Abstract
Virus-like
particle (VLP), a self-assembled multiprotein structure, can stimulate
robust immune responses due to its structure similar to native virions
that curries multiple copies of the target epitopes. Utilizing VLPs as
vaccine platforms to present exogenous antigens is a promising and
challenging approach in the vaccine development field. This study aims
to investigate the potential of hepatitis E virus (HEV) truncated capsid
as a VLP platform to present foreign antigens. The S and M domains of
HEV capsid protein were selected as the optimal carrier (CaSM). The
exogenous antigen Seq8 containing three neutralizing epitopes from three
different foot-and-mouth disease virus (FMDV) strains was linked to the
C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The
construct was successfully expressed and purified. Morphological
analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM
VLP (~26 nm in diameter) but smaller than native HEV
virions. Further, the thermal stability and the resistance to enzymatic
proteolysis of Seq8 were enhanced when it was attached to CaSM carrier.
The antigenicity analysis revealed a more robust reactivity against
anti-FMDV antibodies when Seq8 was presented on the CaSM particles. Upon
injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared
earlier, increased faster, and maintained higher levels for a longer
time than those induced by Seq8 alone or the inactivated FMDV vaccine.
This study demonstrated the potential of utilizing HEV truncated capsid
as an antigen-presenting platform for the development of chimeric VLP
vaccines.
Keywords: Hepatitis E virus; exogenous antigen carrier;
Virus-like particle; Immunogenicity;
Introduction
A virus-like particle (VLP) is a vacant self-assembled multiprotein
structure that does not contain any viral genome 1,2.
VLPs can stimulate robust humoral and cellular immune responses because
of their relatively large size similar to native virions and the
presence of multiple copies of the target epitopes3-5. Given these unique characteristics, utilizing
VLPs as platforms for the presentation of exogenous antigens becomes a
promising approach for the development of effective recombinant vaccines4,6.
Hepatitis E virus (HEV) is an icosahedral spherical virus, with a
diameter of 27-32nm, belonging to the Orthohepevirus genus of theHepeviridae family 7,8. The HEV capsid protein
(encoded by the ORF2) contains three domains named: S domain (residues
129-319), M domain (residues 320-455), and P domain
(residues
456-606). These three domains play different structural and functional
roles in the HEV capsid 9. The S and M domains form an
internal scaffold shell connected to the P domain through a long
proline-rich hinge. This latter allows the P domain to protrude from the
surface of the HEV particles with a certain flexibility for a better
exposition of the dominant neutralization epitopes and the motifs
critical for the interaction with the host cell receptors. Therefore,
such a structural assembly allows an optimal exposure of the functional
P domains as protruding spikes 9,10. Previously, it
has been reported that a truncated HEV ORF2 protein (aa112-660)
expressed in insects cells could form VLPs similar to the native HEV
virions11. Further, the truncated HEV capsid protein
also self-assembled into VLPs even with the N-terminal truncation up to
residue 125 and the C-terminal truncation up to residue 602, indicating
that the essential elements for the self-assembly were located between
residues 125-602 12 and the S domain was indispensable
for the formation of T=1 partciles 13. However, later
on, an N-terminal truncation up to aa367 has been reported as the
largest truncation that maintained the self-assembly ability as it has
been shown with the recombinant vaccine p239 13,14.
Considering the structural features and the ability of truncated HEV
capsid proteins to maintain the self-assembly property, we investigated
the use of the S and M domains as an antigen-presenting carrier (CaSM)
by replacing the protruding P domain with an exogenous antigens.
Several antigen-presenting platforms, based on the HEV capsid protein,
were previously designed by inserting foreign antigens at different
positions: (1) inserting an 18 amino acids peptide of HIV-1 gp120 into
the HEV capsid between residue 485 and 486 6; (2)
inserting a myc-tag/FLAG-tag/HA-tag between residue 488 and residue 489
to present multiple foreign antigens 15; (3) adding a
B cell epitope tag consisting 11 amino acids to the C-terminal16; (4) linking hepatitis A virus VP1 (aa24–171) to
the C-terminal of the P domain to make a bivalent vaccine17; (5) fusing HEV ORF3 p70-123 to ORF2 p112-60818. However, in such designs, the inserted fragments
were mere linear epitopes or too short which could not evoke potent
immune responses, and some epitopes were attached to the C-terminal of
the P domain, which may restrict the flexibility and exposure of the
exogenous antigens and/or the P domain 19. Therefore,
in this study, we aimed to overcome these drawbacks by selecting a large
exogenous antigen, substituting
the
entire P domain, and linking it to the M domain through the HEV
intrinsic proline-rich hinge.
Foot-and-mouth disease virus (FMDV), belonging to the genusAphthovirus of the family Picornaviridae , is the causative
pathogen of foot-and mouth disease (FMD), an acute and contagious
disease of clove-hoofed animals with devastating economic repercussions20,21. The genome of FMDV encodes a large polyprotein
that can be cleaved into four structural proteins (VP1-4) and several
non-structural proteins (NSP) 21. Seven FMDV serotypes
have been identified: O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3, with the
Serotype O as the predominant type in Asia; this Serotype O can be
further classified into 11 topotypes according to the characteristics of
the VP1 protein 21-23. Previous studies have revealed
that two immunogenic regions of VP1 aa140-160 (G-H loop) and aa200-213
(C-terminal fragment), can elicit neutralizing antibodies24,25. Further studies revealed that although the G-H
loop could elicit neutralizing antibodies, it had low immunogenicity
because of its short length and the lack of T-cell epitopes25. The immunogenicity of the G-H loop could be
enhanced drastically when it was incorporated with glutaraldehyde,
liposome, polyinosinic cytidylic acid, or when linked with hepatitis B
core protein, and such formulations also conferred complete protection
in the immunized animals 26-28.
In the present work, we aimed to assess the truncated HEV capsid protein
as a carrier of exogenous antigens. Such carrier (named CaSM) contains
the S and M domain of the HEV capsid, and the foreign antigen would
substitute the protruding HEV P domain, yielding a construct ‘S domain-M
domain-exogenous antigens’ (CaSM-EAg). As a foreign antigen, we
constructed the FMDV Seq8 antigen by combining three FMDV G-H loops
(aa140-160) obtained from the three most prevalent FMDV/O topotype
strains: O/HN/CHA/09, O/IRN/2010, and O/Mya/98. Then, we investigated
whether the chimeric CaSM-Seq8 could self-assemble into VLPs presenting
the Seq8 antigen on the surface, and whether this combination would
enhance the physical, biological, and immunological characteristics of
the inserted antigen Seq8.
- Materials and methods
- Construction of the target clones
The plasmid pET28-W2-1 containing the gene coding for the ORF2 protein
of HEV W2-1 isolate (GenBank: JQ655734.1) has been synthesized (Gene
Create Co., Wuhan, China). The pET-28-Seq8 plasmid has been previously
designed, synthesized and stored in our lab. The polymerase chain
reactions (PCR) were performed using 2×Taq Master Mix (Vazyme Co.,
Nanjing, China) to amplify the target genes
(NcoI -CaSM-BamHI , BamHI -Seq8-XhoI ,NcoI -CaSM-XhoI , and NcoI -Seq8-XhoI ). The
products NcoI -CaSM-BamHI and BamHI -Seq8-XhoIwere digested by the BamHI restriction enzyme (Thermo Fisher
Scientific Inc, USA) and linearly ligated by T4 DNA Ligase (Thermo
Fisher Scientific Inc, USA) to constructNcoI -CaSM- Seq8-XhoI . Then, the ligated gene was
further amplified by PCR.
Next, all the target genes (NcoI -CaSM-XhoI ,NcoI -Seq8-XhoI , and NcoI -CaSM-Seq8-XhoI )
were digested by NcoI and XhoI endonucleases (Thermo
Fisher Scientific Inc, USA), and inserted into a linearized plasmid
(NcoI -pET28a-XhoI ) to construct the recombinant plasmids:
pET28a-NcoI CaSMXhoI ,
pET28a-NcoI Seq8XhoI , and
pET28a-NcoI CaSM-Seq8XhoI .
Then, the expression constructs were used to transform competent E. coli
BL21 cells (Vazyme Co., Nanjing, China). For each target protein,
several clones were selected and tested by PCR and DNA sequencing to
verify the correct insertions of the target genes.
Protein expression and purification
The clones containing the genes that encode for CaSM, CaSM-Seq8, and
Seq8 were cultured at 37℃ overnight in Luria–Bertani broth (LB)
containing 1mM Kanamycin. Then, the overnight grown bacteria were
diluted in 1000mL of LB (1:100 dilution) and incubated at 37℃ for 2-4h,
until the OD600 reached 0.6. Then, isopropyl
β-D-1-thiogalactopyranoside (IPTG) was injected into the medium (final
concentration: 1mM), and the protein expression was induced for another
3-4h. After centrifugation, the pellets were suspended in a lysis buffer
(50mM NaH2PO4, 300mM NaCl, 10mM
Imidazole, pH 8.0). The cell lysis was enhanced by lysozyme and
deoxyribonuclease (DNase) (Sigma-Aldrich, USA), and the samples were
centrifuged at 120000×g for 20 min at 4°C. Finally, the supernatants and
pellets were aliquoted and analyzed by SDS-PAGE.
The soluble fractions of the proteins were purified using the Ni-NTA
affinity chromatography column (QIAGEN Sciences, MD, USA) according to
the manufacturer’s instructions. Briefly, the columns were equilibrated
with the lysis buffer, loaded with the prepared supernatants; then,
washed with the washing buffer (50 mM
NaH2PO4, 300 mM NaCl, and 30 mM
imidazole, pH=8.0); the target proteins were eluted with the elution
buffer (50 mM NaH2PO4, 300 mM NaCl, and
250 mM imidazole, pH=8.0). The eluted proteins were analyzed by
SDS-PAGE, and their concentrations were determined by Quick Start™
Bradford Protein Assay Kit (Bio-Rad Laboratories, Inc. USA). The purity
of the target proteins was evaluated by analyzing the SDS-PAGE gels
using the Image J software.
Transmission electron microscopy (TEM)
CaSM, CaSM-Seq8, and Seq8 were diluted to 0.1mg/ml and loaded on the
carbon-coated grids for 10min and negatively stained with 2% uranyl
acetate for another 10min. The grids were dehydrated and observed by
Transmission electron microscope F30 (Philips, The Netherlands).
Further, the shape and diameter of the particles on the TEM micrographs
were analyzed using the Image-Pro Plus 6.0 software.
Computational analysis of the oligomerization states of the
target proteins
The Phyre2 server 29 was used to predict the 3D
structures of CaSM, CaSM-Seq8, and Seq8; and the models were refined
using GalaxyWeb Refine server 30 as previously
described 31. Then, GalaxyWeb Homomer server32 was used to predict the oligomerization state and
assembly pattern based on the monomer’s 3D structures. The predicted
models with the highest TM score (structure-based method) or the highest
Docking score (ab initio docking method) were selected as the optimal
models.
Immunomagnetic separation (IMS) of native HEV virions
Feces samples containing native HEV virus (genotype 4, strain NJ703
[GenBank: AY789228]) were stored at -80℃ in our lab33. Feces supernatant was prepared using
phosphate-buffered saline (PBS). Then, HEV virions were purified from
the feces supernatant by IMS using Dynabeads® Protein A (Nanoeast
Bio-Technique Co. Ltd, China). The HEV 5G5 monoclonal antibody was used
to coat the IMS beads34, and 1.25μL Tween-20 and 500μL
feces supernatant were added into the coated IMS beads. After the HEV
virions were captured by 5G5 antibody, the IMS beads were magnetized and
washed using 0.05% Tween-20-PBS. Then, the beads were eluted three
times using 50μL of 100 mM glycine buffer (pH=2.5). The eluent was
centrifuged and supplemented with 50μL Tris buffer (pH=8.0).
Analysis of the thermal stability and the enzymatic
proteolysis of the target proteins
To study the thermal stability of CaSM, CaSM-Seq8, and Seq8, the samples
were diluted to 0.1mg/ml, aliquoted, and stored at different
temperatures (-80℃, 4℃, and 37℃). After 1, 3, 5, 7 days post-incubation,
the samples were examined by SDS-PAGE to determine the degradation rate.
To explore their resistance to protease digestion, 0.1mg/ml of CaSM,
CaSM-Seq8, and Seq8 were mixed with an equal volume of simulated gastric
fluid (diluted HCl 1.64ml, ddH2O 80ml, pepsin 1g, adjust
with ddH2O to 100ml) or simulated intestinal fluid
(KH2PO4 0.68g, trypsin 1g, adjust with
NaOH to pH=6.8, total volume 100ml). Then, the samples were incubated at
37℃ for 2h and analyzed by SDS-PAGE.
Antigenicity analysis
First, the antigenicity of CaSM, CaSM-Seq8, and Seq8 was evaluated by a
computational approach. Since the antigen Seq8 contains three
neutralizing epitopes (three different VP1 G-H loops), the exposure of
these epitopes in CaSM-Seq8 and Seq8 was evaluated by Ellipro server35. This server calculates the Protrusion Index (PI)
of each amino acid in a given protein’s structural model. The PI values
of these epitopes in both CaSM-Seq8 and Seq8 were compared by a paired
parametric t-test. Moreover, the area under the PI curve (AUC) of each
epitope was calculated as an index evaluating the overall protrusion of
each G-H loop as a whole.
Next, to experimentally evaluate the antigenicity of these proteins, the
immunoreactivity of CaSM, CaSM-Seq8, and Seq8 against anti-FMDV specific
antibodies was tested by an indirect enzyme-linked immunosorbent assay
(ELISA) and Western Blotting. Since the G-H loops in the Seq8 antigen
were obtained from O/HN/CHA/09, O/IRN/2010, and O/Mya/98 strains, the
following anti-FMDV specific antibodies were used: (1) sera of pigs
infected with O/Mya/98+O/HN/CHA/09 strains (sharing the same G-H loops
with Seq8; (2) sera of pigs infected with O/JMS/00+O/GX/09-7 strains to
test the cross-reactivity with different topotypes of FMDV/O serotype;
(3) negative sera obtained from FMDV-free pigs; (4) purified anti-FMDV/O
polyclonal antibodies (pAbs), commercially available for the treatment
of FMD. The reactivity difference between the target proteins was
statistically assessed by a two-way ANOVA, followed by Tukey post-hoc
test.
Mice immunization and IgG detection
All animal experiments were performed according to ARRIVE guidelines
(Table. S1) 36 and in strict accordance with ‘Care and
Use of Laboratory Animals of Southeast University’. Welfare evaluations
and interventions were performed before, during, and after the
experiments.
A total of forty 6-week-old female BALB/c mice were purchased from
Comparative Medicine Center of Yangzhou University. The mice were
randomly divided into 4 groups and kept in a pathogen-free environment.
CaSM, CaSM-Seq8, and Seq8 (final amount 10μg) were mixed with ISA206
adjuvant (SEPPIC, France), and all the mice were immunized by
intramuscular injections (group1: CaSM-Seq8+ISA206, group2: Seq8+ISA206,
group3: 150μl/mouse of the commercial FMDV inactivated vaccine as a
positive control group, group4: 150μl/mouse of saline+ISA206 as a
negative control group). The commercial FMDV inactivated vaccine
(O/Mya98/XJ/2010+O/GX/09-7) was bought from Jinyu Baoling Biological
Medicine Company (Huhehaote, China). Blood samples were collected from
the internal iliac vein before immunization and at weeks 2, 4, 6, 8, and
10 post-immunization; and centrifuged at 12000rpm for 20min. Then, the
sera were
pooled
and stored at -80℃. The induced
anti-FMDV-specific
IgGs in the mice sera were detected by an indirect ELISA. Seq8 was used
as a coating antigen and
HRP-conjugated
goat anti-mouse IgG (Thermo Fisher Scientific Inc, USA) was used as a
secondary antibody. The difference in antibody levels was assessed by
two-way ANOVA followed by Tukey post-hoc test.
By the end of the study, the mice were euthanized by inhaling a lethal
dose of carbon dioxide (CO2) in the mice asphyxiation
chamber (CO2 flowing rate: 2.5L/min). The mice
euthanasia protocol was approved by Institutional Animal Care and Use
Committee of Southeast University.
SDS-PAGE and Western Blotting
All samples were mixed with an equal volume of 2×loading buffer (5%
SDS, 20% Glycerol, and 0.002% Bromophenol Blue; 1.4% v/v of
β-mercaptoethanol), heated to 100℃ for 5min, and then, loaded into
10%-15% gradient polyacrylamide gel. After the completion of
electrophoresis, the gel was stained in Coomassie staining solution
(300ml methanol, 100ml acetic acid, 1.25g of Coomassie R-250, adjust
with ddH2O to 1000ml).
For Western blotting, the SDS-PAGE gel without staining was washed with
the transfer buffer (25mM Tris base, 190mM glycine, 20% v/v of
methanol, pH 8.3). Then, the proteins in the gel were transferred onto
the NC membrane at 200mA for 2h in a transferring cassette (Bio-Rad
Laboratories, Inc. USA). Next, the membrane was incubated in a blocking
buffer (20mM Tris, 137mM NaCl, 0.1ml Tween 20, pH=7.6, supplemented with
5% w/v of skim milk powder) at 37℃ for 2h. The primary antibody was
added to the blocking buffer to a final dilution of 1:500, followed by
overnight incubation with shaking at 4℃. The next day, after washing,
the membrane was incubated in the blocking buffer containing 1:2000 of
the HRP-conjugated secondary antibody for 2h at room temperature.
Finally, the reactive bands were developed by the addition of
3,3′-Diaminobenzidine solution.
Indirect enzyme-linked immunosorbent assays
The assays were performed as previously described 37:
(1) 96-well flat-bottom plates were coated with the target antigens
(200ng/well) in 1M Urea-phosphate buffered saline (PBS) at 4℃ overnight;
(2) after washing with PBS supplemented with 0.5% Tween 20 (PBST),
100μl of the primary antibody, serially diluted (1:100, 1: 200, 1: 400,
1: 800, 1: 1600, 1: 3200, 1: 6400, 1:12800 and 1:25600) in 1%
Casein-PBS, was added into the wells and incubated at 37℃ for 1h; (3)
after washing with PBST, the HRP-conjugated secondary antibody diluted
1: 5000 in 100μl 1% Casein-PBS was added, and the plates were incubated
at 37℃ for 1h; (4) after washing with PBST, the plates were developed by
the addition of 100μl of 3,3′,5,5′-Tetramethylbenzidine chromogenic
substrate. (5) The reaction was stopped by adding 50μl 2M
H2SO4 and after a 20 min incubation at
37℃, the absorbance of each well was read at 450/630 nm.
Software and statistical analysis
SnapGene 2.3.2 (GSL Biotech) was used for the design, editing, and
manipulations of the primers and DNA sequences. PyMol 2.0.4 (Version 2.0
Schrödinger, LLC.) was used for the visualization of the protein
structure models. All the figures were prepared using Adobe Photoshop CC
2019 (Adobe Systems Incorporated), Image J 1.52K (NIH, USA), GraphPad
Prism 8 (Graphpad Software, Inc.), and Image Pro Plus 6.0 (Media
Cybernetics, Inc.). Statistical analysis was performed using IBM SPSS
V.24 (International Business Machines
Corp.).
p≥0.05: no significance (ns);
0.01≤p<0.05:
*; 0.001≤p<0.01: **; 0.0001≤p<0.001: ***; p<0.0001: ****.
- Results
- Design and preparation of the target recombinant proteins
The target genes (NcoI -CaSM-XhoI ,NcoI -CaSM-Seq8-XhoI, and NcoI -Seq8-XhoI )
were successfully amplified and inserted into the pET28a vector. Then,
the constructed plasmids were successfully transformed into competent E.
coli BL21 cells. After the selection of the target clones and DNA
sequencing, the protein expression was induced. The results revealed
that CaSM, Seq8, and CaSM-Seq8 were highly over-expressed, and the
soluble fractions were successfully purified (Fig. 1A and B).
Assessment of the thermal stability and the proteolysis
resistance
Physical properties, like thermal stability and resistance to
proteolysis, are crucial in vaccine design and development. Therefore,
we sought to study whether these properties could be optimized when the
Seq8 antigen was presented on CaSM particles. According to the thermal
stability results (Fig. 1C), CaSM was stable at 4℃ and 37℃, and only a
slight degradation occurred after 5 and 7 days of storage at 37℃.
Likewise, CaSM-Seq8 was also very stable at both 4℃ and 37℃. By
contrast, Seq8 was extremely thermo-labile with a complete degradation
after 1 day at 37℃ and a gradual degradation at 4℃ starting from day 1
to day 7 (Fig. 1D). These results indicated that CaSM could indeed
enhance the thermal stability of the presented foreign antigen.
Next, we studied whether the carrier CaSM could enhance the resistance
of the exogenous antigen to enzymatic digestion (pepsin and trypsin). As
shown in Fig. 1E, CaSM and CaSM-Seq8 were resistant to pepsin but
vulnerable to trypsin. However, Seq8 was vulnerable to both pepsin and
trypsin and was totally digested. This indicated that CaSM carrier could
enhance the resistance of the presented Seq8 antigen to pepsin.
Morphological assessment of the particles formed by the target
proteins
The target proteins were visualized under a transmission electron
microscope. Both CaSM and CaSM-Seq8 formed spherically-shaped VLPs.
According to the radii calculation and frequency analysis, the radius of
CaSM was 13.03±1.739nm (Fig. 2A), and the radius of CaSM-Seq8 particles
was 12.91±1.823nm (Fig. 2B). However, on the Seq8 micrographs, abundant
smaller aggregates (radius: 6.68±1.438nm) and fewer irregular large
particles were observed (Fig. 2C), without the classical VLP shape
observed with CaSM and CaSM-Seq8. As a control, native HEV virions were
also observed, and displayed a radius of 21.36±2.575nm (Fig. 2D).
To further investigate the reasons for these morphological differences,
GalaxyWeb Homomer server was used to compute the assembly patterns of
the studied proteins 32. The results indicated that
CaSM had the ability to form pentamers that assembled into decamers,
like the native HEV T=1/3 VLPs (Fig. 2E). Furthermore, CaSM-Seq8 showed
the same assembly pattern as CaSM and the native HEV T=1/3 VLPs (Fig.
2E). However, Seq8 was predicted to form only dimers (Fig. 2F), which
could explain the observations on the electron micrographs.
Antigenicity analysis
First, we sought to assess the antigenicity of Seq8 and CaSM-Seq8
computationally based on the predicted 3D structure models. According to
the protrusion index (PI) calculation and comparison, most amino acid
residues were more exposed in CaSM-Seq8 than in Seq8 (Fig. 3A&B). Since
the Seq8 antigen contains three different neutralizing epitopes (3 G-H
loops), the AUC of each epitopes was calculated to evaluate the exposure
of each epitope as a whole. The result revealed that the AUC of epitope
I and III were significantly higher in CaSM-Seq8 than in Seq8 (Fig. 3C).
All these results suggested that the epitopes were more protruding in
CaSM-Seq8 than in Seq8, and thus, the CaSM-Seq8 might have a higher
antigenicity than Seq8.
Next, we assessed and compared the antigenicity of CaSM-Seq8 and Seq8
experimentally. The results of the indirect ELISA revealed that at
almost all dilutions, the reactivity of CaSM-Seq8 against O/Mya/98- or
O/HN/CHA/09-induced antibodies (Fig. 4A), O/JMS/00- or O/GX/09-7-induced
antibodies (Fig. 4B), and purified anti-FMDV/O pAbs (Fig. 4C) was
stronger than that of Seq8. Similarly, the Western blotting results
revealed that the reactive bands of CaSM-Seq8 had higher grayscales than
those of Seq8 (Fig. 4E). Altogether, these results were in accordance
with the computational analysis and indicated an enhanced antigenicity
of Seq8 when it was attached to the CaSM protein.
Immunogenicity analysis
After the injection of CaSM-Seq8, Seq8, and FMDV inactivated vaccine
into mice, the induced FMDV-specific IgG levels were monitored up to 10
weeks post-injection using an indirect ELISA (Fig. 5A). Antibody titers
induced by CaSM-Seq8 were higher than those induced in all the other
experimental groups. Moreover, FMDV-specific antibodies appeared earlier
in CaSM-Seq8 immunized mice (at week 2) than in the inactivated vaccine
group or Seq8-immunized mice (at week 4). Additionally, the humoral
response induced by the inactivated vaccine and Seq8 began to decrease
approximately at week 6 and week 8, respectively. By contrast, the
CaSM-Seq8 induced antibodies maintained the highest levels until the end
of the experiment. These results indicated that CaSM platform improved
the immunogenicity of Seq8, which is in accordance with results of the
computational analysis of the antigenicity, where the exposure of the
G-H loops was found enhanced by attaching the Seq8 antigen to CaSM.
Discussion
Hepatitis E virus, an icosahedral virus with a diameter of approximately
27-32nm, is an oral-transmitted virus causing acute viral hepatitis7. Its capsid protein is encoded by ORF2 and contains
three structural domains: S domain (residues 129-319), M domain
(residues 320-455), and P domain (residues 456-606) which forms
protruding spikes outside the VLP (Fig. 5B) 10,38.
In the present study, the HEV truncated capsid (aa112-455) named CaSM,
comprising the S and M domains as well as the flexible proline-rich
hinge linking the P domain, was selected to present the exogenous
antigen Seq8. The selection of this truncated protein was based on the
structural features of these domains reported after the elucidation of
high-resolution structures of the HEV capsid 9,10,38.
The S domain, which is the most conserved region among HEV genotypes33, formed an icosahedral shell serving as the base
for the arrangement of the subsequent domains (M and P)38. Although HEV capsid seemed to share similar
folding and morphology with other viruses such as caliciviruses39,40, the arrangement of the M domain seemed to be
unique to the HEV capsid, because its strong interaction with the S
domain allows the enhancement of the VLPs stability38. Furthermore, unlike the P2 domain of caliciviruses
that is inserted into the P1 domain (M domain) 39, the
HEV capsid P domain is independent from the SM-formed shell because of
the long proline-rich hinge that links it to the C-terminal of the M
domain 9,10,38.
As an exogenous antigen, we designed the Seq8 chimeric protein (Fig. 5C)
by combining three neutralization epitopes (VP1 G-H loops) derived from
3 different FMDV/O strains as previously reported26,27. According to previous studies, these G-H loops
elicited neutralizing antibodies and protective immune responses against
virulent FMDV strains 24,26,28. However, the VP1 G-H
loop had low immunogenicity 24,28 and accordingly,
many approaches were adopted to improve the immunogenicity of this
peptide, including the incorporation of other T and B cell epitopes25-28. Therefore, in the present work, we explored the
use of HEV CaSM to present Seq8 and the improvement of the
immunogenicity of the G-H loops in the chimeric VLPs. In this chimeric
construct, the Seq8 antigen was linked to the C-terminal of the CaSM as
a substitute of the P domain in the HEV capsid (Fig. 5D).
Next, through computational analysis, we predicted the oligomerization
of the three proteins CaSM, CaSM-Seq8 and Seq8 and found that CaSM and
CaSM-Seq8 could form pentamers and decamers, arranged similarly to those
previously described as intermediates in the assembly of HEV VLPs10,38,41. This suggested that the substitution of the
P domain would not interfere with the proper aggregation of the S and M
domains of the CaSM-Seq8 and the formation of the desired chimeric VLPs.
To experimentally confirm these predictions, the three target proteins
were overexpressed in E. coli, purified and visualized by transmission
electron microscopy. On the micrographs, CaSM self-assembled into VLPs
with a diameter of approximately 26nm, similar in shape but smaller in
size than particles observed on the native HEV micrographs. This was
expected since the CaSM protein contains the amino acids that previous
studies have already identified as essential for truncated HEV ORF2
proteins to form T=1 or T=3 VLPs, such as aa111, aa126 and aa36712,13. More interestingly, the CaSM-Seq8 chimeric
protein also formed VLPs with approximately the same diameter and
morphology than CaSM particles. It is to note that the VLPs formed by
CaSM and CaSM-Seq8 were smaller than the native HEV particles, which is
very likely due to the difference in the proteins’ size (38.3 KDa and
47.9 KDa for CaSM and CaSM-Seq8 respectively, and 70.9 KDa for the
native HEV capsid protein). This size parameter could also explain why
Seq8 antigen alone (10.6 KDa) could not assemble into spherically-shaped
VLPs similar to the CaSM and CaSM-Seq8 ones. These results along with
the computational predictions point toward the conclusion that the
substitution of the P domain with the exogenous Seq8 antigen did not
affect the assembly of the HEV S/M domain into particles that present
Seq8 as a protruding spike. However, further determination of the
high-resolution structure of CaSM-Seq8 chimeric VLPs is needed to
confirm these observations.
Further, we sought to evaluate whether the formation of the VLPs
affected the antigenicity and immunogenicity of the inserted Seq8
antigen. The bioinformatics analysis revealed that the three G-H loops
were more exposed when attached to CaSM than when presented on Seq8
alone, suggesting thus, that the antigenicity of Seq8 would be enhanced
in CaSM-Seq8 chimeric VLPs. Indeed, the Western blotting and ELISA
results showed that the immunoreactivity of CaSM-Seq8 against
FMDV-specific antibodies was more robust than that of Seq8 alone.
Likewise, after injection into mice, as chimeric VLPs, CaSM-Seq8 induced
higher anti-FMDV IgG levels that appeared earlier, increased faster, and
lasted longer compared to the humoral responses induced by Seq8 or the
FMDV inactivated vaccine. These results indicated clearly that on one
hand, the formed chimeric VLPs did not interfere with the presentation
of the Seq8 epitopes; and the optimal exposure of these epitopes
enhanced the overall antigenicity/immunogenicity of Seq8 epitopes, on
the other hand. The improved antigenicity and immunogenicity of the Seq8
moiety in the CaSM-Seq8 can be explained by: 1) the intrinsic
characteristics of CaSM-carrier VLPs such as the inclusion of the
proline-rich linker that would allow the protrusion of the foreign
antigen and augment its conformational flexibility for an optimal
interaction with the host immune system (immunogenicity) and the
FMDV-specific antibodies (antigenicity); 2) the self-assembly of
CaSM-Seq8 into relatively large VLPs (compared to Seq8 alone) permitted
carrying repetitive copies of the target epitopes, which can stimulate
more robust immune reactions 42; 3) numerous T cell
epitopes have been located in S and M domains 43,
which participate in activating a stronger cellular immune response.
Furthermore, it has been previously reported that HEV VLPs could be used
as an oral delivery system 6,16. Therefore, we
investigated the thermal stability and proteolysis resistance the
CaSM-Seq8 chimeric VLPs. As a result, both properties were enhanced when
Seq8 was carried by CaSM particles, suggesting thus, the potential of
CaSM antigen-presenting platform in the design of oral vaccines.
However, further investigation is needed to overcome some limitations of
this work: 1) only FMDV Seq8 was used as the exogenous antigen, and
whether CaSM was an excellent VLP platform for other antigens needed
further research; 2) high-resolution structures of the CaSM VLPs,
CaSM-Seq8 VLPs, and Seq8 should be determined to study their structural
and immunological characteristics at the molecular level; 3) since many
other VLP platforms (HBV, HPV, HIV, etc.) were reported previously42,44, a comparative study would be of great interest.
In conclusion, in this study we demonstrated that the S and M domains of
the HEV capsid protein can self-assemble into VLPs, and by substituting
only the P domain by a foreign antigen, this latter was also presented
as a protruding domain in the chimeric VLPs, enhancing therefore its
antigenicity and immunogenicity. This makes the CaSM particles a
potential presentation platform of exogenous immunogens. Furthermore,
the CaSM-Seq8 investigated here, showed promising preliminary results as
chimeric vaccine against FMDV that is worth further investigation to
evaluate the neutralization activity of CaSM-Seq8- induced antibodies
and their in vivo protection against virulent FMDV strains, and to
select the best adjuvant, the optimal dose, and the best administration
route.