Study On Preparation And Immunogenicity Of HIV-1 Consensus Gp120 DNA Vaccine

AIDS is acquired immunodeficiency syndrome,human immunodeficiency virus(HIV)that causes AIDS.HIV,of the genus Lentivirus,is a retrovirus.Two types of HIV have been characterized: HIV-1 and HIV-2.HIV-1 is predominantly common virus in the worldwide than HIV-2.Three major genotypes including subtype B',CRF01_AE,CRF07/08_BC are prevalent in China.The development of a safe and effective vaccine is hampered by the high genetic variability of the virus,the difficulty of generating broadly neutralizing antibodies.In this research/thesis,identified and mass prepared gp120 DNA vaccine that according to HIV-1 main epidemic subtype of consensus antigens and mosaic antigen construction in China,than HIV-1 Env-specific antibody ELISA method was established,assembled HIV-1pseudoviruses and titration by TCID50,evaluation of immune effect of gp120 DNA vaccine under different immunization conditions to mice and rabbits by electroporation in vivo.1.After the optimized ConS and ConTAE gp120 antigen sequences,genes of interest were amplified and then inserted into either a pVAX1 cloning vector or a pVAX1 incorporating murine soluble PD1 before mass preparation,the expression of ConS/msPD1-ConS and ConTAE/msPD1-ConTAE proteins was tested by Western blotting by transfecting each of the DNA plasmid complexes into 293 T cells.The results show that the clones have the strongest expressions,which infected HIV-1 patient's antibodies were able to recognize and be able to emulate natural viral proteins.2.We used the envelope antigen from transfection of 293 T cell with four types of plasmids,to establish ELISA system detected by HIV-1 specific antibody and optimized reaction condition.The one-cycle pseudovirus was packaged with HIV-1 envelope plasmid and pNL fluorescein-conjugated HIV-1 backbone plasmid mixed with PEI in opti-MEM.infection of ghost X4/R5 cell with pesudovirus,the RLU was calculated by the multienzyme-labeled machine,which valued the immune neutralization effect.3.In order to evaluate the immunological effects of different DNA vaccines,BALB/c female mice were immunized three times with the four ConS,ConTAE,msPD1-ConS,msPD1-ConTAE gp120 vaccine via intramuscular injection(i.m.)followed immediately with electroporation of 60 volts(V),10 ms conditions,respectively.ELISA results show that fourconsensus gp120 DNA vaccine were able to elicit high antibody responses against HIV-Env gp120 antibody titers,which were not able to elicit functional neutralizing antibodies against ADA and JRFL,but ConS DNA induced antisera that were able to neutralize CNE40 pseudovirus in murine model.4.The ConS and ConTAE gp120 DNA vaccine were inoculated with the 1-year-old female New Zealand rabbits by electroporation.The rabbits was given topical Imiquimod cream before administration of i.m.followed by 60 V,10 ms and 90 V,10 ms electroporation,immunized three times with 3 week intervals between each vaccination.The two vaccines were not able to elicit functional neutralizing antibodies against ADA and JRFL under different voltage conditions,but ConS DNA induced antisera that were able to neutralize CNE40 pseudovirus.There does not seem to be a particular advantage in using Imiquimod cream or higher voltage for antibody affinity and induce higher neutralization antibodies level.With 60 V,10 ms low-voltage immune conditions,can elicit different DNA vaccines have a high level and persistence of immune response.