PROJECT 1: DNA AND MVA IMMUNOGENS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在比较表达的体外剂量反应曲线和体内免疫原性的剂量反应曲线方面已经做出了很大的努力。在293T细胞中进行了体外表达测试，并使用FACS分析对表达Gag和Env的细胞进行了评分。在BALB/c小鼠身上进行了免疫原性测试，并使用ICS对反应细胞进行了评分。这些研究的结果表明，体外表达试验比体内免疫原性试验更准确地衡量产品浓度的差异。在1000倍的MVA剂量范围内，表达GAG和Env的细胞数量增加了近1000倍。相比之下，在体内MVA启动和增强后，MVA剂量的100倍差异仅导致CD8细胞对GAG的反应增加4倍，而CD4细胞对GAG的反应增加2倍。在DNA PRIME/MVA Boost方案中，MVA剂量增加100倍，CD8细胞对GAG的应答增加6.5倍，而CD4T细胞对GAG的应答没有增加。对Env的反应显示，MVA剂量对CD8或CD4反应没有可检测到的影响。我们的B类和C类HIV-1疫苗中的ADA和IN3包膜蛋白分别在恒河猴身上启动了诱导中和抗体反应的能力的研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Substantial effort has been placed on comparing in vitro dose response curves for expression with in vivo dose response curves for immunogenicity. In vitro expression tests have been done in 293T cells and used FACS analyses to score cells expressing Gag and Env. Immunogenicity tests have been done in BALB/c mice and used ICS to score responding cells. The results of these studies reveal in vitro expression tests being much more accurate for scoring differences in product concentration than the in vivo immunogenicity tests. The numbers of Gag and Env expressing cells increased by close to 1000-fold over a 1000-fold range of MVA dose. In contrast, following in vivo MVA priming and boosting, a 100- fold difference in MVA dose resulted in only a four-fold increase in CD8 cells responding to Gag and 2-fold difference in CD4 cells responding to Gag. In a DNA prime/MVA boost regimen, a 100-fold increase in MVA dose resulted in a 6.5 fold increase in responding CD8 cells to Gag and no increase in responding CD4 T cells to Gag. Responses to Env revealed no detectable effects of MVA dose on the CD8 or CD4 response. Studies have been initiated in rhesus macaques on the ability of the ADA and IN3 Envs in our clade B and clade C HIV-1 vaccines respectively to elicit neutralizing Ab responses.