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-20-0112
CHARACTERIZING THE I MMUNOPHENOTYPE IN
SPLEEN AND LYMPH NOD E OF MICE TREATED
WITH SARS -COV-2 VACCINE CANDIDATES
Version 01
Date: 13 AUG 2020
Reported by
Test item: BNT162a1, BNT162b1, BNT162b2, BNT162c2
Key w ords: Covid -19, SARS -CoV-2, Vaccine, BALB/c mice, immunophenotyping
This R&D report consists of 105 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, investigato rs, 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
LIST OF FIGURE S ................................ ................................ ............... 5
LIST OF TABLES ................................ ................................ ................. 5
LIST OF ABBREVIATION S ................................ ................................ . 8
RESPONSIBILITIES ................................ ................................ ............ 9
1 SUMMARY ................................ ................................ ......................... 10
2 GENERAL INFORMATION ................................ ................................ 12
2.1 Sponsor and Test Facilities ................................ ................................ 12
2.2 Participating Personnel ................................ ................................ ....... 12
2.3 Study Dates ................................ ................................ ........................ 13
2.4 Guidelines and Regulations ................................ ................................ 14
2.5 Changes and Deviations ................................ ................................ ..... 14
2.6 Documentation and Archive ................................ ................................ 20
3 INTRODUCTI ON ................................ ................................ ................ 22
3.1 Background ................................ ................................ ........................ 22
3.2 Objectives ................................ ................................ ........................... 23
3.3 Study Design ................................ ................................ ...................... 23
4 MATERIALS AND METHOD S ................................ ........................... 27
4.1 Test Item ................................ ................................ ............................. 27
4.2 Control Item ................................ ................................ ........................ 27
4.3 Test System ................................ ................................ ........................ 27
4.4 Materials ................................ ................................ ............................. 27
4.5 Methods ................................ ................................ .............................. 34
4.5.1 Animal Care ................................ ................................ ........................ 34
4.5.1.1 General Information ................................ ................................ ............ 34
4.5.1.2 Housing Condition and Husbandry ................................ ..................... 34
4.5.2 Animal Monitoring ................................ ................................ ............... 34
4.5.3 Animal Treatment ................................ ................................ ............... 35
4.5.3.1 Treatment Schedul e, Route of Administration, and Dose ................... 35
4.5.3.2 Immunization ................................ ................................ ...................... 35
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4.5.3.3 Blood Sampling via the Retro -Orbital Venous Plexus or Vena
Facialis ................................ ................................ ............................... 35
4.5.4 Endpoint of Experiment / Termination Criteria ................................ .... 36
4.5.4.1 Dissection of Animals and Organ Collection ................................ ....... 36
4.5.5 Preparation of Splenocyte Single Cell Suspensions ........................... 36
4.5.6 Preparation of Lymph Node Single Cell Suspensions ........................ 37
4.5.7 RNA Electroporation ................................ ................................ ........... 37
4.5.8 ELISpot Assay ................................ ................................ .................... 37
4.5.9 xCELLigence Cytotoxicity Assay ................................ ........................ 38
4.5.10 Cytokine Multiplex Protein Quantification ................................ ........... 38
4.5.11 Flow Cytometry ................................ ................................ ................... 39
4.5.11.1 Restimulation of T cells for functional T cell analysis in the spleen and
dLN ................................ ................................ ................................ ..... 39
4.5.11.2 Functional T cell analysis in the spleen and dLN ................................ 39
4.5.11.3 Phenotypic T cell analysis in the spleen and dLN ............................... 42
4.5.11.4 Phenotypic T cell analysis in the blood ................................ ............... 44
4.5.11.5 B cell analysis in the spleen and dLN ................................ ................. 47
4.5.11.6 Myeloid cell analysis in the spleen ................................ ...................... 50
4.5.12 Statistical Analysis ................................ ................................ .............. 51
5 RESULTS ................................ ................................ ........................... 53
5.1 ELISpot assay ................................ ................................ ..................... 53
5.2 Flow Cytometry ................................ ................................ ................... 54
5.3 Cytokine Multiplex Assay ................................ ................................ .... 59
5.4 xCELLigence Cytotoxicity Assay ................................ ........................ 61
6 CONCLUSION ................................ ................................ .................... 64
7 DOCUMENT HISTORY ................................ ................................ ...... 65
8 REFERENCES ................................ ................................ ................... 66
9 APPENDIX ................................ ................................ ......................... 67
9.1 Animal Monitoring ................................ ................................ ............... 67
9.2 Animal Monitoring - Observations ................................ ....................... 67
9.3 ELISpot – Raw dat a ................................ ................................ ............ 73
9.4 Cytokine multiplex analysis – Assay detection ranges ........................ 75
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9.5 Cytokine multiplex analysis – Raw data and calculated data .............. 76
9.6 Certificates of Analysis BNT162a1 ................................ ..................... 88
9.7 Certificates of Analysis BNT162b1 ................................ ..................... 90
9.8 Certificates of Analysis BNT162b2 ................................ ..................... 93
9.9 Certificates of Analysis BNT162c2 ................................ ...................... 96
9.10 Statistical analysis ................................ ................................ .............. 98
9.11 List of attachments ................................ ................................ ............ 104
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LIST OF FIGURE S
Figure 1: Schematic overview of the S protein structure of the SARS -CoV S protein
................................ ................................ ................................ ............................ 22
Figure 2: Workflow of part 1 of the study (mCorVac#15) ................................ ..... 25
Figure 3: Workflow of part 2 of the study (mCorVac#16) ................................ ..... 25
Figure 4: Analysis and assay overview ................................ ................................ 26
Figure 5: Draining lymph nodes resection for subsequent analysis ..................... 36
Figure 6: ELISpot analysis using splenocytes from animals treated with BNT162a1,
BNT162b1, BNT162b2 or BNT162c2 ................................ ................................ .. 53
Figure 7: Analysis of lymphocyte frequen cies in the blood of BNT162a1,
BNT162b1, BNT162b2 or BNT162c2 vaccinated mice ................................ ........ 55
Figure 8: Analysis of T cell activation in the blood of BNT162a1, BNT162b1,
BNT162b2 or BNT162c2 vaccinated mice ................................ ........................... 56
Figure 9: Analysis of T cell counts in the dLNs of BNT162a1, BNT162b1,
BNT162b2 or BNT162c2 vaccinated mice ................................ ........................... 57
Figure 10: Analysi s of B cell counts in the dLNs of BNT162a1, BNT162b1,
BNT162b2 or BNT162c2 vaccinated mice ................................ ........................... 57
Figure 11: Analysis of T FH and B cell counts in the spleen of BNT162a1, BNT162b1,
BNT162b2 or BNT162 c2 vaccinated mice ................................ ........................... 58
Figure 12: Quantification of cytokine secreting T cells upon S peptide restimulation
in the spleen of BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccinated
mice ................................ ................................ ................................ ..................... 59
Figure 13: Quantification of cytokine secretion upon S peptide restimulation of
splenocytes of BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccinated
mice ................................ ................................ ................................ ..................... 61
Figure 14: Cytotoxicity towards S protein expressing CT26 cells by CD8+
splenocytes from BNT162a1 or BNT162b1 vaccinated mice (mCorVAC#15). .... 62
Figure 15: Cytotoxicity towards S prot ein expressing CT26 cells by CD8+
splenocytes from BNT162b2 or BNT162c2 vaccinated mice (mCorVAC#16). .... 63
LIST OF TABLES
Table 1: Changes and deviations to R&D study plan ................................ .......... 15
Table 2: Clinical stage SARS -CoV-2 vaccine candidates developed at
BioNTech ................................ ................................ ................................ ............. 23
Table 3: Materials ................................ ................................ ................................ 28
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Table 4: Peptide pool for restimulation of splenocytes and dLN cells for ELISpot
assays, flo w cytometry and cytokine multiplex assay ................................ .......... 31
Table 5: RNAs used for CT26 electroporation ................................ ..................... 32
Table 6: Software ................................ ................................ ................................ 33
Table 7: Mac hines ................................ ................................ ............................... 33
Table 8: Flow cytometry antibody master mixes for functional T cell analysis in the
spleen and dLN (mCorVAC#15 and mCorVAC#1 6). ................................ ........... 40
Table 9: Flow cytometry antibody master mixes for phenotypic T cell analysis in
the spleen and dLN (mCorVAC#15 and mCorVAC#16) . ................................ ..... 42
Table 10: Flow cytometry antibody master mixes for phenotypicT cell analysis in
the blood (mCorVAC#15 and mCorVAC#16). ................................ ..................... 45
Table 11: Flow cytometry antibody master mixes for B cell analysis in the spleen
and dLN (mCorVAC#15 and mCorVAC#16). ................................ ....................... 48
Table 12: Flow cytometry antibody master mixes for myeloid cell analysis in the
spleen (mCorVAC#15 and mCorVAC#16). ................................ ......................... 50
Table 13: Statistical analyses ................................ ................................ .............. 51
Table 14: Parameters for experimental animal monitoring (single animal
assessment) ................................ ................................ ................................ ........ 67
Table 15: Record of body weights of mCorVAC#15 animals during study .......... 69
Table 16: Record of animal monitoring during CorVac#15 study ......................... 70
Table 17: Record of body weights of CorVac#16 animals during study ............... 71
Table 18: Record of animal monitoring during CorVac#16 study ......................... 72
Table 19: ELISpot raw data. ................................ ................................ ................ 73
Table 20: Detection ranges of the ProcartaPlex immunoassay for mCorVAC#15
and mCorVAC#16. ................................ ................................ .............................. 75
Table 21: Cytokine raw data and calculated data for mCorVAC#15, part 1 of 6 (SP)
................................ ................................ ................................ ............................ 76
Table 22: Cytokine raw data and calculated data for mCorVAC#15, part 2 of 6 (SP)
................................ ................................ ................................ ............................ 77
Table 23: Cytokine raw data and calculated data for mCorVAC#15, part 3 of 6 (SP)
................................ ................................ ................................ ............................ 78
Table 24: Cytokine raw data and calculated data for mCorVAC#15, part 4 of 6 (SP)
................................ ................................ ................................ ............................ 79
Table 25: Cytokine raw data and calculated data for mCorVAC#15, part 5 of 6 (SP)
................................ ................................ ................................ ............................ 80
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Table 26: Cytokine raw d ata and calculated data for mCorVAC#15, part 6 of 6 (LN)
................................ ................................ ................................ ............................ 81
Table 27: Cytokine raw data and calculated data for mCorVAC#16, part 1 o f 6 (SP)
................................ ................................ ................................ ............................ 82
Table 28: Cytokine raw data and calculated data for mCorVAC#16, part 2 of 6 (SP)
................................ ................................ ................................ ............................ 83
Table 29: Cytokine raw data and calculated data for mCorVAC#16, part 3 of 6 (SP)
................................ ................................ ................................ ............................ 84
Table 30: Cytokine raw data and calculated data for mCorVAC#16, part 4 of 6 (SP)
................................ ................................ ................................ ............................ 85
Table 31: Cytokine raw data and calculated data for mCorVA C#16, part 5 of 6 (LN)
................................ ................................ ................................ ............................ 86
Table 32: Cytokine raw data and calculated data for mCorVAC#16, part 6 of 6 (LN)
................................ ................................ ................................ ............................ 87
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LIST OF ABBREVIATION S
Ab Antibody
CP Cytoplasmic domain
dLNs Draining lymph nodes
EDTA Ethylenediaminetetraacetic acid
FBS Fetal bovine serum
FM Fluorescence minus
FP Fusion peptide
GC Germinal center
HR1,
HR2 Heptad repeats 1 and 2
i.m. Intramuscular
IFNγ Interferon gamma
Ig Immunoglobulin
IL Interleukin
IL Iliac
IN Inguinal
LD LiveDead viability dye
LN Lymph node
LNPs Lipid nanoparticles
Lot Lot number
MM Master mix
modRNA Nucleoside modified mRNA
NEAA Non-essential amino acids
PO Poplitea l
RBD Receptor binding domain
RBM Receptor binding motif
S Spike protein
saRNA Self-amplifying RNA
SP Signal peptide
TFH Follicular helper T cells
TH T helper cells
TM Transmembrane domain
TNF Tumor necrosis factor
uRNA Non-modified uridine -conta ining mRNA
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1 SUMMA RY
BioNTech is developing RNA -based vaccines designed to protect against the novel
coronavirus disease that emerged in 2019 (COVID -19). The project involves testing
three RNA platforms , which are under development at BioNTech , with the surface or
spike (S) protein of the novel severe acute respiratory syndrome coronavirus 2
(SARS -CoV-2) as the viral antigen.
In the present study, T - and B -cell responses as well as the ability of CD8+ T cells to
kill viral antigen -presenting cells induced by four clinical SA RS-CoV-2 vaccine
candidates were characterized.
The study was divided into two parts , with the first part characterizing the vaccine
candidates BNT162a1 and BNT162b1 , and the second part characterizing BNT162b2
and BNT162c2. For each part, eight BALB/c mi ce per group were vaccinated with 5 µg
of RNA encapsulated in lipid nanoparticles (LNPs) or buffer control on day 0 by
intramuscular injection. T and B cells were analyzed seven days after vaccination in
the blood. Serum for optional determination of SARS -CoV-2 specific IgG responses
was stored , spleen and the draining lymph nodes (dLNs) were analyzed after 12 days
(BNT162a1, BNT162b1 and BNT162b2 ), or 27 days (BNT162c2). Splenocytes were
used for IFNγ ELISpot assay and xCELLigence cytotoxicity assay, and cell
suspensions prepared from dLN s and spleen were analyzed by flow cytometry .
Cytokines produced by restimulated dLN and spleen cells were analyzed by
Procarta Plex cytokine multiplex assay .
IFNγ ELISpot revealed a strong S protein specific T -cell responses particularly in
BNT162b2 , BNT162b1 and BNT162c2 and to a lesser extent in BNT162 a1 treated
groups. In line, CD8+ and CD4+ T cells in dLNs were significantly increased after
BNT162b2 treatment , the former already detectable at day 7 i n the blood . A trend for
increased T cell numbers was detected in the BNT162b1 and BNT162 a1 groups.
Particular BNT162b1 and BNT162b 2 treatment resulted in T cell activation (CD44,
CD38, PD1 and ICOS expression of T c ells in blood) and antigen specific secretion of
cytokines by splenocytes . In those groups, a predominant T H1 phenotype was detected
with increased numbers of T -bet+ CD4+ T cells, high secretion of T H1 type cytokines
(IFNγ, IL -2, TNF) and low secretion of T H2 type cytokines (IL -4, IL-5). In all analyzed
compartments BNT162b 1, BNT162b 2 and BNT162c2 treatment mediated the increase
and activation of TFH cells, a cell type known for its crucial support of B cell responses.
B cell numbers in dLNs were significantly elevated after BNT162b 1 and BNT162b 2
treatment with higher numbers of antibody producing p lasma B cells, class switched
and germinal center B cells essential for affinity maturation of antibodies .
Due to the prominent induction of both T and B cell responses , these results particularly
support further clinical evaluation of the S ARS-CoV-2 vaccine candidates BNT162b1
and BNT162b2 .
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2 GENERAL INFORMATION
2.1 Sponsor and Test Facilities
Sponsor
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Germany
Test Facility
BioNTech SE
An der Goldgrube 12
55131 Mainz
Germany
2.2 Participating Pers
Responsible p erson:
(as defined in SOP -100-024)
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Author:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
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Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech SE
Experimenter:
BioNTech SE
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
h
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
BioNTech RNA Pharmaceuticals GmbH
2.3 Study Dates
Start of experiments: 06 MAY 2020
Completion of experiments: 04 JUN 2020
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2.4 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, Amendme nt from
24.04.2020 (approved 30.04.2020).
SOP -010-015 Pipetten und Dispenser
SOP -010-017 Brutschränke - Biolytics
SOP -010-028 Vi -Cell XR
SOP -010-045 Brutschrank HERAcell 150i
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 -010-128 FACSCelesta
SOP -020-009 Ansetzen von Medien und Zusätzen für die Zellkultur
SOP -030-038 Standardisierte K ultivierung von Zellen
SOP -030-041 Auftauen von Zellen
SOP -030-050 Elektroporation von Zellen
SOP -030-051 Selektion mit MACS MicroBeads
SOP -030-054 Extrazelluläre Färbung für Durchflusszytometrie
SOP -030-071 Abtöten von Mäusen
SOP -030-072 Fixiergriff und O hrmarkierung bei Mäusen
SOP -030-073 Betäubung bei Mäusen
SOP -030-074 Blutentnahme bei Mäusen
SOP -030-078 Isolierung muriner Splenozyten
SOP -030-079 Intramuskuläre Applikation bei Mäusen
SOP -030-110 IFN γ ELISpot (murin)
SOP -090-013 Biological safety in labo ratories
SOP -110-022 Entsorgung von Biostoffabfällen
2.5 Changes and Deviations
This R&D study was conducted according to R&D plan P-20-0112 . Table 1
summarizes all changes and deviations to the R&D plan.
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Table 1: Changes and deviations to R&D study plan
Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
mCorVAC#15 xCELLigen
ce
cytotoxicity
assay Duplicates for all
samples Duplicates only for some
samples Not enough ce lls per
mouse Some samples were
assayed in
singlicates
mCorVAC#15 Flow
cytometry Functional and
phenotypic T
cell analysis,
dLN: 2 × 106
cells/well Functional and phenotypic T cell
analysis, dLN: 1 × 106 cells/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: 2 × 106/well Phenotypic T cell analysis, SP:
4 × 106/well Improve the quality of
the results None.
mCorVAC#15 Flow
cytometry B cell analysis,
dLN, SP:
2 × 106/well B cel l analysis, dLN:
2.5 × 105/well. SP: 1 × 106/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: Use cells in
200 µL for
acquisition Phenotypic T cell analysis, SP:
Group 1 mous e 1: 160 µL
sample acquired by device
without recording. To the
remaining sample volume, 160
µL flow buffer were then added
and the sample re -acquired and
recorded. Deviation Total recordable
living cell number for
Group1 mouse 1 was
133,209 instead of
the intended
minimum of
1,000,000.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Analysis of SP
and dLNs on
day 12 (day 27) Analysis of blood 7 days after
immunization Gain further information
on the T and B cell
immunophenotype
early after immunization Blood phe notyping
data available for 7
days after
immunization.
mCorVAC#15,
mCorVAC#16 ELISpot Use
MultiScreenHTS
plates (Millipore) Precoated ELISpot plates were
used (Mabtech) bring in line with
previous mCorVac
experiments None.
mCorVAC#15,
mCorVAC#16 Flow
cytometry,
cytokine
multiplex
assay Restimulation
with S peptide
mixes at 0.1
µg/mL per
peptide Restimulation with S peptide
mixes at 0.2 µg/mL per peptide
(mCorVAC#15 and
mCorVAC#16) and at 0.5 µg/mL
per peptide (flow cytometry,
mCorVAC#16) Increase peptide
concentration to
increase sensitivity and
pick up low responses Direct quantitative
comparison of
cytokine profiles
derived from
multiplex assay of
mCorVac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no longer
advisable
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(functional
T cell
analysis),
cytokine dLN: All
samples dLN: Not all samples included in
assay Not enough cells per
mouse Lower sample
numbers for this
assay
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Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
multiplex
analysis
mCorVAC#15,
mCorVAC#16 Flow
cytometry Brefeldin A an d
GolgiStop in
medium were
added to a final
concentration of
10 μg/mL
brefeldin A and
a dilution of
GolgiStop of
1:500. GolgiStop and GolgiPlug in
medium were added to a final
dilution of GolgiStop of 1:1,500
and GolgiPlug of 1:1,000. GolgiPlug contains
brefeldin A and
replaced brefeldin A
solution. Dilution of
GolgiStop was lowered
as this lower dilution
was found to be
sufficient for its
purpose. None.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Extracellular and
intracellular/intra
nuclear staining
for phenot ypic
and functional T
cell analysis was
performed alike
and was the
same for
mCorVAC#15
and
mCorVAC#16.
B and myeloid
cell staining was
not described in
detail. Extracellular and
intracellular/intranuclear staining
for phenotypic and functional T
cell ana lysis varied compared to
the study plan and was again
altered between mCorVAC#15
and mCorVAC#16 with respect
to antibody panels, washing
procedures, fixation procedures
and centrifugation conditions
(see sections 4.5.11.2, 4.5.11.3
and 4.5.11.4 for details ).
B and myeloid cell staining was
performed according to section
4.5.11.5 and 4.5.11.6. Improve the quality of
the results.
Detailed stianing
protocol for B and
myeloid cells. Direct quantitative
comparison of T and
B cell flow cytometry
data of mCorV ac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no longer
advisable.
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(myeloid
cell
analysis) dLN: All
samples dLN: No samples included in
assay Not enough cells per
mouse Assay was not
performed for dLN
mCorVAC#15,
mCorVAC#16 Cytokine
multiplex
assay dLN:
5 × 105/well dLN: 4 × 105/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#16 Flow
cytometry Phenotypic T
cell analysis,
dLN: 2 × 106
cells/well. Phenotypic T ce ll analysis, dLN:
1.5 × 106 cells/well Not enough cells per
mouse. Lower sample
numbers for this
assay
mCorVAC#16 Flow
cytometry Phenotypic T
cell analysis,
SP: 2 × 106/well Phenotypic T cell analysis, SP:
4 × 106/well Improve the quality of
the results. None.
mCorVAC#16 Flow
cytometry B cell analysis,
dLN, SP:
2 × 106/well B cell analysis, dLN:
2.5 × 105/well. SP: 1 × 106/well Not enough cells per
mouse. Lower sample
numbers for this
assay
mCorVAC#16 xCELLigen
ce
cytotoxicity
assay Using CT26
cells
electroporated S RNA electroporated CT26
cells were loaded with S peptide
mix after electroporation Improve the quality of
the results None.
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Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
with S RNA as
targets
mCorVAC#15 xCELLigen
ce
cytotoxicity
assay Duplicates for all
samples Duplicates only for some
samples Not enough cells per
mouse Some samples were
assayed in
singlicates
mCorVAC#15 Flow
cytometry Functional and
phenotypic T
cell analysis,
dLN: 2 × 106
cells/well Functional and phenotypic T cell
analysis, dLN: 1 × 106 cells/well Not enough ce lls per
mouse Lower sample
numbers for this
assay
mCorVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: 2 × 106/well Phenotypic T cell analysis, SP:
4 × 106/well Improve the quality of
the results None.
mCorVAC#15 Flow
cytometry B cell analysis,
dLN, SP:
2 × 106/well B cell analysis, dLN:
2.5 × 105/well. SP: 1 × 106/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: Use cells in
200 µL for
acquisition Phenotypic T cell analysis, SP:
Group 1 mouse 1: 160 µL
sample acquired by device
without recording. To the
remaining sample volume, 160
µL flow buffer were then added
and the sample re -acquired and
recorded. Deviation Total recordable
living cell number for
Group1 mouse 1 was
133,209 instead of
the intended
minimum of
1,000,000.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Analysis of SP
and dLNs on
day 12 (day 27) Analysis of blood 7 days after
immunization Gain further information
on the T and B cell
immunophenotype
early af ter immunization Blood phenotyping
data available for 7
days after
immunization.
mCorVAC#15,
mCorVAC#16 ELISpot Use
MultiScreenHTS
plates (Millipore) Precoated ELISpot plates were
used (Mabtech) bring in line with
previous mCorVac
experiments None.
mCo rVAC#15,
mCorVAC#16 Flow
cytometry,
cytokine
multiplex
assay Restimulation
with S peptide
mixes at 0.1
µg/mL per
peptide Restimulation with S peptide
mixes at 0.2 µg/mL per peptide
(mCorVAC#15 and
mCorVAC#16) and at 0.5 µg/mL
per peptide (flow cytometry,
mCorVAC#16) Increase peptide
concentration to
increase sensitivity and
pick up low responses Direct quantitative
comparison of
cytokine profiles
derived from
multiplex assay of
mCorVac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no lon ger
advisable
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(functional
T cell
analysis),
cytokine dLN: All
samples dLN: Not all samples included in
assay Not enough cells per
mouse Lower sample
numbers for this
assay
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Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
multiplex
analysis
mCorVAC#15,
mCorVAC#16 Flow
cytometry Brefeldin A and
GolgiStop in
medium were
added to a final
concentration of
10 μg/mL
brefeldin A and
a dilution of
GolgiStop of
1:500. GolgiStop and GolgiPlug in
medium were added to a final
dilution of GolgiStop of 1:1,500
and GolgiPlug of 1:1 ,000. GolgiPlug contains
brefeldin A and
replaced brefeldin A
solution. Dilution of
GolgiStop was lowered
as this lower dilution
was found to be
sufficient for its
purpose. None.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Extracellular and
intracellular/intra
nuclear staining
for phenotypic
and functional T
cell analysis was
performed alike
and was the
same for
mCorVAC#15
and
mCorVAC#16.
B and myeloid
cell staining was
not described in
detail. Extracellular and
intracellular/intranuclear staining
for phenotypi c and functional T
cell analysis varied compared to
the study plan and was again
altered between mCorVAC#15
and mCorVAC#16 with respect
to antibody panels, washing
procedures, fixation procedures
and centrifugation conditions
(see sections 4.5.11.2, 4.5.11 .3
and 4.5.11.4 for details).
B and myeloid cell staining was
performed according to section
4.5.11.5 and 4.5.11.6. Improve the quality of
the results.
Detailed stianing
protocol for B and
myeloid cells. Direct quantitative
comparison of T and
B cell f low cytometry
data of mCorVac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no longer
advisable.
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(myeloid
cell
analysis) dLN: All
samples dLN: No samples included in
assay Not enough cells per
mouse Assay was not
performed for dLN
mCorVAC#15,
mCorVAC#16 Cytokine
multiplex
assay dLN:
5 × 105/well dLN: 4 × 105/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#16 Flow
cytometry Phenotypic T
cell analysis,
dLN: 2 × 106
cells/well. Phenotypic T cell analysis, dLN:
1.5 × 106 cells/well Not enough cells per
mouse. Lower sample
numbers for this
assay
mCorVAC#16 Flow
cytometry Phenotypic T
cell analysis,
SP: 2 × 106/well Phenotypic T cell analysis, SP:
4 × 106/well Improve t he quality of
the results. None.
mCorVAC#16 Flow
cytometry B cell analysis,
dLN, SP:
2 × 106/well B cell analysis, dLN:
2.5 × 105/well. SP: 1 × 106/well Not enough cells per
mouse. Lower sample
numbers for this
assay
mCorVAC#16 xCELLigen
ce
cytotox icity
assay Using CT26
cells
electroporated S RNA electroporated CT26
cells were loaded with S peptide
mix after electroporation Improve the quality of
the results None.
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Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
with S RNA as
targets
Experiment Assay Plan Change/Deviation Reason for
change/devi ation Implications
mCorVAC#15 xCELLigen
ce
cytotoxicity
assay Duplicates for all
samples Duplicates only for some
samples Not enough cells per
mouse Some samples were
assayed in
singlicates
mCorVAC#15 Flow
cytometry Functional and
phenotypic T
cell analys is,
dLN: 2 × 106
cells/well Functional and phenotypic T cell
analysis, dLN: 1 × 106 cells/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCorVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: 2 × 106/well Phenotypic T cell analysis , SP:
4 × 106/well Improve the quality of
the results None.
mCorVAC#15 Flow
cytometry B cell analysis,
dLN, SP:
2 × 106/well B cell analysis, dLN:
2.5 × 105/well. SP: 1 × 106/well Not enough cells per
mouse Lower sample
numbers for this
assay
mCo rVAC#15 Flow
cytometry Phenotypic T
cell analysis,
SP: Use cells in
200 µL for
acquisition Phenotypic T cell analysis, SP:
Group 1 mouse 1: 160 µL
sample acquired by device
without recording. To the
remaining sample volume, 160
µL flow buffer were then add ed
and the sample re -acquired and
recorded. Deviation Total recordable
living cell number for
Group1 mouse 1 was
133,209 instead of
the intended
minimum of
1,000,000.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Analysis of SP
and dLNs on
day 12 (day 27) Analy sis of blood 7 days after
immunization Gain further information
on the T and B cell
immunophenotype
early after immunization Blood phenotyping
data available for 7
days after
immunization.
mCorVAC#15,
mCorVAC#16 ELISpot Use
MultiScreenHTS
plates (Millipo re) Precoated ELISpot plates were
used (Mabtech) bring in line with
previous mCorVac
experiments None.
mCorVAC#15,
mCorVAC#16 Flow
cytometry,
cytokine
multiplex
assay Restimulation
with S peptide
mixes at 0.1
µg/mL per
peptide Restimulation with S peptid e
mixes at 0.2 µg/mL per peptide
(mCorVAC#15 and
mCorVAC#16) and at 0.5 µg/mL
per peptide (flow cytometry,
mCorVAC#16) Increase peptide
concentration to
increase sensitivity and
pick up low responses Direct quantitative
comparison of
cytokine profiles
derived from
multiplex assay of
mCorVac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no longer
advisable
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(functional dLN: All
samples dLN: Not all samples included in
assay Not enough cells per
mouse Lower sample
numbers for this
assay
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Experiment Assay Plan Change/Deviation Reason for
change/deviation Implications
T cell
analysis),
cytokine
multiplex
analysis
mCorVAC#15,
mCorVAC#16 Flow
cytometry Brefeldin A and
GolgiStop in
medium were
added to a final
concentration of
10 μg/mL
brefeldin A and
a dilution of
GolgiStop of
1:500. GolgiStop and GolgiPlug in
medium were added to a final
dilution of GolgiStop of 1:1,500
and GolgiPlug of 1:1,000. GolgiPlug contains
brefeldin A and
replaced brefeldin A
solution. Dilution of
GolgiStop was lowered
as this lower dilution
was found to be
sufficient for its
purpose. None.
mCorVAC#15,
mCorVAC#16 Flow
cytometry Extracellular and
intracellular/intra
nuclear staining
for phenotypic
and functional T
cell analysis was
performed alike
and was the
same for
mCorVAC#15
and
mCorVAC#16.
B and myeloid
cell staining was
not described in
detail. Extracellular and
intracellular/intranuclear staining
for phenotypic and functional T
cell analysis varied compared to
the study plan and was again
altered between mCorVAC#15
and mCorVAC#16 with respect
to antibody panels, washing
procedures, fixation procedures
and centrifugation conditions
(see sections 4.5.11.2, 4.5.11.3
and 4.5.11.4 for details).
B and myeloid cell staining was
performed according to section
4.5.11.5 and 4.5.11.6. Improve the quality of
the res ults.
Detailed stianing
protocol for B and
myeloid cells. Direct quantitative
comparison of T and
B cell flow cytometry
data of mCorVac#15
(BNT162a1 and
BNT162b1) and
mCorVac#16
(BNT162b2 and
BNT162c2) no longer
advisable.
mCorVAC#15,
mCorVAC#16 Flow
cytometry
(myeloid
cell
analysis) dLN: All
samples dLN: No samples included in
assay Not enough cells per
mouse Assay was not
performed for dLN
mCorVAC#15,
mCorVAC#16 Cytokine
multiplex
assay dLN:
5 × 105/well dLN: 4 × 105/well Not enough cells per
mouse Lower sample
numbers for this
assay
2.6 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_Pr eclinic \00_Pharmacology \mCorVac#15_modRNA_uRNA_V5_dLN
_SP
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P:\BioNTechRNA \RN9391R00_CoV -
VAC\04_Preclinic \00_Pharmacology \mCorVac#16_saRNAV9_modRNAV9_d
LN_SP
Lab book #1934, page 16-80
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3 INTRODUCTION
3.1 Background
In December 2019, an outbreak of pneumonia of unk nown cause in Wuhan, Hubei
province in China , started. The disease sprea d rapidly and in January 2020, the agent
was identified. By July 27th 2020, infection with the novel Coronavirus SARS -CoV-2
was confirmed in approximately 16,100, 000 people with more t han 640,000
casualties1. A vaccine is urgently needed against the elicited coronavirus disease 19
(COVID -19) and B ioNTech decided to initiate a rapid vaccine project based on the
surface or spike (S) protein of the virus as the viral antigen. The S p rotein is a trimer
and during viral egress, the precursor protein is cleaved in to S1 an d S2 ( Figure 1).
While the S1 domain recognizes the host receptor, the S2 domain is essential for
membrane fusion of the viral envelop e and the endosomal membrane. To initiate
membrane fusion, the S2 domain undergoes a conformational change within the
central helix domain.
Figure 1: Schematic overview of the S protein structure of the SARS -CoV S protein
The se quence within the S1 subunit consists of the signal peptide (SP) and the receptor binding domain (RBD) with
its receptor binding motif (RBM). The S2 subunit contains the fusion peptide (FP) for membrane fusion, heptad
repeats (HR1 and HR2 ), the transmembra ne domain (TM) and a cytoplasmic domain (CP) . Source: modified from
Song et al. 2019 .
Based on these features, the S protein is the target of neutralizing antibodies that bind
predominantly the receptor -binding domain (RBD) of the S protein.
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 company in
RNA technology, which has been gained over the last decade. The core innovati on is
based on in vivo delivery of a pharmacologically optimized, antigen -coding RNA
vaccine to induce robust neutralizing antibodies and concomitant T -cell response s to
achieve protective immunization with minimal vaccine doses (Vogel et al. 2018 , Pardi
et al. 2017 , Moyo et al. 2019 ).
At BioNTech, there are three different RNA platforms under development, which are
non-modified uridine containing mRNA (uRNA), nucleosi de modified mRNA
(modRNA) and self -amplifying RNA (saRNA). It is unknown today which RNA vaccine
1 Coronavirus diseas e (COVID -2019) situation report 189, World Health Organization;
https://www.who.int/emergencies/diseases/novel -coronavirus -2019/situation -reports
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platform performs best in terms of activation and duration of a potent immune
response. Initial studies in mice demonstrated the induction of T -cell responses as well
as SARS -CoV-2-specific (neutralizing) IgG antibodies with vaccine candidates o f all
platforms. Four of th ese candidates are currently tested in clinica l trials ( Table 2).
The BNT162 vaccine candidate RNA is encapsulated into lipid n anoparticle s (LNPs),
which protect the RNA from degradation and enable transfectio n of host cells after
intramuscular (i.m.) injection. For all of the BNT162 vaccine candidates , the same LNP
formulation is used.
Table 2: Clinical stage SARS-CoV-2 vaccine candidates developed at BioNTech
ID RNA platform Antigen
BNT162a1 uRNA RBD of S1S2 protein (V5)
BNT162b1 modRNA RBD of S1S2 protein (V5)
BNT162b2 modRNA S1S2 full -length protein, sequence variant (V9)
BNT162c2 saRNA S1S2 full -length protein, sequence variant (V9)
This report covers a mouse study characterizing the immunophenotype in the blood,
spleen and lymph node s of mice treated with these four SARS -CoV-2 vaccine
candidates.
3.2 Objectives
The objective o f this study was to further characterize the four clinical SARS -CoV-2
vaccine candidates to support fast clinical development and approval. In particular, the
goal of this study was to:
Characterize T - and B -cell responses in the spleen , lymph nodes and bl ood.
Analysis include d a thorough phenotypic and functional (cytokine secretion on
the cellular level) characterization of cells by ELISPot and flow cytometry , and
defin ition of the cytokine profile by multiplex protein quantitation. In particular,
the sub type of SARS -CoV-2-specific CD4+ T cells ( TH1, T H2, T FH) and the
abundance of plasma and germinal center (GC) B cells were of interest.
Characterize changes in the myeloid cell compartment.
Determine the ability of CD8+ T cells to kill cells presenting th e vaccine -
encoded antigen .
Collect serum of mice to determine (ne utralizing) antibody responses
(collection was performed, analysis of samples may be performed in the future ,
if required) .
3.3 Study Design
The study was separated into two parts characterizing the vaccine candidates
BNT162a1 and BNT162b1 (mCorVac#15, Figure 2) and BNT162b2 and BNT162c2
(mCorVac#16, Figure 3). Each part compare d the effects of vaccinated mice to a
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control group receiving buffer only. Eight BALB/c mice per group were vaccinated once
(day 0) and blood analyzed 7 days later. S erum and tissues were analyzed 12 days
later. Since T -cell responses of mice vaccinated with saRNA (BNT162c2) take longer
to dev elop, the analysis time point for serum and tissues was postponed to day 27 after
vaccination.
Blood, spleen and draining lymph nodes (dLNs) were harvested from mice. Figure 4
shows an overview of the subsequent analytical methods including sample allocation
to the respective assays .
Serum was obtained from blood and stored frozen for optional determination of
SARS -CoV-2 specific IgG responses.
Splenocytes were tested for recognition of an S protein -specific peptid e mix or
S RNA -electroporated CT26 cells by secretion of IFNγ (IFNγ ELISpot assay ).
A fraction of splenocytes (N=3 only for the control group, N=8 for treatment
groups) was restimulated overnight with an S protein -specific peptide mix and
recombinant IL -2, and isolated CD8+ T cells were challenged on the next day
for killing of S RNA -electroporated CT26 colon carcinoma cells (xCELLigence
cytotoxicity assay).
Splenocytes and dLN (popliteal, iliac and inguinal, pooled) cells were analyzed
for T- (CD4+ TH1, T H2, T FH, CD8+ T cells) and B -cell phenotype (GC, plasma,
memory B cells), T-cell cytokine secretion after restimulation with an S protein -
specific peptide mix , and myeloid cell subsets (flow cytometry ).
dLN and spleen cells were restimulated for 48 h with an S protein -specific
peptide mix to analyze T-cell secreted cytokines in the supernatant
(ProcartaPlex cytokine multiplex assay) .
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Figure 2: Workflow of part 1 of the study (mCorVac#15)
dLN, draining lymph node. GC, germinal cen ter. Gr, group. N, number of mice per group. TH, T helper cells. T FH,
follicular T helper cells.
Figure 3: Workflow of part 2 of the study (mCorVac#16)
Study dates for group 3 are depicted in red. dLN, draining lymph node. GC, g erminal center. Gr, group. N, number
of mice per group. TH, T helper cells. T FH, follicular T helper cells.
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Figure 4: Analysis and assay overview
Schematic depiction of sample allocation to different analysis methods and their timing during analysis days 1 to 3 .
dLN, draining lymph node. EP, electroporated.
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4 MATERIALS AND METHOD S
4.1 Test Item
BNT162a1 (ATM): For CoAs see Appendix 9.6
RNA batch: RNA -SK200305 -01
Polymun batch RBL063.3 LNP with the lot: CoVVAC/090320
Dilution buffer: 0.9% NaCl
Concentration: 0.5 mg/mL
BNT162b 1 (ATM): For CoAs see Appendix 9.6.
RNA batch: RN A-RF200304 -03
Polymun batch RBP020.3 LNP with the lot: CoVVAC/100320
Dilution buffer: 0.9% NaCl
Concentration: 0.5 mg/mL
BNT162b2 (ATM): For CoAs see Appendix 9.8.
RNA batch: RNA -RF200321 -06
Polymun batch RBP20.2 LNP with the lot: CoVVAC/270320
Dilution buffer: 0.9% NaCl
Concentration : 0.5 mg/mL
BNT162c2 (ATM): For CoAs see Appendix 9.9.
RNA batch: RNA -RF200310 -01
Polymun batch RBS004.2 LNP with the lot: CoVVAC/170320
Dilution buffer: 0.9% NaCl
Concentration: 0.3 mg/mL
Test items are diluted to 0.25 mg/mL with sterile 0.9% NaCl before administration.
4.2 Control Item
0.9% NaCl
4.3 Test System
48 female BALB /c mice with approximately nine weeks of age at study start
4.4 Materials
For antibodies used in flow cytometry, refer to Section 4.5.11 .
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Table 3: Materials
Product name Application/specific
ation 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
4mL Sample Cup Cell counting NC9756824 N/A Beckman Coulter
GmbH
8-channel manifold Polypropylene BR704526 -
1EA N/A Sigma -Aldrich
Chemie GmbH
96-well Microplate Clear round bottom
TC-treated
microplate, with lid,
sterile 3799 N/A Corning Holding
GmbH
ACK lysis buffer Flow cytometry
(blood) A10492 -01 1x Gibco
Ammonium chloride NH 4Cl A0988,5000 N/A AppliChem GmbH
Brilliant Stain Buffer Flow cytometry 563794 N/A BD Bioscience
Brilliant Stain Buffer Plus Flow cytometry 566385 N/A BD Biosciences
Capillary pipettes minicaps®, blood
sampling, 4 µL/
10 µL, not
heparinized 9000104/
9000110 N/A Hirschmann
Laborgeräte
GmbH & Co.KG
Cell culture flask 250 ML,
75 cm² Cell culture 658175 N/A Greiner Bio -One
GmbH
CD8a (Ly -2) MicroBeads CD8 T cell
purification 130-117-044 N/A Miltenyi Biotec
Collagenase D Lymphnode
preparation 1108886600
1 1 mg/ml Merck KGaA
Combitips advanced® Biopur®, 50 mL 0030089693 N/A Eppend orf
Vertrieb
Deutschland
GmbH
Concanavalin A from Canavalia
ensiformis (Jack
bean, 5mg),Type IV -
S, lyophilized C0412 -5MG 2 µg/mL Sigma -Aldrich
Chemie GmbH
Dimethyl sulfoxide Cell culture A3672,0100 N/A AppliChem GmbH
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Product name Application/specific
ation Article no. Working
dilution Provider
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
Electroporation cuvette Electroporation 732-1137 N/A VWR
International
GmbH
E-Plate VIEW 96 PET xCelligence 300600910 N/A ACEA
Biosciences
Eppendorf saf e-lock tubes 0.5 mL/ 1.5 mL/
2.0 mL/ 5.0 mL,
Eppendorf Quality™ 0030121023/
0030120086/
0030120094/
0030119401 N/A Eppendorf
Vertrieb
Deutschland
GmbH
Ethylenediaminetetraacetic
acid solution EDTA 03690 -
100ML N/A Sigma -Aldrich
Chemie GmbH
Fetal Bovine Serum Non-USA origin,
sterile -filtered F7524 N/A Sigma -Aldrich
Chemie GmbH
Filtration unit for medium
flasks High Performance,
PES, 0.45 µm,
1000 mL 514-0301 N/A VWR
International
GmbH
FoxP3/Transcription Factor
Staining Buffer Set Flow cytometry 00-5523 -00 N/A Thermo Fisher
Scientific
GolgiStop Flow cytometry
(Restimulation) 554724 1:1,500 BD Biosciences
GolgiPlug Flow cytometry
(Restimulation) 555029 1:1,000 BD Biosciences
Heparin Tubes Flow cytometry
(Blood ) 20.1309 N/A Sarstedt AG &
Co.
HEPES 1 M 15630 -056 N/A Gibco
Insulin syringes BD Micro -Fine™+,
30 G, 0.3 mL 324826 N/A Becton Dickinson
GmbH
Ionomycin, 10 µg/µL Flow cytometry
(Restimulation) I9657 1 µg/mL Sigma
Isoflurane Anesthesia 9714675 N/A Piramal Critical
Care
Isotonic saline Injection solu tion 06173569 N/A Fresenius Kabi
Deutschland
GmbH
LS columns CD8 T cell
purification 130-042-401 N/A Miltenyi Biotec
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Product name Application/specific
ation Article no. Working
dilution Provider
MEM Non -Essential Amino
Acids Solution (100X) Cell culture 11140 -035 1X Gibco
Mouse IFN -γ ELISpotPLUS
kit Kit for enumeration
of cells secreting
mouse IFN -γ 3321 -4APT -2 N/A Mabtech
PBS dry substance No calcium, no
magnesium L182 -10 N/A Merck KGaA
Penicillin -Streptomycin 10,000 U/mL 15140 -122 N/A Gibco
PepMix™ against RBD ELISpot N/A
(customized) 0.0625mg
per
peptide/
vial JPT
Pipette tips ep Dualfilter
T.I.P.S.®, PCR clean
und sterile, 0.1 –
10 µL/2–100 µL/50 –
1000 µL/50 –
1250 µL/0.1 –5 mL 0030077512/
0030077547/
0030077555/
0030077792/
0030077750/
0030078616 N/A Eppendorf
Vertrieb
Deutsch land
GmbH
PMA, 1 µg/µL Flow cytometry
(Restimulation) P1585 0.5 µg/mL Sigma
Potassium bicarbonate KHCO 3 A2375,1000 N/A AppliChem GmbH
ProcartaPlex
mouse T H1/T H2 cytokine
11-plex kit Cytokine multiplex
assay
Lot. No. 232634 -004 EPX110 -
20820 -901
N/A Thermo Fisher
Scientific
Proleukin S Cell culture N/A 100 U/ml Clinigen
Reservoir 25 mL, 100 mL 613-
1174/613 -
1171 N/A VWR
International
GmbH
RotiHistofix Flow cytometry P087.1 2% Roth
Round bottom 5 -mL tubes Flow cytometry
(blood) 10579511 N/A Thermo Fishe r
Scientific
RPMI 1640 Medium GlutaMAX™
Supplement 61870 -010 N/A Gibco
Serological pipettes 5 mL, 10 mL, 25 mL,
50 mL 606180/6071
80/601180/7
68180 N/A Greiner Bio -One
GmbH
Serum Tubes Serum preparation 20.1344 N/A Sarstedt AG &
Co.
Single -use syringe Injekt® Solo 5 mL 4606051V N/A B. Braun
Melsungen AG
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Product name Application/specific
ation Article no. Working
dilution Provider
Sodium acide (10%) Flow cytometry
(blood) 13553.00100 0.01% Morphisto
Sodium Pyruvate 100 mM 11360 -039 N/A Gibco
StemPro™ Accutase™ Cell
dissociation reagent Cell culture A1110501 N/A Gibco
Sterile fil ters 0.45 µm 514-4123 N/A VWR
International
Vi-CELL™ XR Quad Pak For Vi -CELL™ XR
Cell Viability
Analyzer 383722 N/A Beckman Coulter
GmbH
X-VIVO 15, serum -free Electroporation BE02 -060Q N/A Lonza Group Ltd
Table 4: Peptide pool fo r restimulation of splenocytes and dLN cells for ELISpot assays , flow cytometry
and cytokine multiplex assay
S protein-specific peptides
Name Sequence
2019 -nCoV S.wt
With a total of 315
overlapping peptides
(15mers overlapping by
11 amino acids )
GenBank: QHD43416.1
Batch: 43000LHB -1 and
43000LHB -2
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQ
DLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWI
FGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEF
RVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTP I
NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA
AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTS
NFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY
NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN
YKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFE RDISTEIYQA
GSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCG
PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRD
PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPT
WRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRA
RSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTIS VTTEILPVSMTKTSVDCT
MYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTP
PIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAA
RDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM
QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVN
QNAQALNTLVKQLSSNFGAISSVL NDILSRLDKVEAEVQIDRLITGRLQSLQTY
VTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHG
VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE
PQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD
LGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLG
FIAGLIAIVMVTIM LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHY
T
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Table 5: RNAs used for CT26 electroporation
Name Sequence (open reading frame)
S RNA
(Full construct
name:
JR206_mRNA_
pST4_m1ѱ_wts
ec_SARS -CoV-
2-S (Sino CDS)
C∆19 ) atgtttgtgttcctggtgctgctg ccactggtgtccagccagtgtgtgaacctgaccaccaggacccaacttcctcctgcctaca
ccaactccttcaccaggggagtctactaccctgacaaggtgttcaggtcctctgtgctgcacagcacccaggacctgttcctg
ccattcttcagcaatgtgacctggttccatgccatccatgtgtctggcaccaatggcaccaagaggtttgacaaccctgtgctgc
cattcaatgatggagtctactttgcca gcacagagaagagcaacatcatcaggggctggatttttggcaccaccctggacag
caagacccagtccctgctgattgtgaacaatgccaccaatgtggtgattaaggtgtgtgagttccagttctgtaatgacccattc
ctgggagtctactaccacaagaacaacaagtcctggatggagtctgagttcagggtctactcctctgccaacaactgtaccttt
gaatatgtgagccaaccattcctgatggactt ggagggcaagcagggcaacttcaagaacctgagggagtttgtgttcaag
aacattgatggctacttcaagatttacagcaaacacacaccaatcaacctggtgagggacctgccacagggcttctctgcctt
ggaaccactggtggacctgccaattggcatcaacatcaccaggttccagaccctgctggctctgcacaggtcctacctgaca
cctggagactcctcctctggctggacagcaggagcagcagcc tactatgtgggctacctccaaccaaggaccttcctgctga
aatacaatgagaatggcaccatcacagatgctgtggactgtgccctggacccactgtctgagaccaagtgtaccctgaaatc
cttcacagtggagaagggcatctaccagaccagcaacttcagggtccaaccaacagagagcattgtgaggtttccaaacat
caccaacctgtgtccatttggagaggtgttcaatgccaccaggtttgcctctg tctatgcctggaacaggaagaggattagca
actgtgtggctgactactctgtgctctacaactctgcctccttcagcaccttcaagtgttatggagtgagcccaaccaaactgaa
tgacctgtgtttcaccaatgtctatgctgactcctttgtgattaggggagatgaggtgagacagattgcccctggacaaacagg
caagattgctgactacaactacaaactgcctgatgacttcacaggctgtgtgattgc ctggaacagcaacaacctggacagc
aaggtgggaggcaactacaactacctctacagactgttcaggaagagcaacctgaaaccatttgagagggacatcagca
cagagatttaccaggctggcagcacaccatgtaatggagtggagggcttcaactgttactttccactccaatcctatggcttcc
aaccaaccaatggagtgggctaccaaccatacagggtggtggtgctgtcctttgaactgctccatgcc cctgccacagtgtgt
ggaccaaagaagagcaccaacctggtgaagaacaagtgtgtgaacttcaacttcaatggactgacaggcacaggagtgc
tgacagagagcaacaagaagttcctgccattccaacagtttggcagggacattgctgacaccacagatgctgtgagggac
ccacagaccttggagattctggacatcacaccatgttcctttggaggagtgtctgtgattacacctggcaccaacaccagca a
ccaggtggctgtgctctaccaggatgtgaactgtactgaggtgcctgtggctatccatgctgaccaacttacaccaacctgga
gggtctacagcacaggcagcaatgtgttccagaccagggctggctgtctgattggagcagagcatgtgaacaactcctatg
agtgtgacatcccaattggagcaggcatctgtgcctcctaccagacccagaccaacagcccaaggagggcaaggtctgtg
gcaagccagag catcattgcctacacaatgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatc
ccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatct
gtggagacagcacagagtgtagcaacctgctgctccaatatggctccttctgtacccaacttaacagggctctgacaggcatt
gctgtggaacaggacaag aacacccaggaggtgtttgcccaggtgaagcagatttacaagacacctccaatcaaggactt
tggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtccttcattgaggacctgctgttcaac
aaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatttgtgc
ccagaagttcaatggactgacagtgctgc ctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctg
gcaccatcacctctggctggacctttggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggttcaat
ggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattc
aggactccctgtccagcacagcctctgccctgggcaa actccaagatgtggtgaaccagaatgcccaggctctgaacacc
ctggtgaagcaactttccagcaactttggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggc
tgaggtccagattgacagactgattacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgct
gagattagggcatctgccaacctggctgccaccaagatgagtgagtgt gtgctgggacaaagcaagagggtggacttctgt
ggcaagggctaccacctgatgagttttccacagtctgcccctcatggagtggtgttcctgcatgtgacctatgtgcctgcccag
gagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcactttccaagggagggagtgtttgtgagcaatg
gcacccactggtttgtgacccagaggaacttctatgaaccacagattatcaccacag acaacacctttgtgtctggcaactgt
gatgtggtgattggcattgtgaacaacacagtctatgacccactccaacctgaactggactccttcaaggaggaactggaca
aatacttcaagaaccacaccagccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaa
ggagattgacagactgaatgaggtggctaagaacctgaatgagtccctgattgacctccaagaact gggcaaatatgaac
aatacatcaagtggccatggtacatctggctgggcttcattgctggactgattgccattgtgatggtgaccataatgctgtgttgta
tgacctcctgttgttcctgtctgaaaggctgttgttcctgtggctcctgttgttgatga
Irrelevant RNA
(Batch: RNA -
KG200106 -06c) atgggcgccatggcccctagaacattgctcctgctgctggccgctgccctggc ccctacacagacaagagctggacctggc
ggctctggaggaggcggctccggaggcggaggatccggtggtggcggcagcggcggcgtgatcgtggctgtgctgggcg
tgctgggagccatggccatcattggagccgtggtggccttcgtgatgaagcggagaagaaacaccggcggcaagggcgg
cgattacgctctggctcctggcagccagtccagcgagatgagcctgagagactgcaaggcctagtaa
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Table 6: Software
Product name Application Provider
BD FACSDiva software
version 9.1 and 8.0.1.1 Flow cytometry BD Biosciences
Excel Raw data Microsoft Corp.
FlowJo software version
10.6 Flow cytometry FlowJo LLC, BD
Bioscience s
GraphPad Prism software
version 8 Statistical analysis GraphPad Software Inc.
ImmunoCapture 7.0.7.0 ELISpot assay Cellular Technology Ltd
ImmunoSpot® analysis
software version 57.0.17.0 ELISpot assay Cellular Technology Ltd
ProcartaPley Analyst
software version 1 Cytokine multiplex assay Thermo Fisher Scientific
RTCA Data analysis
software xCELLigence cytotoxicity assay ACEA Biosciences
xCELLigence RTCA
Software Pro xCELLigence cytotoxicity assay ACEA Biosciences
Table 7: Machines
Product name Application Provider
BD Symphony A3 Flow cytometry BD Biosciences
BD Celesta Flow cytometry BD Biosciences
Bioplex200 s ystem Cytokine multiplex assay Bio-Rad
Centrifuges Centrifugation Eppendorf
CTL ELISPOT reader
ImmunoSpot® S6 Core
Analyzer ELISpot assay Cellular Technology
Limited
Electroporation system Electroporation BTX
Vi-CELL XR Cell counting Beckman Coulter GmbH
xCELLigence RTCA MP xCELLigence cytotoxicity assay ACEA Biosciences
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4.5 Methods
4.5.1 Animal Care
4.5.1.1 General Informati on
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 local authorities (local welfare committee), condu cted
according to the FELASA recommendations and in compliance with the German
animal welfare act and Directive 2010/63/EU. Only animals with an unobjectionable
health status were selected for testing procedures.
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 labelled with a cage card indicating the mouse strain, gender,
date of birth and number of animals per cage. At th e start of an experiment additional
information was added such 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 Conditi on and Husbandry
Mice were housed at BioNTech SE's animal facility under barrier and SPF conditions
(An der Goldgrube 12, 55131 Mainz) 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 was kept at 20 -24°C and 45 -55%, respectively, and the air change
(AC) rate in the cages at 75 AC/h. The cages with dust -free bedding made of debarked
choppe d aspen wood (Abedd LAB & VET Service GmbH, Vienna, Austria, product
code: LTE E -001) and additional nesting material were changed weekly. Autoclaved
ssniff M -Z food (sniff Spezialdiäten GmbH, Soest, Germany; product code: V1124) and
autoclaved water (tap water) were provided ad libitum and changed 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
animals and control of food and water supplies. Each animal's health was closely
assessed at least once weekly and the results documented in health monitoring
sheets. The general physical condition was assessed with regard to the following
parameters:
Body weight change
Macroscopic assessment of activi ty level/ behavior
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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 indicated by
reduced blo od circulation
Macroscopic 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 fi ghting
4.5.3 Animal Treatment
4.5.3.1 Treatment Schedule, Route of Administration, and Dose
The test compounds were administered i.m. once at a dose of 5 µg (see Figure 2 and
Figure 3). The control group was treated with buffer only.
4.5.3.2 Immunization
For immunization, prior anesthesia by inhalation of 2.5% isoflurane in oxygen, the
injection site (hind leg) was shaved. Buffer or dissolved test item was applied i.m. into
the musculus gastrocnem ius in a volume of 20 µL. After immunization and a short
recovery phase from anesthesia, the animals were observed for any immediate signs
of discomfort following the immunization procedure.
4.5.3.3 Blood Sampling via the Retro -Orbital Venous Plexus or
Vena Facial is
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 held
tightly. A thin glass capillary (29 G) was inserted gently through the retro -orbital sin us
membrane and blood was collected into either Serum tubes (serum preparation) or
Heparin tubes (flow cytometry analysis ). After careful removal of the glass capillary,
the restraining grip was loosened. Alternatively, blood collection was performed via t he
vena facialis according to SOP -030-074. In short, without prior anesthesia, mice were
held tightly and using a lancet, the vena facialis was punctured in a precise and short
movement. Blood was collected into either Serum tubes (serum preparation) or
Heparin tubes (flow cytometry analysis) , and the restraining grip was loosened. Blood
samples were centrifuged at 10,000 x g and ambient temperature for 5 min and serum
transferred to a pre -labeled 0.5 mL reagent tube, to be stored at -20°C.
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4.5.4 Endpoint of Exp eriment / Termination Criteria
Animals were euthanized in accordance with §4 of the German animal welfare act and
the recommendation of GV -SOLAS by cervical dislocation or by exposure to carbon
dioxide. Additionally, termination criteria 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 on
their own suffic ient 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 incisio n. The spleen and dLNs (popliteal (PO),
iliac ( IL) and inguinal (I N), see Figure 5) were collected , pooled and stored in PBS on
ice for subsequent sing le cell preparations.
Figure 5: Draining lymph nodes resect ion for subsequent analy sis
Depicted are the predicted draining lymph nodes after i.m. injection into the gastrocnemius muscle used for further
analysis. Figure adopted according to Harrell et al. 2008 . IL, iliac. IN, inguinal. PO, popliteal .
4.5.5 Preparation of Splenocyte Single Cell Suspensions
Single cell suspensions from collected spleens were prepared according to SOP -030-
078. To this end, spleens were squeezed throu gh 70 µm cell strainer s using the
plunger of a syringe to release the splenocytes in to a 50 mL tube. Splenocytes were
washed with an excess volume of PBS followed by centrifugation at 300 x g for 6 min
at ambient temperature and discarding the supernatants. Erythrocytes were lysed with
erythrocyte lysis buffer (154 mM NH 4Cl, 10 mM KHCO 3, 0.1 mM EDTA) for 5 min at
ambient temperature. The reaction was stopped with an excess volume of PBS. After
another washing step, cells were resuspended in DC medium (RPMI medium1640 (1x)
+ GlutaMAX -I [Life Technologies ], 10% FBS, 1% NEAA, 1% sodium -pyruva te, 0.5%
penicillin/streptomycin, 50 µM 2 -mercaptoethanol), passed through a 70 µm cell
strainer again, counted according to SOP -010-028, and stored at 4 °C until further use.
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4.5.6 Preparation of Lymph Node Single Cell Suspensions
The popliteal, iliac and inguin al dLNs (Figure 5) were stored together in a plastic tube
containing 450 µL PBS at ambient temperature in the dark until single cell preparation.
50 µL collagenase D (10 mg/mL) w ere added to yield a final concentrat ion of 1 mg/mL,
the dLNs were thoroughly cut into pieces using forceps or scissors, and incubated for
10 min at 37 °C. Cells were passed through a 70 µm cell strainer placed on a 50 mL
plastic tube and minced using the plunger of a 5 mL syringe. The cell st rainer is
subsequently rinsed using 5 mL of PBS and the cell solution counted according to
SOP -010-028.
4.5.7 RNA Electroporation
CT26 colon carcinoma cells (ATCC) were washed once with 10 mL of serum -free X -
Vivo 15 medium, centrifuged (300 ×g, 6 min, ambient t emperature), taken up in 1 –
2 mL of X -Vivo 15 medium, counted (SOP -010-028), and diluted to a concentration of
25 × 106 cells/mL. S Protein encoding modRNA or irrelevant modRNA (10 µg in 40 µL
of X -Vivo 15 medium each) was carefully placed at the bottom of a 4 mm
electroporation cuvette, topped up with 200 µL of cells (corresponding to 5 × 106 cells)
and shortly mixed by pipetting up and down. Electroporation was then performed with
a BTX™ ECM™ 830 Square Wave Electroporator applying one 300 V pulse for 15 ms.
Immediately after electroporation, cells were transferred to a 15 mL tube containing 1 –
2 mL of DC medi um, counted, and diluted to 4 × 105 cells/mL for the cytotoxicity assay ,
and 5 × 105 cells/mL for the IFN γ ELISpot assay (Section 4.5.8 ).
4.5.8 ELISpot Assay
IFNγ ELISpot assay was performed according to SOP -030-110 (with minor
modifications as descri bed below) using the mouse IFN -γ ELISpotPLUS kit. Briefly, 96 -
well ELISp ot plates were washed with PBS and blocked with serum -containing medium
(DC medium) for at least 30 min at 37°C. After blocking , 100 µl of the splenocyte
solution (5 x 105 cells) as well as 100 µl electroporated CT26 cells (5 x 104 cells) or
100 µl S pepti de mix (final concentration per well: 0.1 µg/ml) were added yielding a
final volume per well of 200 µL. No peptide or irrelevant RNA transfected cells were
used as controls . Plates were incubated overnight in a 37°C humidified incubator with
5% CO 2. After approximately 18 h cells were discarded and a second biotinylated anti -
mouse IFN -γ antib ody incubated for 2 h at ambient temperature . The plate was then
developed by addition of Streptavidin -ALP for 1 h at ambient temperature in the dark
followed by additi on of BCIP®/NBT substrate for 5 –7 min at ambient temperature in
the dark. Spots were counted on a CTL ELISPOT reader ImmunoSpot® S6 Core
Analyzer according to SOP -010-099.
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4.5.9 xCELLigence Cytotoxicity Assay
Preparation of targets:
Of DC medium, 50 µL per well were added to a 96 -well PET E -plate to perform a blank
measurement at an xCELLigence RTCA MP, Real Time Cell Analyzer. Tumor cells
(50 µL of a 4 × 105 cells/mL suspension, corresponding to 2 × 104 cells) electroporated
with S RNA or irrelevant RNA (10 µg each ) were subsequently added to the E -plate.
After allowing the cell suspension to settle down for 30 min at ambient temperature,
the E -plate was transferred to the xCELLigence device and measurement was
continued.
Peptide loading of targets:
In mCorVac#1 6, 100 µl S peptide mix (final concentration per well: 0.1 µg/ml) was
added to S RNA electroporated tumor cells one hour prior T cell addition. After one
hour of incubation, the medium was carefully aspirated and the wells were washed
with PBS twice . Befor e adding the effector cells, 100 µl of DC medium was dispensed
per well.
Addition of effectors:
On the same day, splenocytes were transferred to a T25 cell cult ure flask at a density
of 1.5 –2 × 106 cells/cm2. S peptide mixes and recombinant IL -2 (Proleu kin) were added
to yield a final concentration of 0.1 µg/mL and 100 U/mL, respectively, and the cell
suspension was kept at 37 °C, 5% CO 2 overnight. On the day after, restimulated
splenocytes were transferred to a 15 mL plastic tube, the T25 flask was rinse d with
5 mL of MACS buffer and added to the same tube. Subsequently, CD8+ cells were
isolated from restimulated splenocytes using CD8a (Ly -2) MACS® MicroBeads
according to the manufacturer’s instructions. Labeled cells were eluted from MACS LS
columns, cen trifuged (5 min at 460 ×g), taken up in 1 –2 mL of warm (approximately
37°C) DC medium, counted (SOP -010-028) and diluted with DC medium to a
concentration of 6 × 106 cells/mL. CD8+ cells (100 µL) , DC medium or Staurosporin
(4 µM final concentration) were a dded in duplicate to the targets in the E-plate and the
xCELLigence measurement was continued for at least three days. RTCA Data analysis
software or xCELLigence RTCA Software Pro ( both ACEA Biosciences) were used for
data analysis.
4.5.10 Cytokine M ultiplex Prot ein Quantification
Cytokine concentrations were determined in supernatants derived from ex vivo
restimulated splenocytes and dLN cells. 5 × 105 splenocytes or dLN cells in 100 µL
medium/well were transferred to a 96 -well U -bottom plate, and 100 µL medium
supplemented with S peptide mixes to a final concentration of 0.2 µg/mL /peptide /well,
or cell culture medium only (negative control ) were added and mixed. For each group,
three samples were treated with 100 µL PMA and i onomycin to a final concentration of
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0.5 µg/mL and 1 µg/mL/well, respectively (positive controls). Cells were incubated for
48 h at 37°C, 5% CO 2. Supernatants were harvested and st ored at -20°C for the
cytokine multiplex assay .
Cytokine concentrations in supernatants of restimulated splenocyt es and dLN cells
were determined from thawed cell culture supernatants using a bead -based, 11 -plex
Th1/Th2 mouse ProcartaPlex immunoassay according to the manufacturer’s
instructions. Analytes included in the assay were IFNγ, IL -12p70, IL -13, IL -1β, IL -2, IL-
4, IL-5, IL-6, TNF , GM -CSF, and IL-18.
Fluorescence was measured with the Bioplex200 system and analyzed with
ProcartaPlex Analyst 1.0 softw are (Thermo Fisher Scientific).
4.5.11 Flow Cytometry
All flow cytometric d ata were acquired on a BD Symphony A3 or BD Celesta (B cell
analysis ) flow cytometer using BD FACSDiva software version 9.1 or 8.0.1.1,
respectively, and analy zed with FlowJo 10.6 (FlowJo LLC, BD Biosciences ).
4.5.11.1 Restimulation of T cells for functional T cell analysis in the
spleen and dLN
For functio nal analysis, splenocytes and dLN cells were ex vivo restimulated. 4 × 106
splenocytes and 1 × 106 (mCorVAC#15) or 2 × 106 (mCorVAC#16) dLN cells in 100 µL
DC medium/well were transferred to a 96 -well U -bottom plate. To each well, 50 µL
medium were added, supplemented with either S peptide mix es to a final concentration
of 0. 2 µg/mL /peptide /well (mCorVAC#15) or 0.5 µg/mL/peptide/well (mCorVAC#16) ,
or medium only (negative controls) , and mixed. To one sample per group, 50 µL PMA
and i onomycin to a final conc entration of 0.5 µg/mL and 1 µg/mL/well, respectively,
were added (positive controls). Three additional wells of any group were added as
unstained controls.
Cells were quickly spun down (30 s, 460 × g) and incubated for 1 h at 37 °C, 5% CO 2.
To each well, 50 µL GolgiStop and GolgiPlug in medium were added to a final dilution
of GolgiStop of 1: 1,500 and GolgiPlug of 1:1,000 , mixed, and cells were furt her
incubated for 4 h at 37 °C, 5% CO 2.
4.5.11.2 Functional T cell analysis in the spleen and dLN
For mouse functional T cell analysis, restimulated cells (see 4.5.11.1 ) were cent rifuged
(5 min, 300 × g) and supernatants discarded . Flow cytometry master mixes (MM) for
functional T cell analysis are depicted in Table 8 and Table 9.
Cells were stained with fixable viability dye and extracellularly with antibodies against
CD3, CD4, CD8α, CD44, PD -1, CD40L, CD62L and CXCR5 (mCorVAC#15, MM1 a),
or CD4, CD8α, CD44, CD45, PD-1, CD40L, CD62L and CXCR5 mCorVAC#16, MM1 b)
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in Brilliant Stain Buffer Plus diluted 1:5 in flow buffer (PBS supplemented with 2% FCS,
2 mM EDTA , 0.01% sodium azide ) in a total volume of 50 µL for 30 min at 2-8 °C. After
washing cells once with 200 µL flow buffer (5 min, 300 × g), cells were stained with
streptavidin Brilliant Stain Buffer Plus diluted 1:5 in flow buffer (MM2). After washing
cells once with 200 µL flow buffer, cells were fixed with 200 µL 2% RotiHistofix for
15 min at room temperature (mCorVAC#15) and resuspended in 200 µL Perm /Wash
buffer (FoxP3/Transcription Factor Staining Buffer Set) overnight at 2 -8 °C
(mCorVAC#15) , or overnight at 2 -8 °C (mCorVAC#16) . After washing cells once with
200 µL Perm/Wash buffer (5 min, 500 × g) (mCorVAC#16) , permeabilized cells were
intracellulary treated with 25 µL Fc block (diluted 1:50) for 10 min at 2-8 °C before IL-
4, TNF, Bcl -6, IFNγ, T -bet and IL -2 antibodies (mCorVAC#15, MM 3a) or IL-4, TNF,
IFNγ, T -bet, IL -2 and CD3 (mCorVAC#16, MM 3b) in Perm/Wash bu ffer in a total
volume of 25 µL were added, and cells incubated for 30 min at 2 -8 °C (staining volume:
50 µL). After washing cells twice with 200 µL Perm/Wash buffer (5 min, 500 × g), cells
were resuspended in 200 µL flow buffer. Fluorescence minus 10 (FM1 0) controls were
stained for viability and with antibodies against CD3, CD8a , CD4 and CD62L
(mCorVAC#1 5), or CD3, CD8a, CD4, CD45 and CD62L (mCorVAC#1 6) only.
Table 8: Flow cytometry antibody master mixes for functional T cell anal ysis in the spleen and dLN
(mCorVAC#15 and mCorVAC#16).
Cat, catalog number. LD, LiveDead viability dye. Lot, lot number. MM, master mix.
MM1a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV395 CD3 145-
2C11 BD 563565 9204644 31.05.2022 100 0,5
BUV563 CD44 IM7 BD 741227 0119427 30.04.2021 2,500 0,1
BV421 CXCR5 L138D7 BioLegend 145512 B281252 L138D7 50 1
BV480 CD4 RM4 -5 BD 565634 9016508 31.05.2020 250 0,2
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1
BV785 CD62L MEL-14 BioLegend 104440 B272550 N/A 200 0,25
FITC CD8 30-F11 BD 553079 6197750 31.08.2021 200 0,25
Biotin CD40L MR1 BD 553657 8186567 12.04.2024 100 0,5
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,000 0,05
MM1b mCorVAC#16
Fluorochro me Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV563 CD44 IM7 BD
Biosciences 741227 0119427 30.04.2021 2,500 0,02
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BUV737 CD45 53-6.7 BD
Biosciences 564297 9030634 N/A 200 0,25
BV421 CXCR5 L138D7 BioLegend 145512 B281252 N/A 100 0,50
BV480 CD4 RM4 -5 BD
Biosciences 565634 9016508 31.05.2020 250 0,20
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1,00
BV785 CD62L MEL-14 BioLegend 104440
B258213 N/A 200 0,25
FITC CD8 30-F11 BD
Biosciences 553079 6197750 31.08.2021 200 0,25
Biotin CD40L MR1 BD
Biosciences 553657 8186567 12.04.2024 100 0,50
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,000 0,05
MM2 mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
PE Streptavidin N/A BioLegend 405203 B170498 N/A 200 0,25
MM3a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BV711 IL-4 11B11 BD 564005 9276915 21.03.2021 100 0,5
BB700 TNF MP6-XT22 BD 566510 0021825 31.03.2021 5,000 0,01
PE Bcl-6 K112 -91 BD 5615 22 9165931 30.06.2022 50 1
PE-Cy7 IFNγ XMG1.2 eBioscience 25-
731182 E07672 -
1632 09.2014 1,000 0,05
AF647 T-bet 4B10 biolegend 644804 B248741 N/A 5,000 0,01
APC-R700 IL-2 JES6 -5H4 BD 565186 9303906 31.03.2021 5,000 0,01
MM3b mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV395 CD3 145-2C11 BD
Biosciences 563565 9204644 31.05.2022 500 0,10
BV711 IL-4 BD
Biosciences 564005 9276915 21.03.2021 100 0,5
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BB700 TNF MP6-XT22 BD
Biosciences 566510 0021825 31.03.2021 5,000 0,01
PE-Cy7 IFNγ XMG1.2 eBiosc ience 25-
731182 E07672 -
1632 09.2014 1,000 0,05
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 5,000 0,01
APC-R700 IL-2 JES6 -5H4 BD
Biosciences 565186 9303906 31.03.2021 5,000 0,01
4.5.11.3 Phenotypic T cell analysis in the spleen and dLN
For mouse phenotypic T cell analysis in the spleen and dLNs, 4 × 106 splenocytes and
1 × 106 (mCorVAC#15) or 1.5 × 106 (mCorVAC#16) dLN cells/well were transferred to
a 96 -well U bottom plate, c entrifuged (3 min, 300 × g, 2–8°C) and supernatants
discarded . Flow cytometry MM for phenotypic T cell analysis are depicted in Table 9.
Cells were stained with fixable viability dye and extracellularly with antibodies against
CD3, CD4, CD8 α, CD25, CD44, PD -1, CD62L, ICOS, CD19 and CXCR5
(mCorVAC#15, MM1 a), or CD4, CD8α, CD25, CD44, CD45, PD -1, CD62L, ICOS,
CD19 and CXCR5 ( mCorVAC#16, MM1 b) in Brilliant Stain Buffer Plus diluted 1:5 in
flow buffer ( PBS supplemented with 2% FCS, 2 mM EDTA , 0.01% sodium azide ) in a
total volume of 50 µL for 30 min at 2-8 °C. After washing cells twice with 200 µL flow
buffer (5 min, 300 × g), cells were resuspended in 200 µL 2% RotiHistofix, immediately
centrifuged (5 min, 300 × g) and fixed again with 200 µL Fix/Perm buffer
(FoxP3/Transcription Factor Staining Buffer Set) overnight at 2 -8 °C (mCorVAC#15) ,
or for 20 min at 2 -8 °C and incubated in 200 µL Perm/Wash buffer overnigh t at 2 -8°C
(mCorVAC#16) . After washing cells once with 200 µL Perm/Wash buffer (5 min,
500 × g) (mCorVAC#15) , permeabilized cells were intracellular y treated with 25 µL Fc
block (diluted 1:50) for 10 min at room temperature before T-bet, GATA3 , FoxP3 and
Bcl-6 antibod ies (mCorVAC#15, MM 2a) or T-bet, GATA3, FoxP3 and CD3
(mCorVAC#16, MM 2b) in Perm/Wash buffer in a total volume of 25 µL, and cells
incub ated for 30 min at 2 -8 °C (staining volume: 50 µL). After washing cells twice with
200 µL Perm /Wash buffer (5 min, 500 × g), cells were resuspended in 200 µL flow
buffer. Fluorescence minus 10 (FM10) controls were stained for viability and with
antibodies against CD3, CD8a , CD4, CD62L and CD19 only.
Table 9: Flow cytometry antibody master mixes for phenotypic T cell analysis in the spleen and dLN
(mCorVAC#15 and mCorVAC#16).
Cat, catalog number. LD, LiveDead viability dye. Lot, lot number. MM, master mix.
MM1a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV395 CD3 145-2C11 BD
Biosciences 565992 9204644 31.05.2022 100 0,50
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BUV563 CD44 IM7 BD
Biosciences 741227 119427 30.04.2021 2,500 0,02
BV421 CXCR5 L138D7 BioLegend 145512 B281252 N/A 100 0,50
BV480 CD4 RM4 -5 BD
Biosciences 565634 9016508 31.05.2020 250 0,20
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1,00
BV711 CD25* PC61 BD
Biosciences 740714 119426 30.04.2021 500 0,10
BV785 CD62L MEL-14 BioLegend 104440 B272550 N/A 200 0,25
FITC CD8 53-6.7 BD
Biosciences 553031 9143776 31.08.2021 200 0,25
PerCPeF710 ICOS 7E.17G9 Invitrogen 46-9942 -
82 2029789 30.04.2021 50 1,00
AF700 CD19 6D5 BioLegend 115528 B261756 N/A 100 0,50
ef780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,000 0,05
MM1b mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV563 CD44 IM7 BD
Biosciences 741227 0119427 30.04.2021 2,500 0,02
BUV737 CD45 53-6.7 BD
Bioscience s 564297 9030634 N/A 200 0,25
BV421 CXCR5 L138D7 BioLegend 145512 B281252 N/A 100 0,50
BV480 CD4 RM4 -5 BD
Biosciences 565634 9016508 31.05.2020 250 0,20
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1,00
BV711 CD25 PC61 BD
Biosciences 740714 0119426 30.04.2021 500 0,10
BV785 CD62L MEL-14 BioLegend 104440 B272550 N/A 200 0,25
FITC CD8 30-F11 BD
Biosciences 553031 9143776 31.08.2021 200 0,25
PerCPeF710 ICOS 7E.17G9 Invitrogen 46-9942 -
82 2029789 30.04.2021 50 1,00
AF700 CD19 6D5 BioLegend 115528 B261756 N/A 100 0,50
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,000 0,05
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MM2a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
PE Bcl-6 BD
Biosciences 561522 9165931
30.06.2022 100 0,5
PECF594 FoxP3 MF23 BD
Biosciences 562466 9276149 28.20.2021 200 0,25
PE-Cy7 GATA3 TWAJ Invitrogen 25-9966 -
42 2142972 N/A 25 2
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 100 0,5
MM2b mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/te st
[µL]
BUV395 CD3 145-2C11 BD
Biosciences 563565 9204644 31.05.2022 50 0,50
PE-CF594 FoxP3 MF23 BD
Biosciences 562466 9276149 28.20.2021 200 0,25
PE-Cy7 GATA3 TWAJ Invitrogen 25-9966 -
42 2142972 N/A 25 2,00
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 5,000 0,01
4.5.11.4 Phenotypic T cell analysis in the blood
For mouse phenotypic T cell analysis in peripheral blood, 50 µL freshly drawn blood
were transferred to round bottom 5 -mL tubes, washed once with 500 µL PBS (Gib co)
(300 × g, 8 min) and the cell pellet was resuspended in 2 mL ACK lysing buffer (Gibco)
and incubated for 3 min at room temperature. Flow cytometry master mixes (MM) for
phenotypic T cell analysis are depicted in Table 10.
Cells were washed twice with 1 mL flow buffer (300 × g, 8 min) and stained with fixable
viability dye and anti -CXCR5 (rat IgG2a) antibody in the presence of Fc block diluted
1:100) in flow buffer in a total volume of 50 µL for 20 min at room temperature (MM1) .
After washing cells twic e with 1 mL flow buffer ( 8 min, 300 × g), cells were stained with
anti-rat IgG2a biotin in flow buffer in a total volume of 50 µL for 20 min at 2-8 °C (MM2) .
After washing cells twice with 1 mL flow buffer ( 8 min, 300 × g), cells were stained
extracellular ly with antibodies against CD3, CD4, CD8 α, CD25, CD38, CD44, PD -1,
CD62L, ICOS, CD127 (4 -1BB), CD19 and streptavidin (mCorVAC#15 , MM3a ), or CD4,
CD8α, CD25, CD38, CD44, PD -1, CD62L, ICOS, CD127 (4 -1BB), CD19 and
treptavidin (no CD3, mCorVAC#16 , MM3b ) in Brilliant Stain Buffer Plu diluted 1 5 in
flow buffer in a total volume of 50 µL for 20 min at 2 -8 °C. After washing ce lls once with
1 mL flow buffer (5 min, 300 × g), cells were fixed in 200 µL 2% RotiHistofix for 15 min
at room temperature (mCorVAC#15), or centrifuged immediately after mixing (5 min,
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300 × g) and fixed again with 200 µL Fix/Perm buffer (FoxP3/Transcription Factor
Staining Buffer Set) for 20 min at 2 -8 °C (mCorVAC#16) . After centrifugation ( 5 min,
500 × g), cells were resuspended in 200 µL Perm /Wash buffer (FoxP3/Transcription
Factor Staining Buffer Set) and incubated over night at 2 -8 °C. Permeabilized cells
were centrifuged ( 5 min, 500 × g) and intracellulary treated with 25 µL Fc block (diluted
1:50) in Perm/Wash buffer for 10 min at 2-8 °C before T-bet and GATA3 antibod ies
(mCorVAC#15 , MM4a ) or CD3, FoxP3, T-bet and GATA3 antibod ies (mCorVAC#16 ,
MM4b ) in Perm /Wash buffer in a total volume of 25 µL were added , and cells incubated
for 30 min at 2 -8 °C (staining volume: 50 µL). After washin g cells twice with 1 mL
Perm /Wash buffer ( 5 min, 500 × g), cells were resuspended in 150 µL flow buffer.
Fluorescence minus 10 (FM10) controls were stained for viability and with antibodies
against CD3, CD8a , CD4, CD62L , CD19 and streptavidin only.
Table 10: Flow cytometry antibody master mixes for phenotypic T cell analysis in the blood (mCorVAC#15
and mCorVAC#16).
Cat, catalog number. LD, LiveDead viability dye. Lot, lot number. MM, master mix.
MM1
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
Purifi ed CXCR5 2G8 BD
Biosciences 551961 9143926 28.02.2027 100 0,50
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1000 0,05
N/A Fc block 2.4G2 BD 553142 0028326 31.05.2027 100 0,50
MM2
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
Biotin IgG2a RG7/1.30 BD
Biosciences 553894 9288614 31.05.2024 100 0,50
MM3a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV395 CD3 145-2C11 BD 56356 5 9204644 31.05.2022 100 0,50
BUV563 CD44 IM7 BD
Biosciences 741227 0119427 30.04.2021 2,500 0,02
BUV737 CD8a 53-6.7 BD
Biosciences 564297 9030634 N/A 200 0,25
BV421 Streptavidin N/A BD
Biosciences 563259 9197684 31.12.2021 200 0,25
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BV480 CD4 RM4 -5 BD
Biosciences 565634 9016508 31.05.2020 250 0,20
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1,00
BV711 CD25 PC61 BD
Biosciences 740714 0119426 30.04.2021 500 0,10
BV785 CD62L MEL-14 BioLegend 104440 B258213 N/A 200 0,25
AF488 CD38 90 BioLegend 102714 B298187 N/A 100 0,50
PerCPeF710 ICOS 7E.17G9 Invitrogen 46-
9942 -82 2029789 30.04.2021 50 1,00
PE 4-1BB 17B5 eBioscience 12-
1371 -82 E01500 -
1632 N/A 100 0,50
PE-Cy7 GATA3 TWAJ Invitrogen 25-
9966 -42 B2142972 N/A 25 2,00
AF647 T-bet 4B10 BioLegen d 644804 B248741 N/A 5000 0,01
AF700 CD19 6D5 BioLegend 115528 B261756 N/A 100 0,50
MM3b mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV563 CD44 IM7 BD
Biosciences 741227 0119427 30.04.2021 2500 0,02
BUV737 CD8a 53-6.7 BD
Biosciences 564297 9030634 N/A 200 0,25
BV421 Streptavidin N/A BD
Biosciences 563259 9197684 31.12.2021 200 0,25
BV480 CD4 RM4 -5 BD
Biosciences 565634 9016508 31.05.2020 250 0,20
BV605 PD-1 29F.1A12 BioLegend 135219 B281806 N/A 50 1,00
BV711 CD25 PC61 BD
Biosciences 740714 0119426 30.04.2021 500 0,10
BV785 CD62L MEL-14 BioLegend 104440 B258213 N/A 200 0,25
AF488 CD38 90 BioLegend 102714 B298187 N/A 100 0,50
PerCPeF710 ICOS 7E.17G9 Invitrogen 46-
9942 -82 2029789 30.04.2021 50 1,00
PE 4-1BB 17B5 eBioscience 12-
1371 -82 E01500 -
1632 N/A 100 0,50
PE-Cy7 GATA3 TWAJ Invitrogen 25-
9966 -42 B2142972 N/A 25 2,00
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 5000 0,01
AF700 CD19 6D5 BioLegend 115528 B261756 N/A 100 0,50
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MM4a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
PE-CF594 FoxP3 MF23 BD
Biosciences 562466 9276149 28.02.2021 200 0,25
PE-Cy7 GATA3 TWAJ Invitrogen 25-9966 -
42 B2142972 N/A 25 2,00
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 5,000 0,01
MM4b mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BUV395 CD3 145-2C11 BD
Biosciences 563565 9204644 31.05.2022 50 0,50
PE-CF594 FoxP3 MF23 BD
Biosciences 562466 9276149 28.02.2021 200 0,25
PE-Cy7 GATA3 TWAJ Invitrogen 25-9966 -
42 B2142972 N/A 25 2,00
AF647 T-bet 4B10 BioLegend 644804 B248741 N/A 5,000 0,01
4.5.11.5 B cell analysis in the spleen and dLN
For mouse B cell analysis in the spleen and dLNs, 1 × 106 splen ocytes and 2.5 × 105
dLN cells/well were transfer red to a 96 -well V bottom plate, centrifuged (5 min, 300 × g,
2–8 °C) and supernatants discarded. Flow cytometry MM for B cell analysis are
depicted in Table 11.
Cells were treated with Fc block (diluted 1: 50) in 50 µL flow buffer for 15 min at 2-8 °C
and cells were stained with fixable viability dye and extracellularly with antibodies
against CD19, CD45R/B220, IgD, CD138, IgM, CD38, CD95/FAS, IgG1, IgG2a, GR -
1, F4/80, CD4 and CD8a (mCorVAC#15, MM1 a) in Brilliant St ain Buffer in a total
volume of 50 µL for 20 min at 2 -8 °C (staining volume: 100 µL); or cells were directly
treated with fixable viability dye and extracellularly with antibodies against CD19,
CD45R/B220, IgD, CD138, IgM, CD38, CD95/FAS, GR -1, F4/80, CD4 and CD8a
(mCorVAC#16, MM1b), in Brilliant Stain Buffer in a total volume of 100 µL for 20 min
at 2-8 °C (staining volume: 100 µL). In addition, cells were treated with Fc block (diluted
1:50) in 50 µL flow buffer for 15 min at 2-8 °C and stained with fixab le viability dye and
extracellularly with antibodies against with PD-L2, CD45R/B220, CD19, CD73, IgM,
CD80, GR-1, F4/80, CD4 and CD8a in Brilliant Stain Buffer in a total volume of 50 µL
(mCorVAC#15 , MM3 ) (staining volume: 100 µL); or cells were directly t reated with
fixable viability dye and extracellularly with MM3 (mCorVAC#16) in Brilliant Stain Buffer
in a total volume of 100 µL for 20 min at 2 -8 °C (staining volume: 100 µL). After
washing cells twice with 200 µL flow buffer ( 5 min, 400 × g, 2–8 °C), cells were fixed
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with 200 µL 2% RotiHistofix and incubated over night at 2 -8 °C. After washing cells
once with 200 µL flow buffer (5 min, 400 × g), cells were resuspended in 100 µL flow
buffer (mCorVAC#15), or stained intracellularly with antibodies against IgG1 and
IgG2a (MM2) in Perm/Wash buffer in a total volume of 50 µL for 30 min at 2-8 °C
(staining volume: 50 µL), before being resuspended in 100 µL flow buffer
(mCorVAC#16) . Fluorescence minus Fas/CD138 (FM Fas/CD138) controls were
stained for MM1 a and M M1b excluding CD95/FAS and CD138 ; fluorescence minus
IgG2a (FM IgG2a) controls were stained for MM1 a excluding IgG2a ; fluorescence
minus IgG1 (FM IgG1) controls were stained for MM1 a excluding IgG1 ; fluorescence
minus 34 (FM 34) controls were stained for M M1b excluding CD138 and CD95/FAS;
fluorescence minus CD73 and CD80 (FM 73/80) controls were stained for MM 3
excluding CD73 and CD80; fluorescence minus PD -L2 (FM PD -L2) controls were
stained for MM 3 excluding PD -L2; and fluorescence minus 35 (FM 35) contro ls were
stained for MM3 excluding PD -L2, CD73 and CD80.
Table 11: Flow cytometry antibody master mixes for B cell analysis in the spleen and dLN (mCorVAC#15
and mCorVAC#16).
Cat, catalog number. LD, LiveDead viability dye. Lot, lot number. MM, master mix.
MM1a mCorVAC#15
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/t
est [µL]
FITC FAS/CD95 Jo2 BD
Biosciences 561979 8296755 30.11.2023 100 1
PE CD38 90 Thermo
Fisher 12-0381 -
82 2150667 25.04.2021 400 0,25
PerCPCy 5.5 Gr1 RB6-8C5 BioLegend 108428 B278340 N/A 800 0,12
PerCPCy5.5 F4/80 BM8 BioLegend 123128 B276793 N/A 800 0,12
PerCPCy5.5 CD4 RM4 -5 BioLegend 100540 B261856 N/A 800 0,12
PerCPCy5.5 CD8 53-6.7 BD
Biosciences 551162 9098816 31.05.2023 800 0,12
PE-Cy7 IgM R6-60.2 BD
Biosciences 552867 9269114 18.07.2021 200 0,5
AF647 CD45R/B22
0 RA3-6B2 BioLegend 103226 B243962 N/A 1,500 0,07
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,600 0,06
BV421 IgD 11-26c.2a BioLegend 405725 B280598 N/A 2,500 0,04
BV510 IgG1 A85-1 BD 746811 0115095 30.04.2021 200 0,5
BV605 CD138 281-2 BioLegend 142531 B299222 N/A 200 0,5
BV711 IgG2a R19-15 BD 744533 0115092 30.04.2021 200 0.5
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BV785 CD19 1D3 BD
Biosciences 563333 0023948 30.06.2021 1,000 0.1
MM1b mCorVAC#16
Fluorochrom e Marker Clone Company Cat Lot Expiry Dilution Volume
/test
[µL]
FITC FAS/CD95 Jo2 BD
Biosciences 561979 8296755 30.11.2023 100 1
PE CD38 90 Thermo
Fisher 12-0381 -
82 2150667 25.04.2021 400 0,25
PerCPCy5.5 Gr1 RB6-8C5 BioLegend 108428 B278340 N/A 800 0,12
PerCPCy5.5 F4/80 BM8 BioLegend 123128 B276793 N/A 800 0,12
PerCPCy5.5 CD4 RM4 -5 BioLegend 100540 B261856 N/A 800 0,12
PerCPCy5.5 CD8 53-6.7 BD
Biosciences 551162 9098816 31.05.2023 800 0,12
PE-Cy7 IgM R6-60.2 BD
Biosciences 552867 9269114 18.07.2021 200 0,5
AF647 CD45R/B220 RA3-6B2 BioLegend 103226 B243962 N/A 1,500 0,07
eF780 LD N/A eBioscience 65-0865 -
14 2178170 N/A 1,600 0,06
BV421 IgD 11-26c.2a BioLegend 405725 B280598 N/A 2,500 0,04
BV605 CD138 281-2 BioLegend 142531 B299222 N/A 200 0,5
BV785 CD19 1D3 BD
Biosciences 563333 0023948 30.06.2021 1,000 0.1
MM2 mCorVAC#16
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume
/test
[µL]
BV510 IgG1 A85-1 BD 746811 0115095 30.04.2021 400 0,125
BV711 IgG2a R19-15 BD 744533 0115092 30.04.2 021 400 0,125
MM3
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume
/test
[µL]
BV421 PD-L2 TY25 BD
Biosciences 564245 9204505 30.11.2021 600 0.2
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BV605 CD45R/B22
0 RA3-6B2 BioLegend 103244 B305934 N/A 800 0,12
BV786 CD19 1D3 BD
Bioscienc es 563333 0023948 30.06.2021 1,000 0,1
PE CD73 TY/11.8 BioLegend 127206 B267137 N/A 600 0,2
PerCPCy5.5 Gr1 RB6-8C5 BioLegend 108428 B278340 N/A 800 0,12
PerCPCy5.5 F4/80 BM8 BioLegend 123128 B276793 N/A 800 0,12
PerCPCy5.5 CD4 RM4 -5 BioLegend 100540 B261856 N/A 800 0,12
PerCPCy5.5 CD8 53-6.7 BD
Biosciences 551162 9098816 31.05.2023 800 0,12
PE-Cy7 IgM R6-60.2 BD
Biosciences 552867 9269114 18.07.2021 200 0,5
AF647 CD80 16-10A1 BioLegend 104718 B278896 N/A 400 0,25
eF780 LD N/A eBioscience 65-0865 -14 2178170 N/A 1,600 0,06
4.5.11.6 Myeloid cell analysis in the spleen
For mouse myeloid cell analysis in the spleen, 2 × 106 splenocytes/well were
transferred to a 96 -well U bottom plate, c entrifuged (3 min, 460 × g) and supernatants
discarded. Flow cytometry MM fo r myeloid cell analysis is depicted in Table 12.
Cells were stained with Fc block and fixable viability dye in PBS in a total volume of
100 µL (MM1) for 15 min at 2 -8 °C. After washing cells once with 200 µL PBS (3 min,
460 × g), cells were stained extracellularly with antibodies against CD8, CD45, BST2,
CD86, XCR1, MHC class II, CD11b, PD -L1, CD103, F4/80, CD11c and GR -1 in Brilliant
Stain Buffer in a total volume of 50 µL (MM2) for 30 min at 2-8 °C (staining volum e:
50 µL). After washing cells once with 200 µL PBS (3 min, 460 × g), cells wer e fixed
with 1 00 µL Fix/Perm buffer (FoxP3/Transcription Factor Staining Buffer Set) for
30 min at 2-8 °C. After washing cells twice with 200 µL Perm/Wash buffer (3 min,
460 × g), cells were resuspended in 200 µL Perm/Wash buffer and incubated overnight
at 2-8 °C. Permeabilized cells were centrifuged (3 min, 460 × g) and intracellular ly
treated with CD206 antibody in Perm/Wash buffer in a total volume of 50 µL (MM3) for
30 min at 2-8 °C (staining volume: 50 µL). After washing cells twice with 200 µL
Perm/Wash buffer (3 min, 460 × g), cells were resuspended in 200 µL flow buffer.
Table 12: Flow cytometry antibody master mixes for myeloid cell analysis in th e spleen (mCorVAC#15 and
mCorVAC#16).
Cat, catalog number. LD, LiveDead viability dye. Lot, lot number. MM, master mix.
MM1
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
BV605 -like LD N/A ThermoFish
er L34959 1921586 N/A 800 0,06
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N/A Fc block 2.4G2 BD 553142 0028326 31.05.2027 100 0,50
MM2
Fluorochrom
e Marker Clone Company Cat Lot Expiry Dilution Volume/tes
t [µL]
BUV395 CD45 30-F11 BD
Biosciences 564279 9016570 N/A 100 0,50
BUV737 CD8 53-6.7 BD
Biosciences 564297 90306 34 N/A 100 0,50
eF450 BST2 eBio927 invitrogen 48-3172 -82 2055199 N/A 100 0,50
BV510 CD86 GL-1 BioLegend 105039 B264604 N/A 100 0,50
BV650 XCR1 ZET BioLegend 148220 B265588 N/A 100 0,50
BV786 MHC II M5/114.15.
2 BD
Biosciences 742894 9333783 30.11.2020 500 0,10
FITC CD11b M1/70 BD
Biosciences 553310 8295813 31.08.2024 200 0,25
PerCP -Cy5.5 PD-L1 10F.9G2 BioLegend 124333 B286738 N/A 100 0,50
PE CD103 Invitrogen 12-1031 -83 2054351 26.12.2021 N/A 400 0,13
PE-Dazzle594 F4/80 BM8 BioLegend 123145 B268244 N/A 100 0,50
APC CD11c N418 Miltenyi 130-119-
802/130 -
102-493 5200308676/
25200308676 24.3.2021/1
3.12.2015 100 0,50
APC-Cy7 GR-1 RB-8C5 BioLegend 108423 B209677 N/A 800 0,06
MM3
Fluorochrome Marker Clone Company Cat Lot Expiry Dilution Volume/test
[µL]
PE-Cy7 CD206 C068C2 BioLegend 141719 B260552 N/A 400 0,13
4.5.12 Statistical Analysis
GraphPad Prism 8 Software (La Jolla, USA) was used for statistical analysis and figure
generation. The following tests were used for data analysis:
Table 13: Statistical analyses
Data set Comparison Statistical test
Flow cytometry, immune cell
subsets Test groups vs. control group One-way ANOVA and Dunnett’s
posttest
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ELISpot assay Test groups vs. control group Repeated measurement o ne-way
ANOVA and Sidak’s posttest
Th1/Th2 cytokines Test groups vs. control group Two-way ANOVA and Sidak ’s
posttest
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5 RESULTS
5.1 ELISpot assay
BALB/c mice were euthanized on day 12 ( BNT162a1, BNT162b1, BNT162b2 ) or day
27 (BNT162c2) after vaccination. Isolated splenocytes w ere restimulated with S -
specific overlapping peptide mixes (S peptide) or CT26 cells electroporated with RNA
encoding the full -length S protein (S RNA). Recognition of S RNA transfected cells
served as an additional proof for successful processing of S -specific epitopes. Cells
cultivated without the presence of a peptide (No peptide) or control RNA electroporated
CT26 cells (Control RNA) served as control. Statistical significance was assessed by
repeated measurement one -way ANOVA and Sidak’s multiple compa rison post -test.
Raw data can be found in Table 19.
Figure 6: ELISpot analysis using splenocytes fro m animals treated with BNT162a1, BNT162b1 , BNT162b2
or BNT162c2
ELISpot assay of splen ocytes from BNT162a1 or BNT162b1 ( a) or BNT162b2 or BNT162c2 ( b) vaccinated mice
(n=8 per group) . Cells were isolated on day 12 (BNT162a1 , BNT162b1 , BNT162b2) or day 27 (BNT162c2) after
vaccination . Splenocytes were restimulated with S -specific overlapping peptide mixes and IFN γ secretion was
measured to assess T -cell responses. Mean spot counts per mouse are shown by dots; group mean values are
indicated by bars. One sample in the BNT162b2 group in response to S peptide and S RNA restimulation yielded
results that were too numerous to count ; these values were set to 1,500.
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Saturating amounts of IFNγ spots were detected in groups receiving BNT162b1,
BNT162b2 or BNT162c2 after restimulation with either S peptide or S RNA. Mean spot
counts were as high as 750 for BNT162b1 and exceeded 1,000 for BNT162b2 and
BNT162c2. Low but significant spot counts were detected for BNT162a1, reaching a
mean of 100 after S peptide restimulation and 36 after S RNA restimulation.
5.2 Flow Cytometry
Flow cytometry was applied to furth er characterize T - and B -cell numbers, activation
status , functional profile and subtypes after vaccination in the blood, spleen and dLNs .
dLNs were analyzed for functionality but are not further described in this report. Myeloid
cell subsets in the spleen were analyzed but are not further described in this report.
dLNs were not assayed for myeloid cell subsets due to insufficient cell numbers ( for
further details see Section 2.5). Statistical significance compari ng the vaccinated
groups to the respective control group was determined by one -way ANOVA and
Dunnett’s multiple comparison post -test. Raw data for analyzed immune cell subsets
including tissues and subsets not described here can be found in Attachment I . Gating
strategies can be found in Attachment II.
Phenotypic T - and B -cell analysis in the b lood
Blood was analyzed 7 days after vaccination. The CD8+ T cell percentage among CD3+
T cells in the blood was significantly increased around 45% to a mean of 34% f or
BNT162b2 treated mice with a corresponding decrease in CD4+ T cells ( Figure 7a,b).
No change in the percentage of CD8+ or CD4+ T cells among CD3+ T cells was
observed in any other group . A significant increase of TFH cells among CD4+ T cells
was observed in the BNT162b1, BNT162b2 and BNT162c2 groups ( Figure 7c).
Highest T FH levels with a mean of 1.34% were found for BNT162c2 followed by
BNT162b2 (0.53%) and BNT162b1 (0.48%) .
Among lymphocytes, B cell levels were significantly reduced in all groups, suggesting
a redistribution from the blood into secondary lymphoid organs ( Figure 7d).
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Figure 7: Analysis of ly mphocyte frequencies in the blood of BNT162a1, BNT162b1, BNT162b2 or BNT162c2
vaccinated mice
Flow cytometry analysis of blood 7 days post BNT162a1, BNT162b1, BNT162b2 or BNT162c2 treatment (N=8 per
group) . Buffer treated mice served as control. For BNT162 c2, the control group of mCorVAC#15 served as control
(sample processing and acquisition on the same day). Cell fractions per mouse are shown by dots; group mean
values are indicated by bars .
The fraction of activated T cells was particularly elevated whe n mice were treated with
BNT162b 1 or BNT162b2 . In these groups, CD8+ T cells significantly upregulated
CD44, CD38, PD -1 as well as ICOS ( Figure 8a). ICOS expression was also elevated
among CD4+ T cells ( Figure 8b). The fraction of ICOS+ TFH cells was increased in all
vaccinated groups but most significantly for B NT162b1, BNT162b2 and BNT162c2
(Figure 8c).
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Figure 8: Analysis of T cell activation in the blood of BNT162a1, BNT162b1, BNT162b2 or BNT162c2
vaccinated mice
Flow cytometry analysis of blood 7 days after BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccination (N=8
per group) . Buffer treated mice se rved as control. Cell fractions per mouse are shown by dots; group mean values
are indicated by bars.
Phenotypic T - and B -cell analysis dLNs
dLNs were analyzed 12 days (BNT162a1, BNT162b1, BNT162b2 ) or 27 days
(BNT162c2) after vaccination . As shown for the frequ ency among CD3+ T cells in the
blood ( Figure 7a), CD8+ T cell counts in the dLNs were significantly elevated in the
BNT162b2 group ( Figure 9a). CD4+ T cells as well as T FH cells were significantly
increased in mice treated with BNT162b1 or BNT162b2 (Figure 9b,c). T H1 T cell
increase was most pronounced in the BNT162b1 ( P=0.0134) and BNT162b2
(P=0.0531) groups (Figure 9d).
In line with increased T FH cell counts, B cell numbers were highest in BNT162b1
(P=0.0053) and BNT162b2 ( P>0.0001) vaccinated mice ( Figure 10a). Among B cells,
antibody secreting plasma B cells, class switched B cells and germinal center B cells
crucial for affinity maturation of antibodies were significantly expanded ( Figure 10b-d).
In BNT162a1, BNT162b1 and BNT162b2 groups only, germinal center B c ells
demonstrated a class switch to IgG1 (BNT162a1, BNT162b1 and BNT162b2) or IgG2a
(BNT162b1 and BNT162b2) (Figure 10e,f).
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Figure 9: Analysis of T cell counts in the dLNs of BNT162a1, BN T162b1, BNT162b2 or BNT162c2 vaccinated
mice
Flow cytometry analysis of T cells in the dLNs after BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccination
(N=8 per group) . Cells were isolated on day 12 (BNT162a1, BNT162b1, BNT162b2) or day 27 (BNT162c2) after
vaccination . Buffer treated mice se rved as control. Cell counts per mouse are shown by dots; group mean values
are indicated by bars.
Figure 10: Analysis of B cell counts in the dLNs of BNT162a1, BNT162b1, BNT162b2 or BNT162c2
vaccinated mice
Flow cytometry analysis of B cells in the dLNs after BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccination
(N=8 per group) . Cells were isolated on day 12 (BNT162a1, BNT162b1, BNT162b2) or day 27 (BNT162c2) after
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vaccination . Buffer treated mi ce se rved as control. Cell counts per mouse are shown by dots; group mean values
are indicated by bars.
Phenotypic T - and B -cell analysis in the spleen
Analysis of T cells and B cells in the spleen revealed similar but less pronounced
results compared to blood and dLNs. T FH cells, germinal center B cells and class
switch ed B cells were sig nificantly increased upon BNT162b1 or BNT162b2
vaccination ( Figure 11).
Figure 11: Analysis of T FH and B cell counts in the spleen of BNT162a1, BNT162b1, BNT162b2 or BNT162c2
vaccinated mice
Flow cytometry analysis of TFH cells (a), germinal center B cells ( b) and class switched B cells ( c) in the spleen after
BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccination (N=8 per group) . Cells were isolated on day 12
(BNT162a1, BNT162b1, BNT162b2) or day 27 (BNT162c2) after vaccination . Buffer treated mice se rved as control.
Cell counts per mouse are shown by dots; group mean values are indicated by bars.
Function al T-cell analysis in the spleen
Splenocytes were analyzed by i ntracellular cytokine staining 12 days ( BNT162a1,
BNT162b1, BNT162b2 ) or 27 days (BNT162c2) after vaccination, to quantify antigen -
specific T cells via flow cytometry. Secretion of IFNγ, IL -2 or TNF w as determined in
unstimulated or S peptide restimulated samples . Responses without stimulation were
subtracted from S peptide stimulated samples from the same mouse and depicted for
each treatment group. Cytokine responses in vaccinated animals were compared to
buffer treated mice (Control) (Figure 12).
In line with ELISpot data ( Figure 6), significant antigen -specific secretion of IFNγ
among CD8+ T cells was detectable in splenocytes of BNT162b1, BNT162b2 and
BNT162c2 vaccinated animals. CD8+ T cells from BNT162b1 and BNT162b2
vaccinated mice also showed significant release of IL -2 and TNF ( Figure 12a).
Significant numbers of CD4+ T cells from BNT162b1 vaccinated mice secreted the T H1
cytokines IFNγ and IL-2, but not the T H2 cytokine IL -4 (Figure 12b). Although numbers
were generally low and the spread between treat ed groups high, significant antigen -
specific secretion of IFNγ among T FH cells w as detected in the BNT162b 2 group
(Figure 12c).
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Figure 12: Quantification of cytokine secreting T cells upon S peptide restimulation in the spleen of
BNT162a1, BNT162b1, BNT162b2 or BNT1 62c2 vaccinated mice
Flow cytometry analysis of cytokine secreting CD8+ (a), CD4+ (b) and T FH cells (c) upon S peptide restimulation.
Splenocytes of BNT162a1, BNT162b1, BNT162b2 or BNT162c2 vaccinated mice (N=8 per group) were isolated on
day 12 (BNT162a1, BNT162b1, BNT162b2) or day 27 (BNT162c2) after vaccination . Splenocytes of b uffer treated
mice se rved as control. Cytokine positive c ell counts per mouse are shown by dots; group mean values are indicated
by bars. Values represent S peptide restimulated s amples subtracted by unstimulated samples from the same
mouse.
In summary, particularly BNT162b1 , BNT162b2 and BNT162c2 vaccination mediated
a potent T -cell response demonstrated by overall increased T -cell numbers,
expression of molecules related to T -cell activation and the production of effector
cytokines. Mainly BNT162b1 and BNT162b2 mediated a T FH response in the dLNs , B
cell proliferation , and the generation of significant numbers of plasma B cells and
germinal center B cells undergoing Ig class switc h and affinity maturation .
5.3 Cytokine M ultiplex Assay
Complimentary to the analysis of cytokine secretion by IFNγ ELISpot and flow
cytometry, sple en and LN cells were restimulated for 48 h with S peptide mixes or
without peptide , and the release of cytokines quantified by a bead -based multiplex
assay. Buffer treated animals served as control group. Unstimulated samples (cell
culture medium) were compared to S peptide restimulated samples and P -values were
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determined by two-way ANOVA and Sidak's multiple comparisons test . Detection
ranges are provided in Table 20. Raw data including tissues and cytokines not shown
in Figure 13 can be found in Table 21 to
Table 32.
Significant antigen -specific release of the T H1 cytokines IFNγ and IL -2 was observed
in the BNT162b1, BNT162b2 and BNT162c2 vaccinated groups ( Figure 13a).
Splenocyte s from BNT162a1 treated mice mediated a significan t IL-2 response and a
weak IFNγ release in three of eight mice. Highest response s for both cytokines
surpassing the upper limit of quantification for IFNγ were found in the BNT162b2 and
BNT162c2 groups encoding the full -length S protein. Comparably weak or no secretion
of the T H2 cytokines IL -4 and IL -5 was measured (Figure 13b). Low but significant
release of IL -4 and IL -5 was shown for BNT162b2 and BNT162c2 . IL-4 but not IL -5
was detected in the supernatant of sple nocytes from BNT162b1 vaccinated mice.
Besides T H1 cytokines, high amounts of proinflammatory IL -18 were released in the
BNT162b2 and BNT162c2 vaccinated groups , and to lesser extent in the BNT162b 1
and BNT162 a1 vaccinated groups ( Figure 13c). Additional proinflammatory cytokines
were significantly elevated , such as GM -CSF (Figure 13d) or IL -6 (not shown) ,
particularly in the BNT162b1, BNT162b2 and BNT162c2 vaccinated groups .
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Figure 13: Quantification of cytokine secreti on upon S peptide restimulation of splenocytes of BNT162a1,
BNT162b1, BNT162b2 or BNT162c2 vaccinated mice
Cytokine multiplex analysis of supernatants of splenocytes upon S peptide restimulation. Splenocytes of BNT162a1,
BNT162b1, BNT162b2 or BNT162c2 vaccinated mice ( N=8 per group) were isolated on day 12 (BNT162a1,
BNT162b1, BNT162b2) or day 27 (BNT162c2) after vaccination . Splenocytes of buffer treated mice (N=3) served
as control. Dots indicate individual values, group mean values are indicated by bars , horizontal d otted lines indicate
the upper limit of detection (ULOQ). Values below the lower limit of quantification (LLOQ) were set to zero. Values
above the upper limit of quantifi cation (ULOQ) were set to the ULOQ.
5.4 xCELLigence Cytotoxicity Assay
Isolated CD8+ splenocytes were probed for the ir capacity to kill CT26 cells
electroporated with S RNA (mCorVac#15) and additionally pulsed with S peptide mixes
(mCorVac#16) . CD8+ T cells stimulated with CT26 cells electroporated with irrelevant
RNA served as negative control. Complete tumor cell lysis was modeled by addition of
Staurosporin to the S RNA electroporated or S peptide mix loaded CT26 cells. Raw
data can be found in Attachment III.
In line with weak antigen -specific cytokine release ( Figure 6, Figure 12, Figure 13), no
relevant CT26 cell lysis was observed in the BNT162a1 group. For the BNT162b1
vaccinated group, a tendency for cell killing was observed in four out of eight mice (3-
2, 3-3, 3-4 and 3 -6) given that the Normalized Cell Index of CT26 cells electroporated
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with irrelevant RNA was higher than for S RNA electroporate d cells ( Figure 14). More
pronounced tumor cell lysis in eight out of eight mice was observed for splenocytes of
mice vaccinated with BNT162b2 or BNT162c2 , which encod e the full -length S protein
(Figure 15). Overall , the detected effects were rather weak and warrant further
optimization of the assay. No quantitative and statistical analysis of this dataset was
performed.
Figure 14: Cytotoxicity towards S protein expressing CT26 cells by CD8+ splenocytes from BNT162a1 or
BNT162b1 vaccinated mice (mCorVAC#15) .
Splenocytes of BNT162a1 or BNT162b1 vaccinated mice ( N=8 per group) were cultured over night with S peptide
and recombinant IL -2 and subsequently CD8+ cells were isolated via magnetic bead based separation (MACS).
CT26 cells electroporated with S RNA or irrelevant RNA were cult ured in xCELL igence plates for 24 h prior to
addition of isolated CD8+ T cells . CT26 cell numbers were quantified via impedance measurement (Normalized Cell
Index, higher values indicate more viable CT26 cells, normalization was performed at the time point of T cell
addition). Staurosporin treatment modeled complete tumor cell lysis. CT26 cells transfected wi th irrelevant RNA
served as negative control. Depicted is the Normalized Cell Index over time for individual mice.
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Figure 15: Cytotoxicity towards S protein expressing CT26 cells by CD8+ splenocytes from BNT162b2 or
BNT162c2 vac cinated mice (mCorVAC#16).
Splenocytes of BNT162b2 or BNT162c2 vaccinated mice ( N=8 per group) were cultured over night with S peptide
and recombinant IL -2 and subsequently CD8+ cells were isolated via magnetic bead based separation (MACS).
CT26 cells elec troporated with S RNA or irrelevant RNA were cult ured in xCELLigence plates for 24 h . Prior to
addition of isolated CD8+ T cells , S RNA transfected CT26 cells were pulsed with S peptide . CT26 cell numbers
were quantified via impedance measurement (Normaliz ed Cell Index, higher values indicate more viable CT26 cells,
normalization was performed at the time point of T cell addition). Staurosporin treatment modeled complete tumor
cell lysis. CT26 cells transfected with irrelevant RNA served as negative control . Depicted is the Normalized Cell
Index over time for individual mice.
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6 CONCLUSION
This study aimed at characterizing T - and B -cell responses induced by the COVID -19
vaccine candidates BNT162a1, BNT162b2 , BNT162b1 and BNT162c2 in detail.
Overall, the r esults of the different assay types pointed towards similar conclusions ,
highlighting the validity of the obtained data. IFNγ ELISpot assay , flow cytometry
analysis and multiplexed quantification of cytokines suggested that particularly
BNT162b1 , BNT162b2 and BNT162c2 vaccination induced a potent T -cell response
demonstrated by overall increased T -cell numbers, expression of molecules related to
T-cell activation and the potential of T cells to produce cytokines. T-cell responses
showed primarily a T H1 phen otype with increased numbers of T -bet+ CD4+ T cells
(mainly BNT162b1 and BNT162b2) and high secretion of T H1 type cytokines (IFNγ, IL -
2, TNF) and low secretion of T H2 type cytokines (IL -4, IL-5). Mainly BNT162b1 and
BNT162b2 mediated a T FH response in the dLNs , B cell proliferation and the generation
of significant numbers of antibody producing plasma B cells a nd germinal center B
cells unde rgoing Ig class switch and affinity maturation.
The results of this stu dy are in agreement with prior studies investigating the number
of IFNγ specific T cells by ELISpot and IgG titers by ELISA 28 days after vaccination
(R-20-0040, R -20-0042, R -20-0053, R -20-0085) . Similarly to this study, responses of
BNT162b1 and BNT162b2 were much stronger compared to BNT162a1 in those
studies .
Since the kinetics of expression for the vaccine encoded protein of BNT16 2c2 differ s
from the o ther three vaccine candidates , the analysis time point was set on day 27
instead of day 12 after vaccination . It is possible that the selected time point was
suboptimal and missed the peak expansion of lymphocytes . BNT162c2 induced a
potent T -cell response (IFNγ ELISpot, intracellular cytokine staining by flow cytometry
and multiplexed protein quantification) including the highest T FH cell responses
amongst all tested candidates in the blood on day 7 after treatment. However, in the
dLNs on day 27 after vac cination , the impact on T FH cells and B cells was weak to
undetectable. Effects of BNT162c2 on both T and B cells might be stronger when
analyzed at an earlier time point. Direct comparison of BNT162c2 to BNT162a1,
BNT162b2 or BNT162b1 is therefore difficu lt and might underestimate the potential of
BNT162c2.
Due to the prominent induction of both T - and B -cell responses, these results
particularly support further clinical evaluation of the COVID -19 vaccine candidates
BNT162b1 and BNT162b2 and warrant furth er evaluation of BNT162c2 .
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7 DOCUMENT HISTORY
First version / no change.
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8 REFERENCES
Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, et al. From SARS to MERS, Thrusting
Coronaviruses into the Spotlight. Viruses. 2019;11(1).
Vogel AB, Lambert L, Kinnear E, Bus se D, Erbar S, Reuter KC, et al. Self-Amplifying
RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at
Much Lower Doses. Mol Ther [Internet]. 2018;26(2):446 –55. Available from:
https://doi.org/10.1016/j.ymthe.2017.11.017
Pardi N , Hogan MJ, Pelc RS, Muramatsu H, Andersen H, DeMaso CR, et al. Zika virus
protection by a single low -dose nucleoside -modified mRNA vaccination. Nature.
2017;543(7644):248 –51.
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. Mol Ther Methods Clin Dev. 2019 Mar 15;12:32 –46.
Harrell MI, Iritani BM, Ruddell A. Lymph node mapping in the mouse. J Immunol
Methods. 2008;332(1 –2):170 –4.
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9 APPENDIX
9.1 Animal Monitoring
9.2 Animal Monitoring - Observations
Table 14: Parameters for experimental animal monitoring (single animal assessment)
The table is separated in immediate euthanasia criteria (end of experiment ) and criteria , which , solitarily observed , do not lead to an immediate termination , but result in higher
monitoring frequency of re-assessment . BCS, body conditioning score.
Observation (if applicable, categorize a):
Code Parameter Renew assessment within < 24 h. Attention:
evaluate accumulated parameters Immediate euthanasia criteria
1 Bodyweight b. Take into account
BCS c Body weight loss >5 –10%, or BCS transition 3 to 2 Body weight loss >15 -20%, or BCS 2
2 Activity Moderate deviation from normal or unusual
beha vior (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, s trongly neglected grooming. Eye lids
narrowed, eyes closed and sticky.
4 Body cavities & body fluids Slight to moderate damp & sticky cavities Clinical signs of disease (diarrhea, distinct sticky)
5 Body temperature & blo od
circulation ears - Body temperature low, ears appear white and hardly
noticeable blood vessels
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Observation (if applicable, categorize a):
Code Parameter Renew assessment within < 24 h. Attention:
evaluate accumulated parameters Immediate euthanasia criteria
6 Posture Moderate deviation of normal physiological
posture i.e., short pause in hunched posture Abnormal posture, hunched, abnormally stretched (belly
touches ground) or cramps
7 Reaction to stimulus d Delayed reaction to unconditioned stimulus,
moderate deviation from normal behavior (e.g.
slight to moderate apathy) Abnormal (distinct delayed reaction to unconditioned
stimulus). Winding and enduring sound utterance
("pain"), aggressiveness to touch
8 Automutilation - Noticable b urden , i.e. missing extremities, continuous
nibbling, biting and gnawing, open wounds
9 Bites (tail, vibrissae, reproductive
organs…), other wounds Open and bleeding wounds (take care of wounds
and separate from others ) Noticable b urden , 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 com bination 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 a nd 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 15: Record of body weights of mCorVAC#15 animals during study
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 7
SBIO-15337 BIO-LO78 BALB/cJRj f 03.03.20 1 20.4 20.3 20.5 20.6 20.3 20.8 20.6
SBIO-15337 BIO-LO79 BALB/cJRj f 03.03.20 1 22.1 22.6 22.5 22.3 22.4 23.5 22.7
SBIO-15337 BIO-LO80 BALB/cJRj f 03.03.20 1 20.9 21.1 20.9 20.8 20.9 21.6 21.3
SBIO-15337 BIO-LO81 BALB/cJRj f 03.03.20 1 21.7 21.5 21.4 21.0 21.2 22.5 22.1
SBIO-15338 BIO-LO82 BALB/cJRj f 03.03.20 1 19.6 19.8 20.2 20.4 20.7 20.5 21.2
SBIO-15338 BIO-LO83 BALB/cJRj f 03.03.20 1 20.9 20.7 21.2 21.0 21.6 20.9 21.3
SBIO-15338 BIO-LO84 BALB/cJRj f 03.03.20 1 19.7 19.5 19.5 19.3 19.9 20.3 19.9
SBIO-15338 BIO-LO85 BALB/cJRj f 03.03.20 1 18.9 18.6 18.3 18.4 19.0 18.9 18.9
SBIO-15339 BIO-LO86 BALB/cJRj f 03.03.20 2 20.9 20.6 20.9 21.2 20.8 21.1 21.2
SBIO-15339 BIO-LO87 BALB/cJRj f 03.03.20 2 21.3 19.3 20.2 22.7 21.4 21.1 20.7
SBIO-15339 BIO-LO88 BALB/cJRj f 03.03.20 2 23.2 20.5 21.9 22.5 22.4 22.9 22.9
SBIO-15339 BIO-LO89 BALB/cJRj f 03.03.20 2 19.8 18.9 20.0 20.8 20.3 21.0 20.7
SBIO-15340 BIO-LO90 BALB/cJRj f 03.03.20 2 22.5 20.9 21.3 21.7 21.6 21.7 21.6
SBIO-15340 BIO-LO91 BALB/cJRj f 03.03.20 2 20.9 19.2 20.6 21.6 20.8 20.8 20.9
SBIO-15340 BIO-LO92 BALB/cJRj f 03.03.20 2 21.8 21.1 21.5 22.1 21.8 21.5 22.1
SBIO-15340 BIO-LO93 BALB/cJRj f 03.03.20 2 22.7 20.6 21.8 22.5 22.5 22.2 22.8
SBIO-15341 BIO-LO94 BALB/cJRj f 03.03.20 3 19.3 18.2 18.9 19.0 18.9 18.9 18.9
SBIO-15341 BIO-LO95 BALB/cJRj f 03.03.20 3 21.1 21.6 20.6 21.1 21.2 21.9 21.1
SBIO-15341 BIO-LO96 BALB/cJRj f 03.03.20 3 20.3 19.3 20.2 20.5 20.8 20.3 20.2
SBIO-15341 BIO-LO97 BALB/cJRj f 03.03.20 3 22.9 22.0 23.0 23.4 23.3 22.9 22.3
SBIO-15342 BIO-LO98 BALB/cJRj f 03.03.20 3 21.1 21.0 21.7 21.7 22.6 23.1 23.3
SBIO-15342 BIO-LO99 BALB/cJRj f 03.03.20 3 19.9 18.9 19.3 19.7 19.2 19.9 19.2
SBIO-15342 BIO-LP00 BALB/cJRj f 03.03.20 3 22.1 21.0 22.3 22.3 20.8 22.1 21.9
SBIO-15342 BIO-LP01 BALB/cJRj f 03.03.20 3 20.6 19.8 21.1 21.4 22.1 21.1 21.3Bodyweight (grams)
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Table 16: Record of animal monitoring during CorVac#15 study
12: swelling of injection site muscle
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 7
SBIO-15337 BIO-LO78 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO79 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO80 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO81 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO82 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO83 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO84 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO85 BALB/cJRj f 03.03.20 1 NAD NAD NAD NAD NAD NAD NAD
SBIO-15339 BIO-LO86 BALB/cJRj f 03.03.20 2 NAD 3+;12+ 12+ NAD 12+ NAD NAD
SBIO-15339 BIO-LO87 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ NAD 12+ NAD NAD
SBIO-15339 BIO-LO88 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ 12+ NAD NAD NAD
SBIO-15339 BIO-LO89 BALB/cJRj f 03.03.20 2 NAD 3+;12+ 12+ 12+ NAD NAD NAD
SBIO-15340 BIO-LO90 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ NAD 12+ NAD NAD
SBIO-15340 BIO-LO91 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ NAD 12+ NAD NAD
SBIO-15340 BIO-LO92 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ 12+ 12+ NAD NAD
SBIO-15340 BIO-LO93 BALB/cJRj f 03.03.20 2 NAD 3+;12++ 12++ 12+ NAD NAD NAD
SBIO-15341 BIO-LO94 BALB/cJRj f 03.03.20 3 NAD 3+;12+ 12+ NAD NAD NAD NAD
SBIO-15341 BIO-LO95 BALB/cJRj f 03.03.20 3 NAD 3+;12+ 12+ NAD NAD NAD NAD
SBIO-15341 BIO-LO96 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ 12+ NAD NAD NAD
SBIO-15341 BIO-LO97 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12+ NAD NAD NAD NAD
SBIO-15342 BIO-LO98 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ NAD NAD NAD NAD
SBIO-15342 BIO-LO99 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ 12+ NAD NAD NAD
SBIO-15342 BIO-LP00 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ 12++ 12+ NAD NAD
SBIO-15342 BIO-LP01 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12+ 12+ NAD NAD NADAnimal Monitoring - Observations
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Table 17: Record of body weights of CorVac#16 animals during study
n/a: not available ( Treatment group 1+2: no weight measurement performe d as treatment had just occurred [day 15] ; Treatment group 3: Weekly weight measurement sufficient )
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 7 Day 15 Day 16 Day 17 Day 18 Day 19 Day 22
SBIO-15337 BIO-LO78 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 21.1 20.8 20.8 21.1 21.5 21.0
SBIO-15337 BIO-LO79 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 21.3 21.2 20.9 21 0 21.9 21.9
SBIO-15337 BIO-LO80 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 20.0 20.2 20.2 20 3 21.2 20.7
SBIO-15337 BIO-LO81 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 22.5 22.4 21.9 21 9 23.1 22.7
SBIO-15338 BIO-LO82 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 21.9 22.4 22.3 22.1 22.1 22.6
SBIO-15338 BIO-LO83 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 20.2 20.5 20.6 20.6 20.7 21.1
SBIO-15338 BIO-LO84 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 20.5 21.1 20.8 21 2 21.7 20.8
SBIO-15338 BIO-LO85 BALB/cJRj f 03 03 20 1 n/a n/a n/a n/a n/a n/a n/a 21.9 22.6 22.3 21 9 22.6 22.3
SBIO-15339 BIO-LO86 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 22.5 21.7 22.6 23 2 22.9 23.1
SBIO-15339 BIO-LO87 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 22.1 21.3 21.9 22 5 23.2 22.1
SBIO-15339 BIO-LO88 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 21.7 20.9 21.6 21.6 22.2 22.1
SBIO-15339 BIO-LO89 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 21.5 21.2 22.6 22.7 23.2 22.7
SBIO-15340 BIO-LO90 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 21.9 20.3 20.5 21.1 21.4 21.1
SBIO-15340 BIO-LO91 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 21.1 20.3 20.4 22.7 21.1 20.6
SBIO-15340 BIO-LO92 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 22.4 21.7 23.9 23 8 23.8 22.5
SBIO-15340 BIO-LO93 BALB/cJRj f 03 03 20 2 n/a n/a n/a n/a n/a n/a n/a 23.3 21.7 22.4 20 9 22.7 22.2
SBIO-15341 BIO-LO94 BALB/cJRj f 03 03 20 3 21 8 20.8 21 5 22.1 21.9 22.1 22.1 n/a 23.4 n/a n/a n/a 22.8
SBIO-15341 BIO-LO95 BALB/cJRj f 03 03 20 3 20 8 19.3 20 3 21.2 20.6 21.1 21.5 n/a 21.7 n/a n/a n/a 22.1
SBIO-15341 BIO-LO96 BALB/cJRj f 03 03 20 3 22.4 20.1 21.4 22.5 22.1 22.2 22.1 n/a 23.3 n/a n/a n/a 22.8
SBIO-15341 BIO-LO97 BALB/cJRj f 03 03 20 3 19.1 17.6 17 8 19.1 18.5 19.4 20.5 n/a 19.5 n/a n/a n/a 19
SBIO-15342 BIO-LO98 BALB/cJRj f 03 03 20 3 18.7 17.2 18 0 18.6 18.5 18.8 18.2 n/a 20.1 n/a n/a n/a 20.6
SBIO-15342 BIO-LO99 BALB/cJRj f 03 03 20 3 20 5 19.2 20.4 21.2 21.1 21.7 20.9 n/a 22.1 n/a n/a n/a 21.5
SBIO-15342 BIO-LP00 BALB/cJRj f 03 03 20 3 19.6 17.6 19.1 19.8 19.9 19.9 19.9 n/a 22.3 n/a n/a n/a 22.8
SBIO-15342 BIO-LP01 BALB/cJRj f 03 03 20 3 18.1 16.8 17.4 17.9 18.2 18.2 18.1 n/a 19.8 n/a n/a n/a 19Bodyweight (grams)
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Table 18: Record of animal monitoring during CorVac#16 study
12: swelling of injection site muscle
n/a: not available (no weight m easurement performed as treatment had just occurred [day 15])
Cage Mouse ID Strain Gender Date of birth Treatment Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 7 Day 15 Day 16 Day 17 Day 18 Day 19 Day 22
SBIO-15337 BIO-LO78 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO79 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO80 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15337 BIO-LO81 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO82 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO83 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO84 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15338 BIO-LO85 BALB/cJRj f 03.03.20 1 n/a n/a n/a n/a n/a n/a n/a NAD NAD NAD NAD NAD NAD
SBIO-15339 BIO-LO86 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12+ 12+ NAD NAD NAD
SBIO-15339 BIO-LO87 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12++ 12+ NAD NAD
SBIO-15339 BIO-LO88 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12+ NAD NAD NAD
SBIO-15339 BIO-LO89 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12+ NAD NAD NAD
SBIO-15340 BIO-LO90 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12+ 12+ NAD NAD
SBIO-15340 BIO-LO91 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12++ NAD NAD NAD
SBIO-15340 BIO-LO92 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12+ 12+ NAD NAD NAD
SBIO-15340 BIO-LO93 BALB/cJRj f 03.03.20 2 n/a n/a n/a n/a n/a n/a n/a NAD 12++ 12++ 12+ NAD NAD
SBIO-15341 BIO-LO94 BALB/cJRj f 03.03.20 3 NAD 3++;12++ 3++;12++ 3++;3++ 12++ 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15341 BIO-LO95 BALB/cJRj f 03.03.20 3 NAD 3++;12+++ 3+;12++ 3+; 12+ 12+ NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15341 BIO-LO96 BALB/cJRj f 03.03.20 3 NAD 3++;12++ 3+;12++ 3+;12+ 12+ NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15341 BIO-LO97 BALB/cJRj f 03.03.20 3 NAD 3++;12+++ 3++;12++ 3+; 12++ 12++ 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15342 BIO-LO98 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ 12+ 12+ NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15342 BIO-LO99 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ 12+ 12++ 12+ NAD NAD NAD NAD NAD NAD NAD
SBIO-15342 BIO-LP00 BALB/cJRj f 03.03.20 3 NAD 3+;12++ 12++ NAD 12+ NAD NAD NAD NAD NAD NAD NAD NAD
SBIO-15342 BIO-LP01 BALB/cJRj f 03.03.20 3 NAD 3++;12+++ 3+;12+++ 3+;12+ 12++ 12+ NAD NAD NAD NAD NAD NAD NADAnimal Monitoring - Observations
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9.3 ELISpot – Raw data
Table 19: ELISpot raw data.
TNTC, too numerous to count (these values are set to 1 ,500 in Figure 6). Thousa nds were not separated by commas.
Stimulation (well 1 | well 2)
Group Mouse No peptide S peptide Control RNA S RNA
Control
(mCorVac#15) 1 2 1 4 3 0 1 3 1
2 3 2 2 2 6 11 2 1
3 3 1 8 11 7 3 6 3
4 5 6 6 4 2 4 3 4
5 6 4 11 15 5 9 6 3
6 6 5 9 13 5 7 4 5
7 3 5 8 14 12 14 11 6
8 8 4 18 15 5 6 4 1
BNT162a1
1 13 13 118 127 7 6 57 63
2 12 9 128 148 12 7 98 101
3 23 17 75 86 5 9 39 40
4 14 21 51 48 5 5 38 34
5 20 18 87 107 13 9 43 51
6 17 23 132 156 11 22 48 84
7 15 14 69 65 7 3 38 41
8 18 42 96 121 13 18 64 67
BNT162b1
1 42 44 658 645 19 21 676 615
2 11 16 456 440 21 14 399 322
3 21 23 889 977 8 9 1124 1218
4 26 21 871 918 11 12 779 751
5 22 26 873 834 15 9 841 881
6 33 16 733 746 12 12 758 842
7 16 24 861 837 16 11 825 702
8 17 18 837 772 9 8 628 598
Control
(mCorVac#16 )
1 21 9 7 57 12 11 8 11
2 4 15 7 28 28 31 18 16
3 13 5 12 23 11 7 6 9
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Stimulation (well 1 | well 2)
Group Mouse No peptide S peptide Control RNA S RNA
4 19 19 26 38 4 9 6 7
5 8 9 19 31 8 8 7 2
6 22 13 26 28 19 12 11 17
7 17 15 21 24 17 20 12 13
8 14 11 37 62 12 15 16 26
BNT162b2 1 6 14 1267 1296 13 13 1674 1628
2 20 17 1196 1147 15 20 1281 1268
3 17 20 1503 1404 39 37 1278 1117
4 11 13 1311 1289 20 17 1226 1324
5 21 21 911 881 23 12 1171 1391
6 15 25 1126 1173 11 13 1143 1427
7 9 14 1128 1096 15 16 1435 1334
8 33 24 TNTC TNTC 59 62 TNTC TNTC
BNT162c2
1 7 6 1315 1328 9 18 1348 1263
2 11 13 1315 1328 24 4 1222 1089
3 7 5 1328 1267 12 14 1351 1206
4 21 16 877 1135 24 15 1188 1173
5 12 9 1371 1199 19 11 1504 1246
6 6 14 1025 786 5 20 1143 1232
7 11 15 1218 1132 21 22 1034 911
8 4 6 1275 1054 13 6 973 1092
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9.4 Cytokine multiplex analysis – Assay d etection ranges
Table 20: Detection ranges of the ProcartaPlex immuno assay for mCorVAC #15 and mCorVAC #16.
Depicted are lower limt of quantification (LLOQ) and upper limit of quantification (ULOQ) for each analyte. LN, lymph node. SP, spleen.
[pg/mL] IFNγ IL-12p70 IL-13 IL-1β IL-2 IL-4 IL-5 IL-6 TNFα GM-CSF IL-18
mCorVAC #15
(SP, LN) 1.1-4,800 1.5-409.3 2.1-8,650 1-4,350 1.2-5,250 4.8-4,950 1.9-8,000 4.7-19,500 2.8-731.2 2.4-9,950 50.5-207,000
mCorVAC #16
Plate 1 (SP) 1.1-4,800 1.5-102.3 2.1-2,162.5 1-1,087.5 1.2-1,312.5 1.2-4,950 7.8-2,000 4.7-4,875 2.8-731.2 9.7-2,487.5 202.1 -51,750
mCorVAC #16
Plate 2 (LN) 1.1-4,800 1.5-102.3 2.1-8,650 1-4,350 1.2-5,250 1.2-4,950 1.9-8,000 4.7-19,500 2.8-731.2 2.4-9,950 202.1 -51,750
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9.5 Cytokine multiplex analysis – Raw data and calculated data
Table 21: Cytokine raw data and calculated data for mCorVAC#15 , part 1 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (>ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. Gr, group. M, mouse ID. SP, spleen.
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
2 11 Medium SP 187,5 1,26 1,26 11 <=0 0 24 <=0 0 17 0,28 0 754 14,82 14,82
3 12 Medium SP 487 3,99 3,99 13 <=0 0 25 <=0 0 18 0,3 0 583 11,34 11,34
4 13 Medium SP 25 0,24 0 10 <=0 0 21 <=0 0 17 0,28 0 152 2,85 2,85
5 14 Medium SP 54 0,39 0 11 <=0 0 21,5 <=0 0 18 0,3 0 813,5 16,05 16,05
6 15 Medium SP 27 0,25 0 9 <=0 0 21 <=0 0 16 0,26 0 333 6,38 6,38
7 16 Medium SP 118 0,77 0 11 <=0 0 27 0,03 0 13 0,2 0 1012,5 20,24 20,24
8 17 Medium SP 46,5 0,35 0 11 <=0 0 59,5 1,34 0 14 0,22 0 915 18,17 18,17
9 18 Medium SP 124 0,81 0 10 <=0 0 28 0,07 0 12 0,18 0 737,5 14,48 14,48
10 21 Medium SP 1249,5 14,42 14,42 20 <=0 0 44 0,70 0 19 0,32 0 1778 37,67 37,67
11 22 Medium SP 165,5 1,10 1,10 14,5 <=0 0 41,5 0,60 0 15 0,24 0 539 10,45 10,45
12 23 Medium SP 219 1,51 1,51 13 <=0 0 30 0,14 0 21 0,36 0 406,5 7,82 7,82
13 24 Medium SP 50 0,37 0 11 <=0 0 33 0,26 0 16 0,26 0 470 9,08 9,08
14 25 Medium SP 2466 40,87 40,87 30 <=0 0 123 4,11 4,11 26 0,47 0 1123 22,62 22,62
15 26 Medium SP 455 3,66 3,66 12 <=0 0 62 1,44 0 16 0,26 0 730 14,32 14,32
16 27 Medium SP 162,5 1,08 0 12 <=0 0 28 0,07 017,5 0,29 0 1605 33,53 33,53
17 28 Medium SP 327 2,42 2,42 17 <=0 0 31 0,18 0 19 0,32 0 1560 32,47 32,47
18 31 Medium SP 2160,5 33,01 33,01 36 <=0 0 43 0,66 045,5 0,91 0 1498,5 31,04 31,04
19 32 Medium SP 446 3,57 3,57 15 <=0 0 33 0,26 0 18 0,30 0 1318 26,93 26,93
20 33 Medium SP 380,5 2,92 2,92 14 <=0 0 48 0,86 0 20 0,34 0 755 14,84 14,84
21 34 Medium SP 265 1,88 1,88 15 <=0 0 63 1,49 0 20 0,34 0 657,5 12,84 12,84
22 35 Medium SP 154 1,02 016,5 <=0 0 98,5 3,02 3,02 18 0,30 0 1112 22,38 22,38
23 36 Medium SP 128 0,84 0 12 <=0 0 46 0,78 0 15 0,24 0 1013 20,25 20,25
24 37 Medium SP 77 0,52 0 11 <=0 0 26 0,00 0 16 0,26 0 1116 22,47 22,47
25 38 Medium SP 347 2,61 2,61 14 <=0 0 115 3,75 3,75 18 0,30 0 902 17,90 17,90IFN-gamma IL-12p70 IL-13 IL-1beta IL-2
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Table 22: Cytokine raw data and calculated data for mCorVAC#15 , part 2 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. Gr, group. M, mouse ID. SP, spleen .
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
2 11 Medium SP 37 <=0 0 10 <=0 0 21 2,36 0 36 0,90 0 9 0,31 0 25 16,07 0
3 12 Medium SP 48 <=0 0 10 <=0 0 27 3,34 0 30 0,72 0 12 0,42 0 48 61,82 61,82
4 13 Medium SP 24 <=0 0 11 <=0 0 24,5 2,93 0 26 0,60 0 6 <=0 0 15 <=0 0
5 14 Medium SP 22 <=0 0 10 <=0 0 23 2,68 0 27 0,63 0 9 0,31 0 14,5 <=0 0
6 15 Medium SP 12 <=0 0 11 <=0 0 18 1,89 0 27 0,63 0 7 0,24 0 15 <=0 0
7 16 Medium SP 15 <=0 0 10 <=0 0 22,5 2,60 0 25 0,58 0 7 0,24 0 22 9,40 0
8 17 Medium SP 24 <=0 0 15 <=0 0 22 2,52 0 29 0,69 0 15,5 0,55 0 15 <=0 0
9 18 Medium SP 25 <=0 0 11 <=0 0 22 2,52 0 33 0,81 0 9,5 0,33 0 22 9,40 0
10 21 Medium SP 131,5 0,82 0 12 <=0 0 81 13 37 13,37 43 1,11 0 21 0,75 0 108 169,05 169,05
11 22 Medium SP 153 1,18 0 12,5 <=0 0 70 11,20 11,20 32 0,78 0 10 0,35 0 25 16,07 0
12 23 Medium SP 148 1,09 0 10 <=0 0 56,5 8,61 8,61 31 0,75 0 11 0,39 0 29 24,53 0
13 24 Medium SP 164 1,37 0 12 <=0 0 55 8 33 8,33 23 0,52 0 9 0,31 0 16 <=0 0
14 25 Medium SP 149 1,11 0 14,5 <=0 0413,5 92,66 92,66 909 35,52 35,52 24 0,85 0200,5 324,27 324,27
15 26 Medium SP 44 <=0 0 16,5 <=0 0 48 7,03 7,03 32 0,78 0 11 0,39 0 44 54,22 54,22
16 27 Medium SP 111 0,5 0 11 <=0 0 62 9,65 9,65 42,5 1,09 0 21 0,75 0 24 13,88 0
17 28 Medium SP 111 0,5 0 10 <=0 0 78 12,77 12,77 40 1,02 0 14 0,50 0 36 38,68 0
18 31 Medium SP 119 0,62 0 12 <=0 0 762 192,57 192,57 1697 77,07 77,07 27 0,96 0 189 305,29 305,29
19 32 Medium SP 83,5 0,11 0 12 <=0 0 59 9,08 9,08 40 1,02 0 22 0,78 0 44 54,22 54,22
20 33 Medium SP 303 4,01 0 11 <=0 0 99,5 17,13 17,13 36 0,90 0 17 0,60 0 39 44,57 0
21 34 Medium SP 275 3,44 0 12,5 <=0 0 157 29,48 29,48 32 0,78 0 13 0,46 0 32 30,67 0
22 35 Medium SP 385 5,72 5,72 29 0,43 0 154 28,82 28,82 33 0,81 0 13 0,46 0 26 18,22 0
23 36 Medium SP 153,5 1,19 0 16,5 <=0 0 107 18,68 18,68 28 0,66 0 15 0,53 0 22 9,40 0
24 37 Medium SP 77 0,03 0 11 <=0 0 45 6,48 6,48 23 0,52 0 11 0,39 0 16 <=0 0
25 38 Medium SP 195 1,92 0 10,5 <=0 0 137 25,08 25,08 38 0,96 0 24 0,85 0 37 40,65 0IL-18 IL-4 IL-5 IL-6 TNF-alpha GM-CSF
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Table 23: Cytokine raw data and calculated data for mCorVAC#15 , part 3 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. Gr, group. M, mouse ID. PMAIono , PMA and Ionomycin (positive control). SP, spleen .
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
26 11 S peptide SP 386,5 2,98 2,98 12 <=0 0 24 <=0 8650 19 0,32 0 610 11,88 11,88
27 12 S peptide SP 75 0,51 0 13 <=0 0 39 0,50 0 16 0,26 0 955 19,01 19,01
28 13 S peptide SP 178 1,19 1,19 10 <=0 0 30 0,14 0 15 0,24 0 246,5 4,69 4,69
29 14 S peptide SP 35 0,29 0 11 <=0 0 19 <=0 16 0,26 0 190,5 3,60 3,60
30 15 S peptide SP 203 1,38 1,38 9,5 <=0 0 25 <=0 15 0,24 0 365 7,01 7,01
31 16 S peptide SP 94 0,62 0 11 <=0 0 20,5 <=0 13 0,20 0 321 6,14 6,14
32 17 S peptide SP 586,5 5,09 5,09 12 <=0 0 21 <=0 11 0,16 0 885 17,54 17,54
33 18 S peptide SP 318 2,34 2,34 14 <=0 0 23 <=0 13 0,20 0 1203,5 24,38 24,38
34 21 S peptide SP 4966,5 149,75 149,75 35 <=0 0 266 10,75 10,75 27 0,49 0 2348 52,26 52,26
35 22 S peptide SP 5474 186,09 186,09 32 <=0 0 224,5 8,78 8,78 24 0,42 0 1414 29,10 29,10
36 23 S peptide SP 4423,5 117,49 117,49 30 <=0 0 336 14,14 14,14 30 0,56 0 1907 40,84 40,84
37 24 S peptide SP 2160 33,00 33,00 21 <=0 0 107 3,40 3,40 17 0,28 0 1130,5 22,78 22,78
38 25 S peptide SP 7059 356,73 356,73 47,5 <=0 0 682,5 31,94 31,94 32 0,60 0 2363,5 52,68 52,68
39 26 S peptide SP 8699,5 707,98 707,98 62 <=0 01080,5 54,24 54,24 31 0,58 0 3508 87,45 87,45
40 27 S peptide SP 2327 37,18 37,18 21 <=0 0 134,5 4,62 4,62 19 0,32 0 2460 55,32 55,32
41 28 S peptide SP 3945 93,72 93,72 29 <=0 0 178 6,61 6,61 22 0,38 0 2176 47,70 47,70
42 31 S peptide SP 12251 5435,41 4800 113 0,53 0 1666 90,58 90,58 53 1,09 1,09 2323 51,59 51,59
43 32 S peptide SP 11207 2540,72 2540,72 67 <=0 0 446,5 19,64 19,64 32 0,60 0 1969,5 42,40 42,40
44 33 S peptide SP 13878 55904,59 4800 123 0,67 02112,5 121,31 121,31 50 1,02 1,02 2650 60,65 60,65
45 34 S peptide SP 8838 752,35 752,35 79,5 0,05 0 1314 68,22 68,22 34,5 0,66 0 2477 55,79 55,79
46 35 S peptide SP 10020 1309,58 1309,58 77,5 0,02 0 1206 61,67 61,67 35 0,67 0 1807 38,38 38,38
47 36 S peptide SP 7982 521,41 521,41 55 <=0 0 882 42,87 42,87 27 0,49 0 1849,5 39,42 39,42
48 37 S peptide SP 9172,5 873,94 873,94 58,5 <=0 0 648 30,09 30,09 28 0,51 0 1861 39,70 39,70
49 38 S peptide SP 8488 646,01 646,01 59 <=0 0 1148 58,21 58,21 29 0,53 0 2611 59,54 59,54
52 16 PMA Iono SP 3238 65,07 65,07 338 3,83 3,83 10885 3220,14 3220,14 82 1,81 1,81 16603 5,85E+07 5250
51 17 PMA Iono SP 3585 78,26 78,26 349,5 4,01 4,01 11523 4700,47 4700,47 95 2,15 2,15 17179,5 5,85E+07 5250
50 18 PMA Iono SP 3246 65,35 65,35 319 3,55 3,55 10442 2593,95 2593,95 67 1,43 1,43 16984 5,85E+07 5250
60 25 PMA Iono SP 4643 129,80 129,80 324 3,62 3,62 10730 2975,64 2975,64 118 2,75 2,75 15930 9,10E+04 5250
59 26 PMA Iono SP 4585 126,45 126,45 371,5 4,35 4,35 13072 93260,89 8650 115,5 2,69 2,69 17962 5,85E+07 5250
58 27 PMA Iono SP 5308 173,46 173,46 306,5 3,36 3,36 10521,5 2691,16 2691,16 84,5 1,87 1,87 18176,5 5,85E+07 5250
66 31 PMA Iono SP 4589,5 126,71 126,71 304,5 3,33 3,33 11181 3790,84 3790,84 86 1,91 1,91 16344 1,67E+06 5250
67 34 PMA Iono SP 3133 61,38 61,38 348,5 3,99 3,99 12338 9811,27 8650 105 2,41 2,41 16468,5 5,85E+07 5250
68 37 PMA Iono SP 4499 121,62 121,62 334 3,77 3,77 11511 4662,32 4662,32 100,5 2,29 2,29 17556 5,85E+07 5250
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Table 24: Cytokine raw data and calculated data for mCorVAC#15 , part 4 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software ( ccalc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
sepa rators. Thousand were not separated by commas. Gr, group. M, mouse ID. PMAIono, PMA and Ionomycin (positive control). SP, spleen .
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
26 11 S pep ide SP 88 0,16 0 9 <=0 0 32 4,18 0 33 0,81 0 11 0,39 0 41 48,45 0
27 12 S pep ide SP 252 2,99 0 10 <=0 0 85 14,17 14,17 36 0,90 0 10 0,35 0 18 <=0 0
28 13 S pep ide SP 22 <=0 0 8,5 <=0 0 28 3,51 0 23 0,52 0 9 0,31 0 27 20,34 0
29 14 S pep ide SP 16,5 <=0 0 10 <=0 0 17,5 1,81 0 21 0,46 0 6,5 0,22 0 15 <=0 0
30 15 S pep ide SP 45,5 <=0 0 9 <=0 0 33 4 36 0 29 0,69 0 9 0,31 0 29 24,53 0
31 16 S pep ide SP 23,5 <=0 0 9 <=0 0 17,5 1,81 0 20 0,43 0 8 0,27 0 19 2,11 0
32 17 S pep ide SP 81 0,07 0 9 <=0 0 38 5,23 5,23 34 0,84 0 12 0,42 0 50 65,59 65,59
33 18 S pep ide SP 78 0,04 0 10 <=0 0 32 4,18 0 41 1,05 0 12 0,42 0 33 32,69 0
34 21 S pep ide SP 302 3,98 0 15 <=0 0 243 49,41 49,41 151 4,63 4,63 90 3,06 3,06 436,5 706,91 706,91
35 22 S pep ide SP 308,5 4,12 0 33 0,59 0 172 32,85 32,85 87 2,50 0 89,5 3,05 3,05 508 822,51 822,51
36 23 S pep ide SP 452 7,19 7,19 17 <=0 0 222 44,41 44,41 107 3,15 3,15 75 2,57 2,57 375,5 608,48 608,48
37 24 S pep ide SP 267,5 3,30 0 10,5 <=0 0 112 19,73 19,73 56 1,51 0 26 0,92 0 170 273,80 273,8
38 25 S pep ide SP 446,5 7,06 7,06 26 0,31 0 303 64,13 64,13 165 5,12 5,12 152,5 5,08 5,08 674,5 1094,22 1094,22
39 26 S pep ide SP 723 13,65 13,65 44 1,05 0 397 88 29 88,29 182 5,71 5,71 264 8,61 8,61 911,5 1491,08 1491,08
40 27 S pep ide SP 290,5 3,75 0 17 <=0 0 176 33,75 33,75 77 2,17 0 59 2,04 0198,5 320,97 320,97
41 28 S pep ide SP 269 3,33 0 13 <=0 0178,5 34,32 34,32 144 4,40 4,40 87 2,96 2,96 350 567,33 567,33
42 31 S pep ide SP 553 9,50 9,50 34 0,63 01090,5 298,53 298,53 836 32,17 32,17 850 27,38 27,38 1542 2640,12 2640,12
43 32 S pep ide SP 297,5 3,89 0 22 0,16 0 184 35,57 35,57 230 7,41 7,41 497,5 15,98 15,98 1248 2084,32 2084,32
44 33 S pep ide SP 1721 43,68 43,68 33 0,59 0 869 225,90 225,90 383 13,13 13,13 1383 45,75 45,75 1735,5 3029,31 3029,31
45 34 S pep ide SP 1594,5 39,34 39,34 55 1,52 0 845 218,32 218,32 217,5 6,97 6,97 461,5 14,84 14,84 936 1532,98 1532,98
46 35 S pep ide SP 921 18,85 18,85 17 <=0 0552,5 130,83 130,83 224 7,20 7,20 613 19,67 19,67 1182 1964,71 1964,71
47 36 S pep ide SP 429,5 6,69 6,69 17 <=0 0285,5 59,77 59,77 182 5,71 5,71 419 13,50 13,50 864 1410,36 1410,36
48 37 S pep ide SP 382,5 5,66 5,66 17 <=0 0207,5 41,01 41,01 183,5 5,76 5,76 615,5 19,75 19,75 1009 1658,93 1658,93
49 38 S pep ide SP 605 10,74 10,74 20 0,08 0 375 82 52 82,52 205,5 6,54 6,54 483 15,52 15,52 831 1354,65 1354,65
52 16 PMA Iono SP 5638 283,37 283,37 4671,5 414,22 414,22 1682 515,81 515,81 4643 470,16 470,16 6211 441,29 441,29 1531,5 2619,57 2619,57
51 17 PMA Iono SP 4136 161,80 161,80 5052 470,40 470,40 1556 466,69 466,69 4764 518,54 518,54 6473 501,59 501,59 1571,5 2698,16 2698,16
50 18 PMA Iono SP 4102 159,57 159,57 7938,5 1092,17 1092,17 1234 5 348 31 348,31 4334,5 378,81 378,81 5295 296,02 296,02 1397,5 2362,01 2362,01
60 25 PMA Iono SP 1610 39,87 39,87 6150,5 661,29 661,29 2882 1065,19 1065,19 4961 626,92 626,92 4831 245,44 245,44 1761 3082,15 3082,15
59 26 PMA Iono SP 3768,5 138,70 138,70 7970 1101,46 1101,46 4470 2065,48 2065,48 5630,5 1732,84 731,2 5489 320,78 320,78 1775 3111,32 3111,32
58 27 PMA Iono SP 1620,5 40,22 40,22 3044,5 219,53 219,53 2258 760,46 760,46 5527 1732,84 731,2 4976,5 260,16 260,16 1813 3191,10 3191,1
66 31 PMA Iono SP 1616,5 40,09 40,09 5670 572,11 572,11 1719 530,52 530,52 4811,5 540,57 540,57 5771,5 361,79 361,79 1645 2844,76 2844,76
67 34 PMA Iono SP 2902,5 92,23 92,23 4899 447,25 447,25 3504,5 1415,19 1415,19 5162 832,46 731,2 6452 496,30 496,30 1568 2691,25 2691,25
68 37 PMA Iono SP 1295 29,70 29,70 4521 393,25 393,25 2322 789,75 789,75 5321 1977,29 731,2 6617 540,43 540,43 1864 3299,55 3299,55
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Table 25: Cytokine raw data and calculated data for mCorVAC#15 , part 5 of 6 (SP)
Acquired MFI val ues (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ LN, lymph node. Commas are used
as decimal separators. Thousand were not separated by commas. Gr, group. M, mouse ID. PMAIono, PMA and Ionomycin (positive control).
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
53 21 Medium LN 39,5 0,31 0 10 <=0 0 22 <=0 6 <=0 0 269 5,13 5,13
54 22 Medium LN 44 0,33 0 10 <=0 0 22 <=0 7 0,08 0 432,5 8,34 8,34
55 23 Medium LN 28 0,25 0 9 <=0 0 14 <=0 6 <=0 0 234,5 4,45 4,45
56 31 Medium LN 62,5 0,43 0 9 <=0 0 16 <=0 6 <=0 0 355 6,81 6,81
57 33 Medium LN 235 1,63 1,63 9 <=0 0 20 <=0 7 0,08 0 166 3,12 3,12
61 34 Medium LN 68 0,47 0 9 <=0 0 14 <=0 8 0,1 0 204 3,86 3,86
62 35 Medium LN 457 3,68 3,68 12 <=0 0 18 <=0 7 0,08 0 786 15,48 15,48
63 37 Medium LN 57 0,40 0 9 <=0 0 17 <=0 7 0,08 0 157,5 2,95 2,95
64 38 Medium LN 99 0,65 0 8,5 <=0 0 19 <=0 6 <=0 0 782 15,39 15,39
69 21 S peptide LN 561,5 4,81 4,81 11 <=0 0 33 0,26 0 6 <=0 0 208,5 3,95 3,95
70 22 S peptide LN 1013 10,70 10,70 14 <=0 0 118 3,88 3,88 7 0,08 0 1873,5 40,01 40,01
71 23 S peptide LN 938,5 9,61 9,61 12 <=0 0 53 1,07 0 7 0,08 0 436 8,41 8,41
72 31 S peptide LN 678 6,17 6,17 12 <=0 0 31 0,18 0 8 0,1 0 703 13,77 13,77
73 33 S peptide LN 916 9,30 9,30 11,5 <=0 0 40 0,54 0 7 0,08 0 331 6,34 6,34
77 34 S peptide LN 1924 27,51 27,51 14 <=0 0 43 0,66 0 7 0,08 0 723,5 14,19 14,19
78 35 S peptide LN 1095 11,94 11,94 13,5 <=0 0 34,5 0,32 0 7 0,08 0 367 7,04 7,04
79 37 S peptide LN 1686 22,46 22,46 13 <=0 0 51 0,98 0 6 <=0 0 701 13,73 13,73
80 38 S peptide LN 564 4,83 4,83 10 <=0 0 19,5 <=0 0 6 <=0 0 658,5 12,86 12,86
74 21 PMA Iono LN 4496,5 121,48 121,48 294 3,17 3,17 6532 662,77 662,77 59 1,24 1,24 17418 5,85E+07 5250
75 23 PMA Iono LN 4808,5 139,72 139,72 406 4,88 4,88 8767 1352,13 1352,13 66 1,41 1,41 19327 5,85E+07 5250
76 27 PMA Iono LN 3138,5 61,57 61,57 258 2,63 2,63 8778 1357,23 1357,23 51 1,04 1,04 16182 3,09E+05 5250
81 33 PMA Iono LN 2999 56,86 56,86 232 2,25 2,25 6309,5 618,58 618,58 51 1,04 1,04 16058,5 1,54E+05 5250
65 34 PMA Iono LN 3287 66,84 66,84 248 2,49 2,49 8188,5 1114,99 1114,99 57 1,19 1,19 17501 5,85E+07 5250
1 37 PMA Iono LN 3505 75,08 75,08 281 2,98 2,98 7593,5 921,89 921,89 66,5 1,42 1,42 16859,50 5,85E+07 5250IFN-gamma IL-12p70 IL-13 IL-1beta IL-2
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Table 26: Cytokine raw data and calculated data for mCorVAC#15 , part 6 of 6 (LN)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. LN, lymph node. Gr, group. M, mouse ID. PMAIon o, PMA and Ionomycin (positive control).
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
53 21 Medium LN 9 <=0 0 25 0,27 0 12 0,99 0 28 0,66 0 9 0,31 0 15 <=0 0
54 22 Medium LN 7 <=0 0 32,5 0,57 0 15 1,43 0 21 0,46 0 7 0,24 0 14 <=0 0
55 23 Medium LN 6,5 <=0 0 19 0,05 0 11 0,85 0 21 0,46 0 7 0,24 0 13 <=0 0
56 31 Medium LN 7 <=0 0 10 <=0 0 12 0,99 0 22 0,49 0 7 0,24 0 15 <=0 0
57 33 Medium LN 10 <=0 0 20 0,08 0 13 1,13 0 29 0,69 0 10 0,35 0 26 18,22 0
61 34 Medium LN 9 <=0 0 13 <=0 0 14 1,28 0 33 0,81 0 6 <=0 0 14 <=0 0
62 35 Medium LN 15 <=0 0 10 <=0 0 12 0,99 0 26 0,60 0 8 0,27 0 41,5 49,42 0
63 37 Medium LN 13 <=0 0 14 <=0 0 15 1,43 0 29 0,69 0 6 <=0 0 14,5 <=0 0
64 38 Medium LN 12 <=0 0 15 <=0 0 15 1,43 0 26 0,60 0 7 0,24 0 17 <=0 0
69 21 S pep ide LN 12,5 <=0 0 30 0,47 0 13 1,13 0 34 0,84 0 11 0,39 0 52,5 70,27 70,27
70 22 S pep ide LN 46,5 <=0 0 215 9,08 9,08 23 2,68 0 46,5 1,22 0 47 1,64 0 83,5 126,35 126,35
71 23 S pep ide LN 11 <=0 0 96,5 3,38 3,38 15 1,43 0 33 0,81 0 20 0,71 0 82 123,69 123,69
72 31 S pep ide LN 32 <=0 0 20 0,08 0 14 1,28 0 31 0,75 0 17 0,60 0 66,5 95,97 95,97
73 33 S pep ide LN 25 <=0 0 16 <=0 0 13 1,13 0 30,5 0,74 0 17,5 0,62 0 76 113,04 113,04
77 34 S pep ide LN 44,5 <=0 0 15 <=0 0 21 2 36 0 54 1,45 0 31 1,09 0 156 250,44 250,44
78 35 S pep ide LN 54 <=0 0 18 0,01 0 15 1,43 0 40 1,02 0 21 0,75 0 91 139,53 139,53
79 37 S pep ide LN 49,5 <=0 0 27,5 0,37 0 18,5 1,96 0 45 1,17 0 26 0,92 0136,5 217,66 217,66
80 38 S pep ide LN 20 <=0 0 14 <=0 0 15 1,43 0 26 0,60 0 11 0,39 0 52 69,33 69,33
74 21 PMA Iono LN 1662 41,64 41,64 8746 1353,47 1353,47 359,5 78,50 78,50 5696 1732,84 731,2 3332,5 133,64 133,64 1848 3265,35 3265,35
75 23 PMA Iono LN 1720 43,64 43,64 11982 3124,01 3124,01 383 84,61 84,61 6322,5 1732,84 731,2 3394 137,20 137,20 1879,5 3332,84 3332,84
76 27 PMA Iono LN 1056,5 22,63 22,63 10201 1974,51 1974,51 569 135,52 135,52 5226 962,86 731,2 1981 68,61 68,61 1547 2649,93 2649,93
81 33 PMA Iono LN 737 14,00 14,00 7712 1027,35 1027,35 257,5 52,91 52,91 4908 592,68 592,68 3682 154,80 154,80 1503 2564,06 2564,06
65 34 PMA Iono LN 853 17,02 17,02 7043,5 854,66 854,66 435 98,41 98,41 5309 1469,86 731,2 5271,5 293,18 293,18 1475,5 2510,88 2510,88
1 37 PMA Iono LN 943,5 19,47 19,47 7862 1069,89 1069,89 491 113,65 113,65 5508,5 1732,84 731,2 4949 257,31 257,31 1666 2887,17 2887,17IL-4 IL-5 IL-6 TNF-alpha GM-CSF IL-18
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Table 27: Cytokine raw data and calculated data for mCorVAC#16 , part 1 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ . Commas are used as decimal
separators. T housand were not separated by commas. Gr, group. M, mouse ID. SP, spleen .
PlateSample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mLcfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL MFIccalc
[pg/mL]cfin
[pg/mL]
1 1 11 Medium SP 98,5 2,06 2,06 13 <=0 0 35 2,39 2,39 23 1,64 1,64 971,5 59,72 59,72
1 2 12 Medium SP 20 0,17 0 10 <=0 0 24 0,70 0 17 0,94 0 106 9,49 9,49
1 3 13 Medium SP 76,5 1,50 1,50 13 <=0 0 41 3,25 3,25 16 0,82 0 646,5 42,56 42,56
1 4 14 Medium SP 78 1,54 1,54 11 <=0 0 29 1,49 0 22 1,53 1,53 692,5 45,05 45,05
1 5 15 Medium SP 26 0,30 0 9 <=0 0 23 0,53 0 18 1,06 1,06 404,5 28,98 28,98
1 6 16 Medium SP 236 5,80 5,80 13 <=0 0 42 3,40 3,40 18 1,06 1,06 354 25,99 25,99
1 7 17 Medium SP 259,5 6,47 6,47 13 <=0 0 28 1,34 018,5 1,12 1,12 426 30,24 30,24
1 8 18 Medium SP 53 0,92 0 15 <=0 0 23 0,53 0 20 1,30 1,30 776 49,50 49,50
1 9 21 Medium SP 44 0,71 0 10 <=0 0 24 0,70 0 15 0,69 0 245 19,22 19,22
1 10 22 Medium SP 275 6,91 6,91 17 <=0 0 59 5,72 5,72 16 0,82 0 955 58,87 58,87
1 11 23 Medium SP 161 3,71 3,71 26,5 0,5 0 95 10,38 10,38 19 1,18 1,18 848 53,29 53,29
1 12 24 Medium SP 228 5,57 5,57 17 <=0 0 52 4,78 4,78 16 0,82 0 773 49,34 49,34
1 13 25 Medium SP 1728 57,82 57,82 27 0,53 0 93 10,12 10,12 18 1,06 1,06 1119,5 67,33 67,33
1 14 26 Medium SP 54 0,95 0 12 <=0 0 22 0,35 0 17 0,94 0 226,5 18,02 18,02
1 15 27 Medium SP 128,5 2,84 2,84 13 <=0 0 47 4,09 4,09 15 0,69 0 953,5 58,79 58,79
1 16 28 Medium SP 2259,5 80,30 80,30 27,5 0,55 0 68 6,91 6,91 20 1,30 1,30 1375,5 80,35 80,35
1 17 31 Medium SP 79 1,56 1,56 10 <=0 0 27 1,18 0 18 1,06 1,06 132 11,47 11,47
1 18 32 Medium SP 79 1,56 1,56 12 <=0 0 29 1,49 0 19 1,18 1,18 296 22,45 22,45
1 19 33 Medium SP 84,5 1,70 1,70 11 <=0 0 25 0,86 0 23 1,64 1,64 142,5 12,24 12,24
1 20 34 Medium SP 919,5 27,52 27,52 22 0,27 0 80 8,47 8,47 22 1,53 1,53 956,5 58,95 58,95
1 21 35 Medium SP 322,5 8,30 8,30 16 <=0 0 60 5,86 5,86 33 2,72 2,72 869 54,39 54,39
1 22 36 Medium SP 130 2,88 2,88 12,5 <=0 0 44 3,68 3,68 19,5 1,24 1,24 374 27,18 27,18
1 23 37 Medium SP 108,5 2,32 2,32 12 <=0 0 32,5 2,02 0 22 1,53 1,53 189,5 15,54 15,54
1 24 38 Medium SP 168 3,90 3,90 13 <=0 0 39,5 3,04 3,04 17 0,94 0 1218 72,35 72,35IL-12p70 IL-13 IL-1beta IL-2 IFN-gamma
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Table 28: Cytokine raw data and calculated data for mCorVAC#16 , part 2 of 6 (SP)
Acquired MFI values (MFI) were converted into concentrations by Procarta Plex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. Gr, group. M, mouse ID. SP, spleen.
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mLcfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
1 11 Medium SP 106,5 5,5 5,5 10 <=0 0 48 26,82 26,82 49 5,51 5,51 7 <=0 0 21 <=0 0
2 12 Medium SP 21,5 0,8 0 11 <=0 0 18 5,99 5,99 26 2,49 0 5 <=0 0 13 <=0 0
3 13 Medium SP 91 4,7 4,7 12 <=0 0 43,5 23,94 23,94 34 3,61 3,61 8 <=0 0 17 <=0 0
4 14 Medium SP 83 4,2 4,2 11 <=0 0 43 23,61 23,61 44 4,90 4,90 8 <=0 0 17 <=0 0
5 15 Medium SP 23 0,9 0 11 <=0 0 29 14,20 14,20 26,5 2,57 0 5,5 <=0 0 14 <=0 0
6 16 Medium SP 429 22,0 22,0 19 0,2 0129,5 73,96 73,96 41 4,52 4,52 7 <=0 0 29 114,63 0
7 17 Medium SP 63 3,2 3,2 10 <=0 0 30 14,90 14,90 38 4,14 4,14 8 <=0 0 32 144,62 0
8 18 Medium SP 50 2,4 2,4 9 <=0 0 185 103,35 103,35 652,5 56,48 56,48 7 <=0 0 16,5 <=0 0
9 21 Medium SP 86,5 4,4 4,4 12 <=0 0 34 17,65 17,65 32 3,34 3,34 7 <=0 0 13 <=0 0
10 22 Medium SP 487,5 24,9 24,9 12 <=0 0 122 69,87 69,87 77 8,68 8,68 19 4,13 0 38,5 202,86 202,86
11 23 Medium SP 1087,5 55,5 55,5 12 <=0 0 297 159,65 159,65 69 7,81 7,81 14 2,36 0 29 114,63 0
12 24 Medium SP 458 23,4 23,4 28 2,39 0 128 73,14 73,14 57 6,45 6,45 8 <=0 0 27 92,89 0
13 25 Medium SP 1096 55,9 55,9 15 <=0 0292,5 157,45 157,45 67 7,59 7,59 19,5 4,29 0 162 929,78 929,78
14 26 Medium SP 206,5 10,7 10,7 14 <=0 0 60,5 34,59 34,59 34 3,61 3,61 6 <=0 0 14 <=0 0
15 27 Medium SP 294,5 15,2 15,2 9 <=0 0 105 60,46 60,46 51,5 5,81 5,81 11 0,87 0 21,5 14,31 0
16 28 Medium SP 395 20,3 20,3 12 <=0 0 163 91,86 91,86 529 47,07 47,07 17 3,48 0 198 1098,85 1098,85
17 31 Medium SP 69,5 3,5 3,5 10 <=0 0 33 16,97 16,97 32 3,34 3,34 7 <=0 0 16 <=0 0
18 32 Medium SP 114 5,9 5,9 13 <=0 0 39 21,00 21,00 37 4,01 4,01 7 <=0 0 17 <=0 0
19 33 Medium SP 63 3,2 3,2 12 <=0 0 34,5 17,99 17,99 37 4,01 4,01 8 <=0 0 18 <=0 0
20 34 Medium SP 608,5 31,0 31,0 12 <=0 0 174 97,63 97,63 64 7,25 7,25 18 3,81 0 78 479,51 479,51
21 35 Medium SP 345 17,7 17,7 14 <=0 0 122 69,87 69,87 51 5,75 5,75 10 <=0 0 34 163,35 0
22 36 Medium SP 219 11,3 11,3 9 <=0 0 92 53,11 53,11 36 3,88 3,88 8 <=0 0 21 <=0 0
23 37 Medium SP 211,5 11,0 11,0 17 <=0 0 69,5 40,01 40,01 41 4,52 4,52 9 <=0 0 20 <=0 0
24 38 Medium SP 144 7,5 7,5 14 <=0 0 47 26,18 26,18 41 4,52 4,52 8 <=0 0 23,5 48,81 0IL-4 IL-5 IL-6 TNF-alpha GM-CSF IL-18
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Table 29: Cytokine raw data and calculated data for mCorVAC#16 , part 3 of 6 (SP)
Acquired MFI values (MFI) were converte d into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Gr, group. M, mouse ID. PMAIono,
PMA and Ionomycin (positive control). SP, spleen.
PlateSample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mLcfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL MFIccalc
[pg/mL]cfin
[pg/mL]
1 25 11 S peptide SP 60,5 1,10 1,10 10 <=0 0 24 0,70 0 19 1,18 1,18 346 25,51 25,51
1 26 12 S peptide SP 70 1,34 1,34 10 <=0 0 24 0,70 0 16 0,82 0 101 9,10 9,10
1 27 13 S peptide SP 53 0,92 0 9,5 <=0 0 21 0,17 0 14 0,56 0 122,5 10,76 10,76
1 28 14 S peptide SP 182,5 4,30 4,30 12 <=0 0 27 1,18 020,5 1,36 1,36 348 25,63 25,63
1 29 15 S peptide SP 46 0,75 0 14 <=0 0 31 1,80 015,5 0,76 0 151 12,85 12,85
1 30 16 S peptide SP 781,5 22,82 22,82 17,5 0,03 0 66,5 6,72 6,72 19 1,18 1,18 697 45,29 45,29
1 31 17 S peptide SP 276 6,94 6,94 12 <=0 0 23 0,53 0 18 1,06 1,06 331 24,60 24,60
1 32 18 S peptide SP 248,5 6,15 6,15 14 <=0 0 29 1,49 0 25 1,87 1,87 351 25,81 25,81
1 33 21 S peptide SP 13171 5808,01 4800 174 7,19 7,19 3368 382,28 382,28 48 4,21 4,21 1871 105,49 105,49
1 34 22 S peptide SP 14077 5808,01 4800 184,5 7,65 7,65 5418 676,64 676,64 65 5,80 5,80 3257 179,56 179,56
1 35 23 S peptide SP 15533 5808,01 4800 210 8,78 8,78 4642 555,60 555,60 97 8,64 8,64 1595 91,47 91,47
1 36 24 S peptide SP 13978 5808,01 4800 180 7,45 7,45 4989,5 608,07 608,07 58 5,16 5,16 2848 156,78 156,78
1 37 25 S peptide SP 13461 5808,01 4800 164 6,75 6,75 3984,5 462,82 462,82 52 4,59 4,59 2266,5 125,84 125,84
1 38 26 S peptide SP 13336,5 5808,01 4800 169 6,97 6,97 4404 521,10 521,10 61 5,44 5,44 2555 141,01 141,01
1 39 27 S peptide SP 12383 5808,01 4800 170 7,01 7,01 5234 646,64 646,64 52 4,59 4,59 2893,5 159,27 159,27
1 40 28 S peptide SP 17133 5808,01 4800 236 9,93 9,93 4725 567,90 567,90 89,5 7,99 7,99 1762 99,94 99,94
1 41 31 S peptide SP 13734 5808,01 4800 121,5 4,86 4,86 505 57,29 57,29 58 5,16 5,16 1366 79,87 79,87
1 42 32 S peptide SP 13464 5808,01 4800 119 4,75 4,75 653 73,40 73,40 54 4,78 4,78 1191 70,98 70,98
1 43 33 S peptide SP 13090,5 5808,01 4800 112 4,44 4,44 610 68,74 68,74 59 5,25 5,25 954 58,82 58,82
1 44 34 S peptide SP 15944 5808,01 4800 159 6,53 6,53 1533 168,50 168,50 76 6,80 6,80 2185 121,61 121,61
1 45 35 S peptide SP 14532,5 5808,01 4800 152 6,22 6,22 2293,5 253,39 253,39 87 7,77 7,77 1549 89,14 89,14
1 46 36 S peptide SP 15439 5808,01 4800 151 6,17 6,17 891 99,08 99,08 61 5,44 5,44 2981 164,08 164,08
1 47 37 S peptide SP 13213,5 5808,01 4800 124 4,98 4,98 1358 149,45 149,45 58 5,16 5,16 1839,5 103,88 103,88
1 48 38 S peptide SP 13278 5808,01 4800 104 4,08 4,08 414 47,28 47,28 53 4,69 4,69 1581 90,76 90,76
1 49 11 PMA Iono SP 4486 201,33 201,33 350 15,01 15,01 9735 1951,80 1951,80 97 8,64 8,64 16018 5,7E+04 1312,5
1 50 12 PMA Iono SP 4369,5 193,67 193,67 399,5 17,25 17,25 11225 4223,94 2162,5 108 9,58 9,58 15895 5,7E+04 1312,5
1 51 13 PMA Iono SP 4254 186,26 186,26 389 16,77 16,77 10268 2346,54 2162,5 104 9,24 9,24 15356 5,7E+04 1312,5
1 52 21 PMA Iono SP 4815 223,94 223,94 348 14,92 14,92 11244 4327,85 2162,5 134,5 11,80 11,80 16167 5,7E+04 1312,5
1 53 22 PMA Iono SP 6032 323,14 323,14 271 11,48 11,48 11185 4031,79 2162,5 99 8,81 8,81 15750,5 5,7E+04 1312,5
1 54 23 PMA Iono SP 5693 292,66 292,66 281,5 11,94 11,94 10321 2395,89 2162,5 111 9,84 9,84 13797 5,7E+04 1312,5
1 55 31 PMA Iono SP 4782,5 221,64 221,64 403,5 17,43 17,43 11140 3848,56 2162,5 120 10,60 10,60 16203,5 5,7E+04 1312,5
1 56 32 PMA Iono SP 5422 270,00 270,00 323 13,80 13,8 10867 3124,27 2162,5 103 9,16 9,16 15723 5,7E+04 1312,5
1 57 33 PMA Iono SP 5531 278,95 278,95 261,5 11,06 11,06 9846 2022,03 2022,03 98 8,73 8,73 14422 5,7E+04 1312,5IFN-gamma IL-12p70 IL-13 IL-1beta IL-2
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R&D Report R -20-0112 Version 01 Page 85 of 105
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Table 30: Cytokine raw data and calculated data for mCorVAC#16 , part 4 of 6 (SP)
Acquired MFI values (MFI) were convert ed into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. Gr, group. M, mouse ID. PMAIono, PMA and Ionomycin (positive control). SP, spleen .
Sample
IDGrMRestimulation Tissue MFIccalc
[pg/mL]cfin
[pg/mL MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mLcfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
25 11 S pep ide SP 47 2,3 2,3 9 <=0 0 41 22,31 22,31 52 5,87 5,87 7 <=0 0 15 <=0 0
26 12 S pep ide SP 68 3,4 3,4 11,5 <=0 0 32 16,29 16,29 32 3,34 3,34 8 <=0 0 16 <=0 0
27 13 S pep ide SP 58 2,9 2,9 10 <=0 0 37 19,67 19,67 28,5 2,86 2,86 6 <=0 0 14 <=0 0
28 14 S pep ide SP 121 6,3 6,3 9 <=0 0 65,5 37,61 37,61 44 4,90 4,90 8 <=0 0 25 69,01 0
29 15 S pep ide SP 320 16,5 16,5 9 <=0 0 89,5 51,68 51,68 33 3,48 3,48 7 <=0 0 16 <=0 0
30 16 S pep ide SP 545 27,8 27,8 14 <=0 0 183 102,32 102,32 53 5,99 5,99 15 2,76 0 71 435,82 435,82
31 17 S pep ide SP 167 8,7 8,7 10 <=0 0 50 28,08 28,08 38 4,14 4,14 7 <=0 0 30 124,92 0
32 18 S pep ide SP 171 8,9 8,9 9,5 <=0 0 69 39,71 39,71 245,5 24,38 24,38 10 <=0 0 35 172,42 0
33 21 S pep ide SP 3097 175,0 175,0 80,5 11,50 11,5 1939 921,39 921,39 748,5 63,72 63,72 1357 133,31 133,31 1730 6283,48 6283,48
34 22 S pep ide SP 2500 135,8 135,8 178,5 25,53 25,53 1790 850,48 850,48 719 61,50 61,50 1401 136,55 136,55 2007,5 7154,03 7154,03
35 23 S pep ide SP 2530 137,7 137,7 120,5 17,47 17,47 2662 1280,76 1280,76 882 73,74 73,74 2013 180,57 180,57 2382 8351,97 8351,97
36 24 S pep ide SP 2212,5 118,2 118,2 321 43,79 43,79 1915,5 910,15 910,15 694 59,61 59,61 1559 148,08 148,08 1846,5 6647,80 6647,80
37 25 S pep ide SP 2365,5 127,5 127,5 271 37,56 37,56 2038 969,03 969,03 601,5 52,61 52,61 840 93,57 93,57 1806 6521,00 6521,00
38 26 S pep ide SP 1640 85,2 85,2 146,5 21,15 21,15 1583,5 753,58 753,58 830 69,84 69,84 1562,5 148,33 148,33 1889,5 6782,64 6782,64
39 27 S pep ide SP 2880 160,3 160,3 200 28,40 28,4 2046 972,90 972,90 827 69,61 69,61 1447 139,92 139,92 1678,5 6122,76 6122,76
40 28 S pep ide SP 1653 85,9 85,9 87,5 12,58 12,58 2760,5 1332,05 1332,05 2128,5 173,56 173,56 2153,5 190,53 190,53 2511 8773,84 8773,84
41 31 S pep ide SP 919 46,8 46,8 24 1,52 0 620 312,22 312,22 657 56,82 56,82 460 60,65 60,65 1982 7073,58 7073,58
42 32 S pep ide SP 839,5 42,7 42,7 29 2,60 0488,5 251,16 251,16 680 58,56 58,56 509 65,21 65,21 1791 6474,08 6474,08
43 33 S pep ide SP 477 24,4 24,4 133 19,26 19,26 334 177,69 177,69 551,5 48,80 48,80 368 51,71 51,71 1829,5 6594,56 6594,56
44 34 S pep ide SP 787,5 40,0 40,0 40 4,74 0 786 388,32 388,32 1065 87,50 87,50 1142 117,21 117,21 2457,5 8598,20 8598,20
45 35 S pep ide SP 1429 73,6 73,6 71 10,01 10,01 1561 743,11 743,11 803 67,81 67,81 860 95,18 95,18 2058 7313,69 7313,69
46 36 S pep ide SP 668,5 34,0 34,0 60 8,22 8,22 624 314,07 314,07 1027 84,63 84,63 954 102,67 102,67 2260 7957,81 7957,81
47 37 S pep ide SP 825 42,0 42,0 78 11,11 11,11 494 253,73 253,73 587 51,51 51,51 656 78,24 78,24 1859,5 6688,55 6688,55
48 38 S pep ide SP 454 23,2 23,2 34 3,60 0 294 158,18 158,18 631,5 54,89 54,89 393 54,19 54,19 1951 6975,93 6975,93
49 11 PMA Iono SP 2056 108,9 108,9 6519,5 872,65 872,65 3366 1662,56 1662,56 5721 4142,40 731,2 5495 461,34 461,34 1671 6099,37 6099,37
50 12 PMA Iono SP 3480 202,3 202,3 9358,5 1609,56 1609,56 4770 2563,19 2563,19 5510,5 1256,15 731,2 5864 502,13 502,13 1411 5288,61 5288,61
51 13 PMA Iono SP 1521 78,6 78,6 7790,5 1147,31 1147,31 4659 2483,32 2483,32 5649,5 1891,68 731,2 5892,5 505,47 505,47 1638 5996,47 5996,47
52 21 PMA Iono SP 1759 91,8 91,8 4315,5 516,09 516,09 4106 2109,67 2109,67 5675 2216,16 731,2 6114 532,52 532,52 1684 6139,92 6139,92
53 22 PMA Iono SP 1338 68,7 68,7 5758 735,82 735,82 3354 1655,73 1655,73 5067 752,98 731,2 6086 528,98 528,98 1184 4576,36 4576,36
54 23 PMA Iono SP 1183 60,5 60,5 5530,5 698,08 698,08 3337 1646,08 1646,08 4612 557,74 557,74 5094 421,22 421,22 1063 4192,38 4192,38
55 31 PMA Iono SP 1795,5 93,9 93,9 10589 2139,77 2000 4271 2217,20 2217,20 6012 4142,40 731,2 5849 500,39 500,39 1660,5 6066,62 6066,62
56 32 PMA Iono SP 870 44,2 44,2 11204,5 2499,94 2000 3363,5 1661,14 1661,14 5047 741,29 731,2 5963 513,87 513,87 1215,5 4675,72 4675,72
57 33 PMA Iono SP 1056,5 53,9 53,9 8771 1415,41 1415,41 2883 1396,74 1396,74 4732 598,42 598,42 4594 375,76 375,76 1068 4208,33 4208,33IL-18 IL-4 IL-5 IL-6 TNF-alpha GM-CSF
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Table 31: Cytokine raw data and calculate d data for mCorVAC#16 , part 5 of 6 (LN)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. LN, lymph node. Gr, group. M, mouse ID. PMAIono, PMA and Ionomycin (posit ive control).
Plate Sample
IDGrMRestimulat
ionTissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL]
2 1 12Medium LN 30 0,06 0 10 <=0 032,5 2,58 2,58 6 0,27 0 172 14,15 14,15
2 2 13Medium LN 35,5 0,23 0 9 <=0 0 32 2,52 2,52 6 0,27 0 520 33,53 33,53
2 3 15Medium LN 32 0,12 0 10 <=0 0 29 2,18 2,18 7 0,40 0 51 5,29 5,29
2 7 21Medium LN 93 1,76 1,76 9 <=0 0 31 2,41 2,41 6 0,27 0 190 15,30 15,30
2 8 22Medium LN 159 3,37 3,37 10 <=0 0 24 1,60 0 7 0,40 0275,5 20,43 20,43
2 9 23Medium LN 194 4,20 4,20 8 <=0 0 24 1,60 0 8 0,52 0 130 11,36 11,36
2 10 24Medium LN 427 9,61 9,61 11 <=0 0 33 2,63 2,63 7 0,40 0 247 18,76 18,76
2 11 25Medium LN 86 1,58 1,58 11 <=0 0 35 2,85 2,85 7 0,40 0 503 32,66 32,66
2 12 26Medium LN 106 2,08 2,08 10 <=0 063,5 5,86 5,86 7 0,40 0693,5 42,09 42,09
2 13 27Medium LN 452,5 10,20 10,20 11 <=0 0 85 8,01 8,01 8 0,52 0 373 25,86 25,86
2 14 28Medium LN 275 6,10 6,10 11 <=0 0 43 3,73 3,73 7 0,40 0 407 27,68 27,68
2 4 31Medium LN 88 1,63 1,63 10 <=0 0 27 1,95 0 7 0,40 0 247 18,76 18,76
2 5 32Medium LN 118 2,38 2,38 9 <=0 0 27 1,95 0 6 0,27 0 227 17,57 17,57
2 6 35Medium LN 3862,5 112,04 112,04 28 0,72 065,5 6,06 6,06 21 1,83 1,83 722 43,46 43,46
2 15 12S peptide LN 27 <=0 0 9 <=0 030,5 2,35 2,35 6 0,27 0 144 12,31 12,31
2 16 13S peptide LN 24 <=0 0 9 <=0 024,5 1,66 0 6 0,27 0 42,5 4,52 4,52
2 17 15S peptide LN 38 0,30 0 9 <=0 0 23 1,48 0 6 0,27 0 39 4,19 4,19
2 21 21S peptide LN 6019 228,22 228,22 43 1,39 01158 98,84 98,84 13 1,06 1,06 3419 169,20 169,20
2 22 22S peptide LN 9299 625,33 625,33 71 2,57 2,57 1738 147,12 147,12 17 1,45 1,45 3274 161,90 161,90
2 23 23S peptide LN 12071 1686,02 1686,02 80 2,94 2,94 1253 106,68 106,68 25,5 2,23 2,23 2581 128,41 128,41
2 24 24S peptide LN 6145 237,22 237,22 45 1,47 01326 112,68 112,68 13 1,06 1,06 3027 149,72 149,72
2 25 25S peptide LN 5060 168,77 168,77 50 1,69 1,69 2139 181,51 181,51 12 0,96 0 3253 160,86 160,86
2 26 26S peptide LN 3496 97,61 97,61 28,5 0,74 0 672 58,84 58,84 10 0,74 0 2548 126,87 126,87
2 27 27S peptide LN 4729 151,42 151,42 42 1,34 03101 268,62 268,62 14 1,16 1,16 2190 110,32 110,32
2 28 28S peptide LN 7910 405,40 405,40 54 1,86 1,86 1622 137,39 137,39 19 1,64 1,64 2899,5 143,55 143,55
2 18 31S peptide LN 1803,5 43,49 43,49 13 <=0 0 41 3,51 3,51 7 0,40 0701,5 42,47 42,47
2 19 32S peptide LN 3364,5 92,70 92,70 20 0,34 0 87 8,20 8,20 8 0,52 0553,5 35,21 35,21
2 20 35S peptide LN 4311 131,42 131,42 27 0,67 0 138 13,06 13,06 33 2,87 2,87 1182,5 64,83 64,83
2 29 11PMA Iono LN 12326 1874,34 1874,34 196 7,52 7,52 5467 527,14 527,14 37 3,20 3,20 16127 8492,61 5250
2 30 21PMA Iono LN 12488 2008,41 2008,41 333 12,79 12,79 8110 953,50 953,50 65 5,36 5,36 18009 5,2E+05 5250
2 31 22PMA Iono LN 13892 3937,75 3937,75 232 8,91 8,91 7472 830,47 830,47 57,5 4,80 4,80 17831 1,5E+05 5250
2 32 31PMA Iono LN 13092,5 2635,94 2635,94 287 11,02 11,02 7797 891,10 891,10 65 5,36 5,36 18563 1,7E+07 5250IFN-gamma IL-12p70 IL-13 IL-1beta IL-2
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Table 32: Cytokine raw data and calculated data for mCorVAC#16 , part 6 of 6 (LN)
Acquired MFI values (MFI) were converted into concentrations by ProcartaPlex Analysis software (c calc). Final concentrations (c fin) meet the following criteria: Values below the
lower limit of quantification (<LLOQ, blue)) are set to zero. Values above the upper limit of quantification (> ULOQ, red) are set to the ULOQ. Commas are used as decimal
separators. Thousand were not separated by commas. LN, lymph node. Gr, group. M, mouse ID. PMAIono, PMA and Ionomycin (positive control).
Sample
IDGrMRestimulat
ionTissue MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mL]cfin
[pg/mL] MFIccalc
[pg/mLcfin
[pg/mL MFIccalc
[pg/mL]cfin
[pg/mL]
1 12Medium LN 5 <=0 0 94 13,14 13,14 17 7,72 7,72 37,5 4,19 4,19 6 1,14 0 14 <=0 0
2 13Medium LN 6 0,23 0 67 9,43 9,43 15 6,36 6,36 32 3,57 3,57 5 <=0 0,00 13 <=0 0
3 15Medium LN 8 0,37 0107,5 14,94 14,94 11 3,47 0 27 2,99 2,99 5 <=0 0,00 12 <=0 0
7 21Medium LN 17 0,95 0 44 6,09 6,09 15 6,36 6,36 28 3,11 3,11 7 1,50 0 18 <=0 0
8 22Medium LN 21 1,19 0 16 1,55 0 14 5,66 5,66 55 6,01 6,01 7 1,50 0 21 51,55 0
9 23Medium LN 11,5 0,61 0 12 0,77 0 17 7,72 7,72 32 3,57 3,57 6 1,14 022,5 70,19 0
10 24Medium LN 28 1,60 1,60 14 1,17 0 17 7,72 7,72 38 4,24 4,24 8 1,83 0 41 224,61 224,61
11 25Medium LN 33,5 1,90 1,90 39 5,33 5,33 17 7,72 7,72 47 5,20 5,20 7 1,50 0 19 19,32 0
12 26Medium LN 18,5 1,04 0 29 3,76 3,76 13 4,95 4,95 35,5 3,96 3,96 9 2,12 0 17 <=0 0,00
13 27Medium LN 23 1,31 1,31 126 17,36 17,36 16 7,04 7,04 48 5,30 5,30 16 3,78 3,78 42 231,36 231,36
14 28Medium LN 28 1,60 1,60 23 2,77 2,77 21 10,35 10,35 39 4,35 4,35 8 1,83 0 32 158,79 0
4 31Medium LN 6 0,23 0 33 4,40 4,40 11 3,47 0 25 2,74 0 5 <=0 0,00 18 <=0 0,00
5 32Medium LN 5 <=0 0,00 44 6,09 6,09 12 4,22 0 27 2,99 2,99 7 1,50 0 18 <=0 51750
6 35Medium LN 80 4,32 4,32 19 2,09 2,09 183 94,27 94,27 112 11,24 11,24 11 2,65 2,65 327 1366,33 1366,33
15 12S peptide LN 6 0,23 0 77 10,83 10,83 12 4,22 0 29 3,22 3,22 6 1,14 0 11 <=0 0,00
16 13S peptide LN 6,5 0,27 0 37 5,02 5,02 12 4,22 0 23 2,50 0 6 1,14 0 13 <=0 0,00
17 15S peptide LN 7 0,30 0 79 11,10 11,10 11 3,47 0 22,5 2,43 0 5 <=0 0,00 12 <=0 0,00
21 21S peptide LN 585 26,67 26,67 306 39,25 39,25 50,5 27,77 27,77 185 17,18 17,18 208,5 24,46 24,46 525 1945,60 1945,60
22 22S peptide LN 620 28,14 28,14 108 15,01 15,01 68,5 37,55 37,55 249 22,03 22,03 246,5 27,42 27,42 932 3012,15 3012,15
23 23S peptide LN 315 15,07 15,07 123,5 17,03 17,03 100 53,89 53,89 327,5 27,72 27,72 362 35,69 35,69 1379 4111,96 4111,96
24 24S peptide LN 722 32,50 32,50 124 17,10 17,10 51 28,04 28,04 190,5 17,61 17,61 326 33,21 33,21 566 2060,06 2060,06
25 25S peptide LN 676 30,52 30,52 838,5 98,03 98,03 41 22,40 22,40 242 21,51 21,51 333 33,69 33,69 466 1780,70 1780,70
26 26S peptide LN 374 17,63 17,63 172 23,18 23,18 35 18,92 18,92 114 11,41 11,41 176 21,79 21,79 304 1292,04 1292,04
27 27S peptide LN 388 18,23 18,23 3354,5 371,05 371,05 44 24,12 24,12 185,5 17,22 17,22 543 47,36 47,36 421 1650,50 1650,50
28 28S peptide LN 835 37,33 37,33 112 15,53 15,53 72 39,41 39,41 231,5 20,73 20,73 537 46,99 46,99 748 2542,37 2542,37
18 31S peptide LN 18 1,01 0 41 5,64 5,64 20 9,70 9,70 66 7,09 7,09 10 2,39 0145 731,35 731,35
19 32S peptide LN 22 1,25 1,25 137 18,77 18,77 17 7,72 7,72 76 8,03 8,03 17 3,99 3,99 256 1136,68 1136,68
20 35S peptide LN 83 4,47 4,47 23 2,77 2,77 185 95,21 95,21 188 17,41 17,41 40 7,78 7,78 375 1513,77 1513,77
29 11PMA Iono LN 802 35,92 35,92 6819 837,42 837,42 951 425,96 425,96 5191,5 528,45 528,45 1086 78,50 78,50 1598 4645,82 4645,82
30 21PMA Iono LN 930 41,41 41,41 11126 1777,66 1777,66 1430 628,21 628,21 6317 924,47 731,2 4143,5 273,05 273,05 2253 6278,43 6278,43
31 22PMA Iono LN 494 22,76 22,76 7001 866,85 866,85 1107 491,32 491,32 6276 901,77 731,2 3748 241,36 241,36 2246 6260,47 6260,47
32 31PMA Iono LN 1641 73,08 73,08 6896,5 849,87 849,87 2801 1244,18 1244,18 6159,5 842,57 731,2 3033,5 190,98 190,98 2431 6740,05 6740,05IL-5 IL-6 TNF-alpha GM-CSF IL-18 IL-4
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9.6 Certificates of Analysis BNT162a1
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(b) (6)
(b) (6)
(b) (4)
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(b) (6)
(b) (6)
(b) (4)
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9.7 Certificates of Analysis BNT162b1
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(b) (6)
(b) (6)
(b) (4)
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(b) (4)
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9.8 Certificates of Analysis BNT162b2
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(b) (4)
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(b) (6)
(b) (4)
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9.9 Certificates of Analysis BNT162c2
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9.10 Statistical analysis
ELISpot – details on statistical analysis performed with GraphPad Prism 8.
Related to Figure 6
Group Sidak's multiple
comparisons test Mean Diff, 95,00% CI of
diff, Significant? Summary Adjusted P
Value
Control
(mCorVac#15) No peptide vs. S
pepide -4.938 -8.321 to -
1,554 Yes ** 0.0041
Control RNA vs. S
RNA 2.125 -1.258 to 5 .508 No ns 0.2696
BNT162a1
No peptide vs. S
pepide -82.81 -103.4 to -
62.26 Yes **** <0.0001
Control RNA vs. S
RNA -47.13 -67.67 to -
26.58 Yes **** <0.0001
BNT162b1
No peptide vs. S
pepide -748.2 -894.1 to -
602.3 Yes **** <0.0001
Control RNA vs. S
RNA -734.5 -880.4 to -
588.6 Yes **** <0.0001
Control
(mCorVac#16 ) No peptide vs. S
peptide -14.50 -22.73 to -
6.270 Yes *** 0.0007
Control RNA vs. S
RNA 2.438 -5.793 to 10.67 No ns 0.7333
BNT162b2 No peptide vs. S
peptide -1177 -1314 to -1041 Yes **** <0.0001
Control RNA vs. S
RNA -1311 -1448 to -1175 Yes **** <0.0001
BNT162c2
No peptide vs. S
peptide -1174 -1293 to -1056 Yes **** <0.0001
Control RNA vs. S
RNA -1171 -1290 to -1053 Yes **** <0.0001
Flow cytometry – details on statistical analysis performed with GraphPa d Prism
8.
Related to Figure 7
Group Dunnett's
multiple
comparisons test Mean
Diff. 95.00% CI
of diff. Significant? Summary Adjusted
P Value
CD8+ T cells Control vs.
BNT162a1 -1.250 -3.386 to
0.8864 No ns 0.3001
Control vs.
BNT162b1 -1.225 -3.361 to
0.9114 No ns 0.3130
Control vs.
BNT162b2 -10.63 -13.87 to -
7.377 Yes **** <0.0001
Control vs.
BNT162c2 -0.4321 -3.794 to
2.930 No ns 0.9348
CD4+ T cells Control vs.
BNT162a1 1.188 -0.9863 to
3.361 No ns 0.3445
Control vs.
BNT162b1 1.425 -0.7488 to
3.599 No ns 0.2302
Control vs.
BNT162b2 11.06 7.473 to
14.65 Yes **** <0.0001
Control vs.
BNT162c2 -0.7571 -4.473 to
2.959 No ns 0.8463
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TFH cells Control vs.
BNT162a1 -0.04500 -0.1701 to
0.08005 No ns 0.6086
Control vs.
BNT162b1 -0.3726 -0.4977 to -
0.2476 Yes **** <0.0001
Control vs.
BNT162b2 -0.3828 -0.7397 to -
0.02579 Yes * 0.0350
Control vs.
BNT162c2 -1.190 -1.560 to -
0.8208 Yes **** <0.0001
B cells Control vs.
BNT162a1 7.813 5.540 to
10.08 Yes **** <0.0001
Control vs.
BNT162b1 7.900 5.628 to
10.17 Yes **** <0.0001
Control vs.
BNT162b2 14.64 9.503 to
19.77 Yes **** <0.0001
Control vs.
BNT162c2 9.921 4.607 to
15.24 Yes *** 0.0005
Related to Figure 8
Group Dunnett's
multiple
comparisons
test Mean
Diff. 95.00% CI of
diff. Significant? Summary Adjusted
P Value
CD44+CD38+PD1+
CD8+ T cells
Control vs.
BNT162a1 -0.2575 -1.951 to
1.436 No ns 0.9102
Control vs.
BNT162b1 -6.015 -7.708 to -
4.322 Yes **** <0.0001
Control vs.
BNT162b2 -28.05 -33.72 to -
22.37 Yes **** <0.0001
Control vs.
BNT162c2 -1.344 -7.222 to
4.533 No ns 0.8115
ICOS+ CD8+ T
cells
Control vs.
BNT162a1 -0.8663 -3.073 to
1.341 No ns 0.5578
Control vs.
BNT162b1 -8.401 -10.61 to -
6.194 Yes **** <0.0001
Control vs.
BNT162b2 -40.48 -47.07 to -
33.89 Yes **** <0.0001
Control vs.
BNT162c2 -4.713 -11.54 to
2.109 No ns 0.1998
ICOS+ CD4+ T
cells
Control vs.
BNT162a1 -0.5650 -1.427 to
0.2973 No ns 0.2304
Control vs.
BNT162b1 -2.551 -3.414 to -
1.689 Yes **** <0.0001
Control vs.
BNT162b2 -2.981 -5.174 to -
0.7890 Yes ** 0.0078
Control vs.
BNT162c2 1.218 -1.051 to
3.488 No ns 0.3555
ICOS+ Tfh cells
Control vs.
BNT162a1 -10.11 -18.24 to -
1.987 Yes * 0.0143
Control vs.
BNT162b1 -26.43 -34.55 to -
18.30 Yes **** <0.0001
Control vs.
BNT162b2 -12.49 -21.24 to -
3.733 Yes ** 0.0054
Control vs.
BNT162c2 -19.20 -27.95 to -
10.45 Yes **** <0.0001
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Related to Figure 9
Group Dunnett's
multiple
comparisons
test Mean
Diff. 95.00% CI of
diff. Significant? Summary Adjusted
P Value
CD8+ T cells
Control vs.
BNT162a1 -337552 -833529 to
158426 No ns 0.2084
Control vs.
BNT162b1 -420848 -916825 to
75130 No ns 0.1019
Control vs.
BNT162b2 -683950 -1112484 to -
255415 Yes ** 0.0021
Control vs.
BNT162c2 -75551 -504086 to
352984 No ns 0.8818
CD4+ T cells
Control vs.
BNT162a1 -749301 -1913693 to
415091 No ns 0.2411
Control vs.
BNT162b1 -1246977 -2411369 to -
82585 Yes * 0.0352
Control vs.
BNT162b2 -1850559 -2886016 to -
815102 Yes *** 0.0007
Control vs.
BNT162c2 -216563 -1252020 to
818895 No ns 0.8389
TFH cells Control vs.
BNT162a1 -2366 -7903 to
3171 No ns 0.5051
Control vs.
BNT162b1 -14242 -19780 to -
8705 Yes **** <0.0001
Control vs.
BNT162b2 -46173 -60706 to -
31640 Yes **** <0.0001
Control vs.
BNT162c2 -4251 -18783 to
10282 No ns 0.7150
TH1 cells
Control vs.
BNT162a1 -7820 -18193 to
2552 No ns 0.1541
Control vs.
BNT162b1 -13043 -23416 to -
2671 Yes * 0.0134
Control vs.
BNT162b2 -2268 -4564 to
28.31 No ns 0.0531
Control vs.
BNT162c2 297.1 -1999 to
2593 No ns 0.9339
Related to Figure 10
Group Dunnett's
multiple
comparisons
test Mean
Diff. 95.00% CI of
diff. Significant? Summary Adjusted
P Value
B cells
Control vs.
BNT162a1 -415609 -1302980 to
471763 No ns 0.4459
Control vs.
BNT162b1 -12666 93 -2154065 to -
379321 Yes ** 0.0053
Control vs.
BNT162b2 -761299 -1089182 to -
433417 Yes **** <0.0001
Control vs.
BNT162c2 26360 -301523 to
354242 No ns 0.9738
Plasma cells
Control vs.
BNT162a1 -244.3 -18256 to
17768 No ns 0.9992
Control vs.
BNT16 2b1 -20130 -38142 to -
2117 Yes * 0.0278
090177e194f89529\Approved\Approved On: 22-Se
…[truncated]