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EXHIBIT 

Article

BNT162b vaccines protect rhesus macaques
from SARS-CoV-2
https://doi.org/10.1038/s41586-021-03275-y
Received: 1 September 2020
Accepted: 20 January 2021
Published online: 1 February 2021
Check for updates

Annette B. Vogel1,10, Isis Kanevsky2,10, Ye Che3,10, Kena A. Swanson2, Alexander Muik1,
Mathias Vormehr1, Lena M. Kranz1, Kerstin C. Walzer1, Stephanie Hein1, Alptekin Güler1,
Jakob Loschko2, Mohan S. Maddur2, Ayuko Ota-Setlik2, Kristin Tompkins2, Journey Cole4,
Bonny G. Lui1, Thomas Ziegenhals1, Arianne Plaschke1, David Eisel1, Sarah C. Dany1,
Stephanie Fesser1, Stephanie Erbar1, Ferdia Bates1, Diana Schneider1, Bernadette Jesionek1,
Bianca Sänger1, Ann-Kathrin Wallisch1, Yvonne Feuchter1, Hanna Junginger1,
Stefanie A. Krumm1, André P. Heinen1, Petra Adams-Quack1, Julia Schlereth1, Stefan Schille1,
Christoph Kröner1, Ramón de la Caridad Güimil Garcia1, Thomas Hiller1, Leyla Fischer1,
Rani S. Sellers2, Shambhunath Choudhary2, Olga Gonzalez4, Fulvia Vascotto5,
Matthew R. Gutman6, Jane A. Fontenot7, Shannan Hall-Ursone4, Kathleen Brasky4,
Matthew C. Griffor3, Seungil Han3, Andreas A. H. Su1, Joshua A. Lees3, Nicole L. Nedoma3,
Ellene H. Mashalidis3, Parag V. Sahasrabudhe3, Charles Y. Tan2, Danka Pavliakova2,
Guy Singh2, Camila Fontes-Garfias8, Michael Pride2, Ingrid L. Scully2, Tara Ciolino2,
Jennifer Obregon2, Michal Gazi9, Ricardo Carrion Jr4, Kendra J. Alfson9, Warren V. Kalina2,
Deepak Kaushal4, Pei-Yong Shi8, Thorsten Klamp1, Corinna Rosenbaum1, Andreas N. Kuhn1,
Özlem Türeci1, Philip R. Dormitzer2, Kathrin U. Jansen2 & Ugur Sahin1,5ಞᅒ

A safe and effective vaccine against COVID-19 is urgently needed in quantities
that are sufficient to immunize large populations. Here we report the preclinical
development of two vaccine candidates (BNT162b1 and BNT162b2) that contain
nucleoside-modified messenger RNA that encodes immunogens derived from the
spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1
encodes a soluble, secreted trimerized receptor-binding domain (known as the
RBD–foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein,
locked in its prefusion conformation by the substitution of two residues with proline
(S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of
the RBD–foldon bind to human ACE2 with high avidity. Approximately 20% of the
S(P2) trimers are in the two-RBD ‘down’, one-RBD ‘up’ state. In mice, one intramuscular
dose of either candidate vaccine elicits a dose-dependent antibody response with
high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell
responses. Prime–boost vaccination of rhesus macaques (Macaca mulatta) with the
BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are
8.2–18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine
candidates protect macaques against challenge with SARS-CoV-2; in particular,
BNT162b2 protects the lower respiratory tract against the presence of viral RNA and
shows no evidence of disease enhancement. Both candidates are being evaluated in
phase I trials in Germany and the USA1–3, and BNT162b2 is being evaluated in an
ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Owing to the effects of the current pandemic of coronavirus disease 2019 (COVID-19) on human health and society, several collaborative research programmes have been launched and have generated
insights and progress in vaccine development. Soon after emerging in
December 2019, SARS-CoV-2 was identified as a betacoronavirus with
high sequence similarity to bat-derived SARS-like coronaviruses4,5. The
fast availability of vaccines is critical in the pandemic, and the rapid
globalized response is mirrored by the upload of over 212,000 viral

genome sequences as of 23 November 2020 to the Global Initiative on
Sharing All Influenza Data.
The trimeric S of SARS-CoV-2 is a key target for virus-neutralizing
antibodies6 and the prime candidate for vaccine development. S binds
its cellular receptor ACE2 through an RBD, which is part of S1 (the
N-terminal furin cleavage fragment of S)7,8. On S, the RBDs are in ’up’
positions, in which the receptor-binding sites and their dense cluster
of neutralizing epitopes are exposed, or in ‘down’ positions, in which

A list of affiliations appears at the end of the paper.

Nature | Vol 592 | 8 April 2021 | 283

Article
a
5′ UTR

SP RBD Foldon
Cap analogue
BNT162b2 RNA
S1
S2

5′ UTR

b

BNT162b1 RNA
3′ UTR

SP

AAAA
Poly(A) tail
3′ UTR
AAAA
Poly(A) tail

RBD

Cap analogue

Relative fluorescence units

the receptor-binding sites are buried but some S neutralizing epitopes
on and off the RBDs remain available9–12. S rearranges to translocate the
virus into cells by membrane fusion9,13. The C-terminal furin cleavage
fragment of S (S2) contains the fusion machinery14.
Messenger RNA (mRNA) technology allows the versatile design of
vaccine antigens as well as highly scalable and fast manufacturing. With
efficient lipid-nanoparticle-formulation processes, RNA vaccines are
highly suited to the rapid development and supply needed during a
pandemic15. RNA generated from DNA templates by a highly productive,
cell-free in vitro transcription process is molecularly well-defined and
free of materials of animal origin. Here we report the preclinical development of lipid-nanoparticle-formulated, N1-methyl-pseudouridine
(m1Ψ) nucleoside-modified mRNA (modRNA) BNT162b vaccine candidates (BNT162b1 and BNT162b2) that encode immunogens derived from
the S of SARS-CoV-2 (Fig. 1a). The m1Ψ modification dampens innate
immune sensing and—together with optimized noncoding sequence
elements—increases the efficiency of RNA translation in vivo16–18. Vaccines based on modRNA have proven to be immunogenic for several
viral targets19,20.
Both of the BNT162b vaccines are being evaluated in phase I clinical trials in the USA (NCT04368728) and Germany (NCT04380701,
EudraCT: 2020-001038-36), and BNT162b2 is being evaluated in a global
phase II/III safety and efficacy study1–3.

S K986P
V987P

c

BNT162b1 RNA
BNT162b2 RNA

40,000
30,000
20,000
10,000

0
1,600 1,800 2,000 2,200
Time (s)

d

e
RBD

RBD
Q498/
N501

f

ACE2

ACE2

B0AT1

B0AT1

Y41 Q42
R357

Y453
K353
H34

K417

D30
N487
Q24

g

Construct design and analysis of expressed antigen
BNT162b1 RNA encodes the RBD with the SARS-CoV-2 S signal peptide
fused to its N terminus (to enable endoplasmic reticulum translocation and secretion) and with the trimerization domain (foldon) of T4
fibritin21 fused to its C terminus for multimeric display. BNT162b2 RNA
encodes full-length S that is stabilized in the prefusion conformation
by substitution of residues 986 and 987 to proline (that is, S(P2))10,22,23
(Fig. 1a). The microfluidic capillary electrophoresis profiles of both
of the RNAs show single sharp peaks that are consistent with their
calculated lengths, indicating high purity and integrity (Fig. 1b). We
detected robust expression of RBD–foldon or S(P2) by flow cytometry
upon transfection of HEK293T cells with BNT162b1 RNA or BNT162b2
RNA formulated as lipid nanoparticles or mixed with a transfection
reagent, respectively (Extended Data Fig. 1a). In transfected cells, the
BNT162b1-encoded RBD or BNT162b2-encoded S(P2) localized to the
secretory pathway, as shown by immunofluorescence microscopy
(Extended Data Fig. 1b). We performed western blot under denaturing and non-denaturing conditions, and detected a main band of
RBD-containing protein with an apparent molecular mass of more than
75 kDa (together with lesser quantities of a faster-migrating species) in
the medium of cells transfected with BNT162b1 RNA, consistent with
the secretion of trimeric RBD–foldon (which has a predicted molecular
mass of 88.4 kDa) (Extended Data Fig. 1c).
For further structural characterization, we expressed the RBD–
foldon and S(P2) antigens from DNA that corresponds to the RNA
coding sequences. We purified the RBD–foldon from the medium
of transfected Expi293F cells by affinity capture with the peptidase
domain of ACE2 immobilized on agarose beads, which left little residual
RBD–foldon uncaptured from the medium. We obtained evidence
that the RBD–foldon has three RBDs flexibly tethered to a central hub
using electron microscopy, which revealed a variety of conformations
(Fig. 1c). The trimerized RBD bound to the peptidase domain of human
ACE2 with an apparent KD of less than 5 pM, which is 1,000-fold the
reported KD (5 nM) for monomeric RBD and is consistent with the avidity
effect of multivalent binding that is enabled by the flexible tethering
(Extended Data Fig. 1d). Although the flexibility of the RBD–foldon precluded direct structural analysis at high resolution, one RBD per trimer
could be immobilized by binding to a complex of ACE2 and the B0AT1
neutral amino acid transporter (which is chaperoned by ACE2) when
that complex was in the previously reported closed conformation8
284 | Nature | Vol 592 | 8 April 2021

Fig. 1 | Vaccine design and characterization of the expressed antigens.
a, Structure of BNT162b1 and BNT162b2 RNA. UTR, untranslated region; SP,
signal peptide. The proline subsitutions of S(P2) (K986P and V897P) are
indicated. b, Liquid capillary electropherograms of in vitro-transcribed
BNT162b1 and BNT162b2 RNA. Peaks represent individual samples merged into
one graph. c, Representative 2D class averages from electron microscopy of
negatively stained RBD–foldon trimers. Box edge, 37 nm. d, Two-dimensional
class average from cryo-EM of the ACE2–B0AT1–RBD–foldon trimer complex.
Long box edge, 39.2 nm. Peripheral to the relatively well-defined density of
each RBD domain bound to ACE2, there is diffuse density that we attribute to
the remainder of the flexibly tethered RBD–foldon trimer. A detergent micelle
forms the density at the end of the complex opposite the RBD–foldon.
e, Density map of the ACE2–B0AT1–RBD–foldon trimer complex at 3.24 Å,
after focused refinement of the ACE2 extracellular domain bound to a RBD
monomer. Surface colour-coding is by subunit. The ribbon model refined to
the density shows the RBD–ACE2 binding interface.Residues that potentially
mediate polar interactions are labelled. f, A 3.29 Å cryo-EM map of S(P2) with
fitted and refined atomic model, viewed down the threefold axis towards the
membrane (left) and viewed perpendicular to the threefold axis (right). The
map is coloured by protomer. g, Mass density map of TwinStrep-tagged S(P2)
produced by 3D classification of images extracted from cryo-EM micrographs
with no symmetry averaging, showing the class in the one-RBD-up and
two-RBD-down position.

(Fig. 1d). The size and symmetry of the RBD–foldon–ACE2–B0AT1 ternary complex aided image reconstruction by cryo-electron microscopy
(cryo-EM), and we determined the structure of the RBD in the complex
to a resolution of 3.24 Å (Fig. 1e, Extended Data Table 1, Supplementary Fig. 2). One copy of the RBD was resolved for each bound trimer.
The binding interface between the resolved RBD and the extracellular domain of ACE2 was fitted to a previously reported structure7,
and showed good agreement. The high-avidity binding to ACE2 and
well-resolved structure in complex with ACE2 demonstrate that the
recombinant RBD–foldon authentically presents the ACE2-binding
site that is targeted by many SARS-CoV-2-neutralizing antibodies11,24.
We affinity-purified the trimeric S(P2) from detergent-solubilized
protein via a C-terminal TwinStrep tag. S(P2) bound the peptidase

a

b
1,000

BNT162b2
0.2 μg

Control

P < 0.0001

5 μg

1 μg

Control

P = 0.0004

1,000

0.2 μg

1 μg

Control
5 μg BNT162b1
5 μg BNT162b2

1

100
P = 0.0381

pVNT50

10

5 μg

1,000

P = 0.0005

100
pVNT50

IgG (μg ml–1)

BNT162b1

P < 0.0001

10

P = 0.0002
P = 0.0015
100
P = 0.0678
10

<0.1
10
20
Time (d after vaccination)

300

300

200

200

200
P = 0.0077

P = 0.0162
100

200

500

P = 0.0045

100

P = 0.0062

100

P = 0.0012

P = 0.0689
0

0

M
ed
i
pe um
pt
id
e
M
ed
S iu
pe m
pt
id
e
S

M
ed
i
pe um
pt
id
e
M
e
S diu
pe m
pt
id
e

300

P = 0.0037

0

S

IL-13

0

0

M
ed
i
pe um
pt
id
e
M
ed
S iu
pe m
pt
id
e

0

30

S

250

IL-5

M
ed
i
pe um
pt
id
e
M
e
S diu
pe m
pt
id
e

500

P = 0.0002

400

1,000

IL-4

IL-2
600

15
20
25
Time (d after vaccination)

S

750

1,500

10

M
ed
i
pe um
pt
id
e
M
e
S diu
pe m
pt
id
e

P < 0.0001

1,000

P = 0.0374
P = 0.0142

30

S

1,250

Cytokine (pg ml–1)

1,500

15
20
25
Time (d after vaccination)

M
ed
i
pe um
pt
id
e
M
ed
S iu
pe m
pt
id
e

P < 0.0001

10

IFNγ

d
IFNγ spots per 5 × 105 cells

c

30

S

0

Fig. 2 | Mouse immunogenicity. We injected BALB/c mice (n = 8)
intramuscularly with a single dose of one of the BNT162b vaccine candidates or
a buffer control. Geometric means of each group ± 95% confidence interval are
shown. Day-28 P values compared to control (multiple comparison of
mixed-effect analysis using Dunnett’s multiple comparisons test) for the single
time points and groups are provided in a, b. a, Levels of RBD-specific IgG in sera
of mice immunized using 5 μg of BNT162b1 or BNT162b2, determined by
enzyme-linked immunosorbent assay (ELISA). For day-0 values, a prescreening
of randomly selected mice was performed (n = 4). Extended Data Figure 3a, b
shows IgG levels with lower BNT162b doses and sera testing for detection of S1.
b, Pseudovirus-based VSV-SARS-CoV-2 50% neutralization titres (pVNT50) in
sera of mice immunized using BNT162b1 (left) or BNT162b2 (right). Extended
Data Figure 3g–i provides the number of infected cells per well with serum

samples drawn 28 d after injection and titre correlation to a SARS-CoV-2 virus
neutralization assay. For cellular response analysis in c, d, splenocytes of
BALB/c mice (n = 8, unless stated otherwise) injected intramuscularly
with BNT162b1 (green) or BNT162b2 (pink) were restimulated ex vivo with
full-length S peptide mix or cell culture medium. Symbols represent individual
mice. Heights of bars indicate the mean. P values compare immunized groups
with the control (parametric, two-tailed paired t-test). c, IFNγ ELISpot of
splenocytes 12 d after injection with 5 μg of one of the BNT162b vaccines.
d, Cytokine production by splenocytes 28 d after injection with 0.2 μg
BNT162b1 or 1 μg BNT162b2, determined by bead-based multiplex analysis
(BNT162b2: n = 7 for IL-4, IL-5 and IL-13, one outlier removed by the ROUT
method (Q = 1%) for the S peptide stimulated samples).

domain of human ACE2 and a human anti-RBD neutralizing antibody
(B38) with high affinity25 (an apparent KD of 1 nM for each) (Extended
Data Fig. 1e, f). Our structural analysis by cryo-EM produced a mass
density map at a nominal resolution of 3.29 Å, into which we fitted
and rebuilt a previously published atomic model10 (Fig. 1f, Extended
Data Fig. 2a, b, Extended Data Table 1). The rebuilt model showed
good agreement with previously reported structures of prefusion
full-length wild-type S and its ectodomain with the P2 mutations9,10.
Three-dimensional classification of the dataset showed a class of particles that was in a one-RBD up (accessible for receptor binding), two-RBD
down (closed) conformation; this class represented 20.4% of the trimeric molecules (Fig. 1g, Extended Data Fig. 2c). The remainder of the
trimeric molecules were in an all-RBD down conformation. The RBD
in the up conformation was less well-resolved than the other parts of
the structure, which suggests conformational flexibility and a dynamic
equilibrium between the RBD up and RBD down states, as has previously
been suggested9,26. Our binding and structural analyses indicate that
the BNT162b2 RNA sequence encodes a recombinant S(P2) that can
authentically present the ACE2-binding site and other epitopes that
are targeted by SARS-CoV-2-neutralizing antibodies.

dose-level-dependent manner (Fig. 2a, Extended Data Fig. 3a–d); these
titres increased more steeply for BNT162b2. On day 28 after injection
with 5 μg BNT162b1 or BNT162b2, geometric mean endpoint titres of
RBD-binding serum IgG were 752,680 or 434,560, respectively. Polyclonal IgG elicited by either of the candidate vaccines had strong apparent binding affinity for a recombinant RBD target antigen (geometric
mean apparent KD of 717 pM for BNT162b1 and 993 pM for BNT162b2),
with a low apparent off-rate and a high apparent on-rate (Extended Data
Fig. 3e). Serum samples from buffer-immunized control mice had no
detectable RBD- or S1-specific IgG (Fig. 2a, b, Extended Data Fig. 3a–d),
and neither did serum samples from mice injected up to two times with
equivalent modRNA, formulated in lipid nanoparticles, that encoded
a SARS-CoV-2 irrelevant antigen (data not shown).
We measured the inhibition of virus entry by BNT162b-immunized
mouse serum in a neutralization assay using vesicular stomatitis virus
(VSV)-based SARS-CoV-2 pseudovirus. As with the antigen-specific IgG
geometric mean titres (GMTs), 50% pseudovirus-neutralization GMTs
increased steadily after injection of 5 μg of either candidate vaccine, and
reached 1,056 for BNT162b1 and 296 for BNT162b2 on day 28 after injection (Fig. 2b, Extended Data Fig. 3f, g). We tested a random selection
of samples in a SARS-CoV-2 neutralization assay, which demonstrated
strong correlation of pseudovirus and SARS-CoV-2 neutralization (Pearson correlation of 0.9479 between the tests) (Extended Data Fig. 3h). In
summary, each candidate vaccine induced a high functional antibody
response in mice, and BNT162b1 induced higher titres than BNT162b2
after one injection.
Our characterization of antigen-specific responses of splenic T cells
in mice at 12 and 28 days after injection with the BNT162b vaccines

BNT162b-elicted immunogenicity in mice
To study vaccine immunogenicity, we characterized B and T cell
responses in a series of experiments in BALB/c mice after a single intramuscular injection of 0.2, 1 or 5 μg of BNT162b1 or BNT162b2, or of a
buffer control. A single immunization using either of the candidate
vaccines induced high titres of RBD- and S1-binding serum IgG in a

Nature | Vol 592 | 8 April 2021 | 285

Article
a

BNT162b1
30 μg
105

20,962
13,575
8,280
4,444

RBD-binding IgG (U ml–1)

104

854
926

b

BNT162b2

100 μg

30 μg

48,575
32,644
21,125

2,994
1,238

BNT162b1

100 μg

30 μg

26,170
23,781
16,316
12,293
10,898
8,172
5,442 602
2,846
2,759
1,488
1,859
1,436

30 μg

100 μg

104
1,689
1,277
1,007

1,714
809

637

94
283

282
247
201

VNT50

103

962

975
564

768

103

102

BNT162b2
100 μg

285

58

81

65

18

102

47

33

1.7

54

18

101
10

101

100
C 14 21 28 35 42 56 14 21 28 35 42 14 21 28 35 42 56 14 21 28 35 42 56 HCS

C 14 21 28 35 42 56 14 21 28 35 42 14 21 28 35 42 56 14 21 28 35 42 56 HCS

Day

IFNγ spots per 1 × 106 cells

4,000

0 μg

30 μg

100 μg

3,000
1,500

d

4,000
IL-4 spots per 1 × 106 cells

c

Day

1,000

500

0 μg

30 μg

100 μg

3,000
1,500

1,000

500

0

0
0 14 28 42

0 14 28 42

0 14 28 42

0 14 28 42

Day

0 14 28 42

0 14 28 42

Day
−1

Fig. 3 | Macaque immunogenicity. Male macaques (2–4 years old) were
injected on day 0 and day 21 (arrows below the x-axes indicate the day of second
injection) with 30 μg or 100 μg of BNT162b1 (green) or BNT162b2 (pink)
(n = 6 each). Additional macaques received saline (control (C), n = 9)). Human
convalescent sera (HCS) were obtained from patients infected with SARS-CoV-2
at least 14 d after PCR-confirmed diagnosis and at a time when acute COVID-19
symptoms had resolved (n = 38). The HCS panel is a benchmark for serology
studies in this Article and previous publications1–3. a, Concentrations (in
arbitrary units) of IgG that binds recombinant SARS-CoV-2 RBD (lower limit of

detection (LLOD) = 1.72 U ml ). b, SARS-CoV-2 50% virus-neutralization titres
(VNT50) (LLOD = 20). c, d, Peripheral blood mononuclear cells collected on
days 0, 14, 28 and 42 after first injection of BNT162b2 were restimulated ex vivo
with full-length S peptide mix. Arrows below the x-axis indicate the days of
dose 1 and dose 2. c, IFNγ ELISpot. d, IL-4 ELISpot. Heights of bars indicate the
geometric (a, b) or arithmetic (c, d) means for each group, and values are
written above the bars (a, b). Whiskers indicate 95% confidence intervals (a, b)
or s.e.m. (c, d). Each symbol represents one macaque. Horizontal dashed line
marks the LLOD. Values below the LLOD were set to 1/2 the LLOD.

revealed a high fraction of CD4+ and CD8+ T cells that produced IFNγ and
CD8+ cells that produced IL-2, as shown by enzyme-linked immunospot
assay (ELISpot) or intracellular-cytokine-staining flow cytometry analysis after ex vivo restimulation with a full-length S peptide pool (Fig. 2c,
Extended Data Fig. 4a, b). Total splenocytes collected on day 28 and
restimulated with the full-length S peptide pool secreted high levels of
the T-helper-1 (TH1) cytokines IL-2 or IFNγ, and minute or undetectable
levels of the T-helper-2 (TH2) cytokines IL-4, IL-5 or IL-13, as measured
in multiplex immunoassays (Fig. 2d). Overall, the patterns of CD4+ and
CD8+ T cell responses were similar for the two vaccine candidates, with
a somewhat stronger IFNγ-producing CD8+ T cell response in mice
immunized with BNT162b2.
We assessed vaccine-induced effects on the proliferation and
dynamics of immune-cell populations in injection-site draining
lymph nodes (to evaluate the principal immune-educated compartments for proficient T and B cell priming) as well as in blood and
spleen (to evaluate systemic effects of the vaccines). We observed
higher numbers of plasma cells, class-switched IgG1+ and IgG2a+
B cells, and germinal-centre B cells in draining lymph nodes, and
higher numbers of class-switched IgG1+ and germinal-centre B cells in
spleens of mice at 12 days after injection with 5 μg of either vaccine as
compared to control (Extended Data Fig. 4c, d). Vaccine-immunized
mice had significantly fewer circulating B cells than did control mice
as measured in blood at day 7 after injection (Extended Data Fig. 4e),
which may imply that B cell homing to lymphoid compartments

contributed to augmented B cell counts in the draining lymph nodes
and spleen.
The draining lymph nodes from BNT162b1- or BNT162b2-immunized
mice also displayed significantly higher counts of CD8+ and CD4+ T cells
(as compared to buffer-immunized mice) at 12 days after injection, which
were most pronounced for T follicular helper (TFH) cells—including
the ICOS+ subsets that are essential for the formation of germinal centres (Extended Data Fig. 4c). Both of the BNT162b vaccines increased TFH
cell counts in the spleen and blood, whereas an increase in circulating
CD8+ T cells was detected only in BNT162b2-immunized mice (Extended
Data Fig. 4d, e). In aggregate, these data indicate a strong induction of
SARS-CoV-2-pseudovirus neutralization titres and systemic CD8+ and
TH1-driven CD4+ T-cell responses by both of the vaccine candidates,
and a somewhat more-pronounced cellular response to BNT162b2.

286 | Nature | Vol 592 | 8 April 2021

BNT162b-elicted immunogenicity in macaques
To assess the immunogenicity of BNT162b1 and BNT162b2 in nonhuman primates, we intramuscularly injected groups of six macaques
(male, 2–4 years old) with 30 or 100 μg of BNT162b1, BNT162b2 or
saline control on day 0 (dose 1) and day 21 (dose 2). RBD-binding IgG
was readily detectable by day 14 after dose 1, and levels had increased
further 7 days after dose 2 (day 28) (Fig. 3a). On day 28, geometric mean
concentrations of RBD-binding IgG were 20,962 units (U) ml−1 (at 30-μg
dose level) and 48,575 U ml−1 (at 100-μg dose level) for BNT162b1, and

b
1010

Sentinel
Control
BNT162b1
BNT162b2

Viral RNA (copies)

7/9

108
4/8
2/6

106
104
0/6 0/9 0/6 0/6 0/6

0/6 0/6

0/6 0/6 0/3 0/6 0/6 0/6

1010
Viral RNA (copies)

a

Pre

104

3
6
Day after challenge

2/9

4/9
2/6

104
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10/EOP

d
Control

104

0/5

0/6

0/6 0/6 0/6

3
6
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1

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BNT162b1

0/6 0/6

0/6 0/6

7-23/EOP

BNT162b2

103
VNT50

103
VNT50

4/9

5/9
5/6

106

102

102

c

Sentinel
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BNT162b2

108

102

102
101

101
Pre 3 6 7 8 14 15 16 21 22 23
Day after challenge

Pre

3

6

10/EOP

Day after challenge

Fig. 4 | Virological and serological evidence of protection of macaques
from challenge with infectious SARS-CoV-2. Macaques that had been
immunized using 100 μg of BNT162b1 or BNT162b2 (n = 6 each) or
mock-immunized with saline challenge (control) (n = 9) were challenged with
1.05 × 106 total plaque-forming units of SARS-CoV-2 split equally between the
intranasal and intratracheal routes. Additional macaques (sentinel) (n = 6) were
mock-challenged with cell culture medium. Macaque assignments to cohorts
and schedules of immunization, challenge and sample collection are provided
in Extended Data Fig. 6, Extended Data Table 2. Viral RNA levels were detected
by RT–qPCR. a, Viral RNA in bronchoalveolar lavage fluid. ‘Pre’, before
challenge; 10/EOP, day 10 or end of the project. b, Viral RNA in nasal swabs;
7–23/EOP, days 7–23 or end of project. Symbols represent individual macaques.
Ratios above bars indicate the number of viral-RNA-positive macaques among
all macaques in a group with evaluable samples. Heights of bars indicate

geometric mean viral RNA copies; whiskers indicate geometric s.d. Dotted
lines indicate LLOD. Values below the LLOD were set to 1/2 the LLOD. Two-sided
statistical significance by a nonparametric test (Friedman’s test) of differences
in viral RNA detection after challenge between six BNT162b1-immunized and
six mock-immunized macaques (challenge cohorts 1 and 2) was P = 0.0152 for
bronchoalveolar lavage fluid and P = 0.0048 for nasal swab; between six
BNT162b2-immunized macaques and three mock-immunized macaques
(challenge cohort 3), the statistical significance was P = 0.0014 for
bronchoalveolar lavage fluid and P = 0.2622 for nasal swabs. Serum samples were
assayed for SARS-CoV-2 VNT50. c, BNT162b1-immunized macaques and controls
(challenge cohorts 1 and 2). d, BNT162b2-immunized macaques and controls
(challenge cohort 3). Symbols represent titres from individual macaques.
Horizontal dashed line indicates the lower limit of quantification of 20.

23,781 U ml−1 (30-μg dose level) and 26,170 U ml−1 (100-μg dose level)
for BNT162b2. For comparison, the geometric mean concentration of
RBD-binding IgG of a panel of 38 SARS-CoV-2-convalescent human sera
was 602 U ml−1, which is lower than the geometric mean concentration
of the immunized macaques after one or two doses.
Fifty per cent virus-neutralization GMTs—measured by a SARSCoV-2-neutralization assay27 (rather than a pseudovirus-neutralization
assay)—were detectable in the sera of most BNT162b1-immunized
macaques by day 21 after dose 1, and in all BNT162b2-immunized
macaques by day 14 after dose 1 (Fig. 3b). There was a strong boosting
effect; comparable peak measured GMTs were elicited by BNT162b1
(768 for 30 μg and 1,714 for 100 μg) and BNT162b2 (962 for 30 μg and
1,689 for 100 μg), as measured in sera drawn 7 or 14 days after dose 2.
For BNT162b2, sera were available up to day 56 after dose 1 (28 days
after dose 2); robust GMTs of 285 for 30-μg and 283 for 100-μg dose
levels persisted to this time point. For comparison, the neutralization
GMT of the human convalescent sera was 94, which is substantially
lower than the GMTs of macaque sera drawn 21 or 35 days after dose 2.
We analysed the S-specific T cell responses of BNT162b2- or salineinjected macaques using peripheral blood mononuclear cells collected
before immunization and at the times indicated after doses 1 and 2.
ELISpot demonstrated strong IFNγ, but minimal IL-4, responses after
dose 2 (Fig. 3c, d, Extended Data Fig. 5a). Intracellular cytokine staining confirmed that BNT162b2 elicited a high frequency of CD4+ T cells
that produced IFNγ, IL-2 or TNF, but a low frequency of CD4+ T cells
that produced IL-4, which indicates a TH1-biased response (Extended
Data Fig. 5b, c). Intracellular cytokine staining also demonstrated that
BNT162b2 elicited circulating S-specific CD8+ T cells that produced
IFNγ (Extended Data Fig. 5d).

that had been immunized using 100 μg BNT162b2, were challenged with
1.05 × 106 plaque-forming units of SARS-CoV-2 (strain USA-WA1/2020)
split equally between the intranasal and intratracheal routes, as previously described28 (Extended Data Fig. 6, Extended Data Table 2).
In addition, nine age-matched macaques (controls) that had been
mock-immunized with saline received the same SARS-CoV-2 challenge, and six age-matched macaques (sentinels)—three of which had
been immunized using 30 μg BNT162b2—were mock-challenged with
cell culture medium. We collected nasal, oropharyngeal and rectal
swabs, and performed bronchoalveolar lavage at the times indicated
(Extended Data Table 2). We then tested samples for SARS-CoV-2 RNA
(genomic RNA and subgenomic transcripts) using reverse-transcription
quantitative polymerase chain reaction (RT–qPCR). All personnel who
performed clinical, radiological, histopathological or RT–qPCR evaluations were blinded to the group assignments of the macaques.
Viral RNA was detected in bronchoalveolar lavage fluid from seven of
nine control macaques on day 3; from four of eight control macaques
on day 6 after challenge (with one indeterminant result); and from
none of the six control macaques that underwent bronchoalveolar
lavage at the end of project (EOP; days 7–23 after challenge) (Fig. 4a).
Viral RNA was detected in the bronchoalveolar lavage fluid of two of
six BNT162b1-immunized macaques on day 3 after challenge, and from
none thereafter. Viral RNA was not detected in bronchoalveolar lavage
fluid from the BNT162b2-immunized, SARS-CoV-2 challenged macaques
at any of the time points we sampled.
In nasal swabs obtained on the day after challenge, viral RNA was
detected from control-immunized macaques (4 of 9) and BNT162b2immunized macaques (5 of 6), but not from BNT162b1-immunized
macaques (Fig. 4b). In subsequent nasal swabs, viral RNA was detected
from some of the control-immunized macaques at each sampling time
point (5 of 9 on day 3, 4 of 9 on day 6 and 2 of 9 on days 7–23), from some
BNT162b1-immunized macaques at only one sampling time point (2 of 6
on day 6) and from none of the BNT162b2-immunized macaques at any
sampling time point. Similar patterns were seen in oropharyngeal and

BNT162b-elicted protection in macaques
Forty-one to fifty-five days after dose 2, six of the 2–4-year-old
macaques that had been immunized using 100 μg BNT162b1, and six

Nature | Vol 592 | 8 April 2021 | 287

Article
rectal swabs: viral RNA was more often detected in control-immunized
macaques than in BNT162b1- or BNT162b2-immunized macaques, and
there was more persistence of viral RNA in rectal swabs than in oropharyngeal swabs (Extended Data Fig. 7a, b).
At the time of challenge, SARS-CoV-2-neutralizing titres ranged
from 208 to 1,185 in the BNT162b1-immunized macaques and from
260 to 1,004 in the BNT162b2-immunized macaques. Neutralizing titres
were below the limit of detection in the control macaques (Fig. 4c, d).
The control macaques responded to challenge with infectious virus
with an increase in SARS-CoV-2-neutralizing titres, consistent with
an immune response to viral infection. However, there was no trend
towards increasing SARS-CoV-2-neutralizing titres in response to
viral challenge in the BNT162b1-immunized or BNT162b2-immunized
macaques, consistent with their immunization suppressing SARS-CoV-2
infection. The maximum SARS-CoV-2-neutralizing titre elicited by virus
challenge of control macaques remained below 150 through to the time
of necropsy, whereas all immunized macaques maintained neutralizing
titres greater than 150 throughout the challenge experiment.
None of the challenged macaques—whether immunized or not—
showed clinical signs of illness (Extended Data Fig. 7c–f). Radiographic
abnormalities were generally minimal or mild, and were not consistently associated with viral challenge (Extended Data Fig. 8a, b). The
histopathology of necropsy specimens obtained 7–8 days after challenge revealed localized areas of pulmonary inflammation that were
limited in extent even in the control macaques challenged after mock
immunization with saline (Extended Data Fig. 8c). We conclude that
the 2–4-year-old male-macaque challenge model is primarily a model
of SARS-CoV-2 infection rather a model than of COVID-19 diseae.

Discussion
We demonstrate that the candidate vaccines BNT162b1 or BNT162b2—
lipid-nanoparticle-formulated, m1Ψ nucleoside-modified mRNAs that
encode secreted, trimerized SARS-CoV-2 RBD or prefusion-stabilized S,
respectively—induce strong antigen-specific immune responses in mice
and macaques. The RBD–foldon coding sequence directs the expression
and secretion of a flexible, trimeric protein that binds to ACE2 with high
affinity and has structurally intact ACE2 receptor-binding sites. We confirmed that protein expressed from DNA with the BNT162b2-encoded
S(P2) amino acid sequence was in the prefusion conformation using
cryo-EM. This analysis showed that the antigenically important RBD
can assume the up conformation, in which the receptor-binding site
that is rich in neutralizing epitopes is accessible in a proportion of
the molecules24. The alternative states observed probably reflect a
dynamic equilibrium between RBD up and down positions10,26. The
binding of expressed and purified S(P2) to ACE2 and a neutralizing
monoclonal antibody further demonstrates the conformational and
antigenic integrity of this prefusion-stabilized S.
In mice, a single sub-microgram immunization using either of the
BNT162b candidates rapidly induced high antibody titres that inhibited pseudovirus entry in the range of—or above—recently reported
neutralizing titres that are elicited by other candicate vaccines
against SARS-CoV-229,30. The candidate vaccines discussed in this
Article also induced strong TFH and TH1-type CD4+ T cell responses,
the latter of which are thought to be a more general effect of lipidnanoparticle-formulated modRNA vaccines against SARS-CoV-230.
Both CD4+ T cell types are known to support antigen-specific antibody
generation and maturation. In some animal models of respiratory virus
infection, a TH2-type CD4+ T cell response has previously been associated with vaccine-associated enhanced respiratory disease31,32. Therefore, a TH1-type response to immunization is preferred as it may reduce
the theoretical risk of enhanced pulmonary disease during subsequent
viral infection. Immunization with the vaccine candidates triggered
redistribution of B cells from the blood to lymphoid tissues, where
antigen presentation occurs. In humans, TFH cells in the circulation
288 | Nature | Vol 592 | 8 April 2021

after vaccination with a VSV-vectored Ebola vaccine candidate have
previously been correlated with a high frequency of antigen-specific
antibodies33. After vaccination of mice with BNT162b1 or BNT162b2,
high numbers of TFH cells were present in both the blood and lymph
nodes, a potential correlate for the generation of a strong adaptive
B cell response in germinal centres. In addition to eliciting favourable
CD4+ T cell responses, BNT162b1 and BNT162b2 both elicit CD8+ T cell
responses in mice, and BNT162b2 appears to be somewhat more efficient at eliciting antigen-specific cytotoxic IFNγ CD8+ T cells.
BNT162b1 and BNT162b2 elicit immune profiles in macaques similar to those observed in mice. Seven days after dose 2 (of 100 μg of
the candidate) was administered to macaques (during the expansion
phase of the antibody response), the neutralizing GMTs elicited by
either candidate reached approximately 18× the GMT of a panel of
SARS-CoV-2-convalescent human sera. Neutralizing GMTs declined
by day 56 (35 days after dose 2), consistent with the contraction phase;
however, they remained well above the GMT of the human sera panel.
The duration of the study was not long enough to assess the rate of
decline during the plateau phase of the antibody response. As in mice,
BNT162b2 elicted a strongly TH1-biased CD4+ T cell response and IFNγ+
CD8+ T cell response in macaques.
Limitation and clearance of virus infection is promoted by the interplay between neutralizing antibodies that eliminate infectious particles and CD8+ T cells that target intracellular reservoirs of virus. CD8+
T cells may also reduce the influx of monocytes into infected lung tissue,
which can be associated with undesirable IL-6 and TNF production and
impaired antigen presentation34,35. The responses elicited by the vaccine candidates reflect a pattern that is favourable for vaccine safety
and efficacy, which provides added reassurance for clinical translation36. The contributions of the individual immune effector systems to
human protection from SARS-CoV-2 are not yet understood. Therefore,
it appears prudent to develop COVID-19 vaccines that enlist concomitant cognate B cell, CD4+ T cell and CD8+ T cell responses.
Both candidates protected 2–4-year-old macaques from challenge
with infectious SARS-CoV-2, and there was reduced detection of viral
RNA in immunized macaques as compared to those that received saline.
Immunization with BNT162b2 provided particularly strong RT–qPCR
evidence for protection of the lower respiratory tract, as demonstrated
by the absence of detectable SARS-CoV-2 RNA in serial bronchoalveolar
lavage samples that were obtained starting 3 days after challenge. The
lack of serological response to SARS-CoV-2 challenge in BNT162b1- or
BNT162b2-immunized macaques—despite a neutralizing response to
challenge in control-immunized macaques—suggests suppression
of infection by the vaccine candidates. Clinical signs of disease were
absent, and radiological and pathological abnormalities were generally
mild after challenge. As in other published reports of immunization
and SARS-CoV-2 challenge of nonhuman primates, there was no evidence of vaccine-mediated enhancement of viral replication, disease
or pathology37,38. The interpretation of vaccine-mediated protection in
nonhuman primates is limited by the small number of animals and the
inherent limitations of animal models. Nevertheless, these preclinical
results provided key support for the immunization of large numbers
of clinical-trial participants with BNT162b2.
The selection of BNT162b2 over BNT162b1 for further clinical testing was largely driven by the greater tolerability of BNT162b2 with
comparable immunogenicity in clinical trials3, and the broader range
and MHC diversity of T cell epitopes on the much larger full-length
S. A global phase III safety and efficacy study of immunization with
BNT162b2 (NCT04368728) is ongoing, and may answer open questions
that cannot be addressed by preclinical models.

Online content
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acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code
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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
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© The Author(s), under exclusive licence to Springer Nature Limited 2021

1

BioNTech, Mainz, Germany. 2Pfizer, Pearl River, NY, USA. 3Pfizer, Groton, CT, USA. 4Southwest
National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA.

5

TRON–Translational Oncology at the University Medical Centre of the Johannes Gutenberg
University, Mainz, Germany. 6VCA SouthPaws Veterinary Specialists and Emergency Center,
Fairfax, VA, USA. 7New Iberia Research Center, New Iberia, LA, USA. 8University of Texas
Medical Branch, Galveston, TX, USA. 9Texas Biomedical Research Institute, San Antonio, TX,
USA. 10These authors contributed equally: Annette B. Vogel, Isis Kanevsky, Ye Che. ᅒe-mail:
[e-mail]

Nature | Vol 592 | 8 April 2021 | 289

Article
Methods
No statistical methods were used to predetermine sample size. The
experiments were not randomized, and investigators were not blinded
to allocation during experiments and outcome assessment, except
for the performance of serological assays of nonhuman primates and
RT–PCR-based viral load measurements and the intepretation of radiographs, computed tomography scans, and histopathology specimens.

Ethics statement
All mouse studies were performed at BioNTech SE, and protocols were
approved by the local authorities (local welfare committee) and conducted according to Federation of European Laboratory Animal Science
Associations recommendations. Study execution and housing were in
compliance with the German Animal Welfare Act and Directive 2010/63/
EU. Mice were kept in individually ventilated cages with a 12-h light/
dark cycle, controlled environmental conditions (22 ± 2 °C, 45% to 65%
relative humidity) and under specific-pathogen-free conditions. Food
and water was available ad libitum. Only mice with an unobjectionable
health status were selected for testing procedures.
Immunizations for the nonhuman primate study were performed
at the University of Louisiana at Lafayette-New Iberia Research Centre
(NIRC), which is accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC) (animal assurance
no. 000452). The work was in accordance with United States Department of Agriculture Animal Welfare Act and Regulations and the NIH
Guidelines for Research Involving Recombinant DNA Molecules, and
Biosafety in Microbiological and Biomedical Laboratories. All procedures performed on these macaques were in accordance with regulations and established guidelines, and were reviewed and approved
by an Institutional Animal Care and Use Committee or through an
ethical review process. Challenge of nonhuman primates with infectious SARS-CoV-2 after immunization was performed at the Southwest
National Primate Research Centre (SNPRC), Texas Biomedical Research
Institute (San Antonio), which is also accredited by the AAALAC (animal assurance no. 000246). Animal husbandry followed standards
recommended by AAALAC International and the NIH Guide for the
Care of Use of Laboratory Animals. This study was approved by the
Texas Biomedical Research Institute Animal Care and Use Committee.
Protein and peptide reagents
Purified recombinant SARS-CoV-2 RBD (Sino Biological) or trimeric S
(Acro Biosystems) was used as a target for western blot, and the RBD
tagged with a human Fc (Sino Biological) was used in ELISA to detect
SARS-CoV-2 S-specific IgG. A recombinant SARS-CoV-2 RBD containing a C-terminal Avitag (Acro Biosystems) was used as a target antigen in Luminex immunoassays. Purified recombinant SARS-CoV-2 S1
including a histidine tag (Sino Biological) was used in ELISA to detect
SARS-CoV-2 S-specific IgG in mice. Purified recombinant SARS-CoV-2
S1 and RBD with histidine tags (both Sino Biological) were used for
surface plasmon resonance spectroscopy. A peptide pool of 15-mer
peptides overlapping by 11 amino acids covering the full-length S was
used for restimulation in ELISpot, cytokine profiling and intracellular
cytokine staining followed by flow cytometry. An irrelevant peptide
(SPSYVYHQF, derived from gp70 AH-139) or a cytomegalovirus (CMV)
peptide pool was used as control for ELISpot assays. All peptides were
obtained from JPT Peptide Technologies.
Panel of SARS-CoV-2-convalescent human sera
A previously described1–3 panel of SARS-CoV-2-convalescent human
sera was used as a benchmark for nonhuman primate serology. The
sera (n = 38) were drawn from donors 18–83 years of age, at least 14 days
after PCR-confirmed diagnosis and at a time when the participants were
asymptomatic. Most serum donors had outpatient (35/38) or inpatient
(1/38) COVID-19; 2 of 38 had asymptomatic SARS-CoV-2 infections. Sera

were obtained from Sanguine Biosciences, the MT Group and Pfizer
Occupational Health and Wellness.

Cell culture
HEK293T and Vero 76 cells (both from ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX (Gibco)
supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). Cell
lines were tested for mycoplasma contamination after receipt, before
expansion and cryopreservation. For studies including nonhuman
primate samples, Vero 76 and Vero CCL81 cells (both from ATCC) were
cultured in DMEM (Gibco) containing 2% HyClone fetal bovine and
100 U ml−1 penicillium–streptomycin (Gibco). Expi293F cells were
grown in Expi293 medium and transiently transfected using ExpiFectamine293 (all from Thermo Fisher Scientific).
In vitro transcription and purification of RNA
Antigens encoded by BNT162b vaccine candidates were designed on
a background of S sequences from SARS-CoV-2 isolate Wuhan-Hu-1
(GenBank MN908947.3). The DNA template for the BNT162b1 RNA is
a DNA fragment encoding a fusion protein of the SARS-CoV-2 S signal
peptide (SP) (amino acids 1–16), the SARS-CoV-2 S RBD and the T4 bacteriophage fibritin trimerization motif21 (foldon). The template for the
BNT162b2 RNA is a DNA fragment encoding SARS-CoV-2 S with K986P
and V987P substitutions. BNT162b1 and BNT162b2 DNA templates
were cloned into a plasmid vector with backbone sequence elements
(T7 promoter, 5′ and 3′ UTR, 100 nucleotide poly(A) tail) interrupted
by a linker (A30LA70, 10 nucleotides) for improved RNA stability and
translational efficiency17,40. The DNA was purified, spectrophotometrically quantified and in vitro-transcribed by T7 RNA polymerase in the
presence of a trinucleotide cap1 analogue ((m27,3′-O)Gppp(m2’-O)ApG)
(TriLink) and with N1-methylpseudouridine-5′-triphosphate (m1ΨTP)
(Thermo Fisher Scientific) replacing uridine-5′-triphosphate (UTP)41.
RNA was purified using magnetic particles42. RNA integrity was assessed
by microfluidic capillary electrophoresis (Agilent Fragment Analyzer),
and the concentration, pH, osmolality, endotoxin level and bioburden
of the solution were determined.
Lipid nanoparticle formulation of the RNA
Purified RNA was formulated into lipid nanoparticles using an ethanolic
lipid mixture of ionizable cationic lipid and transferred into an aqueous
buffer system via diafiltration to yield a lipid nanoparticle composition
similar to one previously described43. The lipid nanoparticle contains
RNA, an ionizable lipid, ((4-hydroxybutyl)azanediyl)bis(hexane6,1-diyl)bis(2-hexyldecanoate)), a PEGylated lipid, 2-[(polyethylene
glycol)-2000]-N,N-ditetradecylacetamide and two structural lipids
(1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC])and cholesterol).
The vaccine candidates were stored at −70 to −80 °C at a concentration
of 0.5 mg ml−1.
Transfection of HEK cells
HEK293T cells were transfected with 1 μg RiboJuice transfection
reagent-mixed BNT162b1 RNA or BNT162b2 RNA, or with the vaccine
candidates BNT162b1 (lipid-nanoparticle-formulated BNT162b1 RNA)
or BNT162b2 (lipid-nanoparticle-formulated BNT162b2 RNA) by incubation for 18 h. Non-lipid-nanoparticle-formulated mRNA was diluted
in Opti-MEM medium (Thermo Fisher Scientific) and mixed with the
transfection reagent according to the manufacturer’s instructions
(RiboJuice, Merck Millipore).
Western blot analysis of size fractions of the medium of
BNT162b1-RNA-transfected cells
Medium from cultured HEK293T cells was collected. After 13-fold concentration via Vivaspin 20 centrifugal concentrators with a molecular
weight cut off of 10 kDa, supernatants were applied to a preparative HiLoad 16/600 Superdex 200 pg column (both Sigma Aldrich).

The column was run at 29.8 cm h−1 in phosphate buffered saline (PBS),
and 500-μl fractions were collected (Supplementary Fig. 1). The gel
filtration column was calibrated with well-defined protein standards
separated under identical conditions in a second run. Size-fractioned
FBS-free medium from BNT162b1-RNA-transfected HEK293T cells was
analysed by denaturing (95 °C) and non-denaturating (no heating) PAGE
using 4–15% Criterion TGX Stain-Free Gel (Bio-Rad) and western blot.
Transfer to a nitrocellulose membrane (Bio-Rad) was performed using a
semi-dry transfer system (Trans-Blot Turbo Transfer System, Bio-Rad).
Blotted proteins were detected with a monoclonal antibody that recognizes SARS-CoV-2 S1 (SinoBiological) and a secondary anti-rabbit
horse radish peroxidase (HRP)-conjugated antibody (Sigma Aldrich).
Blots were developed with Clarity Western ECL Substrate (Bio-Rad) and
imaged with a Fusion FX Imager (Vilber) using the Image Lab software
version 6.0.

Vaccine antigen detection by flow cytometry
Transfected HEK293T cells were stained with Fixable Viability Dye
(eBioscience). After fixation (Fixation Buffer, Biolegend), cells were
permeabilized (Perm Buffer, eBioscience) and stained with a monoclonal antibody that recognizes SARS-CoV-2 S1 (SinoBiological). Cells
were acquired on a FACSCanto II flow cytometer (BD Biosciences) using
BD FACSDiva software version 8.0.1 and analysed by FlowJo software
version 10.6.2 (FlowJo, BD Biosciences).
Localization of expressed vaccine antigens by
immunofluorescence
Transfected HEK293T cells were fixed in 4% paraformaldehyde (PFA)
and permeabilized in PBS/0.2% Triton X-100. Free binding sites were
blocked and cells incubated with a rabbit monoclonal antibody that recognizes the SARS-CoV-2 S1 subunit (SinoBiological), an anti-rabbit IgG
secondary antibody ( Jackson ImmunoResearch), labelled lectin HPA
(Thermo Fisher Scientific) and concanavalin A (Fisher Scientific). DNA
was stained with Hoechst (Life Technologies). Images were acquired
with a Leica SP8 confocal microscope and Application Suite LAS-X
Version 3.1.5.
SARS-CoV-2 RBD–foldon and S(P2) expression and purification
To express the RBD–foldon encoded by BNT162b1 for ACE2-binding
analysis and cryo-EM, DNA corresponding to the RNA coding sequence
was cloned into the pMCG1309 vector. A plasmid encoding amino
acids 1–615 of human ACE2 with C-terminal His-10 and Avi tags was
generated for transient expression of the peptidase domain of ACE2
(ACE2 PD) in Expi293F cells. The ACE2–B0AT1 complex was produced by
co-expression of two plasmids in Expi293F cells, one of them encoding
ACE2 amino acids 1–17 followed by haemagglutinin and Strep II tags
and ACE2 amino acids 18–805, and the other containing a methionine
followed by a Flag tag and amino acids 2–634 of human B0AT1. Secreted
ACE2 PD was isolated from conditioned cell culture medium using
Nickel Excel resin (GE Healthcare) followed by gel filtration chromatography on a Superdex200 10/30 column (GE Healthcare) in PBS.
Approximately 5 mg of purified ACE2 PD was covalently attached per
1 ml of 4% beaded agarose by amine coupling using AminoLink Plus
resin (Thermo Fisher Scientific).
The RBD trimer was purified from conditioned medium by affinity
capture with the ACE2 PD crosslinked agarose and was eluted from the
resin with 3 M MgCl2. Following dialysis, the protein was concentrated
and purified by gel filtration using a Superdex200 10/300 column in
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered
saline (HBS) with 10% glycerol. Purification of the ACE2–B0AT1 complex
was based on a previously described procedure8. To form the ACE2–
B0AT1–RBD-trimer complex, ACE2–B0AT1 aliquots were combined
with purified RBD–foldon diluted in size-exclusion chromatography
buffer (25 mM Tris pH 8.0, 150 mM NaCl, 0.02% glyco diosgenin) for a 3:1
molar ratio of RBD trimers to ACE2 protomers. After incubation at 4 °C

for 30 min, the sample was concentrated and resolved on a Superose
6 Increase 10/300 GL column. Peak fractions containing the complex
were pooled and concentrated.
To express SARS-CoV-2 S(P2) encoded by BNT162b2 for characterization by size-exclusion chromatography, ACE2-PD binding, monoclonal antibody binding and cryo-EM, a gene encoding the full length of
SARS-CoV-2 (GenBank MN908947) with two prolines substituted at
residues 986 and 987 (K986P and V987P) followed with a C-terminal
HRV3C protease site and a TwinStrep tag was cloned into a modified
pcDNA3.1(+) vector with the CAG promoter. The TwinStrep-tagged
S(P2) was expressed in Expi293F cells.
Purification of the recombinant protein was based on a previously
described procedure, with minor modifications9. Upon cell lysis, S(P2)
was solubilized in 1% NP-40 detergent. The TwinStrep-tagged protein
was then captured with StrepTactin Sepharose HP resin in 0.5% NP-40.
S(P2) was further purified by size-exclusion chromatography and eluted
as three distinct peaks in 0.02% NP-40, as previously reported9 (chromatogram not shown). A peak that consists of intact S(P2) migrating
at around 150 kDa, as well as dissociated S1 and S2 subunits (which
co-migrate at just above 75 kDa), was used in the structural characterization. Spontaneous dissociation of the S1 and S2 subunits occurs
throughout the course of protein purification, starting at the point of
detergent-mediated protein extraction, so that S(P2) preparations also
contain dissociated S1 and S2.

Binding kinetics of the RBD–foldon trimer and S(P2) to
immobilized human ACE2 and a neutralizing monoclonal
antibody by biolayer interferometry
Binding of purified RBD–foldon to ACE2 PD and of NP-40 solubilized,
purified S(P2) to ACE2 PD and human neutralizing monoclonal antibody
B3825 was measured by biolayer interferometry at 25 °C on an Octet
RED384 (FortéBio). RBD–foldon binding was measured in 10 mM HEPES
pH 7.5, 150 mM NaCl and 1 mM EDTA (EDTA). S(P2) binding was measured in 25 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.02% NP-40.
Avi-tagged human ACE2 PD was immobilized on streptavidin-coated
sensors; B38 antibody was immobilized on protein G-coated sensors.
For an RBD–foldon concentration series, binding data were collected
for 600 s of association and 900 s of dissociation. For an S(P2) concentration series, after initial baseline equilibration of 120 s, the sensors were dipped in a 10 μg ml−1 solution of Avi-tagged ACE2 PD or B38
monoclonal antibody for 300 s to achieve capture levels of 1 nM using
the threshold function. Then, after another 120 s of baseline, binding
data were collected for 300 s of association and 600 s of dissociation.
Biolayer interferometry data were collected with Octet Data Acquisition software version 10.0.0.87 and processed using ForteBio Data
Analysis software version 10.0. Data were reference-subtracted and
fit to a 1:1 binding model with R2 value greater than 0.96 for the RBD
and 0.95 for S(P2). Potential avidity effects for the RBD–foldon and
potential ongoing dissociation of S1 from S(P2) could make the actual
binding events more complicated than represented by 1:1 binding
model. Therefore, we report apparent kinetics and affinity (S(P2)) or
avidity (RBD–foldon) of binding as calculated using Octet Data Analysis
Software v.10.0 (FortéBio). For the RBD–foldon, the dissociation rate
of interaction (kd) with ACE2 PD was slower than the limit of measurement of the instrument, and the apparent minimum binding avidity
(KD) was estimated using an assumed dissociation rate kd of 1 × 10−6 s−1.
Electron microscopy of negatively stained RBD–foldon trimers
Purified RBD–foldon in 4 μl was applied to a glow-discharged copper
grid overlaid with formvar and amorphous carbon (Ted Pella). Negative
staining was performed with Nano-W organotungstate stain (Nanoprobes) according to the manufacturer’s protocol. The sample imaged
using an FEI TF-20 microscope operating at 200 kV, with a magnification of 62,000× and defocus of −2.5 μm. Micrographs were contrast
transfer function (CTF)-corrected in RELION using CTFFIND-4.144. A

Article
small manually picked dataset was used to generate 2D references for
autopicking. The resulting particle set was subjected to 2D classification in RELION 3.0.645.

Cryo-EM of the ACE2–B0AT1–RBD-trimer complex
Cryo-EM was performed using a Titan Krios operating at 300 keV
equipped with a Gatan K2 Summit direct electron detector in superresolution mode at a magnification of 165,000×, for a magnified pixel
size of 0.435 Å at the specimen level.
Purified ACE2–B0AT1–RBD-trimer complex at 6 mg ml−1 in 4 μl was
applied to gold Quantifoil R1.2/1.3 200 mesh grids glow-discharged
in residual air for 30 s at 20 mA using a Pelco Easiglow. The sample
was blotted using a Vitrobot Mark IV for 5 s with a force of −3 before
being plunged into liquid ethane cooled by liquid nitrogen. In total,
7,455 micrographs were collected from a single grid. Data were collected
over a defocus range of −1.2 to −3.4 μm with a total electron dose of
52.06 e− per Å2 fractionated into 40 frames over a 6-s exposure for 1.30
e− per Å2per frame. Initial motion correction was performed in Warp46,
during which super-resolution data were binned to give a pixel size of
0.87 Å. Corrected micrographs were imported into RELION 3.1-beta45
for CTF estimation with CTFFIND-4.144.
Particles were picked using the LaPlacian-of-Gaussian particle-picking
algorithm as implemented in RELION, and extracted with a box size of
450 pixels. References obtained by 2D classification were used for a
second round of reference-based autopicking, yielding a dataset of
715,356 particles. Two of the three RBDs of each particle (the two not
constrained by binding to ACE2–B0AT1) exhibited diffuse density in 2D
classification that reflected high particle flexibility, consistent with the
conformational flexibility of RBD trimers observed by negative-stain
electron microscopy (Fig. 1c, d). This flexibility precluded the inclusion
of all three RBDs in the final structural solution. Particle heterogeneity
was filtered out with 2D and 3D classification with a mask size of 280 Å
to filter out the diffuse density of the two non-ACE2-bound RBD copies in each RBD trimer, yielding a set of 87,487 particles that refined to
3.73 Å with C2 symmetry. Refinement after subtraction of micelle and
B0AT1 density from the particles yielded an improved map of 3.24 Å.
The atomic model from Protein Data Bank code (PDB) 6M178 was
rigid-body-fitted into the 3.24 Å density and then flexibly fitted to the
density using real-space refinement in Phenix47 alternating with manual
building in Coot48. The microscope was operated for image acquisition
using SerialEM software version 3.8.0 beta49. Validation of this model
is shown in Supplementary Fig. 2. Data collection, 3D reconstruction
and model refinement statistics are listed in Extended Data Table 1.
Cryo-EM of S(P2)
For TwinStrep-tagged S(P2), 4 μl purified protein at 0.5 mg ml−1 were
applied to gold Quantifoil R1.2/1.3 300 mesh grids freshly overlaid with
graphene oxide. The sample was blotted using a Vitrobot Mark IV for
4 s with a force of −2 before being plunged into liquid ethane cooled
by liquid nitrogen. We collected 27,701 micrographs from 2 identically
prepared grids. Data were collected from each grid over a defocus
range of −1.2 to −3.4 μm with a total electron dose of 50.32 and 50.12
e− per Å2, respectively, fractionated into 40 frames over a 6-s exposure
for 1.26 and 1.25 e− per Å2 per frame. On-the-fly motion correction, CTF
estimation, and particle-picking and extraction with a box size of 450
pixels were performed in Warp46, during which super-resolution data
were binned to give a pixel size of 0.87 Å. A total of 1,119,906 particles
were extracted. All subsequent processing was performed in RELION
3.1-beta45. Particle heterogeneity was filtered out with 2D and 3D classification, yielding a set of 73,393 particles that refined to 3.6 Å with
C3 symmetry. Three-dimensional classification of this dataset without particle alignment separated out one class with a single RBD up,
representing 15,098 particles. The remaining 58,295 particles, in the
three-RBD-down conformation, were refined to give a final model at
3.29 Å. The atomic model from PDB 6XR89 was rigid-body fitted into

the map density, then flexibly fitted to the density using real-space
refinement in Phenix47 alternating with manual building in Coot48.
The cryo-EM model validation is provided in Extended Data Fig. 2, and
the full cryo-EM data processing workflow and the model refinement
statistics in provided in Extended Data Table 1.

Immunization
Mice. Female BALB/c mice ( Janvier) (8–12 weeks old) were randomly allocated to groups. BNT162b1 and BNT162b2 diluted in PBS with 300 mM
sucrose (Fig. 2a–c, Extended Data Figs. 3 for both BNT162 vaccine candidates; Fig. 2e, Extended Data Fig. 4a for BNT162b2) or 0.9% NaCl (Fig. 2d,
Extended Data Fig. 4b–e for both BNT162 vaccine candidates; Fig. 2e,
Extended Data Fig. 4a for BNT162b1) were injected into the gastrocnemius muscle at a volume of 20 μl under isoflurane anaesthesia. PBS with
300 mM sucrose or 0.9% NaCl served as buffer controls, respectively.
Macaques. Male macaques (2–4 years old) were randomly assigned to
receive BNT162b1 or BNT162b2 on days 0 and 21 or saline control on
days 0 and 21 or 35. Vaccine was administered in 0.5 ml by intramuscular
injection in the left quadriceps muscle. Macaques were anaesthetized
with ketamine HCl (10 mg kg−1; intramuscular) during immunization
and were monitored for adequate sedation.

Phlebotomy and tissue preparation
Mice. Peripheral blood was collected from the retro-orbital venous
plexus under isoflurane anaesthesia or the vena facialis without anaesthesia. For flow cytometry, blood was heparinized. For serum generation, blood was centrifuged for 5 min at 16,000g and the serum was
immediately used for downstream assays or stored at −20 °C. Spleen
single-cell suspensions were prepared in PBS by mashing tissue against
the surface of a 70-μm cell strainer (BD Falcon). Erythrocytes were
removed by hypotonic lysis. Popliteal, inguinal and iliac lymph nodes
were pooled, cut into pieces, digested with collagenase D (1 mg ml−1)
(Roche) and passed through cell strainers.
Macaques. Serum was obtained before, 6 h after and 1, 14, 21, 28, 35 and
42 days after injection with BNT162b1, BNT162b2 or saline (Extended
Data Table 2). For BNT162b2 and challenge cohort 3 controls, serum
was also obtained on day 56, and peripheral blood mononuclear cells
(PBMCs) were obtained before immunization and on days 7, 28, and
42 (except that PBMCs were not obtained from the challenge cohort 3
control macaques on day 28). Blood for serum and PBMCs was collected
in compliance with animal protocol 2017-8725-023, approved by the
NIRC Institutional Animal Care and Use Committee. Macaques were
anaesthetized with ketamine HCl (10 mg kg−1; intramuscular) during
blood collection and were monitored for adequate sedation.

Analysis of S1- and RBD-specific serum IgG
Mice. MaxiSorp plates (Thermo Fisher Scientific) were coated with
recombinant S1 or RBD (1 μg ml−1) in sodium carbonate buffer, and
serum-derived bound IgG was detected using an HRP-conjugated
secondary antibody and tetramethylbenzidine substrate (Biotrend).
Data collection was performed using a BioTek Epoch reader and Gen5
software version 3.0.9. For concentration analysis, an IgG mouse isotype
control was used in parallel in a serial dilution, and the sample signals
were correlated to a standard curve of the isotype control.
Macaques and humans. Recombinant SARS-CoV-2 S1 containing a
C-terminal Avitag (Acro Biosystems) was bound to streptavidin-coated
Luminex microspheres. Bound macaque or human anti-S1 antibodies present in the serum were detected with a fluorescently labelled
goat anti-human polyclonal secondary antibody ( Jackson ImmunoResearch). Data were captured as median fluorescent intensities using a Bioplex200 system (Bio-Rad) and converted to U ml−1
antibody concentrations using a reference standard consisting of 5

pooled SARS-CoV-2-convalescent human serum samples (obtained
>14 days after PCR diagnosis, from the panel described in ‘Panel of
SARS-CoV-2-convalescent human sera’), diluted in antibody-depleted
human serum with arbitrary assigned concentrations of 100 U ml−1 and
accounting for the serum dilution factor.

Surface plasmon resonance spectroscopy of polyclonal mouse
immune sera
Binding kinetics of mouse S1- and RBD-specific serum IgG to recombinant S1 and RBD was determined using a Biacore T200 device (Cytiva)
with 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v surfactant
P20 (HBS-EP running buffer, BR100669, Cytiva) at 25 °C. Carboxyl
groups on the CM5 sensor chip matrix were activated with a mixture
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride and
N-hydroxysuccinimide to form active esters for the reaction with amine
groups. Anti-mouse IgG Fc-antibody ( Jackson ImmunoResearch) was
diluted in 10 mM sodium acetate buffer pH 5 (30 μg ml−1) for covalent
coupling to immobilization level of about 10,000 response units. Free
N-hydroxysuccinimide esters on the sensor surface were deactivated
with ethanolamine.
Mouse serum was diluted 1:50 in HBS-EP buffer and applied at 10 μl
min−1 for 30 s to the active flow cell for capture by immobilized antibody,
and the reference flow cell was treated with buffer. Binding analysis of
captured mouse IgG antibodies to S1–His or RBD–His (Sino Biological)
was performed using a multicycle kinetic method with concentrations
ranging from 25 to 400 nM or 1.56 to 50 nM, respectively. An association
period of 180 s was followed by a dissociation period of 600 s with a
constant flow rate of 40 μl min−1 and a final regeneration step. Apparent binding kinetics for the captured polyclonal IgG were calculated
using a global kinetic fit model (1:1 Langmuir, Biacore T200 Evaluation
Software Version 3.1, Cytiva).
VSV-SARS-CoV-2 S pseudovirus entry-inhibition assay by serum
IgG in mice
A recombinant replication-deficient VSV vector that encodes green
fluorescent protein (GFP) instead of VSV-G (VSV(ΔG–GFP)) was pseudotyped with SARS-CoV-2 S according to published pseudotyping
protocols50,51. In brief, HEK293T/17 monolayers transfected to express
SARS-CoV-2 S truncated of the C-terminal cytoplasmic 19 amino acids
(SARS-CoV-2-S(CΔ19)) were inoculated with VSVΔG-GFP vector (rescued from pVSVΔG-GFP plasmid expression vector; Kerafast). After
incubation for 1 h at 37 °C, the inoculum was removed, and cells were
washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast) was added to neutralize residual input
virus. VSV-SARS-CoV-2 pseudovirus-containing medium was collected
20 h after inoculation, 0.2-μm-filtered and stored at −80 °C.
Vero-76 cells were seeded in 96-well plates. Serial dilutions of mouse
serum samples were prepared and pre-incubated for 10 min at room
temperature with VSV-SARS-CoV-2 pseudovirus suspension (4.8 × 103
infectious units per ml) before transferring the mix to Vero-76 cells.
Inoculated Vero-76 cells were incubated for 20 h at 37 °C. Plates were
placed in an IncuCyte Live Cell Analysis system (Sartorius) and incubated for 30 min before the analysis (IncuCyte 2019B Rev2 software).
Whole-well scanning for bright-field and GFP fluorescence was performed using a 4× objective. The pVNT50 is reported as the reciprocal
of the highest dilution of serum that still yielded a 50% reduction in
GFP-positive infected cell number per well, compared to the mean of
the no-serum pseudovirus positive control. Each serum sample dilution was tested in duplicate.
IFNγ and IL-4 ELISpot
Mice. ELISpot assays were performed with mouse IFNγ ELISpotPLUS
kits according to the manufacturer’s instructions (Mabtech). A total
of 5 × 105 splenocytes was ex vivo restimulated with the full-length S
peptide mix (0.1 μg ml−1 final concentration per peptide) or controls

(gp70-AH1 (SPSYVYHQF)39, 4 μg ml−1; concanavalin A, 2 μg ml−1 (Sigma)).
Streptavidin–alkaline phosphatase and 5-bromo-4-chloro-3′-indolyl
phosphate/nitro blue tetrazolium-plus substrate were added, and spots
counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer
(CTL)). Spot numbers were evaluated using ImmunoCapture Image
Acquisition Software v.7.0 and ImmunoSpot 7.0.17.0 Professional. Spot
counts denoted too numerous to count by the software were set to
1,500. For T cell subtyping, CD8+ T cells and CD4+ T cells were isolated
from splenocyte suspensions using MACS MicroBeads (CD8a (Ly-2) and
CD4 (L3T4) (Miltenyi Biotec)) according to the manufacturer’s instructions. CD8+ or CD4+ T cells (1 × 105) were subsequently restimulated with
5 × 104 syngeneic bone-marrow-derived dendritic cells loaded with
full-length S peptide mix (0.1 μg ml−1 final concentration per peptide),
or cell culture medium as control. The purity of isolated T cell subsets
was determined by flow cytometry to calculate spot counts per 1 × 105
CD8+ or CD4+ T cells.
Macaques. Macaque PBMCs were tested with commercially available nonhuman primate IFNγ and IL-4 ELISpot assay kits (Mabtech).
Cryopreserved macaque PBMCs were thawed in prewarmed AIM-V
medium (Thermo Fisher Scientific) with benzonase (EMD Millipore).
For IFNγ ELISpot, 1.0 × 105 PBMCs, and for IL-4 ELISpot, 2.5 × 105 PBMCs,
were stimulated ex vivo with 1 μg ml−1 of the full-length S overlapping
peptide mix. Tests were performed in triplicate wells and medium containing dimethyl sulfoxide (medium–DMSO), a CMV peptide pool and
phytohemagglutinin (Sigma) were included as controls. After 24 h for
IFNγ and 48 h for IL-4, streptavidin–HRP and 3-amino-9-ethylcarbazole
substrate (BD Bioscience) were added and spots counted using a CTL
ImmunoSpot S6 Universal Analyzer (CTL). Results shown are background (medium–DMSO) subtracted and normalized to spot-forming
cells per 106 PBMCs.

Cell-mediated immunity by flow cytometry
Mice. For T cell analysis in peripheral blood, erythrocytes from 50 μl
freshly drawn blood were lysed (ammonium–chloride–potassium
lysing buffer (Gibco)), and cells were stained with Fixable Viability Dye
(eBioscience) and primary antibodies in the presence of Fc block in flow
buffer (Dulbecco’s phosphate-buffered saline (Gibco) supplemented
with 2% fetal calf serum (FCS), 2 mM EDTA (both Sigma) and 0.01% sodium azide (Morphisto)). After staining with secondary biotin-coupled
antibodies in flow buffer, cells were stained extracellularly against
surface markers with directly labelled antibodies and streptavidin in
Brilliant Stain Buffer Plus (BD Bioscience) diluted in flow buffer. Cells
were washed with 2% RotiHistofix (Carl Roth), fixed (Fix/Perm Buffer,
FoxP3/Transcription Factor Staining Buffer Set (eBioscience)) and permeabilized (Perm Buffer, FoxP3/Transcription Factor Staining Buffer
Set (eBioscience)) overnight. Permeabilized cells were intracellularly
treated with Fc block and stained with antibodies against transcription
factors in Perm Buffer.
For T cell analysis in lymphoid tissues, 1 × 106 lymph node cells (for
BNT162b1) or 1.5 × 106 lymph node cells (for BNT162b2) and 4 × 106
spleen cells were stained for viability and extracellular antigens with
directly labelled antibodies. Fixation, permeabilization and intracellular staining was performed as described for blood T cell staining.
For B cell subtyping in lymphoid tissues, 2.5 × 105 lymph node and
1 × 106 spleen cells were treated with Fc block, stained for viability and
extracellular antigens as described for blood T cell staining, and fixed
with 2% RotiHistofix overnight.
For intracellular cytokine staining of T  cells from BNT162b1immunized mice, 1 × 106 lymph node and 4 × 106 spleen cells were ex
vivo restimulated with 0.2 μg ml−1 final concentration per peptide of
full-length S peptide mix. For intracellular cytokine staining of T cells
from mice immunized using BNT162b2, 4 × 106 spleen cells were ex
vivo restimulated with 0.5 μg ml−1 final concentration per peptide of
full-length S peptide mix or cell culture medium (no peptide) as control.

Article
The cells were restimulated for 5 h in the presence of GolgiStop and GolgiPlug (both BD Bioscience) for 5 h. Cells were stained for viability and
extracellular antigens as described for lymphoid T cell staining. Cells
were fixed with 2% RotiHistofix and permeabilized overnight. Intracellular staining was performed as described for blood T cell staining.
Mouse cells were acquired on a BD Symphony A3 or BD Celesta (B cell
subtyping) flow cytometer (BD Bioscience) using BD FACSDiva software version 9.1 or 8.0.1.1, respectively, and analysed with FlowJo 10.6
(FlowJo, BD Biosciences).
Macaques. For intracellular cytokine staining in T cells, 1.5 × 106 PBMCs
were stimulated with the full-length S peptide mix at 1 μg ml−1 (concentration of all peptides, combined), Staphyloccocus enterotoxin B (2 μg
ml−1) as positive control, or 0.2% DMSO as negative control. GolgiStop
and GolgiPlug (both BD Bioscience) were added. Following 37-°C incubation for 12 to 16 h, cells were stained for viability and extracellular antigens after blocking Fc binding sites with directly labelled antibodies.
Cells were fixed, permeabilized with BDCytoFix/CytoPerm solution (BD
Bioscience), and intracellular staining was performed in the permeabilization buffer for 30 min at room temperature. Cells were washed,
resuspended in 2% FBS/PBS buffer and acquired on an LSR Fortessa.
Data were analysed by FlowJo 10.4.1 (FlowJo, BD Biosciences). Results
shown are background (medium–DMSO) subtracted.

Cytokine profiling in mice by bead-based immunoassay
Mouse splenocytes were restimulated for 48 h with full-length S peptide
mix (0.1 μg ml−1 final concentration per peptide) or cell culture medium
(no peptide) as control. Concentrations of IFNγ, IL-2, IL-4, IL-5 and (for
splenocytes from BNT162b2-immunized mice) IL-13 in supernatants
were determined using a bead-based, 11-plex TH1/TH2 mouse ProcartaPlex multiplex immunoassay (Thermo Fisher Scientific) according
to the manufacturer’s instructions. Fluorescence was measured with a
Bioplex200 system (Bio-Rad) and analysed with ProcartaPlex Analyst
1.0 software (Thermo Fisher Scientific). Values below the lower limit
of quantification were set to zero.
SARS-CoV-2 neutralization by macaque sera
The SARS-CoV-2 neutralization assay used a previously described strain
of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen gene into open
reading frame 7 of the viral genome27. This reporter virus generates
similar plaque morphologies and indistinguishable growth curves
from wild-type virus. Viral master stocks were grown in Vero E6 cells
as previously described52. When testing human convalescent serum
specimens, the fluorescent neutralization assay produced comparable
results to the conventional plaque reduction neutralization assay. Serial
dilutions of heat-inactivated sera were incubated with the reporter virus
(2 × 104 plaque forming units (PFU) per well) to yield an approximately
10–30% infection rate of the Vero CCL81 monolayer for 1 h at 37 °C
before inoculating Vero CCL81 cell monolayers (targeted to have 8,000
to 15,000 cells in the central field of each well at the time of seeding, one
day before infection) in 96-well plates to allow accurate quantification
of infected cells. Cell counts were enumerated by nuclear stain (Hoechst 33342), and fluorescent virus-infected foci were detected 16–24 h
after inoculation with a Cytation 7 Cell Imaging Multi-Mode Reader
(BioTek) with Gen5 Image Prime version 3.09. Titres were calculated in
GraphPad Prism version 8.4.2 by generating a 4-parameter logistical fit
of the per cent neutralization at each serial serum dilution. The VNT50
is reported as the interpolated reciprocal of the dilution yielding a 50%
reduction in fluorescent viral foci.
SARS-CoV-2 challenge of macaques
The SARS-CoV-2 inoculum was obtained from a stock of 2.1 × 106 PFU ml−1
previously prepared at Texas Biomedical Research Institute, aliquoted
into single-use vials and stored at −70 °C. The working virus stock was

generated from two passages of the SARS-CoV-2 USA-WA1/2020 isolate
(a fourth passage seed stock purchased from BEI Resources; NR-52281)
in Vero E6 cells. The virus was confirmed to be SARS-CoV-2 by deep
sequencing that demonstrated identity to a published SARS-CoV-2
sequence (GenBank accession number MN985325.1).
BNT162b1-immunized (n = 6) and BNT162b2-immunized (n = 6)
male macaques, and age-matched male macaques mock-immunized
with saline (n = 9) (control), were challenged with 1.05 × 106 PFU of
SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (0.25 ml) and intratracheal (0.25 ml) routes, as previously
described28. Sentinel age- and sex-matched macaques (n = 6) were
mock-challenged with DMEM supplemented with 10% FCS intranasally
(0.25 ml) and intratracheally (0.25 ml). The macaques were challenged
or mock-challenged at the times relative to immunization indicated in
Extended Data Fig. 6, Extended Data Table 2.
Twelve to nineteen days before challenge, macaques were moved
from the NIRC where they had been immunized to the animal biosafety
level 3 facility at SNPRC. Macaques were monitored regularly by a
board-certified veterinary clinician for rectal body temperature, weight
and physical examination. Specimen collection was performed under
tiletamine zolazepam (Telazol) anaesthesia as previously described28.
Bronchoalveolar lavage, and nasal, oropharyngeal and rectal swab
collection, X-ray and CT examinations and necropsy were performed
at the times indicated in Extended Data Fig. 6, Extended Data Table 2.
The three control macaques in challenge cohort 3 and three sentinel
macaques were not necropsied to allow their subsequent rechallenge (control) or challenge (sentinel). Bronchoalveolar lavage was
performed by instilling 20 ml of saline 4 times. These washings were
pooled, aliquoted and stored frozen at −70 °C.

SARS-CoV-2 viral RNA quantification by RT–qPCR
To detect and quantify SARS-CoV-2 in nonhuman primates, viral RNA
was extracted from bronchoalveolar lavage fluid and from nasal, oropharyngeal and rectal swabs as previously described53–55, and tested
by RT–qPCR as previously described28. In brief, 10 μg yeast tRNA and
1 × 103 PFU of MS2 phage (Escherichia coli bacteriophage MS2) (ATCC)
were added to each thawed sample, and RNA extraction performed
using the NucleoMag Pathogen kit (Macherey-Nagel). The SARS-CoV-2
RT–qPCR was performed on extracted RNA using a 2019-nCoV N1 assay
developed by the United States Centers for Disease Control and Prevention, on a QuantStudio 3 instrument (Applied Biosystems). The cut-off
for positivity (limit of detection) was established at 10 gene equivalents
per reaction (800 gene equivalents per ml). Samples were tested in
duplicate. One bronchoalveolar lavage specimen from the challenge
cohort 2 control group obtained on day 6 after challenge, and one nasal
swab from the BNT162b1-immunized group obtained on day 1 after
challenge, had—on repeated measurements—viral RNA levels on either
side of the LLOD. These specimens were categorized as indeterminate
and excluded from the graphs and the analysis.
Radiology
Thoracic radiographs and computed tomography scans were performed under anaesthesia, as previously described28. For radiographic
imaging, three-view thoracic radiographs (ventrodorsal, right and left
lateral) were obtained at the times relative to challenge indicated in
Extended Data Table 2. The macaques were anaesthetized using telazol
(2–6 mg kg−1) and maintained by inhaled isoflurane delivered through
a Hallowell 2002 ventilator anaesthesia system (Hallowell). Macaques
were intubated to perform end inspiratory breath-hold using a remote
breath-hold switch. Lung field computed tomography images were
acquired using Multiscan LFER150 PET/CT (MEDISO) scanner. Image
analysis was performed using 3D region-of-interest tools available in
Vivoquant (Invicro). Images were interpreted by a board-certified veterinary radiologist blinded to treatment groups. Scores were assigned
to a total of 7 lung regions on a severity scale of 0–3 per region, with

a maximum severity score of 21. Pulmonary lesions evident before
challenge, or those which could not be unequivocally attributed to
the viral challenge (such as atelectasis secondary to recumbency and
anaesthesia), received a score of 0.

Histopathology
Lung histopathology is reported on necropsies performed on
2–4-year-old male macaques at the times after challenge indicated in
Extended Data Fig. 6, Extended Data Table 2. Necropsy, tissue processing and histology were performed by SNPRC. Samples were fixed in 10%
neutral buffered formalin and processed routinely into paraffin blocks.
Tissue blocks were sectioned to 5 μm and stained with haematoxylin and
eosin. Microscopic evaluation of 7 lung tissue sections per macaque (1
sample of each lobe in the left and right lungs) was performed blindly by
SNPRC and Pfizer pathologists. Lungs were evaluated using a semiquantitative scoring system with inclusion of cell types and/or distribution
as appropriate. Inflammation score was based on area of tissue in section involved: 0 = normal; 1 ≤ 10%; 2 = 11–30%; 3 = 30–60%; 4 = 60–80%;
5 ≥ 80%. Each lobe received an individual score, and the final score for
each macaque was reported as the mean of the individual scores. The
pathologists were unblinded to the group assignments after agreement on diagnoses. As indicated in Extended Data Fig. 6, Extended
Data Table 2, the BNT162b1-immunized and control macaques were
challenged and necropsied in parallel (challenge cohorts 1 and 2), and
the BNT162b2-immunized macaques were immunized and challenged
subsequently (challenge cohort 3).
Statistics and reproducibility
No statistical methods were used to predetermine group and samples
sizes (n). All experiments were performed once. P values reported for
RT–qPCR analysis were determined by nonparametric analysis (Friedman’s test) based on the ranking of viral RNA shedding data within each
day. PROC RANK and PROC GLM from SAS 9.4 were used to calculate
the P values. All available post-challenge bronchoalveolar lavage fluid
and nasal, oropharyngeal and rectal swab samples from the necropsied
macaques and all available post-challenge samples through day 10 from
the macaques that were not necropsied were included in the analysis.
Indeterminate results were excluded from this analysis. All remaining
analyses were two-tailed and carried out using GraphPad Prism 8.4.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.

Data availability
The SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank MN908947.3) is the
genetic background of the BNT162b antigens. The cryo-EM maps and
atomic coordinates have been deposited to the Electron Microscopy
Data Bank (EMDB) and PDB with accession numbers EMD-23211 and
7L7F, respectively, for the ACE2–B0AT1–RBD–foldon complex and
EMD-23215 and 7L7K, respectively, for S(P2). The data that support
the findings of this study are available from the corresponding author
upon reasonable request.
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Acknowledgements We thank T. Garretson and D. Cooper for advice on, and M. Cutler for
coordination of, serology studies in nonhuman primates; R. F. Sommese and K. F. Fennell for
technical assistance with molecular cloning and cell-based binding; M. Dvorak, M. Drude,
F. Zehner, T. Lapin, B. Ludloff, S. Hinz, F. Bayer, J. Scholz, A.L. Ernst, T. Sticker and S. Wittig
for their valuable support and assistance, which resulted in a rapid availability of
oligonucleotides and DNA templates; E. Boehm, K. Goebel, R. Frieling, C. Berger, S. Koch,
T. Wachtel, J. Leilich, M. Mechler, R. Wysocki, M. Le Gall, A. Czech and S. Klenk for carrying out
RNA production and analysis, without which this vaccine candidate could not have been
transferred to nonclinical studies with such speed; B. Weber, J. Vogt, S. Krapp, K. Zwadlo,
J. Mottl, J. Mühl and P. Windecker for supporting the mouse studies and serological analysis
with excellent technical assistance; A. K. Voges and E. Clemmons for interpreting radiographs
for the nonhuman primate study; S. Ganatra for radiology services; E. Romero for veterinary
services; K. A. Soileau and the staff of the New Iberia Research Centre for care of the
nonhuman primates; E. J. Dick for veterinary pathology; Polymun Scientific for excellent
formulation services; Acuitas Therapeutics for fruitful discussions; and S. Wigge and
C. Lindemann for scientific writing support and manuscript review. BioNTech is the sponsor of
the study, and Pfizer is its agent. BioNTech and Pfizer are responsible for the design, data
collection, data analysis, data interpretation and writing of the report. The corresponding
authors had full access to all the data in the study and had final responsibility for the decision
to submit the data for publication. This study was not supported by any external funding at
the time of submission.
Author contributions U.S. conceived and conceptualized the work and strategy. S.Hein,
S.C.D., A.A.H.S., C.K., R.d.l.C.G.G., and M.C.G. designed primers, performed oligosynthesis,
cloned constructs and performed protein expression experiments. T.Z., S.F., J.S. and A.N.K.
developed, planned, performed and supervised RNA synthesis and analysis. E.H.M. purified
S(P2). N.L.N. purified RBD trimer and ACE2 peptidase domain. J.A.L. developed ACE2–
B0AT1–RBD trimer formation and purified the complex. P.V.S. developed and performed
biolayer interferometry experiments. J.A.L. and S. Han performed electron microscopy and
solved the structure of the complex. Y.C. supervised the structural and biophysical
characterization and analysed the structures. A.M. and B.G.L. performed surface plasmon
resonance spectroscopy. A.G., S.A.K., S.S., T.H., L.F. and F.V. planned, performed and
analysed in vitro studies. F.B., T.K. and C.R. managed the formulation strategy. A.B.V., M.V.,
L.M.K. and K.C.W. designed mouse studies, and analysed and interpreted data. A.P., S.E.,
D.P. and G.S. performed and analysed the S1- and RBD-binding IgG assays. M.G. designed
and optimized MS2 SARS-nCoV-2 N1 RT–qPCR assay. M.G., R.C. Jr and K.J.A. performed and
analysed viral RT-qPCR data. A.M., B.S. and A.K.-W. performed and analysed pVNT assays.
C.F.-G. and P.-Y.S. performed and analysed VNT assays. D.E., D.S., B.J., Y.F. and H.J.
performed in vivo studies and ELISpot assays. A.B.V., K.C.W., J.L., M.S.M., A.O.-S. and M.V.
planned, analysed and interpreted ELISpot assays. L.M.K., J.L., D.E., Y.F., H.J., A.P.H., M.S.M.
and P.A.-Q. planned, performed and analysed flow cytometry assays. A.B.V., L.M.K., Y.F. and
H.J. planned, performed, analysed and interpreted cytokine release assays. M.R.G. read and
interpreted radiographs and computed tomography scans. O.G. and S.C. read and
interpreted histopathology specimens. R.S.S. and S.C. interpreted histopathology data. I.K.,
K.A.S., K.T., C.Y.T., M.G., D.K. and P.R.D. designed nonhuman primate studies, and analysed
and interpreted data. K.T., M.P., I.L.S. and W.V.K. oversaw immunogenicity and serology
testing of nonhuman primates. S.H.-U. and K.B. provided veterinary services for nonhuman
primates. J.A.F., J.C., T.C. and J.O. managed the nonhuman primate colony. U.S., Ö.T., P.R.D,
L.M.K., A.M. and M.V. contributed to synthesis and integrated interpretation of obtained
data. A.B.V., I.K., Y.C., A.M., M.V., L.M.K., C.T., K.A.S., Ö.T., P.R.D, K.U.J. and U.S. wrote the
manuscript. All authors supported the review of the manuscript.
Competing interests The authors declare that U.S. and Ö.T. are management board members
and employees at BioNTech SE (Mainz, Germany); K.C.W., B.G.L., D.S., B.J., T.H., T.K. and C.R. are
employees at BioNTech SE; A.B.V., A.M., M.V., L.M.K., S. Hein, A.G., T.Z., F.B., A.P., D.E., S.C.D.,
S.F., S.E., F.B., B.S., A.K.-W., Y.F., H.J., S.A.K., S.S., A.P.H., P.A.-Q., J.S., A.A.H.S., C.K., R.d.l.C.G.G.,
L.F. and A.N.K. are employees at BioNTech RNA Pharmaceuticals GmbH (Mainz, Germany);

Article
A.B.V., A.M., K.C.W., A.G., S.F., A.N.K and U.S. are inventors on patents and patent applications
related to RNA technology and COVID-19 vaccines; A.B.V., A.M., M.V., L.M.K., K.C.W., S. Hein,
B.G.L., A.P., D.E., S.C.D., S.F., S.E., D.S., B.J., B.S., A.P.H., P.A.-Q., J.S., A.A.H.S., T.H., L.F., C.K., T.K.,
C.R., A.N.K., Ö.T. and U.S. have securities from BioNTech SE; I.K., Y.C., K.A.S. J.A.L. M.S.M., K.T.,
A,O.-S., J.A.F., M.C.G., S. Han, J.A.L., E.H.M., N.L.N., P.V.S., C.Y.T., D.P., W.V.K., J.O., R.S.S., S.C., T.C.,
I.L.S., M.W.P., G.S., and P.R.D., K.U.J. are employees of Pfizer and may hold stock options; C.F.-G.
and P.-Y.S. received compensation from Pfizer to perform neutralization assays; M.R.G. received
compensation from Pfizer to read and interpret radiographs and computed tomography scans.
J.C., S.H.-U., K.B., R.C., Jr., K.J.A. O.G. and D.K., are employees of Southwest National Primate
Research Center, which received compensation from Pfizer to conduct the animal challenge
work; M.G. is an employee of Texas Biomedical Research Institute, which received

compensation from Pfizer to conduct the RT–qPCR quantification of viral load. The authors
declare no other relationships or activities that could appear to have influenced the submitted
work.
Additional information
Supplementary information The online version contains supplementary material available at
https://doi.org/10.1038/s41586-021-03275-y.
Correspondence and requests for materials should be addressed to U.S.
Peer review information Nature thanks Wolfgang Baumgärtner and the other, anonymous,
reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.

Extended Data Fig. 1 | Antigen expression and receptor affinity of vaccines.
a, Detection of BNT162b1-encoded RBD–foldon and BNT162b2-encoded S(P2)
in HEK293T cells by S1-specific antibody staining and flow cytometry. HEK293T
cells analysed by flow cytometry were incubated with: no RNA (control),
BNT162b RNA formulated as lipid nanoparticles (BNT162b1 and BNT162b2) or
BNT162b RNAs mixed with a transfection reagent (BNT162b1 RNA and
BNT162b2 RNA). Heights of bars indicate the means of technical triplicates.
b, Localization of BNT162b1 RNA-encoded RBD–foldon or BNT162b2
RNA-encoded S(P2) in HEK293T cells transfected as in a, determined by
immunofluorescence staining. Endoplasmic reticulum and Golgi (ER/Golgi)
(red), S1 (green) and DNA (blue). Scale bar, 10 μm. c, Western blot of denatured
and non-denatured samples of size-exclusion chromatography fractions

(chromatogram in Supplementary Fig. 1) of concentrated medium from
HEK293T cells transfected with BNT162b1 RNA. The RBD–foldon was detected
with a rabbit monoclonal antibody against the S1 fragment of SARS-CoV-2 S.
Protein controls (ctrl): purified, recombinant RBD and S. d, Biolayer
interferometry (BLI) sensorgram demonstrating the binding kinetics of the
purified RBD–foldon trimer, expressed from DNA, to immobilized human
ACE2 PD. e, f, Biolayer inferometry sensorgrams showing binding of a
DNA-expressed S(P2) preparation from a size-exclusion chromatography peak
(not shown) that contains intact S(P2) and dissociated S1 and S2 to immobilized
human ACE2 PD (e) and B38 monoclonal antibody (f). Binding data are in
colour; 1:1 binding models fit to the data are in black. Apparent kinetic
parameters are provided in the graphs.

Article

Extended Data Fig. 2 | Cryo-EM evidence for alternative conformers of
S(P2). a, Representative 2D class averages of TwinStrep-tagged S(P2) particles
extracted from cryo-EM micrographs. Box edge, 39.2 nm. b, Fourier shell

correlation curve from RELION gold-standard refinement of the S(P2) trimer.
c, Flowchart for cryo-EM data processing of the complex, showing 3D class
averages.

Extended Data Fig. 3 | See next page for caption.

Article
Extended Data Fig. 3 | BNT162b-elicited antibody responses in mice.
a–d, BALB/c mice (n = 8) were injected intramuscularly with a single dose of one
of the BNT162b vaccine candidate or buffer (control, n = 8 (a, b) and n = 16 (c, d)).
P values of day 28 compared to control (multiple comparison of mixed-effect
analysis (a, b) and one-way analysis of variance (c, d), all using Dunnett’s
multiple comparisons test) are provided. a, b, RBD- and S1-specific IgG
responses (geometric mean of each group ± 95% confidence interval) in sera
obtained 7, 14, 21 and 28 days after injection with BNT162b1 (a) or BNT162b2 (b),
determined by ELISA. For day-0 values, a prescreening of randomly selected
mice was performed (n = 4). c, d, RBD-specific IgG reciprocal serum endpoint
titres 14 (c) and 28 days (d) after injection. The horizontal dotted line indicates
the LLOD. Each data point represents one mouse, and the height of bar
indicates the geometric mean of groups. e, f, Representative surface plasmon
resonance sensorgram of the binding kinetics of His-tagged RBD (e) to

immobilized mouse IgG from serum drawn 28 days after injection with 5 μg
BNT162b candidates. Binding data (in colour) and 1:1 binding model fit to the
data (black) are depicted. e, Number of infected cells per well in a VSV-SARSCoV-2 pVNT50 assay conducted with serial dilutions of mouse serum samples
drawn 28 days after injection with BNT162b1 (f) or BNT162b2 (g). Lines
represent individual sera measured in triplicate. Horizontal dotted lines
indicate geometric mean ± 95% confidence interval (as grey area) of infected
cells in the absence of mouse serum (virus-positive control). h, Pearson
correlation of VSV-SARS-CoV-2 pVNT50 with live SARS-CoV-2 VNT50 for n = 10
random selected serum samples from mice immunized with BNT162b1 and
BNT162b2 each. For several samples, identical pVNT50 and VNT50 values were
measured: blue symbols represent individual mice; red symbols represent
2 mice; green symbol represents 3 mice.

Extended Data Fig. 4 | See next page for caption.

Article
Extended Data Fig. 4 | T cell response and T cell and B cell phenotyping of
BNT162b-immunized mice. BALB/C mice (n = 8 per group, unless stated
otherwise) were injected intramuscularly with one of the BNT162b vaccines or
buffer (control). a, b, Separated T cells from spleen or splenocytes of BALB/c
mice were ex vivo restimulated with full-length S peptide mix or cell culture
medium (medium). Symbols represent individual mice. Height of bars indicate
the mean of each group. a, IFNγ ELISpot of separated splenic CD4+ or CD8+
T cells after immunization using 1 μg of one of the BNT162b vaccines
(BNT162b1, n = 7 for CD4+ T cells, one outlier removed by Grubbs test, α = 0.05).
P values compare immunized groups with the control (parametric, two-tailed
paired t-test). b, CD8+ T-cell-specific cytokine release by splenocytes after

immunization using 5 μg of one of the BNT162b vaccines or buffer (control),
determined by flow cytometry. S-peptide-specific responses are corrected for
background (medium). P values compare immunized groups with the control
(parametric, two-tailed unpaired t-test assuming equal s.d.). c–e, T cell and
B cell phenotype composition after immunization with BNT162b candidates
was determined by flow cytometry. P values were determined by a two-tailed,
unpaired t-test assuming populations to have the same s.d. c, B cell and T cell
numbers in draining lymph nodes (dLN) (popliteal, iliac and inguinal lymph
nodes). For B cell subtyping, control, n = 4; BNT162b2, n = 7. #For per cent ICOS+
cells of TFH cells, only BNT162b2 data are available. d, B cell and TFH cell numbers
in the spleen. e, B cell and T cell numbers in the blood.

Extended Data Fig. 5 | See next page for caption.

Article
Extended Data Fig. 5 | Macaque CD4+ and CD8+ T cell response. Macaques
(n = 6 per group) were injected on day 0 and day 21 with 30 μg or 100 μg
BNT162b2, as in Figs. 3, 4. PBMCs collected on days 0, 14, 28 and 42 after
BNT162b2 injection were ex vivo restimulated with full-length S peptide mix.
a, Scatter plot showing the correlation of IL-4- and IFNγ-secreting cells by
ELISpot of day-42 PBMCs. b, IFNγ, IL-2 or TNF release from CD4+ T cells by flow

cytometry (LLOD = 0.04). c, IL-4 release from CD4+ T cells by flow cytometry
(LLOD = 0.05). d, IFNγ release from CD8+ T cells by flow cytometry
(LLOD = 0.03). Heights of bars indicate the arithmetic means for each group.
Whiskers indicate s.e.m. (b–d). Each symbol represents one macaque.
Horizontal dashed lines mark LLODs. Values below the LLOD were set to 1/2 the
LLOD. Arrows below the x axis indicate the days of doses 1 and 2.

Extended Data Fig. 6 | Schedule of macaque SARS-CoV-2 challenge and
necropsy. Timing in days from dose 2 of vaccine or saline (numbers to the left
of the bars) and of necropsy (numbers inside bars) are presented relative to the
day of SARS-CoV-2 or mock-challenge (day 0). Numbers of macaques
represented by the bars are indicated by red numbers to the right of the bars.

Control, macaques challenged but not immunized with BNT162b. Sentinel,
macaques mock-challenged (cell culture medium only). n/a, macaques not
necropsied. Additional details, including timing of sample collections and
radiographic examinations, are in Extended Data Table 2.

Article

Extended Data Fig. 7 | See next page for caption.

Extended Data Fig. 7 | Viral RNA detection in oropharyngeal and rectal
swabs, and clinical signs in BNT162b-immunized macaques after challenge
with infectious SARS-CoV-2. Macaques immunized using 100 μg of BNT162b1
or BNT162b2 (n = 6 each) and macaques mock-immunized using saline or not
immunized (control, n = 9) (as described in Fig. 4, Extended Data Fig. 6,
Extended Data Table 2) were challenged with 1.05 × 106 total PFU of SARS-CoV-2
split equally between the intranasal and intratracheal routes. Additional
macaques (sentinel, n = 6) were mock-challenged with cell culture medium.
a, b, Viral RNA levels were detected by RT–qPCR in oropharyngeal (a) and rectal
(b) swabs. Ratios above data points indicate the number of viral-RNA-positive
macaques among all macaques that provided evaluable samples in a group.
Heights of bars indicate geometric mean of viral RNA copies; whiskers indicate

geometric s.d. Every symbol represents one macaque. Dotted lines indicate the
LLODs. Values below the LLOD were set to 1/2 the LLOD. The two-sided
statistical significance by Friedman’s nonparametric test of differences in viral
RNA detection between 6 BNT162b1-immunized and 6 contemporaneously
control-immunized macaques (challenge cohorts 1 and 2) after challenge was
P < 0.0001 for oropharyngeal swabs and P = 0.1179 for rectal swabs; between 6
BNT162b2-immunized macaques and 3 contemporaneously controlimmunized macaques (challenge cohort 3) after challenge, the statistical
significance was P = 0.0007 for oropharyngeal swabs and P = 0.2209 for rectal
swabs. c–f, Vital signs were recorded. c, Body weight change. d, Temperature
change. e, Heart rate. f, Oxygen saturation. Each data point represents an
arithmetic mean. Whiskers indicate s.d.

Article

Extended Data Fig. 8 | See next page for caption.

Extended Data Fig. 8 | Radiographic signs and pulmonary histopathology
of macaques immunized with BNT162b1 or BNT162b2 and challenged with
SARS-CoV-2. Macaques were immunized using BNT162b1 or BNT162b2 or
mock-immunized with saline (control) and challenged with SARS-CoV-2, and a
sentinel group was challenged with cell culture medium. The disposition of the
macaques for immunization, infectious challenge, imaging and necropsy are
described in Figs. 3, 4, Extended Data Fig. 6, Extended Data Table 2. Three-view
thoracic radiographs (ventrodorsal, right and left lateral) and lung field
computed tomography images were obtained. The macaques were
anaesthetized and intubated to perform end inspiratory breath-hold. Images
were interpreted by two board-certified veterinary radiologists blinded to
treatment groups. Scores were assigned to 7 lung regions on a severity scale of
0–3 per region, with a maximum severity score of 21. Pulmonary lesions
evident before challenge, or those which could not be unequivocally attributed

to the viral challenge (such as atelectasis secondary to recumbency and
anaesthesia), received a score of 0. a, Thoracic radiograph scores. b, Lung field
computed tomography scores. Each dot represents the summed radiograph or
computed tomography scores for the seven lung lobes of a single macaque.
Two veterinary pathologists blindly performed microscopic evaluation of
formalin-fixed, haematoxylin and eosin-stained lung tissue sections from each
of seven lobes from each macaque that had been necropsied on day 7 or day 8.
Inflammation scores were assigned by consensus between the pathologists on
a scale of 1–5 on the basis of the area of involvement. c, Pulmonary
histopathology scores. Each dot represents the mean inflammation area score
from the seven lung lobes of an individual macaque. In a–c, the height of each
bar indicates the arithmetic mean of the radiograph, computed tomography or
histopathology score, respectively, for the macaques in each group, and
whiskers indicate s.d.

Article
Extended Data Table 1 | Cryo-EM data collection, 3D reconstruction and refinement statistics

Extended Data Table 2 | Schedule of macaque immunization, challenge, sample collection, radiological examination and
necropsy

1

All macaques in the BNT162b1, BNT162b2 and control challenge groups were challenged with SARS-CoV-2. Macaques in the sentinel challenge group were mock-challenged.
‘No immunization’ is indicated by ‘−’.
3
Challenge cohort 2 was challenged with SARS-CoV-2 or mock-challenged one week after challenge cohort 1. Challenge cohort 3 was challenged with SARS-CoV-2 or mock-challenged six
weeks after challenge cohort 2.
2

4

All macaques were challenged with SARS-CoV-2 or mock-challenged, according to their challenge group. The entry for ‘days from dose 2 to SARS-COV-2 or mock challenge’ for macaques that
were not immunized is ‘−’.

Last updated by author(s): Dec 30, 2020

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Corresponding author(s): Ugur Sahin

Statistics
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Software and code
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Western Blot: Fusion FX Imager (Vilber), Image Lab software version 6.0
Flow cytometry data: BD Biosciences, BD FACSDiva V9.1 or 8.0.1
Immunofluorescence: Leica SP8 confocal microscope, Application Suite LAS-X Version 3.1.5.16308
Biolayer interferometry: Octet Data Acquisition software version 10.0.0.87, ForteBio Data Analysis software version 10.0
Cryo-EM: Titan Krios (Thermo Fisher Scientific), SerialEM software version 3.8.0 beta
S1- and RBD-binding IgG assay data; Cytokine profiling: Gen5 V3.0.9 or Bioplex200 system (Bio-Rad),
Surface plasmon resonance spectroscopy: Biacore T200 device (Cytiva), Biacore T200 Evaluation Software Version 3.1
pVNT: IncuCyte Live Cell Analysis system (Sartorius), IncuCyte 2019B Rev2 software
ELISPOT spot count data: ImmunoSpot® S6 Core Analyzer or Universal Analyzer [CTL], Image Aquision V7.0 or the ImmunoSpot 7.0.17.0
Professional
VNT: Cell Imaging Multi-Mode Reader (BioTek), Gen5 Image Prime version 3.09
RT-qPCR: QuantStudio 3 instrument (Applied Biosystems)

Data analysis

Flow cytometry: FlowJo V10.6 or 10.4.1 (FlowJo LLC, BD Biosciences)
Biolayer interferometry: Octet Data Analysis v10.0 (FortéBio)
Cryo-EM: RELION 3.1-beta
Cytokine profiling: ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific)
Data visualisation and statistical analysis: GraphPad Prism V8

April 2020

Data collection

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- Accession codes, unique identifiers, or web links for publicly available datasets
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
The vaccine sequence is based on GenBank: MN908947.3
For P2 S, the atomic model from PDB ID 6XR8 was rigid-body fitted into the map density (https://www.rcsb.org/structure/6XR8).
The cryo-EM maps and atomic coordinates have been deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) with accession
numbers EMD-23211 and PDB 7L7F for the ACE2/B0AT1/RBD-foldon complex and EMD-23215 and PDB 7L7K for P2 S.

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Data

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
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For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

No sample-size calculation was performed.
For mouse studies, a two-sided test with the hypothesis that the mean is a given value, with the shift to be detected a multiple of the
standard deviation was taken into account. For ɲ=0.05 and a desired power of 80%, a group size of n = 8 is required to find significant
differences between groups.
For non-human primate studies, sample size was limited by availability of suitable animals.

Data exclusions

No data was excluded.

Replication

Replication was not attempted. Independent studies and analysis methods to analyse immune responses were performed.

Randomization

Mice or NHP were randomly allocated to groups. No formal randomization was done.

Blinding

Studies were performed unblinded as analyis results could not be manipulated by interpretation. Thoracic radiographs and computed
tomography scan images were interpreted by a board-certified veterinary radiologist blinded to treatment groups. Histopathology slides were
read by veterinary pathologists blinded to treatment groups.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,
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Materials & experimental systems

Methods

n/a Involved in the study

n/a Involved in the study

Antibodies

ChIP-seq

Eukaryotic cell lines

Flow cytometry

Palaeontology and archaeology

MRI-based neuroimaging

Animals and other organisms
Human research participants
Clinical data
April 2020

Dual use research of concern

Antibodies
Antibodies used

Used reagents (specificity/ fluorochrome/ clone/ manufacturer/ catalogue number/lot number/dilution):
Anti-mouse:
B220/CD45R / AF647 / RA3-6B2 / BioLegend / 103226 / B243962 / 1:1500

2

nature research | reporting summary

CD138 / BV605 / 281-2 / BioLegend / 142531 / B299222 / 1:200
CD19 / BV786 / 1D3 / BD Bioscience / 563333 / 0023948 / 1:1000
CD19 / AF700 / 6D5 / BioLegend / 115528 / B261756 / 1:100
CD278/ICOS / PerCPeF710 / 7E.17G9 / Invitrogen / 46-9942-82 / 2029789 / 1:50
CD38 / PE / 90 / eBioscience / 12-0381-82 / 2150667 / 1:400
CD38 / AF488 / 90 / BioLegend / 102714 / B298187 / 1:100
CD3e / BUV395 / 145-2C11 / BD Bioscience / 563565 / 9204644 / 1:100
CD4 / PerCP-Cy5.5 / RM4-5 / BioLegend / 100540 / B261856 / 1:800
CD4 / BV480 / RM4-5 / BD Bioscience / 565634 / 9016508, 0107454 / 1:250
CD44 / BUV563 / IM7 / BD Bioscience / 741227 / 0119427 / 1:5000, 1:2500
CD62L / BV785 / MEL-14 / BioLegend / 104440 / B258213 / 1:200
CD8a / PerCP-Cy5.5 / 53-6.7 / BD Bioscience / 551162 / 9098816 / 1:800
CD8a / BUV737 / 53-6.7 / BD Bioscience / 564297 / 9030634 / 1:200
CD8a / FITC / 53-6.7 / BD Bioscience / 553031 / 9143776 / 1:200
CD95/Fas / FITC / Jo2 / BD Bioscience / 561979 / 8296755 / 1:100
CXCR5 / Purified / 2G8 / BD Bioscience / 551961 / 9143926 / 1:100
CXCR5 / BV421 / L138D7 / BioLegend / 145512 / B281252 / 1:100
F4/80 / PerCP-Cy5.5 / BM8 / BioLegend / 123128 / B276793 / 1:800
FC block CD16/CD32 / Purified / 2.4G2 / BD Bioscience / 553142 / 9060742 / 1:100
GATA3 / PE-Cy7 / TWAJ / Invitrogen / 25-9966-42 / 2142972 / 1:25
GR-1 / PerCP-Cy5.5 / RB6-8C5 / BioLegend / 108428 / B278340 / 1:800
IFNɶ / PE-Cy7 / XMG1.2 / BD Bioscience / 564336 / 9337390 / 1:1000
IgD / BV421 / 11-26c.2a / BioLegend / 405725 / B280598 / 1:2500
IgG1 / BV480 / A85-1 / BD Bioscience / 746811 / 0115095 / 1:200
IgG2a / BV711 / R19-15 / BD Bioscience / 744533 / 0115092 / 1:200
lgG2a / Biotin / RG7/1.30 / BD Bioscience / 553894 / 9288614 / 1:100
IgM (Igh-Ca/Cb) / PE-Cy7 / R6-60.2 / BD Bioscience / 552867 / 9269114 / 1:200
IL-2 / APC-R700 / JES6-5H4 / BD Bioscience / 565186 / 9303906 / 1:5000
PD-1/CD279 / BV605 / 29F.1A12 / BioLegend / 135219 / B303691 / 1:50
T-bet / AF647 / 4B10 / bioLegend / 644804 / B248741 / 1:200
TNF / BB700 / MP6-XT22 / BD Bioscience / 566510 / 0021825 / 1:5000
Anti-rhesus, anti-human:
CD154 / BV605 / 24-31 / BioLegend / 310826 / B250362 / 1:60
CD20 / PE-Cy5.5 / 2H7 / Invitrogen / 35-0209-41 / 2005219 / 1:600
CD3 / AF700 / SP34-2 / BD Bioscience / 557917 / 9277122 / 1:15
CD4 / BF480 / SK3 / BD Bioscience / 566104 / 58964 / 1:60, 1:30
CD8 / BB700 / RPA-T8 / BD Bioscience / 566452 / 16984 / 1:1200
IFNy / FITC / B27 / BD Bioscience / 552887 / 9329760 / 1:15
IL-2 / PE-Cy7 / MQ1-17H12 / Invitrogen / 25-7029-42 / 2136515 / 1:60
IL-4 / BV421 / MP4-25D2 / BD Bioscience / 564110 / 15834 / 1:30
TNF / BUV395 / Mab11 / BD Bioscience / 563996 / 10280 / 1:30
TruStain FcX / Purified / - / BioLegend / 422302 / B298875 / 1:60
Anti-viral:
SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit Mab / AF647 (labelled) / #007 / Sino Biological / 40150-R007 / MA14FE2702 / 1:400
[flow cytometry]
SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit Mab / Purified / #007 / Sino Biological / 40150-R007 / MA14FE2702 / 1:1000
[Western blot]
SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit Mab / AF647 (labelled) / #007 / Sino Biological / 40150-R007 / MA14FE2702 / 1:100
[Immunofluorescence]
B38 monoclonal antibody, SARS-CoV-2 receptor binding domain; produced at Pfizer for internal research (no catalogue number); 10
ђg/mL (biolayer inferometry)
VSV-G Antibody (clone 8G5F11) / purified / monoclonal / Kerafast Inc. / EB0010 / - / 1:2000

Validation

April 2020

Secondary antibodies and others:
Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcɶ fragment specific / HRP / polyclonal / Jackson ImmunoResearch / 115-035-071 /
144460 / 1:15000 [S1- and RBD-specific serum Ab, mouse]
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat / HRP / polyclonal / Sigma Aldrich / A0545 / 028M4755V /
1:10000 [Western blot]
AffiniPure Goat Anti-Rabbit IgG (H+L) / AF488 / polyclonal / Jackson ImmunoResearch / 111-545-003 / 122290 / 1:300
[Immunofluorescence]
AffiniPure Goat Anti-Mouse IgG, Fcɶ fragment specific (min X Hu, Bov, Hrs Sr Prot) / HRP / polyclonal / Jackson ImmunoResearch /
115-005-071 / 107421 / 1:56.7 [SPR spectroscopy of polyclonal mouse immune sera]
goat anti-human polyclonal secondary antibody / R-PE / polyclonal / Jackson ImmunoResearch / 109-115-098 / 147186 / 1:500
Mouse IgG-UNLB / Purified / polyclonal / Southern Biotech / 0107-01 / D2519-N640 / 1:300 - 1:656100 [S1- and RBD-specific serum
Ab, mouse]
Fixable Viability Dye / eF780 / - / eBioscience / 65-0865-14 / 2178170 / 1:1000 and 1:1600 (mouse flow cytometry)
Fixable Viability Dye / eF450 / - / eBioscience / 65-0863-14 / 2143488 / 1:500 (HEK293T/17 flow cytometry)
Fixable Viability Dye / eF780 / - / eBioscience / 65-0865-14 / 2186489 / 1:5000 (rhesus flow cytometry)
Streptavidin / BV421 / - / BD Bioscience / 563259 / 9197684 / 1:200 [mouse flow cytometry]
Concanavalin A / AF594 / - / ThermoFisher Scientific / C11253 / 2160047 / 1:100 [Immunofluorescence]
Lectin GS-II From Griffonia simplicifolia / AF594 / - / ThermoFisher Scientific / L21416 / 2047156 / 1:100 [Immunofluorescence]
Hoechst 33342 / - / - / ThermoFisher Scientific / H3570 / 1733139 / 1:5000 [Immunofluorescence]
Commercial available Western blot, flow cytometry and immunofluorescence antibodies were selected based on their antigen

3

Validation

Eukaryotic cell lines
Policy information about cell lines
Cell line source(s)

Human embryonic kidney (HEK)293T/17, Cercopithecus aethiops kidney Vero 76 and CCL81 cells (all ATCC); human
embryonic kidney Expi293F™ (Thermo Fisher Scientific)

Authentication

Reauthentication of cell lines was performed by short tandem repeat (STR) profiling at supplier

Mycoplasma contamination

All used cell lines tested negative for mycoplasma contamination

Commonly misidentified lines

No commonly misidentified cell lines were used

nature research | reporting summary

specificity and suggested application as described on the manufacturer`s website and data sheets. Secondary antibodies and others
were validated by the manufacturers for the different detection methods. All 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 (flow cytometry) and (ii) a maximal separation of the negative and positive
population (flow cytometry) or (iii) no minimal to no background signal (ELISA, western Blot, immunofluorescence, SPR). The
specificity of the B38 monoclonal antibody was originally described in Wu et al., Science 368:1274-8. The specificity was confirmed in
these experiments by the absence of a background signal by biolayer inferometry and presence of signal when reacted with SARSCoV-2 spike antigens with identities confirmed by structure determination.

(See ICLAC register)

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals

Male rhesus macaques (Macaca mulatta) (2–4 years)
Female BALB/c mice (Janvier; 8-12 weeks)

Wild animals

The study did not involve wild animals.

Field-collected samples

The study did not involve animals collected from the field.

Ethics oversight

All mouse studies were performed at BioNTech SE, and protocols were approved by the local authorities (local welfare committee),
conducted according to Federation of European Laboratory Animal Science Associations recommendations and in compliance with
the German Animal Welfare Act and Directive 2010/63/EU. Only animals with an unobjectionable health status were selected for
testing procedures.
Immunisations for the non-human primate (NHP) study were performed at the University of Louisiana at Lafayette-New Iberia
Research Centre (NIRC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC,
Animal Assurance #: 000452). The work was in accordance with USDA Animal Welfare Act and Regulations and the NIH Guidelines for
Research Involving Recombinant DNA Molecules, and Biosafety in Microbiological and Biomedical Laboratories. All procedures
performed on these animals were in accordance with regulations and established guidelines and were reviewed and approved by an
Institutional Animal Care and Use Committee or through an ethical review process. Infectious SARS-CoV-2 challenge of NHPs
following immunisation was performed at the Southwest National Primate Research Centre (SNPRC), Texas Biomedical Research
Institute, which is also accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, Animal
Assurance #: 000246). Animal husbandry followed standards recommended by AAALAC International and the NIH Guide for the Care
of Use of Laboratory Animals. This study was approved by the Texas Biomedical Research Institute Animal Care and Use Committee.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants
This manuscript describes preclinical studies. As a benchmark for the non-human primate serology, SARS-CoV-2 neutralising
titers and RBD-binding IgG levels of a panel of human SARS-CoV-2/COVID-19 convalescent sera are reproduced, verbatim,
from three clinical publications (Sahin et al., Nature 10.1038/s41586-020-2814-7, 2020; Mulligan et al., Nature 10.1038/
s41586-020-2639-4, 2020; Walsh et al., The New England Journal of Medicine; 10.1056/NEJMoa2027906, 2020). Please refer
to the clinical reports for background to the referenced clinical data.

Recruitment

This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.

Ethics oversight

This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

April 2020

Population characteristics

4

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 This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.
Study protocol

This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.

Data collection

This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.

Outcomes

This manuscript describes preclinical studies. See the clinical reports for background to the referenced clinical data.

Flow Cytometry
Plots

nature research | reporting summary

Clinical data

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
Mice: For flow cytometry or ELISpot, blood or cell suspensions from lymph node and spleen were used directly or after
peptide stimulation. Peripheral blood was collected from the retro-orbital venous plexus or vena facialis under isoflurane
anaesthesia. Spleen single-cell suspensions were prepared in PBS by mashing tissue against the surface of a 70 ђm cell
strainer (BD Falcon) using the plunger of a 3-mL syringe (BD Biosciences). Erythrocytes were removed by hypotonic lysis.
Popliteal, inguinal and iliac lymph nodes were pooled, cut into pieces, digested with collagenase D (1 mg/mL; Roche) and
passed through cell strainers. For intracellular stains, cells were fixed and permeabilized using the eBioscience™ Foxp3/
Transcription Factor Staining Buffer Set.
NHP: Blood for serum and PBMCs was collected in compliance with animal protocol 2017-8725-023 approved by the NIRC
Institutional Animal Care and Use Committee. Animals were anesthetised with ketamine HCl (10 mg/kg; IM) during blood
collection and immunisation, and monitored for adequate sedation.

Instrument

Cell culture cells were acquired on a FACSCanto II flow cytometer (BD Biosciences). Mouse cells were acquired on a BD
Symphony A3 or BD Celesta (B-cell subtyping) flow cytometer (BD Bioscience). NHP cells were analyzed on a LSR Fortessa
X-20

Software

Cell culture cells were analyzed using BD FACSDiva software version 8.0.1 and analysed by FlowJo software version 10.6.2
(FlowJo LLC, BD Biosciences). Mouse cells were analyzed using BD FACSDiva software version 9.1 or 8.0.1.1, respectively, and
analysed with FlowJo 10.6 (FlowJo LLC, BD Biosciences). NHP cells were analyzed using FlowJo (10.4.1).

Cell population abundance

Sorted CD4 CD8 T cells from mouse were confimed following magnetic bead separation.

Gating strategy

The gating strategies are detailed in the supplementary information.
Mouse: Flow cytometry gating strategy for the identification of IFNɶ, IL-2, and TNF secreting CD8+ T cells in the mouse
spleen. was performed after CD8+ T cells were gated within single, viable lymphocytes, excluding CD4+ T cells.
Flow cytometry gating strategy for identification of TFH cells, activated T cells and B cells in lymph nodes and the spleen was
performed by CD3+CD19- T cells gating within single, viable lymphocytes. CD4+ and CD8+ T cells were gated from CD3+ cells;
TFH cells were gated from CD4+ T cells and defined as CD4+ T-bet- GATA3- CD44+ CD62L- PD-1+ CXCR5+ cells.
Flow cytometry gating strategy for the identification of B cells in lymph nodes and the spleen was done by gating activated B
cells within single, viable lymphocytes defined as IgD-Dump (CD4, CD8, F4/80, GR-1)- cells. Plasma cells (PC) were gated from
activated B cells and defined as CD138+ B220low/- cells. Switched B cells were gated from non-PC and defined as CD19+
CD138- IgM-. Germinal centre (GC) and IgG1+ and IgG2a+ B cells were gated from switched B cells and defined as CD19+
IgM- CD38- CD95+ and CD19+ IgM- IgG1+/IgG2a+, respectively.
Flow cytometry gating strategy for the identification of T cells, B cells and TFH cells in peripheral blood was performed by
gating CD3+ CD19- T cells within single, viable lymphocytes. CD4+ and CD8+ T cells were gated from CD3+ CD19- cells. TFH
cells were gated from CD4+ T cells and defined as CD4+ T-bet- GATA3- CD44+ CD62L- PD-1+ CXCR5+ cells.
Rhesus macaque:
Flow cytometry gating strategy for identification of spike-specific SARS-CoV-2 modRNA vaccine BNT162b2-induced T cells
started with events acquired with a constant flow stream and fluorescence intensity, viable cells, lymphocytes and single
events were identified and gated. Within singlet lymphocytes, CD20- CD3+ T cells were identified and gated into CD4+ T cells
and CD8+ T cells. Antigen-specific CD4+ T cells were identified by gating on CD154 and cytokine-positive cells, and CD8+ T
cells were identified by gating on CD69 and cytokine-positive cells. The antigen-specific cells were used for further analysis.

April 2020

Sample preparation

in vitro:

5

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

nature research | reporting summary

Flow cytometry gating strategy for the identification of HEK293T cells transfected with BNT162b1 or BNT162b2, or
BNT162b1-RNA or BNT162b2-RNA using a transfection reagent or no RNA (control) was performed by gating S1+ HEK293T
cells within single, viable HEK293T cells.

April 2020

6