key: cord-0715568-f8yl4uak
authors: Tauzin, Alexandra; Nayrac, Manon; Benlarbi, Mehdi; Gong, Shang Yu; Gasser, Romain; Beaudoin-Bussières, Guillaume; Brassard, Nathalie; Laumaea, Annemarie; Vézina, Dani; Prévost, Jérémie; Anand, Sai Priya; Bourassa, Catherine; Gendron-Lepage, Gabrielle; Medjahed, Halima; Goyette, Guillaume; Niessl, Julia; Tastet, Olivier; Gokool, Laurie; Morrisseau, Chantal; Arlotto, Pascale; Stamatatos, Leonidas; McGuire, Andrew T.; Larochelle, Catherine; Uchil, Pradeep; Lu, Maolin; Mothes, Walther; De Serres, Gaston; Moreira, Sandrine; Roger, Michel; Richard, Jonathan; Martel-Laferrière, Valérie; Duerr, Ralf; Tremblay, Cécile; Kaufmann, Daniel E.; Finzi, Andrés
title: A single dose of the SARS-CoV-2 vaccine BNT162b2 elicits Fc-mediated antibody effector functions and T-cell responses
date: 2021-06-04
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2021.06.001
sha: 0cdaf226ccd8aa41faf8cd53acf192f3f1c3e6d4
doc_id: 715568
cord_uid: f8yl4uak

While the standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart, some public health authorities are spacing these doses, raising concerns about efficacy. However, data indicate that a single dose can be up to 90% effective starting 14 days post administration. To assess the mechanisms contributing to protection, we analyzed humoral and T cell responses three weeks after a single BNT162b2 dose. We observed weak neutralizing activity elicited in SARS-CoV-2 naïve individuals but strong anti-receptor binding domain and Spike antibodies with Fc-mediated effector functions and cellular CD4+ T cell responses. In previously-infected individuals, a single dose boosted all humoral and T-cell responses, with strong correlations between T helper and antibody immunity. Our results highlight the potential role of Fc-mediated effector functions and T-cell responses in vaccine efficacy. They also provide support for spacing doses to vaccinate more individuals in conditions of vaccine scarcity.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the etiological 57 agent of the Coronavirus disease 2019 (COVID- 19) , responsible for the current pandemic that 58 infected over 155 million people and led to more than 3.24 million deaths worldwide (Dong et infection or vaccination (Prévost and Finzi, 2021) . 77

The D614G S mutation appeared very early in the pandemic and is now highly prevalent 78 individuals, we decided to measure Fc-mediated effector functions that were also shown to play 226 an important role against SARS-CoV-2 infection (Chan et al., 2020a; Schäfer et al., 2020; Zohar 227 et al., 2020) . In agreement with the lack of S-specific Abs, SARS-CoV-2 naïve individuals did 228 not have detectable ADCC activity prior to vaccination ( Figure 3B ). The first dose of the vaccine 229 induced a significant increase in ADCC activity, except for one sample, corresponding to the 230 donor who had not developed anti-S Abs. We noted that ADCC activity in vaccinated naïve 231 individuals achieved comparable levels to those of PI individuals before vaccination. Vaccination 232 of this group significantly boosted the ADCC activity ( Figure 3B ). Based on these results it is 233 tempting to speculate that the generation of Abs with Fc-mediated effector functions, but with 234 low neutralizing activity, might be sufficient to provide a certain level of protection. 235

We examined whether prior SARS-CoV-2 infection alters the CD4 + and CD8 + T cell 239 responses to vaccination. To measure SARS-CoV-2-S-specific T cells in the two cohorts of 240 naïve persons and PI individuals, we utilized two complementary methodologies, T cell receptor 241 (TCR) dependent activation induced marker (AIM) assays and intracellular cytokine staining 242 (ICS). We performed the cytokine-independent AIM assays as previously described (Reiss et 243 al., 2017) with some modifications. We stimulated PBMC for 15h with an overlapping peptide 244 pool spanning the S coding sequence and measured upregulation of the markers CD69, 245 CD40L, 4-1BB and OX-40 upon stimulation. We used an AND/OR Boolean combination gating 246 strategy to identify antigen-specific T cell responses ( Figure S3A ) (Niessl et al., 2020a) . We 247 examined three populations of SARS-CoV-2-S-specific AIM + T cells: (i) AIM + total CD4 + T cells 248 CoV-2 infection (Dan et al., 2021) . 252

After vaccination, we observed a significant increase in total S-specific AIM + CD4 + T cell 253 responses in both groups of participants ( Figure 4A ). We observed similar patterns with S-254 specific AIM + cTfh and S-specific AIM + CD8 + T cell responses which significantly increased after 255 vaccination in both groups ( Figure 4B and C) and stronger in the PI group compared to the 256 naïve group. However, the frequencies range of the AIM + CD8 + responses remain significantly 257 lower than that of AIM + CD4 + T cell responses regardless of the time point studied ( Figure S3C) . we measured by ICS the cytokines secreted by CD4 + and CD8 + T cells in response to a 6h 261 stimulation of PBMC with a S peptide pool. T cells were analyzed for expression of CD40L, 262

CD107a, interferon (IFN)-γ, Interleukin (IL)-2, IL-10, IL-17A and tumor necrosis factor (TNF)-α. 263 IL-17A expression was undetectable for most participants in both CD4 + and CD8 + T cell 264 subsets, and CD40L negligible in CD8 + T cells. These subsets were thus not pursued, whereas 265 all other functions were included in further analysis. We defined frequencies of cytokine + CD4 + 266 and CD8 + T cells as percentage of cells positive for one or more cytokines or functional markers 267 between both assays ( Figure S3F ). After vaccination, the ICS + CD4 + T cell responses were 271 significantly increased in the two groups ( Figure 4D ) with stronger responses in PI group 272 compared to the naïve group. ICS + CD8 + T cell responses was also significantly increased in PI 273 individuals ( Figure 4E ) however there were only trends for an increase after this single dose of 274 vaccine in the naïve cohort. 275

To qualitatively assess S-specific T cells in naïve and PI groups for polyfunctional 276 responses after vaccination, we performed coexpression analysis using Boolean gating and 277 examined each combinations of function ( Figure 4F and G). In comparison to naïve individuals, 278 dominant S-specific CD4 + T subsets that were preferentially increased by vaccination in PI 279 included S-specific CD4 + T cells coexpressing CD40L, IFN-γ alone or in combination with other 280 functions (TNF-α, CD107a, IL-2). The frequency of S-specific CD8 + T cells expressing IFN-γ 281 alone or combined with CD107a was also increased in PI compared to naïve participants. Most protective Ab responses are dependent on CD4 + T cell help, which is critical for B 292 cell expansion, affinity maturation and isotype switching. Therefore, we assessed whether pre-293 existing SARS-CoV-2-S-specific CD4 + T cells and cTfh responses were predictive of higher Ab 294 titers and Ab functions, as measured by neutralization capacity and ADCC after vaccination, 295

irrespective of prior infection status ( Figure 5A ). We found that correlations between the 296 function-agnostic AIM + CD4 + T cell measurements and Ab responses were generally stronger 297 than between ICS + CD4 + T cell responses and serological measurements ( Figure 5A ). Notably, 298

the Ig subsets measured after vaccination in the plasma of each participant showed significant 299 positive correlations between pre-existing S-specific CD4 + T cell and cTfh responses on the one 300 hand, and anti-S IgA and IgG post-vaccination on the other hand ( Figure 5C , D, F and G). In 301 contrast, we observed no significant correlations between total S-specific CD4 + T cell responses 302 and anti-S IgM levels ( Figure 5B ) and between S-specific cTfh responses and anti-S IgM levels 303 ( Figure 5E ). At the functional level, we observed significant correlations between all the pre-304 vaccination AIM + S-specific memory CD4 + T cells and cTfh with ADCC and neutralization 305 capacity post-immunization ( Figure 5A) . While the duration of a protective immune response elicited by a single dose of mRNA vaccines 450 is unknown, given that memory is a core function of the immune system it is unlikely to decline 451 within these intervals. Nevertheless, addressing this question will be very important as the larger 452 the interval between doses the easier it will be to maximize the protection globally given the 453 limited vaccine supply worldwide. 454 455

Our results suggest that different antibody functions, beyond neutralization, could play a role in 457 vaccine efficacy three weeks after a single dose of mRNA BNT162b2 vaccine in SARS-CoV-2 458 naïve and previously infected individuals. A limitation of the study is that while we have shown 459 that these humoral responses correlate with CD4 T cell responses, we don't know in which 460 extent CD4 and CD8 T cell responses directly contribute to vaccine efficacy, besides their 461 helper role for antibody immunity. Additional studies will be required to understand the role of T 

and AIM + cTfh responses (E). Positive correlations between IgA against S and AIM + CD4 + T 568 Further information and requests for resources and reagents should be directed to and will be 591 fulfilled by the lead contact, Andrés Finzi (andres.finzi@umontreal.ca) 592 593

All unique reagents generated during this study are available from the Lead contact without 595 restriction. 596 597

The published article includes all datasets generated and analyzed for this study. Further 599 information and requests for resources and reagents should be directed to and will be fulfilled by 600 No specific criteria such as number of patients (sample size), clinical or demographic were used 615 for inclusion, beyond PCR confirmed SARS-CoV-2 infection in adults. Information related to 616 their average age and gender is reported in Table 1 . Table S2 . Table S3 for Ab staining panel). Cells were fixed using 2% paraformaldehyde before 788 acquisition on Symphony cytometer (BD Biosciences). Analyses were performed using FlowJo 

Symbols represent biologically independent samples from SARS-CoV-2 naïve individuals 794 (n=24) and SARS-CoV-2 PI individuals (n=24). Lines connect data from the same donor. 795

Statistics were analyzed using GraphPad Prism version 8.0.1 (GraphPad, San Diego, CA). 796

Every dataset was tested for statistical normality and this information was used to apply the 797 appropriate (parametric or nonparametric) statistical test. Differences in responses for the same 798 patient before and after vaccination were performed using Wilcoxon matched pair tests. 799

Differences in responses between naïve and PI individuals were measured by Mann-Whitney 800 tests. Differences in responses against the SARS-CoV-2 variants for the same patient were 801 measured by Friedman test. P values < 0.05 were considered significant; significance values 802 are indicated as * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001. Line charts were 803 created with Prism 8.4.3 using normalized data and Akima spline interpolation. For correlations, 804

Spearman's R correlation coefficient was applied. Statistical tests were two-sided and p<0.05 805 was considered significant. 806 807

Normalized heatmaps were generated using the complexheatmap, tidyverse, and viridis 809 packages in R and RStudio (R; R studio). Normalizations were done per "Analysis group", e.g., 

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