ISSN 0026-8933, Molecular Biology, 2016, Vol. 50, No. 5, pp. 731–739. © Pleiades Publishing, Inc., 2016.

Published in Russian in Molekulyarnaya Biologiya, 2016, Vol. 50, No. 5, pp. 828–837.

MOLECULAR CELL BIOLOGY

UDC 615.371

Immunoreactivity of Chimeric Proteins Carrying Poliovirus Epitopes

on the VP6 of Rotavirus as a Vector1
X.-X. Pana, #, B.-X. Zhaob, #, Y.-M. Tengb, W.-Y. Xiab, J. Wangb, X.-F. Lia,
G.-Y. Liaob, C. Yanga, and Y.-D. Chenb, *
aKey Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education,
Yunnan Minzu University, Kunming, P. R. China
b
Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Institute of Medical Biology,
Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, P. R. China
*e-mail: chenyd@imbcams.com.cn
Received October 13, 2015; in final form, November 13, 2015

Abstract—Rotavirus and poliovirus continue to present significant risks and burden of disease to children in
developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be
advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought
to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein,
VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites
on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based
PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot,
immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response.
Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited anti-
bodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes
neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the
use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could
be considered for development of epitope-based vaccines against rotavirus and poliovirus.

Keywords: chimeric protein, cross neutralizing activity, poliovirus epitope, rotavirus VP6-based vector
DOI: 10.1134/S0026893316030092

INTRODUCTION lowing international licensure of RV vaccines (Rotarix

Rotavirus (RV) and poliovirus (PV) are major
human pathogens that present significant risks and
high burden to children in developing countries. RV is
the leading pathogenic agent of acute gastroenteritis
worldwide, and group A rotavirus (RVA) is the most
principal causative agent of diarrhea in infants and
young children. The two oral live RV vaccines, Rotarix
and Rotateq, have been licensed in 2006, and proved
effective in all countries, particularly in those with high
diarrhea-related mortality [1, 2]. However, a risk of
adverse effects has been associated with some live vac-
cines, such as intussusception, vaccine-acquired rotavi-
rus disease in infants with severe combined immunodefi-
ciency (SCID) after administration, the presence of
adventitious agents in the vaccine [3–6]. Because live RV
vaccines are found to have intrinsic limitations, it is nec-
essary to develop more efficacious and safe vaccines,
especially in developing countries. WHO supports the
development of alternative RV vaccine candidates fol-
1 The article is published in the original.
# The authors contributed equally to this work.
and Rotateq) to reduce the global RV burden in devel-
oped and developing countries [7].
PV is the causative agent of acute paralytic polio-
myelitis. Following more than twenty years of anti-
polio programs, prevention of PV has made great
progress and wild PV (WPV) remains endemic in only
three countries (Pakistan, Afghanistan and Nigeria).
The last paralysis case associated with WPV type 2
(WPV2) was registered in India in 1999, and WPV type 3
(WPV3) has not been detected in circulation since
November 2012; since 2012, only WPV type 1 (WPV1)
has been detected [8]. During 2014, a total of 359 WPV
cases were detected in nine countries worldwide [9].
However, in the recent years, low vaccine coverage has
allowed occasional spread of wild PV strains from
endemic countries to neighboring or distant countries
where wild PV had disappeared; also pathogenic recom-
binant circulating vaccine-derived PV (cVDPVs)
appeared, some vaccine-derived PV (VDPV) strains
were isolated from paralytic cases as well as healthy
individuals and environments, indicating that the

731
732 PAN et al.

I6 reactive among all RVA. VP6 has the ability to self-

assemble into virus-like particles (VLPs), tubular or
trimer structures, and thus it can be used as a carrier
I1 I2 candidate for foreign epitopes. Furthermore, in the
recent years a few candidate VP6-based vaccines have
been studied and the results showed that VP6 is able to
induce effective humoral and cellular responses
D''́ ́ against RV in mice [14–19].
I5
D' ́ Previous studies revealed that the neutralizing anti-
I3
genic sites (NAgs) of PV are distributed on the surface-
exposed loops in structural proteins VP1, VP2, and
I I4 VP3. NAg1 includes amino acid residues 91–102, 254,
H C D 168 of VP1. NAg2 includes amino acid residues 166–
170 and 270 of VP2, and residues 221–226 of VP1.
A' A'' B H NAg3 includes amino acid residues 285–289 of VP1,
E F 58–60, 71, 73 of VP3, and residue 72 of VP2 [20]. The
amino acid residues 89 to 100, 220 to 222, 286 to 290
G of VP1 are important cross neutralizing epitopes and
I D are able to induce production of cross neutralizing
αg
antibodies, protecting adults from infections by differ-
ent PV serotypes [21].
A In this report, we searched for fine positions in the
surface loops of RV VP6 that can accommodate the
three foreign peptides derived from PV1. Chimeric
αf proteins carrying the PV epitope were constructed,
and then their immunoreactivities were evaluated. It is
αh expected that the chimeric proteins in this form could
αe be considered as alternative vaccines against RV/PV.
C-ter
αc B
N-ter EXPERIMENTAL
βb βa αa Cells and viruses. Fetal rhesus monkey kidney
αd (MA104) cells were maintained in Eagle’s minimal
αc essential medium (MEM) supplemented with 10%
fetal bovine serum (FBS). Vero cells (African green
monkey kidney cells) were maintained in Dulbecco’s
αb
Modified Eagle’s medium (DMEM) supplemented
with 5% FBS.
Human RV strain Wa (G1P [8]) was kept in our
laboratory. Three types of attenuated poliovirus, type 1
Fig. 1. 3D molecular structure of the VP6F vector protein
with six foreign epitope insertion sites on the outer surface;
Sabin SO+1 (PV1), type 2 Sabin SO+1 (PV2), type 3
these sites could be chosen for insertion of foreign epitopes 457 Pfizer RSO1 (PV3) were provided by the World
[22]. Sites I1, I3, and I5 were chosen for epitope insertion Health Organization (WHO). RV strain Wa was
in the present study. adapted to grow in cell culture by serial passage in
MA104 cells and PV strains were adapted in Vero cells.
eradication of PV is still a big problem currently facing Epitopes and construction of recombinant plasmids.
the globe [10–12]. In our previous study, using molecular cloning,
genetic recombination and expression techniques, a
Since RV and PV share similar transmission routes, new foreign epitope presenting a system based on RV
and co-infections by RV and PV are very common VP6 as the vector (VP6F) was developed [22]. Six sites
[13], the prevention of RV and PV at the same time has that could be used for insertion of foreign epitopes
a great importance for optimally utilizing the limited were created on the outer surface of the vector protein
resources and ensuring high immunization coverage. (Fig. 1).
Given the huge efforts underway to eradicate RV and Three NAg epitopes among the NAgs of PV1 were
PV, the most viable alternative vaccine candidates are studied in this paper: the NAg1 epitope, TVDN-
needed to accelerate the development process. PASTTNKDET corresponding to amino acid resi-
VP6, a group antigen protein of RV, is highly con- dues 91–102, 254, 168 of VP1; the NAg2 epitope,
served and antibodies against VP6 are highly cross- QTSPALSAALGD corresponding to amino acid resi-

MOLECULAR BIOLOGY Vol. 50 No. 5 2016

 IMMUNOREACTIVITY OF CHIMERIC PROTEINS CARRYING
dues 166–170 and 270 of VP2, and residues 221–226
of VP1; NAg3 epitope, DYKDGSATRSPHTDT cor-
responding to amino acid residues 285–289 of the
VP1, 58–60, 71, 73 of the VP3, and residue 72 of the
VP2, respectively, were selected from the PV1 strain
Mahoney in order to construct recombinant epitope
chimeric vaccines.
Three pairs of specific oligonucleotide primers
PV1/N1/298F (GGA CCG TGG ATA ACC CAG-
CTT CCA CCA CGA ATA AGG ATG AGC CGC)
and PV1/N1/298R (GGC TCA TCC TTA TTC GTG-
GTG GAA GCT GGG TTA TCC ACG GTC CGC),
PV1/N2/244F (CCA GAC ATC ACC TGC CTT-
ATC GGC AGC ACT AGG TGA CGG TAC) and
PV1/N2/244R (CGT CAC CTA GTG CTG CCG-
ATA AGG CAG GTG ATG TCT GGG TAC),
PV1/N3/174F (CGA TTA CAA GGA TGG TAG-
TGC CAC ACG GAG TCC ACA TAC AGA CAC-
GGA GCT) and PV1/N3/174R (CCG TGT CTG-
TAT GTG GAC TCC GTG TGG CAC TAC CAT-
CCT TGT AAT CGA GCT), corresponding to the
epitopes NAg1, NAg2, NAg3 of the representative PV1
strain Mahoney (CAA24461) were synthesized and
inserted respectively into the cloning sites I5, I3, I1 on
the VP6F protein vector by means of genetic recombi-
nation techniques. Briefly, the VP6F/PV1 epitope chi-
meric protein coding gene was inserted into the plasmid
pETL which was derived from pET-3a, and four recom-
binant plasmids directing the expression of the chimeric
protein were constructed by genetic recombination
techniques as previously described [23], pETP6F,
pETP6F/PV1N1-I5, pETP6F/PV1N2-I3, and
pETP6F/PV1N3-I1. pETP6F carried the vector protein
VP6F gene, pETP6F/PV1N1-I5 carried the VP6F gene
with the NAg1 epitope inserted in the SacII site (I5),
pETP6F/PV1N2-I3 carried the VP6F gene with the
NAg2 epitope inserted in the KpnI site (I3), and
pETP6F/PV1N3-I1 carried the VP6F gene with the
NAg3 epitope inserted in the SacI site (I1).
The recombinant plasmids described above were
transformed into E. coli DH5α and were then grown
on agar plates, and the clones were selected for further
screening by enzyme digestion and PCR for gene
identity and orientation. Recombinant plasmids were
sequenced to ultimately confirm the authenticity of
the RV/PV gene sequences.
Expression and purification of recombinant proteins.
The recombinant plasmids were transformed into
E. coli BL21(DE3) cells. The transformed cells were
cultured in Luria–Bertani (LB) medium (10 g of tryp-
tone, 5 g of yeast extract, 5 g of NaCl per liter) supple-
mented with 200 µg/mL ampicillin at 37°C without any
chemical induction. When the culture grew to be satu-
rated, cells were harvested by centrifugation (3000 rpm,
8 min, 4°C). Harvested cells were lysed by ultrasoni-
cation. The sonicated solution was then centrifuged
(12000 rpm, 30 min, 4°C), and the residue (inclusion
bodies) containing the chimeric protein was dissolved
MOLECULAR BIOLOGY Vol. 50 No. 5 2016
733
in 8 M urea. The expressed proteins were analyzed by
sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (10% SDS-PAGE).
The expressed proteins were purified as previously
described [23]. Briefly, the proteins were separated on
10% SDS-PAGE and dyed by 150 mM KCl. Accord-
ing to the molecular weight of the protein, the band of
gel containing the target chimeric protein was cut off
and put into a column filled with polyacrylamide gel
(15%) on its bottom. Electrophoresis was performed at
20 mA, 4°C for 15 h, and the chimeric protein was
enriched in the vial that was linked to the column bot-
tom. The protein was further dialyzed in 10 mM phos-
phate-buffered saline (PBS) (pH 7.4) for desalination
and renaturation. The protein concentrations were
determined using the Lowry’s method [24].
Animals and immunization. Guinea pigs (5–7 weeks
of age) used in this study were purchased from the
China Medical Primates Center, Kunming, China,
and housed in groups of four in sterile microisolation
cages. All research performed on guinea pigs in this
study complied with national and institutional guide-
lines set forth by the Institute of Medical Biology Ani-
mal Care and Use Ethic Committee. All studies were
approved by the Institute of Medical Biology Animal
Care and Use Ethic Committee.
All guinea pigs were screened for antibodies against
RVA and PV by neutralization test upon arrival to
ensure the absence of a preexisting immunity from
prior exposure or maternally-derived antibodies.
Recombinant vector protein VP6F, chimeric proteins
carrying PV1 epitopes NAg1, NAg2, NAg3, and refer-
ence RV Wa, PV1, PV2, PV3 were used as immuno-
gens separately (n = 4 guinea pigs per immunogen).
For inoculation with VP6F and chimeric proteins car-
rying PV1 NAg1, NAg2, NAg3 epitope, each animal
was inoculated subcutaneously in the hind leg with
120 µg of each immunogen in 100 µL dilution solution
(15 mM Tris, 150 mM NaCl, pH 7.0) in the absence of
adjuvant. For RV Wa and PV1, PV2, PV3 inoculation,
1 × 107 of the 50% tissue culture infective dose
(TCID50) of the virus was used for each animal at each
injection. Four guinea pigs were used as negative con-
trols and were mock-inoculated with 100 µL of dilu-
tion solution. Each animal was inoculated three times
(at 0, 14, and 28 days). Guinea pigs were bled by heart
puncture at the fifth day after the last inoculation and
antibody levels in antiserum were measured by West-
ern blot and neutralization tests.
Immunofluorescence. Immunofluorescence was
performed to detect PV1 and RV antigens in virus
infected cells. To detect the PV1 antigen, Vero cell
monolayers on glass coverslips cultured in MEM were
washed twice with PBS and infected with PV1. The cov-
erslips were taken out 12 hr after infection, washed twice
with PBS, fixed with prechilled methanol, and rehy-
drated for 10 min at 4°C with 70, 30, and 10% prechilled
ethanol. After washing with PBS, coverslips were incu-
734 PAN et al.
bated for 1 h at 37°C with antiserum (1 : 400 dilution in
0.1% of bovine serum albumin) collected from guinea
pigs inoculated with PV1, VP6F, chimeric proteins, or
with the serum collected from pre-immune guinea
pigs or the mock inoculated guinea pigs, respectively.
After washing, the coverslips were incubated at 37°C
for 1 hr with goat anti-guinea pig IgG labeled with
FITC (1 : 100 dilution), and the fluorescence in the
virus-infected cell was detected under the excitation
light/emission filter EX450-490/B-2A on the D-FL
EPI-fluorescence microscope (Nikon Eclipse E600,
Japan). Meanwhile, for control, fluorescence from
infected cells of pre-inoculated or mock inoculated
(negative) guinea pigs was detected. Fluorescence
detection in noninfected cells with pre-inoculated
sera, virus inoculated or negative (inoculated with
PBS) guinea pig sera were also carried out.
To detect the RV antigen, MA104 cells were
infected with the RV strain Wa, and the immunofluo-
rescence assay was performed as described above for
the PV1 antigen.
Western blot. Soluble fractions including chimeric
proteins (about 4 µg each) were subjected to SDS-PAGE.
Samples were suspended in gel loading buffer (2 mM
EDTA, 50 mM Tris, pH 6.8, 10% glycerol, 1% SDS,
1% β-mercaptoethanol, 0.05% bromophenol blue),
heated (95°C, 5 min), and subjected to 10% SDS-
PAGE. Following SDS-PAGE, separated proteins
were transferred to polyvinylidene fluoride (PVDF)
membranes. The membranes were then blocked with
5% skim milk in TBS (25 mM Tris-HCl, pH 7.5, 0.9%
NaCl), then incubated with antiserum from immu-
nized guinea pigs (at a 1 : 400 dilution). After being
washed with 0.1% Tween20 in TBS (TBST), the mem-
branes were incubated with horseradish peroxidase-
conjugated goat anti-guinea pig immunoglobulin G
(IgG; 1 : 2000 dilution; Sigma, St. Louis, MO, USA).
After washing, the membranes were stained with 3,3'-
diaminobenzidine tetrahydrochloride (DAB, Sigma)
to visualize the bound antibodies.
Cell culture and determination of virus infectious
titer. MA104 and Vero cells were used for preparation
and determination of RV and PV infectious doses
respectively. In short, when cells grew to a confluent
monolayer in 96-well tissue culture plates, medium was
removed and cells were washed twice with PBS, the
cells were incubated for 1 h with RV strain Wa which
was pre-treated with acetylated trypsin (10 µg/mL,
37°C for 1 h), or with PV respectively with gentle rock-
ing at 15 min intervals. The virus solution was removed
and cells were then incubated in maintenance MEM
medium (without calf serum) at 37°C in an atmo-
sphere of 5% CO2. When the cytopathic effect (CPE)
appeared in ≥75% of cells, whole cells and supernatant
were harvested, frozen and thawed three times. After cen-
trifugation at 4000 rpm, at 4°C for 30 min, supernatant
was taken and virus infectious doses were determined by
TCID50 using CPE with Kärber’s method [25].
Neutralization test with guinea pig serum. The Neu-
tralization test (NT) was performed in the micro-neu-
tralization format in Vero cells (for PV) and MA104
cell (for RV) in 96-well culture plates with 100 TCID50
of challenge virus according to the standard protocol.
RV strain Wa and three types of attenuated PV
obtained from WHO, type 1 Sabin SO+1 (PV1), type 2
Sabin SO+1 (PV2), type 3 457 Pfizer RSO1 (PV3)
were used to estimate the neutralizing reactivity of
antibodies elicited in the serum of guinea pigs inocu-
lated with the chimeric proteins. Briefly, 50 µL of virus
solution containing 100 TCID50 was mixed with an
equal volume of the guinea pig antiserum at 2-fold
serial dilutions, and then incubated for 1 h at 37°C.
The mixture was then added to MA104 cell monolay-
ers in 96-well plates. Each dilution was done in four
wells. The cell plates were incubated in a 5% CO2
incubator for 48 h, and CPE was observed. Neutraliz-
ing titers were defined as the highest dilution of the
antiserum that protected 50% of the cells from the
viral infection.
RESULTS
Expression and Purification of Chimeric Proteins
The recombinant plasmids pETP6F, pETP6F/
PV1N1-I5, pETP6F/PV1N2-I3, and pETP6F/
PV1N3-I1 were sequenced to confirm that the epitope
sequences were correctly inserted and transformed
into competent BL21(DE3) cells for protein expression.
The recombinant proteins were expressed in the form of
inclusion bodies in the transformed cells (Fig. 2). The
expressed proteins, recombinant vector protein VP6F,
and the chimeric proteins 6F/PV1N1, 6F/PV1N2,
6F/PV1N3 that carry the epitopes of NAg1, NAg2, or
NAg3 had molecular weights of 43.2, 44.8, 44.5,
45.1 kDa (Fig. 2a) as expected, and accounted for
47.8, 47.6, 48.3, and 53.2% of the total protein in the
transformed cells, respectively. No chimeric proteins
were detected in the supernatants of the cell cultures or
sonication lysates (data no shown). The chimeric pro-
teins were purified (Fig. 2b) and used to immunize
guinea pigs.
Immunoreactivity of Chimeric Proteins
The immunological reactivity of the chimeric pro-
teins was detected by Western blot. Results showed
that all of the chimeric proteins carrying the PV1
NAg1, NAg2, or NAg3 epitopes could be specifically
recognized by antibodies in all four sera derived from
guinea pigs inoculated with recombinant vector pro-
tein VP6F (Fig. 3). Antibodies from PV1 immunized
guinea pigs reacted with the chimera proteins, but not
with the vector protein (Fig. 4), indicating that VP1
epitopes that were carried on the vector protein VP6F
have good immunogenicity. While, antibodies from
PV2 or PV3 immunized guinea pigs did not react with
MOLECULAR BIOLOGY Vol. 50 No. 5 2016
 IMMUNOREACTIVITY OF CHIMERIC PROTEINS CARRYING 735

(a) (b)
M 1 2 3 4 M 1 2 3 4

200.0 200.0

116.0 116.0
97.2 97.2

66.4 66.4

44.3
44.3

Fig. 2. SDS-PAGE (10%) of the chimeric proteins expressed in E. coli BL21 (DE3) cell before (a) and after purification (b). Lane
M: Protein molecular weight standard (kDa); lane 1: vector protein VP6F; lane 2: chimeric proteins 6F/PV1N1; lane 3:
6F/PV1N2; lane 4: 6F/PV1N3.

the vector protein and the chimera proteins (data not in Wa infected MA104 cells with negative control or
shown). pre-inoculation sera (Fig. 6i). The results indicated
Antibodies elicited in all four guinea pigs in each that PV1 antigen in PV1 infected Vero cells and RV
group immunized with chimeric proteins carrying a antigen in Wa infected MA104 cell could be detected
single PV1 NAg epitope reacted with the vector VP6F by antibodies in sera from guinea pigs inoculated with
and chimeric proteins carrying the PV1 NAg epitopes chimeric proteins.
(Fig. 5). No immunoreactivity was observed between
sera derived from pre-immune or negative control ani-
M 1 2 3 4
mals and VP6F or the chimeric proteins carrying PV1
VP1 epitopes (data not shown). It is notable that the 200.0
VP1 but not VP2 or VP3 protein of PV1 could only be
recognized by the antibodies against the chimeric pro- 116.0
tein 6F/PV1N1 (Fig. 5, lane 5). No reactions of the 97.2
VP1 protein of PV1 with the antibodies against the
chimeric protein 6F/PV1N2 or 6F/PV1N3 was
observed (date not shown). 66.4

Immunofluorescence Test of PV1 Antigen

in PV1 Infected Cells 44.3
PV1 antigen in PV1 infected Vero cells and RV
antigen in Wa infected Ma104 cells were detected by
the immunofluorescence assay using guinea pig anti-
bodies against the chimeric proteins (Fig. 6). Results
showed that the PV1 antigen in PV1 infected Vero cells
could be detected by antibodies against PV1 (Fig. 6a) and
antibodies against the chimeric proteins (Figs. 6c–6e). 29.0
As a control, no fluorescence was detected in PV1
infected cells when detected with antibodies against
the vector VP6F (Fig. 6b), negative control or pre- Fig. 3. Western blot of chimeric proteins (about 4 μg each)
inoculation sera (Fig. 6f). RV antigen in Wa infected with antibodies from serum collected from VP6F inocu-
lated guinea pigs. Lane M: protein molecular weight stan-
MA104 cells could be detected by antibodies against dard, lane 1: recombinant vector protein VP6F; lane 2:
Wa (Figs. 6g–6i), antibodies against the chimeric pro- chimeric protein 6F/PV1N1; lane 3: chimeric protein
teins (Fig. 6h). No immunofluorescence was detected 6F/PV1N2; and lane 4: chimeric protein 6F/PV1N3.

MOLECULAR BIOLOGY Vol. 50 No. 5 2016

736 PAN et al.
M 1 2 3 4 vector protein VP6F or mock-inoculated with PBS as
200.0
116.0
97.2
66.4
44.3
29.0
Fig. 4. Western blot of chimeric proteins (about 4 μg each)
with antibodies from serum collected from PV1 inoculated
guinea pigs. Lane M: protein molecular weight standard;
lane 1: recombinant vector protein VP6F; lane 2: chimeric
protein 6F/PV1N1; lane 3: chimeric protein 6F/PV1N2; and
lane 4: chimeric protein 6F/PV1N3.
M 1 2 3 4 5
200.0
116.0
97.2
66.4
44.3
29.0
Fig. 5. Western blot of rotavirus proteins with guinea pig
antibodies from serum of guinea pigs inoculated with chi-
meric protein(s) carrying PV1 NAg1 epitope(s). Lane M:
protein molecular weight standard; lane 1: recombinant vec-
tor protein VP6F; lane 2: chimeric protein 6F/PV1N1;
lane 3: chimeric protein 6F/PV1N2; lane 4: chimeric pro-
tein 6F/PV1N3; and lane 5: virus strain PV1. For each
recombinant protein (lanes 1–4) about 4 μg was loaded.
For the virus strain PV1 (lane 5), about 3 × 106 TCID50
was loaded.
Neutralizing Antibodies Activity
Neutralizing antibodies elicited in guinea pigs were
determined. Antibodies in the serum of guinea pigs
inoculated with chimeric proteins neutralized the
infectivity of the PV1 in Vero cells and RV in MA104
cells.
The neutralizing titers in antiserum from guinea
pigs inoculated with the chimeric proteins carrying
NAg1, NAg2, and NAg3 epitopes were 1 : 120, 1 : 67
and 1 : 40 against PV1 infection, and 1 : 320 against RV
infection, respectively. No neutralizing activity was
detected in antisera from guinea pigs inoculated with
negative control (lower than 1 : 2). Meanwhile, there
was no neutralizing activity against infection of PV
type 2 (PV2) or type 3 (PV3) detected in antisera from
all guinea pigs (lower than 1 : 2) (table).
DISCUSSION
VP6, the major structural protein forming the mid-
dle layer in the triple-layered viral capsid of RV, is both
highly immunogenic and antigenic, and the most fre-
quently targeted protein in diagnostic assays to detect
virus particles. VP6 contains cross-reactive epitopes
shared by all the RVA, and the high degree of identity
in the primary amino acid sequences of VP6 protein
from mammalian RV suggests that VP6-based vac-
cines may potentially provide heterotypic protection.
For a long time it was thought that antibodies directed
against VP6 have no neutralizing ability in vitro, but
evidence to the contrary has been mounting [26–29].
The anti-VP6 IgA7D9 can mediate intracellular neu-
tralization by binding to RV double-layered particles
following transcytosis in mice [30, 31]. Some recent
studies showed that llama-derived single-chain anti-
body fragments (VHH) against a RV recombinant VP6
protein are able to generate a broad neutralizing activ-
ity in vitro and confer partial passive protection against
diarrhea in a neonatal mouse model [27, 32–34]. And
antibodies in sera of guinea pigs immunized with
recombinant VP6F could neutralize infectious activity
of Wa and SA11 in vitro [22].
In our previous study a foreign epitope-presenting
system using the RV VP6 as a vector was constructed.
Six sites that could be conveniently used for insertion
of foreign epitopes were created on the outer surface of
the vector protein, and its usefulness in development
of epitope-based vaccines was demonstrated.
In this study, in order to evaluate the possibility for
developing a combined vaccine that has immunoge-
nicity against both RV and PV, three highly conserved
epitopes derived from PV1 were chosen to insert in
three different outer loops on the surface of the RV
VP6 protein as the vector. The results demonstrated
that these chimeric proteins reacted with anti-VP6F
and -PV1 antibodies, elicited against both proteins in
guinea pigs. Antibodies against VP6F neutralized RV
strain Wa and antibodies against the chimeric proteins
neutralized both RV and PV1 infection in vitro.
In the past years there were some efforts to use
purified proteins or synthesized peptides as vaccines,
but their antigenic and immunogenic properties
finally showed to be unsatisfactory. This may be that
these purified proteins or synthesized peptides lack a
conformational structure, their antigenic and immu-
nogenic properties could not be correctly and fully
displayed. In our previous study we used PV1 strain
Mahoney as the vector to construct antigenic chimeras
of PV. Of these chimeras, the NAg1 (residual aa 91–102
MOLECULAR BIOLOGY Vol. 50 No. 5 2016
 IMMUNOREACTIVITY OF CHIMERIC PROTEINS CARRYING 737

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 6. Immuofluorescence analysis of PV1 and RV antigen. PV1 antigen in PV1 infected Vero cells with antibodies against PV1
(a), vector protein VP6F (b), chimeric protein 6F/PV1N1 (c), chimeric protein 6F/PV1N2 (d), and chimeric protein 6F/PV1N3
(e), and antiserum derived from the guinea pig mock inoculated with PBS (f); RV antigen in Wa infected MA104 cells with anti-
bodies against Wa (g), 6F/PV1N1 (h), and antiserum derived from the guinea pig mock inoculated with PBS (i). Bar: 50 µm (a–f),
20 µm (g–i).

of NAg1) of Mahoney was replaced by a correspond-
ing sequence of PV2 Lansing or PV3 strain Leon.
Since it was conformational and correctly displayed
on the virion, the NAg1 derived from Lansing and
Leon elicited a high neutralization activity that pro-
tected infection of the NAg-homotypic PV in vitro
[35], even after the chimera was inactivated [36].
These results indicated that if an epitope, especially a
conformational epitope, is correctly displayed on an
appropriate vector, the antigenic and immunogenic
properties of the epitope and the vector could be very
good and even may be used as a vaccine candidate.

Neutralization titers of antibodies against chimeric proteins carrying N1, N2, and N3 epitope elicited in guinea pigs
Neutralization titer of antibody against immunogen*
Virus
NC** VP6F 6F/PV1N1 6F/PV1N2 6F/PV1N3 PV1 PV2 PV3 Wa
PV1 <2 <2 120 67 40 10240 <2 <2 <2
PV2 <2 <2 <2 <2 <2 <2 10240 <2 <2
PV3 <2 <2 <2 <2 <2 <2 <2 10240 <2
Wa <2 320 320 320 320 <2 <2 <2 10240
*Neutralizing activity of poliovirus type 1, 2, 3 and RV Wa were detected in Vero cells and MA104 cells. Neutralization titers were defined
as the highest dilution of antiserum that protected 50% of cells from virus infection, and arithmetic mean NT titers were expressed;
**NC, negative control. In this group, sera were derived from animals that were pre-immune or mock immunized with PBS.

MOLECULAR BIOLOGY Vol. 50 No. 5 2016

738 PAN et al.
In this study, using the VP6 of RV as a vector three
recombinant chimeric proteins carrying epitopes from
the NAg1, NAg2, and NAg3 of PV1 were constructed.
The epitopes of NAg2 and NAg3 are conformational:
the epitope of NAg2 contains amino acids pertaining
to VP1 and VP2; the epitope of NAg3 contains amino
acids pertaining to VP1, VP2 and VP3. These chimeric
proteins could react with antibodies against RV and
PV1, and induce production of neutralizing antibodies
in animals. The antibodies against these chimeric pro-
teins could recognize antigens in PV1 and RV infected
cells (immunofluorence test), suggesting that the epi-
topes of NAg2 and NAg3 are correctly displayed on
the vector VP6F, and the vector VP6F has its native
immunogenicity and appropriately displays foreign
conformational epitopes on its surface. Meanwhile,
only antibodies against the chimeric protein carrying
NAg1 could recognize the antigen of the VP1 of PV1
by Western blot test, implying that this linear epitope
in the VP1 protein retains its partial conformation
after inactivation. Antibodies elicited in guinea pigs
inoculated with the chimeric protein could neutralize
infections by RV and PV1 in vitro, thus showing that
the chimeric proteins are valuable for developing alter-
native vaccines against both RVA and PV infections.
It is still necessary to further construct the VP6
based chimeric vaccines carrying epitopes including
all three NAgs of all three PV serotypes, and evaluate
whether these chimeric vaccines react with RV and all
three PV serotypes, which can induce production of
antibodies in animals or humans that could neutralize
infection by RV and three PV serotypes. We believe
such experience would provide a valuable perspective
and potential RV/PV combined vaccine candidates,
and make a great contribution to the control or eradi-
cation of infection by RVA and PV.
ACKNOWLEDGMENTS
This work was supported by grants from Natural Sci-
entific Fund of Yunnan Province (nos. 2012FD039,
2013FZ130 and 2014FA036).
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