The Immunological Characteristics Of Schistosomulum Japonicum Cell-type Immunogens And Their Mimotopes

Chapter One Study on protection against challenge infection induced by Schistosomulum cells with different size and by different immunization routesObjectives To observe the difference of susceptivity and pathogenicity to Schistosoma japonicum infection among three strain mice;to compare the immune protective efficacies induced by Schistosomulum cells with different size and by different immunization routes;to explore the possibility of elevating the immune protective efficacy by immunized with different stage Schistosomulum cells.Methods KM,NIH and BALB/c strain of mice,each strain consisting of 10 mice,were infected with 30±1 S.j caracara per mouse, respectively.All mice were sacrificed at 44^thday after infection to evaluate the developmental rate,hepatic granulomas and level of antibody of each group;Hepatic-stage Schistosomula were processed by shearing plus cold digestion to obtain Schistosomulum cells.And then,all cells were separated into three types(Meg cells,Media-size cells and Mini cells)with different centrifugal force;Cercarie tails were removed to obtain derma-stage Schistosomula and part of them were cultured in vitro for 96 hours for collection of pneumono-stage Schistosomula. Afterwards,two stages of cells were prepared by homogenate of worms. KM mice were divided randomly into seven groups,each consisting of 10 mice:A:PBS control(100μl/mouse);B:HSMegCs(2×10⁶/mouse);C: HSMedCs(2×10⁶/mouse);D:HSMiniCs(2×10⁶/mouse);E:DSCs plus PSCs plus HSCs(2×10⁶/mouse);F:HSCs immunized by s.c.;G:HSCs immunized by i.v..All groups were immunized by subcutaneous injection excepted groups G,which were immunized by intravenous injection.Each mouse was immunized three times at one week interval and challenged with 30±1 S.j caracara by abdominal skin penetration 2w post immunization.All mice were sacrificed at 44^thday after infection.The mean worm burden,hepatic eggs were counted,hepatic granuloma and the level of special antibody were measured.Results Significantly difference(P＜0.05)of developmental rate was found between BALB/c mice(50.33%)and either KM mice(69.23%)or NIH mice(64.00%);there was no difference(P＞0.05)between KM mice and NIH mice.However,LPPG.LEPWP,hepatic guanuluma and the level of special antibody showed no difference(P＞0.05)among three strain mice.The protective immunity results showed by parallel comparison with group A,the worm reduction rate,LEPG and LEPWP were 15.08%,14.56%and 9.00%for group B,7.13%,15.61%and 8.38% for group C,16.4%,13.53%and 0.91%for group D,18.23%,14.33% and 0.15%for group E,31.02%,41.61%and 32.89%for group F and 42.12%,55.36%and 27.84%for group G.statistic analysis showed that no difference(P＞0.05)in the worm reduction,LEPG and LEPWP was found between group B and group A,as well as group C,group D and group E,while significant reduction(P＜0.01 or P＜0.05)for group F and group G.Furthermore,there were significant differences(P＜0.01)in worm reduction and LEPG between group F and group G,but no significant differences(P＞0.05)in LEPWP and granuloma areas.Conclusions The susceptivity and pathogenicity to Schistosoma japonicum in KM mice had no difference with NIH mice and BALB/c mice;the mice immunized with HSCs by s.i.showed the best of protective immunity;the protocol of immunization of mice either with different size of cells or different stage Schistosomula cells,didn't produce satifying result.Chapter Two Identification of mimotopes of Schistosomula japonicum cell-type immunogens and their immunoprotectionObjective To comprehend the immunogenicity and immnoprotection of mimotopes of Schitosomula japonicum cell-type immunogens obtained by immunoscreening with IgG from sera of mice immunized with S.j Schistosomula cells.Methods Four rounds of immunoscreening of a random 12-peptide phage display library were carried out with IgG purified from sera of mice immunized with cultured 12-day Schistosomula cells.Specificity of 20 clones randomly picked out was tested and the amino acid sequences of 15 positive clones were deduced by sequencing.Mice were immunized with single and mixed positive clones to get the immune sera.The immunogenicity of positive clones was tested by ELISA and Western blot, respectively.KM mice were immunized the phage clones containing arginine residue(PC-R)or without(PC-NR).Meanwhile M13 phage immunized mice and SCs-12 immunized mice served as control groups. Each mouse was immunized for three times and challenged with 40±1 S.j caracara 2w post immunization.All mice were sacrificed at 42^thday after infection.The mean worm burden and hepatic eggs were counted.Results The bioinformatics analysis result showed that there were 12 clones with distinct sequences among 15 positive phage clones,and they had no homology with known gene sequences of S.j.There were 8 clones containing arginine residue among them.The result of ELISA showed that 11 clones could react with anti-SCs-12 IgG.The sera from mice immunized with 12 respective phage clones could react with JWA-12,excepted clone 6 and clone 9.Western blot showed that JWA-12 could be recognized by sera from mice immunized with clone 5,7,19 and 20.Immunoprotection result showed,as compared with control group, worm burden and LEPG were significantly reduced in mice immunized with phage clones containing arginine residue or not(P＜0.01),but no significant difference was found in the two groups(P＞0.05).Conclusion Mimotopes of S.j Schistosomula cells could be screened out by utilization of phage display technique and could induce determinate immunoprotection against schistosomiasis. Chapter Three The production of McAb against S.j Schistosomula cells membraneObjective To screen and characterize a McAb against Schistosomula cells membrane for providing a work basement and instrument of biological and immunological research on S.j cell.Methods Hepatic-stage Schistosomula were processed by shearing plus cold digestion to obtain Schistosomulum cells,and the cell membrane protein was extracted with the kit of Membrane Protein Extraction.BALB/c mice were immunized with the membrane protein for 4 times and the level of antibody was tested by ELISA.The mouse was sacrificed when the title achieved to 1:1000 and fused its spleen cells with SP2/0 cells.All the hybridoma cells were cultured with HAT and HT selective medium in 96-well cell culture plate.To obtain positive hybrid clones,the cultural supernatants were tested by ELISA and the hybrid clones were subcloned for 3 times.Result All positive clones died or turned to be negative in the process of subclone.So we failed to obtain positive clone strain.Conclusion The McAb against S.j cell membrane protein was failed to be produced.It was probably associated with mutation of the SP2/0 cells.