Generation And Immunogenicity Of Nucleic Acid And Vaccinia Virus Vector Based Vaccines Expressing HIV Multi-epitope Gene

Respecting the currently epidemic of AIDS and its status quo of prevention and therapy, it isimminent to develop a safe and effective AIDS preventative or therapic vaccine. In these years,many scientists had carried out a large number of beneficial trials with various vaccine vectors andmodalities, HIV immunogens, or different routes of vaccination and immunifaction. In thisinvestigation, DNA plasmid and vaccinia virus Tian Tan strain as immunogen delivery systemwere used to launch the trial research of vaccines against HIV.In this study, we firstly constructed a eukaryotic expression vector of HIV multi-epitope genecontaining unmethylated CpG ODN sequence and cholera toxin B subunit (CTB) and expressionin vitro. CpG-CTB gene sequence (CC) was obtained by PCR using the primers of CTB genecontaining CpG ODN and Linker Sequence designed, and then was connected with pVL. HIVmulti-epitope gene (MEGNp24) from pVAX1-MEGNp24was inserted into the downstream of CCgene to construct pVL-CCMp24. After the recombination plasmid pVL-CCMp24was confirmedby enzyme digestion, it transfected into293T cells with liposome. The expression of CCMp24protein was confirmed by RT-PCR and indirect immunofluorescence. The results showed that theCTB and HIV multi-epitope gene were stably expressed in the293T cells transfected. And thenBALB/c mice were injected with the DNA vaccine by intramuscular injection, to evaluate theimmunogenicity of recombinant DNA vaccine. The immune effects induced by pVL-CCMp24were detected by measuring HIV-specific antibody, Th1type and Th2type cytokines in bloodserum, T cells subtype and lymphocyte proliferation. The results suggested that the recombinantDNA vaccine pVL-CCMp24can induced the production of cellular and humoral immunityresponses in some degree.Based on the idea of multivalent vaccines and preliminary findings, this study designed andconstructed a vaccinia virus shuttle vector pSTKE with Triple-gene expression cassette. Thevector used vaccinia virus thymidine kinase gene (TK) as screening marker, and contained threeseparate foreign gene expression cassettes. The derived recombinant vaccinia virus can express atleast three different target genes with high efficiency at the whole early and late stages. In addition,knocking out of TK attenuated the virus and improved the safety of clinical application. UsingChinese vaccinia virus Tian Tan strain (VTT) and vaccinia virus Tian Tan strain deleting E3Lgene (dVTT) as original viruses, and enhanced green fluorescent protein (EGFP), red fluorescentprotein (RFP) and blue fluorescent protein (BFP) as reporter gene, we verified the three expressioncassettes. By plaque screening, two recombinant vaccinia viruses (rVTT-EGFP and rdVTT-EGFP)were obtained, and the expression and genetic stability of recombinant virus and foreign genes were analyzed using PCR, real-time quantitative PCR, and Western blot. Results showedsuccessful construction of vaccinia virus shuttle vector with three-gene expression cassette. Twostrains of two recombinant vaccinia viruses were identified, EGFP gene was expressed in thevaccinia virus with a high level, and there was no interaction between the three expressioncassettes, and the two recombinant viruses had good genetic stability.A HIV immunogen (CCMp24) from pVL-CCMp24was first cloned into MCS1of vacciniavirus shuttle vector pSTKE, and a fragment (LoxP-EGFP-LoxP) containing Loxp sequence andEGFP gene obtained by PCR with the specific primers was inserted into MCS3of shuttle vectorpSTKE, resulting in the recombinant plasmid pCCMp24-LEL. the recombinant plasmid wastransfected into BHK21cells infected with dVTT, and the recombinant virus rddVTT-CCMp24Gwaspurified with plaque isolates expressing green fluorescent protein used as a selection marker, andEGFP gene in the genome of rddVTT-CCMp24Gwas deleted based on Cre/loxp site-specificrecombination, and plaque screeing was performed as described above to generate therecombinant vaccinia virus rddVTT-CCMp24. RT-PCR, indirect immunofluorescence and Westernblot were used to detected the expression of CCMp24in BHK-21cells infected by rddVTT-CCMp24,the results demonsteated that CTB and HIV MEGNp24were expressed in BHK-21cells.In this research, based on vaccinia virus of virulence related gene deletion, the deletion of TKwas implemented by homologous recombination. The virulence of recombinant virusrddVTT-CCMp24was evaluated in inbred BALB/c mice by the daily measurement of body weightchange after the mice were infected intranasally with rddVTT-CCMp24, ddVTT or VTT.Simultaneously, the neurovirulence of rddVTT-CCMp24was assessed by challenging BALB/c micevia intraperitoneal injection. The neurovirulence in infected mice was determined by measuring50%of the intracranial lethal infectious dose (ICLD50). These results suggest that rddVTT-CCMp24and ddVTT were attenuated due to the deletion of E3L and TK genes, and the introduction of CTBand HIV MEGNp24genes doesn’t alter the infectivity and virulence of the vaccinia virus vectors.Subsequently, in BALB/c models, the immunogenicity of rddVTT-CCMp24was analyzedthrough measuring HIV-specific antiboady, cytokines in peripheral blood, cellular surface antigenof splenocytes and the number of T cells secreting IFN-γ. We found that the expression of thisimmunogen was sufficient to facilitate the development of substantial number of antigen-specificT cells secreting IFN-γ, and the number of CD4+and CD8+T cells was significantly improved.And HIV-specific antibody, IL-2and IL-4were induced to produce after mmunization with therecombinant virus. Hence, rddVTT-CCMp24could induce robust immune responses.Then, in order to minimize the development of anti-vector immunity, heterologousprime–boost vaccine strategies have been implemented in mice to enhance the immune responseusing the DNA vaccnie pVL-CCMp24used for priming the immune system and recombinant viralvector vaccine rddVTT-CCMp24as booster. The results of ELISPOT assay had shown that thisprime–boost vaccination could induce to generate more number of antigen-specific IFN-γcompared with either a DNA vector or a live viral vector alone. This graduation thesis provides a foundation for the following empirical study about HIV vaccines.