Preparation And Immunological Experiment Study Of Rabies Virus-like Particles

Rabies is a fatal zoonotic disease, the causative agent is rabies virus(RABV), which mainly infects neurons and proceeds to the central nervous system, the fatality rate is almost 100% and there is no effective therapeutic drug until now. According to the World Health Organisation, at least sixty thousand people die from the disease in the world every year, most of human deaths occur in Asia and Africa. Rabies is mainly transmitted by the dog with rabies scratch and bite,so the control of rabies from the source is the fundamental of the eradication of human rabies. The main measure to control zoon rabies is to vaccinate domestic dogs, cats and wild animals to reduce the risk of infection.Virus-like particles(VLP) formed by viral capsid protein or viral capsid protein and envelope protein which have an inherent property for self-assembly, there was no genetic material in VLP. VLP could mimic the morphology of the natural virus and cause the host infection which is similar to natural virus. In addition, there was no genetic material in VLP, which non-replicative and non-infective. VLP as a new vaccine for the preparation of the immune system which has high immunogenicity and excellent security and could be a vaccine candidates which has great future and manufacture applications. RABV glycoprotein(GP) has been demonstrated to be the principal correlate of protective immunity against rabies via its stimulation of the body to produce virus neutralizing antibody(VNA), GP has a carboxy terminus that inserts into the matrix protein(MP). The MP plays an important role not only in virus entry and membrane fusion but also in virus release and assembly, and it maintains the basic form of RABV, meanwhile it constitutes an influence of space structure of GP on viral envelope surface. Flagellin as a natural agonist for toll-like receptor 5(TLR5), and triggering of the TLR5 signaling pathway ultimately results in innate immune responses, and inducing B cells- and T cells-mediated adaptive immune responses. Escherichia coli heat-labile enterotoxin B subunit(LTB) is nontoxic and mediates high-affinity binding to the GM1 receptor on the surfaces of all mammalian cells, promote the ability of antigen presentation, elicit the proliferation and differentiation of lymphocytes, enhance the immune responses of the body. GP and MP are basic structure proteins of the RABV-VLP and membrane-anchored forms of flagellin or LTB which were as molecular adjuvants. We use recombinant baculovirus which express RABV structural protein and flagellin or LTB co-infection insect cell, synthesized two chimeric rabies VLPs, at the same time, compares and analyzes the immunogenicity of the two chimeric rabies VLPs. In addition, this study also established a double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for quantitative determination of rabies virus glycoprotein.Chapter one, development and application of andouble-antibody sandwich ELISA detection of Rabies virus. In this experiment, in order to establish double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA) for detection of rabies virus, the monoclonal antibody of rabies virus glycoprotein and rabies virus polyclonal antibody was used as the capture antibody and detecting antibody. SRV9 strain of rabies virus have been quantified the 50% of tissue culture infective dose(TCID50) was used as a standard. Optimized the reaction conditions and analyzed the specificity, repeatability and sensitivity, the rabies virus glycoprotein monoclonal antibody coated concentration was 2.5 g/mL at 4℃ overnight, 1% BSA as blocking agent at 37℃ for 2h, 1:200 dilution of polyclonal antibody against rabies virus at 37℃ 1.5h, the enzyme labeled antibody was diluted into 1:5000 at incubated at 37℃ for 1h, the substrate TMB for ELISA being incubated for 30 min at room temperature. This method can detect rabies virus specifically, and have no reaction with canine parvovirus, canine distemper virus and infectious canine hepatitis virus. The linear detection range of this method is 1.0×104-1.0×107TCID50/mL, coefficient of variability percent(CV%) of inter-batch duplicative texts and extro-batch duplicative texts was less than 6% and 8%.Chapter two, preparation and authenticate of chimeric rabies virus-like particle. We subculture recombinant baculovirus which express RABV Evelyn-Rokitnicki-Abelseth(ERA) strain GP and MP and and membrane-anchored forms of flagellin or LTB, which as molecular adjuvants to vitro passage cultivation. Co-infected Sf9 cells at 27℃ for 4-5 days,by the recombinant baculovirus which express GP and MP and flagellin or LTB, the chimeric rabies VLPs were purified through a centrifugal and collecting the supernatant. Byelectron microscopy, chimeric rabies VLP was round or elliptical, and with diameter was approximately 180~200 nm, obvious surface spikes were observed; by western blot, the construction of chimeric rabies VLP were GP, MP and flagellin or LTB. ELISA detection showed that the glycoprotein contents of two chimeric rabies VLPs are similar with natural rabies virus by cell cultured. These results indicated that we successfully designed and constructed two chimeric rabies VLPs containing GP, MP and flagellin or LTB, named them EVLP-F and EVLP-L, respectively.Chapter three, immunogenicity study of chimeric rabies virus-like particle. Co-infected Sf9 cells at 27℃ for 4-5 days,by the recombinant baculovirus which express GP and MP and flagellin or LTB, the chimeric rabies VLPs were purified through a centrifugal and collecting the supernatant. Using intramuscular injection to the mice and dogs by mixture of the EVLP-F and EVLP supernatant and Alhydrogel, Polysaccharide adjuvant and Freund’s adjuvant, then intramuscular injected mice and canine. enhance the immune once after first immunization, and then evaluate the immunological effect of EVLP-F and EVLP-L. Mice immune test result indicated that, after first immunization two weeks, virus neutralization antibody could be tested in mice serum, VNA of EVLP-L group is higher than EVLP-F group, alhydrogel group is higher than amylose adjuvant group and Freunds adjuvant group. After enhanced immunization, VNA of immuned mice serum was improved remarkably, the highest VNA is 23.38IU/mL. Canine immune test result indicated that, first immunization one week later, virus neutralization antibody could be tested in canine serum, VNA of EVLP-L group is higher than EVLP-F group, alhydrogel group is higher than amylose adjuvant group. After enhanced immunization, VNA titers of immuned canine serum was improved remarkably, the highest VNA titers is 53.30IU/mL. These data indicate that two kinds of chimeric rabies virus-like particles possess immunogenicity commendably, induce high VNA titers in organism after immuned, therefore, these rabies VLPs may be developed as high-efficient and low cost rabies vaccine candidates for animals.