Kinetics Of Humoral Immune Responses Induced By An Infectious Clone Derived From An Attenuated EIAV Vaccine And Env Glycosylation Modification Research

Equine infectious anemia virus (EIAV), like HIV, belongs to the Lentivirus genus and Retroviridae family. The virus is similar to HIV in many aspects including its morphology, genomic structure, structure and function of viral encoding proteins, and antigenicity, etc. Chinese EIAV attenuated live vaccine is the only lentivirus vaccine developed and applied on a large-scale worldwide. Therefore, elucidating the immune protective mechanism of EIAV attenuated vaccine, would facilitate the development of HIV vaccine.This study obtained EIAV an attenuated vaccine---pLGFD3-v from an infectious molecular clone which was constructed in the former research. Horses were immunized with pLGFD3-v by subcutaneous injection and with balanced saline as control. Blood samples were collected at the indicated time-points. Seral binding antibodies against envelop protein Gp90 and matrix protein p26 and neutralizing antibodies were quantified. EIAV attenuated vaccine pLGFD3-v was capable of inducing binding antibodies against both EIAV Env Gp90 and Gag p26. Gp90-specific antibodies significantly increased and reached the plateau at two and half months(titer 1:320, 1:320), and remained detectable even at 32 months post inoculation. In accordance with this observation, the neutralizing antibodies displayed a comparable kinetics(about titer 1:2800—1:3000). In contrast, the Gag-specific antibodies peaked at 1-2 months(titer 1:320,1:40) and declined quickly afterwards and almost reached undetectable level within 4 months. These results demonstrated that pLGFD3-v could induce binding antibodies against both EIAV Env Gp90 and Gag p26, and also neutralizing antibodies. The prolonged lasting of Env-specific binding antibodies and neutralizing antibodies may contribute to the immune protection conferred by EIAV attenuated vaccine. To increase the attenuated vaccine immunogenicity for neutralizing antibodies induction, we attempted to delete the glycosylation sites in the V3, V4 andβsheet regions, and investigate the impact of deglycosylation on humoral immunity. First ,we constructed DNA vaccine(pDRVI—none) and vaccinia vector vaccine(rTTV—none) deglycosylated in the V3,V4 andβsheet regions. We designated EIAV env DNA vaccine(pDRVI—env) and vaccinia vector vaccine (rTTV—env) based on the attenuated vaccine envelop protein as control, then we successfully immunized animals using prime/boost strategy. The Balb/c mice group were divided and immunized as follows:(1) pDRVI—env/rTTV—env, (2)pDRVI—none/rTTV-none, (3)pDRVI—sv1.0/rTTV—7521 and (4)balanced saline. Two months later, binding antibodies against envelop protein gp90 and neutralizing antibodies were quantified. The results showed that deglycosylated env(none group) and unmodified env(env group) were no significant variation statistically for the gp90 binding antibodies induction; however, deglycosylated env(none group) decreased frequency and titer of induced neutralizing antibodies. This demonstrated that the glycosylation sites located in the residues 199 and 217 in the V3 and V4 regions were indispensable for effective neutralizing antibodies induction. New strategy should be used to increase the humoral immunity of vaccine.