Immunological Research On Protection Against Challege Infection Induced By Schistosomulum Cells In Mouse Model

Section OneSchistosoma japonicum: Protective Immunity Induced bySchistosomulum-derived Cells in Mouse ModelWe have previously reported that immunization with intact live cellsresourced from Schistosomulum japonicum (S.j) was capable of inducingproduction of partial immunoprotection against challenge infection by S.jcercariae in Kunming strain murine model. In present work, two schemes ofimmune protective experiment were designed with Kunming strain of mice asexperimental model to further validate the immune protective effect inducedby this type of vaccine and explore the optimal immunization protocolincluding the number of inoculations and parasite stages from whichimmunogenic cells derived. In experiment 1 (EXPⅠ), a four timesvaccination program was formulated to compare the protective efficacyamong three antigens including LLC (live cells resourced from18-daypost-infection larval worms), DLC (dead cells derived from 18-daypost-infection larval worms) and DAC (dead cells derived from 42-daypost-infection adult worms). In experiment 2 (EXPⅡ), LLC was selected tovaccinate animals once (V1), twice (V2) and three times (V3) fordetermination of an optimal inoculation number as it was proved to be thebest immunogen in EXPⅠ. Challenge infection with 30±1 S.j cercariae wascarried out one week for EXPⅠand two weeks for EXPⅡafter the lastvaccination. At the day 42 for EXPⅠand day 45 for EXPⅡ, mice weresacrificed and perfused. Afterward, the worm burden and liver egg burdenwere counted, the development status of worm bodies observed and the sizesof egg granulomas in liver tissues measured. PCR showed that there was nocontaminate of host tissue ingredient in both crude cells. Synchronously,primary dynamic levels of specific total antibody classes and IgG subclasseswere observed by ELISAs. Ingredients and characters of effectiveimmunogen were initially identified by using SDS-PAGE and Western-blottechniques, respectively. By parallel comparison with control group, the results from experimental groups revealed that the reduction rate of wormburden and liver egg burden in EXPⅠwere 65.1％and 76.5％for LLC group,54.5％and 66.9％for DLC, -2.4％and -10.3％for DAC. In EXPⅡ, 64.5％and 62.1％worm burden reduction rate and 66.2％and 66.1％liver eggreduction rate were obtained from the mice immunized with LLC twice andthree times, respectively. Moreover, in mice of EXPⅡ, the development ofworms in the day 45 post infection was stunted. The best exterior of livers inEXPⅠwas from LLC group of mice and followed by DLC group, controlgroup and DAC group while the best outward appearance of livers in EXPⅡwas from mice vaccinated twice and three times followed by that vaccinatedonce and controls. The areas of egg granulomas in liver slices from LLCgroup of mice were significantly smaller than that from other three groups.Compared with DAC and control groups, the levels of specific antibodies andthe ratio of IgG2a/IgG1 were elevated in LLC group. Despite theelectrophoresed polypeptide bands of the hepatic stage juvenile worm cellson acrylamide resolving gel were far less than that of worm bodies, butpossessed potent immunogenicity and antigenicity. Furthermore, the bands of18-day larval warm cell preparations were notrecognized by infected micesera. Our results demonstrated that live cells from 18-day old hepatic stage ofS.j could induce production of the higher level of protective efficacy than thatfrom 42-day old adult worms in murine-S.j challenge model. There was nosignificant difference in protective effects and immune response typebetween vaccination for twice and three times animals. In addition, aTh1-biased immune response was indirectly reflected in high protectivegroups as evidenced by an elevated absolute level of IgG subclasses and theratio of IgG2a/IgG1.Section TwoSchistosoma Japonicum: Cultured Schistosomules Cells Induced aPartial Protection Against Challege Infection In Mouse ModelSchistosomiasis is believed to rank the second behind malaria in global importance. A vaccine would contribute to reduction of schistosomiasismorbidity and mortality through induced immuno-modulatory responsesleading to reduced worm burdens, reduced egg production(anti-fecundity)and reduced viability of eggs(anti-embryonation). A novel cell-type vaccinederived from schistosomulues was explored in our laboratory. To furtherdevelop this cell-type vaccine, this research observed through twoexperiments immune protective effects against challenge infection in Kunminstrain mice model induced by m vitro passage cultured 4th generations of livecells from 12-day and 18-day juvenile worms or 30-day adult worms.Immunogens above were abbreviated as JC-12, JC-18 and AC-30,respectively. After identification of without contamination of host constituentin sub-cultured cells with PCR, the mice were vaccinated intraperitoneallywith JC-12 and JC-18, respectively, in experiment I(EXPⅠ). Anotherexperiment (EXPⅡ) was designed according to the result of EXP I and newindexes. The mice were immunized with AC-30 and JC-12 without adjuvantby different injection routes, respectively. In both experiments, animals werechallenged with 40±2 or 30±1 Schistosomajaponicum (Sj) cercariae after thelast booster dose and sacrificed at day 40 post challenge infection for formerexperiment and day 42 post challenge infection for latter experiment.Parasitological and immunological results were reported here. PCR showedthat no contamination of host constituent was found in sub-cultured cells. Themice immunized with JC-12 recovered significant fewer worms and livereggs than those immunized with JC-18 (p＜0.05) or injected with normalsaline (NS) (p＜0.01) in EXPⅠand those immunized with AC-30 (p＜0.01) orinjected with D'hanks (p＜0.01) in EXPⅡ. Parasitological results obtainedfrom animals immunized with AC-30 were not significant different fromthose from controls. Photomicrographs (×100) of hepatic granulomas in EXPⅠshowed a mixed inflammatory reaction. The immunological studies haveshown: 1, High levels of IgG antibodies against soluble adult and juvenileworm antigens were observed in animals immunized with either culturedcells from juvenile or adult worms; 2, A Th1-type immune response tojuvenile antigen was suggested by ratio of IgG1/IgG2a＜1, coinciding withelevated level of interferon-γ, (IFN-γ) production in animals immunized withJC-12; in contrast, a dominant Th2-type immune response, with elevated IgG1 response to adult antigens and coinciding with detectable interleukin 4(IL-4) production, was generated in animals with AC-30 vaccination; 3,Anti-adult worm antigen (anti-AWA) IgE antibodies and anti-juvenile wormantigen (anti-JWA) IgE in sera from animals immunized with JC-12 or withAC-30 were significantly increased; 4, Passive transfer of sera from bothJC-12 and AC-30 immunized mice conferred partial protection to recipientmice; 5, Splenocytes from JC-12 and AC-30 immunized mice induced/nvitro killing effect on S. japonicum cercariae. Totally, identification ofantibody isotypes and cytokine correlates of partial immunity to subsequentinfection in our experiments suggests a great potential for live cultured cellvaccines as a novel approach against schistosomiasis. However, consistenthigh resistance was not demonstrated in EXP I with other batches of JC-12and JC-18, though a significant protection and IgG against 12-day old larvalworm crude soluble antigen was induced by JC-12 and JC-18, respectively,suggesting that the stability of immunogens remains to be further probed into.Section ThreeScreen and Characterization of Mimotopes of SchistosomulumJaponicum Cell-type ImmunogenIn previous study, our group proved that immunization of Kunmingstrain of murine model with sub-cultured live cells from 12-day oldSchistosoma japonicum(Sj)juvenile(JC-12) induced a partial protectionagainst the parasite challenge infection. In an attempt to further clarify theinduced immunoprotection mechanism on molecular level, isolate andidentify the peptides mimicking epitopes on JC-12 and test their protectivepotentiality against S.j, IgG were prepared by purification from polyclonalimmunosera against JC-12 to screen a 12-mer random phage peptide library.Four rounds of bioparming were performed and resulted in an effectiveenrichment. Of 20 randomly picked clones from the fourth elute, fifteenwerefound to be positive by evaluating the binding to anti-JC-12 sera by a reverse ELISA. Amino acid sequences were deduced by sequencing. Twelveclones were found to possess distinct sequences. Bioinformatics analysisamong 12 different clones was carried out with aid of several sorftwares. Bycomparing each peptide sequence with others, the highest 41.6% of identitywas found between Clone-5 and Clone-7. Independent phage clone and twogroups of mixed phage clone, according to whether containing Arginineresidue in peptide sequence or not, were served for immunization of mice,respectively. Induced immune response (antibody levels) and specialty of theresponse were evaluated by a direct ELSA. All, but one, animals developed aimmune response against these phagotops. Clone-10 could induce thestrongest antibody response, and the most intensely reacted with anti-JC-12sera. However, as to react with soluble 12-day old worm antigen,anti-Clone-7 immunosera revealed the strongest response profile. From theELISA results we concluded that Clone 10 and Clone 7 may be twointeresting mimotopes related to JC-12. Both combinatorial immunizationgroups were all induced to product IgG antibody, especially the group witharginine residue in sequences. The protective experiment is being under way,and the results will be reported soon afterward.