Immunogenicity Of DNA Vaccine And Adenovirus Vector Vaccine Containing HIV-1 Env

Scientists have been focusing on the study of HIV vaccines since HIV was identified in 1980's. However, the enormrous genetic diversity and other unique feature of the HIV have thus far thwarted attempts to identify an effective candidate vaccine. The development of an HIV vaccine poses an unprecedented challenge to the scientific community. A safe, effective and accessible HIV vaccine is urgently needed to control and prevent the prevalence of HIV. The main objectives of our studies are genetic analysis of the complete env genes of HIV-1 from paid blood donors in Henan province and construction of DNA and recombinant Adenovirus vector vaccine (rAdV) containing consensus gp160 gene derived from Henan province. It is expected that high level of cellular immune responses and neutralizing antibodies would be induced by combination of those two vaccines.Firstly, complete HIV-1 env genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) DNA of 60 HIV-1 positive paid blood donors in Henan province, and the amplified full-length genes were sequenced. Twenty one full-length env genes were obtained, sequence analysis found that 15 of them had intact open reading frame (ORF). Fourteen sequences were confirmed to be subtype B', their average genetic distance with the international reference sequence RL42 was 4.87%±0.31%. One was subtype B, it's genetic distance with the international reference sequence HXB2 was 5.43%. The amino acid sequences of these env genes were deduced according to their nucleotide sequences and extensive analysis and comparison of important structural motifs were performed. The results indicated that there was no drastic alteration in the number and position of potential N-linked glycosylation sites among these 15 sequences. And the residues involved in forming the CD4 binding site were highly conserved. Genotypic prediction of coreceptor usage based on V3 sequence and net charge suggested that most samples use CCR5 coreceptor. GPGR motif at the tetrapeptide crown in the V3 loop was most common in these samples and it was detected in 40% sequences. The cleavage site of Gp120/Gp41 was highly conserved, so Gp160 precursor of all isolates would be efficiently cleaved into the Gp120 and Gp41 subunits. The known neutralizing antibody binding sites for 2G12, IgG1b12, 4E10 and 2F5 were also highly conserved, it is expected that most of these isolates will be sensitive to neutralization by these antibodies.According to the codon usage of human being, the codons of consensus gp160 sequence derived from the above 15 intact full length gp160 genes were modified. The modified gp160 gene was inserted into the plasmid pVR to obtain the DNA vaccine. Western Blot analysis found that the modified gp160 gene was expressed efficiently.The recombinant Adenovirus vaccine expressing the modified gp160 gene was also constructed.And the Gp160 protein was expressed efficiently in the rAdV vaccine. BALB/c mice were immunized with DNA and rAdV vaccine alone or in different combination strategies. ELISPOT assay and ELISA assay were used to detect the cellular immune responses and humoral immune responses respectively. It showed that the mice primed with DNA vaccine and boosted with rAdV vaccine elicited highest cellular responses at 1 week post immunization (296 SFC/2×105PBMCs). However, the cellular responses decreased dramatically with time. The mice immunized with DNA vaccine alone elicited lower cellular responses than DNA-priming/rAdV-boosting group at 1 week post immunization. But the cellular responses declined more slowly than other groups. Weak humoral immune responses were induced in some tested groups. Among them the mice immunized with rAdV vaccine alone elicited highest binding antibodies. The mean of OD450/630 is 0.408 (1:100 diluted) at 1 week post immunization and 0.864 (1:400 diluted)at 4 week post immunization.Our date indicated that Env protein in DNA and rAdV vaccines only induced potent cellular immune responses. It is difficult to elicited antibodies especially neutralizing antibodies by Env immunogen. An alternative therapeutic and preventive vaccine concept is to introduce gene therapy vectors expressing potent, broadly reactive antibodies in vivo instead of immunogen. We have begun our research on this area.