The nature and combination of subunits used in epitope-based Schistosoma japonicum vaccine formulations affect their efficacy
- Xuefeng Wang†1, 2,
- Lei Zhang†1,
- Ying Chi1,
- Jason Hoellwarth3,
- Sha Zhou1,
- Xiaoyun Wen1,
- Lei He1,
- Feng Liu1,
- Calvin Wu3 and
- Chuan Su1Email author
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 26 August 2010
Accepted: 19 November 2010
Published: 19 November 2010
Schistosomiasis remains a major public health problem in endemic countries and is caused by infections with any one of three primary schistosome species. Although there are no vaccines available to date, this strategy appears feasible since natural immunity develops in individuals suffering from repeated infection during a lifetime. Since vaccinations resulting in both Th1- and Th2-type responses have been shown to contribute to protective immunity, a vaccine formulation with the capacity for stimulating multiple arms of the immune response will likely be the most effective. Previously we developed partially protective, single Th- and B cell-epitope-based peptide-DNA dual vaccines (PDDV) (T3-PDDV and B3-PDDV, respectively) capable of eliciting immune responses against the Schistosoma japonicum 22.6 kDa tegument antigen (Sj22.6) and a 62 kDa fragment of myosin (Sj62), respectively.
In this study, we developed PDDV cocktails containing multiple epitopes of S. japonicum from Sj22.6, Sj62 and Sj97 antigens by predicting cytotoxic, helper, and B-cell epitopes, and evaluated vaccine potential in vivo. Results showed that mice immunized with a single-epitope PDDV elicited either Tc, Th, or B cell responses, respectively, and mice immunized with either the T3- or B3- single-epitope PDDV formulation were partially protected against infection. However, mice immunized with a multicomponent (3 PDDV components) formulation elicited variable immune responses that were less immunoprotective than single-epitope PDDV formulations.
Our data show that combining these different antigens did not result in a more effective vaccine formulation when compared to each component administered individually, and further suggest that immune interference resulting from immunizations with antigenically distinct vaccine targets may be an important consideration in the development of multicomponent vaccine preparations.
Schistosomiasis is one of the most important neglected tropical diseases (NTDs) and remains a major public health problem in endemic countries [1, 2]. Although schistosomiasis can be treated with praziquantel , the high re-infection rate limits the overall success of chemotherapy which typically needs to be readministered multiple times during the first two decades of life [4, 5]. Therefore, the development of a safe, effective vaccine could improve long-term control of schistosomiasis and improve the efficacy of chemotherapeutic interventions [6–8].
Vaccination with radiation-attenuated cercariae induced significant levels of resistance to schistosome challenge via Th1- and Th2-mediated responses in animal models of disease. However, multiple concerns regarding this method make it unsuitable for human use [9, 10]. Considerable efforts have been aimed at the identification of relevant (immunoprotective) schistosome antigens resulting in the identification of potential vaccine targets [6, 11, 12]. The major challenge in the development of anti-schistosome vaccines is to use defined antigens to stimulate the appropriate immune response that lead to protection. Although the S. japonicum Sj22.6 , Sj62 , and Sj97  antigens, which are all important components of schistosome adult worm antigens (SWA), have been shown to be promising vaccine candidates, other approaches have focused on eliciting specific B-cell and Th-cell responses by identifying different antigenic determinants in potential vaccine targets [16, 17]. Epitope-based vaccines offer the prospect of targeted immunity resulting in safer and more effective antigen-specific immune responses . Previously we developed partially protective Th-, and B-cell epitope vaccines derived from the Sj22.6 or Sj62 antigens, respectively. However, the levels of protection induced by both vaccines were limited.
In addition, type I CD8+ T cells (effector CD8+ T cells), which produce INF-γ, have been proposed to play an immunoregulatory role during schistosomiasis by dampening immunopathologic type 2 responses [19, 20]. Studies of the Sm28GST vaccine suggest that both CD4+ and CD8+ T cells might contribute to protection. Activation of Sm28GST-specific CD8+ T cells produced high levels of gamma interferon (IFN-γ) involved in protective immune responses, which suggest that CD8+ T-cell response induced by an antigen from the extracellular parasite S. mansoni may protect the mice from infection [21, 22].
Currently, there are numerous efforts focused on optimizing schistosome vaccines (and vaccines against other infectious agents) using multiple-antigen or multiple-epitope design [23–26]. One strategy consists of designing subunit constructs containing defined B- and T-cell stimulatory epitopes obtained by genetic engineering or by chemical synthesis [27, 28]. In some experimental models, anti-repetitive peptide responses have been able to confer immune protection against infection [29, 30].
In this report we used the full-length S. japonicum vaccine candidates Sj22.6, Sj62 and Sj97 to generate eight distinct computer-based eptiopes identified by their potential for eliciting Tc-, Th-, or B-cell responses, respectively, using computer-based epitope-predicting software. All eight epitopes (named C1, C2, C3, B1, B2, B3, T2 and T3) were synthesized and encapsulated with the corresponding recombinant eukaryotic plasmid DNA encoding the corresponding epitope, respectively, to construct a peptide-DNA dual vaccine (PDDV) that has an antigenic peptide "shell" and a plasmid "nuclei". These pseudotype virus-like particles have revealed tremendous potential as novel delivery systems to enhance cell-specific gene delivery [31, 32] and efficiently stimulate the host immune reponses [33, 34]. We examined whether multicomponent PDDVs consisting of Tc (C)-, Th (T)- and B-cell (B) epitopes were more effective formulations against S. japonicum challenge than T- or B-cell single-epitope PDDVs. Our data showed that vaccination of mice with single-epitope-PDDV elicited corresponding immune responses - i.e., cytotoxicity, proliferation, or antibody production, respectively - and that vaccination with T3- or B3-PDDV induced partial protection. However, vaccination of mice with multicomponent PDDV formulations comprised of multiple epitopes produced variable immune responses that failed to induce better protection than T3- or B3- single-epitope PDDVs.
Epitopes and their encoding DNA sequences
Design of peptides used in the construction of the PDDVs.
Amino acid sequence of 18 Lys and the 18 Lys fused epitopes synthesized for PDDV formulations
Oligonucleotide sequences used for plasmid construction
Sj 97 (489-510)
Sj 22.6 (77-97)
Sj 62 (167-188)
C2- and C3-PDDV induced the cytotoxic effect and elicited antibody and IFN-γ production in C57BL/6 mice
T3-PDDV induced both cellular and humoral immune responses in C57BL/6 mice
B3-PDDV induced the highest antibody response in C57BL/6 mice
Cytokine and antibody responses in mice vaccinated with CTL-, T- and B-PDDV cocktails
PDDV cocktails did not improve the resistance to S. japonicum infection
Worm and egg burdens following vaccination.
Mean worm count ± SD
Mean liver egg count ± SD
Worm reduction rate (%)
Liver egg reduction rate(%)
Compared to the PBS group
Compared to the 18K group
Compared to the PBS group
Compared to the 18K group
31.33 ± 6.66
20900.9 ± 1899.84
24.50 ± 4.23
18558.54 ± 2120.31
14.50 ± 8.39
10270.27 ± 4357.82
23.71 ± 7.18
20308.88 ± 3154.43
20.40 ± 6.47
21513.51 ± 4128.92
18.86 ± 3.35
15366.78 ± 2371.41
24.80 ± 6.98
19243.24 ± 2172.86
18.75 ± 1.73
20000.0 ± 2167.85
22.57 ± 7.14
22162.16 ± 4496.31
25.75 ± 6.70
18648.65 ± 4105.15
20.00 ± 6.40
12540.54 ± 4658.24
21.33 ± 2.83
15855.84 ± 4944.18
23.33 ± 6.41
21711.70 ± 3937.61
23.88 ± 7.91
23243.24 ± 4784.20
Histopathology of egg granulomas in mice livers
The development of vaccines for complex parasites such as schistosomes is a great challenge. Vaccination with radiation-attenuated cercariae induces significant levels of resistance to schistosome challenge and suggests the fact that vaccination induced both B and T cell responses are critical, and combination of different antigens may be an efficient strategy to improve the immunoprotection against schistosome infection. In this study, different schistosome vaccine candidates (alone or mixed) that stimulated either B or T cell immunity were tested in a mouse model of disease. Results showed that combining different antigens in fact did not result in a more effective vaccine formulation when compared to each component administered individually, suggesting that mixed antigens may not be necessary for protection against schistosome infections and that immune interference resulting from the inoculation with multiple antigens is one mechanism responsible for the unexpected lack of increased protection.
Many years devoted to studying the interactions between schistosome infection and the resulting immune responses by hosts has led to the identification of mechanisms believed to play a critical role in protective immunity. Studies have shown that protection elicited by vaccination is not dependent on one immune mechanism but is multifactorial, involving both cellular and humoral elements and can be affected by the host's genetic background and the vaccine regimen [35, 36]. Therefore, to improve anti-schistosome immunity and protection against infection conferred by respective vaccine formulations, various strategies including multiple antigenic peptides (MAPs), i.e. sequential arrangement of epitopes into a single polypeptide and multicomponent formulations, have been tested [17, 23, 24]. These vaccination modalities have been tested in animal models and some formulations have proved successful [37, 38], suggesting that multicomponent formulations can be used to develop effective anti-schistosome vaccines. Recently, we demonstrated that vaccination with PDDV based on a T cell-epitope (P5, in this study named T3) derived from the Sj22.6 tegument antigen or a B cell-epitope (B3) derived from the Sj62 myosin sequence all induced partial protection against S. japonicum challenge in mice [13, 14]. In this study we predicted and selected six additional peptide epitopes derived from full-length Sj22.6, Sj62, and Sj97 antigens shown to have the potential of eliciting protective immunity in various other studies [6, 13, 14, 39–42]. The experiments were aimed at improving the immunoreactivity and protective efficacy of the peptide-based approach by comparing immune responses following vaccinations with either single or multicomponent formulations in mice subsequently infected with S. japonicum. Results from mice vaccinated with Tc-, Th-, or B-PDDV single-epitope formulations showed that not every single-epitope PDDV elicits corresponding cytotoxic, T helper or antibody responses. This finding suggests that at present, computer assisted epitope prediction is not a very effective way of generating epitopes and experimental validation must remain an early step in any vaccine study.
In contrast to the immune response induced by single PDDVs, cytokine release and antibody production induced by multicomponent PDDV formulations showed varied immune response profiles and variability in protective immunity conferred following vaccination with multicomponent formulations was not always more effective than that observed in mice immunized with single component PDDV preparations. For example, among multicomponent PDDV formulations, only C2-T3-B2, C3-T3-B2, and C3-T3-B3-immunized mice induced partial protection in reducing worm or egg burdens, but all multicomponent PDDV formulations containing T3- or B3-PDDV were not more effective in reducing worm and egg burdens nor did multicomponent formulations elicit significant changes in cytokine or antibody production, when compared to mice immunized with single formulations of T3- or B3-PDDV. These data suggest that T3- or B3-PDDV appeared to elicit a protective response against the parasite, but other epitopes or multicomponent formulations failed to induce an adequate immune response to improve the protection. Furthermore, these results also indicate that combinations of different types of antigenic epitopes may result in immune interference resulting in the development of inefficient immune responses incapable of conferring protective immunity [43, 44]. Others have shown that multicomponent vaccines can be more immunogenic and produce significant anti-parasite activity [45–47]. Similarly, DNA vaccines containing multiple antigens also contributed to improved protective responses against Schistosoma. For example, using a DNA vaccine encoding Sj62, Sj28, Sj23, and Sj14-3-3 induced significant Th1-type cellular responses and conferred partial protection against S. japonicum infection . However, others have also reported different results in relation to multicomponent formulations e.g., a multicomponent vaccine based on antigenic epitopes derived from SmTPI, Sm28, Sm97, Sm23, and Smcalpain of S. mansoni did not elicit a response capable of parasite killing in vivo. Several studies have addressed the properties of an epitope-specific regulatory system [50, 51] that selectively controls immune response (such as IgG antibody production) to the individual determinants on a complex antigen. These regulatory responses are commonly believed to regulate the amount, affinity, and isotype composition of antibody responses to individual epitopes on complex antigens by interference .
In this study, even though we failed to demonstrate improved protective efficacy using a multiple component-based vaccine strategy, we still derived insights regarding effective design of S. japonicum vaccines in two ways. First, predicting epitopes with software alone is not sufficient. A combination of epitope prediction and experimental screening is needed. Second, multiple epitope vaccines capable of inducing protective antibody and cell mediated immune responses against different schistosomal developmental stages theoretically may be more effective, however, the multivalent vaccine construct must be well designed to not suffer the effects of epitope interference.
In conclusion, we have developed single PDDVs and multicomponent PDDV anti-S. japonicum formulations. Our experiments demonstrate that mice immunized with single PDDV formulations were partially protected against S. japonicum infection and that mice vaccinated with multicomponent formulations were not necessarily better protected against S. japonicum challenge. These data suggest that immune interference may account for the inefficiency of the multicomponent formulations and that care must be taken in the selection of epitopes identified for vaccine preparations containing multiple epitopes.
Animal studies and antigen preparation
Six-week-old C57BL/6 female mice were provided by the Center of Experimental Animals (Nanjing University, Nanjing, PR China). Oncomelania hupensis harboring S. japonicum cercariae (Chinese mainland snail strain) were purchased from the Jiangsu Institute of Parasitic Diseases (Wuxi, PR China). All animal experiments were performed in accordance with the Chinese laws for animal protection and with permission from the Institutional Review Board. Soluble schistosome worm antigen (SWA) was prepared as previously described .
Identification of antigenic epitopes
Selection of B-cell epitopes was based on predictions made by the Immune Epitope Database and Analysis Resource (IEDB; http://epitope2.immuneepitope.org/home.do)  and ProtScale http://www.expasy.org/cgi-bin/protscale.pl[14, 54]. All putative T-cell epitopes were predicted using GUATIF, TEPITOPE and ANTHIWHIN software [13, 55]. Briefly, the amino acid sequences for Sj22.6 (GenBank Accession No: AAC67308), Sj62 (GenBank Accession No: AAC82332), and Sj97 (GenBank Accession No: Q05870) were analyzed by software designed to predict the epitopes and candidate peptides most likely to elicit Tc-, Th-, or B-cell responses selected based on their respective prediction scores. The 8 selected epitopes containing an 18 Lys (18K) N-terminal tail (epitope-18K fusion peptides) were synthesized and an 18K control peptide was also synthesized and purified (Invitrogen, Shanghai, PR China). The purity of the peptides determined by mass spectrometry was >99%. The DNA sequences encoding each of the 8 identified epitopes were synthesized and purified based on the published S. japonicum DNA sequences, respectively, for Sj22.6 (GenBank Accession No: AF030404), Sj62 (GenBank Accession No: No. AF039187), and Sj97 (GenBank Accession No: EU488866) (Invitrogen). Sal I and EcoR I restriction sites were included in the primer sequences for cloning purposes.
Preparation of PDDVs
PDDVs encoding the eight antigenic epitopes were prepared and confirmed as described previously [13, 14]. The diagram of PDDV preparation was shown in Additional file 1. Briefly, the recombinant expression plasmid pUMVC1-mGM-CSF (a gift from Professor Yuzhang Wu, Institute of Immunology of the Third Military Medical University, Chongqing, PR China) was 4423 base pairs (bp) long and contained the cytomegalovirus (CMV) promoter and the mouse GM-CSF gene. The two complementary single stranded oligonucleotides encoding each respective epitope were annealed and inserted into the pUMVC1-mGM-CSF vector and the resulting plasmids transformed into Escherichia coli DH5α grown in Luria Bertani (LB) broth. The recombinant plasmid DNAs were purified using the QIAGEN Endofree Plasmid Maxi Kit (QIAGEN, Hilden, Germany). Plasmid preparations were resuspended in ddH2O to a final concentration of 1.5-2.0 mg/ml. Agarose gel electrophoresis confirmed that the plasmid preparations were not contaminated with bacterial genomic DNA or RNA. Preparation of the PDDVs was performed by titrating peptide into a solution of DNA containing 10 mM HEPES and 150 mM NaCl as described previously [13, 14]. The cationic poly-lysine in 18K control peptide or epitope-18K fusion peptide was bound to the anionic plasmid DNA (containing the sequence of corresponding epitope) through electrostatic interactions and the peptide-DNA complex (PDDV) was condensed into nanometric pseudotype virus-like particles. Each PDDV was adjusted with phosphate buffered saline (PBS, pH 7.4) to 100 μl containing of 28 μg of peptide and 10 μg of plasmid. The PDDV containing either the control 18K or the Tc-, Th-, or B-cell epitope-18K fusion peptide were designated as 18K-PDDV, C-PDDV, T-PDDV or B-PDDV, respectively. The integrity of the PDDVs was confirmed using the DNA retardation assay, DNase I digestion assay and transmission electron microscopy as described previously [13, 14].
Immunization and challenge infection
For immune response characterization, three independent experiments were carried out. In each experiment, C57BL/6 mice (6 mice per group) were injected subcutaneously (s.c.) in the back with 100 μl of PBS (control 1), 18K-PDDV (control 2), C-, T-, or B-PDDV per mouse, respectively. The immunization was repeated three times at 14-day intervals. One week after the final vaccination, mice were sacrificed for the characterization of cellular and humoral immune response.
For vaccination/challenge trial, two independent experiments were carried out. In each experiment, C57BL/6 mice were divided into fourteen groups consisting of 14 mice per group. Each mouse was injected subcutaneously (s.c.) in the back with 100 μl of PBS (control 1), 18K-PDDV (control 2), C-, T-, B-PDDV, or multicomponent PDDV preparations, respectively. The multicomponent PDDV preparations were prepared by mixing the single PDDVs at a 1:1:1 ratio (33.3 μl each of C, T- and B-PDDVs, respectively) consisting of 28 μg of peptides and 10 μg of plasmids in total. The immunization was repeated three times at 14-day intervals. One week after the final vaccination, six mice from each group were sacrificed for the cytokine and antibody detection. Two weeks after the final vaccination, the remaining eight mice from each group were challenged percutaneously with 40 ± 1 S. japonicum cercariae. Six weeks later the mice were sacrificed and perfused to determine worm burdens and the liver egg burdens. Reductions in worms/liver egg burdens are expressed as a percentage of the burden recorded in the control groups.
Cytotoxicity was determined by a 4 h 5lCr release assay as described previously . Briefly, spleen cells were harvested from PBS, C1-, C2-, C3-PDDV or 18K-PDDV immunized mice and resuspended at a concentration of 1 × 106/ml in complete 1640 medium (containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin) containing 10 μg/ml C1-, C2-, C3-18K fusion peptide, 18K control peptide or medium only. After a five-day incubation at 37°C, the cells were washed and used as effector cells and 5 × 106 p815 (H-2d) cells were labeled with 200 μCi Na2[51Cr]O4 for 1 h. After thorough washing, labeled p815 cells were used as target cells and pulsed with 10 μg/ml C1-, C2-, C3-18K fusion peptide, 18K control peptide or medium only for 2 h at 37°C, washed and resuspended in complete RPMI 1640 at a concentration of 1 × 105/ml. Effector cells were titrated by serial dilution in U-bottom 96-well plates at Effector/Target ratios of 100:1, 20:1, 10:1. 1 × 104 target cells were added, centrifuged for 30 s at 100 × g and the Effector-Target-cell mix incubated at 37°C, 5% CO2, 90% humidity for 4 h. After centrifugation at 250 × g for 10 min, 100 μl/well supernatant from respective wells was removed and CPMs were measured with a gamma counter (Beckman, Fullerton, USA). The percent specific lysis was determined using the following equation: 100 × [(experimental release-spontaneous release)/(maximum release-spontaneous release)], with spontaneous release measured as the counts obtained from target cells incubated in medium alone, and maximum release determined by the counts obtained from target cells exposed to 1% Triton X-100.
Enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected seven days after the last immunization. Standard ELISAs were performed using SWA as the antigen source [39, 52]. Antibody detection in the sera of immunized mice was performed as previously described [13, 14]. IFN-γ and IL-4 levels in the supernatants of splenocytes stimulated by antigens from PBS, 18K-PDDV, C-, T-, B-PDDV, or multicomponent PDDV immunized mice were measured by ELISA using the eBioscience ELISA Ready-set-Go kit (eBioscience, San Diego, USA), according to the manufacturer's instructions.
Splenocyte proliferation assay
[3H] thymidine (3H-TdR) incorporation was used to measure splenocyte proliferation. Seven days after the last immunization, six mice from each group were sacrificed and splenocytes harvested. In 96-well plates, 2 × 105 cells per well were incubated for 72 h in 200 μl of complete media in the presence of the respective epitope-18k fusion peptides (10 μg/ml) or the 18K control (10 μg/ml). After 56 h in culture, [3H] thymidine (0.5 μCi) (Amersham, Burkinghamshire, UK) was added to each well. At the end of the incubation period, the cells were harvested on filters and the incorporated [3H] thymidine counted.
After portal perfusion, livers were dissected and immediately fixed in 10% buffered formalin for morphometric analysis. Liver sections were embedded in paraffin and stained with hematoxylin and eosin (H&E) for microscopic examination of granulomas at 4× (Olympus, Tokyo, Japan) following sectioning. The number of granulomas in the liver of each mouse was counted in 10 random fields. The size of nonconfluent granulomas formed around single eggs was assessed using a video micrometer (Olympus, Tokyo, Japan) in accordance with the manufacturer's instructions.
The statistical analysis was performed using SPSS version 10.1 (Statistical Package for Social Sciences, Chicago, IL statistical software). Statistical significance was determined by Student's t-test with P < 0.05 considered statistically significant.
List of abbreviations
peptide-DNA dual vaccine
cytotoxic T lymphocyte S.japonicum Schistosome japonicum:
soluble schistosome worm antigen.
We are grateful to Profs. Yuzhang Wu and Ying Wan (Institute of Immunology, Third Military Medical University, Chongqing, PR China) for their valuable assistances and critical reading of the manuscript.
This work was supported by grants from the National Basic Research Program of China (973 Program) (No. 2007CB513106), the National Natural Science Foundation of China (No. 30872206), Major National Science & Technology Special Program (NO: 2008ZX10004-011), and the grant 07KJA31023 from Jiangsu Province to Chuan Su.
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