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\begin{document}

\title{A Subunit-based Influenza/SARS-CoV-2 Omicron Combined Vaccine Induced
Potent Protective Immunity in BALB/c Mice}



\author[1]{NARU ZHANG}%
\author[1]{Zihui Ye}%
\author[2]{Cun Li}%
\author[3]{Jie Zhou}%
\author[2]{Wei Xue}%
\author[1]{Luying Xiang}%
\author[1]{Yuewen Chen}%
\author[1]{Shuchang Chen}%
\author[1]{Rouhan Ye}%
\author[1]{Jingyin Dong}%
\author[2]{J. Zhou}%
\author[3]{Shibo Jiang}%
\author[1]{Haijun Han}%
\affil[1]{Hangzhou City University}%
\affil[2]{The University of Hong Kong}%
\affil[3]{Fudan University}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Infection with influenza A virus (IAV) and severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human
life, health, and the global economy. Vaccination is one of the most
effective strategies in the fight against infectious viruses. In this
study, we, for the first time, have evaluated the immunogenicity and
protective effect of an influenza/SARS-CoV-2 Omicron subunit combined
vaccine adjuvanted with MF59 and administered to BALB/c mice. Results
showed that the combined vaccine induced high levels of IgG, IgG
\textsubscript{1}, and IgG \textsubscript{2a} antibodies, as well as
influenza A H1N1/California/2009 virus-specific
hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this
subunit combined vaccine induced high titers of neutralization
antibodies against SARS-CoV-2 Omicron BA.5 pseudovirus and effectively
reduced the viral load of authentic SARS-CoV-2 Omicron BA.5.2 variant in
the cell culture supernatants. These results suggested that this subunit
combined vaccine achieved protective effect against both H1N1
A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is
therefore expected that this study will establish the scientific
foundation for the next-step development of combined vaccines against
other strains or variants of IAV and SARS-CoV-2.%
\end{abstract}%



\sloppy


\textbf{A Subunit-based Influenza/SARS-CoV-2 Omicron Combined Vaccine
Induced Potent Protective Immunity in BALB/c Mice}

Naru Zhang\textsuperscript{1,\#}, Zihui Ye\textsuperscript{1,\#}, Cun
Li\textsuperscript{2,\#}, Jie Zhou\textsuperscript{3,\#}, Wei
Xue\textsuperscript{2}, Luying Xiang\textsuperscript{1}, Yuewen
Chen\textsuperscript{1}, Shuchang Chen\textsuperscript{1}, Rouhan
Ye\textsuperscript{1}, Jingyin Dong\textsuperscript{1}, Jie
Zhou\textsuperscript{2,*}, Shibo Jiang\textsuperscript{3,*}, Haijun
Han\textsuperscript{1,*}

\textsuperscript{1} School of Medicine, Hangzhou City University,
Hangzhou 310015, China

\textsuperscript{2} Department of Microbiology, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Hong Kong, China

\textsuperscript{3} Key Laboratory of Medical Molecular Virology
(MOE/NHC/CAMS), Shanghai Frontiers Science Center of Pathogenic Microbes
and Infection, School of Basic Medical Sciences, Shanghai Institute of
Infectious Disease and Biosecurity, Fudan University, Shanghai 200032,
China

*Correspondence:
\emph{Jiezhou@hku.hk}(J.Z.);\emph{shibojiang@fudan.edu.cn}(S.J.);
\emph{hanhj@hzcu.edu.cn} (H.H.)

\textbf{Abstract:} Infection with influenza A virus (IAV) and severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a
significant risk to human life, health, and the global economy.
Vaccination is one of the most effective strategies in the fight against
infectious viruses. In this study, we, for the first time, have
evaluated the immunogenicity and protective effect of an
influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with
MF59 and administered to BALB/c mice. Results showed that the combined
vaccine induced high levels of IgG, IgG\textsubscript{1}, and
IgG\textsubscript{2a} antibodies, as well as influenza A
H1N1/California/2009 virus-specific hemagglutination-inhibiting
antibodies in BALB/c mice. Moreover, this subunit combined vaccine
induced high titers of neutralization antibodies against SARS-CoV-2
Omicron BA.5 pseudovirus and effectively reduced the viral load of
authentic SARS-CoV-2 Omicron BA.5.2 variant in the cell culture
supernatants. These results suggested that this subunit combined vaccine
achieved protective effect against both H1N1 A/California/07/2009 strain
and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that
this study will establish the scientific foundation for the next-step
development of combined vaccines against other strains or variants of
IAV and SARS-CoV-2.

\textbf{Keywords:} Influenza A virus; SARS-CoV-2; Omicron BA.5; MF59;
subunit vaccines

\textbf{1. Introduction}

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) has had a huge negative impact on
global public health and economic development.\textsuperscript{1} Since
its discovery, SARS-CoV-2 has evolved into several variants, and now
Omicron is a major public health concern owing to its high infectivity
and antibody evasion.\textsuperscript{2}~Omicron variants BA.4 and BA.5
were reported to be more transmissible and resistant to immunity than
previous variants, including Omicron BA.1 and most monoclonal
antibodies.\textsuperscript{2} As of November 2, 2023, SARS-CoV-2 and
its variants had caused 771,679,618 confirmed cases and 6,977,023 deaths
globally.\textsuperscript{3} Influenza A virus (IAV), a common
respiratory infectious virus, causes severe respiratory illnesses in 3
to 5 million people and 290,000 to 650,000 deaths yearly
worldwide.\textsuperscript{4} IAV is one of the most commonly recognized
respiratory viruses identified as coinfected with
SARS-CoV-2.\textsuperscript{5} Studies have shown that coinfection with
SARS-CoV-2 during the acute stage of IAV infection further increases the
risk of multiple tissue or organ disease in patients, leading to the
occurrence of higher rates of severe illness and
mortality.\textsuperscript{6-8} Vaccination is one of the most effective
measures to prevent respiratory infectious viruses.

Seasonal vaccination against IAV could significantly decrease the number
of individuals getting sick or death from the
infection.\textsuperscript{9} Also, COVID-19 vaccination has effectively
reduced severe cases and mortality rate.\textsuperscript{10, 11} Many
dual-vaccination strategies are currently in the developmental stage,
such as the virion-based vaccine,\textsuperscript{12,
13}receptor-binding domain (RBD)-based
vaccine,\textsuperscript{14}RBD-conjugated inactivated
IAV,\textsuperscript{15} mRNA vaccine,\textsuperscript{8} chimpanzee
adenovirus 68 (AdC68)-based vaccine,\textsuperscript{16} and recombinant
VSV-based bivalent vaccine.\textsuperscript{17} Similarly, we imagined
the advantages of a combined SARS-CoV-2/IAV subunit vaccine in the fight
against COVID-19 and influenza, including the safety and ease of
producing such subunit vaccine on a large scale.

Accordingly, in this study, the immunogenicity and protective effect of
an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with
MF59 were evaluated in BALB/c mice. The combined vaccine induced high
levels of IgG, IgG\textsubscript{1}, and
IgG\textsubscript{2a}antibodies, as well as influenza A
H1N1/California/2009 virus-specific hemagglutination-inhibiting
antibodies in BALB/c mice. Furthermore, the subunit combined vaccine
induced high titers of neutralizing antibodies against SARS-CoV-2
Omicron BA.5 pseudovirus and effectively reduced the viral load of
SARS-CoV-2 Omicron BA.5.2 variant in the cell culture supernatants. Our
study further demonstrated that the subunit combined vaccine achieved a
double protective effect against influenza H1N1 A/California/07/2009
virus and SARS-CoV-2 Omicron BA.5.2 variant infection.

\textbf{2. Materials and Methods}

\emph{2.1 Ethics Statement}

Six-week-old specific pathogen-free (SPF) female BALB/c mice were
purchased from Hangzhou Paisiao Biotechnology Co., Ltd., and all mice
were in good health. Animal studies were conducted in strict accordance
with the recommendations of the Institutional Animal Care and Use
Committee (IACUC), and the animal protocols were approved by the Animal
Experimentation Ethics Committee of the School of Medicine, Hangzhou
City University.

\emph{2.2 Proteins, Reagents, Cell Lines and Viruses}

HA, Omicron BA.5 S, and Omicron BA.5 S-RBD proteins were purchased from
SinoBiological. MF59 adjuvant was prepared according to our previously
published paper.\textsuperscript{18} Horseradish Peroxidase
(HRP)-labeled goat anti-mouse IgG and 3,3',5,5'-Tetramethylbenzidine
(TMB) were purchased from Beyotime (Shanghai, China). HRP-labeled goat
anti-mouse IgG\textsubscript{1} and IgG\textsubscript{2a} were purchased
from Abcam (Shanghai, China). VeroE6-TMPRSS2 cells were purchased from
the American Type Culture Collection (ATCC). Dulbecco's Modified Eagle's
Medium (DMEM) was purchased from Meilun (Dalian, China). Fetal bovine
serum (FBS) was purchased from Gibco (New York, USA). Experiments
involving the SARS-CoV-2 Omicron BA.5.2 strain and H1N1
A/California/07/2009 strain were conducted by following the approved
standard operating procedures of the biosafety level 3 facility at the
University of Hong Kong.

\emph{2.3 Animal Vaccination and Sample Collection}

Twenty BALB/c mice were randomly divided into four groups with five mice
in each group. Mice were intramuscularly immunized as follows. Group 1
was immunized with PBS. Group 2 was immunized with 5 \selectlanguage{greek}µ\selectlanguage{english}g HA formulated
with an equal volume of MF59. Group 3 was immunized with 5 \selectlanguage{greek}µ\selectlanguage{english}g SARS-CoV-2
Omicron BA.5 S formulated with an equal volume of MF59. Group 4 was
immunized with 5 \selectlanguage{greek}µ\selectlanguage{english}g HA plus 5 \selectlanguage{greek}µ\selectlanguage{english}g SARS-CoV-2 Omicron BA.5 S formulated
with an equal volume of MF59. As shown in Figure 1, all mice were
immunized three times at two-week intervals. Serum was isolated from the
collected blood one week after each immunization, heat-inactivated at
56\selectlanguage{ngerman}°C for 30 min, and then divided and frozen at -20°C in the
refrigerator for further use.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} . Schematic diagram of immunization strategy and
sample collection. Mice were intramuscularly immunized with PBS, HA,
Omicron S or HA+Omicron S adjuvanted with MF59, respectively, three
times at two-week intervals. Mouse serum samples were collected one week
after each immunization for antibody detection and neutralization assay.

\emph{2.4 Enzyme-linked Immunosorbent Assay (ELISA)}

ELISA was used to detect HA or SARS-CoV-2 Omicron BA.5 S-RBD-specific
IgG, IgG\textsubscript{1} and IgG\textsubscript{2a} antibodies in mouse
sera. ELISA plates were pre-coated with 2 \selectlanguage{greek}µ\selectlanguage{english}g/mL HA or SARS-CoV-2 Omicron
BA.5 S-RBD protein overnight at 4\selectlanguage{ngerman}°C. The plates were washed with PBS
containing 0.05\% Tween-20 (PBST) and then blocked with PBS containing
2\% skimmed milk powder at 37°C for 1 h. After washing the plates with
PBST, serially diluted mouse serum was added into the plates, followed
by incubation at 37°C for 1 h. After again washing with PBST,
HRP-conjugated goat anti-mouse IgG, IgG\textsubscript{1} or
IgG\textsubscript{2a} was added to the plates, followed again by
incubation at 37°C for 1h. After washing, TMB was added to the plates.
Finally, 1N H\textsubscript{2}SO\textsubscript{4} was added to the
plates to terminate the reaction, and the absorbance value was measured
at 450nm by microplate reader. Endpoint titers were expressed as the
highest reciprocal serum dilution exhibiting an absorbance of 450 nm
\textgreater{} 2.1-fold over the background values.

\emph{2.5} \emph{Hemagglutination Inhibition (HAI) Assay}

HAI of immunized mouse sera was performed as previously
described.\textsuperscript{14}~First, serum samples were treated with
receptor-destroying enzymes (Denka Seiken) overnight at 37°C and
heat-inactivated at 56°C for 30 min. Second, 25 \selectlanguage{greek}µ\selectlanguage{english}L of 8 HAU of
A/California/07/2009 (H1N1) virus were added to an equal volume of
two-fold serially diluted mouse sera in wells of a U-bottom 96-well
microtiter plate and incubated for 45 min to 1 h. Finally, 0.55\% turkey
erythrocytes were added into the wells, followed by further incubation
at room temperature for 30 min. HAI titers were defined as the
reciprocal of the highest serum dilution required for complete
hemagglutination inhibition.

\emph{2.6 SARS-CoV-2 Omicron BA.5 Pseudovirus Production and
Neutralization Assay}

Generation of pseudotyped SARS-CoV-2 Omicron BA.5 was performed as
previously described.\textsuperscript{19, 20} Briefly, the backbone
plasmid of pNL4-3.Luc.R-E- was cotransfected with the plasmid of
pcDNA3.1-SARS-CoV-2 Omicron BA.5-S into HEK-293T cells by using VigoFect
transfection reagent (Vigorous Biotechnology, Beijing, China). Cell
culture supernatant containing the pseudoviruses after centrifuging at
3000 rpm for 10 min was harvested 48 h post-transfection. The
pseudotyped viruses were stored at -80 \selectlanguage{ngerman}°C until use. Detection of
neutralizing antibodies against pseudotyped SARS-CoV-2 Omicron BA.5
variant was conducted by neutralization assay. Briefly, mouse sera were
heat-inactivated at 56 °C for 30 min. Huh-7 cells were seeded in wells
of a 96-well cell culture plate at a density of
1x10\textsuperscript{4}/well. Serially diluted mouse sera were incubated
with the pseudotyped SARS-CoV-2 Omicron BA.5 for 30 min. Then, the
mixture of mouse sera and pseudoviruses was transferred to a 96-well
cell culture plate. After 12 h, the cell culture supernatant was
discarded, and cell culturing continued with fresh DMEM containing 5\%
FBS for 48 h. Then, 1X lysis buffer was added to the wells for 30 min,
the lysate was transferred to a 96-well half-area white plate, and
luciferase activity was detected by using a Firefly luciferase assay kit
(Promega, Madison, WI, USA). Luciferase values were measured using a
SpectraMax i3x multi-mode microplate reader (Molecular Devices, USA).
Neutralizing antibody titers (NT\textsubscript{50}) were defined as the
serum dilutions that could reduce 50\% relative luminescence units
compared to virus control wells.

\emph{2.7 SARS-CoV-2 Omicron BA.5.2 Viral Load Reduction Assay}

Viral load reduction assay was performed on VeroE6-TMPRSS2 cells.
Three-fold serially diluted mouse serum samples were preincubated with
1,000 PFU of authentic SARS-CoV-2 Omicron BA.5.2 at 37°C for 1 h.
Following preincubation, a 100 \selectlanguage{greek}μ\selectlanguage{english}L serum-virus mixture was added gently
to VeroE6-TMPRSS2 cells. The inoculated cells were incubated at 37 for
1 h. Afterwards, the infectious inoculum was aspirated, washed and
replaced with fresh medium containing the serially diluted mouse serum.
Forty-eight hours later, supernatant from the infected cells was
harvested for qRT-PCR analysis of viral load. Briefly, 50 \selectlanguage{greek}µ\selectlanguage{english}L of viral
supernatant were lysed with 200 \selectlanguage{greek}µ\selectlanguage{english}L of AVL buffer and then extracted for
total RNA using the QIAamp viral RNA mini kit (Qiagen, Germany).
Real-time one-step qRT-PCR was used for quantitation of viral load using
the QuantiNova Probe RT-PCR kit (Qiagen, Germany) with the LightCycler
480 real-time PCR system (Roche). Each 20 \selectlanguage{greek}µ\selectlanguage{english}L reaction mixture contained
10 \selectlanguage{greek}µ\selectlanguage{english}L of 2 x QuantiNova Probe RT-PCR master mix, 1.2 \selectlanguage{greek}µ\selectlanguage{english}L of RNase-free
water, 0.2 \selectlanguage{greek}µ\selectlanguage{english}L of QuantiNova Probe RT-Mix, 1.6 \selectlanguage{greek}µ\selectlanguage{english}L each of 10 \selectlanguage{greek}µ\selectlanguage{english}M forward
and reverse primer, 0.4 \selectlanguage{greek}µ\selectlanguage{english}L of 10 \selectlanguage{greek}µ\selectlanguage{english}M probe, and 5 \selectlanguage{greek}µ\selectlanguage{english}L of extracted RNA as
the template. Reactions were incubated at 45\selectlanguage{ngerman}°C for 10 min for reverse
transcription and 95°C for 5 min for denaturation, followed by 45 cycles
of 95 °C for 5 s and 55 °C for 30 s. Signal detection and measurement
were performed in each cycle after the annealing step. The cycling
profile ended with a cooling step at 40°C for 30 s. Primers and probe
sequences were against the RNA-dependent RNA polymerase/Helicase
(RdRP/Hel) gene region of SARS-CoV-2 Omicron BA.5.2: forward primer: 5\selectlanguage{english}'
-CGCATACAGTCTTRCAGGCT-3\selectlanguage{english}'; reverse primer:
5\selectlanguage{english}'-GTGTGATGTTGAWATGACATGGTC-3\selectlanguage{english}'; specific probe: 5\selectlanguage{english}'-FAM
TTAAGATGTGGTGCTTGCATACGTAGAC-IABkFQ-3'.

\emph{2.8 Statistical Analysis}

All statistical analyses were performed with GraphPad Prism 8 software.
Statistical significance was calculated by two-way ANOVA. P-values less
than 0.05 were considered statistically significant.

\textbf{3. Results}

3.1 \emph{Influenza/SARS-CoV-2 Omicron Subunit Combined Vaccine Induced
High Levels of IgG Antibody Titers}

BALB/c mice were intramuscularly immunized with PBS, HA, SARS-CoV-2
Omicron BA.5 S or HA+SARS-CoV-2 Omicron BA.5 S adjuvanted with MF59,
respectively, three times at two-week intervals. Serum samples were
collected one week after each immunization for HA or SARS-CoV-2 Omicron
BA.5 S-RBD-specific IgG detection. Based on mouse sera collected on day
21, one week after the first boost immunization, as shown in Figure 2A,
a high level of HA-specific IgG was observed in mice immunized with HA
with no significant difference between the HA and HA+SARS-CoV-2 Omicron
BA.5 S immunized groups. The level of IgG was significantly increased on
day 35, one week after the second boost immunization, in mice immunized
with HA and HA+SARS-CoV-2 Omicron BA.5 S, respectively. Similarly, based
on mouse sera collected on day 21, as shown in Figure 2B, a high level
of SARS-CoV-2 Omicron BA.5 S-RBD-specific IgG was observed in mice
immunized with SARS-CoV-2 Omicron BA.5 S with no significant difference
between the SARS-CoV-2 Omicron BA.5 S and HA+SARS-CoV-2 Omicron BA.5 S
immunized groups. The level of IgG was significantly increased on day 35
in mice immunized with SARS-CoV-2 Omicron BA.5 S and HA+SARS-CoV-2
Omicron BA.5 S, respectively. As shown in Figure 2C and 2D, mouse sera
collected on day 35 showed good binding ability with coated HA and
Omicron S-RBD proteins.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} . HA-specific IgG and Omicron BA.5 S-RBD-specific IgG
detection in immunized mouse sera. (A) HA-specific IgG antibody titers
in the serum of mice in each group on days 21 and 35 antibody titers;
(B) Omicron BA.5 S-RBD-specific IgG in serum of mice in each group on
days 21 and 35 antibody titers. (C) Curve of HA-specific IgG in
immunized mice on day 35. (D) Curve of Omicron BA.5 S-RBD-specific IgG
in immunized mice on day 35. Geometric mean was calculated for each set
of data, as shown, and compared. Statistical significance was determined
by two-way ANOVA and is indicated as follows: \emph{** p} \textless{}
0.01, \emph{*** p} \textless{} 0.001,\emph{**** p} \textless{} 0.0001.
Error bars represent SD.

3.2

\emph{Influenza/SARS-CoV-2 Omicron Subunit Combined Vaccine Induced High
Levels of IgG\textsubscript{1} and IgG\textsubscript{2a} Antibodies in
Mice}

Endpoint titers of HA or SARS-CoV-2 Omicron BA.5 S-RBD-specific
IgG\textsubscript{1} and IgG\textsubscript{2a} in immunized mouse sera
on day 21 and 35 were also detected by ELISA. As shown in Figure 3A, a
quite high level of HA-specific IgG\textsubscript{1} was observed in
mice immunized with HA with no significant difference between the HA and
HA+SARS-CoV-2 Omicron BA.5 S immunized groups. The level of IgG1 was
significantly increased on day 35 in mice immunized with HA and
HA+SARS-CoV-2 Omicron BA.5 S, respectively. As shown in Figure 3B, a
high level of SARS-CoV-2 Omicron BA.5 S-RBD-specific
IgG\textsubscript{1} was observed in mice immunized with SARS-CoV-2
Omicron BA.5 S. The level of IgG\textsubscript{1} was significantly
increased on day 35 in mice immunized with SARS-CoV-2 Omicron BA.5 S and
HA+SARS-CoV-2 Omicron BA.5 S, respectively. As shown in Figure 2C and
2D, HA or SARS-CoV-2 Omicron BA.5 S-RBD-specific IgG\textsubscript{2a}in
immunized mouse sera on day 21 was detected. Moreover, the level of
IgG\textsubscript{2a} was significantly increased on day 35 in mice
immunized with HA and SARS-CoV-2 Omicron BA.5 S, respectively.
Importantly, the influenza/SARS-CoV-2 Omicron subunit combined vaccine
induced balanced Th\textsubscript{1}/Th\textsubscript{2} cellular immune
response in BALB/c mice.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} . HA-specific IgG1, IgG2a and Omicron BA.5
S-RBD-specific IgG1, IgG2a detection in immunized mouse sera.
HA-specific IgG\textsubscript{1}(A) and IgG\textsubscript{2a}(C)
endpoint titers on day 21 and day 35 in immunized mouse sera; Omicron
BA.5 S-RBD-specific IgG\textsubscript{1}(B) and IgG\textsubscript{2a}(D)
endpoint titers on day 21 and day 35 in immunized mouse sera.
Statistical significance was determined by two-way ANOVA and is
indicated as follows: \emph{* p}\textless{} 0.05, \emph{** p}
\textless{} 0.01, \emph{*** p} \textless{} 0.001, \emph{**** p}
\textless{} 0.0001. Error bars represent SD.

3.3\emph{Influenza/SARS-CoV-2 Omicron Subunit Combined Vaccine Induced
High Levels of H1N1 A/California/07/2009 Virus-specific
Hemagglutination-inhibiting Antibodies in Mice}

The H1N1 A/California/07/2009 virus-specific hemagglutination-inhibiting
antibody titers in immunized mouse sera on day 21 and 35 were detected
by HAI assay. As shown in Figure 4, a quite high level of H1N1
A/California/07/2009 virus-specific HAI titer was detected in mice
immunized with HA with no significant difference between the HA and
HA+SARS-CoV-2 Omicron BA.5 S immunized groups. HAI titer was
significantly increased on day 35 in mice immunized with HA, again with
no significant difference between the HA and HA+SARS-CoV-2 Omicron BA.5
S immunized groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} . H1N1 A/California/07/2009 virus-specific
hemagglutination-inhibiting antibodies in immunized mouse sera on days
21 and 35. Statistical significance was determined by two-way ANOVA and
is indicated as follows: \emph{* p} \textless{} 0.05, \emph{**
p}\textless{} 0.01.

\emph{\textbf{3.4} Influenza/SARS-CoV-2 Omicron Subunit Combined Vaccine
Induced High Titers of Neutralizing Antibodies against SARS-CoV-2
Omicron BA.5 Pseudovirus and Effectively Reduced the Viral Load of
Authentic SARS-CoV-2 Omicron BA.5.2 in the Cell Culture Supernatants}

We next detected the neutralizing antibodies of immunized mouse sera
collected on days 21 and 35 against pseudotyped SARS-CoV-2 Omicron BA.5
variant. As shown in Figure 5A, Omicron S and HA+Omicron S immunized
mouse sera collected on day 21 elicited high titers of Omicron
BA.5-specific neutralizing antibodies (2577 and 1977, respectively). The
neutralizing antibody titers in the sera of mice immunized with Omicron
S or HA+Omicron S collected on day 35 increased significantly (8924 and
6679, respectively) (Figure 5B). We further detected the antiviral
effect of mouse sera collected on day 35 against SARS-CoV-2 Omicron
BA.5.2 strain on VeroE6-TMPRSS2 cells. As shown in Figure 5C, both
Omicron S and HA+Omicron S immunized mouse sera effectively reduced the
viral load of SARS-CoV-2 Omicron BA.5.2 in cell culture supernatants on
day 35, and such reduction was dose-dependent.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} . Neutralization assay against pseudotyped SARS-CoV-2
Omicron BA.5 and viral load reduction assay in immunized mouse sera on
day 35 against authentic SARS-CoV-2 Omicron BA.5.2. Neutralization assay
was conducted by vaccinated mouse sera on days 21 and 35 against
pseudotyped SARS-CoV-2 Omicron BA.5. VeroE6-TMPRSS2 cells were used for
authentic viral infection, and virus copies in the cell culture
supernatants were quantified by real-time one-step qRT-PCR. (A)
Neutralizing antibodies against SARS-CoV-2 Omicron BA.5 pseudovirus by
mouse sera collected on day 21. (B) Neutralizing antibodies against
SARS-CoV-2 Omicron BA.5 pseudovirus by mouse sera collected on day 35.
(C) Viral load reduction assay on SARS-CoV-2 Omicron BA.5.2 by mouse
sera collected on day 35. Error bars represent SD.

\textbf{4. Discussion}

The COVID-19 pandemic caused by SARS-CoV-2 and its variants continues to
pose a threat to human health globally. Although vaccination brought the
pandemic under control to some extent, the large number of breakthrough
infection cases may require annual COVID-19 vaccination. IAV is a common
respiratory infectious virus that causes severe respiratory illnesses
worldwide. WHO updates vaccines to prevent seasonal influenza epidemics
every year. It was reported that the coinfection rate of SARS-CoV-2 and
IAV during the COVID-19 pandemic was as high
as~49.8\%.\textsuperscript{21} To date, no clinically effective
prophylactics are available for the prevention of both SARS-CoV-2 and
IAV infections, highlighting a need for the development of combined
vaccines. The production of a combined influenza/SARS-CoV-2 Omicron
vaccine capable of protecting from both IAV- and SARS-CoV-2-induced
disease may reduce the cost, inconvenience, and administration burden of
multiple independent vaccines. In our study, the immunogenicity and
protective effect of the influenza/SARS-CoV-2 Omicron subunit combined
vaccine adjuvanted with MF59 were evaluated in BALB/c mice. Data showed
that the combined vaccine induced high levels of IgG,
IgG\textsubscript{1}, and IgG\textsubscript{2a} antibodies, as well as
influenza A H1N1/California/2009 virus-specific
hemagglutination-inhibiting antibodies in BALB/c mice. Furthermore, the
subunit combined vaccine induced high titers of neutralizing antibodies
against SARS-CoV-2 Omicron BA.5 pseudovirus infection and effectively
reduced the viral load of authentic SARS-CoV-2 Omicron BA.5.2 variant in
the cell culture supernatants.

In conclusion, our study educed evidence showing the
influenza/SARS-CoV-2 Omicron subunit combined vaccine to be a potential
interventional candidate against COVID-19 and pandemic influenza
coinfection, thus deserving of further translational development.

\textbf{Author Contributions:} N.Z., H.H., S.J., and J.Z. conceived the
idea and designed the experiments. Z.Y., C.L., J.Z., W.X., L.X., Y.C.,
R.Y. and J.D. performed the experiments. N.Z. and Z.Y. analyzed the
data. N.Z. wrote the draft, and J.Z., S.J. and H.H. revised the
manuscript. All authors have read and agreed to the published version of
the manuscript.

\textbf{Funding:} This work was supported by the Scientific Research
Foundation (grant No. J-202106) and Innovation Training Program (grant
No. X202301126) of Hangzhou City University to N.Z.

\textbf{Conflicts of Interest:} The authors declare no conflict of
interest.

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