| Trichinellosis is an important foodborne zoonotic disease which infects humanand more than100mammals. Human infection occurs by eating raw or undercookedmeats containing infective larvae, such as pork and horsemeat. In some countries dueto changes in diet and cooking habits, trichinellosis is regarded as an emerging orre-emerging disease. It is regularly reported of human outbreaks in different parts ofthe world. Because trichinellosis has major impacts on animal husbandry and foodsafety, development of a vaccine defence against Trichinella spiralis (T. spiralis)larvae infection in domestic animals and humans would provide a valuable tool fordisease prevention and control.T. spiralis crude antigen preparations, excretory-secretory (ES) products,attenuated Salmonella or recombinant proteins-based vaccines have been reported inanimal models. In the past decade, several proteins which are secreted by T. spiralis orrelated to establishing parasitism/infection have been studied as composition of therecombinant vaccine and induce partial protection against T. spiralis larvae infection.However, due to the parasite antigens changes during the development in the host andvariety of molecules are involved in establishment of parasitic process, it is difficult toinduce high levels of protection with a single antigen. Therefore, combination ofmultiple recombinant proteins may induce effective protection.The most common method to elicit protection against pathogenic microorganismis native immunization with immunogenic native or recombinant proteins which areimportant for pathogenicity. Our laboratory had immunoscreened T. spiralis intestinalphase infective larvae (inML),3-day-old adults (Ad3) and newborn larvae (NBL)cDNA library with T. spiralis infected26days pig serum and cystatin-like protein of T.Spiralis inML stage (Ts-clp), serine protease gene of adult stage (Ts-Adsp) and serineprotease gene of NBL stage (Ts-NBLsp) were successfully screened; and musclelarvae (ML) cDNA library was screening with T. spiralis infected60days pig serum, a gene encoding serine protease inhibitor (Ts-serpin) was successfully screened. Thisresearch chooses four high-abundance genes from different cDNA libraries, and usesthe four recombinant proteins in expression vector pET-28a which have beenconstructed by our laboratory. Its reactogenicity and immunogenicity was analyzed inorder to develop a highly efficient and safe vaccine which combination of multiplerecombinant proteins.First, the four recombinant proteins were induced, expressed and purified byaffinity chromatography. After identified with SDS-PAGE, purified recombinantproteins each appeared as a single band and the molecular mass was consistent withthe expected size. The reactogenicity of recombinant proteins was identified with pigsand mice sera infected with T. spiralis, the results showed that the purified fourrecombinant proteins were recognized by the sera from T. spiralis infected pig andmouse. It suggested that the recombinant proteins of T. spiralis at differentdevelopment stages had high reactogenicity and can recognize both naturally infectedand laboratory infected T. spiralis antiserum. Polyclonal antibodies were preparedfrom purified recombinant proteins immunized mice, which were added to ESproducts and crude extract antigen of T. spiralis. These results exhibited that therecombinant proteins antiserum could recognize natural Ts-clp, Ts-Adsp, Ts-NBLspand Ts-serpin protein. Therefore, the four recombinant proteins are high reactogenicityantigen and can induce immune response with natural antigens. It has a certain valuein diagnosis and vaccine development of trichinellosis.Secondly, the antigen gene expression characteristics were analyzed byimmunofluorescence techniques, the results exhibited that the antisera of recombinantproteins were expressed on the surface of Ad, NBL and ML of T. spiralis. It suggeststhat these four proteins are involved in host-parasite interaction and are goodcandidates for the control of trichinellosis. We immunized Balb/c mice with the fourhigh reactogenicity antigens, respectively, in combination with Freund’s adjuvant andsubsequently challenged with T. spiralis larvae to determine the immunogenicity andprotection rate of recombinant antigens. The results exhibited that mice immunizedwith rTs-clp, rTs-Adsp, rTs-NBLsp or rTs-serpin exhibited an average reduction in themuscle larvae burden of24.42%,41.31%,22.39%and30.5%, respectively, relative to the control group. Immunization with the recombinant proteins induced both humoraland cellular immune responses, which manifested as elevated specific IgG antibodiesand a mixed Th1/Th2response, as determined by Th1(IFN-γ and IL-2) and Th2(IL-4,IL-10and IL-13) cytokine profiling, with the Th2predominant. Therefore, the fourrecombinant proteins of T. spiralis at different development stages have highimmunogenic and can induce partial protective immunity. They can be used aseffective vaccine candidates defence against trichinellosis.Finally, the immunization scheme was optimized to develop a vaccine whichcombination of four recombinant proteins (with the minimum protection of T. spiralismuscle larvae of rTs-NBLsp as an example). Due to the highly toxic of Freund’sadjuvant and even unsuitable for animal experiments, the study was taken to optimizethe condition of immune adjuvants. The results suggest that alum adjuvant can inducesimilar immune response to Freund’s adjuvant, and it has less toxic, so we choosealum as multi-antigenic vaccine adjuvant. Three different antigen doses (10,20or50μg) were immunized mice, respectively. The results suggested that the20μgrecombinant protein-immunized group could induce a similar immune response to50μg recombinant protein-immunized group, and the type of immune response did notchange. So we chose the dose of20μg for each recombinant protein asmulti-antigenic vaccine immunization dose. According to the optimize schemes above,the high immunogenicity recombinant proteins of T. spiralis at different developmentstages were immunized mice by20μg each. The results showed that the vaccinewhich combination of four recombinant proteins can induce strong humoral andcellular immune responses, and each antigen had a synergistic effect, which inducedfavorable immune response against T. spiralis infection. The vaccine whichcombination of four recombinant proteins can induce a higher reduction in the musclelarvae burden to70.9%, which is greatly increased compared with the singleimmunzed group.In conclusion, this study suggested that immunization with a mixture ofrecombinant antigens could be a very promising tool in immunoprophylaxis oftrichinellosis. Our results suggested that an effective vaccine defence againstTrichinella infection should be composed of a variety of parasite antigens and that the proper proteins could be equally effective, leading to strong protection againstparasite invasion. |
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