Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 27-36
doi:10.1111/j.1745-7270.2007.00243.x
Vaccination against Schistosoma
japonicum Infection by DNA Vaccine Encoding Sj22.7 Antigen
Gan DAI, Shiping WANG*,
Junlong YU, Shaorui XU*, Xianchu PENG, Zhuo HE, Xueqin LIU, Songhua ZHOU, and
Fen LIU
Institute
of Schistosomiasis Research, Xiangya School of Medicine, Central South
University, Changsha 410078, China
Received: July 25,
2006
Accepted: October
17, 2006
This work was
supported by the grants from the China National “Tenth Five-Year
Plan” Important Special Program (2002AA2Z3343), the Hi-Tech Research and
Development Program of China (2004AA2Z3530, 2004AA2Z3522), the Hunan Province
Eleventh Five-Year Plan Important Special Program (2006SK1001) and the Hunan
Province “Tenth Five-Year Key Xueke Plan” (03-985-3-7)
*Corresponding
authors:
Shi-Ping WANG: Tel,
86-731-2355001; E-mail, [email protected]
Shao-Rui
XU: Tel, 86-731-2355001; E-mail, [email protected]
Abstract To observe the in vitro expression
of DNA vaccine pcDNA3-Sj22.7 and its immunological effect in mice, the
recombinant plasmid pcDNA3-Sj22.7 was transfected into HeLa cells with
liposome-mediated method and the expression of Sj22.7 mRNA and protein was
examined using reverse transcription-polymerase chain reaction, sodium
dodecylsulfate-polyacrylamide gel electrophoresis and Western blot,
respectively. Then, the ability of pcDNA3-Sj22.7 to protect against Schistosoma
japonicum challenge infections was analyzed according to worm reduction
rate and egg reduction rate after vaccination of mice. The serum levels of
specific IgG antibody and T lymphocyte proliferation response were also
determined. By the culture of spleen cells after the challenge infection,
Sj22.7-driven interferon (IFN)-g and interleukin (IL)-4 was
also quantified. Results showed that pcDNA3-Sj22.7 could express Sj22.7 mRNA
and protein in vitro. Immunization resulted in a worm reduction rate of
29.70%, egg reduction rate of 47.25% (livers) and 51.73% (intestine), and egg
reduction rate of 25.90% (eggs per female), suggesting induction of significant
anti-fecundity in the pcDNA3-Sj22.7 group. Enzyme-linked immunosorbent assay
and Western blot analysis indicated that immunized mice generated specific IgG
against Sj22.7. T lymphocytes from mice immunized with pcDNA3-Sj22.7 showed a
significant proliferation response to rSj22.7. The culture of spleen cells
showed that secretion of IFN-g increased but IL-4 decreased.
The results indicate that DNA vaccination by pcDNA3-Sj22.7 is sufficient to
elicit significant levels of protective immunity against S. japonicum infection.
The DNA vaccine could induce significant cellular and humoral immune response,
and display predominant T helper cell type 1 type immune responses, which
contribute to the protective immunity against challenge infection in mice.
Key words Schistosoma japonicum; DNA vaccine; Sj22.7; gene
expression; immune protection; immune response
Schistosomiasis is, after malaria, the second most important
parasitic disease in tropic areas. It affects more than 200 million people and
causes more than 500,000 deaths each year [1–4]. A vaccine against this
parasitic infection is desirable to prevent infection. Vaccination can be
targeted towards either the prevention of infection or the reduction of
parasite fecundity. A reduction in worm numbers is the “gold
standard” for anti-schistosome vaccine development. However, as
schistosome eggs are responsible for both pathology and transmission, a vaccine
targeted on parasite fecundity and egg viability seems to be entirely relevant.
Recently, a significant effort has been made to develop a protective vaccine
against schistosome infections, and several vaccine candidates and related
studies have been identified [5–7]. As the efficacy of any of these vaccines
against schistosomiasis remains uncertain, the identification and
characterization of new anti-schistosome vaccine molecules remains a priority.
The development of vaccine remains to be an important long term and challenging
goal in the control of schistosomiasis [8]. DNA vaccine is an attractive and novel immunization strategy against
a wide range of infectious diseases and tumors. Injection of plasmid DNA as
vaccine was first shown to be effective using influenza as a model, where the
results showed that DNA encoding nucleoprotein induced cytotoxic T lymphocytes
and cross-strain protection of mice [9]. The effectiveness of DNA vaccines
against viruses, parasites and cancer cells has been shown in animal models
[10]. It has been shown that DNA immunization induces both antigen-specific
cellular and humoral immune responses [11]. Nucleic acid vaccination against
schistosomiasis has lately been investigated using a panel of plasmids encoding
schistosome antigenic proteins such as Sjc26GST, Sj79 [12,13], Schistosoma
japonicum paramyosin [5,14] and Schistosoma mansoni 23, 28
GST [15]. In the previous study, we identified a gene encoding a
pairing-associated protein identical to the Sj22.7 adult worm antigen using
two-dimensional gel electrophoresis and mass spectrometry [16]. Several
investigators have suggested that the 22.7 kDa-antigens of S. japonicum
adult worms are important vaccine candidates [17]. However, no direct
vaccination/challenge experiments using Sj22.7 had been previously described. Herein, we produced constructed Sj22.7 DNA vaccine pcDNA3-Sj22.7 and
tested its vaccination strategies in mice.
Materials and Methods
Parasites and animals
The snails used for schistosome infection were obtained from Hunan
Provincial Institute of Schistosomiasis Research (Yueyang, China). Cercariae
were collected from exposing infected snails. Female Kunming mice (18–20 g, 6–8 weeks old)
were purchased from the Department of Zoology, Xiangya School of Medicine,
Central South University (Changsha, China). All animals were maintained in our
animal facilities for the duration of the experiments. Animal experiments were
performed according to the protocols approved by Central of Animal Health of
Xiangya Animal Care and Use Committee.
Construction and preparation
of DNA vaccines pcDNA3-Sj22.7
The cDNA containing the entire coding region of Sj22.7 gene (GenBank
accession No. AY815219) was isolated from schistosomula cDNA library. Briefly,
a pair of primers was synthesized according to the DNA sequence of Sj22.7. The
forward primer was P1, 5‘-GCGGTACCACTAACATGGGAGAAGAGA-3‘
and the reverse primer P2, 5‘-ACGGGCCCCCTAAATGTCCTGATTACCTC-3‘
containing KpnI and ApaI restriction sites (italicized),
respectively. The target gene was subcloned into the eukaryotic expression vector
pcDNA3 (Invitrogen, Carlsbad, USA). This vector containing a strong human cytomegalovirus
promoter, an ampicillin-resistance gene and a neomycin-resistance gene was used
as a DNA vaccine vector. The Sj22.7 was amplified using a Hema 480 thermal
cycler in a total volume of 100 ml mixture with 2 mM of each primer (P1 and
P2), 0.2 mg of cDNA library of S. japonicum adult worm as template, 0.2
mM of deoxyribonucleotide triphosphate (TaKaRa, Tokyo, Japan) and 1 U of Taq
polymerase (TaKaRa). The PCR was performed first by 95 ?C for 3 min; then 35
cycles of 94 ?C for 1 min, 55 ?C for 1 min and 72 ?C for 2 min; and finally 72
?C for 8 min. The KpnI/ApaI fragment (622 bp) was purified using
Agar gel DNA purification kit (TaKaRa) and then ligated to the pcDNA3 vector to
obtain recombinant plasmid pcDNA3-Sj22.7. The recombinant plasmid was transformed
into Escherichia coli DH5a competent cells and identified by restriction
enzymes digestion, PCR and sequencing.
In vitro transient transfection of
HeLa cells
To estimate the efficacy of recombinant plasmid pcDNA-Sj22.7 to
produce the 22.7 kDa-protein in mammalian cells, the recombinant plasmid was
transfected into HeLa cells. Briefly, HeLa cells were seeded into 24-well
tissue culture plates at 1.5?105 cells per well and grown to 90% confluence in DMEM (Gibco,
Rockville, USA) supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin (Amresco, Solon, USA) and 10% (V/V) heat
inactivated fetal calf serum (FCS) (Hyclone, Logan, USA) at 37 ?C in 5% CO2. Then, the cells were transfected with the plasmid DNA with
Lipofectamine 2000 (Invitrogen) at the ratio of 1 mg DNA:2 ml lipid per well
in serum-free DMEM at 37 ?C for 12 h. Cells were then grown for 48 h in DMEM
containing FCS. The mRNA and protein levels of Sj22.7 were determined by RT-PCR
and indirect immunofluorescence microscopy, respectively. Briefly, the slides
with transfected HeLa cells were incubated with rabbit anti rSj22.7 serum
(1:100, prepared in our lab) and then with goat anti-rabbit IgG conjugated to
fluorescein isothiocyanate (FITC) (1:10, Boster, Beijing, China). After washing,
slides were examined under a fluorescent microscope (Olympus, Tokyo, Japan).
When yellow green fluorescence appeared in the transfected cells, the reaction
was defined as indirect fluorescent antibody test (IFAT) positive. HeLa cells
transfected with the empty plasmid pcDNA3 and sera from normal mice were used
as negative controls, respectively.
DNA vaccination
The mice were divided randomly into three groups, and each group consisted
of 25 mice. Mice were immunized by intramuscular injection with one kind of the
following regimens in 100 ml of sterilized normal saline: (1) 100 mg of pcDNA3-Sj22.7, (2) 100
mg
of pcDNA3, or (3) control with no plasmid DNA. The mice in the third group
served as a challenge infection control. Twenty-four hours after being injected
with 50 ml of 50 mg/ml bupivacaine hydrochloride in the quadriceps femoris
muscles (hind legs), mice were inoculated intramuscularly with 100 mg of empty
plasmid pcDNA3 or recombinant plasmid pcDNA3-Sj22.7 in the same area. Mice were
immunized 3 times (2 weeks interval).
Expression of the DNA vaccine
in musculature of immunized mice
Two weeks after the last immunization, two mice from each group were
killed and parts of the liver tissues were immediately fixed in 10% buffered
formalin solution and processed in paraffin blocks. The sections of 5 mm were cut on
albuminized glass slides. Expression of Sj22.7 in vivo was examined by
indirect immunofluorescence. Sections were stained by immunofluorescence
method using rabbit anti-rSj22.7 serum (1:100 dilution in PBS-T) and FITC
conjugates of goat anti-rabbit IgG (1:10). Fluorescence was observed with an
immunofluorescence microscope.
Blood sample collection for
antibody assay
Blood samples were collected from tail veins of all mice before
immunization and thereafter at 2, 4 and 6 weeks. Pooled serum samples were
prepared from each group by mixing an equal volume of serum from each group,
then used for an enzyme-linked immunosorbent assay (ELISA) and Western blot
analysis.
Enzyme-linked immunosorbent
assay
To determine the anti-Sj22.7 antibody titer in collected sera from mice
vaccinated with pcDNA3-Sj22.7 and control. The pre-immune and post vaccination
sera were tested for specific IgG antibody level by ELISA. The antigen used in
ELISA was Sj22.7 purified protein [18]. The secondary antibody used in the
ELISA was alkaline phosphatase-conjugated goat anti-mouse IgG (Promega,
Madison, USA). Color reaction was developed by the addition of o-phenylenediamine
(OPD) (Sigma, St. Louis, USA) in citrate phosphate buffer and stopped with 50 ml of 5% sulfuric
acid per well. The absorbance was read at 490 nm using an ELISA reader (BioRad,
Hercules, USA).
Western blot analysis
S. japonicum adult worm antigen (AWA)
was prepared in our lab to confirm the specificity of antibodies to
pcDNA3-Sj22.7 [19]. AWA was separated by 12% sodium
dodecylsulfate-polyacrylamide gel electrophoresis. After electrophoresis, the
gels were either Coomassie brilliant blue R-250-stained to visualize the
protein bands or transferred onto nitrocellulose membrane to analyze using
Western blot. For Western blot, the membrane was blocked with 5% skimmed milk
in PBS containing 0.5% Tween-20 (blocking solution). Vaccinated serum
samples (1:200) were used as the
primary antibody and peroxidase-conjugated goat anti-mouse IgG (1:5000)
(Amresco) was used as the secondary antibody. Specific binding was detected
with H2O2 and diaminobenzidine (DAB) (Sigma) as a chromogenic substrate.
Preparation and cultivation of
spleen cells
Two weeks after the last immunization, three mice from each group
were killed and their spleens were removed under aseptic conditions. The
suspension of single spleen cells was prepared after removing erythrocytes by
hypotonic lysis and resuspended in RPMI 1640 (Gibco) by vigorous pipetting.
The cell suspension was added into the 96-well flat-bottomed tissue culture
plates (Sigma) at 200 ml/well, then cultured at 37 ?C in a humidified atmosphere with 5%
CO2.
T-lymphocyte proliferation
assay
The T-lymphocyte proliferation [20] was detected with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Spleen cell suspensions from immunized and control mice
were prepared in RPMI 1640 supplemented with 10% FCS. Splenocytes were cultured
at 37 ?C with 5% CO2 in a 96-well tissue culture plate at a
concentration of 5?105
cells/well in the presence of medium, 10 mg/ml recombinant antigen or
5 mg/ml
concanavalin A (ConA) (Sigma). The cells were cultured for 3 d followed by
incubation with 10 ml of MTT per well for 4–6 h. After incubation, 100 ml of dimethyl
sulfoxide (DMSO) was added into each well. The plates were shaken slowly for
10 min. The absorption at 570 nm of each well was measured using a microtiter
plate reader (BioRad).
Challenge infection and
parasite loads
Two weeks after the last immunization, mice were challenged with
40±1 normal S. japonicum cercariae by abdominal skin penetration.
All mice were killed and perfused at day 42 after infection, and the
immunoprotection was assessed by worm reduction rate and egg reduction rate.
The egg counts in livers and intestines of the mice were determined by
microscopic examination after digestion with 4% potassium hydroxide for 16 h
at 37 ?C. Fecundity was expressed as the number of eggs per female worm. The
ratio of the liver eggs versus intestinal eggs was also determined. Control
mice were not immunized but treated identically. For each group of mice, total
egg counts were expressed as the number of eggs per gram of mouse liver or
intestine.
Histopathological examination
After killing animals in different groups, parts of the liver
tissues were immediately fixed in 10% buffered formalin solution and processed
in paraffin blocks. The sections of 5 mm were cut on albuminized glass slides and stained
by hematoxylin and eosine for routine histopathological examination for
counting granuloma and size. Liver egg-granulomas were counted in five
successive low power fields (10), and their diameters were measured using
graduated eyepiece lens, considering only lobular granulomas containing ova in
the center. Two perpendicular maximal diameters were measured, getting the mean
diameter for each granuloma, and then calculating the mean granuloma diameter
for the group.
Cytokine detection
Six weeks post-infection, spleen cells were cultured in RPMI 1640
containing 10% FCS on stimulation with 5 mg/ml Con A or 10 mg/ml recombinant
antigen (rSj22.7) at 37 ?C with 5% CO2. Supernatant fluids were
collected at 72 h, and IFN-g and IL-4 assay were carried out using ELISA kits according to the
manufacturer’s instructions (Boster). The cytokine concentrations were
calculated according to the standard curve.
Statistical analysis
Data were expressed as mean±SD. P<0.05 determined by ANOVA or Student's t-test was considered significant.
Results
Construction and
identification of DNA vaccine
The entire coding sequence of the Sj22.7 amplified from cDNA adult
worm library using primers P1/P2 was approximately 622 bp (Fig. 1). The
fragment was cloned into the expression vector pcDNA3 digested with the same
restriction enzymes KpnI and ApaI to construct
recombinant plasmid pcDNA3-Sj22.7. With the pcDNA3-Sj22.7 as a template and P1/P2
as primers, the product of PCR showed a similar size to the insert (Fig. 2).
The identification was confirmed by sequencing.
Transient expression of Sj22.7
The transient expression of Sj22.7 in transfected HeLa cells was determined
using RT-PCR. PCR product of the expected size (622 bp) was obtained using cDNA
derived from transfected cells as a template. To eliminate the possibility
that the Sj22.7 PCR product was produced because of plasmid DNA contamination,
RNA alone was used as a template for PCR. No product was amplified without
reverse transcription of the RNA. Similarly, no product was obtained using cDNA
derived from cells transfected with control plasmids or cells without
undergoing transfection as a template (Fig. 3). The presence of Sj22.7
in the plasma and on the surface of transfected cells was confirmed by
immunofluorescence microscopy (Fig. 4). These results showed that the
pcDNA3-Sj22.7 construct could be expressed in mammalian cells.
Expression of DNA vaccine in
muscle
To determine whether the Sj22.7 protein could be expressed in the
muscle after the injection of pcDNA3-Sj22.7, indirect immunofluorescence assays
were carried out. Results showed that Sj22.7 could express in muscle following
intramuscular injection with pcDNA3-Sj22.7 (Fig. 5).
Antibody response to DNA
vaccination
The presence of antigen-specific IgG antibodies was examined in the
serum from immunized mice using Western blot analysis. An S. japonicum
antigen of 22.7 kDa from AWA was strongly recognized by serum from mice
vaccinated with pcDNA3-Sj22.7 (Fig. 6, lane 1), showing that a specific
humoral response against the Sj22.7 protein. No schistosome antigen was
recognized by the serum from mice vaccinated with pcDNA3 (Fig. 6, lane
2). Serum obtained from mice at weeks 0, 2, 4 and 6 was analyzed quantitatively
by ELISA for the levels of total IgG. In agreement with the results of Western
blot analysis, high levels of anti-Sj22.7 total IgG were detected in the serum
from mice vaccinated with pcDNA3-Sj22.7 after 6 weeks (Fig. 7).
T-lymphocyte proliferation
response
The T-lymphocyte proliferation responses are shown in Fig. 8.
T lymphocytes from mice immunized with pcDNA3-Sj22.7 showed a significant proliferation
response to ConA or rSj22.7 (P<0.01, compared with medium or pcDNA3 group).
Protection induced by DNA
vaccine
Vaccinated and control mice were challenged with 40±1 cercariae each
and the number of worms recovered 6 weeks later was assessed. The results
related to the effect of the DNA vaccine on mouse pathology and parasite
development are summarized in Table 1. At the time of perfusion, the
bodyweight of vaccinated and challenged mice was significantly heavier than
that of non-vaccinated infected control animals, suggesting an overall better
health status. The egg counts showed a significant difference either in terms
of reduced worm burden (–29.70%, P<0.01) or number of present eggs, isolated from the liver or mesentery. The animals showed a significant protection in mice following challenge infection. There was a subsequent reduction in the number of eggs in the liver (–47.25%, P<0.05) and intestine (–51.73%, P<0.05) of the pcDNA3-Sj22.7 immunized group when compared with the pcDNA3 blank vector group. A significant reduction in the fecundity of the parasites (e.g., number of eggs per female worm; –25.90%, P<0.05) was observed in the liver and intestine after vaccination with pcDNA3-Sj22.7. Taken together, the Sj22.7 DNA vaccine permitted a better growth of mice and reduced worm burden, egg number and worm fecundity.
Histopathological changes in
granuloma
Liver sections of both immunized and control groups at 6 weeks post-infection
were studied for granuloma count and size. The histopathological examination
showed a significantly greater number of egg granulomas in the control group
than in the immunized group. The diameter of granuloma was significantly larger
in the control group compared with the immunized group (Fig. 9 and Table
2). In addition, the percentages of degenerated ova were higher in the
immunized group compared with the control groups.Histopathological examination of vaccinated and control infected mice
were carried out using hematoxylin and eosin stain. Sections of group immunized
with pcDNA3-Sj22.7 showed less number and smaller egg granuloma usually formed
by the central egg surrounded by some mononuclear inflammatory cells and few
eosinophils. In contrast, the control groups showed greater number of portal
egg granulomas formed of an ovum surrounded by large number of eosinophils,
neutrophils and histiocytes (Fig. 10).
Cytokine responses
The cytokine profile induced in pcDNA3-Sj22.7 vaccinated or control
animals was measured. ELISA results obtained from culture supernatants
harvested at 72 h showed that, in response to rSj22.7, spleen cells from the
pcDNA3-Sj22.7 group produced higher level of T helper cell type 1
(Th1)-associated cytokine IFN-g and lower level of the T helper cell type 2 (Th2)-associated
cytokine IL-4 (P<0.05) as shown in Fig. 11. Splenocytes of
immunized mice produced an average of 100.67 pg/ml of IFN-g and 47.32 pg/ml
of IL-4 following ConA stimulation (120.67 pg/ml and 68.29 pg/ml
respectively).
Discussion
Nucleic acid immunization can be an effective vaccination technology
that delivers DNA constructs encoding specific immunogens into host cells, inducing
both antigen-specific humoral and cellular immune responses. Since the first
demonstration of protective immunity against viral challenge induced by DNA
vaccination using a plasmid DNA encoding influenza A nucleoprotein, various
degrees of success has been achieved, and the main methods of plasmid-DNA
application are intramuscular injection and intradermal delivery into skin
[21]. In the case of schistosomiasis, vaccination with DNA has shown to induce
immune responses in rats, and partial protection against challenge in mice
[22], underlining the potential of this method of vaccine delivery for this
disease. Previously, we identified a gene encoding a pairing-associated protein
identical to the Sj22.7 AWA by two-dimensional gel electrophoresis and mass
spectrometry [16]. Several investigators have suggested that the 22.7-kDa
antigens of S. japonicum adult worms are important vaccine candidates
[17]. In the present study, we produced the Sj22.7 DNA vaccine pcDNA3-Sj22.7,
tested the pcDNA3-Sj22.7 vaccination strategies in mice and assessed the
immunogenicity and protective efficacy of Sj22.7 as a DNA vaccine.To test whether pcDNA3-Sj22.7 was able to express the schistosome
antigen Sj22.7 in mammalian cells, transfection experiments were carried out
with HeLa cells as recipient cells. The Sj22.7 gene was successfully
transcribed in the transfected HeLa cells. The results of IFAT showed that the
Sj22.7 gene was expressed in HeLa cells transfected with pcDNA3-Sj22.7. The
presence of Sj22.7 in the plasma and on the surface of transfected cells was
also confirmed by immunofluorescence microscopy. These results showed that the
pcDNA3-Sj22.7 construct could be expressed and lead to the direct synthesis of
the immunogen in eukaryotic cells. To determine whether the Sj22.7 protein was
expressed in the muscle after the injection of pcDNA3-Sj22.7, indirect immunofluorescence
assays were carried out. Results showed that Sj22.7 could be expressed in
muscle followed by intramuscular injection with plasmid DNA encoding Sj22.7.
Therefore, the schistosome Sj22.7 was for the first time successfully expressed
in mammalian cells both in vitro and in vivo. To determine if the pcDNA3-Sj22.7 vaccine conferred protection
against S. japonicum, all animals in each groups were challenged
with 40±1 cercariae 2 weeks after the last immunization, and 6 weeks later,
worm/egg burden was analyzed. In all cases, the pcDNA3-Sj22.7 vaccine conferred
a significant protection in mice against challenge infection, which leads to a
worm reduction rate of 29.70% and an egg reduction rate of 47.25% (liver) and
51.73% (intestine). The reduction in worm burden in animals immunized with
pcDNA3-Sj22.7 was also significantly higher than in animals immunized with the
control. The administration of pcDNA3-Sj22.7 by intramuscular inoculation in
the current study resulted in expression in vivo, which induced specific
antibodies in mice as detected by Western blot analysis and ELISA. Vaccination
can be targeted towards the prevention of infection or to the reduction of
parasite fecundity. A reduction in worm numbers is the “gold
standard” for anti-schistosome vaccine development but, as schistosome
eggs are responsible for both pathology and transmission, a vaccine targeted
on parasite fecundity and egg viability also seems to be entirely relevant
[23]. The effective vaccine would prevent the initial infection and reduce egg
granuloma associated pathology [24,25].DNA vaccine pcDNA3-Sj22.7 could induce significant cellular and
humoral immune response. High levels of anti-Sj22.7 total IgG were detected in
the serum from mice vaccinated with pcDNA3-Sj22.7 after 4 weeks. Thus,
pcDNA3-Sj22.7 vaccination successfully induced the production of specific
anti-Sj22.7 antibodies in mice. Simultaneously, T lymphocytes from mice
immunized with pcDNA3-Sj22.7 showed a significant proliferation response to
rSj22.7. To what extent the vaccine-induced humoral or cellular immune
responses are involved in the protective effects needs to be investigated. Both
might be required [26,27]. It is believed that S. japonicum infections
induce an immune response dominated by Th2 cells, whereas a Th1 predominant
response strongly correlates with resistance to infection [13]. A key
distinction between the Th1 versus Th2 pathways lies in the source of
different cytokines involved [28]. Th1 responses are typically characterized
by the secretion of IFN-g and IL-2. However, Th2 responses are characterized by the secretion
of IL-4, IL-5, IL-6 and IL-10. The vaccination-induced Th1-type response plays
an important role in anti-schistosome infection by producing cytokines, such
as IFN-g and IL-2 [29,30]. It has been shown that, at an early stage of
infection, the host’s response against the parasite is a Th1-type one. Epidemiological
surveys of schistosomiasis showed that the individual with a high level of IFN-g was
significantly correlated with resistance to schistosome infection [31]. In
animal models with schistosome infection, it has been observed that IFN-g can suppress
granuloma formation in vivo, and decrease the size of pulmonary
granulomas and the extent of hepatic fibrosis [32,33]. However, the data suggest a role of Th2-type cytokines for hepatic fibrosis in human schistosomiasis mansoni [34]. In the present study, we
found that spleen cells from the pcDNA3-Sj22.7 group produced abundant amounts
of Th1-associated cytokine IFN-g and significant reduction of the Th2-associated cytokine IL-4 (P<0.05). In conclusion, pcDNA3-Sj22.7 is a promising DNA vaccine that can
elicit a protective immunity efficacy against S. japonicum infection
in mice. In addition, the pcDNA3-j22.7 showed potential as a DNA vaccine and
anti-fecundity vaccine. The intramuscular immunization with pcDNA3-Sj22.7 was
able to induce the humoral and cellular immune responses in mice. We have
confirmed that the DNA vaccine could induce strong Th1 responses that enhance
protective immune responses against schistosomiasis. These data suggested a
role for Sj22.7 as a vaccine candidate or as a novel target for
anti-schistosome drugs.
Acknowledgement
We thank Zhongdao WU (Medical college of
Sun Yat-Sen University, Guangzhou, China) for kindly presenting S. japonicum
cDNA library, plasmid and bacteria.
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