Immunogenicity Of Recombinant Rabies Virus Expressing Two Copies Of Glycoprotein As Inactivated Rabies Vaccine

Rabies, caused by Rabies Virus(RABV), is one of the oldest infectious diseases with a long history of more than 5000 years. It is estimated that around 55,000 people die from rabies globally per year, most of which occur in those developing countries in Africa and Asia where canine rabies is endemic. In these countries, more than 95% of human rabies cases are related to dog bites, therefore, massive immunization in dogs is the most effective method to control rabies. Currently, inactivated RABV vaccines are widely used for domestic animals(mainly dogs and cats) vaccination due to their high safety. However, the protective immunity induced by these vaccines is relatively low and multiple shots are generally required, resulting in the long time course and a relative high cost. Hence, to develop affordable and more efficient RABV vaccines capable of inducing robust immunity are highly needed for rabies control, especially in the developing countries.The glycoprotein(G-protein) of rabies virus(RABV) is the primary immunogenic protein to induce virus-neutralizing antibodies(VNA), which is able to restrict the virus infection. Previous studies have confirmed that over-expressing one or two copies of G protein between G and L genes could enhance the production of VNA and provide better protection against virulent RABV challenge. In the present study, two recombinant RABVs(rRABVs) expressing two copies of G protein were generated, with the second G protein inserted between G and L genes(designated as LBNSE-GL-G), and between P and M genes(designated as LBNSE-PM-G), respectively. The growth kinetics of the two rRABVs on BSR cells suggests that the growth curves of both the recombinant viruses are similar with the parent virus. The protein expression and apoptotic analysis demonstrated that the LBNSE-PM-G recombinant virus produced more G protein and induced more apoptosis than LBNSE-GL-G. However, the result from the purified viruses demonstrated that the surface G protein of LBNSE-PM-G was less than LBNSEGL-G, similar to the level of LBNSE. In consistency with the surface G protein level, the in vivo studies showed that inactivated LBNSE-PM-G induced lower level of VNA than that of LBNSE-GL-G and conferred worse protection against CVS-24 challenge in a mouse model. Taken together, our results suggest that expression of one more copy of G gene ahead of M gene does not enhance the incorporation of G protein into the viron; therefore, it cannot enhance the immunogenicity when used as an inactivated vaccine.