| Eastern equine encephalitis virus (EEEV) and western equine encephalitis virus (WEEV) are zoonotic viruses that may cause virulent infectious encephalitis. This disease has a remarkably acute onset with a high mortality rate, therefore, EEEV and WEEV belong to high-risk pathogens, and they have been listed as a category B bioterrorism agent. Since EEEV and WEEV were found, they had caused huge economic losses to the society. At present, no particularly effective vaccine is available for human use for preventing them, currently, traditionally prepared inactivated or attenuated or virus-vectored live EEEV and WEEV vaccines are the primary means of preventing them, however, because of their own shortcomings of the traditional vaccines, especially they are not applicable for persons, few vaccines have high efficacy. In addition, EEEV and WEEV are transmitted by mosquito bites and aerosol action. Changes in the climate and ecological environments, increased international travel, and the highly contagious nature of EEEV and WEEV may cause outbreaks of them at any time. Therefore, it is very necessary to develop new and high effective stockpile vaccines of EEEV and WEEV to prevent them.In recent years, virus like particles (VLPs) as a new vaccine and DNA vaccine have exhibited their own unique advantages, indicating their huge development and application potential. In this study, we investigated the VLPs vaccine of EEEV and WEEV and DNA vaccine of them, and established the neutralization test of EEEV and WEEV pseudoviruses, which can provide some important references for developing of new EEEV and WEEV vaccine. This study was mainly divided into five parts:Part1:The establishment of neutralization test of eastern and western equine encephalitis pseudovirus. In order to effectively evaluate antibody neutralizing and avoid hidden danger on biosafety of EEEV and WEEV as the pathogeny in the neutralization test, here, for the first time, the production of HIV lentiviral vectors pseudotyped with the envelope proteins of EEEV or WEEV was constructed. The identification result showed the pseudoviruses were consistent with EEEV and WEEV reported in the literature on viral morphology and structure, and the result from inhibition assay of pseudovirus infection showed the EEEV and WEEV pseudovirus infection was inhibited by EEEV and WEEV antibodies, respectively. On this basis the neutralization test of EEEV or WEEV pseudoviruses was established, respectively, and testing results indicated the neutralization test was able to effectively evaluate the antibody neutralization.Part2:Construction, identification and immunological experiment study of VLPs vaccine of eastern equine encephalitis virus. In recent years, virus like particles as a new vaccine have exhibited their own many unique advantages, indicating better development prospect. At present, the research of the EEEV VLPs is not yet being seen in the critical literature, therefore, the first recombinant baculoviruses (BmNPV-C-E) were constructed to express structural proteins (C、 E3、E2、6K and E1) of EEEV using Bac to Bac system by means of genetic recombination technology, the identification results showed the EEEV E1proteins and VLPs were expressed by the BmN cells transfected with BmNPV-C-E. Immunological experiment results indicated that EEEV VLPs were able to induce the T cells activation and were able to cause the high levels of IL-2、IFN-γ、TNF-α and neutralizing antibody in mice; In addition, the lymphocytes from the horses vaccinated with EEEV VLPs specifically generated proliferation and activation, and there were high-level antibodies against EEEV effectively neutralizing the EEEV pseudoviruses in the serum from the vaccinated-VLPs horses. Therefore, these results showed that EEEV VLPs were able to stimulate strong cell-mediated immune response and humoral immune response in mice or houses, which indicated that EEEV VLPs had strong immunogenicity.Part3:Construction, identification and immunological experiment study of VLPs vaccine of western equine encephalitis virus. Now, many kinds of VLPs vaccines are successful, but currently, there is no the research of the WEEV VLPs, hence, in this study, the first recombinant baculoviruses (AcMNPV-C-E) were constructed to express structural proteins (C、E3、E2、6K and E1) of WEEV using Bac to Bac system by means of genetic recombination technology, the identification results showed WEEV VLPs were correctly expressed by the SF9cells transfected with AcMNPV-C-E. Immunological experiment results indicated that WEEV VLPs were able to exhibit significantly stronger IFN-gamma responses and may induce the high levels of IL-2、IL-4、IFN-γ from splenocytes and antibodies against WEEV effectively neutralizing the WEEV pseudoviruses in the serum from the vaccinated-VLPs mice; In addition, the lymphocytes from the horses vaccinated with WEEV VLPs specifically generated proliferation and activation, and there were high-level neutralization antibodies against WEEV pseudoviruses in the serum from the vaccinated-VLPs horses. Consequently, these experimental results showed that WEEV VLPs induced strong cell-mediated immune response and humoral immune response in mice or horses, which indicated that WEEV VLPs were able to exhibit strong immunogenicity.Part4:Construction, identification and immunogenicity study of DNA vaccine of eastern equine encephalitis virus. In recent years, DNA vaccine as a developing new vaccine has exhibited a lot of its own unique advantages, indicating great application potential. But, at present, DNA vaccine research of the eastern equine encephalitis is less. Consequently, the pcDNA3.1-C-E-eeev recombinant expression vectors were constructed in this study. The identification results showed that EEEV E1or E2proteins were correctly expressed by the293T cells transfected with pcDNA3.1-C-E-eeev, and the E1or E2proteins were able to package EEEV VLPs that were released into the extracellular environment. In addition, pcDNA3.1-C-E-eeev as a DNA vaccine possessed strong immunogenicity. After two immunizations, the mice vaccinated with pcDNA3.1-C-E-eeev plus mixed CpG/poly (I:C) adjuvant exhibited significantly stronger IFN-gamma responses and generated high-level CD4+cell responses for the cytokines IL-2, IL-4and IFN-y and CD8+T cell responses for the cytokines IL-2and IFN-y compared to the mice vaccinated with the corresponding antigen plus CpG or poly(I:C) alone. In addition, the higher antibody titers against EEEV effectively neutralized the EEEV pseudoviruses in the group immunized with antigen plus mixed CpG/poly (I:C) adjuvant after tertiary immunization. This study demonstrated that the pcDNA3.1-C-E plasmids in conjunction with mixed CpG/poly (I:C) adjuvant priming maximized the cellular immune response and specific antibody generation in mice. Moreover, this mixed adjuvant priming could provide a promising strategy for enhancing the immune effectiveness of a DNA vaccine against EEEV.Part5:Construction, identification and immunogenicity study of DNA vaccine of western equine encephalitis virus. Since DNA vaccine research, the vaccine has exhibited a lot of its own unique advantages, and has huge development potential, but currently, DNA vaccine research of western equine encephalitis is less. In this study, the pcDNA3.1-C-E-weev recombinant expression vectors were constructed by using pcDNA3.1vectors and WEEV structural genes (C、E3、E2、6K and E1). The results of indirect immunofluorescence and electron microscope showed that WEEV E1proteins and VLPs were correctly expressed by the293T cells transfected with pcDNA3.1-C-E-weev, and the VLPs were released outside the cell. The immunological experiment results showed pcDNA3.1-C-E-weev as a DNA vaccine was able to induce the T cells activation and generated the high level IL-2、IL-4、IFN-γ cytokine and antibodies against WEEV effectively neutralizing the WEEV pseudoviruses in vaccinated mice, which suggested that pcDNA3.1-C-E-weev was able to activate cell-mediated immune response and humoral immune response.In sum, these results in this study showed that EEEV or WEEV VLPs prepared using baculovirus expression vector system and DNA vaccine of EEEV or WEEV exhibited strong immunogenicity, indicating the great development potential of vaccines, which was able to provide a good foundation for development of new effective EEEV and WEEV stockpile vaccines. |
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