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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 1Title: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV -2
Challenge in Rhesus Macaques
Study Number: COVID Rh2020- 01 (NI RC study #: 8725-2005)
(SNPRC S tudy #: Covid -1778)
Parent Compound Number(s): PF-07302048
Alternative Compound Identifiers: N/A
Pfizer Vaccine Research and Development
401 N. Middletown Rd.
Pearl River, NY
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
FDA-CBER-2021-5683-0709187
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 2Title: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV -2
Challenge in Rhesus Macaques
PRINCIPAL INVESTIGATOR:
CONTRIBUTING SCIENTIST (S): ;
PREPARED BY:
APPROVED BY:
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
(b) (6)
(b) (6)
(b) (6)
(b) (6)
(b) (6)
FDA-CBER-2021-5683-0709188
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 3Title: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV -2
Challenge in Rhesus Macaques
SYNOPSIS
Prime -boost vaccination of rhesus macaques with BNT162b2 (V9) elicited SARS -CoV -2
neutrali zing geometric mean titers 10.2 to 18.0 times that of a SARS -CoV -2 convalescent
human serum panel. BNT162b2 generated strong T h1 type CD4+ and IFN + CD8+ T cell
responses in rhesus macaques. The BNT162b2 vaccine candidate protected the lungs of
immuniz ed rhesus macaques from infectious SARS -CoV -2 challenge , with no evidence of
vaccine -elicited disease enhance ment .
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FDA-CBER-2021-5683-0709189
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
LIST OF FIGURES ................................ ................................ ................................ ................... 5
1. OBJECTI VES ................................ ................................ ................................ ........................ 6
2. INTRODUCTION ................................ ................................ ................................ ................. 6
3. MATERIAL S AND M ETHODS ................................ ................................ ........................... 7
3.1. I mmunogenicit y Study Design ................................ ................................ .................. 7
3.2. Test Article Information ................................ ................................ ............................ 8
3.3. General Formulation I nstructions ................................ ................................ .............. 8
3.3.1. RNase Reduction Measures ................................ ................................ .......... 8
3.3.2. Source of Study Materials ................................ ................................ ............ 9
3.3.3. Vaccine Preparation ................................ ................................ ...................... 9
3.4. Pre -Screen ................................ ................................ ................................ ............... 10
3.5. Anesthesia ................................ ................................ ................................ ............... 10
3.6. Vaccine Administration................................ ................................ ........................... 10
3.7. Daily Observations ................................ ................................ ................................ ..10
3.8. Sample Collection and Handling................................ ................................ ............. 10
3.8.1. Serum ................................ ................................ ................................ .......... 10
3.8.2. PBMCs................................ ................................ ................................ ........ 11
3.9. Shipping and Storage Conditions ................................ ................................ ............ 11
3.10. I mmunological Assay s ................................ ................................ .......................... 11
3.10.1. SARS -CoV -2 S1-Binding IgG Luminex Assay ................................ .......11
3.10.2. SARS -CoV -2 Neutralization Assay ................................ ......................... 11
3.10.3. IFNγ and IL- 4 ELISpot Assay s ................................ ................................ 12
3.10.4. Flow Cy tometry Intracellular C ytokine Staining (ICS) Assay ................. 12
3.11. SARS -CoV -2 Challenge of Rhesus Macaques ................................ ..................... 13
3.12. Chest X -rays and Computed Tomography Scans ................................ ................. 14
3.13. Reverse -transcription Quantitati ve Poly merase Chain Reaction .......................... 14
3.14. Macroscopic and Microscopic Pathology ................................ ............................. 14
4. RESUL TS AND DI SCU SSION ................................ ................................ .......................... 15
5. CONCLUSION ................................ ................................ ................................ .................... 25
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 56. DEVIATIONS ................................ ................................ ................................ ..................... 26
7. REFERENCES ................................ ................................ ................................ .................... 26
8. APPENDIX ................................ ................................ ................................ .......................... 28
LIST OF TABLES
Table 1. Immunization Study Desi gn................................ ................................ .......7
Table 2. Analy tical Characterization of BNT162b2 (V9) Drug Product.................. 8
Table 3. Dilution Scheme for BNT162b2 (V9) [Concentration = 0.5 mg/mL]...... 10
Table 4. Blood Volume Collection Guidelines ................................ ....................... 11
Table 5. Pathology Cohorts ................................ ................................ .................... 15
LIST OF FIGURES
Figure 1. S1-binding IgG Concentrations Elicited by Immunization of Rhesus
Macaques with BNT162b2 (V9) ................................ .............................. 16
Figure 2. 50% Serum Neutralizing Titers Elicited by Immunization of Rhesus
Macaques with BNT162b2 (V9) ................................ .............................. 17
Figure 3. IFNγ and IL- 4 ELISpot Results in BNT162b2 -and Control -
Immunized Animals................................ ................................ .................. 18
Figure 4. S-specific CD4 and CD8 T- cell Responses in BNT162b2 -and
Control -Immunized Animals as Measured by ICS Assay ........................ 19
Figure 5. Viral RNA in BAL Fluid, Nasal Swabs, and Orophary ngeal Swabs
of Rhesus Macaques after Infectious SARS -CoV -2 Challenge ............... 21
Figure 6. S1-binding IgG and 50% Serum Neutralization Responses in
Rhesus Macaques after Infectious SARS- CoV -2 Challenge .................... 22
Figure 7. Clinical Signs in Rhesus Macaques after Immunization with
BNT162b2 and Challenge with I nfectious SARS -CoV -2........................ 23
Figure 8. Radiograph and CT Scores of Rhesus Macaque Lungs after
Infectious SARS- CoV -2 Challenge................................ .......................... 24
Figure 9. Lung Inflammation Area Score after IN/IT SARS- CoV -2 Challenge ..... 25
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 6Title: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV -2
Challenge in Rhesus Macaques
Study Number: COVID Rh2020-01
(Associated S tudy Numbers: NIRC study #: 8725 -2005 ;
SNPRC Study #:Covid -1777 and Covid -1778 )
Functional Area: Vaccine Research and Development
Test Facility : Pfizer Vaccine Research, 401 North Middletown Road,
Pearl River, NY 10965
Immunizations In-Life Test Facility: New Iberia Research Center (NIRC),
4401 W.Admiral Doy le Drive ,
New Iberia, LA 70560
Challenge In -Life Test Facility: Southwest National Primate Center (SNPRC),
8715 W. Military Dr.
San Antonio, TX 78227 -5302
Neutralization Assay Test Facility: University of Texas Medical Branch (UTMB)
Galveston, TX 77555
Study/Testing Initiation Date: 07Apr2020
Study/Testing Completion Date: 01Nov2020
1.OBJECTIVES
The purpose of this stud y was to evaluate BNT162b2 (V9) -elicited immune responses and
the ability ofthe vaccine to protect against SARS -CoV -2 challenge in rhesus macaques
(Macaca mulatta ).
2.INTRODUCTION
The coronavirus disease 2019 (COVID -19) vaccine (BioNTech code number BNT162, Pfizer
code number PF -07302048) is aninvestigational vaccine intended toprevent COVID -19,
which is caused bysevere acute respiratory syndrome coronavirus 2 (SARS -CoV -2).The
vaccine candidate BNT162b2, otherwise known as BNT162b2 (V9 ),is a m1Ψ nucleoside
modified mRNA (modRNA) expressing full- length S with two proline mutations (P2) to lock
the transmembrane protein in an antigenicall y optimal prefusion conformation .1,2The
vaccine is formulated in lipid nanoparticles (LNPs).
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 7BNT162b2 was assessed for immunogenicity and for protection against an infectious
SARS -CoV -2 challenge in rhesus macaques. SARS- CoV -2 infection in humans manifests as
both asy mptomatic infection and as the disease COVID- 19, with diverse signs, s ymptoms,
and levels of severit y. Based on published reports, SARS -CoV -2 challenged rhesus
macaques develop an acute, transient infection in the u pper and lower respiratory tract and
have evidence of viral replication in the gastrointestinal tract, similar to humans.3,4The
human and rhesus ACE -2 receptor have 100% amino acid identity at the critical binding
residues, which may account for the fidelity of this SARS -CoV -2 animal model.5
3.MATERIALS AND METHODS
3.1.Immunogenicity Study Design
The study was performed in 2–4 year old, male rhesus macaques (Macaca mulatta ) designed
with 3groups as shown in Table 1. Animals were vaccinated with 30 µ g or 100 µ g of
BNT162b2 (n=6 per group) or with saline control (n=6)on day s 0 and 21 , administered in a
0.5 mL dose volume b ythe intramuscular (IM) route .Serum and peripheral blood
mononuclear cells (PBMCs) were collected at the indicated times post immunization.
Immuni zations were performed at the University of L ouisiana at Lafay ette-New Iberia
Research Center (NIRC), which is accredited b y 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 Recombin ant DNA Molecules, and Biosafet y in
Microbiological and Biomedical Laboratories. All procedures performed were in accordance
with regulations and established guidelines and were reviewed and approved by an
Institutional Animal Care and Use Committee or thr ough an ethical review process.
Table 1. Immunization Study Design
Gp# No. of
Rhesus
MacaquesAnimal
IDsImmunogen
DescriptionVaccine
Encoded
AntigenDose
(µg)Dose Vol /
RouteVax
(Day)Bleed
(Week)
1 6 A16N100
A17N102
A17N037
A16N140
A16N020
A16N193Saline
(0.9% sodium
chloride
(Lot#10-106-JT)- - 0.5 mL/IM 0, 21 Prea, 6hr,
24hr, 1, 2a,
3, 4, 5, 6a
2 6 A17N143
A17N149
A17N138
A17N125
A17N107
A17N134BNT162b2 ( V9)(Lot
# CoVVAC/270320)Spike
Protein P2
variant30 0.5 mL/ IM 0, 21 Prea, 6hr,
24hr, 1, 2a,
3, 4a, 5, 6a, 8
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 8Table 1. Immunization Study Design
Gp# No. of
Rhesus
MacaquesAnimal
IDsImmunogen
DescriptionVaccine
Encoded
AntigenDose
(µg)Dose Vol /
RouteVax
(Day)Bleed
(Week)
3 6 A17N109
A17N139
A17N167
A17N105
A17N113
A17N114BNT162b2 V9 (Lot #
CoVVAC/270320)Spike
Protein P2
variant100 0.5 mL/ IM 0, 21 Prea, 6hr,
24hr, 1, 2a,
3, 4a, 5, 6a, 8
a.PBMC collection timepoints
3.2. Test Article Information
The BNT162b2 (V9) drug product was provided by BioNTech (Mainz, Germany ). Anal ytical
testing of the drug product was performed at Pfizer VRD Earl y Bioprocess and Development
in Pearl River, NY.
Table 2. Analytical Characterization of BNT162b2 (V9) Drug Product
Lot Number Description
ofRNACap
(%)PolyA
(%)Integrity
(%)Endotoxin
(EU/mL)Spike Protein Expression
(%)a
CoVVAC/270320 BNT162b2 V9
3.3. General Formulation Instructions
All test articles were opened under aseptic conditions. The LNP formulations were handled
with care to prevent potential RNase contamination. Prior to dose preparation, the frozen
modRNA L NP vials were completel y thawed at ambient temperature and diluted to the
corresponding target concentrations at 60 and 200 µ g/mL by using saline sol ution. The
diluted modRNA L NP article swere well mixed by gentle swirling and/or inversion to ensure
a homogeneous mixture.
3.3.1. RNase Reduction M easures
All preparation steps were performed under a laminar flow hood or PCR Dead Air Box. After
disinfection with Terralin® liquid (alcohol- based disinfectant or similar surface disinfectant)
all work surfaces, gloves, instruments and equipment were treated with RNase Zap™.
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 93.3.2. Source of Study Materials
Materials provided b y BioNTech:
BNT162 RNA LNP vial(s), 0.5 mg/mL RNA , 0.5 mL extractable volume,
stored at -70 °C ± 10 °C
Materials provided b y NIRC:
0.9% Sodium Chloride (referred to dilution buffer) ICU Medical, 1 L bag,
0.9% sodium chloride, Injection, USP, NDC 0990 -7983 -09, Lot 10-106- JT,
Exp 01 Oct 2021
3.3.3. Vaccine Preparation
1. Test vials were removed from -70°C ± 10 °C storage and warm ed to room temperature
(approximately 5-10 minutes) under a laminar flow hood.
2.While the test materials thawed, sterile, R Nase free glass vials were prepared with the
appropriate volume o f dilution buffer (0.9% sterile sodium chloride/saline) . A similar
empty vial was prepared for the pooling of BNT162b2 (V9).
3.Vials containing test materials were gentl y invert edthree to five times to ensure thorough
mixing .
4. F lip cap sand rubber stoppers were carefully removed on the BNT162 b2 (V9) test item
vials.
5.Using sterile, RNase -free pipet tips, the volume from each vial was pooled to provide a
sufficient volume of homogeneous material.
6.After pooling, the appropriate volume of BNT 162b2 (V9)was transferred into the 5 mL
glass vials that contained the buffer that was added in step two. Exact volumes can be
found in the dilution scheme below.
7. V ials were carefull y closed and g ently invert edto ensure a homogeneous mixture.
8.Syringes we re aseptically filled and transported on ice to the animal facility . In the
interest of animal welfare, the sy ringes were warmed to room temperature immediately
prior to administration. All animals were injected within two hours of vaccine
preparation.
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 10Table3.Dilution Scheme for BNT162b2 (V9) [Concentration = 0.5 mg/mL]
Group BNT 162b2 (V9)
Application Dose
[µg/0.5mL]Factor of
DilutionDilution Step 1
Volume BNT162 Test Item
(mL)Volume Dilution Buffer
(mL)
2 30 8.33 0.48 3.52
3 100 2.5 1.6 2.4
3.4.Pre-Screen
Rhesus macaques were selected b ased on pre- study physical exams and body weights were
recorded .Selected macaques were identified by unique body tattoo s prior to beginning any
study related procedure .
3.5.Anesthesia
All vaccinations and peripheral blood draws w ere performed with the macaques
appropriatel y sedated using Ketamine HCl (10 mg/kg), administered as an intramuscular
(IM) injection.
3.6.Vaccine Administration
Vaccine s were administered as a single 0.5 mL intramuscular injection in the left quadricep
muscle. Sites w ereshaven and prepped per NI RC standard operating procedures ( SOPs) prior
to injection.
3.7.Daily Observations
Animals were observed daily for any abnormal clinical signs and/or signs of illness,
behaviors departing from species specific behavior, or distress starting upon assignment to
study . Any abnormal observations would have been reported to the Stud y Director and Stud y
Veterinarian. Evaluation of vaccine administration sites were included in th e daily
observations for signs of redness, swelling, and/or localized reactions.
3.8.Sample Collection and Handling
3.8.1. Serum
Blood was collected into serum separator tubes with volumes determined based on body
weight, according to Table 4. Samples were centrifuged at 3000 rpm/ 2095 RCF (x g) for
10minutes, per NIRC SOPs for serum separation and harvest .Samples w ere barcoded,
recorded and electronic files were sent with each shipment . Each s erum sample was divid ed
into 4 x 0.25 mL aliquots and an y remaining volume was stored at approximately 1.0 mLper
barcoded cry ovial and stored at -70 °C until shipment . For SARS -CoV -2 neutralization assay
testing at the UTMB BSL -3 facilit y, one of the four 0.25 mL aliquots were heat -inactivated
(56°C for 30 minutes in a water bath ) and shipped directly to UTMB. All samples were
handled in a manner to maintain sterility .
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 11Table 4.Blood Volume Collection Guidelines
Body Weight Range (kg) Collection Volume (mL)
<4.5 5.0
4.6-5.5 8.5
5.6-7.2 12.0
>7.3 17.0
kg, kilogram; mL, milliliter
3.8.2. PBMCs
Whole blood w ascollected from each animal at specified time point sin EDTA vacutainer
tubes . PBMCs w ere retained at room temperature then processed per NIRC SOP 8725-06.07.
After processing, cells were frozen at5 x 106cells/m Lcell concentration in liquid nitrogen .
No less than 5 x 106cells/mL or more than 1 x 107cells/mL were frozen per vial. Plasma
from individual animals was aliquo tedand stored at -70 °C.
3.9.Shipping and Storage Conditions
Test material s were shipped from Pfizer (Pearl River) to NIRC in a manner to maintain
frozen conditions during transport. Test material s were inventoried and s tored at -70C upon
arrival. Serum samples were shipped over night on dry ice with Temptale included. PBMC
samples for each animal were split into two boxes, send and retains. Cryoshippers were used
to transport PBMCs to the Pearl River P fizer facility .
3.10. Immunological Assays
3.10.1. SARS -CoV -2 S1-Binding IgG Luminex Assay
Adirect binding Luminex immunoassay (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 L uminex microspheres. Bound
nonhuman primate S1 -binding IgG was detected with a R- Phycoery thrin-conjugated goat
anti-human poly clonal secondary antibody (Jackson L abs). Data were captured as median
fluorescent intensities (MFIs) using a Luminex reader and converted to U/mL antibod y
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.
3.10.2. SARS -CoV -2 Neutralization Assay
The SARS- CoV -2 neutralization assay used a previously described strain of SARS -CoV -2
(USA_WA1/2020) that had been rescued b y reverse genetics and engineered by the insertion
of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome.6This
reporter virus generates similar plaque morphologies and indistinguishable growth curves
from wild ty pe virus. Viral master stocks were grown in Vero 76 cells as previously
described.7When testing human convalescent serum specimens, the fluorescent
neutralization assay produced comparable results as the conventional plaque reduction
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 12neutralization assay . Serial dilutions of heat -inactivated sera were incubated with the reporter
virus (2 x 104PFU per well) to y ield approximately a 10 -30% infection rate of the Vero
CCL81 monolay er for 1 hour at 37 °C before inoculating Vero CCL 81 cell monolay ers
(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 b y nuclear stain (Hoechs t 33342) and fluorescent virall y
infected foci were detected 16- 24 hours after inoculation with a Cy tation 7 Cell I maging
Multi -Mode Reader (Biotek) with Gen5 Image 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 (VNT50) was
reported as the interpolated reciprocal of the dilution y ielding a 50% reduction in fluorescent
viral foci.
3.10.3. IFNγ and IL -4 ELISpot Assays
PBMCs were tested with commerciall y available nonhuman primate IFNγ and IL -4 ELISpot
assay kits (Mabtech, Sweden ). Cry opreserved rhesus macaque 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.
Pre-coated PVDF 96 -well microplates were washed three times with PBS and blocked with
AIM-V. PBMCs were added at 1.0x 105cells/well for IFNγ and 2.5 x 105cells/well for IL-4.
PBMCs were stimulated with a peptide pool spanning the entire S protein (15 mers, 11 amino
acidoverlap, JPT, Germany ) at 1μg/mL for 24 hours for IFNγ and 48 hours for IL -4 at 37 °C
in 5% CO 2. Tests were performed in triplicate wells and medium-DMSO, a CMV peptide
pool (JPT, Germany ) and PHA (Sigma, USA) were included as controls. Cells were removed
and plates washed six times with PBS. Biotiny lated detection antibody diluted to a
concentration of 1 g/mL in PBS/0.5% BSA was added to theplates and incubated for two
hours at room temperature. Plates were washed six times with PBS and S treptavidin -HRP
diluted at 1:1000 in PBS/0.5% BSA was added and incubated for one hour at room
temperature. Plates were washed six times with PBS and AEC substrate (BD, US) was added
and incubated for 10 minutes for IFN -γ and 30 minutes for IL -4 at room temperature until red
spots were developed. The plates were washed with distilled water, dried for 1 -2 hours at
room and scanned and counted using a CTL Im munoSpot S6 Universal Analy zer (CTL, US).
Results shown are background (Media- DMSO) subtracted and normalized to SFC/106
PBMCs.
3.10.4. Flow Cytometry Intracellular Cytokine S taining (ICS) Assay
PBMCs were thawed as above ,rested for 3 to 4 hours , and were stimula ted with apeptide
pool spanning the entire S protein (15 mers, 11 amino acid overlap, JPT, Germany ) at
1μg/mL , Staphy lococcus enterotoxin B (SEB; 2 μg/mL) as positive control, or 0.2 % DMSO
as negative control, in AIM -V medium in 96- well plates. CD107a monoclonal antibody
(mAb) (BioLegend; clone H4A3, APC), GolgiStop, and GolgiPlug were added to each well
and cells were incubated at 37 °C for 12 to 16 h. Following incubation, cells were stained
with Viability Dye eFluor 780 (eBioscience™) and Fc block ad ded prior to surface staining
with mAbs specific for CD4 (clone SK3, BV480), CD3 (clone SP34.2, Alexa 700), and CD8
(clone RPA -T8, BB700). Cells were then washed once with 2% FBS/PBS buffer, fixed and
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
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Page 13permeabilized with BD CytoFix/Cy toPerm solution, washed twice in BD P erm/Wash buffer
and intracellular staining performed with the following mAbs: CD154 (BioLegend; clone
24-31, BV605), IFN -γ(clone B27, FITC), IL-2 (eBioscience™; clone MQ1 -17H12,
PE-Cy7), IL -4 (clone MP4- 25D2, BV421), TNF -α (clone Mab11, BUV395), CD3 (clone
SP34.2, Alexa 700) in perm /wash buffer for 30 min at RT. Cells were washed, resuspended
in 2% FBS/PBS buffer and acquired on a L SR Fortessa. All mAbs are from BD Biosciences,
except mentioned. Data analy zed by FlowJo (10.4.1). Cy tokine -expressing cells were gated
within the CD154+ CD4 T cells and CD69+ CD8 T cells. Results shown are background
(medium-DMSO) subtracted .
3.11. SARS -CoV -2 Challenge of Rhesus Macaques
Infectious SARS -CoV -2 challenge was performed on the BNT162b2- immunized animals
(100 µg dose level; n =6) and age- and sex -matched saline -immunized animals (n=3; Animal
ID# A17N118, A17N157, A17N128) at the Southwest National Primate Research Center.
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.
The SARS- CoV -2 inoculum was obtained from a stock of 2.1 × 106PFU/mL previously
prepared at Texas Biomedical Research Institute (San Antonio, TX), 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 4th passage seed stock purchased from BEI
Resources; NR -52281) in Vero 76 cells. The virus was confirmed to be SARS -CoV -2 by
deep sequencing and identical to the published sequence (GenBank accession number
MN985325.1). Approximately two weeks prior to challenge, animals w ere moved to the
Southwest National Primate Research Center (SNPRC; San Antonio, TX) and into the
ABSL -3 facility . BNT162b2 -immunized (n=6) and age -matched saline control -immunized
(n=3) male rhesus macaques (control) were challenged with 1.05 × 106plaque forming units
of SARS -CoV -2 USA -WA1/2020 isolate, split equally between the intranasal (IN) and
intratracheal (IT) routes as previousl y described.8The challenge was performed 55 day s after
the second BNT162b2 immuni zation. A separate sentinel group of age -and sex -matched
animals (n=3) from the 30 µg BNT162b2- immunized group was mock challenged with cell
culture medium ( DMEM supplemented with 10% FCS ). Animals were monitored regularl y
by a board- certified veterinary clinician for rectal body temperature, weight and phy sical
examination. Specimen collection was performed under tiletamine zolazepam (Telazol)
anesthesia as described.8Nasal and orophary ngeal swabs were collected from all macaques
pre and at Day s, 1, 3, and 6 (relative to the day of challenge), from BNT162b2 -immunized
macaques on Day 7 or 8, and from control and sentinel macaques on Day 10.
Bronchoalveolar lavage (BAL) was performed on macaques the week before challenge and
on Day s 3 and 6 post -challenge and on BNT162b2 -immunized macaques on Day 7 or 8.
BAL was performed b y instilling four times 20 mL of saline. These washings were pooled,
aliquoted and stored frozen at -70 °C. Necrops y was performed on BNT162b2 -immunized
animals on Day 7 or 8. Control and sentinel animals were not necropsied to allow further use
in a separate study .See Appendix ,Supportive Table 8.5for a summary of individual
animals.
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Page 143.12. Chest X -rays and Computed Tomography Scans
X-rays and computed tomograph y (CT)scans were performed under anesthesia as previousl y
described .9,8For 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 (D ay 7/8) or Day 10. High -resolution CT was performed one week prior to
challenge and post- challenge on Day 3 post -challenge for BNT162b2- immunized and control
animals and end of project (Day 7/8) or Day 10for all groups . The animals were anesthetized
using Telazol (2 -6 mg/kg) and maintained by inhaled isoflurane delivered through a
Hallowell 2002 ventilator anesthesia s ystem (Hallowell, Pittsfield, MA). Animals were
intubated to perform end inspiratory breath- hold using a remote breath -hold switch. L ung
field CT images were acquired using Multiscan LFER150 PET/CT (MEDISO I nc., Budapest,
Hungary ) scanner. Image anal ysis was performed using 3D ROI tools available in Vivoquant
(Invicro, Boston, MA). Images were interpreted by 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
thatcould not be unequivocally attributed to the viral challenge (such as atelectasis
secondary to recumbency and anesthesia) received a score of “0”.
3.13. Reverse -transcription Quantitative Polymerase Chain Reaction
To detect and quantify SARS -CoV -2 in rhesus macaques , viral RNA was extracted from
nasal swabs, OP swabs, and BAL spe cimens as previously described10,11,12and tested b y
RT-qPCR as previousl ydescribed.8Briefl y, 10 μg yeast tRNA and 1 × 103PFU 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 CDC -developed
2019 -nCoV_N1 assay on a QuantStudio3 instrument (Applied Bios ystems). The cut- off for
positivity (limit of detection, L OD) was established at 10 gene equivalents (GE) per reaction
(800 GE/mL). Samples were tested in duplicate. Any specimens that had, on repeated
measurement, viral RNA levels on either side of the LLOD, were categori zed as
indeterminate and excluded from the graphs and the anal ysis.
3.14. Macroscopic and Microscopic Pathology
Necrops y, tissue processing, and histology were performed b y SNPRC. Histopathological
assessments 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
histopathology animal cohort (n=3; Table 5 ). 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. Tis sues were fixed
in 10% neutral buffered formalin and routinel y processed into paraffin blocks , sectioned to 5
µm and stained with hematox ylin and eosin.
Microscopic evaluation was performed independently bya SNPRC and a Pfizer pathologist ,
both blinded to treatment group. Lungs were evaluated using a semi -quantitative scoring
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Page 15system with inclusion of cell ty pes and/or distribution as appropriate. An inflammation area
score, based on the estimated area of the lung section with inflammation, was used to grad e
each lung lobe: 0=normal; 1=<10%; 2=11 -30%; 3=30- 60%; 4= 60 -80%; 5=>80%. Samples
were unblinded after agreement on diagnoses and severit y grades. For each animal, the
inflammation area score for each lung lobe was averaged to generate a single inflammati on
area score for that animal. That score was used to evaluate the severit y of respiratory disease
after SARS -CoV -2 challenge .
Table 5.Pathology Cohorts
Group Number of Animals (Animal ID)
Controla3
(A16N193, A17N037, A17N102 )
BNT162b2 6
(A17N109, A17N139, A17N167, A17N105, A17N113, A17N114 )
a.Age-and sex -matched control (saline -immunized and challenged )animals
4.RESULTS AND DISCUSSION
To assess BNT162b2- mediated protection in non -human primates, groups of six male,
2-4year old rhesus macaques were immunized IM with 30 or 100 µg of BNT162b2 or saline
control on Day s 0 and 21. S1- binding IgG was readily detectable b y Da y 14after Dose 1, and
levels increased further after Dose 2 ( Figure 1). Seven day s after Dose 2 (Day 28), the GMCs
of S1- binding IgG were 30,339 units (U)/mL (30 µg dose level) and 34,668 U/mL (100 µg
dose level). For comparison, the S1- binding IgG GMC of a panel of 38 SA RS-CoV -2
convalescent human sera was 631 U/mL , substantially lower than the GMCs of the
immunized rhesus macaques after one or two doses.
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Page 16Figure 1.S1-binding IgG Concentrations Elicited by Immunization of Rhesus
Macaque s with BNT162b2 ( V9)
Numbers on the x -axis indicate the day post first immunization. Heights of bars indicate GMCs, which are
written above the bars; whiskers indicate 95% CIs; dots represent individual monkey IgG concentrations.
Dotted line indicates th e lower limit of quantification (LLOQ =1.267
U/ml). Values at or below LLOQ were set
to ½ LLOQ. C –saline -immunization control; HCS – human convalescent serum panel .
Fifty percent virus neutralization GMTs , measured by an authentic SARS -CoV -2
neutralizati onassay ,6were detectable in rhesus macaque sera b y Da y 14after Dose 1 and
peaked at a GMT of 962 (Day 35, 14 day s after Dose 2 of 30 µg) or 1,689 (Day 28, 7 day s
after Dose 2 of 100 µg;Figure 2). Robust GMTs of 285 for 30 µ g and 310 for 100 µg dose
levels persisted to at least Day 56. For comparison, the neutralization
GMT of the human
convalescent serum panel was 9 4.A summary of the S1 -binding IgG GMCs and
SARS -CoV -2 neutralization GMTs are described in Appendix ,Supportive Table 8.1.
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Page 17Figure 2.50% Serum Neutralizing Titers Elicited by Immunization of Rhesus
Macaques with BNT162b2 ( V9)
Numbers on the x -axis indicate the day post first immunization. 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 se t to ½ LLOQ. Abbreviations as in Figure 1.
S-specific T- cell responses were anal yzedby ELISpot and intracellular cy tokine staining
(ICS). Peripheral blood mononuclear cells (PBMCs) were collected before immuniz ation and
at the times indicated after Doses 1 and 2. In BNT162b2 -immunized animals, s
trong IFN
but minimal IL -4 responses were detected by ELISpot after Dose 2 (day 28 and 42)
(Figure 3).ICS anal ysis confirmed that BNT162b2 elicited strong S -specific IFNγ producing
T cell responses, including a high frequency of CD4+T cells that produced IFN , IL-2, or
TNF -but a low frequency of CD4+cells that produced IL -4, indicating a T h1-biased
response ( Figure 4Ato Figure 4B). BNT162b2 also elicited S -specific IFN+producing
CD8+T cells ( Figure 4E).
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Page 18Figure 3.IFNγ and IL -4 ELISpot R esults in BNT162b2 -and Control -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
saline (Control ).Numbers on the x -axis indicate the day post first immunization (a Day 28 sample was not
available from the Control group) . Height of bars indicates the mean, whiskers indicate the standard error of
mean (SEM), every symbol represents one animal. Dotted lines mark the lower limit of det ection. (A) IFNγ
ELISpot analysis . (B) IL -4 ELISpot analysis. (C) Correlation of frequency of IFNγ or IL -4 producing cells at
Day 42 (21 days post dose 2) .
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Page 19Figure 4. S- specific CD4 and CD8 T -cell Responses in BNT162b2 -and
Control -Immunized Animals as Measured by ICS Assay
Numbers on the x -axis indicate the day post first immunization. Height of bars indicates the mean, whiskers
indicate the standard error of mean (SEM), every symbol represents one animal. (A) Frequency of IFNγ + CD4
T cells. (B) Frequency of IFNγ /IL-2/TNF -+ CD4 T cells (C) Frequency of IL -4+ CD4 T cells. ( D) Correlation
of frequency of IFNγ +with IL-4+ CD4 T cells at Day 42 (21days post dose 2) . (E) Frequency of IFNγ + CD8
Tcells.
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Page 20The s ixrhesus macaques that had received two immuni zations with 100 µg BNT162b 2 and
three age -matched macaques that had received saline were challenged with 1 .05 × 106plaque
forming units of SARS -CoV -2 (strain USA -WA1/2020), split equally between intranasal and
intratracheal rou tes, as previously described.8Three additional non-immuniz ed, age-matched
rhesus macaques (sentinels) were mock -challenge dwith cell culture mediu m. At the time of
challenge, SARS -CoV -2 neutralizing titers ranged from 260 to 1,004 in the BNT162b2
(V9) -immunized animals. Neutralizing titers wer e undetectable in animals from the control -
immunized and sentinel groups. Nasal and orophary ngeal (OP) swab s were collected and
bronchoalveolar lavage (BAL) was performed at the times indicated, and samples were tested
for SARS -CoV -2 RNA (genomic RNA or subgenomic transcripts) by reverse -transcription
quantitative poly merase chain reaction (RT -qPCR ; Figure 5).All personnel performing
clinical, radiological, histopathological, or RT -qPCR evaluations were blinded to the group
assignments of the macaques.
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 (Figure 5A).At no time point sampled was viral
RNA detected i n BAL fluid from the BNT162b2 -immunized and SARS -CoV -2 challenged
macaques . The difference in viral RNA detection in BAL fluid between
BNT162b2 -immuniz ed and control -immuniz ed rhesus macaques after challenge is highl y
statistically significant (by a nonpa rametric test, p=0.0014).
From control -immunized macaques, viral RNA was detected in nasal swabs obtained on
Days 1, 3, and 6 after SARS -CoV -2 challenge; from BNT162b2 -immuniz ed macaques, viral
RNA was detected onl y in nasal swabs obtai ned on Day 1 after c hallenge and not in swabs
obtained on Day 3 or subsequently (Figure 5B).The pattern of viral RNA detection from OP
swabs was similar to that for nasal swabs (Figure 5C).The difference in the proportion of
animals with detectable viral RNA between BNT162b 2-immunized and control -immunized
animals, based on samples obtained after immunization, is statisticall y significant for OP
swabs (p=0.0007) but not for nasal swabs (p=0. 2622) .
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Page 21Figure 5.Viral RNA in BAL Fluid , Nasal Swabs, and Oropharyngeal Swabs of
Rhesus Macaques after Infectious SARS -CoV -2 Challenge
Pre 3 6 10/EOP1021041061081010Bronchoalveolar Lavage
Day relative to challenge
0/3 0/3 0/6 0/3 2/3 0/6 n/a n/a 0/6 0/3 1/3 0/6 Sentinel
Control
BNT162b2 V9
Pre 1 3 6 10/EOP1021041061081010Nasal swab
Day relative to challenge
0/3 0/3 0/6 0/3 2/3 5/6 0/3 0/3 0/6 0/3 1/3 0/6 0/3 2/3 0/6
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Page 22Rhesus macaques were challen ged by the intranasal and intratracheal routes with 1.05 × 106plaque forming
units of SARS -CoV -2.Viral RNA levels were detected by RT -qPCR. a, Viral RNA in bronchoalveolar lavage
(BAL) fluid. b, Viral RNA in nasal swabs. c, Viral RNA in OP sw abs. Ratios above data points indicate the
number of viral RNA positive animals among all animals per group. Dotted lines indicate the low er limits of
detection (LLOD). 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 t he 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.
The control animals responded to infectious virus challenge with an increase in S1 -binding
IgG and SARS -CoV -2 neutralizing ti ters; however, there was no trend toward increasing IgG
levels or SARS -CoV -2 neutralizing titers in response to viral challenge in the
BNT162b2 -immunized animals, providing further evidence that the immuni zation
suppressed SARS -CoV -2 infection ( Figure 6).
Figure 6.S1-binding IgG and 50% Serum Neutralization Responses in Rhesus
Macaques after Infectious SARS -CoV- 2 Challenge
S1-binding IgG concentrations (panels A & C) and 50% serum neutralization titers (panels B & D) were
obtained just prior to challenge (Pre) and 3, 6, and either end of project (EOP ; Days 7/8 for
BNT162b2 -immunized ) or 10 days after challenge (Control anima ls).Each line represents the kinetics of the
response of an individual rhesus macaque that was either immunized twice with 100 µg of BNT162b2 V9
(n=6, blue) or Control ( saline )(n=3, gray). All animals were challenged by the intranasal and intratracheal
routes with 1.05 × 106plaque forming units of SARS -CoV -2. Horizontal dotted line represents theLLOQ.
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Page 23Despite 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 7).
Lung radiograph ( Figure 8A) and computerized tomograph y (CT) (Figure 8B) scores were
determined b y twoboard -certif ied veterinary radiologist who w ereblinded to treatment
group. Data in Figure 8represent the average of the two scores. Radiographic evidence of
pulmonary abnormalit y was observed in challenged control s but not inchallenged
BNT162b2 -immunized animals nor in unchallenged sentinels . No radiographic evidence of
vaccine -elicited enhanced disease was observed.
Figure 7.Clinical Signs in R hesus Macaques after Immu nization with B NT162b2 and
Challenge with I nfectious SARS -CoV -2.
Rhesus macaques were imm unised with BNT162b2 (V9), or saline, and challenged with SARS -CoV -2 or cell
culture medium as described in the Fig ure5legend. Clinical signs were recorded on the days indicated.
EOP, end of project. BNT162b2 -immuni zed(n=6), control (n=3), and sentinel (n=3) macaques. A, Body weight
change . B,Temperature change. C , Oxygen saturation (SpO 2). D,Heart rate.
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Page 24Figure 8.Radiograph and CT Sc ores of Rhesus Macaque Lungs after Infectious
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 macaques 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 in dicate the
standard deviation .
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Page 25At necrops y on Day 7or 8 after virus challenge, there were no significant gross pathology
findings in an y organs. Microscopicall y, the main finding in the lung was inflammation. The
lung inflammation area score was similar between saline -immunized and BNT162b2 -
immunized animals, and there was no evidence of enhanced respiratory disease.
Inflammatory cell infiltrates included macrophages, neutrophils, ly mphocy tes, plasma cells ,
and some eosinophils. There were no other sign
ificant microscopic findings in other tissues.
Figure 9.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 represents 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).
5.CONCLUSION
We demonstrate that BNT162b2 (V9) , an LNP -formulated ,m1Ψ nucleoside -modified
mRNA encoding SARS -CoV -2 S captured in a pref usion conformation is highl y
immunogenic in rhesus macaques .
The immunogenicit y of BNT162b2 in rhesus macaques paralleled its immunogenicity in
mice. Seven day s after Dose 2 of 100 µg, the neutralizing GMT reached 18 -times that of a
human SARS -CoV -2 conval escent serum panel remained 3.3 -times higher than this
benchmark five weeks after the last immuni
zation. The strongly T h1-biased CD4+T cell
response and IFN+CD8+T-cell response to BNT162b2 is a pattern favoured for vaccine
safet y and efficacy, providing added reassurance for clinical translation.13BNT162b2
protected 2- 4 year old rhesus macaques from infectious SARS -CoV -2 challenge, with
reduced detection of viral RNA in immunized animals compared to those that received saline
and with no radiological , microscopic, or clinical evidence of exacerbation. Strong RT-qPCR
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Page 26evidence for lower respiratory tract protection was demonstrated by the absence of detectable
SARS -CoV -2 RNA in serial BAL samples obtained starting 3 day s after challenge of
BNT162b2 -immunized rhesus macaques.
6.DEVIATIONS
Not applicable.
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Page 2712. Mehra S, Alvarez X, Didier PJ, et al. Granuloma correlates of protection against
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PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge
in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 288.APPENDIX
8.1. SARS -CoV -2 Neutralizing Titers and Anti -S1 IgG Levels Elicited b y BNT162b2
(V9) Immunization of Rhesus Macaques ................................ ................. 28
8.2. Frequencies of Cy tokine Expressing CD4 and CD8 T cells Measured by ICS................ 29
8.3. Frequencies of Cy tokine Secreting Cells Measured by ELISpot ................................ ......29
8.4. Frequencies of Cy tokine Expressing CD4 and CD8 T cells Measured by ICS................ 30
8.5. Challenge Stud y Design ................................ ................................ ................................ ....31
8.1.SARS -CoV -2 Neutralizing Titers and Anti -S1 IgG Levels Elicited by BNT162b2
(V9) Immunization of Rhesus Macaques
NT50 Geometric Mean Titer (GMT)
(95% CI)Anti S1 IgG Geometric Mean Concentration
(GMC) U/mL (95% CI)
Day Control 30 µg
BNT162b2100 µg
BNT162b2Control 30 µg
BNT162b2100 µg
BNT162b2
0 10
(10, 10)10
(10, 10)10
(10, 10)0.8
(0.5, 1.0)0.9
(0.4, 2)1.0
(0.4, 5)
14 10
(10,10)47
(31, 73)54
(35, 82)0.8
(0.5, 1.0)2,143
(1186, 3874)3,917
(2190 , 7006 )
21 11.3
(8.2, 15.6)65
(40,104)81
(56, 118)0.6
(0.6, 0.6)1,921
(1180, 3126)2,698
(1475, 4936)
28 10
(10,10)809
(462, 1415)1689
(1068, 2673)0.6
(0.6, 0.6)30,339
(15690, 58665)34,668
(21650, 55514)
35 10
(10,10)962
(529, 1750)1277
(821, 1986)0.8
(0.5, 1)14,978
(6975, 32163)18,603
(11624, 29775)
42 10
(10,10)637
(356, 1141)1007
(675, 1504)0.8
(0.5, 1)10,288
(5418, 19533)12,879
(7840, 21155)
56 10
(10,10)285
(136, 598)310
(175, 549)No data
available4,236
(1380, 13003)6,317
(3877, 10291)
HCS 94 631
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
FDA-CBER-2021-5683-0709214
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 298.2. F requencies of Cytokine Expressing CD4 and CD8 T cells Measured by ICS
CD4+ IFN -γ
(% of CD4 T cells)CD4+ IL-4
(% of CD4 T cells)CD4+ IFN -γ/IL-2/TNF -α
(% of CD4 T cells)CD8+ IFN -γ
(% of CD8 T cells)
Day Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2
0 0.001 ±
0.00060.001 ±
0.00110.000 ±
0.00040.001 ±
0.00040.000 ±
0.00010.000 ±
0.00000.013 ±
0.00530.013 ±
0.00170.003 ±
0.00230.005 ±
0.00230.003 ±
0.00150.002 ±
0.0014
14 0.001 ±
0.00040.006 ±
0.00340.010 ±
0.00360.000 ±
0.00010.001 ±
0.00060.004 ±
0.00120.011 ±
0.00150.128 ±
0.02890.137 ±
0.04160.004 ±
0.00230.009 ±
0.00720.028 ±
0.0179
28 NT 0.078 ±
0.01440.110 ±
0.0287NT 0.017 ±
0.00330.036 ±
0.0070NT 0.470 ±
0.08080.529 ±
0.1107NT 0.033 ±
0.01010.032 ±
0.0156
42 0.001 ±
0.00070.045 ±
0.00920.080 ±
0.01830.001 ±
0.00050.011 ±
0.00310.020 ±
0.00510.014 ±
0.00380.262 ±
0.04430.339 ±
0.05280.009 ±
0.00380.023 ±
0.01030.047 ±
0.0257
NT, not tested
Values reported are m ean ± standard error of the mean ( SEM )of 6 animals within each group
8.3.Frequencies of Cytokine Secreting Cells Measured by ELISpot
IFNγ SFC/106PBMCs (Mean±SEM) IL-4 SFC/106PBMCs (Mean±SEM)
Day Control 30μg BNT162b2 100μg BNT162b2 Control 30μg BNT162b2 100μg BNT162b2
0 41±6 35±0 35±0 5±1 5±1 4±0
14 35±0 159±92 88±27 4±0 16±6 7±2
28 NT 947±472 765±151 NT 202±90 179±32
42 35±0 710±227 850±202 4±0 154±54 121±32
PBMCs, peripheral blood mononuclear cells; SEM, standard error of the mean; NT, not tested
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
FDA-CBER-2021-5683-0709215
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 308.4. F requencies of Cytokine Expressing CD4 and CD8 T cells Measured by ICS
CD4+ IFN -γ
(% of CD4 T cells)CD4+ IL-4
(% of CD4 T cells)CD4+ IFN -γ/IL-2/TNF -α
(% of CD4 T cells)CD8+ IFN -γ
(% of CD8 T cells)
Day Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2Control 30μg
BNT162b2100μg
BNT162b2
0 0.001 ±
0.00060.001 ±
0.00110.000 ±
0.00040.001 ±
0.00040.000 ±
0.00010.000 ±
0.00000.013 ±
0.00530.013 ±
0.00170.003 ±
0.00230.005 ±
0.00230.003 ±
0.00150.002 ±
0.0014
14 0.001 ±
0.00040.006 ±
0.00340.010 ±
0.00360.000 ±
0.00010.001 ±
0.00060.004 ±
0.00120.011 ±
0.00150.128 ±
0.02890.137 ±
0.04160.004 ±
0.00230.009 ±
0.00720.028 ±
0.0179
28 NT 0.078 ±
0.01440.110 ±
0.0287NT 0.017 ±
0.00330.036 ±
0.0070NT 0.470 ±
0.08080.529 ±
0.1107NT 0.033 ±
0.01010.032 ±
0.0156
42 0.001 ±
0.00070.045 ±
0.00920.080 ±
0.01830.001 ±
0.00050.011 ±
0.00310.020 ±
0.00510.014 ±
0.00380.262 ±
0.04430.339 ±
0.05280.009 ±
0.00380.023 ±
0.01030.047 ±
0.0257
NT, not tested
Values reported are m ean ± standard error of the mean ( SEM )of 6 animals within each group
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
FDA-CBER-2021-5683-0709216
PF-07302048: BNT162b2 (V9) Immunogenicity and Evaluation of Protection against SARS -CoV- 2 Challenge in Rhesus Macaques
VR-VTR -10671, Ver. 2.0
PFIZER CONFIDENTIAL
Page 318.5.Challenge Study Design
Challenge
GroupAnimal
IDImmunization DOB Serum
collection
relative to
immunizationPre challenge
serum
collection
week relative
to first
immunizationSample collections relative to challenge Necropsy
Day
(post
challenge)
Nasal, Oral,
Rectal
SwabChest X -ray Chest
CTBAL Serum
BNT162b2 A17N114 BNT162b2
100 µg5/19/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/7 pre/1/3/6/7 pre/3/7 pre/3/6/7 pre/3/6/7 7
A17N113 BNT162b2
100 µg5/19/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/7 pre/1/3/6/7 pre/3/7 pre/3/6/7 pre/3/6/7 7
A17N139 BNT162b2
100 µg6/1/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/7 pre/1/3/6/7 pre/3/7 pre/3/6/7 pre/3/6/7 7
A17N167 BNT162b2
100 µg6/14/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/7 pre/1/3/6/7 pre/3/7 pre/3/6/7 pre/3/6/7 7
A17N105 BNT162b2
100 µg5/18/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/8 pre/1/3/6/8 pre/3/8 pre/3/6/8 pre/3/6/8 8
A17N109 BNT162b2
100 µg5/19/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/8 pre/1/3/6/8 pre/3/8 pre/3/6/8 pre/3/6/8 8
Control A17N118 Saline 5/22/2017 Pre, 6h, 24h,
W1, 2, 3 6 pre/1/3/6/10 pre/1/3/6/10 pre/3/10 pre/3/6 pre/3/6/10 not
necropsied
A17N157 Saline 6/12/2017 Pre, 6h, 24h,
W1, 2, 3 6 pre/1/3/6/10 pre/1/3/6/10 pre/3/10 pre/3/6 pre/3/6/10
A17N128 Saline 5/29/2017 Pre, 6h, 24h,
W1, 2, 3 6 pre/1/3/6/10 pre/1/3/6/10 pre/3/10 pre/3/6 pre/3/6/10
Sentinel A17N125 BNT162b2
30 µg5/27/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/10 pre/1/3/6/10 10 pre/3/6 pre/3/6/10
A17N107 BNT162b2
30 µg5/18/2017 Pre, 6h, 24h,
W1, 2, 3, 4, 5, 6,
8 10 pre/1/3/6/10 pre/1/3/6/10 10 pre/3/6 pre/3/6/10
090177e1959904f1\Approved\Approved On: 23-Nov-2020 23:06 (GMT)
FDA-CBER-2021-5683-0709217
Document Approval Record
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FDA-CBER-2021-5683-0709218