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\begin{document}

\title{Permutation of M2e affects the effectiveness of universal influenza
nanovaccine}



\author[1]{Peiyang Ding}%
\author[2]{Gaiping Zhang}%
\author[1]{Yumei Chen}%
\author[1]{Hongliang Liu}%
\author[3]{Yunchao Liu}%
\author[2]{Rui Jia}%
\author[1]{Yanwei Wang}%
\author[1]{Ge Li}%
\author[2]{Aiping Wang}%
\affil[1]{Affiliation not available}%
\affil[2]{Zhengzhou University}%
\affil[3]{Henan Acad Agr Sci}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Influenza A virus (IAV), a deadly zoonotic pathogen, occasionally
cross-species transmission among humans, swine and avian. The ectodomain
of matrix protein 2 (M2e) is highly conserved in IAV and has been widely
concerned in the development of universal influenza vaccines. Due to low
immunogenicity, multi-copy M2e are usually displayed on the surface of
nanoparticles to constitute universal nanovaccines. Here, we report that
the permutation of the M2e affects the immune effect of the nanovaccine.
Three M2e derived from humans, swine and avian IAV were inserted into
the C-terminal of the Cap protein of porcine circovirus type 2 (PCV2) to
form self-assembled nanovaccine. Immunoprotective effects of different
M2e arrangements were explored in mice. Results showed that the M2e
closest to the surface of nanoparticle induced the most efficient
protection against IAV derived from corresponding species. The results
will help in the development of more effective universal influenza
vaccines, especially for specific species.%
\end{abstract}%



\sloppy


Permutation of M2e affects the effectiveness of universal influenza
nanovaccine

Running title: Universial influenza nanovaccines

Peiyang Ding\textsuperscript{1}, Gaping Zhang\textsuperscript{1, 2},
Yumei Chen\textsuperscript{1}, Hongliang Liu\textsuperscript{1, 2},
Yunchao Liu\textsuperscript{2}, Rui Jia\textsuperscript{2}, Yanwei
Wang\textsuperscript{1, 2}, Ge Li\textsuperscript{2}, Aiping
Wang\textsuperscript{1*}

\textsuperscript{1}School of Life Sciences, Zhengzhou University,
Zhengzhou 450001, China

\textsuperscript{2}Henan Zhongze Biological Engineering Co., Ltd.,
Zhengzhou 450002, China

*To whom correspondence should be addressed:

Aiping Wang E-mail: \emph{pingaw@126.com}

Tel.: +86-037167739345; Fax: +86-037163558998

Address: School of Life Sciences, Zhengzhou University, Zhengzhou
450001, China

\textbf{Summary}

Influenza A virus (IAV), a deadly zoonotic pathogen, occasionally
cross-species transmission among humans, swine and avian. The ectodomain
of matrix protein 2 (M2e) is highly conserved in IAV and has been widely
concerned in the development of universal influenza vaccines. Due to low
immunogenicity, multi-copy M2e are usually displayed on the surface of
nanoparticles to constitute universal nanovaccines. Here, we report that
the permutation of the M2e affects the immune effect of the nanovaccine.
Three M2e derived from humans, swine and avian IAV were inserted into
the C-terminal of the Cap protein of porcine circovirus type 2 (PCV2) to
form self-assembled nanovaccine. Immunoprotective effects of different
M2e arrangements were explored in mice. Results showed that the M2e
closest to the surface of nanoparticle induced the most efficient
protection against IAV derived from corresponding species. The results
will help in the development of more effective universal influenza
vaccines, especially for specific species.

\textbf{Keywords:} influenza A virus; porcine circovirus type 2; M2e;
universal; nanovaccine; permutation

\textbf{1 Introduction}

Influenza A virus (IAV) causes huge economic loss to the global
husbandry industry and poses a significant threat to public health. The
IAV genome is composed of eight single-stranded negative-sense RNA
fragments, resulting in abnormally high frequency of gene mutations and
recombination, which bring great difficulties to the development of
universal vaccines (Lowen, 2017). IAV occasionally cross species
boundaries and pose lethal threats to other species (Long, Mistry,
Haslam, \& Barclay, 2019). In particular, cross-species transmissions
between humans, swine and birds are more frequent, such as 2009 pandemic
H1N1 swine influenza, highly pathogenic H5N1 and H7N9 avian influenza
jumped into humans, causing great panic (Gao, 2018). Therefore, it is
urgent to establish IAV universal vaccines.

Ectodomain of matrix protein 2 (M2e) is the most conservative and
protective viral antigen and can generate hetero-subtype immunity
against multiple virus strains and subtypes. However, M2e is difficult
to be recognized by the immune system due to the low molecular weight,
the low abundance and the steric blocking by HA and NA on the surface of
IAV (Kolpe, Schepens, Fiers, \& Saelens, 2017). Various effective
strategies for improving M2e antibody levels have been proposed, in
particular, some nanoparticle-based nanovaccines show exciting immune
effects, such as ferritin, virus-like particles (VLPs), and gold
nanoparticles(Kolpe et al., 2017). To further improve the level of
M2e-specific antibodies, nanoparticles usually display multi-copy of
M2e. Tandem expression of M2e from human, swine and avian IAV are the
most common pattern (Deng, Chang, et al., 2018; Deng, Mohan, et al.,
2018; Ding, Jin, Chen, et al., 2019; K. H. Kim et al., 2018; M. C. Kim,
Lee, et al., 2013; M. C. Kim et al., 2015; M. C. Kim, Song, et al.,
2013; Petukhova et al., 2013; Qi et al., 2018; Y. Wang et al., 2020;
Yong, Yeap, Ho, Omar, \& Tan, 2015). However, due to the uncertainty of
the crystal structure of M2e and immune systems tend to efficiently
recognize protruding domains on the surface of nanoparticles, it is
worth investigating whether the permutation of M2e of IAV from different
species has an impact on the immune effect of nanovaccines.

In this research, we displayed the M2e derived from IAV of humans, swine
and avian in different order (six orders) at the C-terminal of porcine
circovirus type 2 (PCV2) Cap VLPs to explore the effect of different M2e
arrangement on the immune effect of the universal vaccine. Results
showed that the M2e closest to surface of Cap VLPs induced the highest
M2e-specific antibodies and conferred the best protection against IAV of
corresponding species. This result will help to develop more efficient
universal influenza vaccines.

\textbf{2 Methods}

\section*{2.1 Expression and purification of recombinant
proteins}

{\label{expression-and-purification-of-recombinant-proteins}}

Triple M2e peptides derived from human (hM2e), swine (sM2e) and avian
(aM2e) IAV were combined with the C-terminus of Cap protein in different
arrangements using Gly-Gly-Gly-Gly linker. (Figure 1a-1c). The cysteine
of M2e were mutated to serine. These sequences were inserted into the
pET28a vector by BamHI and HindIII digestion enzymes, and transformed
into \emph{E. coli} BL21 (DE3). Then these transformed cells were
induced expression at 20\selectlanguage{ngerman}°C for 15 h by isopropyl-\selectlanguage{greek}β-\selectlanguage{english}d-thiogalactoside
(IPTG) (0.2 mM). These recombinant proteins were purified by using
Ni-NTA His\selectlanguage{ngerman}·Bind Resin and identified by SDS-PAGE and Western blot. The
concentrations of these purified proteins were determined with a BCA
protein assay kit. The endotoxin concentrations were measured by
ToxinSensor Single Tests Kit.

\section*{2.2 Particle characteristics of these Cap-3M2e
VLPs}

{\label{particle-characteristics-of-these-cap-3m2e-vlps}}

These Cap-3M2e proteins were dialyzed into the assembly buffer (10 mM
Tris-HCl, 100 mM NaCl (pH 8.0)) and formed VLPs. The shape, size, size
distribution and zeta potential of these Cap-3M2e VLPs were
characterized by transmission electron microscopy (TEM) and dynamic
light scattering (DLS).

\section*{2.3 Immunization and
challenge}

{\label{immunization-and-challenge}}

Female BALB/c mice (6-8 weeks old) were randomized into groups. Six
groups were subcutaneously immunized with 26.6 \selectlanguage{greek}µ\selectlanguage{english}g (100 \selectlanguage{greek}µ\selectlanguage{english}L) candidate
vaccines, respectively. The Cap VLPs group (20 \selectlanguage{greek}µ\selectlanguage{english}g, Cap molar equivalent)
is serves as positive control. The Carbopol 971P as adjuvant (Ding et
al., 2017). Booster immunization was performed at the 21 and 42 days
post initial immunization (dpi). Serum samples were collected at 63 dpi
and stored at -20 \selectlanguage{ngerman}°C until use. Mice in each group were divided into
three groups (n=9). Mice were lightly anesthetized and intranasally
challenged with lethal dose humans, swine and avian IAV at 64 dpi,
respectively. Survival rates and body weight loss were monitored daily
for 14 days post challenge (dpc). Mice were humanely euthanized if
weight loss of \selectlanguage{english}[?] 25\%.

\section*{2.4 Antibody titer detection}

{\label{antibody-titer-detection}}

M2e- and PCV2-specific antibodies in sera were determined by indirect
ELISA using 1 \selectlanguage{greek}μ\selectlanguage{english}g/mL of synthesized M2e peptides as coating antigens.
Twofold serially diluted immune serum samples were added into the ELISA
plates, followed by HRP-conjugated goat anti-mouse IgG. The highest
dilution which showing over twofold OD450 readout than that of the
control sample as the antibody endpoint titer. For the detection of
PCV2-specific antibodies, commercial PCV2 antibody test kit (BioChek,
Reeuwijk, Holland) was utilized.

\textbf{2.5 Determination of IAV titers}

Four mice from each group were sacrificed at the time when the virus
titers were highest in the lungs. The MDCK cell-based immunoperoxidase
monolayer assay was used for lung virus titration as described elsewhere
(Ding, Jin, Zhou, et al., 2019)\textbf{.}

\section*{2.6 Ethics Statement}

{\label{ethics-statement}}

All BALB/c mice received humane care and all animal procedures in this
study were performed in accordance with the Institutional Animal Care
and Use Committee (IACUC).

\section*{2.7 Statistical analysis}

{\label{statistical-analysis}}

All statistics were performed using GraphPad Prism (ver 7.0) (GraphPad
Software, San Diego, CA, USA). Quantitative data are reported as means \selectlanguage{ngerman}±
SEM. P values were determined by one-way ANOVA analysis. Statistical
significance was determined at p \textless{} 0.05 (*).

\textbf{3 Results}

\textbf{3.1 Characteristics of Cap-3M2e VLPs}

Six different recombinant Cap-3M2e proteins were expressed in \emph{E.
coli} and purified using Ni\textsubscript{2}\textsuperscript{+}-NTA
column. SDS-PAGE and Western blot showed that these recombinant Cap-3M2e
proteins were successfully expressed and purified (Figure 1d). These
recombinant Cap-3M2e proteins can be recognized well with the anti-PCV2
polyclonal antibody and 14C2 monoclonal antibody (Anti-IAV M2 protein),
indicating that these recombinant proteins retained the reactogenicity
of M2e and the Cap protein (Figure S1). Endotoxin levels in these
recombinant proteins were less than 0.18 EU/mg.

TEM images showed that these recombinant Cap-3M2e proteins could
self-assemble into VLPs (Figure 1e and Figure S2). DLS test results
showed that the diameter of these Cap-3M2e VLPs were larger than that of
Cap VLPs (Figure 1f). The zeta potential of these Cap-3M2e VLPs were
lower than that of Cap VLPs, which were caused by the strong negative
charge of M2e molecules (Figure 1g). The particle size and surface
charge of these Cap-3M2e VLPs are consistent, indicating that the
permutation of M2e of IAV from different species does not affect the
morphology and surface charge of these VLPs (Figure 1f and 1g).

\textbf{3.2 Humoral immune effects}

All Cap-3M2e VLPs groups induced high levels of total M2e-specific
antibodies (Figure S3a). There were significant differences in levels of
antibodies against M2e derived from human, swine and avian IAV in sera
of each group. The Cap-hsaM2e VLPs and Cap-hasM2e VLPs groups induced
higher anti-human IAV M2e antibodies than others (Figure 2a). Anti-human
IAV M2e antibody in the Cap-hasM2e VLPs group was 7.3 times that of the
Cap-sahM2e VLPs and Cap-ashM2e VLPs groups (Figure 2a). The Cap-shaM2e
VLPs and Cap-sahM2e VLPs groups induced higher anti-swine IAV M2e
antibodies than other groups (Figure 2b). Anti-swine IAV M2e antibody in
the Cap-shaM2e VLPs group was 6.4 times that of the Cap-ashM2e VLPs
group (Figure 2b). Meanwhile, anti-avian IAV M2e antibodies in the
Cap-ahsM2e VLPs and Cap-ashM2e VLPs were the highest (Figure 2c). The
anti-avian IAV M2e antibody in Cap-ahsM2e VLPs group was 8 times that of
the Cap-sahM2e VLPs group (Figure 2c). However, the PCV2-specific
antibodies in all groups were consistent, indicating that the insertion
of three copies of M2e in different orders did not affect the
immunogenicity of the Cap VLPs (Figure S3b).

\section*{3.3 Protective efficacy of Cap-3M2e VLPs against
IAV}

{\label{protective-efficacy-of-cap-3m2e-vlps-against-iav}}

All mice immunized with Cap VLPs died by 4 to 6 dpc with the highest
virus titers in lungs and over 25\% body weight loss (Figure 2d-2l). The
protective efficacy of groups Cap-hsaM2e VLPs and Cap-hasM2e VLPs were
the highest among all groups, and protected all mice from lethal
infection when challenged with 10 × LD\textsubscript{50} A/Puerto
Rico/8/1934 (H1N1) (Figure 2d-2f). Similarly, the Cap-shaM2e VLPs and
Cap-sahM2e VLPs groups showed the highest protection efficacy, and
conferred complete protection against 10 × LD\textsubscript{50} of
A/swine/Zhucheng/90/2014 (H1N1) (Figure 2g-2i). The Cap-ahsM2e VLPs and
Cap-ashM2e VLPs groups demonstrated the lowest body weight loss,
mortality rate and lung virus titers among all groups, when challenged
with 40 \selectlanguage{greek}µ\selectlanguage{english}L 10\textsuperscript{9} TCID\textsubscript{50}/mL of
A/chicken/Guangzhou/GZ/2005 (H9N2) (Figure 2j-2l).

\section*{4 Discussion}

{\label{discussion}}

The M2e is a highly conserved candidate epitope in different subtypes of
IAV and offers potential to develop universal vaccines, if it can be
appropriately presented and sensed by host immune system. Displaying
multiple copies of M2e molecules on the surface of nanoparticles is an
effective means to increase the level of anti-M2e antibodies. Although
M2e is conserved among IAV, there are still some differences among
strains. Particularly, M2e derived from different species varies
considerably (Schepens, De Vlieger, \& Saelens, 2018). Therefore,
nanoparticles usually display M2e of human, swine and avian IAV in
tandem to increase the broad spectrum.

Affinity maturation of antibodies requires stable and full display of
epitopes rather than transient state (Dormitzer, Ulmer, \& Rappuoli,
2008). However, M2e adopt at least two transformed conformations (Cho et
al., 2016; Cho et al., 2015). In addition, due to the existence of
flexible links between different M2e, the instability of M2e
conformation is exacerbated. Therefore, the relatively stable one in the
multiple copies of M2e is more easily recognized by the immune system
and eventually induces higher levels of antibodies.

VLPs, as a kind of nanoparticles with precisely defined
three-dimensional structure, provide a large number of sites for M2e
that can be accurately inserted (Rodriguez-Limas, Sekar, \& Tyo, 2013).
The N-terminal, C-terminal and loops are usually the prominent sites of
VLPs suitable for insertion into M2e. However, loops are generally
flexible structures and can only tolerate peptides of limited length (D.
Wang et al., 2018). In this study, the C-terminal of Cap protein
protrudes from the surface of Cap VLPs, and participates in the
formation of linear and conformational neutralization epitopes,
indicating that the C-terminus can be efficiently recognized by immune
system (Khayat et al., 2011; Lekcharoensuk et al., 2004). Therefore, it
can be utilized as an insertion site for multiple copies of M2e. Results
showed that the M2e which near the C-terminal induced higher levels of
species-specific anti-M2e antibodies. It was because the immune system
efficiently recognized the C-terminal of Cap protein, thereby
efficiently recognizing adjacent M2e. The level of species-specific
anti-M2e antibodies induced by two M2e that away from the C-terminal are
low and there no significant difference. This is due to the indefinite
spatial conformation of M2e and the wobble of the flexible link leading
to the decline of the immune system's recognition ability.

Therefore, it is necessary to selectively display M2e of IAV of
species-specific in the most prominent and relatively fixed position of
nanoparticles based the immune target of universal IAV vaccines, so as
to induce a more efficient immune effect. For example, when chickens
inject with universal IAV nanovaccines, M2e of the avian IAV need to be
displayed in a prominent and stable position on nanoparticle.

\textbf{Conclusion}

In this study, we studied the effect of the permutation of three M2e
peptides derived from human, swine and avian IAV on the surface of PCV2
VLPs on the immune effect of IAV universal vaccine. Results demonstrate
that the M2e peptide closest to the surface of the nanoparticle induced
the highest immune protection against this species of IAV. This result
suggests that the M2e of IAV of a specific host should be stably
displayed when designing universal nanovaccines.

\section*{Acknowledgments}

{\label{acknowledgments}}

This study was supported by the 1125 Talent Recruitment Program of
Zhengzhou.

\textbf{Conflict of interest}

The authors report no conflicts of interest are associated with this
work.

\textbf{Data availability statement}

Data that support the findings of this study are available from the
corresponding author upon reasonable request.

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Yong, C. Y., Yeap, S. K., Ho, K. L., Omar, A. R., \& Tan, W. S. (2015).
Potential recombinant vaccine against influenza A virus based on M2e
displayed on nodaviral capsid nanoparticles. \emph{Int J Nanomedicine,
10} , 2751-2763. doi:10.2147/IJN.S77405

\section*{Figure 1 Characterization of these Cap-3M2e
VLPs.}

{\label{figure-1-characterization-of-these-cap-3m2e-vlps.}}

\textbf{(a)} Primary pattern structure of six recombinant Cap-3M2e. (b)
M2e sequence of the human, swine and avian IAV which were used in
challenging experiment. (c) Schematic illustration of Cap-3M2e VLPs
nanovaccine. The VLPs model was derived from the PDB database (3R0R).
(d) SDS-PAGE analysis of these purified Cap-3M2e proteins. Lane M:
molecular weight markers; lane 1: Cap; lane 2: Cap-hsaM2e; lane 3:
Cap-hasM2e; lane 4: Cap-shaM2e; lane 5: Cap-sahM2e; lane 6: Cap-ahsM2e;
lane 7: Cap-ashM2e. (e) Transmission electron micrograph (TEM) of
Cap-3M2e VLPs. The image is part of the TEM of Cap-ashM2e VLPs. Scale
bars = 50 nm. (f) Size distribution of these Cap-3M2e VLPs (n=5). (g)
Zeta potential of these Cap-3M2e VLPs (n=5).

\section*{Figure 2 Protective efficacy of these Cap-3M2e VLPs against
IAV.}

{\label{figure-2-protective-efficacy-of-these-cap-3m2e-vlps-against-iav.}}

(a) Human IAV M2e-specific IgG level (n=6). (b) Swine IAV M2e-specific
IgG level (n=6). (c) Avian IAV M2e-specific IgG level (n=6). (d) Mice
body weight changes (n=5), (e) survival rate (n=5) and (f) lung virus
titers (n=4) post 10 \selectlanguage{ngerman}× LD\textsubscript{50} of A/Puerto Rico/8/1934
(H1N1) challenge. (g) Mice body weight changes (n=5), (h) survival rate
(n=5) and (i) lung virus titers (n=4) post 10 × LD\textsubscript{50} of
A/swine/Zhucheng/90/2014 (H1N1) challenge. (j) Mice body weight changes
(n=5), (k) survival rate (n=5) and (l) lung virus titers (n=4) post 40
\selectlanguage{greek}µ\selectlanguage{english}L 10\textsuperscript{9} TCID\textsubscript{50}/mL of
A/chicken/Guangzhou/GZ/2005 (H9N2) challenge. p \textless{} 0.05 (*).\selectlanguage{english}
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