Construction, Preparation And Immune Effect Evaluation Of Rabies Virus-like Particles

Rabies remains an important, highly fatal zoonotic disease，all mammals,including humans, are susceptible to rabies. Rabies occurs in more than150countries and territories, and causes tens of thousands of deaths every year, but morethan95%of human deaths occur in Asia and Africa, where dog rabies potentiallythreatens over3billion people. The causative agent is rabies virus (RABV), whichmainly infects neurons and proceeds to the central nervous system, causing severeand fatal viral encephalitis in animals and humans. Rabies is a vaccine-preventableviral disease. Most developed countries have eliminated canine rabies by vaccinatingdogs on a large scale, which is considered to be the most cost-effective strategy forrabies prevention, as a result, the incidence of human rabies has significantlydecreased. Therefore, the development of a more affordable, safe, and potent rabiesvaccine is advisable and necessary.Virus-like particle (VLP) formed by one or several viral structural proteinswhich have an inherent property for self-assembly, and mimic the morphology of thenatural virus. There was no genetic material in VLP, which non-replicative andnon-infective. VLP display antigenic epitopes in the correct conformation and in ahighly repetitive manner, and leading to highly immunogenic. VLP can take up byprofessional antigen-presenting cells effectively, stimulate innate immunity andfurther elicits adaptive immune and inflammatory responses against infection.Therefore, VLP represent a major advancement in the development of subunitvaccines with enhanced immunogenicity. The VLP of several viruses have beendeveloped as effective vaccine candidates, such as influenza and hepatitis VLPs,which have been progress to clinical trial or approve to commercial production. But,so for, about rabies VLP is rarely reported. Therefore, in this thesis, we usebaculovirus-insect cell expression system to construct and produce rabies VLP, and evaluate the immunogenicity.Chapter one, construction and preparation of rabies virus-like particle. RABVglycoprotein (GP) is a structural protein, which has a carboxy terminus that insertsinto the matrix protein (MP), has been demonstrated to be the principal correlate ofprotective immunity against rabies via its stimulation of the body to produce virusneutralizing antibody (VNA). Meanwhile, the MP plays an important role not only invirus entry and membrane fusion but also in virus release and assembly, and itmaintains the basic form of RABV. Thus, in the present study, we chose these twoproteins as basic elements to construct the rabies VLP. We constructed and rescuedtwo recombinant baculoviruses (rBVs) that express GP and MP, respectively, andco-infected Sf9cells of suspension culture. Culture supernatants were harvested, andthe rabies VLP was purified through a discontinuous sucrose gradient byultracentrifugation. Byelectron microscopy, the rabies VLP was round or elliptical,and with diameter was approximately180~200nm, obvious surface spikes wereobserved; by western blot, the construction of rabies VLP were GP and MP; byimmunoelectron microscopy, the GP was integrated into the rabies VLP. Theseresults suggest that when expressed in Sf9insect cells, the GP and MP of RABVcould self-assemble into rabies VLP, which named it EVLP.Chapter two, construction and preparation of chimeric rabies virus-like particle.Flagellin as a natural agonist for toll-like receptor5(TLR5), and triggering of theTLR5signaling pathway ultimately results in innate immune responses, andinducing B cells-and T cells-mediated adaptive immune responses. Escherichia coliheat-labile enterotoxin B subunit (LTB) is nontoxic and mediates high-affinitybinding to the GM1receptor on the surfaces of all mammalian cells, promote theability of antigen presentation, elicit the proliferation and differentiation oflymphocytes, enhance the immune responses of the body. Therefore, we designedand constructed two chimeric rabies VLPs containing GP and MP of the RABV, andmembrane-anchored forms of flagellin or LTB, which as molecular adjuvants. Weconstructed two chimeric genes containing flagellin or LTB and the transmembrane and cytoplasmic tail regions from the GP, and rescued two rBVs that expressflagellin and LTB chimeric genes, respectively. Three rBVs expressing GP, MP andflagellin or LTB co-infected Sf9cells of suspension culture. Culture supernatantswere harvested, and chimeric rabies VLP was purified through a discontinuoussucrose gradient by ultracentrifugation. Byelectron microscopy, chimeric rabies VLPwas round or elliptical, and with diameter was approximately180~200nm, obvioussurface spikes were observed; by western blot, the construction of chimeric rabiesVLP were GP, MP and flagellin or LTB; by immunoelectron microscopy, theflagellin and LTB were integrated into chimeric rabies VLP, respectively. Theseresults indicated that we successfully designed and constructed two chimeric rabiesVLPs containing GP, MP and flagellin or LTB, named them EVLP-F and EVLP-L,respectively.Chapter three, immunogenicity study of rabies virus-like particle. Theimmunogenicity and potential of three rabies VLPs, EVLP, EVLP-F and EVLP-L, asnovel rabies vaccine were evaluated by intramuscular vaccination in mouse and dogmodels.Mouse studies demonstrated that three rabies VLPs were immunogenic, butcompared with EVLP, both EVLP-F and EVLP-L induced faster and larger VNAresponses and elicited greater numbers of CD4+and CD8+T cells secreting IFN-γ orIL-4. Moreover, two chimeric rabies VLPs recruited and/or activated more B cellsand dendritic cells (DCs) in inguinal lymph nodes. EVLP-F induced a strong,specific IgG2a response but not an IgG1response, suggesting the activation of Th1class immunity; in contrast, Th2class immunity was observed with EVLP-L.Immunization with EVLP only provide a partial protection for mice against rabies,however, the significantly enhanced humoral and cellular immune responses inducedby two chimeric rabies VLPs provided complete protection against lethal challengewith RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L alsoexhibited increased VNA titers in sera and enhanced IFN-γ and IL-4secretion fromperipheral blood mononuclear cells compared with EVLP. These results illustrate thatrabies VLP possess immunogenicity, but membrane-anchored flagellin and LTB display a strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhancedRABV-specific humoral and cellularimmune responses in both mouse and dog.Therefore, these rabies VLPs may be developed as safe and more efficacious rabiesvaccine candidates for animals.Chapter four, immunogenicity of rabies virus-like particle affected bypolysaccharide from the root of Isatis indigotica.An adjuvant is important forvaccines, it can increase the intrinsic immunogenicity of an antigen or a vaccinecandidate and then induce an enhanced immune response for a long-lasting period.One of the main components of the bioactive substances in traditional Chineseherbal medicine (CHM) is polysaccharide, which has wide pharmacologicalactivities, especially in terms of immunomodulatory and anti-tumor effects. In thisstudy, we used rabies virus-like particles (RVLPs) in combination with IIP-A-1as animmunogen to vaccinate mice by intramuscular route, and then evaluated thedetailed immune responses and further determined the adjuvant activity of IIP-A-1.Compared with immunization with RVLPs alone, co-immunization with RVLPs andIIP-A-1significantly enhanced innate and adaptive immune responses, including therecruitment and/or activation of DC and B cells, the proliferation and activation of Tlymphocytes, the differentiation of IFN-γ-or IL-4-secreting CD4+and CD8+T cells,and the production of rabies virus (RABV)-specific neutralizing antibodies.Furthermore, low-dose RVLPs mixed with IIP-A-1elicited similar immunizationresponses as high-dose RVLPs alone. More importantly, the enhanced immuneresponses induced by the RVLP co-immunization with IIP-A-1provided micecomplete protection against RABV infection. These results indicate that the α-glucanIIP-A-1possesses adjuvant activity and could be developed as a novel adjuvantcandidate for VLP vaccines or other types of vaccines.