Vaccine 31 (2013) 18381847

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Vaccine
j our nal home page: www. el sevi er . com/ l ocat e/ vacci ne
Synthetic B- and T-cell epitope peptides of porcine reproductive and respiratory
syndrome virus with Gp96 as adjuvant induced humoral and cell-mediated
immunity
Caiwei Chen
a,b
, Jing Li
a
, Yuhai Bi
a
, Limin Yang
a
, Shanshan Meng
a
, Yuancheng Zhou
a
,
Xiaojuan Jia
a
, Songdong Meng
a
, Lei Sun
a
, Wenjun Liu
a,b,
a
Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
b
University of Chinese Academy of Sciences, Beijing 100101, China
a r t i c l e i n f o
Article history:
Received 21 January 2012
Received in revised form6 January 2013
Accepted 25 January 2013
Available online 7 February 2013
Keywords:
HP-PRRSV
Synthetic peptide vaccine
B-cell epitope
T-cell epitope
Humoral and cell-mediated immunity
Gp96
a b s t r a c t
Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused
huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vac-
cines and attenuated live vaccines, are available but fail to provide sustainable protection, especially
against genetically heterologous strains. Thus several approaches have been used to develop more effec-
tive PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV
vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive
immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and
a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22355 aa of Gp96
(Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated
PRRSV-specic humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specic
cellular immunity induced by TCE-peptides. Moreover, higher levels of IL-12 and TNF- and lower levels
of IL-4 and IL-10 were observed in the serum of Gp96N-vaccinated piglets compared to piglets immunized
with no Gp96N, displaying a predominant Th1 type of immune response induced by Gp96N. Following
challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptides
and Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their
lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infec-
tion. These data provide important bases for the development of PRRSV epitope-based synthetic peptide
vaccines combined with Gp96N as attractive immunomodulators in swine.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV)
has two major genotypes: the European type (type I; the Lelystad
strain as the prototype) and the North American type (type II;
the ATCC VR2332 strain as the prototype), both of which belong
to the Arteriviridae family, order Nidoviridales [1]. PRRSV, espe-
cially highly pathogenic PRRSV (HP-PRRSV), causes more than
two million cases of pig infection annually in China [2] and costs
the US pork industry approximately 664 million dollars in direct
losses per year [3]. Several approaches to develop PRRSV vaccines
have been evaluated, including live attenuated virus, inactivated

Corresponding author at: Center for Molecular Virology, Institute of Microbiol-

ogy, Chinese Academy of Sciences, Beijing 100101, China. Tel.: +86 10 64807497;
fax: +86 10 64807503.
E-mail address: liuwj@im.ac.cn (W. Liu).
virus, recombinant subunit vaccine systems (based upon plasmid
DNA, bacteria, baculovirus, adenovirus, fowlpox virus, and pseu-
dorabies virus carrying several PRRSV structural proteins) and
chimeric viruses [49]. However, commercially available vaccines
have limited effect against heterologous virus challenges with
potential to revert to virulence [410].
It has been shown that synthetic peptides possess the capa-
bility to elicit critical epitope-specic immune responses without
adverse effects fromthe vaccines mentioned above. Advancements
in molecular biology, molecular immunology, and bioinformatics
have enhanced the discovery of antigenic epitopes of PRRSV. To
date, aa 3745 of the major envelope glycoprotein Gp5 is the main
target for viral neutralizing antibodies (NAs) in both the geno-
types of PRRSV [11]. In addition, type I PRRSVs strongly induce NAs
against aa 5768 of Gp4 [12,13], but whether this B-cell epitope
(BCE) from type II PRRSV strains can induce NA remains unclear.
Recently, two T-cell epitopes (TCEs) in Gp4 and three TCEs in the
nucleoprotein (N) of type I PRRSV strains, as well as two TCEs
0264-410X/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.01.049
C. Chen et al. / Vaccine 31 (2013) 18381847 1839
Table 1
Characteristics of the B- and T-cell epitopes previously reported and used in the present study.
Epitope ID Reported information Synthetic peptide sequences in this study
a
AA position and sequences Reference
Gp4-59B
59
SAAQEKISF
67
(
b
I) [12,13]
59
SSPTIRKIS
Gp5-37B
37
SHL/FQLIYNL
45
(
c
II) [11]
37
SHIQLIYNL
Gp4-7T
9
FLLAGAQHI
17
(II) [15]
7
FLLVGFKCF
Gp4-170T
170
CLFAILLAT
178
(II)
170
CLFAILLAI
Gp5-117T
117
LAALICFVIRLAKNC
131
(II) [14,15]
117
LAALICFVIRLAKNC
Gp5-149T
149
KGRLYRWRSPVII/VEK
163
(II)
149
KGRLYRWRSPVIVEK
N-49T
49
KPEKPHFPL
57
(II) [15]
49
NPEKPHFPL
N-63T
63
VRHHLTQTE
71
(II)
63
VRHHFTPSE
N-104T
104
FMLPVAHTVRLIRVTST
120
(II)
104
FSLPTQHTVRLIRATAS
d
PADRE AKFVAAWTLKAAA [27] AKFVAAWTLKAAA
The different aa of the epitope used in the present study are marked by underlining.
a
AA, amino acid.
b
I, type I PRRSV.
c
II, type II PRRSV
d
PADRE, Pan DR T-helper cell epitope.
in Gp5 of both types of strains were identied based on their
ability to induce IFN- secretion in cultures of peripheral blood
mononuclear cells (PBMCs) obtained fromPRRSV-immunized pigs
[14,15]. Regardless, it is still unknown if these two BCE and seven
TCE peptides can induce PRRSV-specic and protective immune
responses in pigs.
Due to the delayed and weak cell-mediated immunity and NA
production of the current PRRSV vaccines [1618], many immune
modulators have beenusedto provoke stronger immune responses
to experimental vaccines in pigs, including CD40L, GM-CSF, HSP70,
and CpG [68,19]. The heat shock protein (HSP) Gp96, as well as
its N-terminal fragments (aa 22355, Gp96N), are able to bind pep-
tides and act as potent immuno-adjuvants to enhance both innate
and adaptive immune responses inmice models [2023]. However,
whether this fragment of porcine Gp96 can play the same role in
pigs remains to be determined.
In this study, bioinformatics were used to align the above BCEs
and TCEs among the type II PRRSVs, and consensus sequences were
obtained. Humoral and cellular immune responses and the protec-
tive efcacy of BCE+TCE-peptides with the Gp96N adjuvant were
investigated in piglets.
2. Materials and methods
2.1. Cell lines, viruses, and antibodies
MARC-145cells were maintainedinDulbeccos modiedEagles
medium(DMEM, GIBCO) supplemented with 10% heat-inactivated
fetal bovine serum (FBS, GIBCO) and were used to propagate and
titer HP-PRRSVisolate JXwn06 (a kind gift of Prof. Hanchun Yang of
China Agricultural University, GenBank accession No. EF641008.1).
HRP-conjugated and TRITC-conjugated anti-swine IgG were pur-
chased fromSanta Cruz.
2.2. Synthetic peptides and purication of Gp95N
According to the aa sequences of each BCE and TCE shown in
Table 1, Gp4-59B (SSPTIRKIS, purity: 98.4%), Gp5-37B (SHIQLIYNL,
purity: 98.6%), Gp4-7T (FLLVGFKCF, purity: 97.8%), Gp4-170T
(CLFAILLAI, purity: 97.4%), Gp5-117T (LAALICFVIRLAKNC, purity:
87.5%), Gp5-149T (KGRLYRWRSPVIVEK, purity: 85.9%), N-49T
(NPEKPHFPL, purity: 96.7%), N-63T (VRHHFTPSE, purity: 97.2%), N-
104T (FSLPTQHTVRLIRATAS, purity: 81.2%), and a Pan DR T-helper
cell epitope (PADRE) peptide (AKFVAAWTLKAAA, purity: 82.0%)
were all synthesized and puried at Scilight Biotech, Beijing, China.
Gp4-59B and Gp5-37B were coupled to the carrier protein keyhole
limpet hemocyanin (KLH) or bovine serum albumin (BSA) by the
same company.
Recombinant Gp96Nwas expressed in Escherichia coli and puri-
ed using nickel-nitrilotriacetic acid (Ni-NTA) chromatography
(GE). Endotoxins in the protein preparation were removed using
a bacterial Endotoxin Kit (Hycult Biotech).
2.3. Pig immunization and HP-PRRSV challenge
Sixteen 4-week-old piglets were obtained from the Beijing
Center for SPF Swine Breeding & Management. All piglets were
negative for anti-PRRSV antibodies as demonstrated by commer-
cial ELISA kit (Herdcheck, IDEXX). The animals were also negative
for porcine circovirus type 2 (PCV2), Classical Swine Fever Virus
(CSFV), Pseudorabies virus (PRV), and PRRSV by PCR and/or RT-
PCR. Then, piglets were randomly separated into four groups (four
piglets per group) and housed at Ceva-China Biotech, Beijing.
Piglets were intramuscularly injected three times at 2-week inter-
vals according to Table 2. Sera and PBMCs separated from the
blood at 0, 14, 28, and 42 days post injection (dpi) were ana-
lyzed by ELISAs, lymphocyte proliferation assays, and cytokine
assays.
Then, the vaccinated piglets were intranasally challenged with
210
5
TCID
50
HP-PRRSV JXwn06 at 42dpi and monitored daily
until they died. Rectal temperatures were measured daily, and the
severity of the clinical symptoms was evaluated based on a point
scale established by Lee et al. (2005) that independently consid-
ers attitude (04 points), respiratory rate (02 points), respiratory
distress (03 points), and cough (01 point) [24]. Sequential blood
samples were collected from all animals at 0, 3, 4, 5 and 7 days
Table 2
Immunization regimens of peptides and Gp95N in piglets.
Group ID PBS Gp BT BT-Gp
Antigens PBS Gp96N
a
BCEs and TCEs peptides
b
BT peptides with Gp96N
Dose 2ml per pig 0.5mg/2ml per pig (0.2mg of eachpeptide withor without 0.5mg Gp96N)/2ml per pig
a
The mixture of two BCE peptides and eight TCE peptides shown in Table 1.
b
The same peptides as the BT group mixed with Gp96N as an adjuvant.
1840 C. Chen et al. / Vaccine 31 (2013) 18381847
post-challenge (dpc). At 4 dpc, one piglet from each group was
euthanized for pathological examination.
All the animal research was approved by the Beijing Association
for Science and Technology, with approval ID SYXK (Beijing) 2010-
0006 and SYXK(Beijing) 2006-0008, and complied with the Beijing
Laboratory Animal Welfare and Ethical Guidelines of the Beijing
Administration Committee of Laboratory Animals.
2.4. Immunouorescence assay (IFA)
IFA was performed according to our previous report with slight
modications [25]. Briey, MARC-145 cells were infected with
PRRSV JXwn06 at a multiplicity of infection (MOI) of 1. Twenty-
four hours later, the cells were washed with PBS, xed in 4%
paraformaldehyde, permeabilized with PBST (0.1% Triton X-100 in
PBS), and then incubated for 1h at 37

C with 4% BSA in PBS. The

cells were incubated with pig antisera (1:100) at 42dpi from the
four groups for 1h at 37

C. After washing ve times with PBST, the

cells were incubated for 1h at 37

C with TRITC-conjugated anti-

swine IgG (1:100). After washing ve times with PBST, the cells
were stained for nuclei by DAPI and observed using a Leica confocal
microscope.
2.5. Purication of PRRS virions
MARC-145 cells were infected with PRRSV JXwn06 isolate for
72h. The infected cells with cell cultures were frozen and thawed
three times and then centrifuged at 3000g at 4

C for half an hour.

The claried culture supernatant (containing virus) was then con-
centrated by centrifugation (Beckman Coulter SW41 Ti rotor) at
100,000g at 4

C for 2h. The precipitate (virions) was resuspended

in PBS. Subsequently, the virus was puried by ultracentrifuga-
tion through a 30% sucrose cushion using a SW41 Ti rotor at
100,000g for 2h, and the puried virions were resuspended in
PBS and heat-inactivated at 56

C for 30min. At the same time,

the uninfected cells were puried with the same methods and
used as negative control (cell control). The concentration of the
puried virions (or cell control) was determined by the Bradford
assay using BSA as a standard, and the virions were stored at
80

C.
2.6. Monitoring antibodies by ELISA
BCE peptide-specic and PRRSV-specic antibody responses
were determinedusinganindirect ELISA(iELISA) withBSA-coupled
BCE peptides or inactivated puried-PRRS virions (as described
above) as coating antigens. The 96-well ELISA plates were coated
overnight at 4

C with 1g/well coating antigens diluted in 100l

of 50mmol/l sodium carbonate buffer (pH 9.6). The plates were
washedthreetimes withPBST(0.05%Tween-20inPBS) andblocked
for 1h at 37

C with blocking buffer (3% BSA in PBS). Then, the

serumsamples frompiglets (including the negative sera collected
at 0dpi) were diluted 1:100 in blocking buffer, added to each well
(100l/well) in duplicate, and incubated for 1h at 37

C. After
six washes, 100l of HRP-conjugated goat anti-swine IgG, diluted
1:5000 in blocking buffer, was added to each well and incubated at
37

C for 1h. After six washes, 100l of substrate solution (0.4M

tetramethylbenzidine and 1mM H
2
O
2
in 100mM acetate buffer,
pH 5.6) was added and incubated for 20min at room tempera-
ture in the dark, and the reaction was stopped by the addition
of 50l 2M H
2
SO
4
to each well. The absorbance was read at
450nm (OD
450
) using an ELISA reader (Melgan). The OD
450
value
above 0.1 was determined as positive while under 0.1 as nega-
tive.
2.7. Lymphocyte proliferative response assay
Lymphocyte proliferation assays were performed using PBMCs
from the immunized piglets. The PBMCs were plated in 96-
well at-bottom plates at 100l RMPI 1640 medium (containing
5% FBS, 100U/ml of penicillin and 100g/ml of streptomycin,
210
5
cells/well) in triplicate. Subsequently, the inactivated
puried-PRRS virions (20g/ml, MARC-145 cells derived anti-
gens as a negative control) or each of the seven TCE peptide
(20g/ml, PBS as a negative control) was added and mixed.
Con A (5g/ml, Sigma) was used as a positive control. The
proliferative activity was measured according to a previously
described method with slight modications [6]. Briey, after a
72h incubation, 5mg/ml 3-(4,5-dimethylthylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS,
Sigma) was added to each well, and the plates were incubated for
anadditional 5h, followedby adding 150l/well DMSOfor another
10min. At the endof the incubation, the plates were readat 570nm.
The stimulation index (SI) was calculated as the ratio of the aver-
age ODvalue of the wells containing antigen-stimulatedcells to the
average OD value of negative control wells.
2.8. Cytokine assay
The levels of IL-4, IL-6, IL-10, IL-12, IFN-, and TNF- in the
sera and in the supernatant of the activated PBMCs were quan-
titatively determined using commercially available cytokine ELISA
Kits (R&D). PBMCs separated fromthe vaccinated piglets at 42dpi
were plated in 96-well at-bottom plates at 100l RMPI 1640
medium(containing 5% FBS and antibiotics as described in Section
2.7, 210
5
cells/well) intriplicate (freshmediumwithout cells was
used as a blank control) and stimulated with puried inactivated-
PRRS virions (or MARC-145 cells derived antigens as a negative
control). After culturing for 72h, the supernatant from each well
was collected and immediately quantied by ELISA. Cytokines in
the sera at 0, 14, 28, and 42 days post primary immunization
were also examined according to the kits procedure. The cytokine
concentration of each sample was expressed as the mean of the
triplicates, while the cytokine quality of each group at the indi-
cated time point was calculated as the meanstandard deviation
(SD) of four piglets.
2.9. Pathological examination
To estimate the severity of pathological lesions, the histologi-
cal pathology of lungs from the pigs at 4 dpc was determined as
described [26].
2.10. Statistical analysis
Statistical analyses and graphical presentation were performed
using GraphPad Prism5.0 software. All of the data are expressed as
the mean of four pigs SD. Differences in cell-mediated immune
responses, cytokine levels and viral titers at each sampling point
were analyzedusing the Wilconxontest. The clinical signs recorded
of pigs for each group were analyzed using the Fishers exact test.
P-values <0.05 were considered statistically signicant.
3. Results
3.1. Antigenic and sequence conservation analysis of the BCEs
and TCEs of PRRSV
Computational methods were appliedtoalignthe BCEs andTCEs
identied by Daz et al. (2009) and Vashisht et al. (2008) among the
type II PRRSV strains (Table 1). In detail, 379 sequences of Gp4,
C. Chen et al. / Vaccine 31 (2013) 18381847 1841
Fig. 1. Antigenic characteristics and sequence conservation analyses of the BCEs and TCEs. Two BCEs and seven TCEs of the viral proteins from the type II PRRSVs were
selected, their antigenic indices were analyzed by DNAStar software, and their sequence conservation was analyzed by Weblogo software.
Fig. 2. BCE peptides induce humoral immune responses in vaccinated piglets. (A) IFAwas used to determine the binding afnity between PRRSVand the sera fromvaccinated
piglets. MARC-145 cells infected with JXwn06 PRRSV (1 MOI) were xed and then incubated with pig sera (1:100) from the PBS, Gp, BT, or BT-Gp group. After washing,
the cells were incubated with TRITC-conjugated anti-swine IgG antibody (1:100) (red), followed by nucleus staining with DAPI (blue), and observed using a Leica confocal
microscope. (B and C) ELISA was performed to detect the BCE peptide-specic (B) and PRRSV-specic (C) antibody responses with the sera collected at 24, 28, and 42dpi
from the immunized pigs. Data are presented as the meanSD of the four pigs fromeach group. (For interpretation of the references to color in this gure legend, the reader
is referred to the web version of the article.)
1842 C. Chen et al. / Vaccine 31 (2013) 18381847
1001 sequences of Gp5, and 226 sequences of N were analyzed by
DNAStar and Weblogo software. As shown in Fig. 1, eight of the epi-
topes were highly conserved, while Gp4-59B was more divergent.
Gp5-37B, Gp4-7T, and Gp4-170T displayed poor antigenic charac-
teristics using the Jameson-Wolf Antigenic analysis, while strong
antigenicity have been showed in Gp5-59B, Gp5-117T, Gp5-149T,
N-49T, N-63T, and N-104T. Thus, the consensus sequences of these
epitopes, together with PADRE, were chosen and synthesized with
KLH or BSA conjugated to Gp4-59B and Gp5-37B.
3.2. Humoral immune responses induced by BCE-peptides
First, IFA was used to determine whether the sera from the
BCE peptide-vaccinated piglets were PRRSV-specic. As shown in
Fig. 2A, JXwn06 PRRSV could be detected with pig sera against BCE
peptides (groups BT and BT-Gp). No staining was detected in the
infected cells with the sera fromthe pigs receiving PBS or Gp96N.
These results demonstrated that the two BCE peptides could stim-
ulate PRRSV-specic antibodies.
Next, the BSA-conjugated BCE peptides or the inactivated
puried-PRRS virions were used as coating antigens to monitor the
IgG responses by ELISA. A very low level of anti-BEC-peptides and
anti-PRRSV antibodies was detected in piglets fromgroup BT after
three immunizations, while a low level of IgG was detected after
the second immunization and increased notably by the third vac-
cination in group BT-GP (Fig. 2B and C). Thus, Gp96N increased the
levels of BCE-specic (Fig. 2B) andPRRSV-specic (Fig. 2C) IgGs. We
alsodeterminedthe level of NAs inthe sera fromimmunizedpiglets
by sera-virus neutralization assays. Unfortunately, no NA could be
detected in the sera of the immunized piglets.
3.3. TCE-induced cell-mediated immunity by TCE peptides
First, inactivated puried PRRS virions were used as a stimu-
latorto perform the lymphocyte proliferation assays. As shown in
Fig. 3A, comparedtothe PBS groupat eachindicatedtime point, sig-
nicant PRRSV-specic lymphocyte proliferative responses were
exhibited in the PBMCs from the peptide-vaccinated piglets only
at 42dpi (P<0.05 of group BT and P<0.001 of BT-Gp,). Moreover,
the PBMCs from group BT-Gp displayed more signicant PRRSV-
specic lymphocyte proliferative responses than that from group
BT (P<0.05). This suggested that three or more immunizations can
induce a PRRSV-specic cell-mediated immune response inpiglets,
which was greatly enhanced by Gp96N.
Next, PBMCs fromthe immunized piglets at 42dpi were seeded
in 96-well plates in triplicate and stimulated with each TCE pep-
tideto performthe lymphocyte proliferation assays. Fig. 3B shows
that each of the TCE peptides can induce a signicant peptide-
specic lymphocyte proliferative response in group BT-Gp but not
in group BT (compared to group PBS, P<0.05).
3.4. Th1-type cytokine response bias by Gp96N in the immunized
piglets
To determine the innate immunity (especially the inamma-
tory responses) inducedby peptides +Gp96N, IL-6andTNF-levels
were measured. In addition, the Th2 type cytokine IL-4, the Th1
type cytokines IL-12 and IFN-, and the regulatory T cell type
(Treg) cytokine IL-10 were also assayed to clarify the T-helper cell
type responses induced by the candidate vaccine. ELISA kits were
employed to detect the production of these cytokines.
To test for the cytokines secreted by activated PBMCs of piglets
in the lymphocyte proliferation assay, PBMCs fromthe immunized
piglets at 42dpi stimulated with inactivated puried PRRS virions;
72h later, the supernatants were assessed by quantitative ELISA.
Compared to the PBS group, results in Fig. 4Ashows that: (1) the IL-
6 recall responses induced by inactivated PRRSV stimulation in the
PBMCs fromthe BT-Gp group were up-regulated relative to the BT
group, while the TNF- recall response was impaired in the PBMCs
from the BT group but not from the BT-Gp group, indicating that
Gp96Nenhanced the production of the inammatory cytokine IL-6
and rescued the TNF- recall response inhibitory effect induced by
peptides. (2) Theup-regulatedIL-12andthedown-regulationrecall
response of IL-4 were found in both the BT and BT-Gp groups, while
Fig. 3. TCE peptides induce cell-mediated immunity in piglets. PBMCs were collected fromthe vaccinated pigletsand stimulated either with inactivated puried PRRS virions
(MARC-145 cells-derived antigen as a negative control) (A) or with each TCE-peptide (PBS as a negative control) (B); ConA is the positive control. After 72h of stimulation,
the cells were treated with MTS, and the OD
570
values were determined 5h later. Data represent the meanSD of four pigs from each group. *P<0.05 and **P<0.001 by
Wilconxon test compared to the PBS group of each indicated time point.
C. Chen et al. / Vaccine 31 (2013) 18381847 1843
Fig. 4. Th1-type cytokine response bias by Gp96N in the peptides-vaccinated piglets. (A) PBMCs were collected and stimulated as mentioned in Fig. 3A. Six cytokines were
tested by quantitative ELISA fromthe supernatants of the PBMC cultures immediately after 72h of stimulation. (B) Cytokines in the serumfromthe pigs of the PBS, Gp, BT,
and BT-Gp groups were detected by quantitative ELISA. Data are shown as the meanSD of four pigs fromeach group. *P<0.05 and **P<0.001 by Wilconxon test compared
to 0dpi of each group.
the level of IL-12 was much higher in the BT-Gp group, suggesting a
Th1-type cytokine response bias induced by Gp96N. (3) The down-
regulationrecall responses of IL-10werealsodetectedinthePBMCs
fromthe BT-Gp group.
Next, the cytokines in the sera fromthe vaccinated piglets were
tested by ELISA. Before immunization (0dpi), all of the detected
cytokine levels greatly varied between the four groups, except
for IFN-, which was less variable (180210pg/ml) (Fig. 4B). In
the sera of the PBS group, all of the above mentioned cytokines
remained unchangeable before and after immunization (Fig. 4B-
i). As shown in Fig. 4B-ii, Gp96N alone signicantly increased the
production of TNF- after the rst immunization (P<0.001, com-
pared to 0dpi, similarly hereinafter) and IL-12 after the second
immunization (P<0.05), with a slightly decreased level of IL-10
after the third immunization (P<0.05). However, Gp96Nalone had
no inuence on IL-4, IFN-, or IL-6 induction. In the BCE+TCE
peptide-immunization group (BT), a large decrease in the produc-
tion of IL-4 (P<0.05) was noted after the second immunization,
while novariationinthe other cytokines was foundbefore andafter
three immunizations (Fig. 4B-iii). In Fig. 4B-iv, IL-12 levels greatly
increased after the rst immunization (P<0.001), but TNF- was
not signicantly increased until the third immunization (P<0.001).
However, the production of IL-10 decreased by half after the third
immunization (P<0.05). In addition, peptides +Gp96N treatment
caused a large decrease in the production of IL-4 after the second
immunization (P<0.05), while no changes in IFN- and IL-6 were
found.
3.5. Clinical signs and viremia monitoring post-challenge
After challenge with virulent HP-PRRSV(JXwn06), all of the pigs
in the PBS and Gp groups displayed high fever (40.5

C) (Fig. 5A)
and lack of appetite from 1 dpc, which became worse in the fol-
lowing 3 days with the appearance of other clinical signs, including
lethargy, reddened skin, dyspnea, and periocular edema (Fig. 5B).
Further, all of the pigs in the PBS and Gp groups died at 4 dpc. Sim-
ilar lowintensity clinical symptoms were observed in the BT group
animals, which survived for one more day than the PBS group. The
pigs in the BT-Gp group displayed slight fever (39.540.4

C) and
a little uctuation in rectal temperatures during 15 dpc (Fig. 5A).
The scores of clinical symptoms in the BT-Gp group were signif-
icantly lower than those in the other groups at 34 dpc (P<0.05,
compared to the PBS group) (Fig. 5B). However, at 67 dpc, all of
the pigs presented with high fever (40.5

C) (Fig. 5A) and similar

clinical symptoms as those in the PBS and Gp groups at 34 dpc,
with death occurring at 7 dpc (Fig. 5B).
Viremia was monitored by viral TCID
50
titers in the serum of
pigs at 0, 3, 4, 5, and 7 dpc. The results demonstrated that the viral
1844 C. Chen et al. / Vaccine 31 (2013) 18381847
Fig. 5. Detection of clinical sign and viremia of the pigs after challenge with HP-
PRRSV isolate JXwn06. (A) Rectal temperature of the pigs, with values expressed
as the meanSD. (B) Clinical sign scores of pigs per group are expressed as the
meanSDof all clinical signs recorded daily post challenge, including attitude (03
points), respiratory rate (02points), respiratory distress (03points), andcoughing
(01points). (C) Theviremiaof thepigs is expressedas theviral titers inseradetected
at 0 (before challenge), 3, 4, 5, and 7 dpc. The data are shown as the meanSD for
four or three pigs per group. *P<0.05 by Fishers exact test (B) or Wilconxon test (C),
compared to the PBS group at each indicated date (3 and 4 dpc).
titers of live PRRSV in pigs in the BT-Gp group were signicantly
lower than that in the PBS or Gp groups at 3 and 4 dpc (P<0.05)
(Fig. 5C). The viral titer reached 10
5
TCID
50
/ml at 5 dpc, which was
no greater than that in the PBS or Gp groups at 4 dpc when all of
the pigs died.
3.6. Pathological examination
At 4 dpc, lungs fromall of the dead pigs inthe PBS and Gp groups
and one pig each from the BT and BT-Gp groups were examined
by Hematoxylin and eosin staining (HE). Results in Fig. 6 showed
that all the pigs fromthe four groups exhibited a signicant pneu-
monia, including inammatory cellular inltration (triangle and
arrow) and pulmonary congestion (hollow arrow). However, the
interstitial pneumonitis observed in the BT group was milder than
that in the PBS and Gp groups, and that in the BT-Gp group was the
mildest.
4. Discussion
Epitope-based vaccines have several advantages, such as safety,
a highdegree of specicity, andtheir ease andsafety of use [27]. The
great success of an epitope-based vaccine for FMDV has attracted
an increasing amount of research on clinical epitope-based vac-
cines for other diseases caused by viruses, such as HIV, HPV, and
dengue virus [2831]. However, no synthetic epitope-based vac-
cine against HP-PRRSV infection is currently available. The present
study represents the rst research into using synthetic conserved
B- and T-cell epitope peptides mixed with Gp96N as a promising
PRRSV vaccine. In addition, the genetic restriction of the vaccine
was overcome by the inclusion of PADRE, which is an articial gen-
eral Th cell epitope containing 13 residues [27]. The PADRE peptide
bothhas the capacity to increase antibody responses andto provide
signicant helper T-cell activity in vivo, suggested that it might be
efcient at generating an immune response with other multivalent
antigens [27]. Piglet immunization experiments showed that this
epitope-based vaccine could induce PRRSV-specic humoral and
cellular immune responses.
It is generally accepted that both cell-mediated immunity
(CMI) and anti-PRRSV NAs can clear PRRSV infection both in vitro
and in vivo. [17,3238]. First, the cellular immune response is
extremely powerful against PRRSV infection [17,3438]. In the
present study, a strong PRRSV-specic lymphocyte proliferative
responsewas exhibitedandenhancedbyGp96Ninpiglets as shown
using the lymphocyte proliferation assay (Fig. 3A), and each of
the seven peptides could induce strong TCE peptide-specic lym-
phocyte proliferative responses with Gp96N as adjuvant (group
BT-Gp) (Fig. 3B). However, the proliferation of the stimulated
PBMCs could partially be other peripheral cells, for instance,
CD3
+
CD8
high
cells [38]. Thus, another technique such as porcine-
IFN- ELISPOT assay should be applied to investigate the cellular
immune responses. But in the mouse model (Supplement mate-
rials Fig.S3), IFN-/ELISPOT assay was performed to determine
the TCE-peptide induced cell-mediated immunity. Notably, the
peptide-immunized mice (especially vaccinated with either Gp4-
170Tor Gp5-117Tor Gp5-149T) showedsignicant PRRSV-specic
IFN--secreting cells (IFN--SC) responses and all the mice with
great PRRSV-specic IFN--SC responses also had strong peptide-
specic IFN--SCresponses. Second, althoughthe two BCEpeptides
(Gp4-59B and Gp5-37B) could induce detectable BCE-specic and
PRRSV-specic IgG responses (Fig. 2), they could not activate the
protective NAs in piglet. However, these two BCE peptides pro-
duced a titer of 1:16 NAs with Gp96N as an adjuvant in mice
(Supplement materials Fig. S2). This phenomenon may be due to
the differences between species (mouse and pig). Thus, based on
results froma similar experiment inmice has suggestedthat mouse
is probable not a perfect animal model to evaluate the humoral
immunity induced by PRRSV vaccines.
To increase the efciency of the vaccine, immune regulatory
molecules (e.g., IL-2, IL-12, CD40 ligand, IFN-, and HSP70) are
co-delivered to up-regulate the immune response of vaccine anti-
gens [68,19,3941]. Like HSP70, Gp96 is a promising adjuvant to
enhance the innate and adaptive immune responses, especially to
exert potent T cell activation activity in designing vaccines against
HBV, HIV, or HPV [2023,42,43]. Upon engagement of its receptor
onantigenpresenting cells (APCs), Gp96 causes maturationof APCs
and secretion of the pro-inammatory cytokines TNF- and IL-12,
which mediate Th1 response enhancement [44,45]. The present
study showed that the Th1 type cytokine IL-12 levels were pro-
moted by Gp96N, but the other Th1 type cytokine IFN-, which
C. Chen et al. / Vaccine 31 (2013) 18381847 1845
Fig. 6. Pathological examination. At 4 dpc, the lungs of the pigs were examined by HE staining in the PBS (A), Gp (B), BT (C), and BT-Gp (D) groups, as well as a healthy lung
from an uninfected pig (E). Pulmonary alveoli were marked with hollowpentagon; solid triangle exhibited that foci inammatory cells inltrated into the pulmonary alveoli,
and inammatory cells inltrated into the lumens as hollow triangle or near the vessel of the bronchus as solid arrow; hollow arrow represented the blood congestion in
lung. Scale bar: 200m.
is related to the cell-mediated immunity against PRRSV, was not
inuenced by peptides, Gp96N, or peptides +Gp96N (Fig. 4B). The
Th2 type cytokine IL-4 was down-regulated by peptides but not
Gp96N, and the combination of peptides +Gp96N suppressed the
productionof IL-4after the secondimmunization(Fig. 4B). Further-
more, Gp96N inhibited the production of IL-10. It should be noted
that IL-10 is a potent immunosuppressive cytokine that can signif-
icantly inhibit production of several pro-inammatory cytokines
and down-modulate APC activities, resulting in inhibition of innate
and adaptive immunity, particularly the cell-mediated immune
responses [46,47]. Concerning the inammatory cytokines, Gp96N
or peptides did not alter the production of IL-6 (Fig. 4B). The other
innate anti-viral cytokine, TNF-, was signicantly up-regulatedby
Gp96N, but impaired by peptides because Fig. 4A showed that pep-
tides might contribute to the down-regulated TNF- in group BT.
In addition, the TNF- level did not greatly increase until the third
immunization (at 42dpi in the BT-Gp group, Fig. 4B-iv). There may
be other unknown reasons that the TCE-peptides would delay the
up-regulation of TNF- by Gp96N. Taken together, Gp96N was an
effective Th1-biased adjuvant to improve the innate and adaptive
immune responses induced by the epitope-based vaccine, which is
consistent with our previous report [25].
To determine the protective immune response induced by the
epitope-based peptides, the pigs were challenged with a high
dose of virulent HP-PRRSV isolate JXwn06. Temperature, clinical
symptoms, and viremia were monitored. Our results illustrate that
BCE+TCE peptides with Gp96N as an adjuvant provided greater
protection than BCE+TCE peptides alone during the rst 5 days
post HP-PRRSV infection. However, PRRSV infection was not fully
prevented in that all of the pigs in the BT-Gp group died at 7 dpc
witha hightiter of live PRRSVintheir sera (Fig. 5C) andserious clin-
ical symptoms (Fig. 5A and B, Fig. 6). One reason for this could be
that HP-PRRSV JXwn06 is a highly pathogenic isolate and causes
severe disease that leads to 100% mortality in 6-week-old pigs
within 513 days and 80% mortality in 12-week-old pigs [48]. In
this study, the piglets were 10 weeks old when challenged, which
might be relatedthe deathof the piglets. Additionally, the highdose
of HP-PRRSV JXwn06 challenge is another probable cause for the
death of the infected piglets. The viral dosage was 210
5
TCID
50
in the present study, which is two times greater than the previous
report [48]. Based on the high level of mortality with 10
5
TCID
50
of HP-PRRSV infect, twice more dosage of virulent challenge virus
may cause much more serious mortality. This is the probable rea-
son for the 100% mortality at 7 dpc in the present study. Thus,
1846 C. Chen et al. / Vaccine 31 (2013) 18381847
we hypothesize that a lower dosage of HP-PRRSV challenge might
better exhibit the protective effect of the epitope-based vaccine
with a much longer infection time course. Furthermore, the lack of
detectable PRRSV-specic NAs and protective cytokines (especially
IFN-) after challenge did not uncover the correlation between the
viral load and the levels of NAs or IFN-.
In conclusion, the present study for the rst time developed
and validated an epitope-based synthetic peptide vaccine using
Gp96N as an adjuvant in the piglet model. The BCEs and TCEs were
designedbasedontheconservedsequences intypeII PRRSVstrains,
which are the critical domains that induce robust cellular immu-
nity. We demonstrated that Gp96N enhances the humoral and
cell-mediatedimmune response witha Th-1type cytokine immune
responsebias. HP-PRRSVchallengeexperiments demonstratedthat
piglets vaccinated with the mixture of peptides and Gp96N exhib-
ited slight fever, lighter clinical signs, lower viremia, and less
pathological lung lesions during the early stage of viral infection
(15 dpc). However, this vaccine could not provide lasting and
effective protection against HP-PRRSV infection. The novel peptide
vaccine may provide some important information for understand-
ing the immune responses against PRRSV infection and may be
helpful for the development of a multi-epitope peptides vaccine.
Furthermore, porcine Gp96N355 may be used as anattractive adju-
vant or immune-targeting strategy to enhance the PRRSV vaccine
responses in swine.
Acknowledgements
We gratefully acknowledge the workers of Daxing Swine Farm
for their helpwithpigimmunizationandsamplecollection. We also
thank Dr. Maorong YuandDr. Chongfeng Xufor helpful suggestions
on our manuscript. This work was supported by grants from the
National High Technology Research and Development Program of
China (863 Program) (2011AA10A215).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.
2013.01.049.
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