Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
PLoS One. 2010 Jan 5;5(1):e8574. doi: 10.1371/journal.pone.0008574.
This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA10101, DRB10401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1x10(5) immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.
本报告描述了 HIV-1 Gag 表位肽的 HLA-DR4 限制性鉴定和生物信息学分析，以及应用树突状细胞介导的 DNA 质粒构建物免疫。BALB/c（H-2d）和 HLA-DR4（DRA10101、DRB10401）转基因小鼠用转染溶酶体相关膜蛋白-1/HIV-1 Gag（pLAMP/gag）嵌合抗原的重组 DNA 质粒转染的未成熟树突状细胞进行免疫。比较了三种免疫方案：1）用电穿孔转染 pLAMP/gag DNA 质粒的 1x10(5)个未成熟树突状细胞进行初次皮下免疫，并用裸 pLAMP/gag DNA 质粒进行第二次皮下免疫；2）如上进行初次免疫，并用跨越 HIV-1 Gag 序列的重叠肽池进行第二次皮下免疫；3）通过两次皮下注射 pLAMP/gag DNA 质粒进行免疫。用 pLAMP/gag 转染的树突状细胞进行初次免疫可在 BALB/c 和 HLA-DR4 转基因小鼠中引发最大数量的肽特异性 T 细胞反应，如通过体外 IFN-γ ELISpot 测定法测量的。pLAMP/gag 转染的树突状细胞引发和裸露 DNA 增强免疫方案也导致 ELISpot 测定法中肽的明显亲和力增加。引人注目的是，在 HLA-DR4 转基因小鼠中，25 个肽特异性 T 细胞反应中的 20 个含有完全或部分对应于 HIV 分子免疫学数据库中列出的 19 个人类 HLA-DR4 表位中的 18 个的序列。通过分析其在所有报告序列中的代表性和变异性，选择最保守的表位肽作为疫苗靶标。这些数据提供了一个模型系统，该系统证明了 a）与裸露 DNA 相比，用 LAMP/gag 质粒 DNA 转染的树突状细胞进行免疫的优越性，b）HLA 转基因小鼠作为鉴定和评估基于表位的疫苗策略的模型系统的价值，以及 c）在公共数据库中报告序列中的变异性分析用于选择历史上保守的 HIV 表位作为疫苗靶标的应用。
