Application Of Branched-chain DNA In Studying Immunity Induced By Equine Infectious Anemia Attenuated Vaccine

Objective: The Chinese attenuated equine infectious anemia virus (EIAV) vaccine is the first successful lentiviral vaccine in the world, and its molecular mechanism on the ability to induce protective immunity remains to be elucidated. Cytokine secretion stimulated by specific antigens is currently the major approach to evaluated cell mediated immunity. However, it is necessary to establish proper methods to detect accurately and effectively multiple cytokines for studies of animal cell medicated immunities. In this study, simultaneous quantitative analysis of equine cytokine mRNAs using branched-chain DNA was established to evaluate the differences in cell immunity to better understand the immunity induced by the vaccine strain and pathogenesis resulted by the virulent strain.Methods: EIAVDLV34, an EIAV virulent strain that effectively proliferate in cultivated cells was selected and characterized by analysis of the genomic structure and evaluation of the virulence. The branched-chain DNA assay, which is an mRNA-based quantitative multiple-cytokine detection technique, was established to measure 17 equine cytokines simultaneously. These cytokines are IL-1α, IL-1β, IL-2, IL-4, IL-10, IL-12α, IFN-γ, TNF-α, TGF-β1, IL-8, MCP-1, MCP-2, MIP-1α, MIP-1β, IP-10, BAX and Bcl-2. The differences in expressing Th1,Th2,Th3,Th17cytokines and chemokines in macrophages induced by the EIAV vaccine strain EIAVDLV124 and the virulent strain EIAVDLV34 were then analyzed.Results: The virulent strain EIAVDLV34 kept the pathogenic ability to horses as its parental strain EIAVLN40. Although both EIAVDLV34 and EIAVDLV124 induced apoptosis on in vitro cultured macrophages, the vaccine strain resulted significant more apoptotic cells than EIAVDLV34 (p<0.05). Expression levels of all 17 species of cytokine mRNAs were in the detectable ranges (0~20000 relative luminescence units,RLU)measured by branched-chain DNA assay. The expression levels of chemokines MCP-1, MCP-2, MIP-1αand MIP-1βinduced by EIAVDLV34 were dramatically higher than those induced by EIAVDLV124 (p<0.05), whereas the vaccine strain induced scientifically higher level of IL-1αi than the virulent strain (p<0.01).Conclusions: An EIAV virulent strain EIAVDLV34 that adapted in cultured cells was selected and characterized. This strain is essential for in vitro studies of biologic characteristics and molecular bases of the EIAV vaccine strain and virulent strain. In addition, for the first time, a valuable quantitative method for simultaneous detection of equine cytokines by branched-chain DNA technology was established, which provides an important approach for studies on equine immunities. Furthermore, statistically significant higher chemokines induced by the EIAV virulent strain implicates the correlation of up-regulated chemokines to acute equine infectious anemia resulted from EIAV virulent strain-induced inflammation. The stronger apoptosis-inducing feature of the vaccine strain is considered due to the adaptation of the strain to the environment of in vitro cultured cells, which actually altered replication of the vaccine in macrophages in the body.