Development Of Virus-like Particles Of Peste Des Petits Ruminants Virus As Antigen To Study Its Immunogenicity

Peste des petitsrumants (PPR) is a highly contagious and economically important viral disease of small ruminants. It is notifiable to the Office International des Epizooties (OIE). For the control of PPR, attenuated vaccines are available commercially. However, a major disadvantage of these attenuated vaccines is that they cannot be used for differentiating infected from vaccinated animals (DIVA) and may be an obstacle for the control and eradiation of PPR. Furthermore, there are risks of virulence recovery. Therefore, safe, efficient and DIVA vaccines against PPR is urged to develop. Virus-like particles (VLPs) recently emerged as alternatives to subunit vaccines; these have the advantage that they mimic the organization and conformation of the native virus capsid and envelope, but lack the viral genome.Given their intrinsic immunogenic properties and high safety profile, VLPs have been generated and have been tested as vaccine candidates for a variety of human and animal viral diseases. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutinin (H) or fusion (F) protein. Moreover, the immunogenicity of these VLPs was evaluated in mice and goats.Baculovirus expression of heterologous genes permits multiple post-translational modifications and the expression of proteins has similar biological activity of the native protein. Given these advantages, the baculovirus expression vector-insect cell system has been utilized extensively for VLP production, and particularly for production of enveloped VLP vaccines. To anazyze the budding mechanism of PPRV, polycistronic baculovirus that expressing several proteins and monocistronic baculoviruses that expressing single protein (M, N, H and F) were constructed in this study, then the insect cells were infected with one polycistronic baculovirus or with one monocistronic baculovirus, the budding process of PPRV was reconstructed and the structural proteins required for budding was determined through detection of virus like particles in the culture medium. The results showed that the expression of M protein was necessary and sufficient for the formation of VLPs; the additional expression of the glycoproteins or nucelocapsid protein allowed formation of budding particles with the typical morphology and size of PPRV and related paramyxoviruses. VLP budding efficiency was enhanced when several structural proteins were expressed. It concluded that M protein is the major force to drive the virus budding. The expression of M protein alone could drive the VLP budding and released from the cells. M protein of PPRV, at the presence of other structural proteins could assemble into VLP and resemble the authentic virion in size and shape.VLPs are mimic the morphology of the native virus and can be uptaken by the immune cells with the route resemble as the native virus, and effectively induce the immune system to produce protective immune response. VLPs have been generated and have been tested as vaccine candidates for a variety of human and animal viral diseases. In order to evaluate the immunogenicity of the PPRV VLP, three types of recombinant baculovirus (rBac-MH, rBac-MF or rBac-MHF) were choosen to contract the PPRV VLP and subcutaneous injection of these VLP into mice and goats. Sera from immunized animals were analyzed for PPRV-specific antibodies by indirect ELISA, and for the detection of PPRV-specific virus neutralization antibody (VNA) titers using a micro-neutralization assay; IFN-y production of mice splenocytes that were stimulated with inactivated PPRV was measured by ELISPOT assays; the proliferative response was determined by MTS assay to evaluate the cell immune response. The results showed that without adjuvants, the PPRV specific IgG antibodies, VNA and lymphocyte proliferation responses were all induced in mice and goats immunized with PPRV VLP. The immune response induced by the PPRV-H VLP and PPRV-HF VLP was comparable to that obtained using equivalent amounts of PPRV attenuated virus vaccine while the immune response induced by the PPRV-F VLP was low in humoral immune response and high in cellular immune response. Taken together, the results suggested that the PPRV VLP could develope both humoral and cellular responses, the VLPs are potential DIVA vaccine candidate for the control and eradiation of PPR.