Effective Preexposure And Postexposure Prophylaxis Of Rabies With Recombinant Rabies Virus Expressing Immunostimulatory Molecules

Rabies is a life-threatening disease caused by an RNA virus that is usually transmitted to human through bites from rabid animals. Each year, approximately 55000 individuals worldwide die of rabies and millions more undergo post-exposure prophylaxis (PEP). Most of the human cases occur in the developing nations of Asia and Africa where dog rabies remains to be the main source for human exposure. In the developed countries, human rabies has dramatically declined as a direct consequence of routine vaccination of pet animals. Current rabies vaccines used for human are inactivated virus vaccine. Although these vaccines are safe and efficacious, multiple doses (at least 4) must be administered over an extended period of time (14 days) to people who are exposed to rabies or suspected rabid animals. In addition, the high cost associated with these inactivated rabies virus (RABV) vaccines prevents their effective use in developing countries where the vaccines are needed most. Live attenuated RABV vaccines or recombinant live vaccines have been licensed, particularly for wild animals. These vaccines have been used for large scale elimination of rabies in Europe as well as in North America. However, they usually induce intensive skin inflammation and not worked well for dogs and skunks. Furthermore, those vaccines are not so effective in oral vaccination for dogs and skunks. Therefore, more efficacious and affordable RABV vaccines are needed, particularly in the developing nations. Once the clinic manifestations of rabies developed, however, treatment options for rabies are limited. To date, only nine individuals have survived rabies virus infection. Unfortunately, most of these patients died either after prolonged illness or developed severe neurogical sequel. Therefore, more effective therapeutics is needed in management of clinical rabies.It was found previously that induction of innate immunity, particularly chemokines and type I interferons, is an important mechanism of rabies virus attenuation. To evaluated the effect of overexpression of chemokines on RABV infection. Macrophage inflammatory protein 1α(MIP-1α, CCL3) was cloned into the genome of attenuated RABV strain HEP-Flury, the recombinant rabies virus (rRABV) was rescued and designated as rHEP-MIP1α. To characterize the rRABV in vitro, virus growth kinetics was examined in NA cells. No significant difference was found between the rRABV and parental virus, indicating that the viral growth was not affected by the insertion of MIP-1α. The ability of rHEP-MIP1αto produce MIP-1αwas determined by measuring MIP-1αin virus-infected cells with ELISA kits. It was found that MIP-1αwas expressed by rHEP-MIP1αin a dose-dependent manner. To determine the effect of chemokine expression on RABV infection, mice were infected with the rRABV by intracerebral route. A transient MIP-1αexpression and inflammatory cell infiltration was induced in the central nervous system (CNS) of mice infected with rHEP-MIP1α, compared with those infected with parental virus. The pathogenicity of rHEP-MIP1αwas more decreased than parental virus. It indicates that overexpression of MIP-1αfurther decreased RABV pathogenicity by inducing a transient innate immune response in the CNS.To investigate the immunogenicity of rHEP-MIP1α, mice were immunized with rRABV by the intramuscular (i.c.) route. The virus neutralizing antibody titer (VNA) in serum was measured by the rapid fluorescent focus inhibiton test (RFFIT). It was found that the level of VNA was significantly higher in mice immunized with rHEP-MIP1αthan that induced by immunization with parent virus. After challenge with virulent CVS-24, more mice immunized with rHEP-MIP1αsurvived than mice immunized with the parental virus. Virus replication, expression of chemokines, and/or recruitment of immune cells at local sites was determined by quantitative real-time PCR (qRT-PCR). There was no difference in virus replication in mice infected with each virus, indicating the induction of adaptive immunity by rHEP-MIP1αis not due to the rate of virus replication at the local site. However, significantly more MIP-1α, CD19, CD11c and IL-4 mRNA was detected in the muscle tissue of mice infected with rHEP-MIP1αthan in mice infected with the parent virus. CD11c dendritic cells (DCs) and B cells were analyzed by flow cytometry. It was found that the recruitment of DCs and B cells in lymph node and peripheral blood was significantly increased in mice immunized with rHEP-MIP1α, than those immunized with parent virus. All the data demonstrate that expression of MIP-1αenhances the immunogenicity by recruiting DCs and B cells to the site of immunization, lymph nodes, and the blood. Therefore, recruitment and/or activation of DCs play an important role in enhancing the protective immune response against RABV. To address the importance of DCs activation for RABV vaccine efficacy, several DCs stimulatory molecules, e.g. granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage derived chemokines (MDC), and MIP-1α, were cloned into the genome of RABV strain SAD L16. The rRABVs were rescued and designated as rLBNSE-GM-CSF, rLBNSE-MDC, and rLBNSE-MIP1α, respectively. It was found that the rRABVs expressed these molecules activate/recruit DCs and enhanced protective immune response.To investigate if the rRABVs expressing the immunostimulatory molecules have the ability to prevent animals from developing rabies, mice were infected with lethal doses of street RABV and then treated with rRABV at different time points after infection by intracerebral (i.c.), intramuscular (i.m.), intradermal (i.d.) or intranasal (i.n.) route. It was found that significantly more mice intracerebrally treated with rRABV expressing MIP-1α, GM-CSF, macrophage-derived chemokine (MDC), or IP-10 as late as day 5 after infection with virulent RABV were protected from developing rabies than sham-treated mice. On the other hand, treatment with UV-inactivated rRABV did not provide protection despite the fact that virus VNA were induced in the periphery. The leakage of fluorescein sodium (NaF) from the circulation into CNS tissues was measured in cerebrum, cerebellum or spinal cord. It was found that BBB permeability to NaF was significantly elevated in the cerebrum and cerebellum of mice treated with rRABVs than sham-treated mice. Expression of chemokines/cytokines, and infiltration of inflammatory cells were measured by qRT-PCR, multiplex ELISA and flow cytometry. Intracerebral treatment of mice with live rRABVs induced significantly higher levels of chemokine/cytokine expression in the CNS and in the periphery, and more infiltration of inflammatory and immune cells into the CNS, than sham-treated mice or mice treated with UV-inactivated rRABV. Most importantly, treatment with a chemokine (chemoattractant protein-1, MCP-1, also termed CCL2) with doses known to enhance BBB permeability increased the protective efficacy of UV-inactivated rRABV. In sum, these studies confirm that chemokines can enhance the BBB permeability, allow infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and to prevent the development of rabies.In the present study, rRABVs expressing the immunostimulatory molecules were constructed. It was found that the viral growth was not affected by the insertion of foreign genes. Overexpression of these molecules further decreased RABV pathogenicity by inducing a transient innate immune response. Recruitment and activation of DCs is important in enhancing the protective immune responses against rabies. These rRBAVs expressing immunostimulatory molecules could have the potential to be used not only for pre- and post-exposure immunization but also for therapy in clinical rabies. The data from this study provides an important theoretical basis for study on pathogenesis of RABV, and selection of more viable and affordable RABV vaccines. It may also provide a new therapeutic of management of clinical rabies.