Study On Construction And Purification And Experimental Immunity Of Therapeutic DNA VaccinE Against HIV

Acquired Immunodeficiency Syndrome(AIDS) is a very serious disease which hazard mankind healthy, it has the complicated immune suppression of various clinical symptom, including CD4+ T progressive decrease, disservice of cellular immune function, and lead to the opportunistic infection, the formation of malignance tumor and central nerve disease, etc. AIDS was caused from Human Immunodeficiency Virus(HIV), since the first detection at USA on the year 1981, there are 60 million infection case. The document published by the AIDS epidemiological report of 2004 showed that the number of people living with HIV/AIDS totaled to 39 million, people newly infected with HIV in 2003 had a total of 4.9 million, and AIDS deaths in 2003 reached 3 million. The new estimates show increasing numbers of people living with HIV/AIDS. And since the first detection from our country of AIDS, there are a total of 890 thousand people infected with HIV. Therefore, it is crucial to research and develop effective vaccines to prevent HIV/AIDS infection and epidemic. This study choose the strategy of HIV-1 dominant antigen epitope according to the concept of therapeutic vaccine, and a novel HIV-1 vaccine antigen expected to treat HIV-1 infection or AIDS was designed. According to the earlier reports about the immunogenic epitopes of HIV-1, we combined three highly conserved and immunogenic B-epitopes and six T-cell epitopes together as a multiple-epitope antigen which covered the main structural and regulating proteins, including HIV-1 Env, Gag, Nef, Pol, etc. All of the selected epitopes showed 80% conservancy in primary amino acid sequences among the HIV subtypes and clades. Considering the spacial structure of some conformational epitopes, we inserted several flexible amino acids into every two epitopes as a linker. In order to enhance the expression yields of the antigen, a Kozak sequence was introduced to the designed antigen gene and the codons were optimized to adapt to the codon usage of the hosts who may highly express the designed antigen gene. Besides, in the hope of stimulating best immune responses, we further introduced ER target signal sequence to the interest gene. Finally, a universal Th cell epitope pan-DR was also incorporated into this gene. The designed gene encoding HIV-1 therapeutic vaccine immunogen is named as MEG. It was chemically synthesized by extending the overlapping oligonucleotides in three sets of PCR reactions. The gene was then cloned into vetor pKS. The recombinant was named as pKS-MEG. Another gene, HIV-1 p24, amplified from pKS-gagI by PCR, was then inserted into MEG, and a recombinant plasmid containing the chemiric gene MEGp24 was obtained. Then constructed new type HIV-1 remedial recombination DNA vaccine(pVAXI-MEG-p24) with the eukaryotic expression vector pVAXI. The study of mammalian cell transfection indicated that pVAXI-MEG-p24 expressed MEGp24 antigen protein successfully. The constructed therapeutic HIV DNA vaccine was amplified in Jm109 strain of Escherichia coli. After fermented in complex medium, the fermentation technology of pVAXI-MEG-p24 was established. The plasmid was pre-purified by alkaline lysis, calcium chloride precipitation, PEG precipitation and so on. Feedstock solution was loaded into a Qsepharose XL column, and plasmid wassuccessfully isolated by continuous salt gradient elution, followed by applying identified pools to Sepharcryl S1000 column for further purification. Final preparation purity of plasmid amounted to 1.86 (OD260/280), with supercoiled form accounting for 93.6 per cent of the total obtained plamid DNA . The total recovery of plasmid per litre bacterium was 37.8mg, and the recovery rate was about 66.3 percent. When it comes to vaccine quality and properties, several specifications were assessed by methods recommended by FDA, including supercoiled form plasmid percentage, transformation efficacy, identity, overall yield , purity(OD260/280). Residual impurities levels such as RNA, host protein, host genomic DNA in final product were also detected. As the result showed, the quality of purified plasmid met regulatory requirements. Thus, the method developed here to purify plasmid DNA lay the basis for large-scale production of DNA vaccine against HIV as well as the others. BALB/c mice were immunized intramuscularly with the purified recombinant plasmid pVAXI-MEG-p24 followed by the detection of cellular and humoral immune level. Anti-HIV-1 specific antibody was detected from the serum of the immunized mice, and spleen T lymphocyte subsets were found. HIV-1 peptide-specific target lysis was detected. The results demonstrated that specific humoral and cellular immune responses could be elicited by the recombinant therapeutic vaccine. Moreover, stronger cellular and humoral immune responses were observed when compared with pVAXIgag-gp120 innoculation, it showed that pVAXI-MEG-p24 may be activate higher immune responses. After monkeys were immunized intramuscularly with purified recombinant plasmid pVAXI-MEG-p24, the anti-HIV-1 specific antibody was detected from the immunized monkeys, and specific T lymphocyte proliferation was observed under the stimulation of HIV-1 T-cell eptitope peptides. HIV-1 peptide-specific target lysis...