key: cord-0707630-izoqsa7g
authors: Sahin, U.; Muik, A.; Vogler, I.; Derhovanessian, E.; Kranz, L. M.; Vormehr, M.; Quandt, J.; Bidmon, N.; Ulges, A.; Baum, A.; Pascal, K.; Maurus, D.; Brachtendorf, S.; Loerks, V. L.; Sikorski, J.; Koch, P.; Hilker, R.; Becker, D.; Eller, A.-K.; Gruetzner, J.; Tonigold, M.; Boesler, C.; Rosenbaum, C.; Heesen, L.; Kuehnle, M.-C.; Poran, A.; Dong, J. Z.; Luxemburger, U.; Kemmer-Brueck, A.; Langer, D.; Bexon, M.; Bolte, S.; Palanche, T.; Schultz, A.; Baumann, S.; Mahiny, A. J.; Boros, G.; Reinholz, J.; Szabo, G. T.; Kariko, K.; Shi, P.-Y.; Fontes-Garfias, C.; Perez, J. L.; Cutler, M.; Cooper, D.
title: BNT162b2 induces SARS-CoV-2-neutralising antibodies and T cells in humans
date: 2020-12-11
journal: nan
DOI: 10.1101/2020.12.09.20245175
sha: e08df4020cee592ca65f5432d23b052785ee86fa
doc_id: 707630
cord_uid: izoqsa7g

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy to prevent coronavirus disease 2019 (COVID-19). Recently, we reported preliminary BNT162b2 safety and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 vaccine trial. We present here antibody and T cell responses from a second, non-randomized open-label phase 1/2 trial in healthy adults, 19-55 years of age, after BNT162b2 prime/boost vaccination at 1 to 30 g dose levels. BNT162b2 elicited strong antibody responses, with S-binding IgG concentrations above those in a COVID-19 human convalescent sample (HCS) panel. Day 29 (7 days post-boost) SARS-CoV-2 serum 50% neutralising geometric mean titers were 0.3-fold (1 g) to 3.3-fold (30 g) those of the HCS panel. The BNT162b2-elicited sera neutralised pseudoviruses with diverse SARS-CoV-2 S variants. Concurrently, in most participants, S-specific CD8+ and T helper type 1 (TH1) CD4+ T cells had expanded, with a high fraction producing interferon-{gamma} (IFN{gamma}). Using peptide MHC multimers, the epitopes recognised by several BNT162b2-induced CD8+ T cells when presented on frequent MHC alleles were identified. CD8+ T cells were shown to be of the early-differentiated effector-memory phenotype, with single specificities reaching 0.01-3% of circulating CD8+ T cells. In summary, vaccination with BNT162b2 at well tolerated doses elicits a combined adaptive humoral and cellular immune response, which together may contribute to protection against COVID-19.

Participants 19-55 years of age were vaccinated with BNT162b2 in Germany (Extended Data 85 Fig. 1 ). Participants' mean age was 40 years; 56% were female, and all were Caucasian 86 (Extended Data Table 1 ). Twelve participants per dose cohort were assigned to receive a 87 priming dose of 1, 10, 20 or 30 μg on day 1 and a booster dose on day 22 (Extended Data Table  88 2). One individual each in the 1 µg dose cohort and the 10 µg dose cohort discontinued prior to 89 the boost. In each dose group, antibody levels and virus neutralisation titers were assessed in 90 10 to 12 participants per timepoint (up to day 85, 63 days post-boost), and peripheral blood 91 mononuclear cells (PBMCs) from 8 to 10 participants were analysed for cellular immune 92 responses at baseline and day 29 (7 days post-boost) (Extended Data Table 2 ). 93 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 7 both S pool 1 and S pool 2 of SARS-CoV-2. One participant had de novo response only against 168 S pool 2. The remaining three participants had de novo responses against S pool 1 and low 169 numbers of pre-existing S pool 2-reactive CD4 + T cells. In two of these three participants, the 170 pre-existing responses against S pool 2 were amplified by vaccination (from 91 and 188 171 spots/10 6 cells pre-vaccination to 1391 and 965 spots after vaccination, respectively), whereas 172 in one of the three participants, the pre-existing responses against S pool 2 remained stable (53 173 to 140 spots/10 6 cells). In conclusion, these data demonstrate that in 94.1% (32/34) of 174 participants, two doses of BNT162b2 induce poly-epitopic CD4 + T cell responses (de novo or 175 amplified) directed against both N-and C-terminal portions of S and thus against epitopes 176 outside the RBD (Extended Data Fig. 5b) . 177

Although for dose levels ≥10 µg the magnitude of CD4 + T cell responses did not appear to be 178 dose-dependent, it varied between individuals. In the strongest responders, the S-specific CD4 + 179 T cell responses were more than 10-fold of the individual memory responses to common viruses 180 and recall antigens (those from cytomegalovirus, Epstein Barr virus, influenza virus and tetanus 181 toxoid) (Fig. 2b, c) . (Fig. 2b, c) . De novo S -specific CD8 + T cell responses were 186 induced in 33 participants, these were either directed against both (22 participants), or one of 187 the S pools (S pool 1 in ten participants, and S pool 2 in two participants), indicating a 188 preponderance of a poly-epitopic response including non-RBD S-specific T cells (Extended 189 Data Fig. 5b) . In seven participants, pre-existing CD8 + T cell responses to S pool 2 were 190 detected that were not further augmented by vaccination. Six out of these seven participants 191 had a concurrent de novo response to pool 1 of S, which in strength did not differ significantly 192 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 8 from those observed in individuals without pre-existing responses to S pool 2 (Extended Data 193 Fig. 5c ). Of note, the strongest responses (higher than third quartile) against S pool 1 among 194 the 34 participants with detectable CD8 + T cell responses were observed in those without pre-195 existing S pool 2-specific responses. 196

The magnitude of S-specific CD4 + T cell responses correlated positively with S1-binding IgG 197 (Extended Data Fig. 6a) , and, in line with the concept of intramolecular help 26 , also with the 198 strength of S-specific CD8 + T cell responses (Extended Data Fig. 6b ). S-specific CD8 + T cell 199 responses also correlated positively with S1-binding IgG (Extended Data Fig. 6c) , indicating a 200 convergent development of the humoral and cellular adaptive immunity. 201

To assess functionality and polarisation of S-specific T cells, cytokines secreted in response to 203 stimulation with S pool 1, S pool 2 and RBD pool were determined by intracellular staining 204 (ICS) for IFNγ, IL-2 and IL-4 specific responses in pre-and post-vaccination PBMCs of 37 205

BNT162b2-immunised participants (Extended Data Table 2 ). A considerable fraction of 206 vaccine-induced, S-specific CD4 + T cells secreted IFN, IL-2, or both, while T cells secreting 207 the TH2 cytokine IL-4 were barely detectable ( Fig. 3a -c, Extended Data Fig. 5d , e). Vaccine-208 induced S-specific CD8 + T cells secreted predominantly IFNγ and lower levels of IL-2 in 209 response to S pool 1 and S pool 2 stimulation. Fractions of IFNγ + CD8 + T cells specific to S 210 pool 1 constituted up to about 1% of total peripheral blood CD8 + T cells (Fig. 3d ). Of note, 211 several of the analysed participants (n=3 in the 20 µg dose cohort and n=3 in the 30 µg dose 212 cohort) displayed pre-existing S pool 2 specific CD8 + T cell responses, which in 5 out of the 213 6 participants were not further amplified after vaccination. A strong pre-existing S pool 2 214 specific IFNγ + CD4 + T cell response was detectable in one participant (20 µg dose cohort) (Fig.  215 3c). 216 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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In both assay systems, cytokine production of CD4 + as well as CD8 + T cells in response to 217 peptide pools comprising the full SARS-CoV-2 S exceeded the responses against the RBD 218 peptide pool, further confirming the poly-epitopic nature of T cell responses elicited by 219

BNT162b2. The mean fraction of BNT162b2-induced S-specific IFN + or IL-2 + CD4 + and 220 CD8 + T cells within total circulating T cells was higher than that detected in eighteen control 221 subjects who had recovered from COVID-19 (HCS) (Fig. 3c, d) . respectively. This approach identified de novo induced CD8 + T cell reactivities against multiple 229 epitopes for each participant, adding up to a total of eight different epitope/MHC pairs spread 230 across the full length of S (Fig. 4a, c) . The magnitude of epitope-specific T cell responses ranged 231 between 0.01-3.09% of peripheral CD8 + T cells, and the most profound expansion was observed 232 for HLA-A*0201 YLQPRTFLL (3.09% multimer + of CD8 + ), HLA-A*2402 QYIKWPWYI 233

(1.27% multimer + of CD8 + ) and HLA-B*3501 QPTESIVRF (0.17% multimer + of CD8 + ). 234

Whereas the pMHC multimer approach probes a discrete subset of potential reactivities, bulk 235 IFNγ + CD8 + T cell responses against full S determined by ELISpot and ICS are considered to 236 comprehensively capture the full poly-epitopic T cell response. However, comparison of both 237 data sets indicated that a functional T cell assay may underestimate the true extent of the cellular 238 immune response (Extended Data Fig. 5f ). 239

Phenotyping of the identified pMHC multimer + S antigen-experienced CD8 + T cell specificities 240 revealed an early differentiated effector memory phenotype characterised by low expression of 241 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 10 CCR7 and CD45RA and high expression of the costimulatory molecules CD28 and CD27. 242 CD8 + T cells also expressed markers associated with cognate activation, such as CD38, HLA-243 DR and PD-1 (Fig. 4b) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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We report here that vaccination with BNT162b2 induces a coordinated immune response with 266 SARS-CoV-2 S-specific neutralising antibodies, CD4 + T cells, CD8 + T cells, and immune-267 modulatory cytokines such as IFNγ. While all CD8 + T cell responses against the S1 subunit of S were de novo and not detected at 279 baseline, pre-existing immune responses against the S2 subunit were identified in several 280 individuals. The S1 fragment has less sequence similarity to the corresponding seasonal 281 coronavirus sequences than the S2 fragment does, indicating that we may have detected pre-282 existing cross-reactive CD8 + T cells 34, 35 . 283 pMHC multimer technology enabled the identification of S epitopes recognised by vaccine-284 induced CD8 + T cells as well as direct quantification of the respective epitope-specific T cells. 285

The cumulative T cell frequencies in each participant exceeded the overall T cell response 286 measured in ELISpot and ICS assays, indicating that those assays underestimate the true 287 magnitude of the poly-epitopic response. Single peptide analyses are well known to yield higher 288 T cell frequencies as compared to functional T cell assays that stimulate with peptide pools, 289 with a multitude of immunogenic epitopes competing. A high proportion of induced CD8 + T 290 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 12 cells were early differentiated effector memory cells. This favourable phenotype has the 291 potential to respond rapidly, but has a limited capacity to produce IFNγ, and thus is less likely 292 to be detected in functional T cell assays. Previous studies have identified epitopes in SARS-293

CoV-2 S against which infected individuals raise CD8 + T cells 36,37 . To our knowledge, this is 294 the first report of epitopes recognised by COVID-19 vaccine-induced T cells. Of note, the 295 immunodominant HLA-A*02:01 restricted peptide YLQPRTFLL identified in our study has 296 previously been described in convalescent COVID-19 patients 36, 37 . 297

In addition to providing new insights into T cell responses, this study reproduces our previous 298 findings in the U.S.A trial 1 , and confirms a benign safety profile and robust induction of 299 antibody responses, with the latter being followed up for a longer period, up to day 85 (63 days 300 post-boost). Prime/boost vaccination with 10 to 30 µg of BNT162b2 elicited GMTs that, after 301 an initial decline, remained stable up to day 85 in the range of, or higher than, GMTs in COVID- is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 13 whether this immune response pattern will protect from SARS-CoV-2 infection and prevent 316 COVID-19. These questions will be addressed by the ongoing clinical program, which includes 317 longer term follow-up of participants in the two ongoing phase 1/2 trials, a dedicated immune 318 biomarker trial to further dissect the composite elements of the immune response, and the 319 ongoing phase 2/3 study with efficacy endpoints. 320

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Funding 365 BioNTech is the Sponsor of the study and responsible for the design, data collection, data 366 analysis, data interpretation, and writing of the report. Pfizer advised on the study and the 367 manuscript, generated serological data, and contracted for the generation of serological data. 368

The corresponding authors had full access to all the data in the study and had final responsibility 369 for the decision to submit the data for publication. All study data were available to all authors. 370

This study was not supported by any external funding at the time of submission. 371

Supplementary Information is available for this paper. 373

Correspondence and requests for materials should be addressed to Ugur Sahin. 374 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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Page 17 The presented data are from the BNT162b2-immunised healthy adults 19 to 55 years of age 387 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Page 18 cell studies were obtained on day 1 (pre-prime) and 29±3 (post-boost) (Extended Data Fig. 1) . 400 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Recombinant SARS-CoV-2 S1 or RBD containing a C-terminal Avitag™ (Acro Biosystems) 451 were bound to streptavidin-coated Luminex microspheres. Heat-inactivated participant sera 452 were diluted 1:500, 1:5,000, and 1:50,000. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint (normally distributed); and is the noise component, of which is Cauchy distributed, and 546

is Student's-t distributed. ensures that each sample is treated as a different batch. 547 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Peptide/MHC multimer staining. 570

In order to select MHC-class I epitopes for multimer analysis, a mass spectrometry-based 571 binding and presentation predictor 42,43 was applied to 8-12 amino acid long peptide sequences 572 from the Spike glycoprotein derived from the GenBank reference sequence for SARS-CoV-2 573 (accession: NC_045512.2, https://www.ncbi.nlm.nih.gov/nuccore/NC_045512) and paired 574 with 18 MHC-class-I alleles with >5% frequency in the European population. Top predicted 575 epitopes were identified by setting thresholds to the binding percent-rank (≤1%) and 576 presentation scores (≥10 -2.2 ). Peptides were manufactured at >90% purity. pMHC complexes 577 were refolded with the easYmer technology (easYmer® kit, ImmuneAware Aps), and complex is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint Page 26 1:1,667; all ThermoFisher Scientific). Cells were fixed for 15 minutes at 4 °C in 1x Stabilization 595 Fixative (BD), acquired on a FACSymphony™ A3 flow cytometer (BD Biosciences) and 596 analysed with FlowJo software version 10.6.2 (FlowJo LLC, BD Biosciences). CD8 + T cell 597 reactivities were considered positive, when a clustered population was observed that was 598 labelled with only two pMHC multimer colors. 599

The sample size for the reported part of the study was not based on statistical hypothesis testing. 601

All participants with data available were included in the safety and immunogenicity analyses. 602

The statistical method of aggregation used for the analysis of antibody concentrations and titers 603 is the geometric mean and the corresponding 95% CI. Employing the geometric mean accounts 604 for non-normal distribution of antibody concentrations and titers spanning several orders of 605 magnitude. Spearman correlation was used to evaluate the monotonic relationship between non-606 normally distributed data sets. 607

All statistical analyses were performed using GraphPad Prism software version 8.4.2. 608

The data that support the findings of this study are available from the corresponding author 610 upon reasonable request. Upon completion of this clinical trial, summary-level results will be 611 made public and shared in line with data sharing guidelines. 612 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine 614

Nucleoside-modified mRNA vaccines induce potent T follicular helper 616 and germinal center B cell responses

New Vaccine Technologies to Combat 618

Expression kinetics of nucleoside-modified mRNA delivered in lipid 620 nanoparticles to mice by various routes

mRNA-based therapeutics -developing a new class 622 of drugs

Nucleoside-modified mRNA immunization elicits influenza virus 624 hemagglutinin stalk-specific antibodies

Zika virus protection by a single low-dose nucleoside-modified mRNA 626 vaccination

Characterization of HIV-1 nucleoside-modified mRNA vaccines in 628 rabbits and rhesus macaques

Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in adults

Improving mRNA-Based Therapeutic Gene Delivery 637 by Expression-Augmenting 3′ UTRs Identified by Cellular Library Screening

Incorporation of pseudouridine into mRNA yields superior 640 nonimmunogenic vector with increased translational capacity and biological stability

Cryo-EM structure of the 2019-nCoV spike in the prefusion 643 conformation

BNT162b vaccines are immunogenic and protect non-human primates against SARS-645

CoV-2

An RNA vaccine drives immunity in checkpoint-inhibitor-treated 647 melanoma

Type I interferons directly 649 regulate lymphocyte recirculation and cause transient blood lymphopenia

Effect of influenza vaccine on markers of inflammation and lipid 652 profile

Development of VAX128, a recombinant hemagglutinin

influenza-flagellin fusion vaccine with improved safety and immune response

RNA-based adjuvant CV8102 enhances the immunogenicity of a 657 licensed rabies vaccine in a first-in-human trial

Statistical positivity criteria for 707 the analysis of ELISpot assay data in HIV-1 vaccine trials

Response definition criteria for ELISPOT assays revisited

Mass Spectrometry Profiling of HLA-Associated Peptidomes in 712

Mono-allelic Cells Enables More Accurate Epitope Prediction

Sequence-based prediction of SARS-CoV-2 vaccine targets using a mass 715 spectrometry-based bioinformatics predictor identifies immunogenic T cell epitopes

Use of 'one-pot, mix-and-read' peptide-MHC class I tetramers and 718 predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle

One-pot, mix-and-read peptide-MHC tetramers

Parallel detection of antigen-specific T-cell responses by 723 multidimensional encoding of MHC multimers

Toxicity grading scale for healthy adult and adolescent volunteers 726 enrolled in preventive vaccine clinical trials

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. Pre  8  22  29  43  50  8  22  29  43  50  85  8  22  29  43  50  85  8  22  29  43  50 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. Each data point represents one study participant (1 µg dose cohort, n=8; 20 µg dose cohort, 854 n=8; 10 and 30 µg, n=10 each). One participant from the 20 µg dose cohort with a strong pre-855 existing CD4 + T cell response to S pool 2 was excluded. f, Antigen-specific CD8 + T cell 856 frequencies determined by pMHC class I multimer staining (% multimer + of CD8 + ), ICS and 857 ELISpot (% IFNγ + of CD8 + ) for the three participants analysed in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. Correlation of S1-specific IgG responses with S-specific CD4 + T cell responses. b, Correlation 866 of S-specific CD4 + with CD8 + T cell responses. c, Correlation of S1-specific IgG responses 867 with S-specific CD8 + T cell responses. 868. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Twelve participants per dose cohort received the priming and the booster dose except for two 875 participants, who discontinued prior the booster dose due to a study drug-unrelated withdrawal 876 by the participant (1 µg dose) and an adverse event (10 µg; upper respiratory syndrome), 877 respectively. Safety analysis: Number of participants for whom 7 days of reactogenicity follow-878 up after both doses was evaluable at data cut-off. Antibody analysis: Numbers of participants 879 for whom virus neutralisation assays and S1-and RBD-binding IgG antibody assays were 880 performed. T cell analysis: Numbers of participants for whom PBMCs were available at data 881 cut-off and IFNγ ELISpot and flow cytometry (in parentheses). N/A, not applicable. *8 and **7 882for CD4 + T cell responses. 883 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.09.20245175 doi: medRxiv preprint