Article

COVID-19 vaccine BNT162b1 elicits human

antibody and TH1 T cell responses

https://doi.org/10.1038/s41586-020-2814-7 Ugur Sahin1,2 ✉, Alexander Muik1, Evelyna Derhovanessian1, Isabel Vogler1, Lena M. Kranz1,
Mathias Vormehr1, Alina Baum3, Kristen Pascal3, Jasmin Quandt1, Daniel Maurus1,
Received: 16 July 2020
Sebastian Brachtendorf1, Verena Lörks1, Julian Sikorski1, Rolf Hilker1, Dirk Becker1,
Accepted: 22 September 2020 Ann-Kathrin Eller1, Jan Grützner1, Carsten Boesler1, Corinna Rosenbaum1, Marie-Cristine
Kühnle1, Ulrich Luxemburger1, Alexandra Kemmer-Brück1, David Langer1, Martin Bexon4,
Published online: 30 September 2020
Stefanie Bolte1, Katalin Karikó1, Tania Palanche1, Boris Fischer1, Armin Schultz5, Pei-Yong Shi6,
Check for updates Camila Fontes-Garfias6, John L. Perez7, Kena A. Swanson7, Jakob Loschko7, Ingrid L. Scully7,
Mark Cutler7, Warren Kalina7, Christos A. Kyratsous3, David Cooper7, Philip R. Dormitzer7,
Kathrin U. Jansen7 & Özlem Türeci1

An effective vaccine is needed to halt the spread of the severe acute respiratory
syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety,
tolerability and antibody response data from an ongoing placebo-controlled,
observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with
BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes
the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present
antibody and T cell responses after vaccination with BNT162b1 from a second,
non-randomized open-label phase I/II trial in healthy adults, 18–55 years of age. Two
doses of 1–50 μg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong
antibody responses, with RBD-binding IgG concentrations clearly above those seen in
serum from a cohort of individuals who had recovered from COVID-19. Geometric mean
titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-μg dose)
to 3.5-fold (50-μg dose) those of the recovered individuals. Immune sera broadly
neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants
had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and
CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific
CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine
responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to
protect against COVID-19 through multiple beneficial mechanisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which transiently expressed and does not integrate into the genome. It is
was identified in China in December 2019, causes coronavirus disease molecularly well defined, free from materials of animal origin, and
2019 (COVID-19)—a severe, acute respiratory syndrome with a complex, synthesized by an efficient, cell-free in vitro transcription process from
highly variable disease pathology. On 11 March 2020, the World Health DNA templates5,9,10. The fast and highly scalable mRNA manufacturing
Organization (WHO) declared the SARS-CoV-2 outbreak a pandemic. As and LNP formulation processes enable rapid production of manyvac-
of 16 September 2020, more than 29 million cases have been reported cine doses6,7,11, making it suitable for rapid vaccine development and
worldwide, with over 930,000 deaths2. The severe and worldwide effect pandemic vaccine supply.
of the pandemic on human society calls for the rapid development of Two phase I/II umbrella trials in Germany and the USA are investigat-
safe and effective therapeutics and vaccines3. ing several LNP-encapsulated RNA vaccine candidates developed in
Lipid nanoparticle (LNP)-formulated mRNA vaccine technology ‘Project Lightspeed’, the joint BioNTech-Pfizer COVID-19 RNA vaccine
allows the delivery of precise genetic information together with an development program. Recently, we reported interim data obtained
adjuvant effect to antigen-presenting cells4. The prophylactic effec- in the USA trial (NCT04368728) for the most advanced candidate,
tiveness of this technology against multiple viral targets has been BNT162b11. BNT162b1 encodes the receptor-binding domain (RBD)
proven in preclinical models5–7. LNP- and liposome-formulated RNA of the SARS-CoV-2 spike protein, a key target of neutralizing antibodies.
vaccines for preventing infectious diseases or treating cancer have The RBD antigen expressed by BNT162b1 is fused to a T4 fibritin-derived
been shown in clinical trials to be safe and well-tolerated8. mRNA is ‘foldon’ trimerization domain to increase its immunogenicity by

1
BioNTech, Mainz, Germany. 2TRON gGmbH–Translational Oncology at the University Medical Center of the Johannes Gutenberg, Mainz, Germany. 3Regeneron Pharmaceuticals, Tarrytown, NY,
USA. 4Bexon Clinical Consulting, Upper Montclair, NJ, USA. 5CRS Clinical Research Services Mannheim GmbH, Mannheim, Germany. 6University of Texas Medical Branch, Galveston, TX, USA.
7
Pfizer, Pearl River, NY, USA. ✉e-mail: [email protected]

594 | Nature | Vol 586 | 22 October 2020

multivalent display12. The RNA is optimized for high stability and trans- RBD-specific IgG
106 Pre
lation efficiency13,14 and incorporates 1-methylpseudouridine instead 9,107 17,051 25,006
3,920
12,431
18,289 1 μg
of uridine to dampen innate immune sensing and to increase mRNA 105 2,015 6,707
1,058 602 10 μg
1,273 1,672
translation in vivo15. In the placebo-controlled, observer-blinded USA 104 265 826 909 755 30 μg

IgG (U ml–1)
50 μg
trial, dosages of 10 μg, 30 μg (prime and boost doses 21 days apart for 103
60 μg
both dose levels) and 100 μg (prime only) were administered. No serious 102 2 3 1 2 1 1 (no boost)
adverse events were reported. Local injection site reactions and sys- 101 HCS

temic events (mostly influenza-like symptoms) were dose-dependent, 100 LLOQ

generally mild to moderate, and transient. RBD-binding immuno- Pre 8 22 29 43 8 22 29 43 8 22 29 43 8 22 29 43 8 22 29 43 HCS

globulin G (IgG) concentrations and SARS-CoV-2 neutralising titres Days after initial vaccination
in sera increased with dose level and after the second dose. Fourteen
days after the boost dose, geometric mean neutralising titres reached Fig. 1 | BNT162b1-induced IgG concentrations. Vaccination schedule and serum
sampling are described in Extended Data Fig. 1. Participants were immunized
1.9- to 4.6-fold those seen in a panel of COVID-19 human convalescent
with BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 μg)
sera (HCS).
(n = 12 per group; from day 22 n = 11 for the 10 μg and 50 μg cohorts). Pre-dose
This study now complements and expands our previous report
responses across all dose levels were combined. COVID-19 convalescent samples
with available data from the German trial (NCT04380701, EudraCT: (HCS, n = 38) were obtained at least 14 days after PCR-confirmed diagnosis and at
2020-001038-36), providing a detailed characterization of antibody a time when the donors were no longer symptomatic. Each serum was tested in
and T cell immune responses elicited by vaccination with BNT162b1. duplicate and GMC plotted. For values below the lower limit of quantification
(LLOQ) = 1.15, LLOQ/2 values were plotted. Arrowheads indicate days of
vaccination. Checked bars indicate that no boost vaccination was performed.
Study design and analysis set Data shown as group GMC (values above bars) with 95% confidence interval (CI).
Between 23 April 2020 and 22 May 2020, 60 participants were vacci-
nated with BNT162b1 in Germany. Twelve participants for each of the Both CRP levels and lymphocyte counts are considered pharmacody-
dose level groups (1 μg, 10 μg, 30 μg, and 50 μg) received the first dose namics markers for the mode-of-action of RNA vaccines.
on day 1 and a booster dose on day 22 (except for one individual in each
of the 10- and 50-μg dose-level cohorts who discontinued participation
for reasons not related to the study drug), and 12 participants received a Vaccine-induced antibody responses
60-μg prime dose on day 1 only (Extended Data Fig. 1). The study popula- Concentrations of RBD-binding IgG and SARS-CoV-2-neutralizing titres
tion consisted of healthy males and non-pregnant females with a mean were assessed at baseline, 7 and 21 days after the BNT162b1 priming
age of 37 years (range 20–56 years) with equal gender distribution. dose (days 8 and 22), and 7 and 21 days after the boost dose (days 29
Most participants were white (96.7%) with one African American and and 43), except for the 60-μg cohort, which received a priming dose
one Asian participant (1.7% each; Extended Data Table 1). Preliminary only (Fig. 1, Extended Data Table 3).
data analysis focused on immunogenicity (Extended Data Table 2). Immunized participants showed a strong, dose-dependent
vaccine-induced antibody response. Twenty-one days after the prim-
ing dose (for the four dose levels ranging from 1 to 50 μg), geometric
Preliminary safety and tolerability data mean concentrations (GMCs) of RBD-binding IgG had increased in a
In brief, there were no serious adverse events and no withdrawals due dose-dependent manner, with GMCs ranging from 265 to 1,672 units
to related adverse events for any dose. Similar to the USA trial, most of (U) ml−1 (Fig. 1). Seven days after the boosting dose (day 29), RBD-binding
the reported solicited systemic events in the 10-μg and 30-μg groups IgG GMCs in participants vaccinated with 1–50 μg BNT162b1 showed
were due to reactogenicity, with a typical onset within the first 24 h of a strong, dose-dependent booster response ranging from 2,015 to
immunization (Extended Data Fig. 2). Injection site reactions within 25,006 U ml−1. On day 43 (21 days after boost), RBD-binding antibody
7 days of the prime or boost doses mainly involved pain and tenderness. GMCs were in the range of 3,920–18,289 U ml−1 in BNT162b1-vaccinated
Reactogenicity was dose-dependent, and was more pronounced after individuals, as compared to a GMC of 602 U ml−1 measured in a panel
the boost dose. The associated symptomatology, such as fever, chills, of convalescent sera from 38 patients who had been infected with
headache, muscle pain, joint pain, injection site pain, and tenderness, SARS-CoV-2. The patients were 18–83 years of age, and sera were drawn
was mostly mild or moderate, with occasional severe (grade 3) mani- at least 14 days after diagnosis confirmed by polymerase chain reaction
festations. In the 30-μg dose level cohort, 2 out of 12 (16.7%) subjects (PCR). In the 60 μg dose-level cohort, which received a priming dose
experienced severe local reactogenicity; 6 out of 12 (50%) subjects only, the RBD-binding IgG GMC was 755 U ml−1 by day 43, indicating
reported severe systemic reactogenicity (primarily headache, chills, that a boosting dose is necessary to increase antibody concentrations.
fatigue or muscle pain); and 1 subject out of 12 (8.3%) reported fever. Geometric mean titres (GMTs) of SARS-CoV-2 neutralizing antibod-
These adverse events were transient, resolved spontaneously or were ies increased modestly in a dose-dependent manner 21 days after the
manageable with simple measures (for example, paracetamol). Because priming dose (Fig. 2a, Extended Data Table 4). Substantially higher
of the reactogenicity reported after the 50-μg boost dose, participants serum-neutralising GMTs were achieved 7 days after the booster dose,
who had received an initial 60-μg dose did not receive a boost injection. reaching 36 (1 μg dose level), 158 (10 μg dose level), 308 (30 μg dose
Although there were no relevant changes in routine clinical labora- level), and 578 (50 μg dose level), compared to 94 for the convalescent
tory values after vaccination with BNT162b1, vaccinated participants serum panel. On day 43 (21 days after the boost), the neutralizing GMTs
showed a transient increase in C-reactive protein (CRP) and a tem- and RBD-binding GMCs decreased (with the exception of the 1 μg dose
porary reduction in blood lymphocyte counts, both of which were group). Serum virus-neutralizing GMTs were strongly correlated with
dose-dependent (Extended Data Fig. 3). CRP is an inflammatory serum RBD-binding IgG GMCs (Fig. 2b), and the vaccine elicited lower ratios
protein that has previously been described as biomarker for various of serum-neutralizing GMT to RBD-binding IgG GMC than did infec-
infectious disease vaccines and an indicator of vaccine adjuvant activ- tion with SARS-CoV-2. In summary, the antibody responses elicited
ity16–19. Our previous clinical experience with RNA vaccines suggests by BNT162b1 in study BNT162-01 largely mirrored those observed in
that the transient decrease in lymphocytes is likely to be attributable the USA study1.
to innate immune stimulation-related redistribution of lymphocytes To demonstrate the breadth of the neutralizing response, we tested
into lymphoid tissues20. Concomitant neutropenia was not observed. sera from vaccinated participants against a panel of 16 SARS-CoV-2 RBD

Nature | Vol 586 | 22 October 2020 | 595

Article
a Virus neutralization titres a CD4+ T cells CD8+ T cells b CD4+ T cells
308 Pre 9/11 Pre Post
578 5,000 10/10

IFNγ spots per 1 × 106 cells

1 μg 7/10 RBD
157
333 4,000 10/10 8/11
94 3,000
158 10 μg 11/11
103 126 2,000 9/11 8/10 CEF
30 μg 1,000
62
36 28 50 μg CEFT
500
VNT50

21 32 60 μg
102 20 400 Control
12 19 17 (no boost)
HCS 300
LLOQ 200 CD8+ T cells
5/9 4/9
10 10 10 10 10 10 Pre Post
101 100
RBD
0
Pre 8 22 29 43 8 22 29 43 8 22 29 43 8 22 29 43 8 22 29 43 HCS CEF

30 g
50 g
μg

30 g
50 g
μg
10 g

μg
t)

t)
bo g

bo g
μ
μ

μ
μ
μ

os

os
o μ

o μ
10
1

1
( n 60

( n 60
Days after initial vaccination CEFT
Immunization dose Control
b c 105 Pseudovirus neutralization titres
4 Spearman r = 0.9452 10 μg 30 μg 50 μg c CD4+ T cells CD8+ T cells
P ≤ 0.0001 5,000
104

IFNγ spots per 1 × 106 cells

log VNT50, day 29

3 4,000
pVNT50

3,000 1 μg
103
2,000 10 μg
2 30 μg
1,500
102 50 μg
LLOQ
1 1,000
101
500
0
Wild-type
Q321L
V341I
A348T
N354D
S359N
V367F
K378R
R408I
Q409E
A435S
K458R

G476S

Y508H
H519P
D614G
I472V

V483A
2 3 4 5 0
log[RBD-specific IgG (U ml–1)], day 29 RBD CEFT RBD CEF
SARS-CoV-2 RBD variants
Fig. 3 | Frequency and magnitude of BNT162b1-induced CD4+ and CD8+ T cell
Fig. 2 | BNT162b1-induced virus neutralization titres. Vaccination schedule responses. The vaccination schedule is described in Extended Data Fig. 1.
and serum sampling are described in Extended Data Fig. 1 and participants PBMCs obtained on day 1 (pre-prime) and on day 29 (7 days after boost for
were immunized as in Fig. 1. a, SARS-CoV-2 50% neutralization titres (VNT50) in cohorts 1 and 10 μg, n = 11 each; 30 and 50 μg, n = 10 each; 28 days after prime for
immunized participants and patients who had recovered from COVID-19 (HCS). the 60 μg cohort, n = 9) were enriched for CD4+ or CD8+ T cell effectors and
Each serum was tested in duplicate and GMT plotted. For values below the separately stimulated overnight with an overlapping peptide pool
LLOQ = 20, LLOQ/2 values were plotted. Arrowheads indicate days of representing the vaccine-encoded RBD for assessment in direct ex vivo IFNγ
vaccinations. Checked bars indicate that no boost vaccination was performed. ELISpot. Common pathogen T cell epitope pools CEF (CMV, EBV, influenza
Data shown as group GMTs (values above bars) with 95% CI. b, Nonparametric virus HLA class I epitopes) and CEFT (CMV, EBV, influenza virus, tetanus toxoid
Spearman correlation of recombinant RBD-binding IgG GMCs (as in Fig. 1) with HLA class II epitopes) served to assess general T cell reactivity and cell culture
VNT50 from sera collected on day 29. c, Pseudovirus 50% neutralization titres medium served as negative control. Each data point represents the normalized
(pVNT50) across a pseudovirus panel with 17 SARS-CoV-2 spike protein variants mean spot count from duplicate wells for one study participant, after
including 16 RBD mutants and the dominant spike protein variant D614G (dose subtraction of the medium-only control (a, c). a, RBD-specific CD4+ and CD8+
level 10 μg, n = 1; dose levels 30 and 50 μg, n = 2 representative day 29 sera). T cell responses for each dose cohort. Ratios above post-vaccination data
Each serum was tested in duplicate and GMT plotted. LLOQ = 40. Data shown as points are the number of participants with a detectable CD4+ or CD8+ T cell
group GMT with 95% CI. response out of the total number of tested participants per dose cohort.
b, Exemplary CD4+ and CD8+ ELISpot images for a 10-μg cohort participant.
c, RBD-specific CD4+ and CD8+ T cell-responses in all participants who received
variants identified through publicly available information21 and the
prime and boost vaccination (n = 42) with a positive response to RBD and their
dominant (non-RBD) spike variant D614G22 in pseudovirion neutraliza-
baseline CEFT- and CEF-specific T cell responses. Horizontal bars indicate
tion assays. Sera collected 7 days after the second dose of BNT162b1
median.
showed high neutralizing titres to each of the SARS-CoV-2 spike variants
(Fig. 2c, Extended Data Table 5).

cohorts, indicating the importance of booster vaccination. No CD4+

Vaccine-induced T cell responses T cell responses were detectable at baseline, except for one participant
CD4+ and CD8+ T  cell responses in individuals immunized with in the 50 μg dose cohort with a low number of pre-existing RBD-reactive
BNT162b1 were characterized before the priming vaccination (day CD4+ T cells, which increased substantially after vaccination (normal-
1) and on day 29 (7 days after the boost vaccination for the 1–50 μg ized mean spot count from 63 to 1,519). For two participants from the
cohorts) using direct ex vivo IFNγ enzyme-linked immunosorbent 1 μg cohort the baseline data could not be evaluated. The strength
spot (ELISpot) assay with peripheral blood mononuclear cells (PBMCs) of RBD-specific CD4+ T cell responses correlated positively with
from 51 participants across the 1 μg to 60 μg dose-level cohorts (Fig. 3). both RBD-binding IgG and SARS-CoV-2-neutralizing antibody titres
In this assay, CD4+ or CD8+ T cell effectors were stimulated overnight (Extended Data Fig. 4a, b), consistent with the concept of intramo-
with overlapping peptides representing the full-length sequence of lecular help23. The two participants immunized with 1 μg BNT162b1
the vaccine-encoded RBD. who lacked a CD4+ response had no detectable virus-neutralizing titres
Of 42 participants who had received prime–boost vaccination (the (VNT50) (Extended Data Fig. 4b).
1 μg to 50 μg cohorts), 40 (95.2%, including all participants treated Among participants who showed any vaccine-induced CD8+ T cell
with 10 μg BNT162b1 or more) mounted RBD-specific CD4+ T cell response (32/42 participants receiving the prime-boost dosing, 76.2%),
responses. Although the magnitude of the response varied between the majority mounted strong responses (Fig. 3a) that were comparable
individuals, participants with the strongest CD4+ T cell responses to with memory responses against CMV, EBV and influenza virus in the
RBD had more than tenfold the memory responses observed in the same participants (Fig. 3b, c). Individuals immunized with a single dose
same participants when stimulated with cytomegalovirus (CMV), of 60 μg had a lower response rate (4/9; 44%) and a weaker CD8+ T cell
Epstein Barr virus (EBV), influenza virus and tetanus toxoid-derived response to RBD. The strength of RBD-specific CD8+ T cell responses
immuno-dominant peptide panels (Fig. 3a–c). In the 60 μg cohort, who correlated positively with vaccine-induced CD4+ T cell responses but
had been treated with the priming dose only, both immunogenicity did not significantly correlate with SARS-CoV-2 neutralizing antibody
rate (5/9; 55.6%) and response strength were lower than for the other titres (Extended Data Fig. 4c, d).

596 | Nature | Vol 586 | 22 October 2020

 a CD8+ T cells CD4+ T cells b CD4+ T cells
100

RBD-specific CD4+ T cells

0.02 0.00 0.01 0.01 0.00 0.00

Percentage of cytokine+
1 μg
10 μg

Pre
75 30 μg

IL-4 APC
0.14 50 μg

IL-2 PE
0.29 0.07
50 60 μg
(no boost)
5 0.02 0.04 0.28 0.33 0.01 0.01
4 25

Post
3
2 0.24 0
1.97 0.37

IL 2 +

IF +

IL 2 +
-4 +

IL 2 +
-4 +

IL 2 +

IF +

IL 2 +
-4 +
IL 2 +

IL 2 +

IL 2 +

IL 2 +

IL 2 +
N IL γ +

N IL γ +

N IL γ +

N IL γ +

N IL γ +
3 4 5

-4

-4
γ+ -

γ+ -

γ+ -

γ+ -

γ+ -
IF FN

IF FN

N
-

-
IFNγ PE-Cy7

IF
I

I
IF

IF

IF
c RBD-specific CD8+ T cells d
TNF IL-1β IL-12p70
IFNγ IL-2
Percentage cytokine+ of CD8+ T cells

5 1.44 0.8 4,000 500 500

0.51 Pre

Cytokine release (pg ml–1)

4 0.28
0.08
3 0.6 Post
2 400 400
1 0.4 * No boost 3,000
0.8 0.25 0.04
300 300
0.6 0.22 0.20 2,000
0.02
0.15 200 200
0.4
0.10 1,000
0.2 0.01 0.01 0.01 100 100
0.02 0.05 0.01
0 0 0 0 0
Pre Post Pre Post Pre Post
10 g
30 g
50 g
60 μg

10 g
30 g
50 g
60 μg
H *
S

H *
S
μg

μg
μ
μ
μ

μ
μ
μ
C

C
1

Immunization dose
RBD-specific CD4+ T cells IL-4 IL-5
Percentage cytokine+ of CD4+ T cells

IFNγ IL-2 IL-4

100 100
0.35 0.06 0.35

Cytokine release (pg ml–1)

0.6 0.11
0.30 0.05 0.30 0.03 80 80
0.5 0.09
0.25 0.25 60 60
0.10 0.4
0.20 0.11 0.20
0.2 0.04 40 40
0.15 0.15
0.06 0.03
0.10 0.02 0.10 0.01 20 20
0.03 0.1
0.05 0.01 0.05 0.01
0.01 0.01 0 0
0.00 Pre Post Pre Post
0 0 0
10 g
30 g
50 g
60 μg

10 g
30 g
μg

60 μg

10 g
30 g
50 g
60 μg
H *
S

H *
S

H *
S
μg

μg

μg
μ
μ
μ

μ
μ

μ
μ
μ
C

C
1

50

Immunization dose

Fig. 4 | Cytokine polarization of BNT162b1-induced T cells. The vaccination CD4+ T cells. Arithmetic mean with 95% CI. CD4 non-responders (<0.03% total
schedule is described in Extended Data Fig. 1. PBMCs from vaccinated cytokine-producing T cells; 1 μg, n = 5; 10 μg, n = 1; 30 μg, n = 2; 50 μg, n = 1; 60 μg,
participants (7 days after boost for cohorts 1 and 10 μg, n = 10 each; 30 μg, n = 12; n = 6) were excluded. c, RBD-specific CD8+ (top) or CD4+ (bottom) T cells
50 μg, n = 9; 28 days after prime for the 60 μg cohort, n = 11) and donors who had producing the indicated cytokine as a percentage of total circulating T cells of
recovered from COVID-19 (HCS, n = 15; c) were stimulated over night with an the same subset. Values above data points indicate mean fractions per dose
overlapping peptide pool representing the vaccine-encoded RBD and analysed cohort. Participants’ PBMCs were tested as single instance (b, c). d, Cytokine
by flow cytometry (a–c) and bead-based immunoassay (d). The gating strategy is release by PBMCs from the 50 μg cohort (n = 5; assay results from remaining
depicted in Supplementary Fig. 1. a, Exemplary pseudocolour flow cytometry samples of this and other cohorts not available at the time). Each data point
plots of cytokine-producing CD4+ and CD8+ T cells from a participant who was represents the mean from duplicate wells subtracted by the DMSO control for
immunized with the 10-μg dose. b, RBD-specific CD4+ T cells producing the one study participant. LLOQs were 6.3 pg ml−1 for TNF, 2.5 pg ml−1 for IL-1β,
indicated cytokine as a percentage of total cytokine-producing RBD-specific 7.6 pg ml−1 for IL-12p70, 11.4 pg ml−1 for IL-4 and 5.3 pg ml−1 for IL-5.

Of note, although at 1 μg BNT162b1 the rates of CD4+ and CD8+ T cell In summary, these findings indicate that BNT162b1 induces
response were lower than for the other doses (9 and 8 out of 11 partici- functional and proinflammatory CD4+ and CD8+ T cell responses in
pants, respectively), the number of vaccine-induced T cells in some almost all participants, with TH1 polarization of the helper response.
participants was almost as high as with 50 μg BNT162b1 (Fig. 3a).
To assess the functionality and polarization of RBD-specific T cells,
we identified cytokines secreted in response to stimulation with Discussion
overlapping peptides representing the full-length sequence of the We observed concurrent production of neutralizing antibodies,
vaccine-encoded RBD by intracellular staining (ICS) for IFNγ, IL-2 and activation of virus-specific CD4+ and CD8+ T cells, and robust release
IL-4 in PBMCs collected before and after vaccination from 52 partici- of immune-modulatory cytokines such as IFNγ, which represents a
pants who had been immunized with BNT162b1. RBD-specific CD4+ coordinated immune response to counter a viral intrusion24. IFNγ is
T cells secreted IFNγ, IL-2, or both, but in most individuals they did not a key cytokine for several antiviral responses. It acts in synergy with
secrete IL-4 (Fig. 4 a–c, Extended Data Table 6). Similarly, fractions of type I interferons to inhibit the replication of SARS-CoV25. Individuals
RBD-specific CD8+ T cells secreted IFNγ+ and IL-2. with polymorphisms in the IFNG gene that impair IFNγ activity have
The mean fraction of RBD-specific T cells within total circulating a fivefold increase in susceptibility to SARS26. The robust elicitation
T cells obtained by BNT162b1 vaccination was substantially higher than of IFNγ-producing CD8+ T cells indicates that a favourable cellular
that observed in fifteen donors who had recovered from COVID-19. immune response with anti-viral and immune-augmenting properties
Fractions of RBD-specific IFNγ+ CD8+ T cells reached up to several per complements the strong neutralizing antibody response.
cent of total peripheral blood CD8+ T cells in immunized individuals The detection of IFNγ, IL-2 and IL-12p70, but not IL-4 or IL-5, indi-
(Fig. 4c). The supernatants of PBMCs from five vaccinated participants cates a favourable TH1 profile and the absence of a potentially del-
were stimulated ex vivo with overlapping RBD peptides and produced eterious TH2 immune response. CD4+ and CD8+ T cells may confer
the proinflammatory cytokines TNF, IL-1β and IL-12p70, but neither long-lasting immune memory against coronaviruses, as indicated in
IL-4 nor IL-5 (Fig. 4d). SARS-CoV-1 survivors, in whom CD8+ T cells persisted for 6–11 years24,27.

Nature | Vol 586 | 22 October 2020 | 597

Article
Some cases of asymptomatic virus exposure have been associated in the future, the versatility of the RNA platform could facilitate fast
with cellular immune response without seroconversion, indicating adaptation to newly emerging viral strains.
that SARS-CoV-2-specific T cells could be relevant in disease control Limitations of our clinical study include the small sample size and its
even in the absence of neutralizing antibodies28. In our study, almost restriction to participants below 55 years of age. Another constraint is
all vaccinated volunteers mounted RBD-specific T cell responses that that we did not perform further T cell analysis (for example, deconvo-
were detected using an ex vivo ELISpot assay, which was performed lution of epitope diversity, characterization of HLA restriction, T cell
without prior expansion of T cells and captures only high-magnitude phenotyping and TCR repertoire analysis) before and after vaccination,
T cell responses. While the strength of the T cell responses varied con- because of the limited blood volumes that were available for biomarker
siderably between participants, we observed no clear dose dependency analyses. Similarly, we did not assess the induction of tissue-resident
of the T cell response strength within the tested dose range (1–50 μg). memory CD8+ T cells. Further, as vaccine-induced immunity can wane
Even with a dose as low as 1 μg, mRNA-encoded immunogen stimulation over time, it is important to study the persistence of potentially pro-
and robust expansion of T cells was accomplished in most subjects. tective immune responses. Samples to assess persistence are not yet
Our results confirm the dose-dependency of RBD-binding IgG and available but are planned in the study protocol and will be reported
neutralization responses and reproduces our previous findings for the elsewhere. The results reported here were obtained from immunization
10 and 30 μg dose levels of BNT162b1 in the USA trial1. The observed with one of four vaccine candidates in the study. Upcoming reports of
strong boost response for BNT162b1 is in line with the absence of a Project Lightspeed will present the data obtained for other COVID-19
limiting anti-vector immunity, which is a characteristic advantage of vaccine candidates, including BNT162b2, the RNA-based vaccine can-
the RNA-based vaccine platform. didate that encodes the full-length SARS-CoV-2 spike glycoprotein and
The ratio of serum virus neutralization GMT to recombinant is being tested in a phase III efficacy trial32.
RBD-binding IgG GMC is lower after immunization with BNT162b1
than after infection with SARS-CoV-2. As noted previously, this differ-
ence may be attributed, in part, to BNT162b1 eliciting antibodies that Online content
bind epitopes that are exposed on the RNA-encoded RBD immuno- Any methods, additional references, Nature Research reporting sum-
gen but buried and inaccessible in the spikes of SARS-CoV-2 virions, maries, source data, extended data, supplementary information,
differentially increasing RBD-binding IgG GMCs after immunization. In acknowledgements, peer review information; details of author con-
addition, infection with SARS-CoV-2 might elicit neutralizing antibod- tributions and competing interests; and statements of data and code
ies that recognize epitopes that are exposed on virions and located availability are available at https://doi.org/10.1038/s41586-020-2814-7.
outside the RBD, differentially increasing the serum neutralizing GMT
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Article
Methods
Human convalescent sera and PBMC panel
Clinical trial design Human SARS-CoV-2 infection/COVID-19 convalescent sera (n = 38)
Study BNT162-01 (NCT04380701) is an ongoing, first-in-human, phase were drawn from donors 18–83 years of age at least 14 days after
I/II, open-label dose-ranging clinical trial to assess the safety, toler- PCR-confirmed diagnosis and at a time when the participants were
ability, and immunogenicity of ascending dose levels of various intra- asymptomatic. The mean age of the donors was 45 years. Neutraliz-
muscularly administered BNT162 mRNA vaccine candidates in healthy ing GMTs in subgroups of the donors were as follows: symptomatic
men and non-pregnant women 18 to 55 years of age (amended to add infections, 90 (n = 35); asymptomatic infections, 156 (n = 3); hospital-
56–85 years of age). Key exclusion criteria included previous clinical ized, 618 (n = 1). Sera were obtained from Sanguine Biosciences (Sher-
or microbiological diagnosis of COVID-19; receipt of medications to man Oaks, CA), the MT Group (Van Nuys, CA) and Pfizer Occupational
prevent COVID-19; previous vaccination with any coronavirus vac- Health and Wellness (Pearl River, NY). Human SARS-CoV-2 infection/
cine; a positive serological test for SARS-CoV-2 IgM and/or IgG; and a COVID-19 convalescent PBMC samples (n = 15) were collected from
SARS-CoV-2 NAAT-positive nasal swab; those with increased risk for donors 22–79 years of age 30–62 days after PCR-confirmed diagnosis
severe COVID-19; and immunocompromised individuals. The primary when donors were asymptomatic. PBMC donors had asymptomatic or
endpoints of the study are safety and immunogenicity. mild infections (n = 13; clinical score 1 and 2) or had been hospitalized
In the part of the study reported here, five dose levels (1 μg, 10 μg, (n = 2; clinical score 4 and 5). Blood samples were obtained from the
30 μg, 50 μg or 60 μg) of the BNT162b1 candidate were assessed at Frankfurt University Hospital (Germany).
one site in Germany with 12 healthy participants per dose level in a
dose-escalation/de-escalation design. Sentinel dosing was performed Cell culture and primary cell isolation
in each dose-escalation cohort. Progression in that cohort and dose Vero cells (CCL-81) and Vero E6 cells (ATCC CRL-1586) were sourced from
escalation required data review by a safety review committee. Partici- the American Type Culture Collection (ATCC), which maintains a quality
pants received a BNT162b1 prime dose on day 1, and a boost dose on day management system commensurate to ISO 9001:2015, ISO 13485:2016,
22 ± 2. Serum for antibody assays was obtained on days 1 (pre-prime), ISO 17025:2017, and ISO 17034:2016. Cells were certified by the vendor
8 ± 1 (post-prime), 22 ± 2 (pre-boost), 29 ± 3 and 43 ± 4 (post-boost). and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
PBMCs for T cell studies were obtained on days 1 (pre-prime) and 29 ± 3 GlutaMAX (Gibco) supplemented with 10% fetal bovine serum (FBS)
(post-boost). Tolerability was assessed by patient diary. One individual (Sigma-Aldrich). Cell lines were tested for mycoplasma contamination
in the 10 μg cohort and one in the 50 μg cohort left the study before after receipt and before expansion and cryopreservation. PBMCs were
the boosting immunization owing to withdrawal of consent for private isolated by Ficoll-Hypaque (Amersham Biosciences) density gradient
reasons. centrifugation and cryopreserved before subsequent analysis.
The presented data comprise the BNT162b1-immunized cohorts
only and are based on a preliminary analysis with a data extraction RBD-binding IgG assay
date of 23 July 2020, focused on analysis of vaccine-induced immuno- A recombinant SARS-CoV-2 RBD containing a C-terminal Avitag
genicity (secondary endpoint) descriptively summarized at the various (Acro Biosystems) was bound to streptavidin-coated Luminex micro-
time points and on reactogenicity. All participants for whom data were spheres. Heat-inactivated participant sera were diluted to 1:500,
available were included in the immunogenicity analyses. 1:5,000, and 1:50,000. Following overnight incubation at 2–8 °C while
The trial was carried out in Germany in accordance with the shaking, plates were washed in a solution containing 0.05% Tween-20.
Declaration of Helsinki and Good Clinical Practice Guidelines and with A secondary R-PE-labelled goat anti-human IgG polyclonal antibody
approval by an independent ethics committee (Ethik-Kommission of (1:500; Jackson Labs) was added for 90 min at room temperature while
the Landesärztekammer Baden-Württemberg, Stuttgart, Germany) and shaking, before plates were washed once more in a solution containing
the competent regulatory authority (Paul-Ehrlich Institute, Langen, 0.05% Tween-20. Data were captured as median fluorescent intensities
Germany). All participants provided written informed consent. (MFIs) using a Bioplex200 system (Bio-Rad) and converted to U/ml
antibody concentrations using a reference standard curve
Manufacturing of RNA (reference standard composed of a pool of five convalescent serum
BNT162b1 incorporates a Good Manufacturing Practice (GMP)-grade samples obtained more than 14 days after COVID-19 PCR diagnosis
mRNA drug substance that encodes the trimerized SARS-CoV-2 spike and diluted sequentially in antibody-depleted human serum) with
glycoprotein RBD antigen. The RNA is generated from a DNA template arbitrarily assigned concentrations of 100 U/ml and accounting for
by in vitro transcription in the presence of 1-methylpseudouridine- the serum dilution factor. Three dilutions were used to increase the
5′-triphosphate (m1ΨTP; Thermo Fisher Scientific) instead of uridine- likelihood that at least one result for any sample would fall within the
5′-triphosphate (UTP). Capping is performed co-transcriptionally using useable range of the standard curve. Assay results are reported in U/ml
a trinucleotide cap 1 analogue ((m27,3′-O)Gppp(m2′-O)ApG; TriLink). The of IgG. The final assay results were expressed as the GMC of all sample
antigen-encoding RNA contains sequence elements that increase RNA dilutions that produced a valid assay result within the assay range.
stability and translation efficiency in human dendritic cells13,14. The
mRNA is formulated with lipids to obtain the RNA–LNP drug product. SARS-CoV-2 neutralization assay
The vaccine was transported and supplied as a buffered-liquid solution The neutralization assay used a previously described strain of
for intramuscular injection and was stored at −80 °C. SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics
and engineered by the insertion of an mNeonGreen (mNG) gene into
Proteins and peptides open reading frame 7 of the viral genome33. This reporter virus gener-
A pool of 15-mer peptides that overlapped by 11 amino acids and cov- ates similar plaque morphologies and indistinguishable growth curves
ered the whole sequence of the BNT162b1-encoded SARS-CoV-2 RBD from wild-type virus. Viral master stocks (2 × 107 PFU/ml) were grown
was used for ex vivo stimulation of PBMCs for flow cytometry, IFNγ in Vero E6 cells as previously described33. With patient convalescent
ELISpot and cytokine profiling. CEF (CMV, EBV, influenza virus; human sera, the fluorescent neutralization assay produced comparable results
leukocyte antigen (HLA) class I epitope peptide pool) and CEFT (CMV, to the conventional plaque reduction neutralization assay34. Serial
EBV, influenza virus, tetanus toxoid; HLA class II epitope peptide pool) dilutions of heat-inactivated sera were incubated with the reporter
(both JPT Peptide Technologies) were used as controls for general T cell virus (2 × 104 PFU per well to yield a 10–30% infection rate of the Vero
reactivity. CCL81 monolayer) for 1 h at 37 °C before inoculating Vero CCL81 cell
monolayers (targeted to have 8,000 to 15,000 cells in a central field of cells were stimulated for 16–20 h with an overlapping peptide pool
each well at the time of seeding, 24 h before infection) in 96-well plates representing the vaccine-encoded RBD. Bound IFNγ was visualized
to allow accurate quantification of infected cells. Total cell counts per using a secondary anti-IFNγ antibody directly conjugated with alkaline
well were enumerated by nuclear stain (Hoechst 33342) and fluorescent phosphatase (1:250; ELISpotPro kit, Mabtech) followed by incuba-
virally infected foci were detected 16–24 h after inoculation with a Cyta- tion with a 5-bromo-4-chloro-3′-indolyl phosphate (BCIP)/nitro blue
tion 7 Cell Imaging Multi-Mode Reader (BioTek) with Gen5 Image Prime tetrazolium (NBT) substrate (ELISpotPro kit, Mabtech). Plates were
version 3.09. Titres were calculated in GraphPad Prism version 8.4.2 scanned using an AID Classic Robot ELISPOT Reader and analysed by
by generating a four-parameter (4PL) logistical fit of the percentage AID ELISPOT 7.0 software (AID Autoimmun Diagnostika). Spot counts
neutralization at each serial serum dilution. The 50% neutralization were summarized as mean values of each duplicate. T cell responses
titre (VNT50) was reported as the interpolated reciprocal of the dilution stimulated by peptides were compared to effectors incubated with
yielding a 50% reduction in fluorescent viral foci. medium only as a negative control using an in-house ELISpot data
analysis tool (EDA), based on two statistical tests (distribution-free
VSV-SARS-CoV-2 spike variant pseudovirus neutralization assay resampling) as described35,36, to provide sensitivity while maintaining
Vesicular stomatitis virus (VSV)-SARS-CoV-2-S pseudoparticle gen- control over false positives.
eration and neutralization assays were performed as previously To account for varying sample quality reflected in the number of spots
described21. In brief, human codon-optimized SARS-CoV-2 spike (Gen- in response to anti-CD3 antibody stimulation, a normalization method
Bank: MN908947.3) was synthesized (Genscript) and cloned into an was applied to enable direct comparison of spot counts/strength of
expression plasmid. SARS-CoV-2 complete genome sequences were response between individuals. This dependency was modelled in a
downloaded from GISAID nucleotide database (https://www.gisaid. log-linear fashion with a Bayesian model including a noise component
org) on 20 March 2020, as described previously21. Sequences were (unpublished). For a robust normalization, each normalization was
curated and the genetic diversity of the spike-encoding gene was sampled 10,000 times from the model and the median taken as normal-
assessed across high-quality genome sequences using custom pipe- ized spot count value. Likelihood of the model logλE = αlogλP + logβj + σε,
lines. Amino acid substitutions were cloned into the spike expression where λE is the normalized spot count of the sample, α is a stable factor
plasmid using site-directed mutagenesis. HEK293T cells (ATCC CRL- (normally distributed) common among all positive controls λP, βj is
3216) were seeded (culture medium: DMEM high glucose (Life Technolo- a sample j-specific component (normally distributed) and σε is the
gies) supplemented with 10% heat-inactivated FBS (Life Technologies), noise component, of which σ is Cauchy distributed and ε is Student’s
90.1 units/ml penicillin, 90.1 μg/ml streptomycin and 0.26 mg/ml t-distributed. βj ensures that each sample is treated as a different batch.
l-glutamine (Life Technologies)) and transfected the following day
with spike expression plasmid using Lipofectamine LTX (Life Technolo- Flow cytometry
gies) following the manufacturer’s protocol. At 24 h post-transfection Cytokine-producing T cells were identified by intracellular cytokine
at 37 °C, cells were infected with the VSVΔG:mNeon/VSV-G diluted in staining. PBMCs thawed and rested for 4 h in OpTmizer medium sup-
Opti-MEM (Life Technologies) at a multiplicity of infection of 1. Cells plemented with 2 μg/ml DNase I (Roche) were restimulated with a pep-
were incubated for 1 h at 37 °C, washed to remove residual input virus tide pool representing the vaccine-encoded SARS-CoV-2 RBD (2 μg/
and overlaid with infection medium (DMEM high glucose supplemented ml/peptide; JPT Peptide Technologies) in the presence of GolgiPlug
with 0.7% low IgG BSA (Sigma), 1 mM sodium pyruvate (Life Technolo- (BD) for 18 h at 37 °C. Controls were treated with DMSO-containing
gies) and 0.05 μg/ml gentamicin (Life Technologies)). After 24 h at medium. Cells were stained for viability and surface markers (CD3
37 °C, the supernatant containing VSV-SARS-CoV-2-S pseudoparticles BV421, 1:250; CD4 BV480, 1:50; CD8 BB515, 1:100; all BD Biosciences)
was collected, centrifuged at 3,000g for 5 min to clarify and stored at in flow buffer (DPBS (Gibco) supplemented with 2% FBS (Biochrom),
−80 °C until further use. 2 mM EDTA (EDTA; Sigma-Aldrich) for 20 min at 4 °C. Afterwards,
For pseudovirus neutralization assays, Vero cells (ATCC CCL-81) samples were fixed and permeabilized using the Cytofix/Cytoperm
were seeded in 96-well plates in culture medium and allowed to reach kit according to the manufacturer’s instructions (BD Biosciences).
approximately 85% confluence before use in the assay (24 h later). Sera Intracellular staining was performed in Perm/Wash buffer for 30 min
were serially diluted 1:2 in infection medium starting with a 1:40 dilu- at 4 °C (CD3 BV421, 1:250; CD4 BV480, 1:50; CD8 BB515, 1:100; IFNγ
tion. VSV-SARS-CoV-2-S pseudoparticles were diluted 1:1 in infection PE-Cy7, 1:50; IL-2 PE, 1:10; IL-4 APC, 1:500; all BD Biosciences). Samples
medium for a fluorescent focus unit (ffu) count in the assay of ~1,000. were acquired on a fluorescence-activated cell sorter (FACS) VERSE
Serum dilutions were mixed 1:1 with pseudoparticles for 30 min at room instrument (BD Biosciences) using BD FACSuite software version
temperature before addition to Vero cells and incubation at 37 °C for 1.0.6 and analysed with FlowJo software version 10.5.3 (FlowJo LLC,
24 h. Supernatants were removed and replaced with PBS (Gibco), and BD Biosciences). RBD-specific cytokine production was corrected for
fluorescent foci were quantified using the SpectraMax i3 plate reader background by subtraction of values obtained with dimethyl sulfoxide
with MiniMax imaging cytometer (Molecular Devices). Neutralization (DMSO)-containing medium. Negative values were set to zero. Cytokine
titres were calculated in GraphPad Prism version 8.4.2 by generating a production in Fig. 4b was calculated by summing the fractions of all
4PL fit of the percentage neutralization at each serial serum dilution. CD4+ T cells positive for IFNγ, IL-2 or IL-4, setting this sum to 100% and
The pVNT50 was reported as the interpolated reciprocal of the dilution calculating the fraction of each specific cytokine-producing subset
yielding a 50% reduction in fluorescent viral foci. thereof. Pseudocolour plot axes are in log10 scale.

IFNγ ELISpot Cytokine profiling

IFNγ ELISpot analysis was performed ex vivo (without further in vitro Human PBMCs were restimulated for 48 h with SARS-CoV-2 RBD pep-
culturing for expansion) using PBMCs depleted of CD4+ and enriched tide pool (2 μg/ml final concentration per peptide). Stimulation with
for CD8+ T cells (CD8+ effectors), or depleted of CD8+ and enriched DMSO-containing medium served as negative controls. Concentrations
for CD4+ T cells (CD4+ effectors). Tests were performed in duplicate of tumour necrosis factor (TNF), IL-1β, IL-12p70, IL-4 and IL-5 in super-
and with a positive control (anti-CD3 monoclonal antibody (1:1,000; natants were determined using a bead-based, 11-plex TH1/TH2 human
Mabtech)). Multiscreen filter plates (Merck Millipore) pre-coated ProcartaPlex immunoassay (Thermo Fisher Scientific) according to the
with IFNγ-specific antibodies (ELISpotPro kit, Mabtech) were washed manufacturer’s instructions. Fluorescence was measured with a Bio-
with PBS and blocked with X-VIVO 15 medium (Lonza) containing 2% plex200 system (Bio-Rad) and analysed with ProcartaPlex Analyst 1.0
human serum albumin (CSL-Behring) for 1–5 h. Per well, 3.3 × 105 effector software (Thermo Fisher Scientific). RBD-specific cytokine production
Article
was corrected for background by subtraction of values obtained with Acknowledgements We thank M. Dolsten for advice during drafting of the manuscript;
C. Anders, C. Anft, N. Beckmann, K. Bissinger, G. Boros, P. Cienskowski, K. Clarke, C. Ecker,
DMSO-containing medium. Negative values were set to zero. A. Engelmann, Y. Feuchter, L. Heesen, M. Hossainzadeh, S. Jägle, L. Jeck, O. Kahl, M. Knezovic,
T. Kotur, M. Kretschmer, O. Pfante, J. Reinholz, L.-M. Schmid, R. Schulz, B. Stock, C. Müller,
Statistical analysis S. Murphy, G. Szabó and M. Vehreschild for technical support, project management and
advice; A. Finlayson and M. Rao for editorial assistance; P. Koch and F. Groher for data
The sample size for the reported part of the study was not based on management and analysis; S. Liebscher and O. Kistner for expert advice; J. Absalon for
statistical hypothesis testing. All participants with data available were manuscript advice; the CRS Team (Mannheim and Berlin) for study conduct: S. Baumann,
included in the safety and immunogenicity analyses. The statistical M. Berse, M. Casjens, B. Ehrlich, and F. Seitz; the Pfizer Vaccines Clinical Assays Team and the
Pfizer Aviation Team for technical and logistical support of serology analyses; and the GISAID
method of aggregation used for the analysis of antibody concentrations Nucleotide database for sharing of SARS-CoV-2 complete genome sequences. BioNTech is the
and titres is the geometric mean and the corresponding 95% CI. Using sponsor of the study and responsible for the design, data collection, data analysis, data
the geometric mean allows us to account for non-normal distribu- interpretation and writing of the report. Pfizer advised on the study and the manuscript,
generated serological data and contracted for the generation of serological data. The
tion of antibody concentrations and titres spanning several orders of corresponding authors had full access to all the data in the study and had final responsibility
magnitude. Spearman correlation was used to evaluate the monotonic for the decision to submit the data for publication. All study data were available to all authors.
relationship between non-normally distributed data sets. All statistical This study was not supported by any external funding at the time of submission.

analyses were performed using GraphPad Prism software version 8.4.2. Author contributions U.S. conceived and conceptualized the work and strategy, supported by
The experiments were not randomized and the investigators were not Ö.T. Experiments were planned or supervised by E.D., C.F.-G., C.A.K., L.M.K., U.L., A.M., J.Q.,
blinded to allocation during experiments and outcome assessment. P.-Y.S. and I.V. A.B., D.C., M.C., C.F.-G., W.K., K.P., J.Q., I.L.S. and P.-Y.S. performed experiments.
D.B., S. Brachtendorf, E.D., P.R.D., J.G., K.U.J., A.-K.E., L.M.K., M.-C.K., V.L., A.M., J.Q., J.S., I.V. and
M.V. analysed data. D.M. planned and supervised dashboards for analysis of clinical trial data.
Reporting summary R.H. was responsible for data normalization and adaption. C.B. and C.R. were responsible for
Further information on research design is available in the Nature biomarker and R&D program management. K.K. optimized the mRNA. M.B., S. Bolte, B.F.,
A.K.-B., D.L., T.P. and A.S. coordinated operational conduct of the clinical trial. J.L.P. advised on
Research Reporting Summary linked to this paper. the trial, and J.L. and K.A.S. advised on experiments. U.S. and Ö.T., supported by M.B., E.D.,
P.R.D., K.U.J., L.M.K., A.M., I.V. and M.V., interpreted data and wrote the manuscript. All authors
supported the review of the manuscript.
Data availability
Competing interests All authors have completed the International Committee of Medical
The data that support the findings of this study are available from the Journal Editors (ICMJE) uniform disclosure form at https://www.gisaid.orgwww.icmje.org/coi_
corresponding author upon reasonable request. SARS-CoV-2 complete disclosure.pdf` and declare: U.S. and Ö.T. are management board members and employees at
BioNTech SE (Mainz, Germany); D.B., C.B., S. Brachtendorf, E.D., A.-K.E., B.F., J.G., R.H., M.-C.K.,
genome sequences were downloaded from the GISAID nucleotide data- U.L., V.L., D.M., C.R., J.S. and T.P. are employees at BioNTech SE; K.K., L.M.K., I.V., A.M., J.Q. and
base (https://www.gisaid.org) on 20 March 2020, as described previ- M.V. are employees at BioNTech RNA Pharmaceuticals GmbH; M.B. is an employee at Bexon
ously21. Upon completion of this clinical trial, summary-level results Clinical Consulting LLC. A.B., C.A.K. and K.P. are employees of Regeneron Pharmaceuticals
Inc; K.K., A.M., U.S. and Ö.T. are inventors on patents and patent applications related to RNA
will be made public and shared in line with data sharing guidelines. technology and COVID-19 vaccine; D.B., C.B., S. Bolte, E.D., J.G., K.K., R.H., A.K.-B., L.M.K., D.L.,
U.L., A.M., C.R., U.S., Ö.T., I.V. and M.V. have securities from BioNTech SE; D.C., M.C., P.R.D., K.U.J.,
33. Xie, X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 27, 841–848.e3 W.K., J.L., J.L.P., I.L.S. and K.A.S. are employees at Pfizer and may have securities from Pfizer;
(2020). C.A.K. is an officer at Regeneron Pharmaceuticals, Inc; A.B., C.A.K. and K.P. have securities
34. Muruato, A. E. et al. A high-throughput neutralizing antibody assay for COVID-19 from Regeneron Pharmaceuticals, Inc; C.F.-G. and P.-Y.S. received compensation from Pfizer to
diagnosis and vaccine evaluation. Nat. Commun. 11, 4059 (2020). perform the neutralization assay; no other relationships or activities that could appear to have
35. Moodie, Z., Huang, Y., Gu, L., Hural, J. & Self, S. G. Statistical positivity criteria for the influenced the submitted work.
analysis of ELISpot assay data in HIV-1 vaccine trials. J. Immunol. Methods 315, 121–132
(2006).
36. Moodie, Z. et al. Response definition criteria for ELISPOT assays revisited. Cancer Additional information
Immunol. Immunother. 59, 1489–1501 (2010). Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
37. U.S. Department of Health and Human Services. Toxicity grading scale for healthy adult 2814-7.
and adolescent volunteers enrolled in preventive vaccine clinical trials. https://www.fda. Correspondence and requests for materials should be addressed to U.S.
gov/regulatory-information/search-fda-guidance-documents/ toxicity-grading-scale- Peer review information Nature thanks Barbra Richardson and the other, anonymous,
healthy-adult-and-adolescent-volunteers-enrolled-preventive-vaccine-clinical reviewer(s) for their contribution to the peer review of this work.
(2007). Reprints and permissions information is available at http://www.nature.com/reprints.
 Prime Boost

Day -30 -1 1 8 22 29 43

Screening
Antibody analysis X X X X X
T cell analysis X X

Extended Data Fig. 1 | Schedule of vaccination and assessment. Study

participants received a prime immunisation with BNT162b1 on day 1 (all dose
levels), and a boost immunisation on day 22 ± 2 (all dose levels except 60 μg).
Serum was obtained on day 1 (pre-prime), 8 ± 1 (post-prime), 22 ± 2 (pre-boost),
29 ± 3 and 43 ± 4 (post-boost). PBMCs were obtained on day 1 (pre-prime) and
29 ± 3 (post-boost).
Article
a Swelling Redness

1 g 10 g 30 g 50 g 60 g 1 g 10 g 30 g 50 g 60 g None
12 12
Mild
10

Number of subjects
10
Number of subjects

Moderate
8
8 Severe
6 6

4 4

2 2

0 0
Day

24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28
Day

7
24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28
7

Pain
1 g 10 g 30 g 50 g 60 g
12
10
Number of subjects

8
6
4
2
0
Day
24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28
7

b Fatigue Chills
1 g 10 g 30 g 50 g 60 g 1 g 10 g 30 g 50 g 60 g
12 12
10 10
Number of subjects

Number of subjects
None
8 8
Mild
6 6 Moderate
4 4 Severe
2 2
0 0
Day Day
24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22

24
26

26
28
22
26
28

1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28
7

Headache Joint pain

1 g 10 g 30 g 50 g 60 g 1 g 10 g 30 g 50 g 60 g
12 12

10 10
Number of subjects
Number of subjects

8 8

6 6

4 4

2 2

0 0
Day Day
24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28
24
26

28
26
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24

24
26
22

22
26
28

7
7

Vomiting Diarrhea
1 g 10 g 30 g 50 g 60 g 1 g 10 g 30 g 50 g 60 g
12 12
10 10
Number of subjects

Number of subjects

8 8
6 6
4 4
2 2
0 0
Day Day
24
26

26
28

24
26

26
28
22

22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9

1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3

3
1

5
7

5
7
24

24
26

24

24
26
22

22

22

22
26
28

26
28
7

Muscle pain Fever

1 g 10 g 30 g 50 g 60 g 1 g 10 g 30 g 50 g 60 g
12 12
10 10
Number of subjects

Number of subjects

8 8
6 6
4 4
2 2
0 0
Day Day
24
26

26
28
22
1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
3
1

5
7

24
26

26
28
22
24

24
26

1
3
5

22

1
3
5
7

24

1
3
5

1
3
5
7
9
22

22

3
1

5
7
26
28

24

24
26
22

22
26
28
7

None Mild Moderate Severe

(<38.0 °C) (38.0-38.4 °C) (38.5-38.9 °C) (39.0-40.0 °C)

Extended Data Fig. 2 | Solicited adverse events. Number of participants with reasons. Grey shading indicates number of participants at each time point. As per
local (a) or systemic solicited adverse events (AEs) (b). Participants were protocol, AEs were recorded up to 7 days after each immunisation (days 1-7 and
immunised with BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 22-28) to determine reactogenicity; for some participants 1-2 additional days of
60 μg). n = 12 subjects were injected per group, from day 22 on n = 11 for the 10 μg follow-up were available. Grading of AEs was performed according to US Food
and 50 μg cohort due to discontinuation of patients due to non-vaccine related and Drug Administration (FDA) recommendations37.
 a CRP b Lymphocytes c Neutrophils
25 Pre 4 Pre Day 2 Day 8 10 Pre Day 2 Day 8
Day 2
20 Day 8 8
3

[Counts/nL]
[Counts/nL]
[mg/L] 15 6
2
10 4
1
5 2

0 0 0
µg

µg

µg

µg

µg

µg

µg

µg

µg

µg

µg

µg

µg

µg

µg
10

30

50

60

10

30

50

60

10

30

50

60
1

1
Extended Data Fig. 3 | Pharmacodynamic markers. Participants were counts. c, Kinetics of neutrophil counts. Dotted lines indicate upper and lower
immunised with BNT162b1 on days 1 (all dose levels) and 22 (all dose levels limit of reference range. For values below the lower limit of quantification
except 60 μg) (n = 12 per group, from day 22 on n = 11 for the 10 μg and 50 μg (LLOQ) = 0.3, LLOQ/2 values were plotted (a).
cohort). a, Kinetics of C-reactive protein (CRP) level. b, Kinetics of lymphocyte
Article
a b c d
Spearman r = 0.4469 Spearman r = 0.48 Spearman r = 0.3299

[log(IFN spots per 1 x 106 cells)]

Spearman r = 0.70

[log(IFN spots per 1 x 106 cells)]

[log(IFN spots per 1 x 106 cells)]

[log(IFN spots per 1 x 106 cells)]
4 P = 0.0038 4 P = 0.0135 4 P <0.0001 4 P = 0.0652

CD8+ T-cell response

CD4+ T-cell response

CD8+ T-cell response

CD4+ T-cell response

3 3 3
3
2 2 2
2
1 1 1

0 0 1 0
2 3 4 5 1 2 3 4 1 2 3 4 1 2 3 4
RBD-specific IgG log(U/mL), day 29 log(VNT50, day 29) CD4+ T-cell response log(VNT50, day 29)
[(IFN spots per 1 x 106 cells)]

Extended Data Fig. 4 | Correlation of antibody and T cell responses. IgG responses (as in Fig. 1) with CD4+ T cell responses on day 29 (as in Fig. 3).
Participants were immunised with BNT162b1 on days 1 (all dose levels) and 22 r = 0.4829, P = 0.0014. b, Correlation of VNT50 (as in Fig. 2a) with CD4+ T cell
(all dose levels except 60 μg) (n = 12 per group, from day 22 on n = 11 for the 10 μg responses (as in Fig. 3). r = 0.48, P = 0.0057. c, Correlation of CD4+ with CD8+
and 50 μg cohort). Data are plotted for all prime/boost vaccinated participants T cell responses (n = 51 as in Fig. 3a) from day 29 in dose cohorts 1 to 60 μg.
(cohorts 1, 10, 30 and 50 μg) with data points for participants with no r = 0.7, P < 0.0001. d, Correlation of VNT50 (as in Fig. 2a) with CD8+ T cell
detectable T cell response (open circles; a, b, d) excluded from correlation responses (as in Fig. 3) on day 29. r = 0.3299, P = 0.0652.
analysis. Nonparametric Spearman correlation. a, Correlation of RBD-specific
Extended Data Table 1 | Demographic characteristics

N, number of subjects in the specified group. This value is the denominator for the percentage calculations. n, number of subjects with the specified characteristics.
Article
Extended Data Table 2 | Subject disposition and analysis sets

Antibody analysis: Values indicate number of participants for whom virus neutralisation assays and RBD binding IgG antibody assays were performed. T cell analysis: Values indicate number of
participants for whom IFNγ ELISpot and flow cytometry (values in parentheses) were performed. N/A, not applicable. *9 for CD4+ response data.
Extended Data Table 3 | BNT162b1-induced geometric mean RBD-binding IgG concentrations and 95% confidence intervals

Geometric mean RBD-binding IgG concentration values and 95% confidence intervals by cohort and sampling time-point as displayed in Fig. 1. CI, confidence interval; N, Sample number; HCS,
Human COVID-19 convalescent sample.
Article
Extended Data Table 4 | BNT162b1-induced virus geometric mean 50% neutralization titers and 95% confidence intervals

Geometric mean 50% virus neutralisation titer values (VNT50) and 95% confidence intervals by cohort and sampling time-point as displayed in Fig. 2a. CI, confidence interval; N, Sample num-
ber; HCS, Human COVID-19 convalescent sample.
Extended Data Table 5 | BNT162b1-induced geometric mean 50% pseudovirus neutralization titers and 95% confidence
intervals

Geometric mean 50% pseudovirus neutralisation titer (pVNT50) values and 95% confidence intervals by tested SARS-CoV-2 spike protein variants as displayed in Fig. 2c. CI, confidence interval;
N, Sample number; HCS, Human COVID-19 convalescent sample; Variant, SARS-CoV-2 spike protein variant.
Article
Extended Data Table 6 | BNT162b1-induced mean cytokine values and 95% confidence intervals

Mean values and 95% confidence intervals for the individual cytokines tested as in Fig. 4b. CI, confidence interval; N, Sample number.
 nature research | reporting summary
Corresponding author(s): Ugur Sahin
Last updated by author(s): Sep 16, 2020

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Policy information about availability of computer code
Data collection Flow cytometry data was collected using the FACS VERSE instrument (BD Biosciences) and FACSSuite software version 1.0.6 and analysed
with FlowJo software version 10.5.3 (FlowJo LLC, BD Biosciences).
ELISpot plates were scanned using an AID Classic Robot ELISPOT Reader and analysed by AID ELISPOT 7.0 software (AID Autoimmun
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For SARS-CoV-2 neutralisation assay, total cell counts per well were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally
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Data analysis Flow cytometry data was analysed using FlowJo software version 10.5.3 (FlowJo LLC, BD Biosciences).
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October 2018

(EDA), based on two statistical tests (distribution-free resampling) according to Moodie et al. (refer to Material&Methods section in the
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1
 titre (VNT50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in fluorescent viral foci.
Cytokine profiles in PBMC supernatants were analysed using ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific).

nature research | reporting summary

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Life sciences study design

All studies must disclose on these points even when the disclosure is negative.
Sample size In the part of the clinical study reported here five dose levels (1 μg, 10 μg, 30 μg, 50 μg or 60 μg) of the BNT162b1 vaccine candidate were
assessed at one site in Germany with 12 healthy volunteers per dose level in a dose escalation and de-escalation design. Sentinel dosing was
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Data exclusions Clinical data available until data extraction date of 13JUL2020 were included. All participants with data available were included in the safety
and immunogenicity analyses.
For serology/cell-mediated immunity correlation analyses (Ext. Data Fig. 4 a/b/d), data were only plotted for prime/boost vaccinated
participants (excluding the 60 μg dose level cohort) with detectable T-cell response.
All participants with sufficient PBMC material available were included in the ICS analyses. In Fig. 4b, CD4 non-responders (<0.03% total
cytokine producing T cells; 1 μg, n=5; 10 μg, n=1; 30 μg, n=2; 50 μg, n=1; 60 μg, n=6) were excluded.
All participants with sufficient PBMC material available were included in the ELISPOT analyses. In Fig.3c, participants without a T-cell response
were excluded, and data from the 60 μg cohort were excluded. In Ext. Data Fig. 4c, only data from participants with both CD4+ and CD8+ T-
cell responses were included.
For cytokine analysis in Fig. 4d, only assay results from n=5 participants of the 50 μg cohort were available by the time of submission/re-
submission. The remaining samples from this and other cohorts were prioritized for other analyses.
Data shown are preliminary and not fully source-data verified.

Replication A parallel clinical study of very similar design has been conducted in the USA involving the same populations, vaccine candidates and doses.
The results for safety and immunogenicity align closely. The US study is randomized placebo controlled.
Serology: Participant sera were tested in duplicate and geometric mean concentration (RBD-specific IgG dLIA) or titer (virus neutralisation and
pseudovirus neutralisation assay) were plotted.
T cell immunity: Participant PBMCs were tested as single instance in ICS analyses. Participant PBMCs were tested in duplicates in ELISpot
analyses. Spot counts were summarized as mean values of each duplicate.
Data shown are preliminary and not fully source-data verified.

Randomization Randomization was not performed in order to facilitate operational efficiencies with the sentinel design, also knowing that a parallel
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Blinding This is a non-randomized open-label phase I/II trial. Investigators were not blinded in order to facilitate operational efficiencies with the
sentinel design, also knowing that a parallel randomized, placebo-controlled study was being conducted in the same vaccine constructs in the
October 2018

USA.

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Materials & experimental systems Methods

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n/a Involved in the study n/a Involved in the study
Antibodies ChIP-seq
Eukaryotic cell lines Flow cytometry
Palaeontology MRI-based neuroimaging
Animals and other organisms
Human research participants
Clinical data

Antibodies
Antibodies used Flow cytometry (specificity/host+reactivity/fluorochrome/clone/manufacturer/catalogue number/lot number/dilution/extra- or
intracellular):
CD3/mouse anti-human/BV421/UCHT1/BD Biosciences/562426/9113553/1:250/extracellular+intracellular
CD4/mouse anti-human/BV480/RPA-T4/BD Biosciences/746541/0171955/1:50/extracellular+intracellular
CD8/mouse anti-human/BB515/RPA-T8/BD Biosciences/564526/0037189/1:100/extracellular+intracellular
IFNγ /mouse anti-human/PE-Cy7/B27/BD Biosciences/557643/9332967/1:50/intracellular
IL-2/rat anti-human/PE/MQ1-17H12/BD Biosciences/554566/9337013/1:10/intracellular
IL-4/rat anti-human/APC/MP4-25D2/BD Biosciences/554486/9185677/1:500/intracellular

Fixable Viability Dye/eF780/eBioscience/65-0865-14/2185428/1:1,666

ELISpotPro kit/cat. no. 3420-2APT-10/lot no. 370/Mabtech:

Primary anti-IFNg antibody/clone c1-D1K/pre-coated plates
Secondary anti-IFNg antibody/clone 7-B6-1 (ALP conjugate)/1:250
CD3/clone CD3-2/1:1,000

RBD-binding IgG assay:

goat anti-human IgG/R-PE/polyclonal/Jackson Labs/109-115-098/147186/1:500

Validation Commercially available antibodies were selected based on their antigen specificity and suggested application as described on the
manufacturer`s website and data sheets. The antibody concentrations for staining were optimized by titrating down each
reagent starting at the manufacturer`s recommendation. The optimal amounts of the reagents were defined by (i) minimal
unspecific shift of the negative population and (ii) a maximal separation of the negative and positive population. Individual
antibody validation reports are not evident from the BD Biosciences website.

Eukaryotic cell lines

Policy information about cell lines
Cell line source(s) Vero cells (CCL-81), Vero E6 cells (CRL-1586) and HEK293T (CRL-3216) were obtained from ATCC.

Authentication Vero and Vero E6 cells were sourced from ATCC, which maintains a quality management system commensurate to ISO
9001:2015, ISO 13485:2016, ISO 17025:2017, and ISO 17034:2016. Cells were certified by the vendor and propagated
according to the manufacturer’s instructions.

Mycoplasma contamination All used cell lines were tested negative for mycoplasma contamination after receipt and before expansion and
cryopreservation.

Commonly misidentified lines No commonly misidentified cell lines were used.

(See ICLAC register)

Human research participants

Policy information about studies involving human research participants
Population characteristics Healthy men and non-pregnant women 18 to 55 years (amended to add 56 -85 of age) of age with equal gender distribution.
Most participants were Caucasian (96.7%) with one African American and one Asian subject (1.7% each). Key exclusion criteria
included previous clinical or microbiological diagnosis of COVID-19; receipt of medications to prevent COVID-19; previous
October 2018

vaccination with any coronavirus vaccine; a positive serological test for SARS-CoV-2 IgM and/or IgG at the screening visit; and a
SARS-CoV-2 NAAT-positive nasal swab within 24 hours before study vaccination; those with increased risk for severe COVID-19;
immunocompromised individuals, those with known infection with HIV, hepatitis C virus, or hepatitis B virus and those with a
history of autoimmune disease.

Recruitment Recruitment was performed by teaching investigators according to inclusion and exclusion criteria without any bias. No protocol-
specified methods. The sites are experienced phase 1 units with established rostas of potential subjects who they can invite for
screening for inclusion. Also the sites advertise through their own web-site. Some subjects self-referred via the sponsor.

3
 Ethics oversight The trial was carried out in Germany in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines and

nature research | reporting summary

with approval by an independent ethics committee (Ethik-Kommission of the Landesärztekammer Baden-Württemberg,
Stuttgart, Germany) and the competent regulatory authority (Paul-Ehrlich Institute, Langen, Germany). All subjects provided
written informed consent.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Clinical data
Policy information about clinical studies
All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

Clinical trial registration ClinicalTrials.gov Identifier: NCT04380701, see also manuscript

Study protocol The full clinical study protocol is not published online, but a comprehensive description of the clinical trial design, eligibility
criteria and endpoints is available at https://clinicaltrials.gov/ct2/show/study/NCT04380701.

Data collection Serum for antibody assays was obtained on day 1 (pre-prime), 8±1 (post-prime), 22±2 (pre-boost), 29±3 and 43±4 (post-boost).
PBMCs for T cell studies were obtained on day 1 (pre-prime) and 29±3 (post-boost). Tolerability was assessed by patient diary.
All formal protocol-determined visits were conducted on-site at the investigators premises (in each case a dedicated phase 1
unit). All study procedures such as blood sample, physical examinations, screening checks were conducted at the study sites. The
only exceptions were the completion of the subject diaries, which was done by the subjects at home. Diaries were collected by
the sites at the subjects' next scheduled visits and the data entered on site. There was also dedicated telephone follow-up, 48
hrs following dosing, to ensure subject well-being, which was documented on site by the investigator conducting the call.

Outcomes Primary objective: To describe the safety and tolerability profiles of prophylactic BNT162 vaccines in healthy adults after single
dose (SD; prime only) or prime/boost (P/B) immunization.
Endpoints: Solicited local reactions & solicited systemic reactions (listed in subject diaries, to be graded by subjects) and
unsolicited treatment-emergent adverse events.

Secondary objectives: To describe the immune response in healthy adults after SD or P/B immunization measured by a functional
antibody titer, e.g., virus neutralization test or an equivalent assay available by the time of trial conduct.
Endpoints: Functional antibody responses; fold increasese in functional antibody titers; number of subjects with seroconversion

Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Cytokine-producing T cells were identified by intracellular cytokine staining. PBMCs thawed and rested for 4 hours in OpTmizer
medium supplemented with 2 μg/mL DNAseI (Roche), were restimulated with a peptide pool representing the vaccine-encoded
SARS-CoV-2 RBD (2 μg/mL/peptide; JPT Peptide Technologies) in the presence of GolgiPlug (BD) for 18 hours at 37 °C. Controls
were treated with DMSO-containing medium. Cells were stained for viability and surface markers in flow buffer ((DPBS (Gibco)
supplemented with 2% FCS (Biochrom), 2 mM EDTA (Sigma-Aldrich)) for 20 minutes at 4 °C. Afterwards, samples were fixed and
permeabilized using the Cytofix/Cytoperm kit according to manufacturer’s instructions (BD Biosciences). Intracellular staining
was performed in Perm/Wash buffer for 30 minutes at 4 °C.

Instrument Samples were acquired on a FACS VERSE instrument (BD Biosciences).

Software For data analysis FlowJo software version 10.5.3 (FlowJo LLC, BD Biosciences) was used.

Cell population abundance Bulk PBMCs were used. No cell sorting was performed.
October 2018

Gating strategy The gating strategies are detailed in the respective figure or in the supplementary information. Briefly, singlets were gated based
on their location in the FSC-A/FSC-H plot. Debris was exclduded in the subsequent FSC-A/viability dye plot. Viable cells were
gated from non-debris in the FSC-A/viability dye plot. From viable cells, lymphocytes were gated based on their size and
granularity in the FSC-A/SSC-A plot. From lymphocytes, CD3+ T cells were gated in the CD3/SSC-A plot. From CD3+ T cells, CD4+
and CD8+ T cells were gated in the CD4/CD8 plot. From CD4+ T cells, IFNg+, IL-2+, IL-4+ or IFNg+ IL-2+ T cells were gated by
plotting CD4/IFNg, CD4/IL-2, CD4/IL-4, or IFNg/IL-2. From CD8+ T cells, IFNg+, IL-2+ or IFNg+ IL-2+ T cells were gated by plotting
CD8/IFNg, CD8/IL-2, or IFNg/IL-2.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.