Document text
BioNTec h SE
An der Goldgrube 12
55131 Mainz, Germany
Phone: +49 (0)6131 9084 -0
Telefax: +49 (0)6131 9084 -390
R&D STUDY REPORT N o. R-20-0085
COVID -19: IMMUNOGENI CITY STUDY OF THE LNP-
FORMULATED MODRNA EN CODING THE VIRAL
S PROTEIN -V9
Version 04
Date: 23 NOV 2020
Reported by
Test i tem: BNT162b2 ( animal trial material )
Key words: Coronavirus, COVID -19, modRNA, ATM, mouse, immunogenicity
This R&D report consists of 93 pages.
Confidentiality Statement: The information contained in this document is the property and copyright of
BioNTech RNA Pharmaceuticals GmbH . Therefore, this document is provided in confidence to the recipient (e.g. ,
regulatory authorities, IECs/IRBs, investigators, auditors, inspectors). No information contained herein shall be
published, disclosed , or reproduced without prior written approval of the proprietors.
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TABLE OF CONTENTS
TABLE OF CONTENTS ................................ ................................ ........................ 2
LIST OF FIGURES ................................ ................................ ................................ 3
LIST OF TABLES ................................ ................................ ................................ .. 4
LIST OF ABBREVIATION S ................................ ................................ ................... 5
RESPONSIBILITIES ................................ ................................ .............................. 6
1 SUMMARY ................................ ................................ ........................... 7
2 GENERAL INFORMATION ................................ ................................ .. 8
2.1 Participating Personnel ................................ ................................ ......... 8
2.2 Study Dates ................................ ................................ .......................... 9
2.3 Guidelines and Regulations ................................ ................................ 10
2.4 Changes and Deviations ................................ ................................ ..... 10
2.5 Documentation and Archive ................................ ................................ 11
3 INTRODUCTION ................................ ................................ ................ 12
3.1 Background ................................ ................................ ........................ 12
3.2 Objectives ................................ ................................ ........................... 13
3.3 Study Design ................................ ................................ ...................... 13
4 MATERIALS AND METHOD S ................................ ............................ 15
4.1 Test Item ................................ ................................ ............................. 15
4.2 Control Item ................................ ................................ ........................ 15
4.3 Test System ................................ ................................ ........................ 15
4.4 Materials ................................ ................................ ............................. 15
4.5 Methods ................................ ................................ .............................. 21
4.5.1 ANIMAL CARE ................................ ................................ ................... 21
4.5.1.1 GENERAL INFORMATION ................................ ................................ 21
4.5.1.2 HOUSING CONDITIONS A ND HUSBANDRY ................................ .... 21
4.5.2 ANIMAL MON ITORING ................................ ................................ ...... 21
4.5.3 ANIMAL TREATMENT ................................ ................................ ....... 22
4.5.3.1 TREATMENT SCHEDULE, ROUTE OF ADMINISTRAT ION, AND
DOSE ................................ ................................ ................................ . 22
4.5.3.2 IMMUNIZATION ................................ ................................ ................. 22
4.5.3.3 BLOOD SAMPLING VIA T HE RETRO -ORBITAL VEN OUS PLEXUS
OR VENA FACIALIS ................................ ................................ ........... 22
4.5.4 ENDPOINT OF EXPERIME NT/TERMINATION CRITE RIA ................ 23
4.5.4.1 DISSECTION OF ANIMAL S AND ORGAN COLLECTI ON ................. 23
4.5.5 ELISA ................................ ................................ ................................ . 23
4.5.6 SURFACE PLASMON RESO NANCE SPECTROSCOPY .................. 24
4.5.7 PSEUDOVIRUS -BASED NE UTRALIZATION TEST .......................... 24
4.5.7.1 PRODUCTION OF SARS -COV-2-S PSEUDOTYPED V SV
VECTOR ................................ ................................ ............................. 24
4.5.7.2 TITRATION OF VSV/SAR S-COV -2-S PSEUDOVIRU S ..................... 25
4.5.7.3 PSEUDOVIRUS -BASED NE UTRALIZATION TEST .......................... 25
4.5.8 PREPARATION OF SPLEN OCYTES ................................ ................. 26
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4.5.9 ELISPOT ASSAY ................................ ................................ ................ 27
4.5.9.1 SUBTYPING OF CD8+ VERSUS CD4+ T-CELL RESPONSES .......... 27
4.5.10 LUMINEX ASSAY ................................ ................................ ............... 28
4.5.11 INTRACELLULAR CYTOKI NE STAINING ................................ ......... 28
4.5.12 STATISTICAL ANALYSIS ................................ ................................ .. 29
5 RESULTS ................................ ................................ ........................... 30
5.1 ELISA ................................ ................................ ................................ . 30
5.1.1 WHOLE IGG ELISA ................................ ................................ ............ 30
5.1.2 IGG SUBTYPE -SPECIFIC ELISA ................................ ....................... 35
5.1.3 IGG2A/IGG1 RATIO ................................ ................................ ........... 37
5.2 Binding Kinetics of Antigen -specific IgGs Using SPR ......................... 37
5.3 Pseudovirus -based Neutralization Test ................................ .............. 39
5.4 ELISpot Analysis ................................ ................................ ................. 40
5.5 Luminex Assay ................................ ................................ ................... 43
5.6 Intracellular Cytokine Staining ................................ ............................ 46
5.7 Animal Monitoring ................................ ................................ ............... 49
6 CONCLUSION ................................ ................................ .................... 51
7 DOCUMENT HISTORY ................................ ................................ ...... 52
8 REFERENCES ................................ ................................ ................... 53
9 APPENDIX ................................ ................................ ......................... 54
APPENDIX 1: ANIMAL M ONITORING - OBSERVAT IONS ................................ . 54
APPENDIX 2: CERTIFICATES OF ANAL YSIS ................................ ................... 58
APPENDIX 3: CONTROLS FOR ELISPOT ANALYSI S ................................ ...... 62
APPENDIX 4: SUMMARY OF LUMINEX ASSAY DAT A ................................ ..... 63
APPENDIX 5: DETAILED ICS PROTOCOL ................................ ........................ 65
APPENDIX 6: STATISTI CAL ANALYSIS ................................ ............................ 71
LIST OF FIGURES
Figure 1: Schematic overview of the S protein organization of the SARS -CoV-2
S protein. ................................ ................................ ................................ ............. 12
Figure 2: ELISA screening analysis on days 7, 14, and 21 against the recombinant
S1 protein ................................ ................................ ................................ ............ 30
Figure 3: ELISA screening analysis on days 7, 14, and 21 against the recombinant
RBD ................................ ................................ ................................ ..................... 31
Figure 4: ELISA endpoint titration on day 28 ................................ ....................... 32
Figure 5: Kinetics of the antibody concentration against the viral antigen ........... 33
Figure 6: ELISA endpoint titration (long titration) ................................ ................. 34
Figure 7: Reciprocal serum endpoint titer at day 14 and 28 after immunization .. 35
Figure 8: IgG subtype -specific ELISA on day 28 ................................ ................. 36
Figure 9: ELISA endpoint titration on day 28 (IgG subtypes) ............................... 36
Figure 10: IgG2a/IgG1 subtype ratio on day 28 ................................ ................... 37
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Figure 11: Binding affinities of vaccine -elicited IgG for S1 -His and RBD -His protein
................................ ................................ ................................ ............................ 38
Figure 12: Titers of neutralizing antibodies on days 14, 21, and 28 ..................... 40
Figure 13: ELISpot analysis using fresh splenocytes on day 28 .......................... 41
Figure 14: ELISpot analysis using frozen splenocytes on day 28 ........................ 42
Figure 15: ELISpot analysis using splenocytes of 5 µg BNT162b2 (RBP020.2)
immunized mice on day 28 after MACS cell separation ................................ ...... 43
Figure 16: Cytokine concentrations in supernatants of re -stimulated splenocytes
28 days after immunization ................................ ................................ .................. 45
Figure 17: CD4+ T cell intracellular cytokine staining 28 days after
immunization ................................ ................................ ................................ ....... 47
Figure 18: CD8+ T cell intracellular cytokine staining 28 days after
immunization ................................ ................................ ................................ ....... 48
Figure 19: Body weights of experimental mice during study ................................ 49
Figure 20: Summary of observations made during study’s concomitant animal
monito ring ................................ ................................ ................................ ............ 50
Figure 21: Controls for ELISpot analysis using splenocytes on day 28 ............... 62
Figure 22: T H1 and proinflammatory cytokine concentrations in supernatants of re -
stimulated splenocytes 28 days after immunization ................................ ............. 63
Figure 23: T H2 cytokine and IL -2 concentrations in supernatants of re -stimulated
splenocytes 28 days after immunization ................................ .............................. 64
LIST OF TABLES
Table 1: Study design ................................ ................................ .......................... 14
Table 2: Materials ................................ ................................ ................................ 15
Table 3: Equipment ................................ ................................ ............................. 19
Table 4: Software ................................ ................................ ................................ 20
Table 5: Peptide pools for stimulation of splenocytes for ELISpot assays ........... 20
Table 6: Summary of binding kinetic parameters of vaccin e-elicited IgG for S1 -
His ................................ ................................ ................................ ....................... 38
Table 7: Summary of binding kinetic parameters of vaccine -elicited IgG for RBD -
His ................................ ................................ ................................ ....................... 39
Table 8: Chemokines and cytokines included for multiplex measurement .......... 43
Table 9: Parameters for experimental animal monitoring (single mouse
assessment) ................................ ................................ ................................ ........ 54
Table 10: Record of body weights of experimental mice during study ................. 56
Table 11: Record of animal monitoring for each mouse during study .................. 57
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LIST OF ABBREVIATIONS
AH-1 Irrelevant peptide derived from endogenous retroviral gene product envelope
glycoprotein 70
ATM Animal trial material
BCS Body Conditioning Score
BNT162 BioNTech’s SARS -CoV-2 vaccine candidate
CD Cluster of differentiation
ConA Concanavalin A
COVID -19 Coronavirus disease emerged 2019
DMSO Dimethyl sulfoxide
DPBS Dulbecco ’s phosphate -buffered saline
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme -linked immunosorbent assay
ELISpot Enzyme -linked immune absorbent spot
FBS Fetal bovine serum
GFP Green fluorescent protein
GM-CSF Granulocyte -macrophage colony -stimulating factor
GMP Good manufacturing practice
Hsopt10 Nucleoside optimization protocol 10 based on Homo sapiens databank
ICS Intracellular cytokine staining
IFN Interferon
Ig Immunoglobulin
IL Interleukin
i.m.
KD
koff
kon Intramuscularly
Binding affinity
Dissociation rate constant (off-rate)
Association rate constant (on -rate)
LNP Lipid nanoparticle
LLOQ Lower limit of quantification
MACS Magnetic cell separation
modRNA Nucleoside -modified mRNA
nAb Neutralizing antibody
No. Number
OD Optical density
PBS Phosphate -buffered saline
PMA Phorbol 12 -myristate 13 -acetate
pVNT Pseudovirus -based neutralization test
RBD Receptor -binding domain
RNA Ribonucleic acid
S protein Spike protein
S1 Subdomain 1 of the S protein
S2 Subdomain 2 of the S protein
saRNA Self-amplifying mRNA
SARS -CoV-2 Severe acute respiratory syndrome coronavirus -2
SPR Surface plasmon resonance
TH1/T H2 Type 1/2 helper T cells
TNF Tumor necrosis factor
ULOQ Upper limit of quantification
uRNA Uridine -containing mRNA
V Variant
VSV Vesicular stomatitis virus
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2 GENERAL INFORMATION
Sponsor
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Germany
Test Facility
BioNTech SE
An der Goldgrube 12
55131 Mainz
Germany
2.1 Participating Personnel
Responsible p erson:
(as defined in SOP -100-024)
An der Goldgrube 12
55131 Mainz
Author:
BioNTech SE
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech SE
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Experimenter:
BioNTech SE
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech SE
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech Diagnostics GmbH
Experimenter:
BioNTech Diagnostics GmbH
Experimenter:
BioNTec h RNA Pharmaceuticals GmbH
2.2 Study Dates
Start of experiments: 31 MAR 2020
Completion of experiments: 17 SEP 2020
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2.3 Guidelines and Regulations
All experiments are executed in accordance with the existing standard operating
procedures and described processes from BioNTech SE. Applicable documents are
listed below.
Animal test application approval number: G18 -12-100, amendment from
18.02.2020 (approved 20 FEB 2020)
SOP-010-017 Brutschränke - Biolytics
SOP -010-028 Vi-Cell XR
SOP -010-045 Brutschrank HERAcell 150 i
SOP -010-047 Zentrifuge Eppendorf 5810/5810R
SOP -010-051 Tiefkühlschränke -80 °C
SOP -010-058 Sicherheitswerkbank Klasse II
SOP -010-086 Zentrifuge Thermo Scientific Heraeus Pico und Fresco 17
SOP -010-099 CTL ELISPOT Reader
SOP -020-009 Ansetzen von Medien und Zusätzen für die Zellkultur
SOP -030-043 Kryokonservierung von Zellen
SOP -030-071 Abtöten von Mäusen
SOP -030-072 Fixiergriff und Ohrmar kierung bei Mäusen
SOP -030-073 Betäubung bei Mäusen
SOP -030-074 Blutentnahme bei Mäusen
SOP -030-078 Isolierung muriner Splenozyten
SOP -030-079 Intramuskuläre A pplikation bei Mäusen
SOP -030-110 IFN γ ELISpot (murin )
SOP -030-112 Durchführung eines virusprotein -spezifischen ELISA
SOP -090-013 Biological safety in laboratories
SOP -110-022 Entsorgung von Biostoffabfällen
2.4 Changes and Deviations
This R&D study was conducted according to R&D plan P -20-0085.
A change occu rred in the pVNT. It was planne d to perform this analysis with an external
partner, . However, the CRO had no pVNT or VNT in place when samples
were ready to analyze. Therefore, an internal assay was developed using the VSV -
based pseudovirus to analyze for neutralizing anti bodies.
Furthermore vaccine -induced SARS -CoV-2 specific antibodies were analyzed for their
affinity toward recombinant SARS -CoV-2 S and RBD protein via surface plasmon
resonance (SPR) spectroscopy . Affinity measurements were only conducted with day
28 sera of the 5 µg BNT162b2 dose cohort.
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Another change occurred in the protocol for murine ELISpot, described within SOP -
030-110. The described change resulted in faster dryness of the ELISpot plate and
thus its readiness for the subsequent protocol step; analy sis of spot numbers per well
via ImmunoSpot® S6 Core Analyzer, CTL. This change has no impact on performance
of the protocol.
Furthermore, in a first run with fresh splenocytes a mis calculation of cells in the group
immunized with 5 µg modRNA occurred. Therefore , a second ELISpot run was
included with frozen splenocytes .
Because the utilized major histocompatibility complex (MHC ) I/II blockade was not
effective in determining T -cell subtypes, an additional ELISpot analysis was performed
after separation of CD4+ and CD8+ cells by MACS isolation to identify the responding
T-cell subtype (group 4 only).
Cytokine concentrations in sup ernatants of re -stimulated splenocytes were determined
using a bead -based, T H1/T H2 mouse ProcartaPlex immunoassay. An intracellular
cytokine staining was added for TH1/T H2 cytokine analysis.
2.5 Documentation and Archive
Study plans and reports are stored and archived according to SOP -100-003 Archiving
of Paper -Based Documents.
Raw data and evaluated data are saved at :
P:\BioNTechRNA \RN9391R00_ CoV-VAC\04_Preclinic \00_Pharmacology \
mCorVAC# 11_modRNA -V9
Animal Models & Facility: Lab book No. 1893
Infectious Disea se Vaccines (ELISA): Lab book No. 1858 , 1978
Infectious Disease Vaccines (ICS) : Lab book No. 1937
Immunomodulators: Lab book No. 1935, 1936
Cancer Vaccines: Lab book No. 1934
New Scaffolds: Lab book No. 2009
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3 INTRODUCTION
3.1 Background
In December 2019, an outbreak of pneumonia of unknown cause in Wuhan, Hubei
province in China was reported . The disease spread rapidly and in January 2020, the
agent was identified. By 21 June 2020, infection with the novel coronavirus ( SARS -
CoV-2) was con firmed in over 8,700,000 people with more than 460,000 casualties1.
A vaccine is urgently needed and B ioNTech decided to develop a rapid vaccine project
based on the surface or spike protein (S protein) of the virus as the viral antigen. The
S protein is a trimer and during viral egress, the precursor protein is cleaved in S1 and
S2 (Figure 1). While the S1 domain recognizes the host receptor, the S2 domain is
essential for the membrane fusion of viral envelope and endosomal membrane. To
initiate the membrane fusion, the S2 domain undergoes a conformational change
within the central helix domain.
Figure 1: Schematic overview of the S protein organization of the SARS -CoV-2 S protein.
The sequence within the S1 subunit consists of the signal sequence (SS) and the receptor -binding domain (RBD)
which is the key subunit within the S protein which is relevant for binding to the huma n cellular receptor ACE2. The
S2 subunit contains the S2 protease cleavage site (S2’) followed by a fusion peptide (FP) for membrane fusion,
heptad repeats (HR1 and HR2) with a central helix (CH) domain, the transmembrane domain (TM) and a
cytoplasmic tail (CT); source: modified from ( Wrapp et al. 2020 ).
Based on these features, the S protein is the target of the neutralizing antibody (nAb)
that binds dominantly to the RBD of the S protein. V accine candidates selected for
non-clinical testing include the following vaccine antigens:
A secreted variant of the RBD of the SARS -CoV-2 S protein (called V5)
(Kirchdoerfer et al. 2018 )
Membrane -tethered full -length S protein with two point mutations within the
central helix domain (called V8/V9). Mutation of the two amino acids to proline,
(KV286 -287PP) retains the S protein in an antigenically optimal prefusion
conformation (called V8 or V9) ( Wrapp et al. 2020 , Pallesen et al. 2017 )
1 Coronavirus diseas e (COVID -2019) situation report 153, World Health Organization;
www.who.int/emergencies/diseases/novel -coronavirus -2019/situation -reports
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The development of in vitro transcribed RNA as an active platform for the use in
infectious disease vaccines is based on the extensive knowledge of the com pany in
RNA technology, which has been gained over the last decade. The core innovation is
based on in vivo delivery of a pharmacologically optimized, antigen -coding RNA
vaccine to induce robust neutralizing antibodies and accompanying/concomitant T cell
response to achieve protective immunization with minimal vaccine doses
(Vogel et al. 2017 , Moyo et al. 2018 , Pardi et al. 2017 ).
At BioNTech, there are three different RNA platforms under development, namely non-
modified uridine -containing mRNA (uRNA), nucleoside -modified mRNA (modRNA) ,
and self -amplifying RNA (saRNA). It is unknown today which RNA vaccine platform
performs best in terms of activation and duration of a pote nt immune response.
Therefore, BioNTech has developed a project plan that is based on testing GMP -
produced, available material that has already been tested in clinical trials. The three
vaccine platforms will be tested for each antigen construct in non -clinical mouse
studies and tested for their virus -neutralizing response and the total amount of IgG
antibodies developed against the S protein. Candidates that induce a high fraction of
nAb within the total IgG population are desired. This repor t covers a mou se study
testing mod RNA encoding the antigen variant 9 (V9) of the generated variants of the
S protein.
3.2 Objectives
In this study, the primary objective was to understand the immunogenicity of the
designed construct. For this purpose, a dose titration in BA LB/c mice was performed
with the LNP -formulated modRNA encoding the antigen variant 9 (V9) of the generated
variants of the S protein ( V9 main characteristics: S protein full -length with two point
mutations, opt1 sequence optimization that increases the GC -content of the coding
sequence ). The immune response was analyzed focusing on the antibody immune
response and included the analysis of the IFN - release of splenocytes at the end of
study as well as assessment of cytokine/chemokine responses .
3.3 Study Design
Four groups of eight female BALB/c mice were immunized once (on day 0) with
BNT162 b2 at three different doses, or with the buffer alone (control group).
Immunizations were given intramuscularly (i.m.) in a dose volume of 20 µL. Blood was
collected o nce weekly for three weeks (days 7, 14, and 21) to analyze the antibody
immune response by ELISA and pseudovirus -based neutralization assay (pVNT). At
the end of the study (on day 28), blood was collected for ELISA and pVNT analyses
(all samples), as well as for affinity measurements of vaccine -induced antibodies
toward recombinant SARS -CoV-2 S and RBD via SPR (high -dose cohort samples
only). Animals were then euthanized for spleen collection and additional analysis of
the T -cell response in splenocytes by ELISpot, Luminex assay , and ICS (see Table 1).
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Table 1: Study design
Group
no. No. of
animals Vaccine /
batch Concentrati
on of active
component
[µg/animal] Immunization
day Dose
volume
[µL] /
route Blood
collection
day End
of
study
day
1 8 Buffer - 0 20 / i.m. 7, 14, 21,
28 28
2 8 BNT162b2
/RBP 020.2 0.2 0 20 / i.m. 7, 14, 21,
28 28
3 8 BNT162b2
/RBP 020.2 1 0 20 / i.m. 7, 14, 21,
28 28
4 8 BNT162b2
/RBP 020.2 5 0 20 / i.m. 7, 14, 21,
28 28
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4 MATERIALS AND METHODS
4.1 Test Item
BNT162 b2, animal trial material (ATM): For CoAs see Appendix 2: Certificates of
Analysis .
RNA batch : RNA -RF2003 21-06
Polymun batch RB P020.2 LNP with the lot: CoVVAC/ 270320
4.2 Control Item
PBS+300 mM sucrose (from Polymun)
4.3 Test System
32 female BALB/c mice at an age of 9 weeks at stud y start.
4.4 Materials
Table 2: Materials
Product name Application/
specification Article no. Working
dilution Provider
15 mL/50 mL tube Conical bottom, PP,
30/115 MM, CELLSTAR® 188271/
227261 N/A Greiner Bio -One
GmbH
2 mL tube CRYO.S, round bottom 122278 N/A Greiner Bio -One
GmbH
2-Mercaptoethanol 50 mM 31350 -010 N/A Gibco
8-channel manifold Polypropylene BR704526 -
1EA N/A Sigma -Aldrich
Chemie GmbH
96-well flat -bottom
plate pVNT 655160 N/A Greiner
96-well microplate Clear round bottom TC -
treated microplate, with
lid, sterile 3799 N/A Corning Holding
GmbH
96-well V -bottom plate pVNT 651180 N/A Greiner
AffiniPure goat anti-
mouse IgG SPR 115-005-071 N/A Jackson
ImmunoResearch
Alexa Fluor® 488 anti -
mouse TNF -
antibody, clone MP6 -
XT22 ICS 506313 1:100 BioLegend
Amine coupling kit SPR BR100050 N/A GE Healthcare
Ammonium chloride NH 4Cl A0988,5000 N/A AppliChem GmbH
Anti-rat/hamster Ig,
κ/negative control
(FBS*) Compensation Particles
Set 552845
component
no. 51-90-
9000949 1 drop BD
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Product name Application/
specification Article no. Working
dilution Provider
Anti-VSV-G antibody Clone 8G5F11 EB0010 N/A Kerafast
APC anti -mouse IL -2
antibody ICS 503810 1:100 BioLegend
BD Pharmingen™
purified rat anti-mouse
CD16/CD32 Mouse BD Fc Block™
(2.4G2) 553142 1:100 BD
Blocker™ bovine
serum albumin ( BSA)
in PBS (10×) ICS 7011150 1× ThermoFisher
Brilliant Violet 510™
anti-mouse CD4
antibody ICS 100559 1:200 BioLegend
Brilliant Violet 711™
anti-mouse IL -4
antibody ICS 504133 1:200 BioLegend
Brilliant Violet 785™
anti-mouse CD25
antibody PC61 ICS 102051 1:200 BioLegend
BV421 rat anti -mouse
CD8a antibody ICS 100753 1:200 BioLegend
Capillary pipettes minicaps®, blood
sampling, 4 µL/10 µL, not
heparinized 9000104/
9000110 N/A Hirschmann
Laborgeräte
GmbH & Co.KG
Casein blocking buffer
10× ELISA B6429 -
500ml N/A Sigma -Aldrich
Chemie GmbH
CM5 sensor chip SPR BR100012 N/A GE Healthcare
Combitips advanced® Biopur®, 50 mL 0030089693 N/A Eppendorf
Vertrieb
Deutschland
GmbH
Concanavalin A From Canavalia
ensiformis (Jack bean,
5 mg),Type IV -S,
lyophilized C0412 -5MG N/A Sigma -Aldrich
Chemie GmbH
Cover films ELISA RATI601841
0 N/A VWR International
GmbH
Dimethyl sulfoxide
(DMSO) For cell culture A3672,0100 N/A AppliChem GmbH
DPBS No calcium, no
magnesium 14190 -094 1 × Thermo Fisher
Scientific
Easystrainer 70 µm For 50 mL tubes 542070 N/A Greiner Bio -One
GmbH
eBioscience™ Fixable
Viability Dye eFluor™
780 ICS 65-0865 -18 1:1,000 ThermoFisher
Eppendorf safe -lock
tubes 0.5 mL/ 1.5 mL/ 2.0 mL/
5.0 mL, Eppendorf
Quality™ 0030121023
/003012008
6/00301200
94/0030119
401 N/A Eppendorf
Vertrieb
Deutschland
GmbH
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Product name Application/
specification Article no. Working
dilution Provider
Ethylenediaminetetraa
cetic acid solution EDTA 03690 -
100ML N/A Sigma -Aldrich
Chemie GmbH
Fetal bovine serum
(FBS) Non-USA origin, sterile -
filtered F7524 N/A Sigma -Aldrich
Chemie GmbH
Filtration unit for
medium flasks High Performance, PES,
0.45 µm, 1 ,000 mL 514-0301 N/A VWR International
GmbH
Goat anti -mouse IgG
(POX) Whole IgG Fc y fragment,
secondary antibody, IgG
isotype -specific ELISA 115-035-071 1:15,000 Jackson
ImmunoResearch
via Dianova
Goat anti-mouse IgG
HRP ELISA 115-035-071 1:15,000 Jackson
Immu noResearch
Goat anti -mouse IgG1
(HRP) IgG1 Fc y subtype -
specific , secondary
antibody, IgG isotype -
specific ELISA 115-035-205 1:5,000 Jackson
ImmunoResearch
via Dianova
Goat anti -mouse
IgG2 a (HRP) IgG2 a Fc y subtype -
specific secondary
antibody, IgG isotype -
specific ELISA 115-035-206 1:5,000 Jackson
ImmunoResearch
via Dianova
Goat anti-rabbit IgG
HRP ELISA A0545 -1ml 1:10,000 Sigma -Aldrich
GolgiPlug ICS 555029 1:1,000 BD
GolgiStop ICS 554724 1:1,500 BD
HBS-EP+ buffer 10× SPR BR100669 N/A GE Healthcare
HEPES 1 M 15630 -056 N/A Gibco
Human SARS
coronavirus spike S1
subunit antibody
Anti-COVID -19-S1
Isotype: rabbit IgG ELISA 40150 -RP01 S1:
1:1,000
RBD:
1:2,000 Sino Biological
Insulin syringes BD Micro -Fine™+, 30 G,
0.3 mL 324826 N/A Becton Dickinson
GmbH
Ionomycin ICS I9657 1 µg/m L Sigma
Isoflurane Anesthesia 9714675 N/A Piramal Critical
Care
Isotonic saline Injection solution 06173569 N/A Fresenius Kabi
Deutschland
GmbH
Lipofectamine® LTX &
PLUS™ Transfection reagent 15338 -100 N/A Invitrogen
MACS LS columns MACS 130-042-401 N/A Miltenyi Biotec
MACS® MicroBeads CD8a (Ly -2)/CD4 (L3T4) 130-117-
044/130-
117-043 N/A Miltenyi Biotec
MaxiSorp plate ELISA 439454 N/A Thermo Scientific
MEM non-essential
amino acids (NEAA)
solution 100× 11140 -035 1× Gibco
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Product name Application/
specification Article no. Working
dilution Provider
Mouse IFN -
ELISpotPLUS kit Kit for enumeration of
cells secreting mouse
IFN- 3321 -4APT -
2 N/A Mabtech
Mouse IgG1 -BIOT Clone 15H6, isotype
control for IgG-specific
ELISA 0102 -08 1:100 Southern Biotech
via Biozol
Mouse IgG2a -BIOT Clone HOPC -1, isotype
control for IgG -specific
ELISA 0103 -08 1:100 Southern Biotech
via Biozol
Mouse IgG -UNLB ELISA 0107 -01 Starting
dilution
1:300 Southern Biotech
PBS powder No calcium, no
magnesium L182 -10 N/A Merck KGaA
pcDNA3.1 -derived
expression plasmid VSV vector production V79020 N/A Invitrogen
PE hamster anti-
mouse CD3e clone
145-2C11 ICS 553064 1:200 BD
PE/Cy7 anti -mouse
IFN- antibody, clone
XMG1.2 ICS 505826 1:500 BioLegend
Penicillin -streptomycin 10,000 U/mL 15140 -122 N/A Gibco
Phosphate -buffered
saline (PBS),
powdered ELISA 0780 -10L N/A VWR International
GmbH
Pipette tips ep Dualfilter T.I.P.S.®,
PCR clean und sterile,
0.1–10 µL/2–100 µL/50 –
1,000 µL/50 –
1,250 µL/0.1 –5 mL 0030077512
/003007754
7/00300775
55/0030077
792/003007
7750/00300
78616 N/A Eppendorf
Vertrieb
Deutschland
GmbH
Phorbol 12 -myristate
13-acetate (PMA ) ICS P1585 0.5 µg/m
L Sigma
Potassium
bicarbonate KHCO 3 A2375,1000 N/A AppliChem GmbH
ProcartaPlex assay Bead -based, 11 -plex
TH1/T H2 mouse
immunoassay EPX110 -
20820 -901 N/A Thermo Fisher
Scientific
Recombinant RBD
protein
SARS -CoV-2 (2019 -
nCoV) spike protein
(RBD, Fc Tag) ELISA 40592 -V02H 100 ng/
100 µL SinoBiological
Reservoir 25 mL, 100 mL 613-
1174/613 -
1171 N/A VWR International
GmbH
Roti Histofix, 4%
formaldehyde ICS P087.4 2% Carl Roth GmbH
& Co. KG
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Product name Application/
specification Article no. Working
dilution Provider
RRPMI 1640 medium GlutaMAX™ supplement 61870 -010 N/A Gibco
SARS -CoV-2 (2019 -
nCoV) spike antibody,
rabbit Mab ELISA 40150 -R007 S1:
1:500
RBD:
1:1,000 Sino Biological
SARS -CoV-2 (2019 -
nCoV) spike RBD -Fc
recombinant protein ELISA 40592 -V02H 100 ng/
100 µL Sino Biological
SARS -CoV-2 (2019 -
nCoV) spike RBD -His
recombinant protein SPR 40592 -V08B N/A Sino Biological
SARS -CoV-2 (2019 -
nCoV) spike S1 -His
recombinant protein ELISA, SPR 40591 -V08H N/A Sino Biological
Serological pipettes 5 mL, 10 mL, 25 mL,
50 mL 606180/607
180/601180/
768180 N/A Greiner Bio -One
GmbH
Single -use syringe Injekt® Solo 5 mL 4606051V N/A B. Braun
Melsungen AG
Sodium bicarbonate ELISA S5761 N/A Sigma -Aldrich
Chemie GmbH
Sodium carbonate ELISA S7795 N/A Sigma -Aldrich
Chemie GmbH
Sodium pyruvate 100 mM 11360 -039 N/A Gibco
Sterile filters 0.45 µm 514-4123 N/A VWR International
Sulfuric acid 25%
EMSURE® ELISA 1007161000 N/A VWR International
GmbH
TMB One (3,3’,5,5’-
Tetramethylbenzidin e)
ready -to-use-solution ELISA 4380A N/A Biotrend
Chemikalien
GmbH
Tween 20 ELISA 9127.1 N/A Carl Roth GmbH
& Co. KG
Vero -76 cells Pseudovirus titration CRL-1587 N/A ATCC
Vi-CELL™ XR Quad
Pak For Vi -CELL™ XR Cell
Viability Analyzer 383722 N/A Beckman Coulter
GmbH
VSV-ΔG-GFP vector VSV vector production EH1004 N/A Kerafast
Table 3: Equipment
Product name Application Provider
Biacore T200 SPR analysis Cytiva
Vi-CELL™ XR Cell Viability
Analyzer Splenocyte count Beckman Coulter GmbH
CTL ImmunoSpot S6 Core
Analyzer ELISpot plate reader Cellular Technology Ltd .
BioTek Epoch reader ELISA plate reader BioTek
IncuCyte Live Cell Analysis
system pVNT Sartorius
Celesta Flow cytometry analysis (ICS) BD
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Table 4: Software
Product name Application Provider
Biacore T200 Evaluation
Software 3.1 SPR analysis Cytiva
Excel Animal monitoring , raw data Microsoft Corp.
GraphPad Prism 8 Analysis of ELISpot, ELISA , and pVNT GraphP ad Software Inc.
Gen5 software 3.0.9 ELISA plate read out BioTek
ImmunoCapture 7.0.7.0 ELISpot analysis Cellular Technology Ltd .
ImmunoSpot® analysis
software version 5 7.0.17.0 ELISpot analysis Cellular Technology Ltd .
IncuCyte Live Cell Analysis
system pVNT Sartorius
BD FACSDiva software
version 8.0.1.1 Flow cytometry analysis (ICS) BD
Table 5: Peptide pools for stimulation of splenocytes for ELISpot assays
S protein -specific peptides
Name Sequence
2019 -nCoV S.wt
With a total of 315
overlapping peptides
(Format 15/11)
GenBank: QHD43416.1
Batch: 43000LHB -1 and
43000LHB -2
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQ
DLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI
FGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF
RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI
NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSS GWTAGAA
AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTS
NFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY
NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN
YKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQA
GSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV LSFELLHAPATVCG
PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRD
PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPT
WRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRA
RSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCT
MYICGDSTECSNLLLQYGSFCTQLNRALT GIAVEQDKNTQEVFAQVKQIYKTP
PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAA
RDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM
QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN
QNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTY
VTQQLIRAAEIRASANL AATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHG
VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE
PQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD
LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLG
FIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHY
T
RBD -specific peptides
Name Sequence
2019 -nCoV RBD
With a total of 48
overlapping peptides
(Format 15/11) VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKC
YGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC
VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVE
GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK
Irrelevant peptide control
Name Sequence
AH-1 SPSYVYHQF
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4.5 Methods
4.5.1 Animal Care
4.5.1.1 General Information
BALB/c mice were delivered at the age of at least six weeks. Delivered mice were used
for experiments after approximately one week of acclimatization. All experiments and
protocols were approved by the loc al authorities (local animal welfare committee),
conducted according to the Federation of European Laboratory Animal Science
Associations (FELASA ) recommendations and in compliance with the German animal
welfare act and Directive 2010/63/EU. Only animals w ith an unobjectionable health
status were se lected for testing procedures.
All animals were registered upon arrival in the lab animal colony management system
PyRAT (Scionics Computer Innovation GmbH, Dresden, Germany) and tracked until
death. Each cage was labeled with a cage card indicating the mouse strain, sex, date
of birth , and number of animals per cage. At the start of an experiment additional
information was added suc h as the project and license number, the start of the
experiment and details on interventions. Where necessary for identification, animals
were arbitrarily numbered with earmarks.
4.5.1.2 Housing Condition s and Husbandry
Mice were housed at BioNTech SE ’s animal fa cility (An der Goldgrube 12, 55131
Mainz) under barrier and specific -pathogen -free (SPF) conditions in individually
ventilated cages (Sealsafe GM500 IVC Green Line, TECNIPLAST, Hohenpeißenberg,
Germany; 500 cm²) with a maximum of five animals per cage. The temperature and
relative humidity in the cages and animal unit w ere kept at 20 -24°C and 45 -55%,
respectively, and the air change (AC) rate in the cages was 75 AC/h. Cages contained
dust-free bedding made of debarked chopped aspen wood (Abedd LAB & VET Ser vice
GmbH, Vienna, Austria, product code: LTE E -001) and additional nesting material w as
changed weekly. Autoclaved ssniff M -Z food (sniff Spezialdiäten GmbH, Soest,
Germany; product code: V1124) and autoclaved tap water were provided ad libitum
and change d at least once weekly. All materials were autoclaved prior to use.
4.5.2 Animal Monitoring
Routine animal monitoring was carried out daily and included inspection for dead mice
and control of food and water supplies. The health of e ach mouse was closely
assesse d at least once weekly and the results documented in health monitoring sheets
(see Appendix 1: Animal Monitoring - Observations ). The general physical c ondition of
the mice was assessed according to the following parameters:
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Body weight change
Macroscopic assessment of activity level/behavior
Macroscopic assessment of general discomfort: drop in body temperature
determined by touch and by visual inspection of ears and paws (ears and paws
appear pink in a healthy mouse, white in a mouse with discomfort indicate s
reduced blood circulation )
Macros copic assessment of fur condition and appearance of eyes, inspection
of body cavities/fluids
Macroscopic assessment of irregularities in breathing ability
Indication of pain
Macroscopic assessment for signs of automutilation and /or fighting
Details on anim al monitoring criteria are shown in Appendix 1: Animal Monitoring -
Observations , Table 9.
4.5.3 Animal Treatment
4.5.3.1 Treatment Schedule, Route of Administration, and Dose
The test compound was administered i.m. once at three different doses ( 0.2 µg, 1 µg,
or 5 µg per anima l) to the three test groups of mice on day 0. The control group was
treated with buffer alone.
4.5.3.2 Immunization
Following anesthesia by inhalation of 2.5% isoflurane in oxygen , the injection site on
the hind leg of the mouse was shaved for immunization . Buffer or dissolved test item
was applied i.m. i nto the musculus gastrocnemius in a volume of 20 µL. After
immunization and a short recovery phase from anesthesia , the mice were o bserved for
any immediate signs of discomfort due to the immunization procedure.
4.5.3.3 Blood Sampling via the Retro -Orbital Venous Plexus or
Vena Facialis
Blood was sampled via the retro -orbital venous plexus according to SOP -030-074. In
short, mice were anesthetized by inhalation of 2.5% isoflurane in oxygen and tightly
held for blood collection . A thin glass capillary (29 G) was ins erted gently through the
retro-orbital sinus membrane and blood was collected into an appropriate plastic tube
(Sarstedt, Z -gel included for clotting activation). After careful removal of the glass
capillary, the restraining hold on the mouse was loosened. Alternatively, blood
collection took place via the vena facialis according to SOP -030-074. In short, without
prior anesthesia, mice were tightly held for blood collection, and the vena facialis was
punctured using a lancet in a precise and short movement. Blood was collected into
an appropriate plastic tube (Sarstedt, Z -gel included for clotting activation), and then
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the restraining hold on the mouse was loosened. Blood samples were centrifuged at
10,000 ×g and RT for 5 min and serum transferred to a pre -labeled 0.5 mL reagent
tube for use in subsequent downstream assays or storage at -20°C.
4.5.4 Endpoint of Experiment/Termination Criteria
Animals were euthanized in accordance with §4 of the German animal w elfare act and
the recommendation of the German Society of Laboratory Animal Science (GV-
SOLAS ) by cervical di location or by e po ure to carbon dio ide Additionally,
termination criteria were applied according to the specification within the respective
animal test approval as listed below. Body weight losses exceeding 20%, or a high
severity level in any of the parameters found in Section 4.5.2 were o n their own
sufficient reason for immediate euthanasia.
4.5.4.1 Dissection of Animals and Organ Collection
Following euthanasia, mice were disinfected with 70% ethanol and the dissection was
performed starting with an abdominal incision. The spleen was collected a nd stored in
DPBS on ice for subsequent splenocyte preparation.
4.5.5 ELISA
Serum samples were tested in 96 -well plates for their S -specific antibody concentration
based on SOP -030-112 (with minor modifications as described below) . Briefly, for the
time points 7, 14 , and 21 days after immunization, a screening analysi s was performed
and for day 14 and 28, serum samples were analyzed by endpoint titration.
1. Coat each well of a Maxi Sorp plate with 100 ng/100 µL rec ombinant protein per
well or iso type controls according to plate layout .
Coating buffer: 50 mM sodium carbonate b uffer (1 .696 g Na 2CO 3 +
2.856 g NaHCO 3, top up to 1 L distilled H 2O, pH 9.6 (pH adjustment not
needed ))
2. Cover plates and incubate at 4°C o/n .
3. Wash three times with 300 µL/well PBS with Twee n (PBS-T).
4. Block all wells with 1xBB, 250 µL/well .
5. Incubate at 37°C for 1 h on shaker .
6. Wash three times with 300 µL/well PBS -T.
7. Dilute primary antibodies (samples and positive control) according to schedule .
8. Incubate at 37°C for 1 h on shaker .
9. Wash three times with 300 µL/well PBS -T.
10. Dilute the secondary antib odies according to calculations.
11. Incubate at 37°C for 45 min on shaker .
12. Wash three times with 300 µL/well PBS -T.
13. Add 100 µL/well TMB substrat e.
14. Incubate 8 min at RT (clear ->blue) .
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15. Stop the reaction wi th 100 µL 25% sulf uric acid. (blue -> yellow) .
16. Read on plate reader (450 nm, reference: 620 nm).
For concentration analysis, the signal of the specific samples was correlated to the
isotype control. For analysis of IgG subtypes, the mean ΔOD 450 -620 nm per group
was calculated and the ratio of IgG2 a:IgG1 ratio was calculated.
For reciprocal serum endpoint titer, the serum dilution that emitted the OD exceeding
4-fold background was used. The background was defined as the OD signal given by
the recombinant p rotein incubated with the secondary detection anti -mouse IgG
antibody only.
4.5.6 Surface Plasmon Resonance Spectroscopy
Binding kinetics of murine S1 - and RBD -specific serum IgGs was determined using a
Biacore T200 device with HBS -EP running buffer at 25°C. Car boxyl groups on the CM5
sensor chip matrix were activated with a mixture of 1 -ethyl -3-(3-dimethylaminopropyl)
carbodiimidehydrochloride (EDC) and N -hydroxysuccinimide (NHS) to form active
esters for the reaction with amine groups. Anti -mouse -Fc-antibody wa s diluted in
10 mM sodium acetate buffer pH 5 (30 µg/mL) for covalent coupling to immobili zation
level of ~10,000 response units (RU). Free NHS esters on the sensor surface were
deactivated with ethanolamine.
Mouse serum was diluted 1:50 in HBS -EP buffer and applied at 10 µL/min for 30
seconds to the active flow cell for capture by immobili zed antibody, while the reference
flow cell was treated with buffer. Binding analysis of captured murine IgG antibodies to
S1-His or RBD -His was performed using a multi -cycle kinetic method with
concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively. An
association period of 180 seconds was followed by a dissociation period of
600 seconds with a constant flow rate of 40 μL/min and a final regeneration s tep.
Binding kinetics were calculated using a 1:1 Langmuir global kinetic fit model.
4.5.7 Pseudovirus -based Neutralization Test
For analyzing the amount of functional nAbs in the serum samples, pVNTs were
performed.
4.5.7.1 Production of SARS -CoV-2-S Pseudotyped VSV Vector
Replication -deficient vesicular stomatitis virus (VSV) that lacks the genetic information
for the VSV envelope glycoprotein VSV -G but contains an open reading frame (ORF)
for green fluorescent protein (GFP) was used for SARS -CoV-2-S pseudovirus
generation. VSV pseudotypes were generated according to a published protocol
(Hoffmann et al. 2020 ).
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In brief, HEK293T/17 cells cultured in DMEM supplemented with 10% FBS were
transfected with a pcDNA3.1 -derived expression plasmid (Invitrogen) coding for the
SARS -CoV-2 spike protein (GenBank ID: QHD43416.1) with shortened cytoplasmic
tail, i.e. , pSARS -CoV-2-S-CΔ19, using Lipofectamine® LTX & PLUS™ Reagent
(Invitrogen) following the manufacturer’s instructions. The cytoplasmi c tail was
truncated for the 19 C -terminal amino acids to facilitate a more efficient integration of
SARS -CoV-2-S into VSV virions analogous to SARS -CoV-2-S pseudotyped VSV
(Fukushi et al. 2005 ). At 24 h post transfection, ce lls were inoculated with VSV -G
transcomplemented VSV -ΔG-GFP vector (Indiana strain, de novo generated by
reverse genetics from plasmid ( Lawson et al. 1995 )) at a multiplicity of infection (MOI)
of three and incubated for 2 h at 37°C and 5% CO 2. Next, the inoculum was removed,
cells were washed with PBS, and standard culture medium which contained 0.5 µg/mL
anti-VSV-G antibody (clone 8G5F11) was added to neutralize residual input virus.
Twenty -four hours after infection, VSV/SA RS-CoV-2-S pseudovirus -containing
supernatants were harvested, filtered (0.45 µm) and stored at -80°C in aliquots until
further use.
4.5.7.2 Titration of VSV/ SARS -CoV-2-S Pseudovirus
For titration of VSV/SARS -CoV-2-S pseudovirus, Vero -76 cells (ATCC) were thawed
according to SOP -030-041, diluted to 2.67 × 105 cells/mL in assay medium (DMEM
and 10% FBS) and seeded in 96 -well flat -bottom plates at 4 × 104 cells per well. Cells
were incubated for 4 to 6 h at 37°C and 7.5% CO 2. Meanwhile, two -fold, eight -step
serial di lutions were prepared in 96 -well V -bottom plates beginning with undiluted
pseudovirus supernatant. Vero -76 wells were inoculated with 50 µL of the diluted
pseudovirus supernatant and incubated for 16 to 24 h at 37°C and 7.5% CO 2. Each
dilution was tested i n duplicate wells. After the incubation, the cell culture plates were
removed from the incubator, placed in an IncuCyte Live Cell Analysis system (Essen
Bioscience) and equilibrated for 30 min prior to the analysis. Whole well scanning for
brightfield and GFP fluorescence was performed using a 4× objective. The number of
infected GFP -fluorescent cells per well was plotted as a function of pseudovirus
supernatant dilution using GraphPad Prism. Data (x = logx) were fitted with linear
regression and the derive d slope and y -intercept used to calculate the amount of viral
supernatant needed to obtain 144 infected cells/96 -well (20% excess for virus
neutralization test included) .
4.5.7.3 Pseudovirus -based Neutralization Test
Sera from blood samples collected 14, 21 and 28 days after immunization were tested
using the VSV/SARS -CoV-2-S pseudovirus neutralization test (pVNT). For the pVNT
assay, Vero -76 cells were thawed according to SOP -030-041, diluted to 2.67 × 105
cells/mL in assay medium (DMEM and 10% FBS) and seeded in 96-well flat -bottom
plates at 4 × 104 cells per well. Cells were incubated for 4 to 6 h at 37°C and 7.5%
CO 2. Initial dilutions of mouse serum samples were prepared by adding 10 µL of serum
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to 50 µL assay medium in a 96 -well V -bottom plate. Seven additiona l dilutions were
subsequently prepared in two -fold dilution steps, by iteratively transferring 30 µL of
diluted sera to wells containing 30 µL assay medium. VSV/SARS -CoV-2 pseudovirus
was thawed and diluted to obtain 120 infected cells/25 µL (4.8 × 103 infectious units
[IU]/mL). 30 µL of diluted pseudovirus (corresponds to 144 infected cells; see
Section 4.5.7.2 ) was added to the wells containing the serum dilution series.
Pseudovirus/serum dilution mix was incubated for 5 min at RT on a microplate shaker
at 750 rpm, and additional 5 min at RT without agitation. Pseudovirus/serum dilution
mix was then added to the seeded Vero -76 cells (50 µL mix per well, MOI:0.003),
followed by incubation for 16 to 24 h at 37°C and 5% CO 2. Each dilution of serum
samples was tested in duplicate wells. Vero -76 cells inc ubated with pseudovirus in the
absence of mouse sera were used as positive controls. Vero -76 cells incubated without
pseudovirus were used as negative controls. After the incubation, the cell culture plates
were removed from the incubator, placed in an Inc uCyte Live Cell Analysis system and
incubated for 30 min prior to the analysis. Whole well scanning for brightfield and GFP
fluorescence was performed using a 4× objective. To calculate the neutralizing titer,
infected GFP -positive cell number per well was compared with the no -serum
pseudovirus positive control. Mean values of the no -serum pseudovirus positive control
multiplied by 0.5 represent the pseudovirus neutralization 50% (pVN 50); mean values
of the no -serum pseudovirus positive control multiplied b y 0.1 represent the
pseudovirus neutralization 90%. Serum samples with mean values below this cut -off
exhibit >50% or >90% virus neutralization activity, respectively .
4.5.8 Preparation of Splenocytes
The single cell suspensions from collected spleens were prepa red according to SOP -
030-078. To this end, the spleens were squeezed through 70 µm cell meshes using
the plunger of a syringe to release the splenocytes into a tube. Splenocytes were
washed with an excess volume of DPBS followed by centrifugation at 300 × g for 6 min
at RT and discarding the supernatants. Erythrocytes were then lysed with erythrocyte
lysis buffer (154 mM NH 4Cl, 10 mM KHCO 3, 0.1 mM EDTA) for 5 min at RT. The
reaction was stopped with an excess volume of DPBS. After another washing step,
cells were resuspended in medium (10% FB S, 1% NEAA, 1% sodium pyruvat e, 0.5%
penicillin/streptomycin), passed through a 70 µm cell mesh again, counted according
to SOP -010-028, and stored short -term at 37°C for use on the same day or frozen in
liquid nitrogen , resuspended in 1 mL FBS/10% DMSO . For the use of f rozen
splenocytes in ELISpot analysis after thawing, the amount of cells per well was doubled
(1 × 106 cells). Immediately after thawing, pre -warmed (RT) PBS was added to
splenocytes. Two washing steps using pre-warmed PBS to remove DMSO from
freezing process were performed and splenocytes were counted according to SOP -
010-028. Splenocytes were stored short -term at 37°C for further use .
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4.5.9 ELISpot A ssay
ELISpot assays with fresh or frozen splenocytes were performed according to SOP -
030-110 (with m inor modifications as described below) using the mouse IFN -
ELISpotPLUS kit. Briefly, 96 -well ELISpot plates were washed with PBS and blocked
with medium for at least 30 min at 37°C. 100 µL of the splenocyte solution ( fresh cells:
5 × 105 cells; frozen cells: 1 × 106 cells) were transferred to the respective well of the
96-well ELISpot plate. Another 100 µL of overlapping peptide pools or controls were
added in the following concentrations:
overlapping peptide mix PepMix™ against SARS -CoV-2 S.wt: 0.1 µg/mL final
concentration per peptide
overlapping peptide mix PepMix™ against SARS -CoV-2 RBD: 0. 1 µg/mL final
concentration per peptide
irrelevant peptide (AH -1): 4 µg/mL
Concanavalin A (ConA): 2 µg/mL
For positive control, the splenocytes were stimulated with ConA, for a non -stimulation
control only medium was added and as a negative control to detect unspecific
background signals, the irrelevant peptide was added (AH -1). Plates were incubated
overnight in a 37°C humidified incubator with 5% CO 2 and after approximately 18 h,
cells were removed from the plates and the detection protocol of spots was initiated.
To this end, the detection antibody, Streptavidin -ALP, and the ready -to-use substrate
were added to the wells according to the manufacturer’s protocol. After plate drying for
2–3 h under the laminar flow, an ELISpot plate reader (ImmunoSpot® S6 Core
Analyzer, CTL) was used to count and analyze spot numbers per well.
4.5.9.1 Subtyping of CD8+ versus CD 4+ T-cell Responses
This method was performed with fresh splenocytes (non -frozen). CD8+ or CD4+ T cells
were isolated from splenocyte cell suspensions using MACS® MicroBeads (CD8a
(Ly-2) or CD4 (L3T4)) according to the manufacturer’s instructions. Labeled cells were
eluted from MACS LS columns, centrifuged (5 min at 460 ×g) and taken up at a
concentration of 1 × 106 cells/mL in medium. 100 µL of CD8+ or CD4+ T cells were
subsequently re -stimulated by addition of 50 µL peptide solution (control peptide AH -
1 (2 µg/mL), RBD peptide mix (0. 1 µg/mL per peptide) or S peptide mix (0.1 µg/mL per
peptide)) and 50 µL of bone marrow -derived dendritic cells (1 × 106 cells/mL, cells
were frozen at -80°C prior use and prepared from BALB/c mice according to SOP -030-
080) in an IFN - ELISpot assay (SOP -030-110). Each condition was tested in
duplicate.
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4.5.10 Luminex Assay
1 × 106 previously frozen splenocytes in 100 µL DC medium (part of SOP -030-110)
were transferred to a 96-well flat -bottom cell culture plates. 100 µL of an overlapping
peptide pool or controls were added in the following concentrations:
overlapping peptide mix PepMix™ against SARS -CoV-2 S.wt: 0.1
or 0.03 µg/mL final concentration per peptide (equal to 31.5 or 9.6 µg/mL total
peptide)
overlapping peptide mix PepMix™ against SARS -CoV-2 RBD: 0.66 or
0.2 µg/mL final concentration per peptide (equal to 31.5 or 9.6 µg/mL total
peptide)
PMA: 1 µg/mL and ionomycin: 2 µg/mL
The plates were incubated for 48 h and supernatant thereafter was harvested for
cytokine profil ing. Cytokine concentrations in supernatants of the re-stimulated
splenocytes were determined using a bead -based, 11 -plex T H1/T H2 mouse
ProcartaPlex immunoassay according to the manufacturer’s instructions.
Fluorescence was measured with the Bioplex200 Sys tem (Bio -Rad) and analyzed with
ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific). The following analytes
were measured: IFN-, IL-12p70 , IL-13, IL-1, IL-2, IL-4, IL-5, IL-6, TNF-, GM-CSF,
and IL-18.
4.5.11 Intracellular Cytokine Staining
Briefly, 5 × 105 fresh splenocytes in 100 µL DC medium (part of SOP -030-110) were
transferred to 96 -well flat -bottom cell culture plates. Finally, 100 µL of an overlapping
peptide pool or controls were added in the following concentrations:
overlapping peptide mix Pe pMix™ against SARS -CoV-2 S.wt: 0.1 µg/mL final
concentration per peptide (equal to 31.5 µg/mL total peptide)
overlapping peptide mix PepMix™ against SARS -CoV-2 RBD: 0. 1 µg/mL final
concentration per peptide (equal to 4.8 µg/mL total peptide)
PMA: 1 µg/mL and ionomycin: 2 µg/mL
As a non -stimulation control, only medium was added to detect unspecific background
signals. Plates were incubated for 1 h in a 37°C humidified incubator with 5% CO 2
before adding a GolgiStop+GolgiPlug. After another 4 h, cells were harvested and
transferred to a 96 -well, V-bottom plate for flow cytometry staining. After the staining
procedure, cells were solved in 100 µL FACS buffer (PBS + 0.1% BSA) for flow
cytometry analysis using a FACS Celesta (BD).
A detailed protoc ol is presented in Appendix 5: Detailed ICS Protocol .
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4.5.12 Statistical Analysis
GraphPad Prism 8 Software (La Jolla, USA) was used for statistical analysis an d figure
generation. All test groups were compared to the buffer control group by a one -way
analysis of variance ( ANOVA ) on each measurement day as described in the
respective results section . For Luminex assays, statistical significance was assessed
by mi xed-effects analysis/Sidak’s comparison.
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5 RESULTS
5.1 ELISA
5.1.1 Whole IgG ELISA
IgGs against recombinant S1 protein or RBD were detected by ELISA analysis in
serum samples obtained on study days 7, 14 , and 21. Statistical significance was
assessed by one -way ANOVA and Dunnett ’s multiple comparison s test.
Before immunization, no S1 protein - or RBD -specific IgGs were detected (Pre -
treatment, Figure 2, Figure 3). Treatment with BNT162 b2 induced the formation of
IgGs specific for S1 protein and RBD, while these antibodies were not detected in
samples from buffer control animals independent of the day of sample collection. A
dose -dependent increase in S1 -specific IgGs was observed on all study days
(Figure 2), with statistically significant differences between the treatment groups and
the buffer control group ( p < 0.0001 for all doses and test days ).
Figure 2: ELISA screening analysis on days 7, 14 , and 21 against the recombinant S1 protein
ELISA was performed using serum samples collected on days 7, 14 , and 21 after immunization to analyze antibody
responses. The serum samples were tested against the S1 protein . Individual ΔOD values for each mouse
(measured in duplicates) are shown by dots; group mean values are indicated by horizontal bars (±SEM).
**** p < 0.0001 .
0123
Pre-treatment (1:300) S1OD (450-620nm)
Buffer 0.2 µg 1 µg 5 µg0123
Day 7 (1:900) S1
BNT162b2 (RBP020.2)
Buffer 0.2 µg 1 µg 5 µg0123
Day 14 (1:900) S1
BNT162b2 (RBP020.2)OD (450-620nm)
Buffer 0.2 µg 1 µg 5 µg0123
Day 21 (1:900) S1
BNT162b2 (RBP020.2)**** **** ****
**** **** ******** **** ****
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All test g roups showed a statistically significant increase in RBD -specific IgGs
compared to buffer control ( Figure 3; p < 0.0001 for all doses and test days ).
Figure 3: ELISA screening analysis on days 7, 14, and 21 against the recombinant RBD
ELISA was performed using serum samples collected on days 7, 14 , and 21 after immunization to analyze antibody
responses. The serum samples were tested against the receptor -binding domain (RBD) . Individual ΔOD values for
each mouse (measured in duplicates) are shown by dots; group mean values are indicated by horizontal bars
(±SEM). **** p < 0.0001 .
ELISA endpoint titration was performed on day 28 after immunization to analyze
antibody responses ( Figure 4A, B).
Antibody concentrations in the serum samples were calculated for the individual
sampling days and the kinetics of IgGs against S1 and RBD proteins is shown in
Figure 5. Antibody concentrations against S1 (Figure 5A) and RBD (Figure 5B)
increased in a dose -dependent manner over time in the test groups. Statistical
significance of the differences in IgG concentrations between the test groups and the
control group was assesse d by one -way ANOVA with Dunnett’s multiple comparison
post-test on day 28.
0123
Pre-treatment (1:300) RBDOD (450-620nm)
Buffer 0.2 µg 1 µg 5 µg0123
Day 7 (1:900) RBD
BNT162b2 (RBP020.2)
Buffer 0.2 µg 1 µg 5 µg0123
Day 14 (1:900) RBD
BNT162b2 (RBP020.2)OD (450-620nm)
Buffer 0.2 µg 1 µg 5 µg0123
Day 21 (1:900) RBD
BNT162b2 (RBP020.2)**** **** ****
**** **** **** **** **** ****
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The differences in concentrations of IgGs against S1 and RBD in the test groups
compared to the buffer control group were statistically significant ( S1: p = 0.0259 for
0.2 µg, p < 0.0001 for 1 µg and 5 µg; RBD: p = 0.0072 for 0.2 µg, p < 0.0001 for 1 µg
and 5 µg) on day 28.
Figure 4: ELISA endpoint titration on day 28
Endpoint titration was performed on day 28 after immunization to analyze antibody respons es. The serum samples
were tested against the S1 protein (A) and RBD (B). Group mean values (±SEM) are shown.
1:300 1:9001:2700 1:81001:24300 1:729001:218700uncoated 0123
S1-ELISA
serum dilutionOD (450-620nm)
Buffer control
0.2 µg BNT162b2 (RBP020.1)1 µg BNT162b2 (RBP020.1)
5 µg BNT162b2 (RBP020.1)
1:300 1:9001:2700 1:81001:24300 1:729001:218700uncoated 0123
RBD-ELISA
serum dilutionA B
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Figure 5: Kinetics of the antibody concentration against the viral antigen
For individual ΔOD values, the antibody concentration s in the serum sample s were calculated. The serum samples
were tested against (A) the S1 protein and (B) RBD. Group mean antibody concentrations are shown (±SEM) .
Furthermore, to calculate the reciprocal serum endpoint titer of antibodies we
performed an endpoint titration for day 14 and 28 samples after immunization
exceeding the previously shown dilution steps (Figure 6A, B).
The r eciprocal serum endpoint titer was defined as the first highest dilution step which
emitted an OD exceeding the background signal four -times as shown . Reciprocal
serum endpoint tite r against S1 ( Figure 7A) and RBD ( Figure 7B) were high already
14 days after immunization and increased in a dose -dependent manner over time in
the test groups. Statistical significance of the differences in IgG concentrations
between the test groups and the control group was assessed by a one-way AN OVA
with Tukey’s multiple comparison post -test.
The differences in titers of IgGs against S1 and RBD in the test groups compared to
the buffer control group were statistically significant (S1 , day 28 : p = 0.0082 for 1 µg,
p < 0.0001 for 5 µg; RBD , day 14 : p < 0.0001 for 5 µg and day 28 : p = 0.0109 for 1 µg,
p < 0.0001 for 5 µg).
Buffer
0.2 µg
1 µg
5 µg
pre d7 d14 d21 d280100200300400500
IgG concentrations S1
BNT162b2 (RBP020.2)IgG [µg/mL]
pre d7 d14 d21 d280200400600
IgG concentrations RBD
BNT162b2 (RBP020.2)Buffer
0.2 µg
1 µg
5 µgA B
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Figure 6: ELISA endpoint titration (long titration)
Endpoint titration against the S1 protein (A) and RBD (B) was performed on day 14 (left) and 28 (right) after
immunization to analyze reciprocal serum endpoint titer of antibodies . Group mean values (±SEM) are shown ;
samples were measured in duplicates . Background was defined as the OD value of the recombinant protein
incubated with the secondary anti-mouse IgG detection antibody only and included in the graphs (blue dotted line;
n = 8).
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Figure 7: Reciprocal serum endpoint titer at day 14 and 28 after immunization
For individual OD values, the reciprocal serum endpoint ti ter was calculated. The serum samples were tested
against (A) the S1 protein and (B) RBD. Group mean titer are shown (±SEM) . Significance compared to buffer
control is included, * p ≤ 0.05, ** p ≤ 0.01, **** p < 0.0001 ; LDL = lower detection limit.
5.1.2 IgG Subtype -specific ELISA
IgG1 and IgG2 a subtypes of anti -S1 antibodies were analyzed by IgG subtype -specific
ELISA in serum samples obtained on study day 28. Statistical significance was
assessed by one -way ANOVA followed by a Dunnett ’s multiple comparison post -test.
Treatment with BNT162 b2 induced the formation of IgG1 and IgG2 a specific for
S1 protein, while these antibodies were not detected in samples from buffer control
animals independent of the day of sample collection (Figure 8).
On day 28, all dose groups displayed significantly higher group mean ΔOD values for
IgG1 and IgG2 antibodies than the control animals ( IgG1: p < 0.0001 for all doses,
IgG2a: p = 0.0020 for 0.2 µg, p < 0.0001 for 1 µg and 5 µg).
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Figure 8: IgG subtype -specific ELISA on day 28
ELISA was performed using serum samples collected on day 28 after immunization to analyze (A) IgG1 and (B)
IgG2 a responses. The serum samples were tested against the S1 protein . Individual ΔOD values for each mouse
(measured in duplicates) are shown by dots; group mean values are indicated by horizontal bars (± SEM).
** p ≤ 0.01, **** p < 0.0001.
ELISA endpoint titration was performed on day 28 after immunization to analyze IgG1
and IgG2 a responses ( Figure 9A, B).
Figure 9: ELISA endpoint titration on day 28 (IgG subtypes)
Endpoint titration was performed on day 28 after immunization to analyze IgG1 (A) and IgG2 a (B) responses. Group
mean values (±SEM) are shown.
Buffer 0.2 µg 1 µg 5 µg0123
Day 28 (1:2700) IgG1
BNT162b2 (RBP020.2)OD (450-620nm)
Buffer 0.2 µg 1 µg 5 µg0123
Day 28 (1:2700) IgG2a
BNT162b2 (RBP020.2)A B
**** **** **** ** **** ****
1:9001:2700 1:81001:24300 1:729001:218700 1:656100 uncoated0123
Day 28, IgG1
serum dilutionOD (450-620nm)Buffer
0.2 µg
1 µg
5 µg
1:9001:2700 1:81001:24300 1:729001:218700 1:656100 uncoated0123
Day 28, IgG2A
serum dilutionBuffer
0.2 µg
1 µg
5 µgA B
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5.1.3 IgG2a/IgG1 Ratio
To analyze the ratio between the two IgG subtypes, the ΔOD values were used.
Antibody ratios in the serum samples were calculated for day 28 (Figure 10). Statistical
significance was assessed by one -way A NOVA followed by Tukey’s multiple
comparison post -test to compare all test groups with each other.
While the two higher doses induced a balanced IgG2a /IgG1 response, the lowest dose
induced a higher ignal for IgG1 than IgG2a The difference between the group treated
with 0.2 µg and the groups treated with 1 µg and 5 µg were statistically significant
(p = 0.0004 for 0.2 µg vs 1 µg, p = 0.0041 for 0.2 µg vs 5 µg).
Figure 10: IgG2a/IgG1 subtype ratio on day 28
Based on the 1:2 ,700 dilution step (see Figure 9), the ΔOD the ΔOD for every single sample were used to calculate
the ratio of IgG2a and IgG1. For this purpose, the ΔOD value of IgG2a was divided by the ΔOD values of IgG1 per
mouse. Group mean values (±SEM) are shown. The value of “1” in the graph would give the equal signal between
the two subtypes while ratio > 1 mirror a higher IgG2a subtype d etection. ** p ≤ 0.01, *** p ≤ 0.001 .
5.2 Binding Kinetics of Antigen -specific IgGs Using SPR
To obtain kinetic and affinity information about the binding of vaccine -elicited IgG to
SARS -CoV-2 S1 fragment and RBD, SPR spectroscopy was conducted. Whole IgG
from ser a (n = 8) generated at 28 days after immunization with 5 µg BNT162b2 was
captured by high -affinity anti -IgG antibody immobilized on the sensor chip surface .
Binding analysis of captured murine IgG antibodies to recombinant S1 -His or RBD -His
protein wa s performed using a multi -cycle kinetic method with concentrations ranging
from 25 to 400 nM S1 -His or 1.5625 to 50 nM RBD -His. Kinetic parameters were
calculated by fitting the sensorgram curves with a 1:1 Langmuir global kinetic fit model.
0.2 µg 1 µg 5 µg0.00.51.01.5
BNT162b2 (RBP020.2)Ratio (IgG2a/IgG1)*** **
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At day 28 after immuniz ation, vaccine -elicited IgG had a strong binding affinity for S1 -
His (geometric mean K D = 12 nM), with affinities ranging from 8.06 nM to 34.5 nM
across the 8 serum samples tested (Figure 11A, Table 6). Somewhat higher binding
affinity was detected for RBD -His ( geometric mean K D = 0.99 nM), with affinities
ranging from 0.48 nM to 2.78 nM (Figure 11A, Table 7). Binding to S1 -His and RBD -
His can be characterized by a comparable low disso ciation rate constant (geometric
mean k off = 4×10-4 s-1 vs. 5.97 ×10-4 s-1). However, association of RBD -His to captured
IgG was approximately 20 -fold faster (geometric mean k on = 6.02×105 M-1s-1 vs.
3.33×104 M-1s-1).
Figure 11: Binding affinities of vaccine -elicited IgG for S1 -His and RBD -His protein
Representative SPR sensorgram of the binding kinetics of recombinant S1 -His (A) and RBD -His protein (B) to
immobili zed mouse IgG from serum 28 days after immuni zation with 5 µg BNT16 2b2 (n=8). Actual binding (dark
blue) and the best fit of the data to a 1:1 binding model (thin line in black) is shown .
Table 6: Summary of binding kinetic parameters of vaccine -elicited IgG for S1 -His
Animal no. kon
[1/Ms] koff
[1/s] KD
[nM]
4-1 2.62E+04 9.05E-04 34.5
4-2 3.36E+04 5.70E-04 17.0
4-3 2.91E+04 4.92E-04 16.9
4-4 4.78E+04 5.95E-04 12.5
4-5 2.94E+04 4.54E-04 15.4
4-6 2.45E+04 2.95E-04 12.0
4-7 2.91E+04 3.16E-04 10.9
4-8 3.36E+04 2.71E-04 8.06
Geometric
mean 3.33E+04 4.00E-04 12.0
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Table 7: Summary of binding kinetic parameters of vaccine -elicited IgG for RBD -His
Animal no. kon
[1/Ms] koff
[1/s] KD
[nM]
4-1 4.35E+05 6.79E-04 1.56
4-2 2.89E+05 8.04E-04 2.78
4-3 6.64E+05 7.23E-04 1.09
4-4 4.82E+05 5.82E-04 1.21
4-5 4.64E+05 6.26E-04 1.35
4-6 8.46E+05 4.07E-04 0.481
4-7 6.36E+05 5.55E-04 0.873
4-8 1.06E+06 5.42E-04 0.512
Geometric
mean 6.02E+05 5.97E-04 0.993
5.3 Pseudovirus -based Neutralization Test
Virus -neutralizing antibodies in serum s amples obtained on study days 14, 21 , and 28
were detected by pVNT. Statistical significance was assessed by one -way ANOVA
with Dunnett’s multiple comparison post -test.
Treatment with all tested uRNA doses induced the formation of virus -neutralizing
antibodies with temporally increasing pVN 50 titers (Figure 12). On day 14, several
samples from animals treated with 0.2 µg modRNA displayed pVN 50 titers that were
below the lower limit of quantification. Significantly higher pVN 50 titers were measured
in samples from animals treated with the high dose of 5 µg RNA than in buffer control
samples ( p = 0.0010). On days 21 and 28, the differences of the groups treated with
1 µg and 5 µg BNT162b2 compared to the buffer control group were statistically
signific ant (day 21: p = 0.0036 for 1 µg, p < 0.0001 for 5 µg; day 28: p < 0.0001 for
1 µg and 5 µg).
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Figure 12: Titers of ne utralizing antibodies on days 14, 21 , and 28
Serum samples were collected on days 14, 21 , and 28 after immunizations and titers of virus -neutralizing ant bodies
were determined by pseudovirus -based neutralization test (pVNT). Individual VNT titers are shown by dots; group
mean values are indicated by horizontal bars (±SEM, standard error of th e mean). ULOQ: Upper limit of
quantification, LLOQ: Lower limit of quantification. ** p ≤ 0.01, *** p ≤ 0.001 , **** p < 0.0001 .
5.4 ELISpot Analysis
Mice were euthanized on day 28 and splenocytes were isolated to assess T -cell
responses by ELISpot analysis. S plenocytes were stimulated with S1 - and RBD -
specific overlapping peptide pools (Table 5) and IFN - secretion was detected.
Statistical significance was assessed by one -way ANOVA with Dunnett’s multiple
comparison post -test. Control measurements were performed using an irrelevant
peptide pool, medium only or Concanavalin A (ConA, for exemplary controls for the
assay performed with frozen splenocytes see Appendix 3: Controls for ELISpot
Analysis , Figure 21).
Stimulation of fresh splenocytes with an S protein - or RBD -specific overlapping peptide
pool induced IFN - responses in T cells of immunized animals (Figure 13). After
stimulation with either the S protein -specific or RBD peptide pool , splenocytes of the
groups treated with modRNA displayed significantly higher spot numbers than buffer
Buffer 0.2 µg 1 µg 5 µg816326412825651210242048Day 14
BNT162b2 (RBP020.2)pVN50titer [serum dilu ion-1]
LLOQULOQ
Buffer 0.2 µg 1 µg 5 µg816326412825651210242048Day 21
BNT162b2 (RBP020.2)LLOQULOQ
Buffer 0.2 µg 1 µg 5 µg816326412825651210242048Day 28
BNT162b2 (RBP020.2)pVN50titer [serum dilu ion-1]
LLOQULOQ***** ****
**** ****
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control splenocytes (for S protein: p = 0.0001 for 0.2 µg, p < 0.0001 for 1 µg; RBD: p =
0.0094 for 0.2 µg and p < 0.0001 for 1 µg).
Figure 13: ELISpot analysis using fresh splenocytes on day 28
ELISpot assay was performed using splenocytes isolated on day 28 after immunization. Splenocytes were
stimulated with S protein - or RBD -specific overlapping peptide pools and I FN- secretion was measured to assess
T-cell responses. Individual spot counts are shown by dots; group mean values are indicated by bars (±SEM).
** p ≤ 0.01, **** p < 0.0001 . Note that for the 5 µg group, a miscalculated ce ll number was used in the assay,
therefore this group is not included in the graph.
In the assay with fresh splenocytes, a miscalculation of cells in the group immunized
with 5 µg modRNA occurred. Therefore, a second ELISpot run was included with
frozen splenocytes.
Stimulation of frozen splenocytes with an S protein - or RBD -specific overlapping
peptide pool induced IFN - responses in T cells of immunized animals ( Figure 14).
Frozen splenocytes of the groups treated with modRNA displayed significantly higher
spot numbers than buffer control splenocytes (p = 0.0087 for 0.2 µg, p < 0.0001 for
1 µg and 5 µg) after stimulation with the S protein -specific peptide pool . Stimulation
with the RBD -specific peptide pool induced significantly higher spot numbers in the
groups treated with 1 µg and 5 µg modRNA compared to the buffer control group
(p = 0.0001 for 1 µg, p = 0.0015 for 5 µg).
Buffer 0.2 µg 1 µg050100150RBD protein peptide mix
BNT162b2 (RBP020.2)
**
****
Buffer 0.2 µg 1 µg050010001500S1 protein peptide mix
BNT162b2 (RBP020.2)IFN-+spots/ 5x105 splenocytes
***
****
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Figure 14: ELISpot analysis using frozen splenocytes on day 28
ELISpot assay was performed using previously frozen splenocytes isolated on day 28 after immunization.
Splenocytes were stimulated with S protein - or RBD -specific overlapping peptide pools and IFN - secretion was
measured to assess T -cell responses. Individual spot counts are shown by dots; group mean values are indicated
by bars (±SEM). ** p ≤ 0.01, *** p ≤ 0.001 , **** p < 0.0001 .
To identify the responding T -cell subtype , an additional ELISpot analysi s was
performed after separation of fresh CD4+ and CD8+ cells by MACS isolation using
splenocytes isolated from the group treated with 5 µg RNA. Statistical significance was
assessed by one -way ANOVA with Dunnett’s multiple comparison post -test comparing
cells stimulated with RBD - and S protein -specific peptide pools to cells stimulated with
an irrelevant AH -1-specific peptide pool .
After stimulation with an S protein -specific peptide pool, but not after stimulation with
irrelevant AH -1, both CD4+ and CD8+ cells displayed IFN - responses ( Figure 15). The
differences between cells stimulated with the S protein -specific peptide pool and the
cells stimulated with the AH -1-specific peptide pool were statistically significant
(p < 0.0001 for CD4+ and CD8+ cells). No significant increase in spot numbers was
detected in CD4+ and CD8+ cells after stimulation with an RBD -specific peptide pool.
Buffer 0.2 µg 1 µg 5 µg0100200300400500
S1 protein peptide mix
BNT162b2 (RBP020.2)IFN-+spots/ 1x106 splenocytes
Buffer 0.2 µg 1 µg 5 µg01020304050
RBD protein peptide mix
BNT162b2 (RBP020.2)A B
** **** ******* **
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Figure 15: ELISpot analysis using splenocytes of 5 µg BNT162b2 (RBP020.2) immunized mice on day 28
after MACS cell separation
ELISpot assay was performed using splenocytes isolated on day 28 after immunization from group 4 after magnetic
cell separation MACS. CD4+ splenocytes (A) or CD8+ splenocytes (B) were stimulated with an RBD - or S protein -
specific overlapping peptide pool a nd IFN - secretion was measured to assess T -cell responses. ( C) Splenocytes
were stimulated with an irrelevant peptide or with medium alone or Concanavalin A. IFN - secretion was measured
to assess T -cell responses. Mean values ±SEM are shown. **** p < 0.0001.
5.5 Luminex Assay
Cytokine concentrations in supernatants of re -stimulated splenocytes were determined
using a bead -based, 11 -plex TH1/T H2 mouse ProcartaPlex immunoassay (Table 8).
Table 8: Chemokines and cytokines included for multiplex measurement
T-cell population Analyte s
TH1 IFN-, GM -CSF, TNF -, IL-1, IL-6, IL-12p70, IL -18
TH2 IL-4, IL-5, IL-13
Teff IL-2
For cytokine analysis, frozen splenocytes from immunized animals were stimulated
with eit her medium, PMA and ionomycin, or the S- or RBD -overlapping peptide mix.
Immunization with BNT162b2 induced an increased level of T H1-specific and
proinflammatory analytes. Stimulation of splenocytes with 0. 1 µg/mL per peptide (total
peptide concentration = 31.5 µg/mL) of the S -specific overlapping peptide pool induced
a stronger increase in cytokine concentrations than 0.66 µg/mL per peptide (total
peptide concent ration = 31.5 µg/mL) of the RBD -specific overlapping peptide pool .
Several values were below the lower level of quantification . Therefore, statistical
analysis was assessed by mixed -effects analysis/Sidak’s comparison. Taking the
background of the buffer group, medium control signal into account, a stimulation of
AH1 RBD S0200400600
CD4+ splenocytesIFN-+spots/
5x105 CD4+ cells (flow trough)
AH1 RBD S0200400600
CD8+ splenocytesIFN-+spots/ 1x105 CD8+cells
CD4+ Medium CD8+ MediumCD4+ ConA CD8+ ConA05001000150020002500
ControlsIFN-+spots/ 5x105 splenocytesA B C
********
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IFN-, TNF -, GM -CSF, IL-6, IL-18, and IL -2 was observed for the groups immunized
with 1 µg BNT162b2 (Figure 16). Due to the missing values, statistical significance was
not reached or could in some cases not be calculated for the cytokines shown in
Figure 16 , even though a clear trend was observed. Therefore, statistical significance
is not depicted, but only shown in Appendix 6: Statistical Analysis .
A more detailed summary of the results including PMA controls is shown in Appendix 4:
Summary of Luminex Assay.
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Figure 16: Cytokine concentrations in supernatants of re -stimulated splenocytes 28 days after
immunization
Luminex assay was performed using frozen splenocytes isolated on day 28 after RNA injection to assess
concentrations of the indicated chemokines/cytokines. After 48 h of stimulation with S- or RBD -overlapping peptide
mix, supernatant was collected and secretion of different A) T H1-specific and proinflammatory, B) T eff-specific and
Buffer 0.2 µg 1 µg 5 µg0510152025
IL-4[pg/mL]
BNT162b2 (RBP020.2)
<LLOQ<LLOQ<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
Buffer 0.2 µg 1 µg 5 µg050100150200IL-13
BNT162b2 (RBP020.2)
<LLOQ<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQC
Buffer 0.2 µg 1 µg 5 µg0100200300400500
IL-5
BNT162b2 (RBP020.2)
<LLOQ<LLOQ<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
<LLOQ
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C) T H2 cytokines was determined . Values for individual animals are shown by dots. Although all animals within the
groups were tested, several values were excluded as they were below the lower level of quantification (LLOQ) and
out of standard range. If all values within on e group were <LLOQ, this is marked in the graph. Mean values ±SEM
are shown.
5.6 Intracellular Cytokine Staining
ICS was performed after stimulation of splenocytes with an overlapping peptide pool
of the S protein or controls (Figure 17). Statistical significance was assessed by one -
way ANOVA with Dunnett’ multiple compari on po t te t
Due to a miscalculation of cells in the group immunized with 5 µg modRNA, ICS results
are only shown for buffer control and the groups treated with 0.2 µg and 1 µg
BNT162b2.
A peptide -specific stimulation was observed for specific cytokines. The fraction of IFN -
-expressing CD4+ T cells was significantly higher for animals immunized with 0.2 µg
and 1 µg BNT162c2 (p = 0.0002 for 0.2 µg, p < 0.000 1 for 1 µg, Figure 17A) than for
buffer control animals. No statistically significant increase was observed for IL -4 after
BNT162c2 treatment in comparison to buffer control (Figure 17B). The fraction of TNF -
-expressing CD4+ T cells was significantly higher for animals immunized with 1 µg
modRNA (p < 0.0001, Figure 17C) than for animals treated with buffer control. For IL -
2 expression, the CD4+ T-cell fractions were significantly higher in both treatment
groups than in the buffer control group ( p = 0.0015 for 0.2 µg, p = 0.0001 for 1 µg,
Figure 17D).
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Figure 17: CD4+ T cell intracellular cytokine staining 28 days after immunization
On day 28 after RNA injection, isolated splenocytes were stimulated with either buffer, PMA or a n S protein -
overlapping peptide mix (Pepmix) to assess the detailed T -cell response via flow cytometry. The intracellular
cytokine expression of CD4+ T cells expressing (A) IFN -, (B) IL -4, (C) TNF -, or (D) IL -2 was analyzed. The left
Buffer 0.2 µg 1 µg0.000.050.100.150.200.25
BNT162b2 (RBP020.2)% CD3+CD4+IFN+of single live cells
***
****
Medium PMA Pepmix S02468
Buffer 0.2 µg 1 µg0.060.080.100.120.14
BNT162b2 (RBP020.2)% CD3+CD4+IL4+of single live cells
Medium PMA Pepmix S0.00.20.40.60.81.0
Buffer 0.2 µg 1 µg0.00.10.20.3
BNT162b2 (RBP020.2)% CD3+CD4+TNF+of single live cells
****
Medium PMA Pepmix 020406080100
Buffer 0.2 µg 1 µg0.00.10.20.3
BNT162b2 (RBP020.2)% CD3+CD4+IL2+of single live cells
**
***
Medium PMA Pepmix 0204060TH1 cells
TH2 cellsA
CB
D
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graph in each subfigure shows the fraction after peptide stimulation (measured in duplicates), the right graph shows
each single value for all stimulation conditions (buffer and PMA stimulation was measured in single replicates, th e
Pepmix is t he same data as in the left graph but with all single values). Mean values ±SEM are shown. ** p ≤ 0.01;
*** p ≤ 0.001 ; **** p ≤ 0.0001.
For CD8+ T cells, a statistically significant induction of IFN -, TNF -, and IL -2 was
detectable after pep tide stimulation in the groups im munized with 0.2 µg and 1 µg RNA
compared to buffer control (IFN -: p = 0.0002 for 0.2 µg, p < 0.0001 for 1 µg; TNF -:
p = 0.0013 for 0.2 µg, p < 0.0001 for 1 µg; IL -2: p = 0.0003 for 0.2 µg, p < 0.0001 for
1 µg; Figure 18A, B, and C).
Figure 18: CD8+ T cell intracellular cytokine staining 28 days after immunization
On day 28 after RNA injection, isolated splenocytes were stimulated with either buffer, PMA or a n S protein -
overlapping peptide mix (Pepmix) to assess the detailed T -cell response via flow cytometry. The intracellular
Buffer 0.2 µg 1 µg0.00.20.40.60.81.0
BNT162b2 (RBP020.2)% CD3+CD8+IFN+of single live cells
***
****
Medium PMA Pepmix 01020304050
Buffer 0.2 µg 1 µg0.00.51.0
BNT162b2 (RBP020.2)% CD3+CD8+TNF+of single live cells
**
****
Medium PMA Pepmix 020406080
Buffer 0.2 µg 1 µg0.00.20.4
BNT162b2 (RBP020.2)% CD3+CD8+IL2+of single live cells
***
****
Medium PMA Pepmix 0510152025A
CB
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cytokine expression of CD8+ T cells expressing (A) IFN -, (B) TNF -, or (C) IL -2 was analyzed. The left graph in
each subfigure shows the fraction after peptide stimulation (measured in duplicates), the right graph shows each
single value for all stimulation conditions (buffer and PMA stimulation was meas ured in single replicates, the P epmix
is the same data as in the left gra ph but wi th all single values). Mean values ±SEM are shown. ** p ≤ 0.01;
*** p ≤ 0.001; **** p ≤ 0.0001.
5.7 Animal Monitoring
The animals’ body weight as well as observations regarding fur appearance and
injection site reactions are shown in Figure 19 and Figure 20.
The group mean body weights of animals treated with RNA displayed a development
comparable to the buffer control group (Figure 19). Over the course of the study, a
slight increase in body weights was observed for all groups.
Figure 19: Body weights of experimental mice during study
Experimental animals were weighed at study start and at indicated days , and the change in body weight was
calculated as a percentage of the initial weight of the individual mouse . Group mean values (±SEM) are shown .
No changes in fur appearance (i.e., fur defects, neglected grooming, ruffled) were
observed in animals treated with BNT162 b2 (Figure 20A).
Slight to distinct observations at the injected muscle (i.e., edema formation seen as
swollen muscle without flinching in reaction to touch, indicating absence of pain) in
comparison to the non -injected hind leg were made in animals treated with 1 µg and
5 µg of BNT162 b2 (Figure 20B). By day 2 (1 µg group) or latest by day 4 (5 µg group),
the injection site reactions had fully resolved.
0 5 10 15 20 25 308090100110120
Days post application% of initial body weightBuffer
0.2 µg BNT162b2 (RBP020.1)
1 µg BNT162b2 (RBP020.1)
5 µg BNT162b2 (RBP020.1)
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Additional animal m onitoring details are shown in Appendix 1: Animal Monitoring -
Observations .
Figure 20: Summary of observations made during study’s concomitant animal monitoring
Shown are deviations to normal appearance in (A) fur condition and (B) observations at the injection site (edema
formation) of each mouse . Severity of observations is graded with 0, none; +, slight; ++, moderate ; and + ++, distinct.
A B
1 2 3 4
BIO-LJ26 0 0 0 0
BIO-LJ27 0 0 0 0
BIO-LJ28 0 0 0 0
BIO-LJ29 0 0 0 0
BIO-LJ30 0 0 0 0
BIO-LJ31 0 0 0 0
BIO-LJ32 0 0 0 0
BIO-LJ33 0 0 0 0
BIO-LJ34 0 0 0 0
BIO-LJ35 0 0 0 0
BIO-LJ36 0 0 0 0
BIO-LJ37 0 0 0 0
BIO-LJ38 0 0 0 0
BIO-LJ39 0 0 0 0
BIO-LJ40 0 0 0 0
BIO-LJ41 0 0 0 0
BIO-LJ42 0 0 0 0
BIO-LJ43 + 0 0 0
BIO-LJ44 + 0 0 0
BIO-LJ45 + 0 0 0
BIO-LJ46 + 0 0 0
BIO-LJ47 + 0 0 0
BIO-LJ48 + 0 0 0
BIO-LJ49 + 0 0 0
BIO-LJ50 +++ ++ + 0
BIO-LJ51 ++ ++ + 0
BIO-LJ52 ++ ++ + 0
BIO-LJ53 ++ + 0 0
BIO-LJ54 ++ ++ + 0
BIO-LJ55 +++ ++ + 0
BIO-LJ56 ++ + 0 0
BIO-LJ57 ++ + 0 0Days post applicationTreatment Mouse ID
0.2 µg
BNT162b2
(RBP020.2)
1 µg
BNT162b2
(RBP020.2)
5 µg
BNT162b2
(RBP020.2)Buffer
dpi
1
BIO-LJ26 0
BIO-LJ27 0
BIO-LJ28 0
BIO-LJ29 0
BIO-LJ30 0
BIO-LJ31 0
BIO-LJ32 0
BIO-LJ33 0
BIO-LJ34 0
BIO-LJ35 0
BIO-LJ36 0
BIO-LJ37 0
BIO-LJ38 0
BIO-LJ39 0
BIO-LJ40 0
BIO-LJ41 0
BIO-LJ42 0
BIO-LJ43 0
BIO-LJ44 0
BIO-LJ45 0
BIO-LJ46 0
BIO-LJ47 0
BIO-LJ48 0
BIO-LJ49 0
BIO-LJ50 0
BIO-LJ51 0
BIO-LJ52 0
BIO-LJ53 0
BIO-LJ54 0
BIO-LJ55 0
BIO-LJ56 0
BIO-LJ57 0Treatment Mouse ID
0.2 µg
BNT162b2
(RBP020.2)
1 µg
BNT162b2
(RBP020.2)
5 µg
BNT162b2
(RBP020.2)Buffer
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6 CONCLUSION
Treatment with all tested BNT162b2 doses, namely 0.2, 1 and 5 µg, induced a strong
immune response across the observation period of 28 days after vaccination with a
safe profile in terms of animal monitoring.
Total IgG ELISA show ed that the construct is immunogenic and induced a strong,
dose -dependent generation of antibodies against the S1 antigen and the receptor -
binding domain. First detection of IgG antibodies was possible 7 days after
immunization for all animals throughout t he groups with an increase of total antibody
amount until day 2 8. At day 28 after immuni zation, vaccine -elicited IgG had a strong
binding affinity for S1 (geometric mean K D = 12 nM) and the RBD (geometric mean
KD = 0.99 nM), both had low off -rates.
Profiling the IgG subtypes, a balanced IgG2a/ IgG1 response was detected for the two
higher doses, while the low dose induced a response with higher IgG1 than IgG2
levels . In pVNT analysis, starting 14 days after immunization, a development of
functional ne utralizing antibodies was shown for all animals and the titers increased
until the final study day. The summary of antibody titers at day 28 is as follows:
BNT162b2
0.2 µg BNT162b2
1 µg BNT162b2
5 µg
Anti-S1 protein total IgG [µg/mL] 73.0 ± 10.4 205.9 ± 21.0 392.7 ± 28.9
Anti-RBD protein total IgG [µg/mL] 83.1 ± 12.3 241.7 ± 17.2 448.6 ± 28.6
pVN 50 titer [reciprocal dilution] 33.0 ± 9.8 192.0 ± 31.4 312.0 ±35.1
The ELISpot assay confirmed a strong T -cell activation with the dose of 1 µg resulting
in the strongest reactivity. An additional ELISpot with CD8+- and CD4+-separated
T cells showed both a reactive CD8+ and CD4+ T-cell response . In Luminex analysis,
chemokines and cytokine production after peptide stimulation was confirmed for the
group dosed with 1 µg for analytes that indicate a T H1-driven and proinflammatory
immune response in line with the ELISpot . Similarly, reactive IFN --, TNF--, and IL -
2-secreting CD4+ as well as CD8+ T cells were detected after peptide stimulation in
ICS. Taken together, the cellular analysis revealed that in addition to a cytotoxic CD8+
T-cell response, a T H1-specific response was activated after peptide stimulation.
In summary, the vaccine c andidate was highly immunogenic and induced high IgG and
neutralizing antibody titers against the antigen as well as a desired T H1-driven T -cell
response including a strong cytotoxic T -cell response. Therefore, BNT162b2 is a
promising candidate for further testing in clinical trial.
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7 DOCUMENT HISTORY
Reason s for change s compared to previous version:
Minor editorial changes, such as the correction of typing errors, are not specifically
listed.
Section s Version 01 Version 02 Reason for change
1 - Further
experimental
information
added Added SPR measurements of binding affinities of
BNT162b2 vaccine -induced SARS -CoV-2-specific
antibodies toward recombinant SARS -CoV-2 S
and RBD proteins. 2.4
3.3
4.5.6
5.2
6
4.4 - Further
equipment and
software
information
added Equipment table was added and software table
was updated.
Sections Version 02 Version 03 Reason for change
List of
Abbreviation s - Update of list Additional abbreviation s included .
2.4 - Further
experimental
information
added Reciprocal endpoint serum titer added for day 14
and day 28 serum samples . 4.5.5
5.1.1
4.5.10 - Luminex The used peptide concentration was corrected
and the CoA of the ProcartaPlex was included . 5.5
9
4.5.11 - ICS The used peptide concentration was corrected .
5.2 - SPR Table corrected .
Sections Version 0 3 Version 0 4 Reason for change
4.5.3.1 - Information for
3 doses of
BNT162b2
modified Doses corrected
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8 REFERENCES
Fukushi S, Mizutani T, Saijo M, Matsuyama S, Miyajima N, Taguchi F et al. Vesicular
Stomatitis Virus Pseudotyped with severe acute respiratory syndrome coronavirus
spike protein. J Gen Virol. 2005;86(Pt 8):2269 -2274.
Hoffmann M, Kleine -Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S et al.
SARS -CoV-2 Cell Entry Depends o n ACE2 and TMPRSS2 and Is Blocked by a
Clinically Proven Protease Inhibitor Cell. 2020;181(2):271 -280.
Kirchdoerfer RN, Wang N, Pallesen J, Wrapp D, Turner HL, Cottrell CA et al. Stabilized
coronavirus spikes are resistant to conformational changes induced by receptor
recognition or proteolysis. Sci Rep. 2018;8(1):15701.
Lawson ND, Stillman EA, Whitt MA, Rose JK. Recombinant vesicular stomatitis viruses
from DNA. Proc Natl Acad Sci U S A. 1995;92(10):4477 -81.
Moyo N, Vogel AB, Buus S, Erbar S, Wee EG, Sahin U et al. Efficient Induction of
T Cells against Conserved HIV -1 Regions by Mosaic Vaccines Delivered as Self -
Amplifying mRNA. Molecular therapy. Methods & clinical development. 2018;12,
32-46.
Pallesen J, Wang N, Corbett KS, Wrapp D, Kirchdoerfer RN, Turner HL et al.
Immunogenicity and structures of a rationally designed prefusion MERS -CoV spike
antigen. Proc Natl Acad Sci USA. 2017;114(35):E7348 -E7357.
Pardi N, Hogan MJ, Pelc RS, Muramasu H, Andersen H, DeMaso CR et al. Zika virus
protection by a single low -dose nucleoside -modified mRNA vaccinatio n. Nature.
2017;543 (7644), 248 -251.
Ullman -Culleré MH, Foltz CJ. Body condition scoring: a rapid and accurate method for
assessing health status in mice. Lab Anim Sci. 1999;49(3):319 -23.
Vogel AB, Lamber t L, Kinnear E, Busse D, Erbar S, Reuter KC et al. Self-amplifying
RNA vaccines give equivalent protection against influenza to mRNA vaccines but at
much lower doses. Molecular therapy: the journal of the American Society of Gene
Therapy. 2017;26 (2), 446 -455.
Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O et al. Cryo -EM
structure of the 2019 -nCoV spike in the prefusion conformation. Science.
2020;367(6483):1260 -1263.
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9 APPENDIX
Appendix 1: Animal Monitoring - Observations
Table 9: Parameters for experimental animal monitoring (single mouse assessment)
The table is separated in immediate euthanasia criteria (end of experiment ) and criteria which solitarily observed do not lead to an imme diate termination , but result in shorter
monitoring frequency (re -assessment).
Observation (if applicable , categorize a):
Code Parameter Renew assessment within < 24 h. Attention:
evaluate cumulation Immediate euthanasia criteria
1 Bodyweight b. Take into account
Body Conditioning Score (BCS) c Body weight loss > 5 – 10% or BCS transition 3
2 Bodyweight loss > 15 - 20% or BCS 2
2 Activity Moderate deviation from normal or u nusual
behavior (e.g. , limited, reduced , or hyperactive
movements) Immobility, very slow movements (high grade of
lethargy), self -isolation
3 Appearance (condition) of fur &
eyes Fur defects/ grooming malfunction (reduced or
exaggerated grooming). Moderate orbital
tightening. Distinct scruffy fur, strongly neglected grooming. Eyes
lids narrowed, eyes closed and sticky.
4 Body cavities & body fluids slight - moderate damp & sticky cavities Clinical signs of disease (diarrhea, distinct sticky)
5 Body temperature & blood
circulation ears - Animal ’s body temperatu re low, ears appear white , and
hardly noticeable blood vessels
6 Posture Moderate deviation of normal physi ological
posture i.e., short pause in hunched posture Abnormal posture, hunched, abnormal ly stretched (belly
touches ground) or cramps
7 Reaction to stimulus d Delayed reaction to unconditioned stimulus,
moderate deviation from normal behavior (e.g. ,
slight - moderate apathy) Abnorm al (distinct delayed reaction to unconditioned
stimulus). Winding and enduring sound utterance (“pain” ),
aggressiveness at touch
8 Automutilation - Burden for the animal noticeable i.e. , missing extremities,
continuous nibbling, biting and gnawing, open wounds
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Observation (if applicable , categorize a):
Code Parameter Renew assessment within < 24 h. Attention:
evaluate cumulation Immediate euthanasia criteria
9 Bites (tail, vibrissae, reproductive
organs ), other wounds Open and bleeding wounds (take care of wounds
and separate animal) Burden for the animal noticeable i.e. , inflamed wounds
10 Respiration frequency Moderate deviation of spontaneous breathing
(normal respiration frequency) High frequency, any sign of dyspnea, gasping, flat
stretched posture in combination with strongly retracting
flanks
11 Motor function Weak, loose grip (cage grid) Staggering, circular movement, missing grasp
12 Other abnormalities e - -
a Categories: NAD , no abnormality detected ; +, slight ; ++, moderate ; +++, distinct.
b Calculate ratio bodyweight start of experiment/ bodyweight monitoring day .
c According to Ullman -Culleré and Foltz 1999 .
d Unconditioned = Stimulus to force a reaction e.g. , normal background noise, tapping the cage , and normal handling procedure e.g. , tilt and turns of the cage .
e Description of abnormality (or abnormalities) on monitoring sheet .
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Table 10: Record of body weight s of experimental mice during study
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 7 Day 14 Day 21 Day 28
SBIO-15164 BIO-LJ26 BALB/cJRj f 28.01 20 1 21.4 21.6 21.6 22 0 21.9 21.7 22.1 22.2 24.4
SBIO-15164 BIO-LJ27 BALB/cJRj f 28.01 20 1 22.0 22.4 21.6 22 2 23.9 22.2 23.1 22.9 24.2
SBIO-15164 BIO-LJ28 BALB/cJRj f 28.01 20 1 19.9 20.8 20.7 21.1 20.7 20.3 21.3 20.9 22.7
SBIO-15164 BIO-LJ29 BALB/cJRj f 28.01 20 1 22.3 22.8 23.1 23.4 22.3 22.7 23.2 24.4 24.2
SBIO-15165 BIO-LJ30 BALB/cJRj f 28.01 20 1 21.6 21.2 20.9 21 5 21.4 21.2 23.4 22.4 23.9
SBIO-15165 BIO-LJ31 BALB/cJRj f 28.01 20 1 20.8 20.8 20.8 21 2 21.2 21.7 21.9 23.4 24.0
SBIO-15165 BIO-LJ32 BALB/cJRj f 28.01 20 1 21.8 22.1 22.2 22 8 22.1 22.6 22.8 23.4 24.2
SBIO-15165 BIO-LJ33 BALB/cJRj f 28.01 20 1 22.1 22.1 21.9 22 5 22.5 22.5 22.7 22.8 23.8
SBIO-15166 BIO-LJ34 BALB/cJRj f 28.01 20 2 20.1 20.6 19.9 20 3 20.0 20.6 20.5 21.6 21.8
SBIO-15166 BIO-LJ35 BALB/cJRj f 28.01 20 2 20.6 20.6 19.8 19.7 20.1 20.1 20.3 20.7 21.8
SBIO-15166 BIO-LJ36 BALB/cJRj f 28.01 20 2 19.7 20.7 20.3 20 5 20.0 20.3 20.1 20.7 21.3
SBIO-15166 BIO-LJ37 BALB/cJRj f 28.01 20 2 20.3 20.2 20.2 20 5 20.7 21.9 21.0 22.5 22.8
SBIO-15167 BIO-LJ38 BALB/cJRj f 28.01 20 2 21.2 21.7 21.6 22.7 21.8 21.6 21.9 23.3 24.1
SBIO-15167 BIO-LJ39 BALB/cJRj f 28.01 20 2 21.2 21.7 21.3 22.1 22.3 21.7 21.8 23.1 23.5
SBIO-15167 BIO-LJ40 BALB/cJRj f 28.01 20 2 19.6 20.8 20.9 20 8 20.7 20.6 20.6 21.4 22.6
SBIO-15167 BIO-LJ41 BALB/cJRj f 28.01 20 2 21.0 21.5 21.4 22.1 22.1 21.9 21.0 22.2 22.9
SBIO-15168 BIO-LJ42 BALB/cJRj f 28.01 20 3 19.7 19.7 19.7 19 5 19.6 19.6 19.9 21.1 20.8
SBIO-15168 BIO-LJ43 BALB/cJRj f 28.01 20 3 21.2 22.5 22.0 22 6 23.1 22.2 22.7 23.3 23.5
SBIO-15168 BIO-LJ44 BALB/cJRj f 28.01 20 3 21.7 21.5 21.8 22 5 22.4 20.9 23.6 21.9 24.4
SBIO-15168 BIO-LJ45 BALB/cJRj f 28.01 20 3 20.8 21.1 21.1 21 8 21.4 21.6 22.6 23.5 23.5
SBIO-15169 BIO-LJ46 BALB/cJRj f 28.01 20 3 20.5 20.6 21.0 20 8 20.7 20.9 21.4 22.0 22.6
SBIO-15169 BIO-LJ47 BALB/cJRj f 28.01 20 3 20.2 20.4 20.5 21 6 20.6 20.5 20.7 21.2 22.8
SBIO-15169 BIO-LJ48 BALB/cJRj f 28.01 20 3 19.8 20.9 20.9 21 5 20.1 20.7 21.9 20.9 23.3
SBIO-15169 BIO-LJ49 BALB/cJRj f 28.01 20 3 20.5 20.7 21.2 21 2 21.1 20.9 21.0 21.4 22.2
SBIO-15170 BIO-LJ50 BALB/cJRj f 28.01 20 4 22.6 22.1 22.9 23 6 23.6 22.7 23.2 24.5 24.9
SBIO-15170 BIO-LJ51 BALB/cJRj f 28.01 20 4 22.4 21.5 21.5 22.1 22.3 22.1 23.0 22.8 23.9
SBIO-15170 BIO-LJ52 BALB/cJRj f 28.01 20 4 21.2 20.9 20.3 20 8 21.2 21.6 21.0 21.7 23.2
SBIO-15170 BIO-LJ53 BALB/cJRj f 28.01 20 4 21.8 20.5 21.2 21 8 22.1 22.1 22.0 22.9 25.1
SBIO-15171 BIO-LJ54 BALB/cJRj f 28.01 20 4 20.3 19.7 19.5 20 2 20.1 20.0 20.5 20.8 22
SBIO-15171 BIO-LJ55 BALB/cJRj f 28.01 20 4 20.5 19.2 19.1 22 9 20 20.8 20.4 20.2 22
SBIO-15171 BIO-LJ56 BALB/cJRj f 28.01 20 4 22 20.8 21.2 21 8 22.2 22.3 22.1 22.9 23.4
SBIO-15171 BIO-LJ57 BALB/cJRj f 28.01 20 4 22.8 21.3 21.3 22 9 22.5 21.8 22.6 24.4 24Bodyweight (grams)
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Table 11: Record of animal monitoring for each mouse during study
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 7 Day 14 Day 21 Day 28
SBIO-15164 BIO-LJ26 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15164 BIO-LJ27 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15164 BIO-LJ28 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15164 BIO-LJ29 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15165 BIO-LJ30 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15165 BIO-LJ31 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15165 BIO-LJ32 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15165 BIO-LJ33 BALB/cJRj f 28.01.20 1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15166 BIO-LJ34 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15166 BIO-LJ35 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15166 BIO-LJ36 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15166 BIO-LJ37 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15167 BIO-LJ38 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15167 BIO-LJ39 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15167 BIO-LJ40 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15167 BIO-LJ41 BALB/cJRj f 28.01.20 2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15168 BIO-LJ42 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15168 BIO-LJ43 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15168 BIO-LJ44 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15168 BIO-LJ45 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15169 BIO-LJ46 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15169 BIO-LJ47 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15169 BIO-LJ48 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15169 BIO-LJ49 BALB/cJRj f 28.01.20 3 NAD 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15170 BIO-LJ50 BALB/cJRj f 28.01.20 4 NAD 12+++ 12++ 12+ NAD NAD NAD NAD NAD
SBIO-15170 BIO-LJ51 BALB/cJRj f 28.01.20 4 NAD 12++ 12++ 12+ NAD NAD NAD NAD NAD
SBIO-15170 BIO-LJ52 BALB/cJRj f 28.01.20 4 NAD 12++ 12++ 12+ NAD NAD NAD NAD NAD
SBIO-15170 BIO-LJ53 BALB/cJRj f 28.01.20 4 NAD 12++ 12+ ok NAD NAD NAD NAD NAD
SBIO-15171 BIO-LJ54 BALB/cJRj f 28.01.20 4 NAD 12++ 12++ 12+ NAD NAD NAD NAD NAD
SBIO-15171 BIO-LJ55 BALB/cJRj f 28.01.20 4 NAD 12+++ 12++ 12+ NAD NAD NAD NAD NAD
SBIO-15171 BIO-LJ56 BALB/cJRj f 28.01.20 4 NAD 12++ 12+ NAD NAD NAD NAD NAD NAD
SBIO-15171 BIO-LJ57 BALB/cJRj f 28.01.20 4 NAD 12++ 12+ NAD NAD NAD NAD NAD NADAnimal Monitoring - Observations
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Appendix 2: Certificates of Analysis
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(b) (4)
(b) (6)
(b) (6)
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(b) (4)
(b) (6)
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(b) (6)
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Appendix 3: Controls for ELISpot Analysis
Figure 21: Controls for ELISpot analysis using splenocytes on day 28
ELISpot assay was performed using splenocytes isolated on day 28 after immunization. Splenocytes were
stimulated with the irrelevant peptide AH-1 (left), or with Concanavalin A or medium alone (right). IFN - secretion
was measured to assess T -cell responses. Mean values ±SEM are shown.
Buffer 0.2 µg 1 µg 5 µg01020304050
AH-1
BNT162b2 (RBP020.2)IFN-+spots/ 1x106 splenocytes
ConA
Medium 0500100015002000 Controls
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Appendix 4: Summary of Luminex Assay Data
Figure 22: TH1 and proinflammatory c ytokine concentrations in supernatants of re -stimulated splenocytes
28 days after immunization
Luminex assay was performed using frozen mouse splenocytes isolated on day 28 after RNA injection to assess
concentrations of the indicated chemokines/cytokines. After 48 h of stimulation with medium, PMA plus ionomycin
(PMA) , or the S -/RBD -overlapping pep tide mix, supernatant was used for the analysis of the secretion of different
analytes. Values for i ndividual animals are shown by dots; group mean values are indicated by lines (±SEM).
Several values were excluded, as they were below the LLOD or out of the standard range (<LLOQ) or upper the
limit of quantification (ULOQ). Therefore, no statistical information was included in the figure (for calculation , see
Appendix 6: Statistical Analysis ). Medium , S 0. 1 µg/mL and RBD 0.66 µg/mL per peptide are shown in Figure 16.
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Figure 23: TH2 cytokine and IL -2 concentrations in supernatants of re -stimulated splenocytes 28 days after
immunization
Luminex assay was performed using frozen mouse splenocytes isolated on day 28 after RNA injection to assess
concentrations of the indicated chemokines/cytokines. After 48 h of stimulation with medium, PMA plus ionomycin
(PMA) , or the S -/RBD -overlapping peptide mix, supernatant was used for the analysis of the secretion of different
analytes. Values for i ndividual animals are shown by dots; group mean values are indicated by lines (±SEM). The
RBD peptide mix was not tested for animals treated with 0.2 µg and 1 µg RNA. Several values were excluded , as
they were below the LLOD or out of the standard range (<LLOQ). Therefore, no statistical information was included
in the figure (for calculation , see Appendix 6: Statistical Analysis ). Medium , S 0. 1 µg/mL and RBD 0.66 µg/mL per
peptide are shown in Figure 16. Note after PMA stimulation, all IL -2 concentration s were above the upper limit of
quantification (ULOQ).
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Appendix 5: Detailed ICS Protocol
FACS panel (FACS Celesta)
Wavelengths Markers µL per
50 µL Clones Colors Name Company Catalog
no.
1 450/50 CD8a 0.25 53-6.7 BV421 Brilliant Violet
421™ anti -
mouse CD8a
antibody BioLegend 100753
2 525/50 CD4 0.25 RM4 -5 BV510 Brilliant Violet 510™
anti-mouse CD4
antibody BioLegend 100559
610/20 no BV605
3 710/50 IL-4 0.25 11B11 BV711 Brilliant Violet
711™ anti -
mouse IL -4
antibody BioLegend 504133
4 780/60 CD25 0.25 PC61 BV786 Brilliant Violet
785™ anti -
mouse CD25
antibody PC61 BioLegend 102051
5 530/30 TNF- 0.5 MP6-XT22 Alexa 488 Alexa Fluor® 488
anti-mouse TNF -
ant body, clone
MP6-XT22 BioLegend 506313
6 575/25 CD3 0.25 145-2C11 PE PE Hamster anti-
mouse CD3e clone
145-2C11 BD 553064
7 695/40 no PerCP -Cy5.5
8 780/60 IFN- 0.1 XMG1.2 Pe-Cy7 PE/Cy7 anti -
mouse IFN -
antibody, clone
XMG1.2 BioLegend 505826
9 670/30 IL-2 0.5 JES6 -5H4 APC APC anti -mouse IL -2
antibody BioLegend 503810
10 730/45 no APC-R700
11 780/60 dead 0.05-0.03 eFluor780 eBioscience™
Fixable Viability
Dye eFluor™
780 ThermoFisher 65-0865 -14
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96-well plate s:
Plate 1: against S protein
1 2 3 4 5 6 7 8 9 10 11 12
A pool*1 pool*1 pool*1 pool*1 1-1 1-1 1-1 1-1
B pool*² pool*² pool*² pool*² 1-2 1-2 1-2 1-2
C pool*³ pool*³ pool*³ pool*³ 1-3 1-3 1-3 1-3
D 1-4 1-4 1-4 1-4
E 1-5 1-5 1-5 1-5
F 1-6 1-6 1-6 1-6
G 1-7 1-7 1-7 1-7
H 1-8 1-8 1-8 1-8
Plate 2: against S protein
1 2 3 4 5 6 7 8 9 10 11 12
A 2-1 2-1 2-1 2-1 3-1 3-1 3-1 3-1 4-1 4-1 4-1 4-1
B 2-2 2-2 2-2 2-2 3-2 3-2 3-2 3-2 4-2 4-2 4-2 4-2
C 2-3 2-3 2-3 2-3 3-3 3-3 3-3 3-3 4-3 4-3 4-3 4-3
D 2-4 2-4 2-4 2-4 3-4 3-4 3-4 3-4 4-4 4-4 4-4 4-4
E 2-5 2-5 2-5 2-5 3-5 3-5 3-5 3-5 4-5 4-5 4-5 4-5
F 2-6 2-6 2-6 2-6 3-6 3-6 3-6 3-6 4-6 4-6 4-6 4-6
G 2-7 2-7 2-7 2-7 3-7 3-7 3-7 3-7 4-7 4-7 4-7 4-7
H 2-8 2-8 2-8 2-8 3-8 3-8 3-8 3-8 4-8 4-8 4-8 4-8
Plate 3: against RBD
1 2 4 5 5 6 7 8 9 10 11 12
A 1-1 1-1 2-1 2-1 3-1 3-1 4-1 4-1
B 1-2 1-2 2-2 2-2 3-2 3-2 4-2 4-2
C 1-3 1-3 2-3 2-3 3-3 3-3 4-3 4-3
D 1-4 1-4 2-4 2-4 3-4 3-4 4-4 4-4
E 1-5 1-5 2-5 2-5 3-5 3-5 4-5 4-5
F 1-6 1-6 2-6 2-6 3-6 3-6 4-6 4-6
G 1-7 1-7 2-7 2-7 3-7 3-7 4-7 4-7
H 1-8 1-8 2-8 2-8 3-8 3-8 4-8 4-8
* controls 1No Ab ²CD3+ L/D ³LD + CD3 + CD4 +
CD8+ L/D
Medium only Positive stimulus S protein
RBD
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Mastermixes for stimulation
Mastermix for blocking reagents
Blocking
reagent Volume
needed/well
[µL] Volume
needed/mL
[µL] Total
wells Total volume
needed (ml;
10 µL/well;
incl 10%
spare) Volume
blocking (µL) Volume DC
medium (µL)
GolgiStop 0,13 13 192 2,112 27,46 2042,30
GolgiPlug 0,2 20 192 2,112 42,24
working concentration Stop (1:1500), Plug (1:1000)
10 µL per well
Stimulation protocol:
1. Prepare a 96 -well tissue culture (F -well)
2. Add 100 µL of stimulus (or medium) to the according well; “pool” on plates are FACS
controls – 100 µL medium is sufficient
3. Add 500,000 cells in DC medium per well (100 µL)
4. Incubate plate for 1 h @ 37°C in 5% CO 2
5. Add 10 µL of blocking reagents
6. Swing plates (5 × 8-moves)
7. Incubate for 5 h @37°C in 5% CO 2
8. Proceed with staining protocol or put plates in 4°C o/n
Mastermix for L/D reagents
Live-dead
reagent Volume
needed/well
[µL] Volume
needed/mL
[µL] Total
wells Total volume
needed (ml;
50 µL/well;
incl 5% spare) Volume L/D
(µL) Volume PBS
(µL)
eFluor780 L/D 0,05 1 200 11 11,00 10989,00
working concentration Stop is 1:1000
50 µL per well
Stimulusconcentration
stock (mg/mL)concentration
needed (mg/mL)concentration
needed (mg/mL) *
2Dilution factor total wellstotal volume
needed (mL;
100µl/well; incl
10% spare)Volume stimulus
(µL)Volume DC
medium (µL)
Medium only - - - - 32 3,52 0 3520
PMA 1 0,0005 0,001 1000 32 3,52 3,52
Ionomycin 10 0,001 0,002 5000 32 3,52 0,70
S protein peptide
mix 1158 peptide 15,8 254,84 64,00 7,04 27,63
S protein peptide
mix 2157 peptide 15,7 253,23 64,00 7,04 27,80
RBD peptides 48 peptides 1,2 0,0048 0,0096 125 64 7,04 56,32 6983,68
the S peptide stocks has a concentration of 100µg/mL per peptide; RBD has 25µg/mLPositive stimulus 3515,78
0,0315 0,062 6984,57
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Antibody mix control 1 mastermix
Markers
(extracellular) µL per
reaction
(50 µL) Total
wells Volume Ab (µL) Volume
FACS buffer
BV510 CD4 0,25 4 1,05 207,9 includes 5% spare
volume
BV421 CD8 0,25 4 1,05
Antibody mix 1 mastermix
Markers
(extracellular) µL per
reaction
(50 µL) Total
wells Volume Ab (µL) Volume
FACS buffer
(µL)
BV421 CD8a 0,25 192 50,40
9928,8 includes 5% spare
volume
BV510 CD4 0,25 192 50,40
BV786 CD25 0,25 192 50,40
Staining protocol:
Note: work with a 4°C cooled centrifuge
9. Mix cells by pipetting 3 × up and down and transfer total volume to ‘v’ bottom plate
10. Centrifuge at 350 ×g, 5 min
11. Wash cells once with 150-200 µL cold PBS
12. Centrifuge at 350 ×g, 5 min. Discard supernatant
a. Vortex cells carefully; snap against the wells to support pellet dissolving
13. Stain with L/D reagen t 50 µL to each well in PBS at 4°C for 15 min
14. Add 100 µL PBS
15. Centrifuge at 350 ×g, 5 min. Discard supernatant; vortex/snap
16. Add antibody master mix 1/antibody mix 1 controls /FACS buffer to each well 50 µL
17. Incubate 30 min at 4°C
18. Add 100 µL FACS buffer
19. Centrifuge at 350 ×g, 5 min. Discard supernatant; vortex/snap
20. Add 2% Histofix
21. Incubate following protocol a, b , or c:
a. keep it in 4°C for overnight (up to ~16 h) if not proceeding for intracellular
staining immediately
b. incubate for at least 1 h at 4°C and
i. proceed for intracellular staining or
c. incubate for at least 1 h at 4°C
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i. add 100 µL PBS
ii. centrifuge at 400 ×g, 5 min. Discard supernatant; vortex/snap
iii. add 100 µL PBS and keep it in 4°C until further usage (within ~36 h)
iv. proceed with intracellular staining protocol
Fc Block mastermix
Fc Block µL per
reaction
(25 µL) Total
wells Volume Ab (µL) Volume perm
buffer (µL)
CD16/CD32 0,5 200 105 4935 includes 5% spare
volume
Antibody mix control 2 mastermix
Markers
(extracellular) µL per
reaction
(50 µL) Total
wells Volume Ab (µL) Volume perm
buffer (µL)
PE CD3 0,25 4 1,05 208,95 includes 5% spare
volume
Antibody mix 2 mastermix
Markers
(intracellular) µL per
reaction
(25 µL) Total
wells Volume Ab (µL) Volume perm
buffer (µL)
BV711 IL-4 0,25 192 50,40
4717,44 includes 5% spare
volume
PE CD3 0,25 192 50,4
Alexa 488 TNF- 0,5 192 100,80
Pe-Cy7 IFN- 0,1 192 20,16
APC IL-2 0,5 192 100,80
Intracellular s taining protocol:
1. Centrifuge at 400 ×g, 5 min, 4°C. Discard supernatant, vortex/snap
2. Suspend cells with perm buffer, 150 µL each well
3. Centrifuge at 400 ×g, 5 min, 4°C. Discard supernatant, vortex/snap
4. Add Fc block 25 µL amount, to each well, incubate for 10 min
5. Add 25 µL of the antibody master mix 2/ antibody mix 2 controls/FACS buffer to the
cells, mix carefully and incubate for another 45 min, 2 -8°C
6. Add 100 µL 1× Perm buffer
7. Centrifuge at 400 ×g, 5 min, 4°C. Discard supernatant, vortex/snap
8. Add 200 µL 1× Perm buffer
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9. Centrifuge at 400 ×g, 5 min, 4°C. Discard supernatant, vortex/snap
10. Discard the supernatant carefully
11. Resuspend the cells carefully in 100 µL FACS buffer or FACS flow
12. Mix cells b y pipetting 3 × up and down and transfer total volume to ‘U’ bottom plate for
FACS Celesta/HTS a cquire
13. Store plate at 4°C until time point of measurement/flow cytometry
a. Note that the plates should be acquired the same day as the intracellular
staining may d ecrease in signal.
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Appendix 6: Statistical Analysis
ELISA
Descriptive statistics, ELISA screening analysis, day 7, S1
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,362 0,544 1,35
Maximum 0,00100 1,22 1,59 2,05
Range 0,00100 0,858 1,05 0,704
Mean 0,000125 0,872 1,14 1,74
SD 0,000354 0,321 0,373 0,245
SEM 0,000125 0,113 0,132 0,0867
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ELISA screening analysis, day 14, S1
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,724 1,09 1,71
Maximum 0,0100 1,53 1,80 2,11
Range 0,0100 0,809 0,713 0,407
Mean 0,00325 1,18 1,45 1,88
SD 0,00388 0,299 0,218 0,143
SEM 0,00137 0,106 0,0772 0,0504
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ELISA screening analysis, day 21, S1
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,815 1,36 1,97
Maximum 0,0100 1,67 2,04 2,33
Range 0,0100 0,858 0,672 0,357
Mean 0,00138 1,24 1,67 2,12
SD 0,00350 0,327 0,219 0,122
SEM 0,00124 0,116 0,0775 0,0430
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA screening
analysis, day 7, S1
ANOVA summary
F 55,55
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,8562
Please note that commas are used as decimal separators.
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,8714 -1,213 to -0,5301 Yes **** <0,0001
Buffer vs. 1 µg -1,135 -1,477 to -0,7940 Yes **** <0,0001
Buffer vs. 5 µg -1,745 -2,086 to -1,403 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA screening
analysis, day 14, S1
ANOVA summary
F 131,1
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9335
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -1,179 -1,425 to -0,9326 Yes **** <0,0001
Buffer vs. 1 µg -1,449 -1,695 to -1,202 Yes **** <0,0001
Buffer vs. 5 µg -1,875 -2,121 to -1,629 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA screening
analysis, day 21, S1
ANOV A summary
F 156,3
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9436
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -1,240 -1,496 to -0,9841 Yes **** <0,0001
Buffer vs. 1 µg -1,672 -1,928 to -1,417 Yes **** <0,0001
Buffer vs. 5 µg -2,115 -2,370 to -1,859 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Descriptive statistics, ELISA screening analysis, day 7 , RBD
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,253 0,869 1,51
Maximum 0,00800 1,57 1,84 2,17
Range 0,00800 1,32 0,971 0,661
Mean 0,00288 0,930 1,38 1,85
SD 0,00270 0,473 0,330 0,221
SEM 0,000953 0,167 0,117 0,0783
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ELISA screening analysis, day 14 , RBD
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,920 1,18 1,84
Maximum 0,0130 1,64 1,96 2,39
Range 0,0130 0,720 0,785 0,550
Mean 0,00388 1,28 1,57 2,07
SD 0,00476 0,293 0,265 0,195
SEM 0,00168 0,104 0,0936 0,0690
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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Descriptive statistics, ELISA screening analysis, day 21, RBD
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00100 0,747 1,56 2,04
Maximum 0,00400 1,65 1,98 2,40
Range 0,00300 0,907 0,424 0,362
Mean 0,00250 1,20 1,82 2,21
SD 0,00107 0,392 0,150 0,140
SEM 0,000378 0,138 0,0529 0,0493
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA screening
analysis, day 7 , RBD
ANOVA summary
F 52,18
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,8483
Please note that commas are used as decimal separators.
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,9270 -1,311 to -0,5432 Yes **** <0,0001
Buffer vs. 1 µg -1,381 -1,765 to -0,9975 Yes **** <0,0001
Buffer vs. 5 µg -1,852 -2,236 to -1,468 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA s creening
analysis, day 14 , RBD
ANOVA summary
F 128,0
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9320
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Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -1,274 -1,547 to -1,000 Yes **** <0,0001
Buffer vs. 1 µg -1,571 -1,844 to -1,297 Yes **** <0,0001
Buffer vs. 5 µg -2,065 -2,338 to -1,791 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F-statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA screening
analysis, day 21, RBD
ANOVA summary
F 152,6
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9424
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -1,198 -1,472 to -0,9232 Yes **** <0,0001
Buffer vs. 1 µg -1,813 -2,087 to -1,539 Yes **** <0,0001
Buffer vs. 5 µg -2,211 -2,485 to -1,937 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Descriptive statistics, ELISA, IgG concentrations, day 28, S1
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 35,8 117 312
Maximum 0,00 117 289 536
Range 0,00 81,5 172 224
Mean 0,00 73,0 206 393
SD 0,00 29,3 59,3 81,7
SEM 0,00 10,4 21,0 28,9
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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Descriptive statistics, ELISA, IgG concentrations, day 28, RBD
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 36,7 161 345
Maximum 0,00 129 298 583
Range 0,00 92,8 137 238
Mean 0,00 83,1 242 449
SD 0,00 34,9 48,5 80,9
SEM 0,00 12,3 17,2 28,6
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA, IgG
concentrations, day 28, S1
ANOVA summary
F 86,02
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9021
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -72,97 -138,3 to -7,684 Yes * 0,0259
Buffer vs. 1 µg -205,9 -271,2 to -140,6 Yes **** <0,0001
Buffer vs. 5 µg -392,7 -458,0 to -327,4 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ELISA, IgG
concentrations, day 28, RBD
ANOVA summary
F 123,4
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9297
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Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -83,10 -145,5 to -20,66 Yes ** 0,0072
Buffer vs. 1 µg -241,7 -304,2 to -179,3 Yes **** <0,0001
Buffer vs. 5 µg -448,6 -511,0 to -386,1 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F-statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Tukey ’s multiple comparisons post -test, ELISA, reciprocal
serum endpoint titer , S1
ANOVA summary
F 13,76
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,6276
Tukey ’s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg (day
14) -66525 -470716 to 337666 No ns 0,9987
Buffer vs. 1 µg (day 14) -84750 -488941 to 319441 No ns 0,9949
Buffer vs. 5 µg (day 14) -291300 -695491 to 112891 No ns 0,3063
Buffer vs. 0.2 µg (day
28) -163725 -567916 to 240466 No ns 0,8727
Buffer vs. 1 µg (day 28) -491775 -895966 to -87584 Yes ** 0,0082
Buffer vs. 5 µg (day 28) -983850 -1388041 to -579659 Yes **** <0,0001
0.2 µg (day 14) vs. 1 µg
(day 14) -18225 -422416 to 385966 No ns >0,9999
0.2 µg (day 14) vs. 5 µg
(day 14) -224775 -628966 to 179416 No ns 0,6131
0.2 µg (day 14) vs. 0.2
µg (day 28) -97200 -501391 to 306991 No ns 0,9893
0.2 µg (day 14) vs. 1 µg
(day 28) -425250 -829441 to -21059 Yes * 0,0332
0.2 µg (day 14) vs. 5 µg
(day 28) -917325 -1321516 to -513134 Yes **** <0,0001
1 µg (day 14) vs. 5 µg
(day 14) -206550 -610741 to 197641 No ns 0,7009
1 µg (day 14) vs. 0.2 µg
(day 28) -78975 -483166 to 325216 No ns 0,9965
1 µg (day 14) vs. 1 µg
(day 28) -407025 -811216 to -2834 Yes * 0,0474
1 µg (day 14) vs. 5 µg
(day 28) -899100 -1303291 to -494909 Yes **** <0,0001
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Tukey ’s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
5 µg (day 14) vs. 0.2 µg
(day 28) 127575 -276616 to 531766 No ns 0,9580
5 µg (day 14) vs. 1 µg
(day 28) -200475 -604666 to 203716 No ns 0,7289
5 µg (day 14) vs. 5 µg
(day 28) -692550 -1096741 to -288359 Yes **** <0,0001
0.2 µg (day 28) vs. 1 µg
(day 28) -328050 -732241 to 76141 No ns 0,1837
0.2 µg (day 28) vs. 5 µg
(day 28) -820125 -1224316 to -415934 Yes **** <0,0001
1 µg (day 28) vs. 5 µg
(day 28) -492075 -896266 to -87884 Yes ** 0,0081
One-way ANOVA with Tukey ’s multiple comparisons post -test, ELISA, reciprocal
serum endpoint titer , RBD
ANOVA summary
F 17,63
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,6834
Tukey’ s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg (day
14) -66525 -250933 to 117883 No ns 0,9224
Buffer vs. 1 µg (day 14) -78675 -263083 to 105733 No ns 0,8433
Buffer vs. 5 µg (day 14) -327750 -512158 to -143342 Yes **** <0,0001
Buffer vs. 0.2 µg (day
28) -60450 -244858 to 123958 No ns 0,9498
Buffer vs. 1 µg (day 28) -218400 -402808 to -33992 Yes * 0,0109
Buffer vs. 5 µg (day 28) -491775 -676183 to -307367 Yes **** <0,0001
0.2 µg (day 14) vs. 1 µg
(day 14) -12150 -196558 to 172258 No ns >0,9999
0.2 µg (day 14) vs. 5 µg
(day 14) -261225 -445633 to -76817 Yes ** 0,0012
0.2 µg (day 14) vs. 0.2
µg (day 28) 6075 -178333 to 190483 No ns >0,9999
0.2 µg (day 14) vs. 1 µg
(day 28) -151875 -336283 to 32533 No ns 0,1707
0.2 µg (day 14) vs. 5 µg
(day 28) -425250 -609658 to -240842 Yes **** <0,0001
1 µg (day 14) vs. 5 µg
(day 14) -249075 -433483 to -64667 Yes ** 0,0024
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Tukey’ s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
1 µg (day 14) vs. 0.2 µg
(day 28) 18225 -166183 to 202633 No ns >0,9999
1 µg (day 14) vs. 1 µg
(day 28) -139725 -324133 to 44683 No ns 0,2513
1 µg (day 14) vs. 5 µg
(day 28) -413100 -597508 to -228692 Yes **** <0,0001
5 µg (day 14) vs. 0.2 µg
(day 28) 267300 82892 to 451708 Yes *** 0,0009
5 µg (day 14) vs. 1 µg
(day 28) 109350 -75058 to 293758 No ns 0,5396
5 µg (day 14) vs. 5 µg
(day 28) -164025 -348433 to 20383 No ns 0,1112
0.2 µg (day 28) vs. 1 µg
(day 28) -157950 -342358 to 26458 No ns 0,1385
0.2 µg (day 28) vs. 5 µg
(day 28) -431325 -615733 to -246917 Yes **** <0,0001
1 µg (day 28) vs. 5 µg
(day 28) -273375 -457783 to -88967 Yes *** 0,0007
Descriptive statistics, IgG subtype -specific ELISA, day 28, IgG1
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,498 1,04 1,71
Maximum 0,00300 1,57 1,70 2,68
Range 0,00300 1,07 0,651 0,966
Mean 0,000375 1,00 1,40 2,20
SD 0,00106 0,399 0,229 0,319
SEM 0,000375 0,141 0,0811 0,113
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, IgG subtype -specific ELISA, day 28, IgG2 a
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,00 0,206 0,811 2,21
Maximum 0,00100 1,48 2,37 2,68
Range 0,00100 1,27 1,56 0,462
Mean 0,000125 0,640 1,82 2,48
SD 0,000354 0,399 0,515 0,167
SEM 0,000125 0,141 0,182 0,0589
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, IgG subtype -specific
ELISA, day 28, IgG1
ANOVA summary
F 85,19
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9013
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -1,004 -1,351 to -0,6567 Yes **** <0,0001
Buffer vs. 1 µg -1,396 -1,743 to -1,049 Yes **** <0,0001
Buffer vs. 5 µg -2,197 -2,544 to -1,849 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post-test, IgG subtype -specific
ELISA, day 28, IgG2 a
ANOVA summary
F 88,60
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9047
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,6398 -1,057 to -0,2221 Yes ** 0,0020
Buffer vs. 1 µg -1,821 -2,238 to -1,403 Yes **** <0,0001
Buffer vs. 5 µg -2,475 -2,893 to -2,057 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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Descriptive statistics, ELISA, IgG2a/IgG1 ratio, day 28
0.2 µg 1 µg 5 µg
Number of values 8 8 8
Minimum 0,272 0,777 0,827
Maximum 1,27 1,81 1,56
Range 0,998 1,03 0,737
Mean 0,635 1,29 1,15
SD 0,307 0,311 0,227
SEM 0,109 0,110 0,0804
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Tukey’s multiple comparisons post -test ELISA, IgG2a/IgG1
ratio, day 28
ANOVA summary
F 11,92
P value 0,0003
P valu e summary ***
Significant diff. among means (P < 0.05)? Yes
R square 0,5317
Tukey’ s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
0.2 µg vs. 1 µg -0,6595 -1,018 to -0,3009 Yes *** 0,0004
0.2 µg vs. 5 µg -0,5186 -0,8771 to -0,1600 Yes ** 0,0041
1 µg vs. 5 µg 0,1409 -0,2177 to 0,4995 No ns 0,5907
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Pseudovirus -based neutralization test
Descriptive statistics, pVNT, day 14
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 6,00 6,00 12,0 48,0
Maximum 6,00 96,0 192 192
Range 0,00 90,0 180 144
Mean 6,00 21,8 46,5 90,0
SD 0,00 31,1 59,6 47,6
SEM 0,00 11,0 21,1 16,8
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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Descriptive statistics, pVNT, day 21
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 6,00 6,00 48,0 96,0
Maximum 6,00 96,0 384 384
Range 0,00 90,0 336 288
Mean 6,00 30,8 144 228
SD 0,00 31,3 112 102
SEM 0,00 11,1 39,5 36,0
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, pVNT, day 28
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 6,00 12,0 96,0 192
Maximum 6,00 96,0 384 384
Range 0,00 84,0 288 192
Mean 6,00 33,0 192 312
SD 0,00 27,8 88,9 99,4
SEM 0,00 9,82 31,4 35,1
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, pVNT, day 14
ANOVA summary
F 6,330
P value 0,0021
P valu e summary **
Significant diff. among means (P < 0.05)? Yes
R square 0,4041
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -15,75 -66,89 to 35,39 No ns 0,7862
Buffer vs. 1 µg -40,50 -91,64 to 10,64 No ns 0,1440
Buffer vs. 5 µg -84,00 -135,1 to -32,86 Yes *** 0,0010
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, pVNT, day 21
ANOVA summary
F 14,28
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,6047
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -24,75 -120,6 to 71,14 No ns 0,8582
Buffer vs. 1 µg -138,0 -233,9 to -42,11 Yes ** 0,0036
Buffer vs. 5 µg -222,0 -317,9 to -126,1 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, pVNT, day 28
ANOVA summary
F 35,44
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,7916
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -27,00 -111,5 to 57,54 No ns 0,7684
Buffer vs. 1 µg -186,0 -270,5 to -101,5 Yes **** <0,0001
Buffer vs. 5 µg -306,0 -390,5 to -221,5 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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ELIS pot analysis
Descriptive statistics, day 28, fresh splenocytes, S protein
Buffer control 0.2 µg 1 µg
Number of values 8 8 8
Minimum 6,00 228 858
Maximum 13,7 626 1334
Range 7,67 399 477
Mean 8,54 334 1054
SD 2,72 134 177
SEM 0,961 47,4 62,6
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, day 28, fresh splenocytes, RBD
Buffer control 0.2 µg 1 µg
Number of values 8 8 8
Minimum 1,67 20,0 33,0
Maximum 8,00 73,0 126
Range 6,33 53,0 93,3
Mean 4,46 36,3 87,3
SD 1,90 19,6 29,2
SEM 0,672 6,93 10,3
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28 , fresh
splenocytes, S protein
ANOVA summary
F 139,2
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9299
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -325,0 -477,0 to -173,0 Yes *** 0,0001
Buffer vs. 1 µg -1045 -1197 to -893,3 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28 , fresh
splenocytes, RBD
ANOVA summary
F 33,83
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,7631
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -31,83 -55,90 to -7,763 Yes ** 0,0094
Buffer vs. 1 µg -82,79 -106,9 to -58,72 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Descriptive statistics, day 28 , frozen splenocytes, S protein
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,667 24,7 157 51,7
Maximum 4,67 137 277 232
Range 4,00 112 121 180
Mean 2,13 76,4 206 139
SD 1,49 35,9 52,3 66,7
SEM 0,527 12,7 18,5 23,6
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, day 28, frozen splenocytes, RBD
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,333 1,33 12,3 4,67
Maximum 4,67 20,0 33,0 42,7
Range 4,33 18,7 20,7 38,0
Mean 2,63 9,38 21,8 17,9
SD 1,20 6,72 7,21 12,0
SEM 0,425 2,38 2,55 4,25
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28 , frozen
splenocytes, S protein
ANOVA summary
F 28,56
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,7537
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -74,25 -131,4 to -17,11 Yes ** 0,0087
Buffer vs. 1 µg -203,6 -260,8 to -146,5 Yes **** <0,0001
Buffer vs. 5 µg -136,5 -193,6 to -79,32 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28 , frozen
splenocytes, RBD
ANOVA summary
F 9,726
P value 0,0001
P valu e summary ***
Significant diff. among means (P < 0.05)? Yes
R square 0,5103
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -6,750 -16,43 to 2,933 No ns 0,2210
Buffer vs. 1 µg -19,17 -28,85 to -9,484 Yes *** 0,0001
Buffer vs. 5 µg -15,25 -24,93 to -5,567 Yes ** 0,0015
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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Descriptive statistics, day 28, ELISpot after MACS
CD4 CD8
AH-1 RBD S AH-1 RBD S
Number of values 8 8 8 8 8 8
Minimum 0,00 1,50 66,0 1,00 21,5 173
Maximum 4,50 23,0 155 3,50 62,5 477
Range 4,50 21,5 89,0 2,50 41,0 304
Mean 1,81 13,0 98,8 2,19 43,6 287
SD 1,62 7,75 34,6 0,843 15,6 104
SEM 0,574 2,74 12,2 0,298 5,51 36,9
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28, ELISpot after
MACS, CD4+
ANOVA summary
F 53,66
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,8363
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
AH-1 vs. RBD -11,19 -35,47 to 13,10 No ns 0,4568
AH-1 vs. S -97,00 -121,3 to -72,71 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, day 28, ELISpot after
MACS, CD8+
ANOVA summary
F 51,01
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,8293
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
AH-1 vs. RBD -41,44 -113,6 to 30,70 No ns 0,3119
AH-1 vs. S -284,5 -356,6 to -212,4 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Intracellular cytokine staining
Descriptive statistics, ICS, CD4+, IFN -
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0500 0,110 0,115 0,0730
Maximum 0,104 0,175 0,220 0,240
Range 0,0540 0,0650 0,105 0,167
Mean 0,0858 0,145 0,159 0,119
SD 0,0172 0,0220 0,0317 0,0550
SEM 0,00606 0,00779 0,0112 0,0194
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ICS, CD4+, IL-4
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0750 0,0695 0,0780 0,0590
Maximum 0,110 0,129 0,120 0,184
Range 0,0350 0,0590 0,0420 0,125
Mean 0,0934 0,107 0,101 0,0968
SD 0,0116 0,0180 0,0163 0,0471
SEM 0,00411 0,00638 0,00577 0,0166
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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Descriptive statistics, ICS, CD4+, TNF -
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0740 0,0785 0,150 0,130
Maximum 0,170 0,155 0,250 0,265
Range 0,0960 0,0765 0,100 0,135
Mean 0,0992 0,113 0,190 0,193
SD 0,0305 0,0243 0,0317 0,0514
SEM 0,0108 0,00858 0,0112 0,0182
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ICS, CD4+, IL-2
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0685 0,110 0,130 0,0995
Maximum 0,130 0,235 0,220 0,660
Range 0,0615 0,125 0,0900 0,561
Mean 0,0968 0,158 0,175 0,223
SD 0,0210 0,0365 0,0331 0,195
SEM 0,00741 0,0129 0,0117 0,0689
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD4+, IFN -
ANOVA summary
F 20
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,66
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,059 -0,088 to -0,030 Yes *** 0,0002
Buffer vs. 1 µg -0,073 -0,10 to -0,044 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD4+, IL-4
ANOVA summary
F 1,6
P value 0,2304
P valu e summary ns
Significant diff. among means (P < 0.05)? No
R square 0,13
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. ns: Not significant.
No post -test for insignificant main test.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD4+, TNF -
ANOVA summary
F 23
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,69
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,013 -0,048 to 0,021 No ns 0,5633
Buffer vs. 1 µg -0,091 -0,13 to -0,056 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not signi ficant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD4+, IL-2
ANOVA summary
F 14
P value 0,0001
P valu e summary ***
Significant diff. among means (P < 0.05)? Yes
R square 0,57
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,061 -0,097 to -0,024 Yes ** 0,0015
Buffer vs. 1 µg -0,078 -0,11 to -0,042 Yes *** 0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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Descriptive statistics, ICS, CD8+, IFN -
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0340 0,190 0,435 0,210
Maximum 0,102 0,700 0,875 0,645
Range 0,0680 0,510 0,440 0,435
Mean 0,0594 0,361 0,704 0,463
SD 0,0223 0,162 0,141 0,134
SEM 0,00789 0,0572 0,0500 0,0472
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ICS, CD8+, TNF -
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,104 0,270 0,450 0,270
Maximum 0,205 0,750 0,945 0,670
Range 0,102 0,480 0,495 0,400
Mean 0,150 0,410 0,723 0,524
SD 0,0350 0,162 0,154 0,134
SEM 0,0124 0,0571 0,0543 0,0475
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
Descriptive statistics, ICS, CD8+, IL-2
Buffer control 0.2 µg 1 µg 5 µg
Number of values 8 8 8 8
Minimum 0,0585 0,165 0,170 0,135
Maximum 0,117 0,290 0,400 0,555
Range 0,0580 0,125 0,230 0,420
Mean 0,0840 0,214 0,286 0,268
SD 0,0198 0,0504 0,0794 0,172
SEM 0,00699 0,0178 0,0281 0,0608
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD8+, IFN -
ANOVA summary
F 54
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,84
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,30 -0,45 to -0,15 Yes *** 0,0002
Buffer vs. 1 µg -0,64 -0,79 to -0,50 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD8+, TNF -
ANOVA summary
F 39
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,79
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,26 -0,41 to -0,11 Yes ** 0,0013
Buffer vs. 1 µg -0,57 -0,73 to -0,42 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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One-way ANOVA with Dunnett’s multiple comparisons post -test, ICS, CD8+, IL-2
ANOVA summary
F 27
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,72
Dunnett’s multiple
comparisons test Mean d iff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer vs. 0.2 µg -0,13 -0,20 to -0,064 Yes *** 0,0003
Buffer vs. 1 µg -0,20 -0,27 to -0,14 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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1Von:
Gesendet: Montag, 23. November 2020 14:17
An:
Cc:
Betreff: Final R-20-0085 version 4.0 report, with signatures
Anlagen: R-20-0085 modRNA V9_Report_V4.0_signatures.pdf
Kennzeichnung: Zur Nachverfolgung
Kennzeichnungsstatus: Gekennzeichnet
Hi ,
Attached here is the final updated PDF for R-20-0085 version 4. With this email, I’m giving my approval as Author. I’ll
wet-ink sign this document when I’m next in Mainz. Best,
BioNTech SE
090177e19598ca29\Approved\Approved On: 23-Nov-2020 20:20 (GMT)
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