The Immunity Of Multi-epitope DNA Vaccine Against HIV And The Construction Of The Fowl-pox Viral Transfer Vector

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 malignant tumor and central nerve disease, etc. AIDS was caused by Human Immunodeficiency Virus (HIV), Today, HIV/AIDS is the leading cause of death in sub-Saharan Africa and the fourth biggest killer in the world. At the end of 2006, the global number of adults and children living with HIV/AIDS was estimated by WHO/UNAIDS have reached 42 million with an estimated 23 million HIV-infected persons dying from the disease. The number of people living with HIV/AIDS is still increasing. Therefore, it is crucial to research and develop effective vaccines to prevent HIV/AIDS infection and epidemic.HIV is retrovirus which has high variability. It is a challenge to develop effective vaccine through routine methods. One way to solve this problem is research multiple-epitope vaccine. In this research, the designed multiple-epitope antigen named as MEGN was chemically synthesized by extending the overlapping oligonucleotides in PCR reactions. The gene was then inserted into HIV-1 p24, and a recombinant plasmid containing the chemiric gene MEGNp24 was obtained. Then new type HIV-1 multiple-epitope recombinant DNA vaccine (pVAXI-MEGN-p24) with the eukaryotic expression vector pVAXI was constructed. The multiple-epitope gene covered the genes coding the main structural and regulating proteins, including HIV-1 Env, Gag, Nef, Pol, etc. All the selected epitopes showed 80% conservancy in primary amino acid sequences among the HIV subtypes and clades. In order to produce cross-protection, several HIV-2 epitope antigen were inserted. The result of mammalian cell transfection experiment indicated that MEGNp24 antigen protein was expressed successfully.Balb/c mice were immunized intramuscularly with the recombinant plasmid pVAX1-MEGN-p24 followed by the detection of cellular and humoral immune level. HIV-1 peptide-specific target lysis was detected, suggesting a strong CTL response was achieved by pVAX1-MEGN-p24. Furthermore, the secreted levels of Th1/Th2 cytokines were detected higher in immunized mice than those of controls, suggesting strong humoral and cellular immune responses could be achieved by pVAX1-MEGN-p24. At the same time, Levamisole and chitosan were added to vaccine as adjuvant. The result showed that Levamisole could enhance cellullar immunologic response and chitosan could enhance humoral immunoresponse.One of the strategies experienced over the last decade to increase their immunogenicity was to combine these vaccines in"prime-boost"vaccination regimens, commonly using DNA vaccine candidates for priming followed by live viral vectored vaccines for boosting. So, based on the previous studies of rDNA vaccine, fowlpox virus vector was studied furthermore. Analyzing the first generation fowlpox recombinant transfer vector pUTA-2, repetat nucleotide sequences were found in the bis- lateral wing of TK gene. The redesigned three new TK gene fragments were chemically synthesized by extending the overlapping oligonucleotides in PCR reactions. After the three genes were inserted into pMD18-T vector, the skeleton of fowl pox recombinant transfer vector was got.Promoter plays an important role in the viral vector, because the expression of foreign gene influences immunifaction directly. Up to now, there were no promoter which from fowlpox virus could promote foreign gene in high level. So it is very important to select effective promoter when using fowlpox virus as vector. In this research, three high expressed promoters were chemically synthesized which were named as PE/L, P7.5, PL respectively. PE/L was early and late bidirectional promoter. P7.5 was early and late promoter whose efficiency was 3.5 times of VV promoter. PL was late promoter. The three promoters were connected together to promote the expression of GFP gene. Then the recombinant expression plasmid pMTK-3 wad constructed. The fluorescence was seen under fluorescence microscope, and the tensity was higher in that in controls of pUTA-2. The result showed that the expression of foreign gene was more effective in this new transfer vector than in the first generation vector.The chemiric gene MEGNp24 was inserted into pMTK-3 to take the place of GFP gene. Then the recombinant expression plasmid of pMTK-MEGNp24 was constructed. The recombinant expression plasmid and fowlpox virus 282E4 strain were co-tansfected into embryo fibroblast cells, then the homologous recombination occurred. The recombinant fowlpox virus rFPV-MEGNp24 were screened by using BrdU and identified by RT-PCR and indirect immunofluorescence. The result showed that foreign gene carried by recombinant fowlpox virus could express efficiently in CEF. This study will provide valuable support for the further combined immunization.