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BNT162b2
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Page 1MODULE 2.6.2 PHARMAC OLOGY WRITTEN SUMMARY
This document contains confidential information belonging to BioNTech /Pfizer .Except as may be
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Page 2TABLE OF CONTENTS
LIST OF TABLES ................................ ................................ ................................ ..................... 3
LIST OF FIGURES ................................ ................................ ................................ ................... 3
LIST OF ABBREVIATION S................................ ................................ ................................ ....5
2.6.2. PHARMACOL OGY WRI TTEN SUMMARY ................................ ............................... 7
2.6.2.1. I ntroduction ................................ ................................ ................................ ......... 7
2.6.2.2. SARS -CoV -2 S as a Vaccine Target ................................ ................................ ...7
2.6.2.3. I n Vitro Expression of Antigens from BNT162b2 (V9) RNA ............................ 9
2.6.2.4. Structural and Biophy sical Characterization of P2 S as a Vaccine
Antigen ................................ ................................ ................................ ...................... 11
2.6.2.5. I mmunogenicit y of BNT162b2 (V9) in Mice ................................ ................... 14
2.6.2.6. BNT162b2 (V9) Vaccine Immunogenicit y and Evaluation of Protection
against SARS -CoV -2 Challenge in Rhesus Macaques ................................ ............. 21
2.6.2.6.1. I mmunogenicity in Rhesus Macaques ................................ .................. 21
2.6.2.6.2. SARS -CoV -2 Challenge of BNT162b2 (V9) -Immunized
Nonhuman Primates ................................ ................................ ......................... 26
2.6.2.7. Immunogenicit y Testing of Rats in the GLP Compliant Repeat Dose
Toxicity Studies and Developmental and Reproductive Toxicity Study .................. 31
2.6.2.7.1. Repeat -Dose Toxicity Study of Three LNP -Formulated RNA
Platforms Encoding for Viral Proteins by Repeated Intramuscular
Administration to Wistar Han Rats ................................ ................................ ..31
2.6.2.7.2. 17 -Day Intramuscular Toxicity Study of BNT162b2 (V9) in
Wistar Han Rats With a 3 -Week Recovery ................................ ..................... 33
2.6.2.7.3. A Combined Fertility and Developmental Study (Including
Teratogenicity and Postnatal Investigations) of BNT162b1, BNT162b2
and BNT162b3 b y Intramuscular Administration in the Wistar Han Rat .......33
2.6.2.8. Secondary Pharmacody namics ................................ ................................ .......... 34
2.6.2.9. Safet y Pharmacology ................................ ................................ ......................... 34
2.6.2.10. Pharmacod ynamic Drug Interactions ................................ .............................. 34
2.6.2.11. Discussion and Conclusions................................ ................................ ............ 34
2.6.2.12. I mmunogenicity and Eff icacy Methods ................................ .......................... 35
2.6.2.12.1. SARS -CoV -2 S1 and RBD Direct ELISA ................................ .......... 35
2.6.2.12.2. VSV/SARS -CoV -2 S Pseudovirus Neutralization Assay ................... 35
2.6.2.12.3. SARS -CoV -2 S1-Binding and RBD -Binding Kinetics using
Surface Plasmon Resonance Spectroscop y................................ ...................... 35
2.6.2.12.4. SARS -CoV -2 S1-Binding IgG Luminex Assay ................................ .36
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Page 32.6.2.12.5. SARS -CoV -2 Neutralization Assay ................................ ................... 36
2.6.2.12.6. ELI Spot and Cy tokine Profiling I mmunoassay s in Mice ................... 36
2.6.2.12.7. ELI Spot and Intracellular C ytokine Staining Assay s in NHPs .......... 37
2.6.2.12.8. Quantitative RT- PCR for Detection of SARS -CoV -2 Viral
RNA ................................ ................................ ................................ ................. 38
2.6.2.12.9. L ung Radio graphs and Computed Tomograph y Scans ...................... 38
2.6.2.12.10. Macroscopic and Microscopic Pathology ................................ ......... 38
2.6.2.13. References ................................ ................................ ................................ .......40
LIST OF TABLES
Table 2.6.2 -1. Summary of IgG Concentrations at Day 28 Post Immunization .............. 17
Table 2.6.2 -2. IgG antibod y Concentration [mg/mL] Against the Viral Antigen in
Wistar Han Rats after BNT162b2 (V8) Immunization............................. 32
Table 2.6.2 -3. Group Mean Titers of SARS -CoV -2 Neutralizing Antibodies ................. 33
Table 2.6.2 -4. Group Mean Titers of SARS -CoV -2 Neutralizing Antibodies ................. 34
LIST OF FIGURES
Figure 2.6.2-1. Replication Cy cle of a Coronavirus ................................ ............................ 8
Figure 2.6.2-2. Schematic of the Organization of the SARS- CoV -2 S Gly coprotein ......... 9
Figure 2.6.2-3. Flow Cy tometry Anal ysis of BNT162b2 Transfection Frequency ........... 10
Figure 2.6.2 -4. Immunofluorescence Detection of P2 S in BNT162b2 Transfected
Cells ................................ ................................ ................................ .......... 11
Figure 2.6.2-5. Binding to Cell Surface -Expressed Recombinant P2 S ............................ 12
Figure 2.6.2-6. Biolay er Interferometry Sensorgrams for Binding of P2 S to
ACE2 -PD and B38 mAb ................................ ................................ .......... 13
Figure 2.6.2-7. CryoEM P2 S Structure at 3.29 Å Resolution ................................ .......... 14
Figure 2.6.2-8. Anti- S IgG Response 7, 14, 21, and 28 d after Immunization with
BNT162b2 ................................ ................................ ................................ 15
Figure 2.6.2-9. Binding Kinetics of Murine SARS- CoV -2 S1-and RBD -specific
IgGs ................................ ................................ ................................ ........... 16
Figure 2.6.2-10. BNT162b2 Pseudovirus Neutralizing Titers 14, 21, and 28 d after
Immunization................................ ................................ ............................ 17
Figure 2.6.2-11. ELISpot Analy sis Using Splenocy tes Obtained on Day 28 after One
Immunization................................ ................................ ............................ 18
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Page 4Figure 2.6.2-12. Cytokine Release Anal ysis Using Splenocy tes Obtained on Day 28
after One Immunization ................................ ................................ ............ 19
Figure 2.6.2-13. B- and T -cell Phenoty ping in Ly mph Nodes of BNT162b2
Immunized Mice ................................ ................................ ....................... 20
Figure 2.6.2-14. S1-Binding IgG Levels Elicited by Immunization of Rhesus
Macaques with BNT162b2 ................................ ................................ .......22
Figure 2.6.2-15. 50% Serum Neutralizing Titers Elicited by Immunization of Rhesus
Macaques with BNT162b2 ................................ ................................ .......23
Figure 2.6.2-16. IFNγ and IL- 4 ELISpot Results in BNT162b2 I mmunized Animals .......24
Figure 2.6.2-17. S-specific CD4 and CD8 T- cell Response in BNT162b2 I mmunized
Animals as Measured b y ICS Assay ................................ ......................... 25
Figure 2.6.2-18. Viral RNA in BAL Fluid and Nasal and Oropharyngeal Swabs of
Rhesus Macaques after Infectious SARS- CoV -2 Challenge .................... 27
Figure 2.6.2-19. Clinical Signs in Rhesus Macaques after Immunization with
BNT162b2 and Challenge with I nfectious SARS -CoV -2........................ 29
Figure 2.6.2-20. Radiograph and CT Scores of Rhesus Macaque Lungs after
Infectious SARS- CoV -2 Challenge................................ .......................... 30
Figure 2.6.2-21. Lung Inflammation Area Score after IN/IT SARS- CoV -2 Challenge ..... 31
Figure 2.6.2-22. Pseudovirus Neutralization Activity in Rats after BNT162b2 V8
Immunization................................ ................................ ............................ 32
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Page 5LIST OF ABBREVIATIONS
Abbreviation Term
ACE2 angiotensin converting enzy me 2
BAL Bronchoalveolar lavage
CDC Centers for Disease Control
COVID -19 Coronavirus disease 2019
CT Cytoplasmic tail
DART Developmental and reproductive toxicology
dLIA Direct Luminex immunoassay
DSPC 1,2-distearo yl-sn-glycero-3-phosphocholine
ELISA enzy me-linked immunosorbent assay
ELISpot Enzy me-linked immunospot
FITC Fluorescein isothiocy anate
FP Fusion peptide
GD Gestation day
GFP Green fluorescent protein
GMC Geometric mean concentration
GMT Geometric mean titer
HCS Human convalescent sera
HR Heptad repeat
HRP Horseradish peroxidase
ICOS inducible costimulatory molecule
ICS Intracellular cy tokine staining
IFA immunofluorescence
IFN Interferon
IgG Immunoglobulin G
IL interleukin
IM intramuscular
IN Intranasal
IT intratracheal
kDa kilodalton
LD Lactation day
LLOQ lower limit of quantification
mAb Monoclonal antibody
MERS Middle East respiratory syndrome
mL milliliter
mNG mNeongreen
modRNA Modified mRNA
MW molecular weight
NHP Nonhuman primate
ORF Open reading frame
P2 S stable prefusion S including two prolin esubstitution s
PBS Phosphate -buffered saline
PD Protease domain
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Page 6Abbreviation Term
PFU Plaque forming unit
PND Postnatal day
PVDF Polyvinylidene fluoride
pVNT Pseudoty peneutralization titer
pVN T50 50% pseudovirus neutralizing titer
pVN T90 90% pseudovirus neutralizing titer
RNA ribonucleic acid
RDRP RNA -dependent RNA poly merase
RT-qPCR Reverse transcription -quantitative poly merase chain reaction
S SARS -CoV -2 spike glycoprotein
S1 / S2 SARS -CoV -2 spike gl ycoprotein subdomains 1 / 2
S2′ S2 protease cleavage site
SARS -CoV -2 severe acute respiratory syndrome coronavirus 2
SDS-PAGE Sodium dodecy l sulfate -polyacrylamide electrophoresis
SEC Size exclusion chromatograph y
SS Signal sequence
Tfh T follicular helper cell
TLR Toll-like receptor
TM transmembrane
TNF tumor necrosis factor
µg microgram
ULOQ Upper limit of quantification
US United States
USA United States of America
VEE Venezuelan equine encephalitis virus
VSV Vesicular stomatitis virus
VNT 50 50% virus neutralizing titer
WHO World Health Organization
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Page 72.6.2. PHARMACOLOGY WRITTEN SUMMARY
2.6.2.1. Introduction
BNT162b2 (BioNTech code number BNT162, Pfizer code number PF -07302048) is
BNT162b2 (V9),a nucleoside -modified mRNA (modRNA) vaccine that encodes the
SARS -CoV -2 full -length spike gl ycoprotein (S ). In some preclinical research,
adifferent variant of BNT162b2 was used: BNT162b2 (V8), which has a different codon
optimization but encodes a prot ein with the same amino acid sequence as
BNT162b2 (V9). In this document, “BNT162b2” refers to BNT162b2 (V9), unless otherwise
specified. The gl ycoprotein encoded b y both BNT162b2 variants includes two amino acid
substitutions toprolin e(P2S)locking the transmembrane protein in an antigenically optimal
prefusion conformation (Pallesen et al, 2017 ;Wrapp et al, 2020 ).
The RNA is formulated with functional and structural lipids, which protect the RNA from
degradation and enable transfection of the RNA into host cells after IM injection. The
formulation contains two functional lipids, AL C-0315 and AL C-0159, and, two structural
lipids ,DSPC (1,2- distearoy l-sn-glycero-3-phosphocholine) and cholesterol.
The modRNA comprises a single -stranded, 5 ′-capped mRNA that is translated upon entering
the cell. In addition to the open reading frame ( ORF )encoding the SARS -CoV -2 P2 S
antigen , each modRNA contains common structural elements optimized for high translation al
efficacy of the RNA. ModRNA also contains a substitution of 1 -methy l-pseudouridine for
uridine. This substitution decreases recognition of the vaccine RNA by innate immune
sensors, such as toll -like receptors (TLRs) 7 and 8, resulting in decreased innate immune
activation and increased protein translation (Kariko et al, 2005 ).Vaccination with modRNA
is characterized b y the strong expansion of Th1- skewed antigen -specific T follicular helper
(Tfh) cells, which stimulate and expand germinal center B cells, thereb y resulting in
particularl y strong, long -lived, high -affinity antibody responses (Sahin et al, 2014 ;
Pardi etal, 2018 ). The structural elements of BNT162b2 contain non-coding sequences
optimized for prolonged and strong translation of the P2 S antigen -encoding RNA
component.
2.6.2.2. SARS -CoV -2 S as a Vaccine Target
SARS -CoV -2is an enveloped, positive sense, single -stranded RNA virus that is coated with
S, which gives the virion its characteristic corona or “crown” appearance ( Figure 2.6.2-1 ).
Coronavirus S is a major target of virus neutralizing antibodies and isakey antigen for
vaccine development . S is a transmembrane glycoprotein responsible for receptor
recognition, attachment to the cell, and viral envelope fusion with a host cell membrane
resulting in genom erelease , which is driven b y the S conformation ch ange leading to the
fusion of viral and host cell membranes. For infection, S requires proteol ytic cleavage by two
host proteases, a furin- like protease between the S1 and S2 subunits, and by the serine
protease TMPRSS2 at a conserved site directl y precedi ng the fusion peptide (S2’)
(Figure 2.6.2 -2; Bestle et al , 2020 ; Hoffmann et al , 2020 ). While the membrane -proximal S2
furin cleavage fragment is responsible for membrane fusion, the membrane -distal S1
fragment, with its receptor -binding domain (RBD), recognizes the host receptor and binds to
the target host cell. SARS -CoV S and SARS -CoV -2 Shave similar structural properties and
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Page 8bind to the same host cell receptor, angiotensin conver ting enzy me 2 ( ACE2 )
(Zhou etal,2020 ).
Figure 2.6.2 -1.Replication Cycle of a Coronavirus
Source: de Wit et al, 2016
S is a large, trimeric gl ycoprotein that exists predominantly in a prefusion conformation on
the virion (Ke et al, 2020 ).Spontaneously and during cell entry , the S1 fragment dissociates,
and the S2 fragment undergoes a fold- back rearrangement to the post -fusion conformation in
a process that f acilitates fusion of viral and host cell membranes. S iscritical for the
induction of virus neutralizing antibodies by the host immune sy stem
(Zakhartchouk etal,2007 ;Yong et al, 2019 ).Some monoclonal antibodies against S,
particularl y those directed against the RBD, neutralize SARS -CoV and Middle East
respiratory syndrome (MERS) -CoV infection in vitro and in vivo (Hulswit et al, 2016 ).
Vaccines targeting the S protein aresufficient to induce strong neutralizing immune
responses ( Al-Amri et al, 2017).
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Page 9The RBD forms membrane distal “heads” on the S trimer that are connected to the body by a
hinge. In the native S, the RBD alternates bet ween an open (up ) and closed (down) position.
Although potent neutralizing epitopes have been described when the RBD is in the “heads
down” closed conformation, the “heads up” receptor accessible conformation exposes a
potentially greater breadth of neutra lizing antibody targets (Brouwer et al ,2020 ;
Liuet al,2020; Robbiani et al ,2020) . A P2 mutant (P2 S) variant of S contains two
consecutive prolines introduced at amino acid positions 986 and 987, between the central
helix (CH) and heptad repeat 1 (HR1) ( Figure 2.6.2-2 ).These mu tations lock S in the
prefusion conformation (Pallesen et al, 2017 ; Wrapp et al, 2020). A proportion of P2 S has
one RBD in the “heads up” and two RBDs in the “heads down” position, and t here is
probably a dynamic equilibrium as the heads hinge up and down (Cai et al, 2020 ;
Henderson etal,2020).
Figure 2.6.2 -2. Schematic of the Organization of the SARS -CoV- 2 S Glycoprotein
TheS1furin cleavage fragment includes the signal sequence (SS ), the N terminal domain (NTD), the receptor
binding domain (RBD, which binds the human cellular receptor, ACE -2), subdomain 1 (SD1), and subdomain 2
(SD2) .The furin cleavage site (S1/ S2) separat es S1 from the S2 fragment, which contains the S2 protease
cleavage site (S2 ′) followed by a fusion peptide (FP ),heptad repeats (HR1 and HR2 ),a central helix (CH)
domain, the connector domain (CD), the transmembrane domain (TM) and a cytoplasmic tail (CT ).
Source: modified from Wrapp et al, 2020 .
BNT162b2 ( V9)encodes for a full-length P2 S.TheV9codon optimization variant contains
a higher content of cy tosine ribonucleotides than V8for increased protein expression and is
the focus of this marketing application. The RNA -expressed P2 Sismembrane anchored.
Itelicits ofapotent humoral neutralizing antibody response and Th1-type CD4+and CD8+
cellular response to block virus infection and kill virus infected cells, respectively .
2.6.2.3. In Vitro Expression of Antigens from BNT162b2 (V9) RNA
Different in vitro methods were performed to analyze SARS -CoV -2 P2 S expression. To
assess transfection frequencies in cells expos ed to BNT162b2 RNA mixed with a commercial
transfection reagent or exposed to BNT162b2 (which is L NP-formulated) , flow cy tometry
analysis was performed. Immunofluorescence staining of transfected cells was used to assess
cellular localization .
Flow cy tome tryanalysisof HEK293T cells transfected with either BNT162b2 RNA or
LNP -formulated BNT162b2 led to high frequencies of cells being transfected, with
BNT162b2 -transfected cells being transfected at a slightly higher frequenc y than cells
exposed to BNT162b 2RNA mixed with a commercial transfection reagent (Figure 2.6.2 -3).
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Page 10There were no differences in cell viability after transfection with BNT162b2 RNA or
BNT162b2 compared to non -transfected cells. Furthermore, co -localization of the S protein
antigen with an ER marker was detected b y immunofluorescence experiments in HEK293T
cells expressing BNT162b2 -RNA suggesting the S protein is processed within the ER
(Figure 2.6.2 -4).
Figure 2.6.2 -3.Flow C ytometry Analysis of BNT162b2 Transfection Frequency
HEK 293T cells w ere transfected using RiboJuice™ mRNA transfection reagent (Merck Millipore) with 1 µg
of the RNA encoding BNT162b2 P2 S (V9)(BNT162b2 RNA) or the BNT162b2 (LNP -formulated RNA ).
After 18 h in culture, cells were stained with a viability dye, fixed, permeabilized and stained w ith a monoclonal
rabbit antibody recognizing S1 and labelled with Alexa Fluor647. Non -transfected cells were used as a control.non transfected
BNT162b2-RNA 1 µg
BNT162b2 1 µg
anti-S1-AB positive cells [%]
non transfected
BNT162b2-RNA 1 µg
BNT162b2 1 µg020406080100Viability
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Page 11Figure 2.6.2 -4. Immunofluorescence Detection of P2 S in BNT162b2 T ransfected
Cells
HEK293T cells w ere transfected w ith BNT162b2 (V9) RNA using RiboJuice™ RNA transfection reagent
(Merck Millipore). After 18 h in culture, cells were fixed , perm eabilized and stained for DNA to visualize the
nucleus with Hoechst (blue), for the endoplasmic reticulum and Golgi (ER/ Golgi ) with concanavalin A and
Golgi tracker, both Alexa Fluor™ 594 conjugated (red). Cells were stained for P2 S with a monoclonal anti-S1
antibody and Alexa Fluor® 488 (green). The merged color panels show that the P2 S expressed by BNT162b2
(V9) colocalize swith the ER / Golgi marker (scale: 10 µm). A control of non -transfected cells is shown in the
lower row.
2.6.2.4. Structural and Biophysical Characterization of P2 S as a Vaccine Antigen
For structural characterization, P2 S was expressed in Expi293F cells from DNA that
encodesthe same amino acid sequence as BNT162b2 RNA , with the addition of a C -terminal
TwinStrep tag for affinity purification (VR-VTR -10741) .To confirm surface expression of
untagged P2 S as well as theability of P2 S to bind to human ACE2, flow cy tometry
experiments were performed o n nonpermeabilized cells (Figure 2.6.2-5 ). Antibodies to the
RBD, S1, and S2 were pre -incubated with Alexa -488 anti -IgG Fab for staining, and a nucleic
acid d ye was used to separate live and dead cells. To confirm binding of human ACE2, P2
S-expressing cells were labeled with the extracellular domain of human ACE2 pre -incubated
with a FITC -labeled antibody against an affinity tag on the ACE2. Finall y, anti-RBD human
neutralizing antibodies B38 and H4 isolated from a COVID- 19 convalescent patient
(Wuetal, 2020 ) as well as the anti -RBD therapeutic antibody CR3022 (Yuan et al, 2020 )
weresimilarl y confirmed to bind the surface -expressed P2 S.
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Page 12Figure 2.6.2 -5. B inding to Cell Surface -Expressed Recombinant P2 S
P2 S antigen was over -expressed in Expi293F cells ,and surface expression confirmed by staining with
antibodies against the RBD, S1, and S2 regions of the full -length S protein. Human ACE2 extracellular domain
(ACE2) as w ell as the therapeutic antibody CR3022 and tw o neutralizing antibodies isolated from a COVID -19
convalescent patient, B38 and H4, w ere further confirmed to bind to surface express P2 S. The nucleic acid dye
7-AAD was used iden tify viable cells (low er quadrants in flow plots). Binding to surface expressed P2 S over
background in live cells is quantified across replicates in the bar graph.
Purification of the recombinant P2 S was based on a procedure described previously
(Cai et al, 2020 ), with minor modifications. Upon cell ly sis, P2 S was solubilized in 1%
NP-40 detergent. The TwinStrep -tagged protein was then captured with StrepTactin
Sepharose HP resin in 0.5% NP -40. P2 S was further purified b y size-exclusion
chromatograph y and eluted as three distinct peaks in 0.02 % NP -40 as previously reported
(Cai et al, 2020 ).Protein from the first peak of a size exclusion column, containing intact P2
S and dissociated S1 and S2, wasassay ed by biolayer inter ferometry (Figure 2.6.2-6 ). The
trimeric P2 S bound to the human ACE2 peptidase domain (ACE2- PD), and an anti -RBD
human neutralizing antibody B38 with high affinity (apparent KD= 1nM).
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Page 13Figure 2.6.2 -6. Biolayer Interferometry Sensorgrams for Binding ofP2 S to
ACE2 -PD and B38 mAb
P2 S with a C -terminal T winStrep tag expressed in Expi293F cells, was detergent solubilized and purified by
affinity and size exclusion chromatography . Protein from the first peak of a size exc lusion column, containing
intactP2 S and dissociated S1 and S2 , was assayed by biolayer inter ferometry on an Octet RED384 (FortéBio)
at 25 ° C in running buffer consisting of 25 mM Tris pH 7.5, 150 mM NaCl, 1mM EDTA and 0.02 % NP -40.
Sensorgram sshowing the binding kineti cs of TwinStrep -tagged P2 S to immobilized A,human ACE2 -PDand
B,B38 mo noclo nal antibody. The highest concentration tested for P2 S w as 71 nM with 2 more 3 -fold
dilutions. The binding curves were globally fit to a 1:1 Langmuir binding model with R2values greater than
0.95. Actual binding data (black) and the best fit of the data to a 1:1 binding model ( green ).Apparent kinetic
parameters are p rovided in the graphs.
Purified TwinStrep -tagged P2 S was characterized structurally using cryo-electron
microscop y (cryoEM ). 2D classification of particles from cry oEM data revealed a particle
population that closely resembles the prefusion conformation of SARS -CoV -2 spike protein
(Figure 2.6.2 -7A).Processing and refinement of this dataset y ielded a high -quality 3D map
with a nominal resolution of 3.29 Å (Figure 2.6.2 -7B), into which a previously published
atomic model (PDB ID: 6VSB) was fitted and rebuilt. The rebuilt model shows good
agreement with reported structures of prefusion full -length wild ty pe S (Cai et al, 2020 ) and
its ectodomain with P2 mutations ( Wrapp et al, 2020) . Three -dimensional classification of
the dataset ( Figure 2.6.2 -7C)showed a class of particles that was in the one RBD ‘up’
(accessible for receptor binding), two RBD ‘down” (closed) conformation and represented
20.4% of the trimeric molecules. The re mainder were in the all RBD ‘down’ conformation.
The RBD in the ‘ up’conformation w asless well resolved than other parts of the structure,
suggesting conformational flexibility and a d ynamic equilibrium between RBD ‘up’ and
RBD ‘down’ states as also suggested b y others (Cai et al, 2020 ;Henderson et al, 2020).
The well -resolved trimeric prefusion structure and the high affinity binding to ACE2 and
human neutralizing antibodies demonstrat e that the recombinant P2 S authentically presents
the ACE2 binding site and other epitopes targeted by man y SARS -CoV -2 neutralizing
antibodies.
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Page 14Figure 2.6.2 -7. C ryoEM P2 S Structur e at 3. 29Å Resolution
A. 2D class averages of T winStrep -tagged P2 S particles extracted from cryoEM micrographs. Box size is
39.2 nm in each dimension. B. 3.29Å cryoEM map of T winStrep -tagged P2 S, with fitted atomic model,
showing top (perpendicular to the three -fold axis) and side (parallel to the three -fold axis) views. CryoEM
model is based on PDB 6VSB and w as fitted into the structure using manual rebuilding in Coot and real -space
refinement in Phenix. ~28,000 micrographs were collected using a Titan Krios electron microscope operating at
300 kV accelerating voltage, and image processing and 3D reconstructions were performed using Warp and
RELION. C. Maps of P2 S produced by 3D classification indicate some heterogeneity in positioning of the
RBD domains. Percentages of the particle population represented in each class are indicated below the models.
2.6.2.5. Immunogenicity of BNT162b2 (V9)in Mice
The immunogenicit y of BNT162b2 (V9) inmice was investigated (Report R -20-0085).
Four groups of eight female B alb/c mice were immunized on day 0 with 0.2 µg, 1 µg or 5 µg
RNA/ animal of BNT162b2, or with buffer alone (control group). Blood was collected on
Days 7, 14, 21 a nd 28 after immunization to analyze the antibody response by
SARS -CoV2- RBD or S1 IgG ELISA and pseudoty pe neutralization ( pVNT ) (detailed
methods described in Section 2.6.2.12.1 for ELI SA and Section 2.6.2.12.2 for pVNT) .
Binding kinetics of SARS -CoV -2 S1-and RBD -specific IgGs weredetermined with sera
generated at D ay 28.
Immunization with BNT162b2 induced IgGs that bind S1 and RBD, while these antibodies
were not detected in samples from buffer control animals .A dose -dependent increase in
S1-binding IgGs was observed . Antibody concentrations in the serum samples were
calculated using a mouse IgG monoclonal standard, and the kinetics of IgGs against S1 and
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Page 15RBD are shown in Figure 2.6.2-8 .At Day 28, the differences in concentrations of IgGs
against S1 and RBD in the test groups compared to the buffer control group were statisticall y
significant (S1: p = 0.0259 for 0.2 µg, p < 0.0001 for 1 µg and 5 µg; RBD: p = 0.0072 for
0.2µg, p < 0.0001 for 1 µg and 5 µg) .
Figure 2.6.2 -8.Anti -S IgG R esponse 7, 14, 21, and 28 d after Immunization with
BNT162b2
BALB/c mice were im munized IM once with 0.2, 1 and 5 µg BNT162b2 or buffer. On 7, 14, 21, and 28 days
after immunization, animals were bled .For individual ΔOD values, the antibody concentrations in the serum
samples were calculated. The serum samples were tested by ELISA against (A) recom binant S1 and (B)
recombinant RBD. Group mean antibody concentrations are shown (±SEM). Group size n=8. Statistical
significance of the differences in IgG concentrations between the test groups and the control group w as assessed
by one -way ANOVA test with Dunnett’s multiple comparison post -test on day 28.
At Day 28 after immunization, vaccine -elicited IgG against the S1 domain showed a very
strong binding affinity (geometric mean KD 12 nM) including IgG binding the RBD with
high affinity (geometric mean KD 0.99 nM), both with high on- rate (geometric mean kon:
3.33 x 104/Ms for S1- specific affinity ; 6.02 x 105/Ms for RBD -specific affinity ) and low
off-rate (geometric mean koff: 4.00 x 10- 4/s for S1- specific affinit y; 5.97 x 10 -4/s for
RBD- specific affinity ) (Figure 2.6.2-9 ).
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Page 16Figure 2.6.2 -9. Binding Kinetics of Murine SARS -CoV- 2 S1-and RBD -specific IgGs
BALB/c m ice were immunized IM once w ith 5 µg BNT162b2. On Day 28 after immunization, animals were
bled. IgG in the sera were tested for binding to recom binant histidine -tagged S1 (A) or recombinant histidine
tagged RBD (B) (Sino Biological) using surface plasmon r esonance spectroscopy in multi -cycle mode with
concentrations ran ging from 25 -400 nM (S1 -His) or 1.562 -50 nM (RBD -His). Binding kinetics were calculated
using a global kinetic fit to a 1:1 Lan gmuir model . Binding parameters are given in (C). Actual binding data
(black) and the best fit of the data to a 1:1 binding model (green). One point in the graphs stands for one mouse.
Group size n=8. Mean ± SEM is shown by horizontal bars with whiskers for each group.
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Page 17In pVNT analysis, dose -dependent in creases in neutralizing antibodies were observed
(Figure 2.6.2 -10).
Figure 2.6.2 -10.BNT162b2 Pseudovirus Neutralizing Titers 14, 21, and 28 d af ter
Immunization
BALB/c mice were immunized IM once w ith 0.2, 1 and 5 µg BNT162b2 or buffer. On 14, 21, and 28 d after
immunization, animals were bled . The sera w ere tested for SARS -CoV -2 pseudovirus neutralization. Graphs
depict pVN T50serum dilutions (50% reduction of infectious events, compared to positive controls without
serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group
size n=8. Mean ± SEM is shown by horizontal bars with whiskers for each group. LLOQ, low er limit of
quantification. ULOQ, upper limit of quantification.
The summary of antibody titers on Day 28 is as follows:
Table 2.6.2 -1.Summary of IgG C oncentrations at Day 28 Post Immuni zation
BNT162b2
0.2µgBNT162b2
1µgBNT162b2
5µg
Anti S1 total IgG [µg/m L] 73.0 ± 10.4 205.9 ± 21.0 392.7 ± 28.9
Anti RBD total IgG [µg/m L] 83.1 ± 12.3 241.7 ± 17.2 448.6 ± 28.6
pVN 50titer [reciprocal dilution] 33.0 ± 9.8 192.0 ± 31.4 312.0 ±35.1
In addition, the cellular immune response was analy zed. At Day 28 after one immunization ,
mice were sacrificed and splenocy tes were isolated to test for IFN γ release after antigen
stimulation by ELISpot . Stimulation of fresh splenocy tes with an S -specific overlapping
peptide pool induced IFNγ responses in T cells of immunized animals . Splenocy tes of the
groups immunized with BNT162b2 hadsignificantly higher spot numbers than splenocy tes
from the group sthat received buffer control (Figure 2.6.2-11 ). To identify the T -cell subty pe,
an additional ELI Spot analy sis was performed after separation of fresh CD4+and CD8+cells
by MACS isolation from splenocy tes obtained from the group immunized with 5 µg
BNT162b2 . Both CD4+and CD8+cells display ed IFNγ responses.
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Page 18Figure 2.6.2 -11.ELISpot Analysis Using Splenocytes Obtained on Day 28 after One
Immun ization
****
***
***
ELISpot assay was performed using (A) bulk splenocytes isolated on D ay 28 after IM immunization of mice
with 1 µg BNT162b2 or (B) CD4+ and CD8+ splenocytes after m agnetic cell separation from the 5 µg
BNT162b2 immunized group. Splenocytes were stimulated with S- specific overlapping peptide pools , buffer or
an irrelevant control peptide (ctrl) ,and IFN- γ secretion was measured to assess S-specific T-cellnumber .
Individual spot counts are shown by dots; group mean values are indicated by bars (±SEM). P-values were
determined by one -way ANOVA analysis followed by Dunnett’s multiple comparisons test. *** p < 0.001,
**** p <0.0001.
Furthermore , cytokine release data from the S-peptide mix stimulated splenocy tes was
acquire d28 day s after immunization with 5 µg BNT162b2. H igh levels of the Th1cytokines
IFNγ and IL -2 but minute amounts of the Th2cytokines IL -4, IL -5 and IL -13 in multiplex
immunoassay s weredetected (Figure 2.6.2-12 ).
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Page 19Figure 2.6.2 -12.Cytokine Release Analysis Using Splenocytes Obtained on Day 28
after One Immunization
0.0374
0.0002
0.0045
0.0062
0.0012
A B
Splenocytes of BALB/c mice immunized IM with 1µg BNT162b2 we re stimulated ex vivo with full-length
Speptide mix and c ytokine multiplex analysis of supernatants was performed (n =8 per group) . Splenocytes of
buffer treated mice served as control. Cy tokine production was determined by bead -based multiplex analysis
(n=8 per group, n=7 for IL -4, IL -5 and IL -13 as one outlier was removed via routs test [Q=1%] for the S peptide
stimulated samples). Individual dots indicate results from one animal; group mean values are indicated by bars .
P-values were determined by a tw o-tailed paired t -test.
To dissect the cellular response afte r BNT 162b2 immunization in more detail , mice were
immunized with 5 µg BNT162b2 and 12days after immunization draining lymph nodes
(dLNs) were collected to perform B-cell and T -cell phe notyping analy sis by flow cy tometry
(Figure 2.6.2 -13). Much higher numbers of B cells (including plasma cells, class switched
IgG1 -and IgG2a- positive B cells, and germinal center B cells) were observed in the samples
from mice that received BNT162b2 compared to controls. In addition, dLNs from
BNT162b2 -immunized mice also display ed an elevation in T -cell counts, particularl y
numbers of T folli cular helper ( Tfh) cells, including subsets with ICOS upregulation, which
isknown to play an essential role in the formation of germinal centers (Hutloff 2015).
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Page 20Figure 2.6.2 -13. B- and T -cell Phenotyping in Lymph N odes of BNT162b2 Immunized
Mice
0.1531
0.0002
0.0002
0.0001
0.0153
0.0023
0.0007
0.0012
0.0006
0.0420
<0.0001
<0.0001
Mice (n=8 per group) w ere immunized with 5 µg BNT162b2 or buffer (Control). (A) B-cell and (B) T-cell
numbers 12 days after immunization in the subsets indicated by the y -axis labels were analysed in draining
lymph nodes by flow cytometry. P -values were determined by an unpaired tw o-tailed t -test. The percentage of
ICOS+cells among T follicular helper cells ( Tfh) in draining lymph nodes ( dLNs )is depicted on the low er right .
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Page 21In summar y, BNT162b2 ( V9)induced a strong antibody response , with high total IgG , high
binding affinity to S1 and the RBD, andhigh pVN Ttiters. Both CD4+and CD8+T-cell
responses were detectable 12 and 28 day s after one immunization with an overall significant
increase in T-cell reactivity compared to control animals. Taking the phenoty ping of B and
Tcells in aggregate, the data indicate a strong and concurrent induction of SARS -CoV -2
S-specific neutralizing antibody titers and a Th1-driven T-cell response b y BNT162b 2.
2.6.2.6. BNT162b2 (V9)Vaccine Immunogenicity and Evaluation of Protection against
SARS -CoV -2 Challenge in Rhesus Macaques
The ability of BNT162b2 immunization to protect rhesus macaques from live SARS -CoV -2
challenge was evaluated in 2–4 year old male rhesus macaques (VR-VTR -10671 ).
2.6.2.6.1. Immunogenicity in Rhesus Macaques
Groups of 2- 4 year old male rhesus macaques were immunize d IM with 30 or 100 µg of
BNT162b2 or saline control on Day s 0 and 21. S1- binding IgG was readil y detecta ble after a
single immuni zation, and levels increased further seven day s after the second immuni zation
(Day 28)togeometric mean S1- binding IgG concentrations (GMCs) of30,339 units (U)/mL
(30 µg dose level) and 34,668 U/mL (100 µg dose level) (Figure 2.6.2-14 ). For comparison,
the GMC of a panel of 38 SARS -CoV -2 convalescent human sera was 631 U/mL ,
substantially lower than the GMC of the immuni zed rhesus macaques after one or two doses.
Human convalescent sera (HCS )were drawn from SARS -CoV -2 infected individuals 18 to
83 years of age, at least 14 day s after PCR -confirmed diagnosis, and at a time when
individuals were as ymptomatic. The serum donors predominantly had symptomatic
infections (35/38), and one had been hospitalized. Based on the assumptions that the immune
response to SARS -CoV -2 infection provides some measure of protection from disease upon
subsequent exposure to the virus and that the neutralizing anti body response contribute sto
that protection, the neutralizing antibody titer of the convalescent serum panel provides a
currentl y assess able benchmark to judge the quality of the immune response to the vaccine
candidates.
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Page 22Figure 2.6.2 -14. S1-Binding IgG Levels Elicited by Immunization of Rhesus Macaques
with BNT162b2
S1-binding IgG concentrations elicited by immunization of rhes us macaques with BNT162b2. Numbers on the
x-axis indicate the day post first immunization. Heights of bars indicate geometric mean concentrations ( GMCs )
in arbitrary units , which are written above the bars; whiskers indicate 95% CIs; dots represent individual
monkey IgG concentrations. Dotted line indicates the lower limit of quantification (LLO Q 1.151 U/ml). Values
at or below LLOQ were set to ½ LLOQ. C –saline -immunization control; HCS – human convalescent serum
panel.
Fifty percent neutralization titers (VNT 50), measured b y an authentic SARS- CoV -2
neutralization assay (Muruato et al, 2020) , were detectable in rhesus sera b y Da y 14 after a
single immunization and peaked at geometric mean titers (GMTs) of 962 (on Day 35, 14
days after Dose 2 of 30 µg) or 1,689 (on Day 28, 7 daysafter Dose 2 of 100 µg;
Figure 2.6.2- 15). Robust neutralization GMTs of 285 for 30 µg and 310 for 100 µg dose
levels persisted to at least Day 56 (most recent time point tested). For comparison, the 50%
neutralization GMT of the human convalescent serum panel was 94.
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Page 23Figure 2.6.2 -15. 50% Serum Neutralizing Titers Elicited by Immunization of Rhesus
Macaques with BNT162b2
Numbers on the x -axis indicate the day post first immunizati on. Heights of bars indicate GMTs, which are
written above the bars; whiskers indicate 95% confidence intervals; dots represent individual monkey titers.
LLOQ –20.Titers at or below LLOQ were set to ½ LLOQ. C –saline -immunization control; HCS –human
convalescent serum panel .
Antigen -specific T-cell response splay an important role in generation of antigen -specific
antibody response as well as in elimination of infected cells to mediate protection against
disease. S -specific T- cell responses were anal yzed in animals immunized with 30 µg or
100 µ g of BNT162b2 and unimmunized controls (Control )by ELISpot and intracellular
cytokine staining (I CS). PBMCs were collected before immunization (day 0), 14 day s post
dose 1 (14d PD1), 7 day s post dose 2 (7d PD2) ,and 21 day s post Dose 2 (21d PD2).
S-specific T cells were low to undetectable in naïve animals. Strong IFN ELISpot responses
but minimal IL -4 ELISpot responses were detected after the second 30 or 100 µ g dose of the
vaccine candidate (Figure 2.6.2- 16). ICS confirmed that BNT162b2 elicited strong S -specific
IFNγ producing T cell responses, including a high frequency of CD4+T cells that produced
IFNγ, IL-2, or TNF -but a low frequency of CD4+cells that produce IL -4, indicating a
Th1-biased response ( Figure 2.6.2- 17A through D). BNT162b 2 also elicited S -specific IFNγ
producing CD8+T cell responses ( Figure 2.6.2- 17E).
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Page 24Figure 2.6.2 -16. IFNγ and IL -4 ELISpot Results in BNT162b2 Immunized Animals
Groups of six 2 -4 year old rhesus macaques were immunized on Days 0 and 21 w ith 30 or 100 µg BNT162b2 or
buffer (Placebo). Height of bars indicates the mean ;whiskers indicate the standard error of mean (SEM) ; and
each symbol represent s one animal. Dotted lines mark the lower limit of detection. (A) IFNγ (B) IL -4 ELISpot
analysis. (C) Correlation of frequency of IFNγ or IL -4 producing cells 21 days PD2.
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Page 25Figure 2.6.2 -17. S- specific CD4 and CD8 T -cell Response in BNT162b2 Immunized
Animals as Meas ured by ICS Assay
Height of bars indicates the mean ;whiskers indicate the standard error of mean (SEM) ; and each symbol
represents one animal. (A) Frequency of IFNγ+CD4 T cells. (B) Frequency of IFNγ/IL -2/TNF -+CD4 T c ells
(C) Frequency of IL -4+CD4 T cells. (D) Correlation of frequency of IFNγ or IL -4+CD4 T cells at 21 days PD2.
(E) Frequency of IFNγ+CD8 T cells.
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Page 262.6.2.6.2. SARS -CoV -2 Challenge of BNT162b2 (V9)-Immunized Nonhuman Primates
SARS -CoV -2 infection and COVID -19 in humans present diverse manifestation of signs,
symptoms, and severity . Based on published reports, SARS -CoV -2 challenged rhesus
macaques develop an acute, transient infection in the upper and lower respiratory tract and
have evidence of viral replication in the gastrointestinal tract, similar to humans
(Zouet al, 2020 ; Kim et al , 2020 ). Vary ing degrees of pulmonary inflammation, primarily at
the peak of infection at approximately day 2 to 4 post -challeng e, have been reported in the
literature ( Munster et al , 2020 ). The human and rhesus ACE2 receptor have 100% amino acid
identity at the critical binding residues, which may account for the fidelit y of this
SARS -CoV -2 animal model ( Zhou et al , 2020 ).
The g roups of 2- 4 year old male rhesus macaques that had received two intramuscular
immunizations with 100 µg BNT162b2 (V9)(n=6) or buffer (Control ; n=3) 21 day s apart
(described in Section 2.6.2.6.1 )were challenged 55 day s after the second immunization with
1.05 × 106plaque forming units of SARS- CoV -2 (strain USA -WA1/2020), split equally
between the intranasal and intratracheal routes, as previously described (Singh et al, 2020 ).
Three additional non- immuniz ed, age -matched, rhesus macaques (sentinel) were
mock- challenged with cell culture medium. Nasal and orophary ngeal (OP) swabs were
collected and bronchoalveolar lavage (BAL) was performed at the times indicated, and the
samples were tested for the presence of SARS -CoV -2 RNA (genomic RNA andsubgenomic
transcripts) by reverse -transcription quanti tative poly merase chain reaction (RT -qPCR;
Figure 2.6.2-18 ). All personnel performing the clinical , radiographic, histopathologic, and
RT-qPCR evaluation s were blinded to the group assignments of the macaques
(VR-VTR -10671 ).
All samples obtained before the infectious challenge and all those obtained from sentinel
animals lacked detectable SARS- CoV -2 RNA ( Figure 2.6.2-18 ). Viral RNA was detected in
BAL fluid from 2 of the 3 control- immunized macaques on Day 3 after challenge and from 1
of 3 on Day 6. At no time point sampled was viral RNA detected in BAL fluid from the
BNT162b2 (V9)-immuniz ed and SARS -CoV -2 challenged macaques ( Figure 2.6.2-18 A).
Thedifference in viral RNA detection in BAL fluid between BN T162b2- immuni zed an d
control -immunized rhesus macaques after challenge is statisticall y significant (p=0.00 14).
From control -immuniz ed macaques, viral RNA was detected in nasal swabs obtained on
Days 1, 3, and 6 after SARS -CoV -2 challenge; from BNT162b2 (V9)-immuniz ed macaques,
viral RNA was detected only in nasal swabs obtained on Day 1 after challenge and not in
swabs obtained on Day 3 or subsequentl y (Figure 2.6.2-18B ). The pattern of viral RNA
detection from OP swabs was similar to that for nasal swabs (Figure 2.6.2- 18C).
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Page 27Figure 2.6.2 -18.Viral RNA in BAL Fluid and Nasal and Oropharyngeal Swabs of
Rhesus M acaques after Infectious SARS -CoV -2 Challenge
Viral RNA (log10copies)
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Page 28Groups of 2 -4 year old rhesus macaques were immunized on days 0 and 21 with 100 µg BNT162b2 (V9) (n=6) ,
or buffer (Control ; n=3 ).Fifty -fivedays after the second immunization, the animals were challenged with
1.05× 106pfu of SARS -CoV- 2 split equally between the IN and IT routes. Three age -matched male rhesus
macaques were unimmunized and challenged with cell culture medium only (Sentinel). Viral RNA levels were
detected by RT -qPCR in A) bronchoalveolar lavage, B) nasal swabs, and C) oropharyngeal sw abs.EOP, end of
project. Values below the LLOD set to ½ the LLOD. The viral RNA levels between control -immunized and
BNT162b2 -immunized animals after challenge were compared by a non -param etric analysis (Friedman’s test),
and the p -values are 0.0014 for BAL fluid, 0.2622 for nasal swabs, and 0.0007 for OP sw abs. The Friedman’s
test is a non -param etric analysis based on the ranking of viral RNA shedding data within each day. PROC
RANK and PROC GLM from SAS® 9.4 w ere used to calculate the p -values.
Despite the presence of viral RNA in BAL fluid from challenged control animals, none of the
challenged animals, immunized or control, showed clinical signs of illness (Figure 2.6.2 -19),
indicating that the 2 -4 year old male rhesus monkey challenge model appears to be an
infection model, but not a clinical disease model . Lung radiograph ( Figure 2.6.2 -20A) and
computerized tomography (CT) ( Figure 2.6.2 -20B) scores were dete rmined by two
board -certified veterinary radiologist who were blinded to treatment group. Data in
Figure 2.6.2 -20represent the average of the two score s. Radiographic evidence of pulmonary
abnormality was observed in challenged controls but not in challenged BNT162b2 -
immunized animals nor in unchallenged sentinels. No radiographic evidence of vaccine-
elicited enhanced disease was observed. At necropsy onDay 7 or 8 after virus challenge,
there were no significant gross pathology findings in any organs. Microscopically , the main
finding in the lung was inflammation. The lung inflammation area score was similar between
saline -immunized and BNT162b2- immunize d animals, and there was no evidence of
enhanced respiratory disease ( Figure 2.6.2-21 ).
Overall, t hese data demonstrate that, compared to control, BNT162b2 (V9) immunization
provided complete protection in the lungs from infectious SARS -CoV -2 challenge in rhesus
macaques with n o evidence of vaccine -elicited disease enhancement.
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Page 29Figure 2.6.2 -19.Clinical Signs in Rhesus Macaques after Immunization with
BNT162b2 and Challenge with Infectious SARS -CoV -2
Rhesus macaques were immuni zed with BNT162b2 (V9), o r saline, and challenged with SARS -CoV -2 or cell
culture medium as described in the Figure 2.6.2 -18legend. Clinical signs were recorded on the days indicated.
EOP, end of project. BNT162b2 -immunized (n=6), control (n=3), and sentinel (n=3) macaques. A , Body
weight. B ,Temperature. C, Oxygen saturation. D, Heart rate.
% Weight Change
Baseline 0 1 3 6 10/EOP-10-50510
Day relative to challenge% Temp ChangeSentinel
Control
BNT162b2
SpO2%
Pre 0 1 3 6 10/EOP050100150200250
Day relative to challengeHeart RateA. B.
C. D.
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Page 30Figure 2.6.2-20 .Radiograph and CT Sc ores of Rhesus Macaque Lungs after Infectio us
SARS -CoV -2 Challenge
Fifty -fivedays after the second immunization, BNT162b2 or Control (saline) -immunized animals were
challenged with 1 .05× 106pfu of SARS -CoV- 2 split equally betw een the IN and IT routes. Three age -matched
unimmunized rhesus macaq ues w ere challenged with cell culture medium only (Sentinel). Chest X -rays and CT
scans were performed prior to challenge and at the times indicated on the x -axis. EOP, end of project.
Radiograph ( A) and CT ( B) scores w ere assigned to a total of 7 regions on a scale of 1 -20. Images were
evaluated by two board -certified veterinary radiologist sblinded to treatment group . Individual data points
represent the average of the two scores . The height of the bars indicates the mean score. Error bars indicate the
standard deviation .
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Page 31Figure 2.6.2 -21 .Lung Inflammation Area Score after IN/IT SARS -CoV- 2 Challenge
Graph (left panel): Lung inflammation area score on Day 7 or 8 after IN/IT SARS -CoV -2 challenge. Each data
point represents the mean lung inflammation area score of a single animal (mean score of the 7 lung lobes).
Saline -immunized and challenged animals (Control ; n=3 ) are shown in grey and BNT162b2 -vaccinated and
challenged animals (BNT162b2; n=6) are shown in blue .Each dot repr esents the inflammation mean area score
for an individual animal. Bars indicate the geometric mean area scores w ithin each group. Photomicrographs
(right panel; 2.5x objective, A and C; 20x objective, B and D) of hematoxylin and eosin -stained lung sections
from Control animals (A and B) and lungs from BNT162b2 -immunized and challenged animals (C and D).
2.6.2.7. Immunogenicity Testing of Rats in the GLP Compliant Repeat Dose Toxic ity
Stud ies and Developmental and Reproductive Toxicity Study
Immunogenicit yresults from twoGLP
-compliant repeat -dose toxicity studies, one
(Study 20GR142 ) with BNT162b2 (V9) and one ( Study 38166 ) with its closely related
variant BNT162b2 (V8), as well as a DART stud y (Study 20256434 ) with BNT162b2 (V9)
are presented below.
2.6.2.7.1. Repeat -Dose Toxicity Study of Three LNP -Formulated RNA Platforms
Encoding for Viral Proteins by Repeated Intramuscular Administration to Wistar Han
Rats
The immuno genicit y of BNT162b2 (V8) in the GLP --compliant repeat -dose rat toxicity
study (Study 38166) was analyzed.BNT162b2 (V8) has an alternative coding sequence to
the Phase 2/3 study clinical candidate and subject of this application, BNT162b2 (V9) , with
V9containing a higher content of cy tosine ribonucleotides for increased protein expression.
Both variants, V8 and V9, encode the identical protein ,and in this toxicology study , the V8
was a surrogate for BNT162b2 (V9) .
Male and female Wistar Han rats recei ved three weekl y doses of 100 µg of BNT162b2 (V8) .
Serum samples were collected and anal yzed (5 animals/sex) from main study animals on
Day17, two day safter the 3rdadministration, at the end of the dosing phase as well as from
recovery cohorts at the en d of the study on Day 38.Treatment with theBNT162
b2vaccine
elicited binding IgG against the S1 fragment andthe RBD of SARS -CoV2 S. There was a
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Page 32strong antibod y response at both anal yzed time points . The group mean IgG concentration
against S1 and RBD are given in Table 2.6.2 -2. Antibody concentrations against S1 and RBD
increased over time .
Table 2.6.2 -2.IgG antibody Concentration [mg/mL] Against the Viral Antigen in
Wistar Han Rats after BNT162b2 (V8) Immunization
BNT162b2 ( 100µg)
17days after first immunization Against S1 1.76 ± 0.16
Against RBD 2.33 ± 0.19
38days after first immunization Against S1 3.46 ± 0.52
Against RBD 4.90±0.87
Pseudovirus neutralization results mirror ed the antigen binding results .
Treatment of rats with BNT162 b2(V8) resulted in the elicitation of neutralizing antibodies
against pseudovirus infection. Neutralizing antibody titers in vaccinated animals i ncreased
over time with the recorded neutralizing activity being consistent with the ELISA data shown
above. Serum titers resulting in 50% pseudovirus neutralization exceeded the upper limit of
quantification (ULOQ) of a reciprocal titer of 1536 in more th an 8 out of 10 animals on
Day38, and therefore a neutralization titer of 90% was evaluated as well (Figure 2.6.2-22 ).
Figure 2.6.2 -22.Pseudovirus Neutralization A ctivity in Rats after BNT162b2 V8
Immunization
Wistar Han rats were immunized IM with three w eekly injections of 100 µg BNT162b2. On Day 17 and
Day38,animals were bled ,and the sera w ere tested for titers of pseudovirus neutralizing antibodies. I ndividual
titers resulting in 50% pseudovirus neutralization (pVN T50, left graph ) or 90% pseudovirus neutralization
(pVN T90, right graph ) are shown by dots; group mean values are indicated by horizontal bars and are included
in the figure (±SEM, standard error of the mean). Group size for analysis wasn=5 male and n=5 female rats.
Mean titers are given i n the bars. All control serum samples were below the lower limit of quantification
(LLOQ); ULOQ = upper limit of quantification.
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Page 332.6.2.7.2. 17 -Day Intramuscular Toxicity Study of BNT162b2 (V9) in Wistar Han Rats
With a 3- Week Recovery
The immunogenicit y of the COVID -19 vaccine candidate BNT162b2 (V9) (and BNT162b3c)
in the GL P compliant repeat- dose rat toxicity study (Study 20GR142) was anal yzed. The
summary of the results described below will focus on only the BNT162b2 (V9) candidate.
Wistar Han rats (15/sex/group) were administered I M doses of 0 (saline) or 30 BNT162b2
(V9) µg RNA/dose per animal. Doses were administered once a week for 3 weeks (Day s 1, 8,
and 15). Following the dosing phase, 10 animals/sex from each group were euthan ized 2 days
post last immunization for post- mortem assessments. The remaining 5 animals/sex/group
were euthanized following a 3 -week recovery phase.
Administration of 3 once -weekl y doses of BNT162b2 (V9) elicited SARS -CoV -2
neutralizing antibod y responses in males and females at the end of the dosing (Day 17) and
recovery (Day 21) phases of the study . SARS -CoV -2 neutralizing antibody responses were
not observed in animals prior to vaccine administration or in saline -administered control
animals ( Table 2.6.2 -3).
Table 2.6.2 -3.Group Mean Titers of SARS -CoV -2 Neutralizing Antibodies
Study Day Sex Saline
(0 µg RNA)BNT162b2 (V9)
(30 µg RNA)
Prior to Dosing Initiation (Day -5) Male 5 5
Female 5 5
End of Dosing Phase (Day 17) Male 5 1114
Female 5 2501
End of Recovery Phase (RP Day 21) Male 5 5120
Female 5 5120
RP = Recovery phase.
2.6.2.7.3. A Combined Fertility and Developmental Study (Including Teratogenicity and
Postnatal Investigations) of BNT162b1, BNT162b2 and BNT162b3 by Intramuscular
Administration in the Wistar Han Rat
The immunogenicit y of the COVID -19 vaccine candidate BNT162b2 (V9) (and BNT162b3c)
in the GL P compliant DART ( Study 20256434 ) was anal yzed. The summary of the results
described below will focus on only the BNT162b2 (V9) candidate.
Female Wistar Han rats (44 animals/group) were administere d saline or 30 µg RNA/dosing
day of BNT162b2 (V9) by IM injection for a total of 4 doses (21 and 14 day s prior to mating
and on GDs 9 and 20). On GD 21, half of the females in each group underwent Caesarean
section. The remaining females in each group were allowed to naturall y deliver their pups
and both maternal animals and their offspring were monitored out through the end of
weaning (LD 21/PND 21). SARS -CoV -2 neutralizing antibodies were assessed in maternal
animals prior to mating, on GD 21, and LD 21 as well as in fetuses on GD 21 and in pups on
PND 21.
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Page 34BNT162b2 elicited SARS- CoV -2 neutralizing antibody responses in all of the females just
prior to mating (M 0), at the end of gestation (GD 21), and at the end of lactation (LD 21).
SARS -CoV -2 neutralizin g titers were detected in all offspring (fetuses on GD 21 and pups on
PND 21). SARS -CoV -2 neutralizing antibod y titers were not observed in animals prior to
vaccine administration or in saline-administered control animals ( Table 2.6.2 -4).
Table 2.6.2 -4.Group Mean Titers of SARS -CoV -2 Neutralizing Antibodies
Interval/Occasion Saline
(0 µg RNA)BNT162b2 (V9)
(30 µg RNA)
Prior to Dosing Initiation 5.0 5.3
Just Prior to Mating 5.0 3886.4
Gestation Day 21 (Dams) 5.0 3445.5
Lactation Day 21 5.0 3620.4
Fetuses (Gestation Day 21) 5.0 640.0
Pups (Postnatal Day 21) 5.0 4561.4
2.6.2.8. Secondary Pharmacodynamics
No secondary pharmacody namics studies were conducted with BNT162b2 .
2.6.2.9. Safety P harmacology
No safet y pharmacology studies were conducted with BNT162b2 as they are not considered
necessary according to the WHO guideline ( WHO, 2005 ).
2.6.2.10. Pharmacodynamic Drug Interactions
Pharmacod ynamic drug interaction studies with BNT162b2 have not been conducted.
2.6.2.11. Discussion and Conclusions
The BNT162b2 vaccine candidate encoding the full-length P2 S induc esrobust immune
responses in mice ,rats, and non human primates. SARS -CoV -2 S is a primary target of
neutralizing antibodies ,and the modRNA that encodes the vaccine antigen induces a strong
neutralizing antibod y response ,Th1- type CD4+T-cell response, and a CD8+IFNresponse .
This diversity of elicite d immune mechanisms could block virus infection as a first line of
defense and clear virus -infected cells as a second line of defense.
Arecombinant form of the P2 S antigen encoded by the vaccine and transiently expressed on
the surface of mammalian cells was bound b y a soluble ACE2 receptor and SARS -CoV -2
neutralizing monoclonal antibodies with high affinities. Analy sis of the P2 S trimer structure
by cryoelectron microscopy reveal edhigh similar ity to previousl y reported P2 S structures .
The well -resolved trimeric prefusion structure and the high affinity binding to ACE2 and
human neutralizing antibodies demonstrate that the recombinant full -length P2 S
authentically presents the ACE2 binding sit e and other epitopes targeted b y many
SARS -CoV -2 neutralizing antibodies.
Nonclinical studies in mice and nonhuman primates showed that antigen -binding IgG and
neutralizing antibod y responses were detectable as earl y as 14d post -immunization , with
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Page 35substan tial increases observed in nonhuman primates after the second dose .Similar results
indicating immunogenicity were obtained in an accessory study to the GLP -compliant
repeat -dose toxicology studies in rats ( Study 38166 andStudy 20GR142) and DART study
(Study 20256434 ).In a SARS -CoV -2 rhesus challenge model, BNT162b2 provided partial
protection from infection in the upper airway , andno viral RNA was detected in the lower
airway s, sampled seriall y by BAL starting 3 days after challenge .No evidence of disease
enhancement was observed in BNT162b2 -immunized and SARS -CoV -2 challenged
macaques (VR-VTR -10671 ).
2.6.2.12. I mmunogenicity and Efficacy Methods
2.6.2.12.1. SARS -CoV -2 S1 and RBD D irect ELISA
For preclinical studies in mice, antigen- based direct ELI SAs measured S1- binding (S1
recombinant protein, Sino Biological) and RBD- binding (recombinant RBD, Sino
Biological) IgG levels in serum samples. MaxiSorp plates (Thermo Fisher Scientific) were
coated with recombinant protein (100 ng/100 µL) in sodium carbonate buffer, and bound IgG
was detected using an HRP -conjugated secondary antibod y and TMB substrate (Biotrend).
Data collection was performed using a BioTek Epoch reader and Gen5 software version
3.0.9. For concentration anal ysis, the signal of the specific samples was correlated to a
standard curve of an isotype control.
2.6.2.12.2. VSV/SARS -CoV -2 S Pseudovirus Neutralization Assay
For preclinical immunogenicity studies in rodents , a pseudot ype neutralization assay (pVNT)
was used as a surrogate of virus neutralization (which, for SARS -CoV -2, requires BSL3
containment). The pVNT is based on a recombinant replication -deficient vesicul ar stomatitis
virus (VSV) vector that encodes GFP instead of VSV- G (VSVΔG -GFP). VSVΔG -GFP was
pseudoty ped with SARS -CoV -2 S protein according to published pseudot yping protocols
(Berger & Zimmer 2011 ; Baum et al, 2020 ). Serial dilutions of mouse sera were incubated
with the pseudoty ped reporter virus for 10 minutes at room temperature before inoculating
Vero -76 cell monolay ers in 96 well plates. Virus was added at 300 IU per well and infected
cell counts per well were detected 16 -24 hours after inoculation with an IncuCy te Live Cell
Analy sis sy stem (Sartorius) with I ncuCy te 2019B Rev2 software. The 50% pseudovirus
neutralization titer (pVNT 50) was reported as the reciprocal of the first serum dilution
yieldin g a 50% reduction in GFP -positive infected cell number per well compared to the
mean of the no serum pseudovirus positive control.
2.6.2.12.3. SARS -CoV -2 S1-Binding and RBD -Binding Kinetics using Surface Plasmon
Resonance Spectroscop y
Binding kinetics of murine S1 -and RBD -binding serum IgGs was determined using a
Biacore T200 device (C ytiva). An anti -mouse -Fc antibody (Jackson ImmunoResearch) was
covalentl y coupled to immobiliz ation level of ~10,000 response units (RU) on the CM5
sensor chip matrix. Bulk mouse IgGs were captured from diluted serum and binding anal yses
to histidine -tagged S1 ( S1-His) or histidine -tagged RBD ( RBD -His) (Sino Biological) were
performed using a multi- cycle kinetic method with concentrations ranging from 25 to
400 nM or 1.5625 to 50 nM, r espectivel y. Binding kinetics were calculated using a global
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Page 36kinetic fit to a 1:1 L angmuir model with Biacore T200 Evaluation Software Version 3.1
(Cytiva).
2.6.2.12.4. SARS -CoV -2 S1-Binding IgG Luminex Assay
For nonhuman primate studies, a direct binding Luminex immu noassay (dLIA) was used to
quantify S1-binding serum IgG levels ( VR-MQR -10211 ).Arecombinant SARS -CoV -2 S1
with a C-terminal Avitag™ (Acro Bios ystems) was bound to streptavidin- coated Luminex
microspheres. Bound nonhuman primat eS1-binding IgG was detected with a
R-Phycoery thrin-conjugated goat anti- human polyclonal secondary antibody (Jackson Labs).
Data were captured as median fluorescent intensities (MFIs) using a Luminex reader and
converted to U/mL antibody concentrations using a reference standard curve with arbitrary
assigned concentrations of 100 U/mL and accounting for the serum dilution factor. Assay
results were reported in U/mL of IgG.
2.6.2.12.5. SARS -CoV -2 Neutralization Assay
For nonhuman primate studies, the same authentic S ARS -CoV -2 neutralization assay used
for clinical testing was applied (VR-MQR -10214 ). The SARS- CoV -2 neutralization assay
used a previousl y described strain of SARS -CoV -2 (USA_WA1 /2020) that had been rescued
by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into
open reading frame 7 of the viral genome ( Xie et al, 2020). This reporter virus generates
similar plaque morphologies a nd indistinguishable growth curves from wild- type virus
(Muruato et al, 2020) . Viral master stocks used for the neutralization assay were grown in
Vero E6 cells as previously described ( Xie et al, 2020 ). Serial dilutions of heat i nactivated
sera were incubated with the reporter virus for 1 hour at 37 °C before inoculating Vero
CCL81 cell monolay ers in 96 well plates to allow accurate quantification of infected cells.
Virus was added at 2 x 104PFU per well to yield a target of 10 -30% of infected cells in the
monolay er. Total cell counts per well were enumerated b y nuclear stain (Hoechst 33342) and
fluorescent virally infected foci were detected 16- 24 hours after inoculation with a Cy tation 7
Cell I maging Multi -Mode Reader (Biotek) w ith Gen5 I mage Prime version 3.09. Titers were
calculated in GraphPad Prism version 8.4.2 by generating a 4- parameter (4PL) logistical fit
of the percent neutralization at each serial serum dilution. The 50% neutralization titer was
reported as the interpo lated reciprocal of the dilution y ielding a 50% reduction in fluorescent
viral foci.
2.6.2.12.6. ELISpot and Cytokine Profiling Immunoassays in Mice
Spleen single -cell suspensions were prepared in PBS by mashing tissue against the surface of
a 70 µm cell strainer (BD Falcon). Ery throcy tes were removed by hypotonic ly sis. Popliteal,
inguinal and iliac l ymph nodes were pooled , cut into pieces, digested with collagenase D
(1 mg/mL; Roche) and passed through cell strainers.
ELISpot assay s were performed with mouse IFNγ ELISpotPLUSkits according to the
manufacturer’s instructions (Mabtech). A total of 5 × 105splenocy tes was ex vivo
restimulated with the full -length S peptide mix (0.1 µg/mL final concentration per peptide,
JPT) or controls (gp70 -AH1 [SPSYVYHQF] (Slansky et al, 2000 ),JPT). Streptavidin- ALP
and BCIP/NBT -plus substrate were added, and spots counted using an ELI Spot plate reader
(ImmunoSpot® S6 Core Analy zer, CTL). Spot numbers were evaluated using
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Page 37ImmunoCapture Image Aquision Soft ware V7.0 and I mmunoSpot 7.0.17.0 Professional. For
T-cell subty ping, CD8+T cells were isolated from splenocy te suspensions using MACS
MicroBeads (CD8a [Ly -2], Milteny i Biotec) according to the manufacturer’s instructions.
The flow -through served as a sou rce of CD4+T cells. CD8+or CD4+T cells were
subsequently restimulated with sy ngeneic bone marrow- derived dendritic cells loaded with
full-length S peptide mix (0.1 µg/mL final concentration per peptide) or medium as control.
For c ytokine profiling in mi ce by bead -based immunoassay s, mouse splenocy tes were
re-stimulated for 48 h with full -length S peptide mix (0.1 µg/mL final concentration per
peptide) or cell culture medium (no peptide) as control. Concentrations of IFNγ, IL -2, IL -4,
IL-5 and (for splenocy tes from BNT162b2 -immunised 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 sy stem (Bio -Rad) and analysed with
ProcartaPlex Analy st 1.0 software (Thermo Fisher Scientific). Values below the lower limit
of quantification (LLOQ) were set to zero.
2.6.2.12.7. ELISpot and Intracellular Cytokine Staining Assays in NHPs
Cryopreserved NHP PBMCs were thawed in pre- warmed AIM -V media (Thermo Fisher
Scientific, US) with Benzonase (EMD Millipore, US) ,washed once ,and the concentration
wasadjusted to 2.5 x 106cells/mL in AIM -V.
For ELISpot assays, commerciall y available NHP IFNγ and IL -4 ELISpot assay kits were
used (Mabtech, Sweden). Briefl y, pre-coated PVDF 96- well microplates were washed with
PBS and blocked with AIM- V. PBMCs were added at 1.0x 105cells/well for IFNγ and
2.5x 105cells/well for IL-4. Cells were stimulated with a peptide pool spanning the entire
Sprotein (15 mers, 11aa overlap, JPT, Germany ) at 1μg/mL for 24 hours for IFNγ and
48hours for IL -4 at 37 °C in 5% CO 2. Tests were performed in triplicate wells ;
media-DMSO, a CMV peptide pool (JPT, Germany ) and P HA (Sigma, USA) were included
as controls. Cells were removed, plates washed, and spots detected using a b iotiny lated
detection antibod y followed b y a S treptavidin -HRP secondary antibody and AEC
chromogenic substrate (BD, US) for 10 minutes for IFNγ and 30 minutes for IL -4 at room
temperature until red spots were developed. Dried plates were scanned and counted using a
CTL ImmunoSpot S6 Universal Anal yzer (CTL, US). Reported results are background
(media-DMSO) subtracted and normalized to spot forming cells (SFC)/106 PBMCs.
For intracellular cy tokine staining (ICS) flow cy tometry -based anal ysis, thawed PBMCs
rested for 3 to 4 hours were stimulated in AIM -V medium in 96 -well plates with thepeptide
pool spanning the entire S protein at 1 μg/mL ;Staphy lococcus enterotoxin B (SEB; 2 μg/mL)
was used asapositive control ;and0.2 % DMSO was used as a negative control. An
APC -conjugated CD107a monoclonal antibody , GolgiStop, and GolgiPlug were added to
each well , and cells were incubated at 37 °C for 12 to 16 h. C ellswere then stained with
Viability Dye eFluor 780 and Fc block prior to surface staining with mAbs specific for CD4,
and CD8. Following staining for surface markers, cells were fixed and permeabilized with
BDC ytoFix/Cy toPerm solution, and intracellular staining performed with mAbs specific for
the following proteins, diluted in permeabilization buffer : CD154, IFN γ, IL-2, IL -4, TNF -α,
CD3. Cells were washed, resuspended in 2% fetal bovine serum ( FBS)/phosphate buffered
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Page 38saline ( PBS) buffer and acquired on a LSR Fortessa. Data were analyzed by FlowJo (10.4.1).
Cytokine -expressing cells were gated within the CD154+ CD4+ T cells and CD69+ CD8+
Tcells. Results shown are background (m edia-DMSO) subtracted .
2.6.2.12.8. Quantitative RT-PCR for Detection of SARS- CoV -2 Viral RNA
For quantification of SARS- CoV -2 virus in nonhuman primate challenge model swabs and
bronchoalveolar lavage (BAL) specimens, the US Centers for Disease Control -developed
2019 -nCoV_N1 assay ,a sensitive reverse transcription -polymerase chain reaction
(RT-PCR) -based assay that detects both viral genomic RNA and RNA transcripts, was use d
(Singh et al, 2020 ).
2.6.2.12.9. Lung Radiographs and Computed Tomography Scans
Lung radiographs (X -rays)and computed tomography (CT) scans were performed under
anesthesia as previously described (Singh et al, 2020 ;Kaushal et al, 2015 ).For radiographic
imaging, 3 -view thoracic radiographs (ventrodorsal , right and left lateral) were obtained one
week prior to challenge, and post- challenge on Day s 1, 3, 6 and end of project (Day 7/8) or
Day 10. High -resolution CT was performed one week prior to challenge and post -challenge
on Day s 3 and 6, for BNT162b2- immunized and control NHP and end of project (Day 7/8) or
Day 10 for all groups. The animals were anesthetized using Telazol (2 -6 mg/kg) and
maintained by inhaled isoflurane delivered through a Hallowell 2002 ventilator anesthesia
system (Hallowell, Pittsfield, MA). Animals were intubated to perform end inspiratory
breath -hold using a remote breath -hold switch. Lung field CT images were acquired using
Multiscan LFER150 PET/CT (MEDI SO Inc., Budapest, Hungary ) scanner. Image analy sis
was performed using 3D ROI tools available in Vivoquant (Invicro, Boston, MA). Images
were interpreted b y twoboard -certified veterinary radiologist sblinded to treatment groups.
Scores were assigned to a total of 7 lung regions on a severit y scale of 0 -3 per region, with a
maximum severity score of 21. Pulmonary lesions that could not be unequivocally attributed
to the viral challenge (such as atelectasis secondary to recumbency and anesthesia) received a
score of “0”.
2.6.2.12.10. Macroscopic and Microscopic Pathology
Histopathological assessme nts were performed at Day s 7 or 8 following infectious
SARS -CoV -2 challenge on the BNT162b2 -immunized animals (100 µg dose level; n =6) and
age-and sex -matched saline -immunized and SARS -CoV -2-challenged control animals that
were included in the histopath ology animal cohort (n=3). Tissues collected and
microscopicall y evaluated included lung (7 sections -1 sample of each lobe on L & R),
kidney , liver, spleen, skin, large and small intestine, heart [with coronary arteries], bone
marrow, nasal septum, tongue , trachea, mediastinal ly mph node, and mucocutaneous
junctions. Tissues were fixed in 10% neutral buffered formalin and routinely processed into
paraffin blocks, sectioned to 5 µm and stained with hematoxy lin and eosin.
Microscopic evaluation was performed independently by twopathologist s, both blinded to
treatment group. Lungs were evaluated using a semi -quantitative scoring system with
inclusion of cell ty pes and/or distribution as appropriate. An inflammation area score, based
on the estimated area of t he lung section with inflammation, was used to grade each lung
lobe: 0=normal; 1=<10%; 2=11- 30%; 3=30- 60%; 4= 60- 80%; 5=>80%. Samples were
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Page 39unblinded after agreement on diagnoses and severity grades. For each animal, the
inflammation area score for each lung lobe was averaged to generate a single inflammation
area score for that animal. That score was used to evaluate the severit y of respiratory disease
after SARS -CoV -2 challenge.
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Page 402.6.2.13. References
Al-Amri SS, Abbas AT, Siddiq LA, et al. Immunogenicity of candidate MERS -CoV DNA
vaccines based on the spike protein. Sci Rep 2017;7:44875.
Baum A, Fulton BO, Wloga E, et al. Antibod y cocktail to SARS -CoV -2 spike protein
prevents rapid mutational escape seen with individual antibodies. Science 2020; First release
Berger Rentsch M, Zimmer G. A vesicular stomatitis virus replicon- based bioassay for the
rapid and sensitive determination of multi- species ty pe I interferon. PLoS One 2011;
6(10):e25858.
Bestle D, Heindl MR, Limburg H,et al. TMPRSS2 and furin are both e ssential for
proteoly tic activation and spread of SARS ‐CoV‐2 in human airway epithelial cells and
provide promising drug targets. Available from: https://doi.org/10.1101/2020.04.15.042085.
Accessed: 24 Sep 2020.
Brouwer PJM, Caniels T G, van der Straten KJ. Potent neutralizing antibodies from COVID -
19 patients define multiple targets of vulnerability. Science 2020;369(6504):643-650 .
Cai Y, Zhang J, Xiao T, et al. Distinct conformational states of SARS -CoV -2 spike protein.
Science 2020: 10.1126/science.abd425 1.
de Wit E, van Doremalen N, Falzarano D, et al. SARS and MERS: recent insights into
emerging coronaviruses. Nat Rev Microbiol 2016;14(8)(08):523 -34.
Henderson R, Edwards RJ, Mansouri K, et al. Controlling the SARS -CoV -2 spike
glycoprotein conformation. Nat Struct Mol Biol 2020;(Jul):Online.
Hoffmann M, Kleine- Weber H, Schroeder S, et al. SARS -CoV -2 cell entry depends on
ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020;
181(2):271- 280.e8.
Hulswit RJ, de Haan CA, Bosch BJ. Coronavirus spike protein and tropism changes. Adv
Virus Res 2016;96:29-57.
Hutloff A. Regulation of T follicular helper cells by ICOS. Oncotarget
2015;6(26)(Sep):21785 -6.
Kariko K, Buckstein M, Ni H, et al. Suppression of RNA recognition by Toll-like receptors:
the impact of nucleoside modification and the evolutionary origin of RNA. Immunity
2005;2(Aug):165-75.
Kaushal D, Foreman TW, Gautam US, et al. Mucosal vaccination with attenuated
Mycobacterium tuberculosis induces strong central memo ry responses and protects against
tuberculosis. Nat Commun 2015; 6:8533 .
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Module 2.6.2. Pharmacology Written Summary
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Page 41Ke Z, Oton J, Qu K. et al. Structures, conformations and distributions of SARS -CoV -2 spike
protein trimers on intact virions. Nature 2020;10.1038/s41586-020-2665- 2.
Kim JY, Ko JH, Kim Y, et al. Viral load kinetics of SARS -CoV -2 infection in first two
patients in Korea. J Korean Med Sci 2020;35(7)(Feb):e86.
Liu L, Wang P, Nair MS, et al. Potent neutralizing antibodies against multiple epitopes on
SARS -CoV -2 spike. Nature 2020;584( 7821):450-456.
Munster VJ, Feldmann F, Williamson BN, et al. Respiratory disease and virus shedding in
rhesus macaques inoculated with SARS -CoV -2.Available from:
https://doi.org/10.1101/2020.03.21.001628. Accessed: 24 Sep 2020.
Muruato AE, Fontes -Garfias CR, Ren P, et al. A high -throughput neutralizing antibody assay
for COVID -19 diagnosis and vaccine evaluation. Nat Commun 2020;11(1):4059.
Pallesen J, Wang N, Corbett KS, et al. Immunogenicity and structures of a rationally
designed prefusion MERS- CoV spike antigen. Proc Natl Acad Sci USA
2017;114(35):E7348 -57.
Pardi N, Parkhouse K, Kirkpatrick E, et al. Nucleoside- modified mRNA immunization elicits
influenza virus hemagglutinin stalk -specific antibodies. Nat Commun 2018;9(1)(08):3361.
Report R -20-0085 : Immunogenicity Study of the LNP -Formulated Mod RNA Encoding the
Viral S Protein -V9.
Robbiani DF, Gaebler C,Muecksch F, et al. Convergent Antibody Responses to
SARS ‐CoV‐2 I nfection in Convalescent Individuals. Available from:
https://doi.org/10.1101/2020.0 5.13.092619. Accessed: 24 Sep 2020.
Sahin U, Karikó K, Türeci Ö. mRNA -based therapeutics - developing a new class of drugs.
Nat Rev Drug Discov 2014;13(10):759 -80.
Singh DK, Ganatra SR, Singh B,et al. SARS -CoV -2 infection leads to acute infection with
dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate
species. Available from: https://doi.org/10.1101/2020.06.05.136481 .Accessed: 24 Sep 2020.
Slansky JE,Rattis FM, Boy d LF, et al. Enhanced antigen -specific antitumor immun ity with
altered peptide ligands that stabilize the MHC-peptide- TCR complex. I mmunity 2000;
13(4):529-38.
Study 38166 .Repeat -Dose Toxicity Study of Three LNP -Formulated RNA Platforms
Encoding for Viral Proteins By Repeated Intramuscular Administration to Wistar Han Rats.
Study 20GR142. Giovanelli M. 17- Day Intramuscular Toxicity Study of BNT162B2 (V9)
and BNT162B3C in Wistar Han Rats with a 3 -Week Recovery . Study ongoing.
090177e1962f80a2\Approved\Approved On: 04-Feb-2021 00:09 (GMT)
FDA-CBER-2021-5683-0708883
BNT162b2
Module 2.6.2. Pharmacology Written Summary
CONFIDENTIAL
Page 42Study 20256434 (RN9391R58). Bouressam M. A Combined Fertility and Developmental
Study (Including Teratogenicity and Postnatal Investigations) of BNT162b1, BNT162b2 and
BNT162b3 b y Intramuscular Administration in the Wistar Rat. 22 Dec 2020.
VR-MQR -10211 Method Qualification Report for a Single- Plex Direct Luminex Assay for
Quantitation of IgG Antibodies to SARS -CoV -2 S1 Protein in Human Sera.
VR-MQR -10214 neut assay Qualification of the SARS -CoV -2 mNeonGreen Virus
Microneutralization Assay .
VR-VTR -10671 BNT162b2 (V9) Immunogenicity and Evaluation of Protection against
SARS -CoV -2 Challenge in Rhesus Macaques .
World Health Organization. WHO guidelines on nonclinical evaluation of vaccines. Annex 1.
In: World Health Organization. WHO technical report series, no. 927. Geneva, Switzerland;
World Health Or ganization; 2005:31-63.
Wrapp D, Wang N, Corbett KS, et al. Cry o-EM structure of the 2019 -nCoV spike in the
prefusion conformation. Science 2020;367(6483):1260 -3.
Wu Y, Wang F, Shen C, et al. A noncompeting pair of human neutralizing antibodies block
COVID -19 virus binding to its receptor ACE2. Science 2020; 368 (6496): 1274 -8.
Xie X, Muruato A, Lokugamage KG, et al. An infectious cDNA clone of SARS -CoV -2. Cell
Host Microbe 2020; 27(5):841- 848.e3.
Yong CY, Ong HK, Yeap SK, et al. Recent advances in the vaccin e development against
middle east respiratory syndrome -coronavirus. Front Microbiol 2019;10:1781.
Yuan M, Wu NC, Zhu X, et al. A highl y conserved cry ptic epitope in the receptor binding
domains of SARS -CoV -2 and SARS -CoV. Science 2020; 368(6491):630-3.
Zakhartchouk AN, Sharon C, Satkunarajah M, et al. I mmunogenicit y of a receptor -binding
domain of SARS coronavirus spike protein in mice: implications for a subunit vaccine.
Vaccine 2007;25(1):136 -43.
Zhou M, Zhang X, Qu J. Coronavirus disease 2019 (COVID -19): a clinical update. Front
Med 2020(Apr):1 ‐10.
Zou L, Ruan F, Huang M, et al. SARS- CoV -2 viral load in upper respiratory specimens of
infected patients. N Engl J Med 2020;382(12)(03):1177-9.
090177e1962f80a2\Approved\Approved On: 04-Feb-2021 00:09 (GMT)
FDA-CBER-2021-5683-0708884