Studies On The Protective Efficacies Of Schistosoma Japonicum Heat Shock Protein And Hypoxanthine-guanine Phosphoribosyltransferase DNA Vaccines

Schistosomiasis continues to be a health problem currently in China. Considerable morbidity and mortality results from the affliction of an estimated 200 million people worldwide by several species of schistosomes.More than 700 million are exposed to the disease in 74 different countries.In the past,emphasis has been placed on chemotherapy as the preferred method for the control of schistosomiasis. However,control programs based on chemotherapy are complicated by the rapidity and frequency of re-infection and the difficulties and expense involved in maintaining these programs over a long term.The possibility that the parasite may develop drug resistance must also be considered.Even though other control measures,including public hygiene and snail control are available,the advent of an effective vaccine still remains the most feasible long-term solution to the problem of schistosomiasis.DNA vaccines offer advantages over many of the existing vaccines.However,DNA vaccination alone is limited in that it often generates only weak immune response, particularly the cellular immunity.Thus,efficient adjuvants and delivery systems are suggested in order to improve their protective efficacy.Heat shock proteins(HSPs),are a family of most highly conserved and abundant proteins expressed by prokaryotic and eukaryotic cells.Among the HSP family, HSP70,which has been shown to bind antigenic peptides endogenously,is the most conserved member.In mammalian hosts,it plays two main immunological roles:a) HSP70 can interact with receptors of the innate immune system,b) HSP70 is able to bind to a wide range of proteins and peptides,a property shared by major histocompatibility complex(MHC) molecules.This second property is fundamental for the presentation of antigens to the adaptive immune system.Thus,the ability of HSP70 to chaperone exogenous peptides and introduce them in the endogenous pathway makes HSP70 extremely powerful for the generation of CD8~+ responses. The unique properties of HSP70 as natural adjuvant,together with its ability to elicit antigen-specific MHC classⅡrestricted CD4~+ responses,make the molecule as powerful adjuvants for vaccines development,and has thus attracted more attention in the immunotherapy of cancer or infectious diseases.However,there has been no report related to the modulatory effect using the HSP70 gene as a molecular adjuvant to enhance the DNA vaccine against schistosomasis.Schistosoma japonicum is unable to synthesize purines de novo;therefore,it uses precursors obtained from the host blood.One enzyme of this biochemical pathway is hypoxanthine-guanine phosphoribosyltransferase,which is essential for the synthesis of guanosine monophosphate(GMP) from guanine and/or hypoxanthine.If no GMP is available,no RNA is synthesized and cell death ensues.Hence,it plays an important role during the growth of schistosome,which has long pointed to HGPRT as a potential drug target for treating schistosomiasis.In the present study,genes encoding Schistosoma japonicum heat shock protein 70(SjHSP70) and Schistosoma japonicum hypoxanthine-guanine phosphoribosyl transferase(SjHGPRT) were cloned into plasmid pVAX1 in order to evaluate the protective efficacy.The adjuvant effects of SjHSP70 by SjHGPRT DNA vaccine were also investigated.1 Construction on the DNA vaccine using the full-length gene encoding heat shock protein 70 of Schistosoma japonicumA pair of primers was designed,with the HindⅢrestriction endonuclease site introduced in forward primer and BamHI in reverse primer.Total RNA was isolated from Schistosoma japonicum(Chinese strain) adults and the full-length HSP70 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR).The PCR product was ligated with pGEM-T vector and then cloned into JM109.The target DNA fragments and the pVAX1 plasmids were digested by restriction endonucleases HindⅢand BamHI.The target DNA fragment was purified and cloned properly into the eukaryotic expression vector of pVAX1.The recombinant plasmids were then transformed into competent E.coli DH5αand identified by endonucleases digestion, PCR,agarose gel electrophoresis and sequencing.The deduced amino acid sequence showed to be of 99%identical with target gene.Therefore,the pVAX1-HSP70 DNA vaccine had been constructed successfully,and further studies will be made in different animal models for its immunogenicity.2 Construction on the DNA vaccine using the full-length gene encoding hypoxanthine-guanine phosphoribosyl transferase of Schistosoma japonicumTwo primers were designed,with the BamHI restriction endonuclease site introduced in forward primer and PstⅠin reverse primer.The full-length cDNA of SjHGPRT was amplified by RT-PCR from total RNA extracted from the Schistosoma japonicum adults.The PCR-amplified SjHGPRT DNA fragment were digested with restriction endonucleases and annealed by ligation with T4 DNA ligase to prokaryotic expression plasmid pGEM-T expression vector,referred to be pGEM-T-SjHGPRT. Recombinant plasmid was constructed with the target DNA fragments cut from pGEM-T-SjHGPRT and digested plasmid pVAX1 DNA.The inserted DNA fragments were confirmed experimentally by restriction enzyme digestion and gel electrophoresis,as well as DNA sequencing.3 The protective efficacies of SjHSP70 and SjHGPRT DNA vaccines in mice aganist Schistosoma japonicumA total of 75 C57BL/6 female mice were divided randomly into five groups with fifteen mice per group.Each mouse was vaccinated 3 times by intramuscular injection into the M.quadriceps at both sides with 100μg pVAX1,pVAX1-SjHSP70 or pVAX1-SjHGPRT DNA or 200μg mixed pVAX1-SjHSP70 and pVAX1-SjHGPRT plasmids respectively.Mice in the control group were inoculated with injection of 100μl normal saline.The injections were repeated at 2 week interval.One week after the final boost,mice were challenged percutaneously with 30±1 cercariae.On day 36 after the challenge,adult worms were perfused from hepatic portal system and mesenteric veins after mice sacrificed.The levels of antibody were measured by ELISA,the recovered adult worms from each mouse were individually identified as male,female and total worm burdens per animal.Eggs of parasites were recovered from each mouse liver and counted.Five female C57BL/6 mice(1 mouse/group) were immunized as above and the muscle tissues at injection foci were procceeded for frozen section 72 h after immunization.Indirect fluorescent antibody test(IFAT) was used to investigate the location of specitic antigen expressed in muscle.Results showed that both pVAX1-SjHSP70 and pVAX1-SjHGPRT were expressed in the muscle tissue injection area.ELISA results showed that the level of specific IgG in pVAX1-SjHSP70 and pVAX1-SjHGPRT DNA vaccinated groups were significant higher than that of challenge control group.The worm reduction rates of pVAX1-SjHSP70 DNA vaccinated group and pVAX1-SjHGPRT DNA vaccinated group were 34.85%and 40.25%.The egg reduction rates of the two groups were 34.31%and 26.48%,respectively.The worm reduction rate and the egg reduction rate of the complex injection group were 27.80%and 34.54%,respectively.The results demonstrated that partial protective immunity could be induced in mice immunized with the two DNA vaccines against challenge infection of Schistosoma japonicum. SjHSP70 in this experiment did not show its adjuvant effect potentially as expected and further experiments should be made on the adjuvant effect for DNA vaccines.