Investigation Of Epitope-based PDDV Against Schistosoma Japonicum

Schistosomiasis, the second major parasitic disease in the worldafter malaria, affects mainly developing countries. There are 200 mil-lion people worldwide infected with schistosomes, resulting in morethan 250,000 deaths annually. Although the last half-century of schisto-somiasis control programs have brought down the overall prevalence ofhuman infection with Schistosoma japonicum, the parasitic disease re-mains a serious zoonosis with many problems unsolved, especially re-infection and reemergence of prevalence. An efficacious vaccine againstSchistosomiasis japonica would represent a significant addition to thecurrent arsenal of control tools, particularly in the framework of an in-tegrated control approach. However, the fact remains that after manyyears of trying, a rationally designed effective anti-schistosome vaccinehas yet to be developed. As a prophylactic measure, using vaccinewould be the ideal method for sustainable control of schistosomiasis,alone or in combination with anthelmintic drugs and the development ofan efficient vaccine is required to make the elimination of schistosomi-asis achievable In the past years, many laboratories have attempted to identifythe schistosome antigens that induce the partially protective immuneresponse. Vaccinations with synthetic or recombinant antigens havesuccessfully induced partial protection and/or reduced female fecundityin animal models. However, epitope-based vaccine offers the prospectof targeted immunity resulting in safer and more effective anti-gen-specific immune responses. The host's immunity against schisto-some infection is mainly cellular and humoral immune response, whichis mediated by the specific T cells and B cells. Consequently, identifica-tion of these T/B-cell epitopes as safe effective immunogens representsa pivotal step for the study of pathogenesis and immunity and especiallyfor the development of vaccine against schistosomiasisTo optimize the vaccine design, we undertook studies to identifyT/B epitope on ten protective antigens by bioinformatics in order toidentify the potential protective epitopes, including Sj14, Sj97, Sj14-3-3,Sj22.6, Sj23, Sj26, Sj28, Sj338, Sj62 and SjTPI. Several online T/B cell-related databases and analysis tools were chosen according to the hu-man allele HLA-DRB1*0401, HLA-A*0201 or C57BL/6 mouse H-2b(H-2Kb,H-2Db). And the proteasomal cleavage sites were predictedand excluded to protect the epitope from being digested. The epitopespredicted above were screened on BLAST to exclude the homologousones. Eventually, three epitopes of CTL, Th and B cell, respectively were chosen for further study.To improve the efficacy of the vaccine, peptide-DNA dual vac-cine (PDDV) strategy was used, which was a nanometer-size particleantigen delivery system constructed by combination of the epitope fromthe protective antigen and the plasmid encoding the same epitope. Theepitopes containing 18 lysines which acted as the bridge of the peptideand its control peptide of 18 lysines (18K) were synthesized and puri-fied. The oligonucleotides encoding the epitopes with restrictive en-zyme (RE) Sal I site at 5' terminus and EcoR I site at 3' terminus werealso synthesized and insert into expression plasmidpUMVC1-mGM-CSF. The procedure for preparing PDDV was per-formed by titrating peptide into a solution of the corresponding plasmid.The input molar ratio of peptide to DNA (r) was the ratio of moles oflysine to nucleotide (NH4⁺/PO3^-). PDDVs prepared was identified byDNA retardation assay, DNase I digestion assay and electron micros-copy to choose the best condition of preparation. The optimized PDDVswere prepared for the animal experiments.C57BL/6 mice were divided into 29 groups, 14 mice each group.Each mouse was injected subcutaneously (s.c) in the back with 100μl ofsolution containing 28μg of peptide and 10μg of plasmid pGT or pGrespectively, or PBS (control group). The immunization was repeatedwith 14-day interval. One week after the final vaccination, 6 mice from each group were sacrificed for the detection of the cellular and humoralimmune response. CTL activity of the CD8+ effector cells was assessedin a standard 4-h ⁵¹Cr-release assay. T cell proliferation and cytokineswere detected for evaluation of Th cellular immune response. And anti-bodies were measured for evaluation of humoral immune response. Theresults showed that CTL responses, Th response and antibodies wereinduced by the CTL, Th and B-cell epitope-PDDV respectively. But therelevant immune response was not detected in the groups immunizedwith mixed epitope PDDVs.Two weeks after the last vaccination, 8 mice of each group werechallenged percutaneously with 40 cercariae of S. j aponicum. Six weeksafter challenge, all vaccinated and control mice were sacrificed andperfusion was undertaken with saline containing heparin to recover theadult worms. The livers were digested and the number of eggs was de-termined by microscopic examination. The worm/egg reduction ratewas calculated. After portal perfusion, mice livers were dissected andstained with hematoxylin and eosin (HE) for microscopic examination.The number of egg granulomas was counted and the sizes of noncon-fluent granulomas formed around a single egg containing a maturemiracidium were assessed using a video micrometer. The resultsshowed that several PDDVs induced promising protection. Especially,T3 epitope PDDV induced both cellular and humoral immune responses, achieved 33.8%～44.7% protections against S. japonicum infection anddiminished egg granulomas in the livers of challenged mice.