key: cord-0302682-j8gvgev5
authors: Müller, M.; Volzke, J.; Subin, B.; Müller, S.; Sombetzki, M.; Reisinger, E. C.; Müller-Hilke, B.
title: Single-Dose SARS-CoV-2 Vaccination With BNT162b2 and AZD1222 Induce Disparate Th1 Responses and IgA Production
date: 2021-09-21
journal: nan
DOI: 10.1101/2021.09.17.21263726
sha: b884da62029ec7b8e1bf1e8246023a0cad470f80
doc_id: 302682
cord_uid: j8gvgev5

While vaccination programs against SARS-CoV-2 are globally ongoing, disparate strategies for the deployment of spike antigen show varying effectiveness. In order to explore this phenomenon, we sought to compare the early immune responses against AZD1222 and BNT162b2. SARS-CoV-2 seronegative participants received a single dose of either vaccine and were analyzed for immune cell, effector T cell and antibody dynamics. AZD1222 induced transient leukopenia and major changes among innate and adaptive subpopulations. Both vaccines induced spike protein specific effector T cells which were dominated by Th1 responses following AZD1222 vaccination. A significant reduction of anti-inflammatory T cells upon re-stimulation was also restricted to AZD1222 vaccinees. While IgM and IgG were the dominant isotypes elicited by AZD1222, BNT162b2 led to a significant production of IgG and IgA. Our results suggest that the strategy for spike antigen delivery impacts on how and to what extent immune priming against the main SARS-CoV-2 antigen proceeds.

dose of either vaccine and were analyzed for immune cell, effector T cell and antibody 23 dynamics. AZD1222 induced transient leukopenia and major changes among innate and 24 adaptive subpopulations. Both vaccines induced spike protein specific effector T cells 25 which were dominated by Th1 responses following AZD1222 vaccination. A significant 26 reduction of anti-inflammatory T cells upon re-stimulation was also restricted to 27 AZD1222 vaccinees. While IgM and IgG were the dominant isotypes elicited by 28 AZD1222, BNT162b2 led to a significant production of IgG and IgA. Our results suggest 29 that the strategy for spike antigen delivery impacts on how and to what extent immune 30 priming against the main SARS-CoV-2 antigen proceeds. 31 . CC-BY-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 (which was not certified by peer review) preprint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 21, 2021.

nanoparticles 7,8 . 48 Complete vaccination with either of the vaccines, which includes two doses at varying 49 intervals, was shown to efficiently protect from symptomatic COVID-19 9,10 . Although 50 early data hint at similar efficiencies after a single dose of either vaccine, booster 51 immunization with BNT162b2 achieved somewhat higher rates of thwarting viral 52 breakthrough [9] [10] [11] [12] [13] . With the emergence of SARS-CoV-2 variants that accumulate 53 mutations in the spike glycoprotein [14] [15] [16] , the discrepancies between both vaccines grew 54 . CC-BY-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 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 To further substantiate the time lines of early immune events following AZD1222 and 104

BNT162b2 vaccination, we analyzed the major immune cell populations in more detail. 105 E.g., live monocytes were identified by their sideward scatter properties before 106 alterations of CD14 and CD16 expression patterns were analyzed for the various time 107 points. Fig. 2A illustrates the time line for one participant receiving AZD1222. Fig. 2B  108 documents a transient yet statistically significant increase in CD14 + CD16 + pro-109 inflammatory monocytes on day 2 for the AZD1222 group (p < 0.0001). In contrast, there 110 . CC-BY-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 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 7 was no alteration among the proportions of pro-inflammatory monocytes following 111 vaccination with BNT162b2 (Fig. 2B) . 112 Figs. 1 and 2) . In 116 detail, CD177 -CD11b + among CD14 + CD16granulocytes were significantly elevated on 117 days 2 and 13 following vaccination ( Supplementary Fig. 1 ). By day 20, this 118 subpopulation was still increased to some extent however, due to high variance, the 119 difference to baseline did not reach statistical significance. Interestingly, dynamics of 120 CD177 -CD11bamong CD14 + CD16 + granulocytes followed an opposing trend with 121 . CC-BY-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 . CC-BY-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 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 9 patterns for CD27 and CD127 on day 20 after AZD1222 administration. We thus 145 discovered that CD4 + CD127 -CD27 + effector memory T cells re-expressing RA were 146 enriched towards the end of the observation period ( Supplementary Fig. 4B ). Taken 147 together, we here demonstrated that significant changes among subpopulations of B-148

and T-lymphocytes were observed after vaccination with AZD1222 only. 149 So far we have shown that a single dose of AZD1222 was capable of significantly 154 modifying immune cell compositions. However, we assumed that both vaccines would 155 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint on a small scale induce a specific cellular immune response towards the SARS-CoV-2 156 spike protein, which would become detectable upon re-stimulation with the antigen. We 157 therefore used both, recombinant spike protein and BNT162b2 vaccine and aimed to 158 investigate cytokine profiles as well as the expression of inducible activation markers. In 159 case of the recombinant spike protein, we expected it to be taken up by antigen 160 presenting cells (APCs). Upon processing the protein in lysosomes, respective peptides 161 would predominantly become displayed on human leukocyte antigen (HLA) class II 162 molecules and thus, would be ready to activate spike protein specific T helper cells. By 163 employing the mRNA vaccine, we anticipated its cellular uptake, translation into protein 164 and then both, secretion for uptake by APCs and class II presentation as well as 165 processing the protein for presentation via HLA class I molecules and thereby re-166 stimulating cytotoxic T cells 20 . 167

In order to establish a working protocol, we used PBMCs from fully vaccinated or 168 COVID-19 convalescent blood donors and investigated the expression of the activation 169 marker CD137 on unstimulated cells compared to cells challenged with either the 170 recombinant spike protein or BNT162b2. Indeed, we found a significant activation of 171 CD4 + but not CD8 + cells after providing the recombinant spike protein (Supplementary 172 . CC-BY-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 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 of ELISPOTs, Fig. 4B summarizes all results and indeed shows a significant increase in 179

IFNγ-positive cells after BNT162b2 vaccination (p=0.0059). There was also a trend 180 towards increased IFNγ-positive cells after AZD1222 vaccination however, this did not 181 reach statistical significance (p=0.0968). 182 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint significantly increased percentages of FasL + CD8 + cells after in vitro re-stimulation (Fig. 193 5B). We further detected a significant increase in IFNγ-producing CD8 + T cells from 194 AZD1222-but not from BNT162b2-vaccinated donors (p = 0.0325 vs 0.1514, Fig. 5C ). 195

Finally, re-stimulation with spike mRNA decreased IL-2 + IL-10 + co-expressing regulatory 196 CD8 + cells for both AZD1222 and BNT162b2, the latter short of reaching statistical 197 significance (p = 0.0058 vs 0.0768, Fig. 5D ). 198 (CD4 + CD25 + CD137 + ) were also detected for both vaccine regimens after in vitro re-202 stimulation (Fig. 6A ). In contrast, an increase in pro-inflammatory helper T cells 203 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint 13 producing TNFα was restricted to re-stimulated cultures from day 20 AZD1222 donors 204 ( Fig. 6B ). Of note, neither vaccination induced the expansion of spike specific type 2 205 helper T cells as demonstrated by a lack of inducible IL-4 production by CD4 + cells after 206 re-stimulation (Fig. 6C ). Similar to regulatory CD8 + cells and again restricted to the 207 AZD1222 group, re-stimulation with spike mRNA statistically significantly reduced anti-208 inflammatory IL-2 and IL-10 co-production in a subset of CD4 + cells (Fig. 6D ). 209 In summary, these results demonstrate that single doses of either AZD1222 or 212

BNT162b2 induced the polarization of spike specific CD4 + and CD8 + effector T cells. 213

More pronounced changes were again observed after vaccination with AZD1222 that 214 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint included reduced proportions of IL-2 and IL-10 co-expressing CD4 + and CD8 + cells alike, 215 combined with increased IFNγ-producing CD8 + and TNFα-producing CD4 + cells. 216 2.5 BNT162b2 vaccination led to significantly more spike protein-specific plasma 217

Even though both vaccines led to significant adaptive immune activation, alterations to 219 inducible effector functions followed distinct patterns for AZD1222 and BNT162b2, 220 respectively. We therefore sought to investigate whether these differences resulted in 221 the production of diverse collections of immunoglobulin isotypes. To that extent, spike 222 protein specific IgM, IgG and IgA were assessed for all time points via ELISA. As shown 223 in Fig. 7 , either vaccine induced the production of detectable amounts of antibodies as 224 early as day 13. However, significant differences emerged between AZD1222 and 225

BNT162b2 vaccination concerning the distribution and amounts of spike specific 226 antibody isotypes. In detail, AZD1222 predominantly induced IgM and IgG, while IgA 227 was virtually absent, even at day 20 after vaccination. In contrast, although not 228 significantly different from AZD1222, BNT162b2 elicited little IgM. There were though 229 increased IgG and IgA titers in BNT162b2 vaccinated subjects and these differences 230 reached significances on day 20 for IgG and already on day 13 for IgA ( Fig. 7) CC-BY-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 it is the adenoviral vector rather than the encoded spike protein that elicits a significant 259 immune cell response in the periphery. 260

By comparison, the immune cell response to BNT162b2 vaccination appeared rather 261 bland and thus corroborated the observation that this vaccine is globally well tolerated 262 among first dose recipients 9 . Indeed, the BNT162b2 encoded mRNA bears an m1Ψ 263 modification which attenuates innate immune sensing 32 . We therefore assume that, the 264 relatively small enrichment of peripheral plasmablasts after BNT162b2 vaccination 265 resulted from an adaptive response towards the SARS-CoV-2 spike protein whereas the 266 larger enrichment observed with AZD1222 likely reflects a combined response towards 267 the spike protein and the adenoviral vector. 268

When comparing the specific antibody responses against the spike protein, we did not 269 observe any quantitative differences in total Ig between both vaccines. However, the 270 isotypes produced were significantly different with AZD1222 inducing primarily IgM and 271

IgG compared to predominantly IgG and IgA by BNT162b2. Even though we cannot yet 272 predict whether this trend will be continued beyond the first three weeks after 273 vaccination, a pronounced IgA response following BNT162b2 vaccination may explain 274 its superior effectiveness in preventing symptomatic COVID-19 after both, infection with 275 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint wild type SARS-CoV-2 and its variants 15, 17, 19, 33 . Optimizing existing vaccines might 276 therefore aim at alternative antigen delivery, e.g. towards mucosal sites, in order to not 277 only support IgA production but also tissue-resident effector cells which will help contain 278 viral loads at the nasopharyngeal entry sites 34,35 . 279 Optimizing vaccines may also aim at modifying potential bystander effects. When 280 analyzing the spike protein-specific T cell responses, we observed that both vaccines 281 elicited functional immune responses. Both vaccination regimes expanded effector cells 282 to a comparable degree as documented by significant increases in activated CD25 and 283 CD137 co-expressing CD4 + and CD8 + T lymphocytes as well as FasL expressing CD8 + 284 cells upon re-challenge. However, when looking at intracellular cytokine production, 285 AZD1222 induced a prominent Th1 response as illustrated by significant increases in 286

IFNγ and TNFα, respectively. Because adenoviral vectors have previously been shown 287 to facilitate strong cellular immunity towards the delivered antigen and drive the 288 expansion of type 1 helper (Th1) cells 6 , we believe that this Th1 reaction towards the 289 adenoviral vector exerted some bystander effect on the response against the spike 290 protein. However, an inordinate Th1 response may foster a cytokine layout that is hardly 291 supportive of class-switch recombination towards IgA 36 . On the other hand, we 292 assessed neither IL-5 nor TGFβ and are therefore not yet able to verify whether 293 BNT162b2 indeed induced more IgA promoting cytokines. 294

Of note, our ELISPOT experiments addressing spike protein-specific Th1 cells before 295 and after vaccination found an even stronger induction of IFNγ-producing T helper cells 296

among BNT162b2 compared to AZD1222 vaccinees. Even though these results 297 seemingly contradict the intracellular cytokine readout of stimulated vs unstimulated day 298 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint 18 20 T cells, they show that both vaccines induced Th1 responses. In addition, we found a 299 reduction of IL-2 and IL-10 co-producing CD4 + and CD8 + T cells upon in vitro re-300 stimulation which however, only reached significance in the case of AZD1222 301 vaccination. IL-10 is a hallmark of regulatory T cells and exerts anti-inflammatory effects 302 via suppressing not only effector T cells, but also antigen presentation and the secretion 303 of inflammatory cytokines by APCs 37-39 . We like to speculate that this significant 304 reduction of IL-10 expression is not restricted to in vitro re-challenge but may also occur 305 after booster immunization and perpetuate a Th1 response that impedes an IgA 306 promoting cytokine milieu. 307

This study has a few limitations, among them the small samples sizes. Nonetheless, our 308 results depict for both vaccines significantly disparate effects on the peripheral immune 309 layout and on the regulation of T cell effector molecules. Another limitation is the lack of 310 assaying neutralizing SARS-CoV-2 specific antibodies. However, previous studies have 311 already demonstrated that total spike-binding Ig titers strongly correlate with the 312 amounts of neutralizing antibodies and are therefore a suitable measure for humoral 313 protection from SARS-CoV-2 infection 5,40-42 . In summary, we consider the description of 314 disparate vaccine effects on the immediate immune response the strength of our study 315 and we believe that our results will be of use for further optimization of vaccination 316 strategies. 317

Study participants were recruited from the coordination center for clinical studies at the 320 Rostock University Medical Center. Individuals with a study-independent appointment at 321 . CC-BY-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. per sample) and concatenated data sets was performed using the FlowJo plugin for the 361 algorithm "uniform manifold approximation and projection" (UMAP) 43 . 362

PBMCs from d0 and d20 were thawed, centrifuged and suspended in complete RPMI 364 medium containing 10% FCS, 1% Penicillin/Streptomycin, 2 mM L-Glutamine (Thermo 365 Fisher), 10 mM HEPES and 1 mM sodium pyruvate (PAN-Biotech, Aidenbach, 366 Germany). Cell counts were determined cytometrically on the Cytek ® Aurora (Cytek 367 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint 21 Biosciences) using DAPI (Biolegend) as a live/dead discriminator. Five hundred 368 thousand PBMCs were pipetted into a 96-well U-bottom plate and centrifuged for 5 min 369 at 4 °C and 400 × g. Subsequently, supernatants were removed by carefully blotting the 370 plate on a paper tissue. Cells were then suspended in 36 µL complete RPMI medium 371 containing 0.2 µg of the SARS-CoV-2 trimeric spike protein (R&D Systems, 372 Minneapolis/MN, United States). Afterwards, PBMCs were transferred into a 96-well 373 enzyme-linked immune absorbent spot (ELISpot) assay plate coated with capture 374 antibodies specific for human interferon (IFN)γ (R&D Systems). After incubating the cells 375

for 30 min at 37 °C, 164 µL of complete RPMI medium was added to all wells followed 376 by 24 h incubation at 37 °C in a CO 2 incubator (Binder, Tuttlingen, Germany). The 377

ELISpot assay was then performed according to the manufacturer's guidelines. The 378 numbers of IFNγ producing cells were determined by automated counting using the 379 ImmunoSpot ® analyzer running on the ImmunoSpot ® Software version 5.0.9.15 (CTL 380 Europe, Bonn, Germany). The counts of IFNγ-positive cells were normalized to their 381 respective paired sample from d0. 382

For the establishment of T cell re-stimulation and intracellular cytokine staining assays, 384 blood was collected from six fully vaccinated subjects and one patient who had 385 recovered from COVID-19 at least 2 weeks after the second vaccination and infection, 386 respectively. PBMCs were isolated and frozen until further use in the same fashion as 387 described above. For assaying the vaccinated study participants, day 20 PBMCs were 388 used. Upon thawing, PBMCs were counted as described above and aliquots of 0.8 389 million were seeded into single wells of 96-well U-bottom plates. Every sample was 390 . CC-BY-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 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 20. Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics -developing a new 500 class of drugs. Nat. Rev. Drug Discov. 13, 759-780 (2014) . Immunol. 199, 9-16 (2017) . or BNT162b2 (n = 18, right). All FACS analyses were on CD19 + CD45RA + B cells. p-581 values resulting from one-way ANOVAs were < 0.0001 for both AZD1222 analyses. p-582 values resulting from Kruskal-Wallis and Dunn´s multiple comparisons tests were 0.0008 583 . CC-BY-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 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint 31 for the comparison of plasmablast and 0.6998 for the comparison of late memory B cells 584 for the BNT162b2 analyses. *p < 0.05, **p < 0.01, ***p < 0.0001 585 producers were increased (C) and IL-2 and IL-10 co-expressing cells were significantly 605 reduced among AZD1222 vaccinees only (D). *p < 0.05, **p < 0.01 resulting from paired 606 . CC-BY-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 21, 2021. ; https://doi.org/10. 1101 /2021 

Rostock University Medical Center) for her help during flow cytometry 634 panel design and setup. Organizational and documentary support was provided by the 635 coordination center for clinical studies and Manja Ehmke (Division of Tropical Medicine 636 and Infectious Diseases

Blood samples were also collected by Anxhela Muca and Stefanie Brigitte 638

This study was financially supported by the Federal State of 640

Pomerania via the "Sondervermögen des MV Schutzfonds Säule 641

We thank all our participants for taking part in this study and providing blood samples. 632The authors want to particularly thank Wendy Bergmann (Core Facility for Cell Sorting 633

stimulated at least in duplicates. After centrifugation, cells were re-suspended and 391 stimulated in a total volume of 36 µL complete RPMI medium with either 1 µg of the 392 BNT162b2 vaccine (BioNTech, Mainz, Germany) or with 0.2 µg of the SARS-CoV-2 393 trimeric spike protein or left without any stimulation. PMA (10 ng/ mL) and Ionomycin (1 394 µg/mL) stimulated samples were processed in parallel as positive controls. After adding 395 164 µL of complete RPMI medium, cells were incubated for 20 h under 5 % CO 2 396 atmosphere at 37°C. One microgram of Brefeldin A (Sigma-Aldrich) was added 397 thereafter followed by incubation for another 4 h. Successive incubation steps were 398 performed in the dark. Duplicate samples were pooled, washed in PBS (Thermo Fisher), 399 suspended in PBS containing 2,000-fold diluted ZombieNIR dye (Biolegend) and 400 incubated for 20 min at room temperature. Thereafter, cells were washed and 401 suspended in autoMACS ® Running Buffer (RB, Miltenyi Biotec, Bergisch Gladbach, 402 Germany Biolegend) and 0.13 µg TNFα:PE/Cy7 (Mab11, Thermo Fisher) were added and 416 incubated for 30 min at room temperature. Finally, cells were washed twice in RB. Data 417 acquisition and analysis of expression data was performed as described above. The 418 gating scheme for the intracellular cytokine staining assay is shown in Supplementary 419 Fig. 7 . 420

Plasma samples from all time points were thawed on ice and centrifuged at 10,000 × g 422 in order to remove precipitates. For the detection of SARS-CoV-2 trimeric spike protein 423 specific immunoglobulin (Ig)G and IgA levels, plasma was diluted 101-fold. The enzyme-424 linked immunosorbent assays (ELISA) for these isotypes was conducted after the 425 manufacturer's specifications (Euroimmun, Lübeck, Germany). In order to determine IgM 426 levels, plasma samples were diluted 1,000-fold and the ELISA was performed to the 427 manufacturer's instructions (Thermo Fisher). The absorbance was detected at 450 nm 428 (A450) on the Infinite ® 200 automated plate reader (Tecan, Männedorf, Switzerland). 429Absorbance readouts were normalized to calibrator values and were reported as 430 arbitrary units. According to the manufacturer's guidelines, calibrated sample values 431 between 0.8 and 1.1 were considered borderline and above 1.1 were considered clearly 432 positive. Samples from individuals with arbitrary unit values of less than 0.8 were 433 considered non-responders. 434

Data analyses were performed using R (version 3.5.1) and InStat version 3.10 436 (GraphPad, San Diego/CA, United States). Data sets were evaluated for Gaussian 437 . CC-BY-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 . CC-BY-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) preprintThe copyright holder for this this version posted September 21, 2021. ; https://doi.org/10.1101/2021.09.17.21263726 doi: medRxiv preprint t-tests. # p < 0.05, ### p < 0.001 resulting from Wilcoxon signed rank tests for matched 607 pairs. 608 Sample sizes were n = 18 for AZD1222 and n = 18 for BNT162b2, respectively. p-values 625 resulting from one-way ANOVA and Tukey-Kramer multiple comparisons tests were < 626 0.0001 for all three isotype analyses. ***p < 0.001 627 . CC-BY-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 . CC-BY-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) preprintThe copyright holder for this this version posted September 21, 2021. ; https://doi.org/10. 1101 /2021