key: cord-0949366-hg5annzg
authors: Torres, I.; Albert, E.; Gimenez, E.; Alcaraz, M. J.; Botija, P.; Amat, P.; Remigia, M. J.; Beltran, M. J.; Rodado, C.; Huntley, D.; Olea, B.; Navarro, D.
title: B and T cell immune responses elicited by the BNT162b2 (Pfizer BioNTech) COVID-19 vaccine in nursing home residents
date: 2021-04-22
journal: nan
DOI: 10.1101/2021.04.19.21255723
sha: e688ab51b1a5528db52db1090a4e9b3db31e2b92
doc_id: 949366
cord_uid: hg5annzg

Objectives: The immunogenicity of the BNT162b2 COVID-19 vaccine is understudied in elderly people with comorbidities. We assessed SARS-CoV-2-S-targeted antibody and T cell responses following full vaccination in nursing home residents (NHR). Methods: We recruited 60 NHR (44 female; median age, 87.5 years), of whom 10 had previously had COVID-19, and 18 healthy controls (15 female; median age, 48.5 years). Pre- and post-vaccination blood specimens were available for quantitation of total antibodies binding RBD and enumeration of SARS-CoV-2-S-reactive IFN-{gamma}; CD4+ and CD8+ T cells by flow cytometry. Results: The seroconversion rate in presumably SARS-CoV-2 naive NHR (95.3%), either with or without comorbidities, was similar to controls (94.4%). A robust booster effect was documented in NHR with prior COVID-19. Plasma antibody levels were higher in convalescent NHR than in individuals across the other two groups. A large percentage of NHR had SARS-CoV-2 S-reactive IFN-{gamma}; CD8+ and/or CD4+ T cells at baseline, in contrast to healthy controls. Either CD8+ and/or CD4+ T-cell responses were documented in all control subjects after vaccination. Contrariwise, the percentage of NHR exhibiting detectable SARS-CoV-2 IFN-{gamma}; CD8+ or CD4+ T-cell responses (or both), irrespective of their baseline SARS-CoV-2 infection status, dropped consistently after vaccination. Overall, SARS-CoV-2 IFN-{gamma}; CD8+ and CD4+ T-cell responses in NHR decreased in post-vaccination specimens. Conclusion: The BNT162b2 COVID-19 vaccine elicits robust SARS-CoV-2-S antibody responses in NHR. Nevertheless, the frequency and magnitude of detectable SARS-CoV-2 IFN-{gamma}; T-cell responses after vaccination was lower in NHR compared to controls.

The BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine, a nucleoside-modified 55 messenger RNA that encodes the full-length transmembrane S glycoprotein locked in its 56 perfusion conformation, elicits high levels of serum neutralizing antibodies (NtAb), 57 mainly targeting the SARS-CoV-2 receptor-binding domain (RBD), and strong TH 1 -58 skewed functional CD4 + and CD8 + T cell responses in experimental models and humans 59 [1] [2] [3] [4] . The efficacy of the vaccine has been shown to approach 95% in preventing severe 60 heparinized whole blood (0.5 mL) was simultaneously stimulated for 6 h with two 114 sets of 15 mer overlapping peptides (11 mer overlap) encompassing the 115 SARS CoV 2 Spike (S) glycoprotein (S1, 158 peptides and S2, 157 peptides) at a 116 concentration of 1 μ g/mL per peptide, in the presence of 1 μ g/ml of costimulatory 117 monoclonal antibodies (mAbs) to CD28 and CD49d. Peptide mixes were obtained from 118 JPT peptide Technologies GmbH (Berlin, Germany). Samples mock-stimulated with 119 phosphate buffered saline (PBS)/dimethyl sulfoxide and costimulatory antibodies were 120 run in parallel. Brefeldin A (10 μ g/mL) was added for the last 4 h of incubation. 121

Blood was then lysed (BD FACS lysing solution) and frozen at −80°C until tested. On 122 . 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 April 22, 2021. ; https://doi.org/10.1101/2021.04.19.21255723 doi: medRxiv preprint the day of testing, stimulated blood was thawed at 37°C, washed, permeabilized (BD 123 permeabilizing solution) and stained with a combination of labeled mAbs 124 (anti IFNγ FITC, anti CD4 PE, anti CD8 PerCP Cy5.5, and anti CD3 APC) 125 for 1 h at room temperature. Appropriate positive (phytohemagglutinin) and isotype 126 controls were used. Cells were then washed, resuspended in 200 μ L of 1% 127 paraformaldehyde in PBS, and analyzed within 2 h on an FACSCanto flow cytometer 128 using DIVA v8 software (BD Biosciences Immunocytometry Systems, San Jose, CA). 129 CD3 + /CD8 + or CD3 + /CD4 + events were gated and then analyzed for IFN γ production. 130

All data were corrected for background IFN-γ production and expressed as a percentage 131 of total CD8 + or CD4 + T cells. Representative flow cytometry plots are shown in 132

Supplementary Figure 1 . 133

Frequency comparisons for categorical variables were carried out using the Fisher exact 135 test. Differences between medians were compared using the Mann-Whitney U-test or 136 the Wilcoxon test for unpaired and paired data, when appropriate. Two-sided exact P-137 values were reported. A P-value <0.05 was considered statistically significant. The 138 analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA). 139

No serological evidence of prior SARS-CoV-2 infection was found in 49 (83%) of the 142 59 NHR at baseline. Pre-vaccination plasma was not available from one patient. In 143 addition, these subjects had been tested at least once for presence of SARS-CoV-2 RNA 144 in nasopharyngeal specimens since the beginning of the epidemic, as a part of a local 145 public health policy for nursing homes, systematically returning negative results. Ten 146 . 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. 

Analysis of pre-vaccination blood specimens revealed the presence of SARS-CoV-2 S-175 reactive IFN-γ CD8 + and CD4 + T cells in 18 (36.7%) and 30 (61.2%) of 49 naïve NHR, 176 and in 6 (60%) and 8 (80%) out of 10 infected NHR; these figures were substantially 177 lower in healthy controls (17.6% for CD8 + and 29% for CD4 + T cells) ( Table 2) . 178

Following the second vaccine dose, all control subjects had detectable SARS-CoV-2 S-179 reactive IFN-γ CD4 + T cells and 88% had both IFN-γ CD4 + and CD8 + T cells. 180

Conversely, the percentage of NHR exhibiting detectable SARS-CoV-2 IFN-γ CD8 + or 181 CD4 + T cell responses (or both), independently of their baseline SARS-CoV-2 infection 182 status, dropped consistently after vaccination (except for CD8 + T cells in NHR without 183 prior infection), as shown in Table 2 . Both loss and de novo acquisition of detectable 184 SARS-CoV-2 IFN-γ CD8 + or CD4 + T-cell responses were observed in some 185 individuals, particularly in CD8 + T cells. Overall, the magnitude of SARS-CoV-2 IFN-γ 186 CD8 + or CD4 + T cell responses in NHR, irrespective of their SARS-CoV.2 infection 187 status, decreased consistently in post-vaccination specimens, as can be seen in Table 3 . 188

The opposite was observed for healthy controls. The same differential kinetics pattern 189 between NHR and controls was noticed when individuals with detectable T cell 190 responses at baseline were analyzed separately ( Figure 2 ). 191

Overall, the rate and magnitude of detectable SARS-CoV-2 S-reactive IFN-γ CD8 + and 192 CD4 + T cell responses following vaccination were comparable in NHR with or without 193 comorbidities (Supplementary Table 1) . 194

. 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. Here, we quantified total antibodies binding SARS-CoV-2 RBD by means of an ECLIA 208 normalized to the first WHO international standard [9], which strongly correlate with 209 neutralizing antibody titers [12, 13] . In turn, SARS-CoV-2-S-reactive IFN-γ-producing 210 CD8 + and CD4 + T cell were enumerated using a whole blood flow cytometry assay 211

[10,11] at a median of 2-3 weeks after the second vaccine dose. Most NHR recruited 212 (median age, 87.5 years) had one or more comorbidities (84%), and were either with or 213 without a SARS-CoV-2 diagnosis by serological and molecular assay prior to 214 vaccination. The main findings of the study are summarized as follows. 215

First, overall, the SARS-CoV-2-S seroconversion rate was similar in NHR (95.2%) and 216 controls (94.4%), with no significant differences in median antibody levels across 217 groups. To rule out the presence of antibodies targeting epitopes outside RBD in plasma 218 specimens from NHR and controls testing negative, these were run with a SARS-CoV-2 219 TrimericS IgG assay, which also returned negative results. Second, a dramatic booster 220 . 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. prior SARS-CoV-2 infection displaying T cell responses at baseline (60% for CD4 + T 243 cells). Nevertheless, we cannot rule out that some of the current study participants, 244 NHR in particular, could have been asymptomatically infected and failed to mount 245 . 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 April 22, 2021.

[17]. This is plausible, as several SARS-CoV-2 outbreaks were declared in both NH 247 during 2020. Likewise, healthy controls in this study were laboratory employees or staff 248 at the Microbiology unit, and could have been exposed and infected. The current study has several limitations that must be underlined. Firstly, the number of 271 participants was relatively limited; second, the possibility that NHR displayed SARS-272

CoV-2-S-reactive T cells with functional specificities other than IFN-γ production was 273 not explored. Additionally, a whole-blood flow cytometry assay was used to assess T-274 cell immunity: it is uncertain whether employing isolated peripheral blood mononuclear 275 cells instead would increase sensitivity. Finally, no attempt was made to differentiate 276 between truly SARS-CoV-2-specific and cross-reactive T cells, which can be 277 accomplished according to recent reports, although this need further validation [20, 21] . 278

In summary, we were able to document robust SARS-CoV-2-S antibody responses 279 equivalent to those of healthy controls in NHR following complete vaccination, 280

irrespective of SARS-CoV-2 infection status and presence or absence of comorbidities. 281

Nevertheless, our data point to differential vaccine effectivity between NHR and 282 controls in terms of eliciting SARS-CoV-2 IFN-γ T-cell responses. In this context, the 283 potential detrimental effect of pre-existing bona fide or cross-reactive SARS-CoV-2 284 immunity seen in NHR merits further investigation. 285

We are grateful to all personnel who work at NHR affiliated to the Health Department 

This work received no public or private funds. 294 . 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 April 22, 2021. CoV-2 antibody. WHO/BS/2020.2403, December 10, 2020. 327 two rapid high-throughput automated electrochemiluminescence immunoassays 338 . 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) . 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 preprint this version posted April 22, 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 preprint this version posted April 22, 2021. ; https://doi.org/10.1101/2021.04.19.21255723 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. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 22, 2021. ; https://doi.org/10.1101/2021.04.19.21255723 doi: medRxiv preprint Table 3 . 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)

The copyright holder for this preprint this version posted April 22, 2021. ; https://doi.org/10.1101/2021.04.19.21255723 doi: medRxiv preprint

COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. 304 targeting total antibodies to the SARS-CoV-2 nucleoprotein and spike protein receptor 339 binding domain

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on 342 T cell immunity in naïve and COVID-19 recovered individuals

Poor antigen-specific responses to the second BNT162b2 mRNA vaccine dose 346 in SARS-CoV-2-experienced individuals medRxiv

T cell 349 response to SARS-CoV-2 infection in humans: A systematic review

The authors declare no conflicts of interest. 296