The Role Of Antigen Diversity And Super-infection Resistance In Protective Immunity Induced By The Chinese EIAV Attenuated Vaccine Strain

Equine infectious anemia virus (EIAV) is a member of lentivirus can infect equine animals. The Chinese EIAV attenuated vaccine strain, designed since1970s, induces excellent immune protection against the infection of different EIAV virulent strains. It is the only lentiviral vaccine that successfully applied to control the spread of a disease in a country. The principle of how the EIAV vaccine reduced the virulence and obtained the ability to induce solid protective immunity should provide valuable information for the study on other lentiviral vaccines, inducing vaccines to AIDS. In this study, we chose the fetal donkey dermal cells adapted vaccine strain (EIAVFDDV13) of EIAV vaccine, which induced stronger protective immunity than the other EIAV vaccine strains, to intensively investigate the biological characterizations of this attenuated Chinese vaccine. Specifically, we focused on the relationship between genomic structure of EIAVFDDV13and its ability to induce protective immunity, and between the super-infection resistance induced by EIAVFDDV13and the innate immunity activation and the following construction of adaptive immunity too.First, the attenuated equine infectious anemia virus (EIAV) vaccine strain EIAVFDDV13and a proviral derivative of the vaccine EIAVFDDV13-8were compared with respect to differences in inducing protective immunity. Although these two strains replicated equally well in vitro and in vivo, the proviral strain induced significantly less protection from disease and infection caused by viral challenge and significantly lower specific neutralizing capability. These findings indicated that the proviral strain had lost the ability to stimulate immune protection compared to the parental vaccine strain. A further comparison of innate immunity activation in monocyte-derived macrophage (MDM) in vitro induced by infection of these two attenuated strains showed different mRNA expression on Toll like receptors and type Ⅰ interferons (IFNα/β), and these mRNA expression difference had been proved to have relationship with the subsequent development of adaptive immunity to virus. The results indicated these two strains might induce different humoral immunity by activating a different innate immunity. Then, what's kind of mechanism utilized by these two strains to cause a different inducing should be study intensively. Besides the influence elements from the host, to the viruses themselves, a further analysis of the envelope gp90gene variation revealed that compared to the proviral strain, the vaccine strain displayed wide sequence diversity in antigen composition. Considering the recent preclinical and clinical experiments have suggested that the multi-envelope vaccine had potential advantages in the induction of immune responses over immunogens with a single envelope protein, especially because the combinatorial use of diverse immunogens can result in an expanded spectrum of neutralizing antibodies that are induced by these vaccines, we tent to infer that the differences in antigen composition might be the major cause for the failure of the proviral derivative to elicit the immune protection induced by the parental strain. Furthermore, we also need to prove whether the difference of antigen composition is related to the different innate immunity activation induced by these different attenuated strains or not.Second, we focused on the super-infection resistance phenomenon (SIR) induced by the Chinese EIAV attenuated vaccine strain. SIR is a capacity of a cell which was infected by a virus showed an ability to resist second infection by a same or similar virus with the virus infected already. The lentiviruses included HIV and EIAV can induce SIR in host cells. In order to clarify whether the attenuated lentivirus can induce a different SIR compared with the virulent strain or not, in this study, we used the Chinese EIAV vaccine strain EIAVFDDV13and an infectious molecular clone strain EIAVUK3from a wild type EIAV strain EIAVwyoming, as representatives of an attenuated strain and a virulent strain, to infect MDM and detect the SIR induced by these two strains. Based on the quantitive PCR and a new RNA situ hybridization technique, viewRNA technique which can differentiate these two EIAV strains specifically, we determined that EIAVFDDV13induced a predominant SIR against EIAVUK3in MDM in vitro, and the intensity of this SIR was stronger than the SIR against EIAVFDDV13induced by EIAVUK3.Next, to answer what's kind of mechanism is responsible for this different inducing of SIR by these two EIAV strains, we used branched DNA technology to compare the mRNA expression levels of several intracellular proteins, including ELR1, ELR1-IN, TLR3, TLR7/8, TLR9, IFNα1/β, Apobecs, Trim5α, SAMHD1and Tetherin. These molecules are related with SIR or anti-lentivirus directly or indirectly as they can play important roles in influencing lentiviruses to entry the cells or suppressing replication of lentiviruses in host cells. According the detection results, we found that compared with the MDMs infected by EIAVUK3, the mRNA expression of ELR1-IN, TLR3, INFP, Trim5a and Tetherin were uploaded in MDMs after infected by EIAVFDDV13.Interestingly, we also can mimic a similar mRNA expression uploading levels of these four proteins in MDMs by dealing MDMs with suitable dose of Poly I:C, a synthetic analog of dsRNA can activate TLR3. Furthermore, the MDMs treated with Poly I:C showed an obvious resistance to EIAV infection. So, it inferred that the activation of TLR3-dependent pathway took part in the stronger SIR inducing by EIAVFDDV13. As same time, considering the relationship among TLR3-dependent pathway, the main product IFNβ induced after this pathway activation, innate immunity activation and adaptive immunity construction, what and how the EIAV vaccine strain and virulent strain did in inducing different SIR, innate immunity and adaptive immunity should be elucidated intensively in future. In conclusion, all the results obtained in this study are important information for understanding of the essential feature of attenuated Chinese EIAV vaccines. These results also established a solid base for further studies on how the EIAV vaccine induces protective immunity to different pathogenic strains.