| More than 200 million people in 74 tropical countries or regions of Africa,Asia,and South America are infected with one or two species of schistosomes,making schistosomiasis one of the world's major public health problems.For the past half-century,schistosomiasis control activities in China have won a big success,but the disease staged a comeback in recent years.Nowadays China has more than 670 thousand people infected with Schistosomajaponicum(Sj),and most of them suffer from the chronic sequelae of schistosomiasis.Thus the requirements of curing advanced schistosomiasis are increasing.Schistosomiasis is an immune disease.Antigens released from different worm stages can cause immune responses in host,which is characterized by egg-induced granulomatous inflammation and cumulative fibrosis,the main pathological manifestation in advanced schistosomiasis.The development of hepatic fibrosis,portal hypertension and other complications are the main causes of death to those people. Current anti-fibrosis measurements involve etilogical treatment, anti-oxygen and anti-inflammation,collagen metabolism modulation, microcirculation disturbance and dysmetabolism improvement, complication risk reduction,etc.,but lack complete efficient drugs or treatments.Developing new way to slow down or reverse the fibrosis proceeding is of great importance.Fibrosis is a progressive pathological process in which wound-healing happened and myofibroblasts of the liver respond to injury by promoting replacement of the normal hepatic tissue with a scar-like matrix composed of crosslinked collagens.CD4+T cells play an important role in the progression of fibrosis and the opposing effects of Th1- and Th2-type cytokine responses have also been substantiated by recent microarray experiments:studies investigating the gene-expression profiles(transcriptomes)of the diseased tissues found that markedly different programmes of gene expression are induced when chronic inflammatory responses are dominated by Th1- or Th2-type cytokines. Two main groups of genes were identified in Th1-polarized mice,which were involved in the acute phase reaction and apoptosis.This might explain the large amount of cell death and tissue damage observed when Th1-cell responses continue unrestrained.By contrast,the transcription of several genes that are known to be involved in the mechanisms of wound-healing and fibrosis is upregulated by Th2-type cytokines.The regulation and function of a few of these genes,including those that encode procollagen-â… ,procollagen-â…¢,arginase,lysyl oxidase,matrix metalloproteinase 2(MMP2),MMP9 and tissue inhibitor of matrix metalloproteinase 1(TIMP1),have been investigated in some detail. Similar to the research finding of Schistosoma mansoni(Sm),the hepatic granulomatous response to Sj eggs begin as a Th1-type response and then it is rapidly driven by egg antigens to a Th2-type dominant response, which suggests that the fibrotic process is highly dependent on Th2-type cytokines.Therefore,during the fibrotic progression how to both upregulate Th1-type cytokine responses and down-regulate Th2-type cytokine responses to recover the Th1/Th2 balance may play the key role in slowing down or reversing the fibrosis.Li GF and Wang XJ et al have screened and identified several Th1-type epitopes for C57BL/6 mice from Sj vaccine candidates, including P4 for Sj22.6(22.6kDa membrane protein of Sj)and P6 for Sj28GST(28kDa Glutathione S-transferases of Sj),as well as T cell epitopes including P18 for SjTPI(triose phosphate isomerase of Sj)and P22 for Sj97(97kDa paramyosin of Sj)in the execution of Natural Science Foundation of China(NSFC No.30271166).This study is to further identify the P18 and P22 epitopes as the Th1 epitopes.Our laboratory has also screened CpG ODN(CpG oligodeoxynucleotide)1826 as the Th1 response immune adjuvant with species-specific for mice C57BL/6,which can induce higher Th1 response than other types of CpG ODN.The present study is to apply the four Th1 epitopes to construct four PDDV(peptide-DNA dual vaccine)separately.Each PDDV collocated the antigen with encoding gene and CpG ODN1826 being combined in expression vector pCI-neo,and then we used the four PDDV isodose mixture to immune C57BL/6 mice to upregulate Th1 response producing higher endogenous IFN-γ,in order to slow down hepatic fibrosis caused by Sj infection.Partâ… .Further immunological identification of Th1-type epitope in triose-phosphate isomerase and paramyosin of Schistosoma japonicum to promote the design of epitope-based vaccine.Triose-phosphate isomerase(TPI)of Sm is a putative vaccine candidate antigen.Its presence appears to maintain an IFN-γ-producing population of cells even in a strong Th2 microenvironment,and recombinant TPI(rTPI)was used to determine specific T and B epitopes recognized by two strains of mice(C57BL/6J and CBA/J)by Roynolds et al.Among them,SmTPI-P18 is defined as T epitope specific to both C57BL/6J and CBA/J mice,but T epitope SmTPI-P9 is only specific to CBA/J mice.In addition,the homology of nucleotide sequence between SmTPI and SjTPI is 84%,naive TPI,recombination TPI and TPI DNA vaccine all showed efficient immunomodulatory and antifibrogenic effects.Wang XJ et al in previous research of our laboratory designed the corresponding epitope SjTPI-P18 and its control epitope SjTPI-P9 according to DNA sequence of SmTPI-P18 and SmTPI-P9,and identified that SjTPI-P18 was the T epitope specific to C57BL/6 mice.In this present study C57BL/6 mice were immunized twice with recombinant rSjTPI-P18 emulsified with Freund's adjuvant,and the levels of IFN-γand IL-4 in the supernatant of cultured lymphocytes stimulated with the recombinant or synthetic SjTPI-P18 were detected;then mice were immunized twice with SjTPI-P18 emulsified with Freund's adjuvant,and so was the cytokines detection.Paramyosin,an invertebrate myofibrillar protein,has shown to be a promising vaccine target and thus was selected by WHO as one of vaccine candidates against schistosomiasis.The previews study showed that paramyosin could elicit higher humoral and cellular immunity responses,which induced anti-infection and anti-pathology protection positively correlated to the dominated Thl response.Wang XJ et al obtained five T cell epitope candidates of Sj97 using T cell epitope predictive algorithms SYFPEITHI and found out that Sj97-P22 was the best T epitope specific for C57BL/6 mice.The immunogenicity of synthetic epitope SjTPI-P18 is better than that of recombinant epitope rSjTPI-P18 at the same dose in the previews study.Thus in this study, C57BL/6 mice were immunized twice with Sj97-P22 emulsified with Freund's adjuvant,and the cell proliferation was detected by 3H-TdR incorporation assay after the primed lymphocytes were incubated with Sj97-P22;the concentrations of IL-2,IFN-γand IL-4 in the cultured lymphocytes supernatant were detected by ELISA.Results:Both SjTPI-P18 and rSjTPI-P18 were able to effectively stimulate lymphocytes to secret higher level of IFN-γ(P＜0.05)and lower IL-4 when the mice were immunized twice with rSjTPI-P18 or SjTPI-P18,emulsified with Freund's adjuvant.SjTPI-P18 was able to effectively stimulate the spleen cells of the mice immunized with SjTPI-P18 twice to secret higher level of IFN-γ(P＜0.05)and lower IL-4 (P＜0.05).Therefore the immunogenicity of synthesis epitope is better than that of recombinant epitope at the same dose.In the immunological identification of Sj97,Sj97-P22 was an efficient stimulator for lymphocytes proliferation and can effectively stimulate lymphocytes to secret higher level of IL-2 and IFN-γ(P＜0.05),and lower IL-4 when the mice were immunized twice with Sj97-P22 emulsified with Freund's adjuvant.As a result,it is evident that the epitope candidates SjTPI-P18 and Sj97-P22 are Thl-type epitopes.Partâ…¡.Construction of PDDV mixture with four Th1-type epitopes and immune adjuvant CpG ODN1826,and then identification of its immunological characteristics.The concept PDDV is initiated by Wu YZ et al using polylysine,a widely used self-assembly nonviral vector for gene therapy,as a bridge between antigenic peptide and vector DNA which has harbored the correspondent encoding gene.The partâ… and previous studies in our laboratory have screened four Th1-type epitopes(P4,P6,P18,P22)and Th1 response immune adjuvant CpG ODN1826.In this part study,we synthesized four different bifunctional linear cationic peptides([K]18P4, [K]18P6,[K]18P 18,[K]18P22)and connected them separately to the recombinant vector pCI-neo DNA,with the four corresponding encoding gene and CpG ODN1826 in certain conditions,to constructed four kinds of PDDV.Furthermore,C57BL/6 mice were immunized with the four PDDV isodose mixtures to observe its capacity to induce a Th1-type response. Results:By DNA Precipitation Assays,Gel retardation Assay, DNaseâ… Digestion Assay and Electron Microscopy Examination, PDDV was constructed successfully with a diameter of about 20 nm as the key parameter for gene delivery and expression,at 150 mM NaCl and 40 mM HEPES concentrations and a charge ratio(NH4+/PO4-)of 4.The four PDDV isodose mixture could efficiently stimulate lymphocytes both proliferation and secreting higher level of IL-2 and IFN-γ(P＜0.05),and lower IL-4 as C57BL/6 mice immunized twice with the mixture.As a result,it is evident that the four PDDV isodose mixtures can induce dominant Th1-type response in C57BL/6 mice.Partâ…¢.Study of the effect of PDDV mixture on attenuating the development of hepatic fibrosis caused by schistosomiasis japonica in C57BL/6 mice.Fibrosis is a progressive pathological process involving many complicated factors and the radical point is that the deposition of ECM is more than its degradation.The activated hepatic stellate cell(HSC)is the main collagen-producing cell,and the equilibrium expression of matrix metalloproteinase / tissue inhibitor of matrix metalloproteinase (MMPs/TIMPs)plays an essential role in both fibrogenesis and fibrolysis, among which MMP9/TIMP1 balance is important.Many researches demonstrated that Th2-type cytokines and the lost balance between Th1 and Th2 responses are responsible for fibrosis caused by schistosomiasis by stimulation of ECM accumulation and HSCs activation.By contrast, IFN-γcan suppress the phenotype of activated HSCs and diphasic regulate MMP9 expression to down-regulate the development of fibrosis. Therefore,increasing Thl response to recover the Th1/Th2 balance may play a significant role in slowing down the development of fibrosis through upregulating MMP9/TIMP1 equilibrium and suppressing the phenotype of activated HSCs. In study partâ…¡,the PDDV mixture inducing Th1-type response dominated by IFN-γin C57BL/6 mice have been constructed successfully. In this part study the PDDV mixture was applied to immunize C57BL/6 mice to observe the fibrosis degree measured by fibrosis scoring and the size of non-confluent granulomas;to view the expression of certain proteins in liver by immunohistochemistry,includingα-SMA,Collagenâ… , Collagenâ…¢,TIMP-1,MMP-9,IFN-γ;to know some mRNA expression in liver by reverse transcriptase PCR,includingα-SMA,Collagenâ… , Collagenâ…¢,IL-4,IL-13,TGF-β1,IFN-γ;to observe the cellular and humoral response in different time points(0 w,10 w,14 w,25 w)by detecting IFN-γand IL-4 concentrations in sera and SWAP-specific anti-IgG;IgG1,IgG2a,IgG1/IgG2 value,and the possible mechanism involved were discussed.Results:The anti-fibrosis effect of the mixed epitope-CpG ODN1826 PDDVs(PDDV mixture)group and IFN-γtreatment group (IFN-γgroup)are obviously better than that of other groups,and the anti-fibrosis effect of non epitope-CpG ODN1826 PDDV(CpG PDDV) group is only better than that of solvent group without statistical significance.The PDDV mixture immunized mice showed a slow resorption of liver fibrous tissue with obvious drop in the protein expression ofα-SMA,Collagen I,TIMP-1 and the obvious rise in the protein expression of MMP-9 with statistical significance(P＜0.05),and the increase in IFN-γand the decrease in Collagenâ…¢protein expression but no statistical significance.Furthermore,the drop ofα-SMA, Collagenâ…¢,TGF-β1,IL-13 expression at mRNA level in the PDDV mixture-immunized mice were statistically significant(P＜0.05),and the increase in IFN-γand the decrease in Collagenâ… had no statistical significance.In addition,IFN-γconcentration in sera was gradually increasing accompanied with no notable change of IL-4 concentration at four different time points,and at 25 w the PDDV mixture immunized mice secreted higher level of IFN-γand lower IL-4 than other groups but no statistical significance.At the same time in the sera of the PDDV mixture-immunized mice,the SWAP-specific anti-IgG and IgG2a level increased gradually at the four time points,and the SWAP-specific anti-IgG1 level increased significantly at 14 w compared to that at 10 w but was similar to that at 25 w,thus at 25 w IgG1/IgG2a value was lower than that of 14 w.In summary,the PDDV mixture-immunized mice showed a down-regulating effect on hepatic fibrosis,accompanied with the decrease expression ofα-SMA(a marker protein of activated HSC)and the shifting of balance in favor of MMP9/TIMP1 expression,and also upregulating the equilibrium in favor of Th1/Th2 response in cell immune and humoral responses.In conclusion,we have successfully constructed the PDDV mixture, which can elicit high production of IFN-γ,showing Thl-type response, and therefore attenuated the development of hepatic fibrosis caused by schistosomiasis japonica.This is also of great importance to the study on other chronic liver injury causing fibrosis.
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