The Immunogen Preparation And Immunogenicity Study Of Recombinant Virus RCVS-11-G

Rabies is a fatal encephalomyelitis caused by a single-stranded RNA virus, to which all warm blooded animals and human are susceptible. Because the lethality of rabies is nearly 100% once clinical signs appear, the best way to prevent rabies is inoculation with a vaccine. The most widely used rabies vaccines in China are inactivated vaccines. Although these inactivated rabies vaccines are safe, but they own a low immunogenicity. There are two ways to increase the effect of inactivated vaccine; one is to use virus strains with a better immunogenicity, the second is to use a adjuvant to boost and accelerate the immune response.The culturing cell adaption and identification of recombinant rabies virus r CVS-11-G. The extra G gene of r CVs-11-G was stablely expressed when cultured in BSR and Vero cells. The titers of r CVS-11-G collected from BSR were between 5×108TCID50/m L and 109TCID50/m L. The titers of r CVS-11-G collected from Vero were about 108 TCID50/m L. The results indicate that r CVS-11-G could replicate well in BSR and Vero cells, which made it possible to construct a immunogen with r CVS-11-G.In the present study, a recombinant rabies virus, r CVS-11-G, were inactivated, then mixed with either polysaccharide IIP-A-1 and IIP-2 isolated from Isatis indigotica root or polysaccharide PCP-II which was isolated from Poria cocos as immunogen to immune mice via the intramuscular route. Blood samples were collected to determine the virus-neutralizing antibody titer by FAVN. Peripheral blood mononuclear cells were isolated to determine the activation of B and T lymphocytes in blood. Inguinal lymph node samples were collected, and the proliferation of B lymphocytes was measured by flow analysis. Spleens were collected and splenocytes were isolated, the IL-4 and IFN-γsecreting cells were measured by ELISpot assays, intracellular cytokine staining. The amount of IL-2, IL-4, IL-10 and IFN-γsecreted by the splenocytes were determined by ELISAs. The results of the challenge test and post-exposure test show that the polysaccharides could protect more mice than aluminum hydroxide when they were mixed with inactivated rabies vaccine. The results revealed that all three polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 and PCP-II strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. All the data suggest that all three polysaccharides possess excellent adjuvant activity and enhances both antigen-specific cellular and humoral immunity in mice.We chose the polysaccharide PCP-II, which owns best adjuvant activities of the three polysaccharides. Dogs were immunized with r CVS-11-G together with aluminum hydroxide or PCP-II as an adjuvant; the control group was injected with a commercial rabies vaccine. Serum samples were collected, and the virus-neutralizing antibody titers were measured. The results of both the booster immune test and the single-dose immune test indicate that PCP-II is an excellent candidate adjuvant for inactive rabies vaccines in the veterinary setting.In conclusion, these results suggest that the polysaccharides evaluated here can significantly enhance cellular immune responses and accelerate antibody responses to an inactivated rabies vaccine. Aluminum hydroxide could only induce a slow antibody response that included the secretion of only a few cytokines. Thus, our results suggested that plant root polysaccharides may be more effective for improving antibody responses than traditional adjuvants when used in conjunction with an inactivated rabies vaccine. This increased effectiveness accelerates immune protection and reduces the required vaccine dosage. Overall, our results indicated that all three of the polysaccharides tested in this study, in particular PCP-II, are excellent adjuvants candidates for veterinary rabies vaccine. Finally,Our work highlights the potential of using polysaccharides to develop a more efficient and affordable post-exposure vaccine for use in humans.