Construction, Purification And Experimental Immunization Of New HIV Multi-Epitope Vaccines

Acquired Immunodeficiency Syndrome (AIDS) is caused by Human Immunodeficiency Virus (HIV), and it can damage humoral and cellular immune function by deletion of CD4~+ T cells, and lead to the opportunistic infection, the formation of malignance tumor, etc.The AIDS epidemiological report of 2008 showed that the number of people living with HIV/AIDS totaled to 33.2 million. As at the end of September 2000, there are 260 thousand people infected with HIV in China, 77 thousand infected people have developed AIDS, and 24 thousand AIDS patients have been dead. People newly infected with HIV in 2007 had a total number of 5 million, in other words there are 140 people were infected with HIV everyday. Therefore, it is crucial to research and develop effective vaccines to prevent HIV/AIDS infection and epidemic.This study we incorporated two efficient co-stimulatory molecules 4-1BBL and OX40L as molecular adjuvant into downstream of the MEGNp24 gene (including HIV-1 p24 gene and 26 highly conserved and immunogenic T-cell and B-cell epitope genes of Env, Gag. Nef, Pol, Rt, Vpr and Tat) of the DNA vaccine pVMEGNp24 which was constructed and confirmed effective by animal experiments previous studies. By this means, dual costimulatory molecules DNA vaccine pVMEGNp24-4-1BBL-OX40L and single co-stimulatory molecule DNA vaccine pVMEGNp24-OX40L were obtained. In order to enhance the biological function of the co-stimulatory molecules, furin restriction site genes were introduced to the intervals of co-stimulatory molecules, so they could be divided by furin enzyme in cells immediately after expression. Considering the spacial structure of some conformational epitopes, we inserted rigid amino acids between epitopes and co-stimulatory molecules as a linker.The study of mammalian cell BHK21 transfection indicated that the newly constructed vaccine could be expressed successfully.The DNA vaccine (pVMEGNp24-4-1BBL-OX40L) was amplified in coli Jm109. After established the fermentation congdition, the Feedstock solution of plasmid was obtained by alkaline lysis after the collection of bacterial by hollow fiber cross flow filtration system. Feedstock solution was loaded into a Sepharose 6 FF column for the removal of RNA. followed by applying collected fluid to PlasmidSelect Xtra column for the removal of linear plasmid, finally the plasmid was further purified by SOURCE30Q. The vaccine quality and properties, including super coiled form plasmid percentage, identity, overall yield, purity and residual impurities were assessed by methods recommended by SFDA. As the result showed, the quality of purified plasmid met regulatory requirements.We transferred reconstructed plasmid DNA vaccines to monocyte-derived DCs by liposome, and then the transfected DCs were cocultured with autologous Lymphocytes. After 2 days of coculture, immune effects were detected by measuring cytokine accumulation in cultures, intracellular cytokine staining and cytolytic activity. Significant enhancement of IL-2 and IL-4 accumulation were detected in the culture of co-stimulatory molecules groups, but IL-10 levels are lower than pVMEGNp24 group. So we suppose that co-stimulatory molecules have more facilitative effects on cell-mediated immunity. The effect of the co-stimulatory molecules on humoral immunity should be further examined in animal experiments. Synergistic effects were observed in the dual co-stimulatory molecules DNA vaccine. Dual co-stimulatory molecules DNA vaccine showed maximal up-regulation the numbers of IFN-γproducing antiviral CD8 T cells and CD4 T cells, and the most robust antigen-specific cytolytic activity.To evaluate the immunogenicity and protective effect against SHIV-KB9 challenge, 16 Chinese-origin Rhesus Macaques were immunized with prime/boost strategy and challenged. Animals were followed immunological detection and viral RNA loads. ELISA and ELISPOT assay were introduced to detecte HIV-specific antibody titers and CTLs responses respectively. Vaccine-specific T cells numbers of all immunization groups were significantly enhanced after immunized with rFPV. Compared to the non co-stimulatory molecules group, the co-stimulatory molecules groups raised more number of vaccine-specific T cells, and the co-stimulatory molecules groups were able to induce stronger memory responses after SHIV-KB9 challenge.After SHIV-KB9 challenge, all immunized animals had achieved varying degrees of control of the infection with postponed the coming of peakpoint viral load, declined peakpoint viral load and rapid decline of viral load. The animals in single co-stimulatory molecules group had achieved better control of the infection, one animal (M10) in single co-stimulatory molecules group was complete protected. It was indicated that the rapid, appropriate and persistent humoral and cell-mediated immune responses might contributed to the clearance of the SHIV-KB9 challenge.Single co-stimulatory molecules vaccine which could provide the potent, appropriate and persistent immunity necessary to protect against SHIV-KB9 is a more potential Vaccine Candidate. This study provides a foundation for developing potent HIV vaccines and its preclinical study.