Brief Report
BNT162b2 Vaccine Encoding the SARS-CoV-2 P2 S Protects
Transgenic hACE2 Mice against COVID-19
Rui-Ru Ji
1,†
, Yajin Qu
2,†
, Hua Zhu
2,†
, Yumei Yang
1
, Annette B. Vogel
3
, Ugur Sahin
3,4
, Chuan Qin
2,
*
and Aimin Hui
1,
*
Citation: Ji, R.-R.; Qu, Y.; Zhu, H.;
Yang, Y.; Vogel, A.B.; Sahin, U.; Qin,
C.; Hui, A. BNT162b2 Vaccine
Encoding the SARS-CoV-2 P2 S
Protects Transgenic hACE2 Mice
against COVID-19. Vaccines 2021, 9,
324. https://doi.org/10.3390/
vaccines9040324
Academic Editors: Soo-Hong Lee,
Hansoo Park, Jagathesh
Chandra Rajendran and K.
S. Jaganathan
Received: 5 March 2021
Accepted: 24 March 2021
Published: 1 April 2021
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Attribution (CC BY) license (https://
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4.0/).
1
Shanghai Fosun Pharmaceutical Industrial Development, Co. Ltd., 1289 Yishan Road,
Shanghai 200233, China; ruiruji@msn.com (R.-R.J.); yangyumei@fosunpharma.com (Y.Y.)
2
NHC Key Laboratory of Human Disease Comparative Medicine, Beijing Key Laboratory for Animal Models
of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of
Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100021, China;
quyajin@pumc.edu.cn (Y.Q.); zhuhua@pumc.edu.cn (H.Z.)
3
BioNTech, An der Goldgrube 12, 55131 Mainz, Germany; Annette.Vogel@biontech.de (A.B.V.);
Ugur.Sahin@biontech.de (U.S.)
4
TRON gGmbH—Translational Oncology at the University Medical Centre of the Johannes Gutenberg University,
Freiligrathstraße 12, 55131 Mainz, Germany
* Correspondence: qinchuan@pumc.edu.cn (C.Q.); aimin.hui@fosunpharma.com (A.H.)
† These authors contributed equally to this work.
Abstract:
BNT162b2 is a highly efficacious mRNA vaccine approved to prevent COVID-19. This
brief report describes the immunogenicity and anti-viral protective effect of BNT162b2 in hACE2
transgenic mice. Prime-boost immunization with BNT162b2 elicited high titers in neutralizing
antibodies against SARS-CoV-2, which correlated with viral clearance and alleviated lung lesions in
these mice after viral challenge.
Keywords: SARS-CoV-2; COVID-19; mRNA vaccine; efficacy; immunogenicity; challenge study
1. Introduction
SARS-CoV-2 infects cells through the binding of the spike (S) viral membrane protein
to the human angiotensin converting enzyme 2 (hACE2) on target cells [
1
]. A transgenic
C57BL/6 mouse, expressing the hACE2 gene under the control of the mouse Ace2 pro-
moter, was first developed as a model system to study severe acute respiratory syndrome
(SARS) caused by SARS-CoV [
2
]. Using the hACE2 transgenic mouse model, SARS-CoV-2
replication was observed in the lungs of infected mice, and viral antigens were detected in
bronchial epithelial cells, macrophages, and alveolar epithelia. The typical histopathology
was interstitial pneumonia with infiltration of considerable numbers of macrophages and
lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveo-
lar cavities. By contrast, wildtype mice challenged with SARS-CoV-2 did not develop these
symptoms [
3
]. These data suggested that the hACE2 transgenic mouse is a valuable model
for the evaluation of vaccines and antiviral therapeutic agents fighting COVID-19.
BNT162b2 is a lipid nanoparticle (LNP) formulated nucleoside-modified mRNA vac-
cine expressing the full-length S protein stabilized in the prefusion conformation (P2 S,
containing K986P and V987P) [
4
–
6
]. BNT162b2 is being tested in several clinical trials
including global Phase 2/3 trials (NCT04380701, NCT04368728) to investigate its safety,
immunogenicity, and efficacy in humans [
7
,
8
]. Until 16th December 2020, BNT162b2 has
received 16 emergency approvals. Our previous preclinical studies demonstrated that
immunization of wildtype BALB/c mice with a single dose of BNT162b2 intramuscu-
larly induced a fast and dose-dependent increase in total IgG response and pseudo-virus
neutralization titers. In rhesus macaques, two vaccinations elicited high SARS-CoV-2
neutralization titers. In both animal models, a strong T helper type 1 (Th1) bias with a
Vaccines 2021, 9, 324. https://doi.org/10.3390/vaccines9040324 https://www.mdpi.com/journal/vaccines
Vaccines 2021, 9, 324 2 of 7
high IFN
γ
+
CD8
+
T-cell response was observed, and BNT162b2 fully protected the lungs of
immunized rhesus macaques from the SARS-CoV-2 challenge [
9
]. Addressing the limited
utility of rhesus macaques as a disease model, we report the preclinical efficacy evaluation
of BNT162b2 in a challenge study using hACE2 transgenic mice.
2. Materials and Methods
2.1. Ethics Statement
This mouse study was performed in an animal biosafety level 3 (ABSL3) facility
using HEPA-filtered isolators. All procedures in this study involving mice were reviewed
and approved by the Institutional Animal Care and Use Committee of the Institute of
Laboratory Animal Science, Peking Union Medical College (GH20012). All experiments
complied with all relevant ethical regulations.
2.2. Cell lines and Virus
The human Vero E6 cell line was cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with ten percent fetal bovine
serum (Gibco, Grand Island, NE, USA) and incubated at 37
◦
C and five percent carbon
dioxide. The SARS-CoV-2 isolate SARS-CoV-2/human/CHN/WH-09/2020 (GenBank:
MT093631.2) [10] was used.
2.3. Construction of the BNT162b2 Vaccine
BNT162b2 was generated as described elsewhere (Vogel et al., 2020). Briefly, a full-
length SARS-CoV-2 spike protein (GenBank: MN908947) including the K986P and V987P
mutations was designed (P2 S) [
4
–
6
].
In vitro
transcription was analyzed using T7 RNA
polymerase in the presence of a trinucleotide cap1 analogue ((m
2
7,3
0
-O
)Gppp(m
2
0
-O
)ApG;
TriLink, San Diego, CA, USA) and with N1-methylpseudouridine-5
0
-triphosphate (m1
Ψ
TP;
Thermo Fisher Scientific, Schwerte, Germany) replacing uridine-5
0
-triphosphate (UTP) [
11
].
The purified RNA was formulated into lipid nanoparticles (LNPs) using an ethanolic lipid
mixture of ionizable cationic lipid [
12
]. The vaccine candidate was stored at
−
70 to
−
80
◦
C
at a concentration of 0.5 mg/mL.
2.4. Study Design Animal Experiments
Mouse studies were performed in an animal biosafety level 3 (ABSL3) facility using
HEPA-filtered isolators. Table 1 contains the animal grouping and testing information, and
Supplemental Figure S1 shows a schematic view of the study design. C57BL/6 female and
male hACE2 knockin mice [
3
] approximately 6 weeks of age at study start were randomly
assigned to three groups: control (injection dilution buffer), medium dose (1
µ
g/animal),
and high dose (5 µg/animal). The animal body weights were 20–23 g.
Table 1. Summary of study key parameters and results.
Dose
Group.
Number
of Mice
Dose Per
Mouse
Infection
Neutral. Ab
Titer
Viral Load
Log
10
Alveolar Score Blood Vessel Score
Normal Mild Mod. Normal Mild Mod.
High 10 5 µg
10
5
TCID
50
/50
µ
L
1024 * 0+/−0 * 2 7 1 6 4 0
Medium 10 1 µg >1024 * 0+/−0 * 1 7 2 3 7 0
Control 8 - <8 6+/−0.22 0 2 6 3 5 0
* p << 0.001 compared to control. Listed from left to right are, for each dose group, number of animals per group; vaccine dose per animal
per shot; viral infection dose per animal; titers of neutralizing antibodies with <8 being below the lower limit and >1024 being higher than
upper limit of quantification; viral load given as log10 of the viral particle count; and the number of animals per lung lesion score (see
Figure 1 for definition of scores).
Vaccines 2021, 9, 324 3 of 7
Vaccines 2021, 9, x 3 of 7
Figure 1. Antibody immune response after BNT162b2 immunization (a+b) and analysis of protection after viral challenge
(c–i). (a) Titration of anti-S1 IgG antibodies analysis by enzyme-linked immunosorbent assay (ELISA); (b) titers of 50%
virus-neutralizing antibodies; (c) viral load in lung tissue and associated pairwise p values 5 days after challenge; (d)
percentage (y axis) and number (data labels) of mice grouped by lung symptom and dose group. Lung symptom was
scored as normal (green), mild lesion (orange), or moderate lesion (red), for alveolar septum (left) and tissue around blood
vessels (right); (e–i) representative Hematoxylin and Eosin (H&E) stained lung tissue slices used to identify histopatho-
logical changes. Scalebar is 250 µm, HE ×100. Classification criteria for alveolar septum: mild lesion—mild thickening of
the alveolar septum; moderate lesion—obvious thickening of the alveolar septum, with the lesion range greater than ½.
Classification criteria of inflammatory cells infiltration around blood vessels: mild lesion—lesion range less than 1/4 of the
lung tissue section; moderate lesion—lesion range being 1/4 to 2/4 of the lung tissue section.
Routine animal monitoring such as body weight and macroscopic assessment of an-
imal activity and behavior was carried out daily throughout the study period. Primary
and second immunizations occurred on day 0 and day 21 via intramuscular injection (i.m.)
at an injection volume of 20 µL per injection per animal. On day 28 blood samples were
collected from all mice, and sera of every 2~3 mice in each group were mixed to detect
total IgG and neutralizing antibodies.
After being intraperitoneally anaesthetized by 2.5% Avertin (tribromoethanol) with
0.02 mL/g body weight, mice were challenged intranasally on day 42 with 50 µL viral
suspension of strain SARS-CoV-2/WH-09/human/2020/CHN at 10
5
TCID
50
per animal. The
mice were observed continuously for 5 days after challenge, and the weight changes were
recorded. Five days after challenge (day 47), all mice were sacrificed for viral load detec-
tion and pathological examination of lung tissues.
2.5. S1 Binding IgG Assay
The 96-well plates were coated with recombinant S1 (100 ng/100 µL, Sino Biological,
Beijing, China) in sodium carbonate buffer, and bound IgG was detected using an HRP-
conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Cambridge, UK)
and TMB substrate (Sino Biological, Beijing, China). Data collection was performed using
a Multiskan MK3 reader (Thermo Fisher, Waltham, MA, USA). The OD value (450–630
nm) was calculated.
Figure 1.
Antibody immune response after BNT162b2 immunization (a+b) and analysis of protection after viral challenge
(
c
–
i
). (
a
) Titration of anti-S1 IgG antibodies analysis by enzyme-linked immunosorbent assay (ELISA); (
b
) titers of 50%
virus-neutralizing antibodies; (
c
) viral load in lung tissue and associated pairwise p values 5 days after challenge; (
d
)
percentage (y axis) and number (data labels) of mice grouped by lung symptom and dose group. Lung symptom was scored
as normal (green), mild lesion (orange), or moderate lesion (red), for alveolar septum (left) and tissue around blood vessels
(right); (
e
–
i
) representative Hematoxylin and Eosin (H&E) stained lung tissue slices used to identify histopathological
changes. Scalebar is 250
µ
m, HE
×
100. Classification criteria for alveolar septum: mild lesion—mild thickening of the
alveolar septum; moderate lesion—obvious thickening of the alveolar septum, with the lesion range greater than
1
/2
.
Classification criteria of inflammatory cells infiltration around blood vessels: mild lesion—lesion range less than 1/4 of the
lung tissue section; moderate lesion—lesion range being 1/4 to 2/4 of the lung tissue section.
Routine animal monitoring such as body weight and macroscopic assessment of
animal activity and behavior was carried out daily throughout the study period. Primary
and second immunizations occurred on day 0 and day 21 via intramuscular injection (i.m.)
at an injection volume of 20
µ
L per injection per animal. On day 28 blood samples were
collected from all mice, and sera of every 2~3 mice in each group were mixed to detect total
IgG and neutralizing antibodies.
After being intraperitoneally anaesthetized by 2.5% Avertin (tribromoethanol) with
0.02 mL/g body weight, mice were challenged intranasally on day 42 with 50
µ
L viral
suspension of strain SARS-CoV-2/WH-09/human/2020/CHN at 10
5
TCID
50
per animal.
The mice were observed continuously for 5 days after challenge, and the weight changes
were recorded. Five days after challenge (day 47), all mice were sacrificed for viral load
detection and pathological examination of lung tissues.
2.5. S1 Binding IgG Assay
The 96-well plates were coated with recombinant S1 (100 ng/100 µL, Sino Biological,
Beijing, China) in sodium carbonate buffer, and bound IgG was detected using an HRP-
conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Cambridge, UK)
and TMB substrate (Sino Biological, Beijing, China). Data collection was performed using a
Multiskan MK3 reader (Thermo Fisher, Waltham, MA, USA). The OD value (450–630 nm)
was calculated.
Vaccines 2021, 9, 324 4 of 7
2.6. SARS-CoV-2 Virus Neutralisation Test
SARS-CoV-2 strain SARS-CoV-2/human/CHN/WH-09/2020 (GenBank: MT093631.2)
was used in the virus neutralization test (VNT), and the serum samples were incubated at
56
◦
C for 30 min for thermal inactivation. Dulbecco’s modified Eagle’s medium (DMEM)
was used to continuously dilute each serum sample. The dilution ratio was 2 or 3 times,
depending on OD value or sample quantity. The staring dilution was 1:8 for BNT162b2
sera. Serum dilution was mixed with the same volume of diluted virus and incubated
at 37
◦
C for 1 h. The Vero E6 cells in the 24-well plate were incubated with the serum
virus mixture at 37
◦
C. After 1 h, DMEM containing 2.5% FBS and 0.8% carboxymethyl
cellulose was used to replace the mixed culture medium of serum virus in the wells. They
were fixed with 8% paraformaldehyde and dyed with 0.5% crystal violet 3 days later. All
samples were repeated, and the neutralization titer was defined as a serum dilution ratio
that resulted in a reduction in plaque by at least 50%.
2.7. RNA Extraction and Reverse-Transcription Quantitative Polymerase Chain Reaction
The virus load was analyzed by RT-qPCR. Total RNA was extracted from organs
using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and reverse transcription was
performed using the PrimerScript RT Reagent 203 Kit (TaKaRa, Kusatsu, Japan) following
the manufacturer instructions. Quantitative real-time reverse transcription-PCR (qRT-
PCR) reactions were performed using the PowerUp SYBG Green Master Mix Kit (Applied
Biosystems, USA), in which samples were processed in duplicate using the following
cycling protocol: 50
◦
C for 2 min, 95
◦
C for 2 min, followed by 40 cycles at 95
◦
C for
15 s and 60
◦
C for 30 s, and then 95
◦
C for 15 s, 60
◦
C for 1 min, and 95
◦
C for 45 s.
The primer sequences used for qRT-PCR are targeted against the envelope (E) gene of
SARS-CoV-2 and are as follows: forward: 5
0
-TCGTTTCGGAAGAGACAGGT-3
0
; reverse:
5
0
-GCGCAGTAAGGATGGCTAGT-3
0
. The PCR products were verified by sequencing
using the dideoxy method on an ABI 3730 DNA sequencer (Applied Biosystems, Foster
City, CA, USA). During the sequencing process, amplification was performed using specific
primers. The sequencing reads obtained were linked using DNAMAN, and the results were
compared using the Megalign module in the DNAStar software package. The SYBR green
real-time PCR standard curve was generated by serial tenfold dilutions of recombinant
plasmid with a known copy number (from 1.47
×
10
9
to 1.47
×
10
1
copies per
µ
L). These
dilutions were tested and used as quantification standards to construct the standard curve
by plotting the plasmid copy number against the corresponding threshold cycle values
(Ct). Results were expressed as log10-transformed numbers of genome equivalent copies
per mL of sample.
2.8. Histopathology
Autopsies were performed in an animal biosafety level 3 (ABSL3) laboratory. Major
organs were grossly examined and then fixed in 10% buffered formalin solution.
The lung tissue was fixed in 10% buffered formalin solution, and paraffin sections
(3–4
µ
m in thickness) were prepared routinely. Hematoxylin and Eosin (H&E) stain was
used to identify histopathological changes. The histopathology of the lung tissue was
observed by light microscopy and scored based on the severity of lesions (thickening of
alveolar septum and infiltration of inflammatory cells, and perivascular inflammatory cell
infiltration).
2.9. Statistical Analysis
All data were analyzed with GraphPad Prism 8.0 software. Statistically significant
differences between two groups were determined using unpaired Student’s t-tests. A
two-sided p < 0.05 was considered to be statistically significant.
Vaccines 2021, 9, 324 5 of 7
3. Results and Discussion
Transgenic hACE2 mice were immunized twice, three weeks apart, with either a
medium (1
µ
g) or high (5
µ
g) dose of BNT162b2 or dilution buffer (control) (Table 1 and
see Supplementary Materials, Figure S1). During the immunization period and prior to
virus challenge, the animals in all groups had similar normal food and water intake and
steady weight gain. No clinically significant abnormal findings were observed.
Seven days after the second immunization (day 28), mice were bled to quantify S1
specific antibodies. BNT162b2 was able to induce high serum IgG titers in immunized mice
also using 1
µ
g (Figure 1a). The levels of neutralizing antibody in the BNT162b2 group all
reached the upper limit of quantification (ULOQ) at a titer of 1024 (Figure 1b). By contrast,
the control group had no detectable anti-S1 IgG binding nor neutralizing antibodies.
Three weeks after the second immunization (day 42), animals were challenged in-
tranasally with 10
5
TCID
50
SARS-CoV-2, continuously observed for five days, then sacri-
ficed for final analysis of viral load and histopathology of lung tissues. After virus challenge,
every experimental group experienced a drop in body weight (see Supplementary Materi-
als, Table S1). The weight losses were 6.58% in the control, 5.57% in the 1
µ
g, and 7.88%
in the 5
µ
g dosed groups. There was no statistically significant difference in weight loss
among the groups (p > 0.05). Five days after virus challenge, the mean viral load in the
control group was 10
6.01
copies/mL. By contrast, no viral RNA could be detected in the
lung tissues in BNT162b2-treated mice, suggesting that viral replication was completely
inhibited in those animals (Figure 1c and see Supplementary Materials, Table S2). Taken
together with the virus neutralization test (VNT) data, the complete suppression of viral
replication coincided with the high titer of neutralizing antibodies in the mice.
Scoring of lung lesions of these mice is summarized in Figure 1d (see Supplementary
Materials, Table S3), and representative staining of lung tissues from every group are
shown in Figure 1e–i. After viral challenge, all hACE2 mice in the control group developed
mild or moderate interstitial pneumonia, characterized by thickening of alveolar septum
and infiltration of inflammatory cells in the lungs. BNT162b2 vaccination at both doses
ameliorated these lung lesions. For example, two animals in the medium-dose group
developed moderate thickening of alveolar septum and infiltration of inflammatory cells,
seven had mild phenotype, and one had no lesion. The high-dose group showed further
improvement: two were free of any lesions, seven had mild, and one had moderate lesions
(Figure 1d).
Although we have observed improved lung lesions in vaccinated groups, the pheno-
typic manifestations of virus infection were variable among these animals and not directly
correlated with the clearance of the virus or neutralizing antibody titers. For example,
BNT162b2 vaccination cleared the virus completely in the lungs; however, these mice
still exhibited various degrees of lung lesions. The lack of a strong correlation among
these measures such as viral load, weight changes, and lung lesions limits our ability to
rationalize the observations.
There are clear limitations to this mouse model, such as the limited tissue distribution
of hACE2 expression and a relatively mild histopathology [
2
]. A very different approach
using hACE2-transduced wildtype mice [
13
] also showed that mouse models of the disease,
while critical tools, have significant limitations as a means to assess the full spectrum of the
potential protective effect of vaccines and therapeutic agents.
These results support the decision to move BNT162b2 toward market authorization.
4. Conclusions
The reported hACE2-transduced mouse study using BNT162b2 for vaccination demon-
strates that this market authorized COVID-19 vaccine is highly immunogenic and an
effective mean for anti-viral protection.