new and potentially more dangerous SARS-CoV-566 2 variants leads the binding and neutralization assays available at any given time. In this 567 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 17, 2022. ; https://doi.org/10.1101/2022.01.17.22269081 doi: medRxiv preprint BNT162b2 vaccine monitoring in naive healthcare workers perspective, running neutralization assays with VoC RBD could give deeper insights in the breadth 568 of the vaccine-induced functional immunity. Finally, long-term sustainability of the vaccine-569 induced immune response can only be truly considered when looking even beyond the three month 570 timepoint. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Acknowledgments 591The authors would like to thank the people from BIOGNOST CV, Heule (Belgium) for their 592 support. 593 10 References 594