Research on calpain of Schistosoma japonicum as a vaccine candidate

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.     

Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis.

, , , and 1992. Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. International Journal for Parasitology 22: 1187–1191. Paramyosin from the blood fluke, Schistosoma mansoni, has shown promise as a vaccine candidate for schistosomiasis mansoni. Here we report the cloning and partial nucleotide sequence of a cDNA encoding paramyosin from the related human parasite, Schistosoma japonicum. Affinity purified antibodies to this clone recognized a S. japonicum antigen of molecular weight 97 kDa, equivalent to the reported size of S. mansoni paramyosin. Alignment of the cDNA sequence with that of S. mansoni paramyosin revealed 90% identity. Comparison of the predicted amino acid sequences revealed 95% identity. Although these two parasites differ in many characteristics, the substantial homology demonstrated here between S. mansoni and S. japonicum paramyosin could have important implications for the development of a S. japonicum vaccine.     

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.     

Outer membrane vesicles as a candidate vaccine against edwardsiellosis

Infection with Edwardsiella tarda, a gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.     

Cimetidine enhances the protective effect of GST DNA vaccine against Schistosoma japonicum

Cimetidine (CIM), a histamine-2-receptor antagonist, has a long history of safe use in gastric acid-mediated gastrointestinal disorders. In this study, we used CIM, as an adjuvant, with pEGFP-Sj26 GST (the recombinant plasmid containing enhanced green fluorescent protein gene and the gene encoding 26 kDa glutathione S-transferase of Schistosoma japonicum) DNA vaccine to immunized mice and attempted to enhance the protective effect against S. japonicum. The results showed that the reduction rate of worm and egg burdens in the pEGFP-Sj26GST plus CIM group were 79.0% and 68.4%, respectively, significantly higher than that in pEGFP-Sj26GST alone group (27.0% and 22.5%, P < 0.01). Compared with the pEGFP-Sj26GST alone group, mice immunized with pEGFP-Sj26GST plus CIM showed an elevated level of IFN-γ and IL-12 and a low level of IL-10 in splenocytes, while the levels of IL-4 and IL-5 showed no difference between the two groups. Our data also demonstrated that the percentage of CD4⁺CD25⁺ regulatory T cells (Tregs) was significantly decreased in the spleens of mice immunized with pEGFP-Sj26GST plus CIM. All these findings suggest that CIM as a potential schistosome DNA vaccine adjuvant can enhance the protective effect of pEGFP-Sj26GST vaccine.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

Recombinant BCG as a vaccine vehicle to protect against tuberculosis

Mycobacterium bovis Bacille Calmette Guérin (BCG) was first administered to humans in 1921 and has subsequently been delivered to an estimated 3 billion individuals, with a low incidence of serious complications. The vaccine is immunogenic and is stable and cheap to produce. Additionally, the vaccine can be engineered to express foreign molecules in a functional form, and this has driven the development of BCG as a recombinant vector to protect against infectious diseases and malignancies such as cancer. However, it is now clear that the existing BCG vaccine has proved insufficient to control the spread of tuberculosis, and a major focus of tuberculosis vaccine development programs is the construction and testing of modified forms of BCG. This review summarizes the strategies employed to develop recombinant forms of BCG and describes the potential of these vaccines to stimulate protective immunity and protect against Mycobacterium tuberculosis infection.     

Schistosoma mansoni egg-derived extracellular vesicles: A promising vaccine candidate against murine schistosomiasis

Extracellular vesicles (EVs) are protein-loaded nano-scaled particles that are extracellularly released by eukaryotes and prokaryotes. Parasite’s EVs manipulate the immune system, making them probable next-generation vaccines. Schistosomal EVs carry different proteins of promising immunizing potentials. For evaluating the immune-protective role of Schistosoma mansoni (S. mansoni) egg-derived EVs against murine schistosomiasis, EVs were isolated from cultured S. mansoni eggs by progressive sequential cooling ultra-centrifugation technique. Isolated EVs were structurally identified using transmission electron microscope and their protein was quantified by Lowry’s technique. Experimental mice were subcutaneously immunized with three doses of 20 μg EVs (with or without alum adjuvant); every two weeks, then were challenged with S. mansoni cercariae two weeks after the last immunizing dose. Six weeks post infection, mice were sacrificed for vaccine candidate assessment. EVs protective efficacy was evaluated through parasitological, histopathological, and immunological parameters. Results showed significant reduction of tegumentally deranged adult worms, hepatic and intestinal egg counts reduction by 46.58%, 93.14% and 93.17% respectively, accompanied by remarkable amelioration of sizes, numbers and histopathology of hepatic granulomata mediated by high interferon gamma (IFN γ) and antibody level. Using sera from vaccinated mice, the molecular weight of EVs’ protein components targeted by the antibody produced was recognized by western immunoblot. Results revealed two bands of ~ 14 KDa and ~ 21 KDa, proving that EVs are able to stimulate specific antibodies response. In conclusion, the present study highlighted the role of S. mansoni-egg derived EVs as a potential vaccine candidate against murine schistosomiasis mansoni.     

One step membrane incorporation of viral antigens as a vaccine candidate against HIV

Liposomes can been used as potential immunoadjuvants, because they have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. For all these purposes, liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices and, or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles. A second group of preparation procedures uses suitable detergents, e.g., bile salts or alkylglycosides. A third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase. The concentration of the organic solvent is then reduced by suitable procedures. Here we present a new technique for the preparation of liposomes with associated membrane proteins, where lipid vesicles are formed immediately after injection into a micellar protein solution. The model membrane protein used for these studies is a truncated recombinant gp41 produced in E. coli. This viral membrane antigen is a possible candidate protein for the establishment of HIV-vaccines. The data presented here, show an efficient and reproducible one step membrane protein encapsulation procedure into liposomes in a closed and sterile containment. We examined encapsulation efficiency, membrane protein conformation and immunogenicity of this possible liposomal vaccine candidate, which can be produced in GMP-compliant quality with the described technique.     

The Role of Microscopy in the Investigation of Paramyosin as a Vaccine Candidate against Schistosoma japonicum

There has been growing interest in paramyosin as a vaccine component to combat schistosomiasis. Immunological and molecular techniques have been used in the past to investigate the effectiveness of a paramyosin vaccine as an anti-schistosomal treatment. However, recent localization studies at ultrastructural and morphological levels have highlighted a number of questions concerning the role of paramyosin within schistosome parasites. Debates about how a non-surface protein such as paramyosin might provide protection against schistosome infections have recently been addressed by microscopy results. Immunolocalization studies have indicated multiple functions of paramyosin within the parasite and provided insights into how a vaccine may target the parasite, as discussed here by Geoffrey Gobert.     

Schistosoma japonicum: The design and experimental evaluation of a multivalent DNA vaccine

The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO₂SjFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO₂, pVIVO₂Sj23, pVIVO₂SjFABP and pVIVO₂SjFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO₂SjFABP-23, pVIVO₂Sj23 or pVIVO₂SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO₂SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.     

Fasciola hepatica: tegumental alterations as a consequence of lectin binding

Adult flukes, Fasciola hepatica, incubated in Hedon - Fleig saline containing concanavalin A (Con A) for 10 and 45 min, respectively, exhibited severe alterations to tegumental morphology involving increased secretory activity, blebbing of the apical plasma membrane, increased total surface area, and swelling of the basal infolds . The effects of Con A were prevented by the addition of alpha-methyl-D-mannoside to the incubating medium. Similar, but less pronounced, effects were caused by wheat germ agglutinin (WGA) binding. Con A and WGA binding indicate the presence of mannose, glucosamine, or glucose moieties and of N-acetylglucosamine. The effects of lectin binding were similar to the early effects of antibody attachment, and it was considered that accelerated membrane turnover was occurring in both cases. Swelling of the basal infolds was thought to be a result of increased apical surface membrane and/or increased permeability due to lectin binding.     

Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed “clean linear B cell epitopes,” and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.     

The insulin receptor is a transmission blocking veterinary vaccine target for zoonotic Schistosoma japonicum

Insulin receptors have been previously identified in Schistosoma japonicum that can bind human insulin. We used the purified recombined protein of the ligand domain of S.japonicum insulin receptor 2 (SjLD2) in three independent murine vaccine/challenge trials. Compared with controls, vaccination of mice with SjLD2 resulted in a significant reduction in faecal eggs, the stunting of adult worms and a reduction in liver granuloma density in all three trials. Furthermore, in the final trial, in which mature intestinal eggs were also quantified, there was a reduction in their number. These results suggest that development of a vaccine based on rSjLD2 for preventing transmission of zoonotic schistosomiasis is feasible.