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The Optimization of HIV-1 DNA Vaccine and Immunization Approaches

Author: ZhangZhou
Tutor: LiXingGuo；ShaoYiMing
School: Shandong Agricultural University
Course: Developmental Biology
Keywords: DNA vaccines Optimal design Bicistronic In vivo electroporation
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 41
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Abstract

Vaccines are the most effective and most economical means of prevention and control of infectious diseases. DNA vaccine as the most potential for development of a new vaccine strategy, can effectively induce cellular immune response and humoral immune responses can induce long-term immune memory. However, the weak immunogenicity has been a bottleneck restricting the industrialization of the DNA vaccine. In this study, the integrated use of carrier transformation, gene modification and plasmid delivery means, and greatly improve the immunogenicity of the HIV-1 DNA vaccine. This laboratory experience shows that the the vaccine plasmid size exceeds the 9KB makes production difficult to significantly improve significantly reduced, and the plasmid into the cell's efficiency. This topic in order to build a polyvalent HIV DNA vaccine DNA vaccine vector pDRVI1.0, starting by removing approximately 2Kb nonessential plasmid backbone sequence constructed pDRVI4.0 plasmid vector, as the basis for building HIV DNA vaccine. The animal immunization results show that, as vector vaccine induced humoral and cellular immune response was no significant difference compared with the DNA vaccine based on pDRVI1.0 pDRVI4.0. Will cover four of the HIV-1 epidemic strain CN54 6 antigen gene genetic code to optimize the immune Gene Env, Pol, Gag and TNR (encoding Tat-Nef-Rev fusion protein) were cloned into pDRVI4.0 carrier, get 4 a monocistronic DNA vaccine plasmid p4.0-Env the p4.0-Pol, p4.0-Gag and p4.0-TNR. To further reduce the vaccine production and cost, from pDRVI4.0 carrier starting build two pairs shun anti-child DNA vaccine plasmid p4.0-Env/Pol and p4.0-Gag/TNR. Bicistronic vaccines and single cis anti-child vaccine parallel immunized mice, the results show that the bicistronic DNA vaccine induced immune response monocistronic DNA vaccine was no significant difference. To further enhance the dual-cis, anti-child immunogenicity of DNA vaccines, the subject of Pol, Gag, and TNR antigen gene the transmembrane modification, anchored in the expression of cell surface antigen gene, and build a double Shun carry these genes The anti child the DNA vaccine p4.0-Env/Pol-TM and p4.0-Gag/TNR-TM. The mice immune results show that the antigen the transmembrane modified from the general enhanced humoral and cellular immune responses induced by DNA vaccine. In order to study the expression and orientation of bicistronic DNA vaccine immunogenicity, this topic has constructed two expression cassettes are arranged direction the opposite bicistronic DNA vaccine plasmid p4.0-Env/Pol-TM-trans of and p4.0-Gag/TNR-TM-trans. The the immune results show that expression boxes arranged in the direction of the bicistronic DNA vaccine induced humoral and cellular immune responses had no effect. Secondly, the present study, in vivo electroporation technology to enhance efficiency in the delivery of DNA vaccines. We kept in our laboratory carrying luciferase Luciferase gene reporter plasmid p1.0-Luc and p1.0-Env DNA vaccine plasmid carrying the HIV-1 CN54 Env gene by intramuscular injection, intramuscular injection and electroporation two Different Methods injected mice, the IVIS? real-time detection of the in vivo imaging system Luciferase reporter gene expression in vivo, with ELISA detection of HIV-1 env-specific antibody response, with the IFN-γELISpot detection HIV-1 Env-specific T cell immune response . The results show that in vivo electroporation technology can significantly improve the Luciferase expression levels in mice, and enhance the rate of 35 times. Env-specific immune results show 8μg plasmid dose electroporation pathways induced by strong humoral and cellular immune responses in a dose of 40μg plasmid simplex the intramuscular group; vivo electroporation immunized 2 times 3 times with the intramuscular route alone immunization induced humoral and The cellular immune response comparable level. The results of the above two parts of the consolidated dicistronic DNA vaccine can without sacrificing significantly reduce the number of DNA vaccine plasmid and the plasmid size vaccine immunogenicity premise; transmembrane modified antigen gene can significantly enhance DNA vaccine-induced humoral and cellular immune responses; vivo electroporation technology as a new gene delivery pathways can significantly enhance the level of target gene expression and DNA vaccine immunogenicity.

The Optimization of HIV-1 DNA Vaccine and Immunization Approaches

Abstract

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