COMBINED APPROACH TO BROADLY PROTECTIVE AIDS VACCINES: I

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 异种“Prime-Boost”免疫可诱导对多种灵长类慢病毒的保护性免疫，包括SIVmne、SHIVIIIB和SHIV89.6P。然而，比较不同优质-助推剂组合的保护效果的研究相对较少。该项目的目标是研究“初始-增强”免疫策略，以确定诱导抗体和CD8+T细胞介导的针对灵长类慢病毒的免疫反应的组合。 在这项研究的前两个阶段，我们使用SHIVSF162 P4比较了重组痘苗病毒或DNA疫苗启动，然后DNA、蛋白质或甲病毒增强，以防止CCR5黏膜攻击的能力。用表达HIV-1SF162全长env和SIVmac239gag/Poll的重组痘苗病毒(第1-3组)或表达相同抗原的DNA质粒(第5-7组)对24只猪尾猕猴进行免疫。静息一段时间后，每组6只动物分别用下列免疫原之一加强免疫：第1组和第5组，表达密码子优化的SF162 env gp140或SIVmac239 gag-Poll的DNA质粒；第2组和第6组，表达相同抗原的蛋白免疫原SIVmac239 gag-Pol颗粒和SF162 Env gp140蛋白；或表达相同抗原的重组病毒载体(第3组为α病毒复制体，第7组为痘苗病毒重组体)。对照动物只接受母体痘苗病毒作为基础和空载体或佐剂作为加强免疫。在最后一次免疫后，所有试验组的动物都产生了SIV和HIV特异性抗体反应。与其他试验组的动物相比，用蛋白质增强的动物(第2组和第6组)具有显著更高的SIV特异性和HIV-1特异性gp120抗体效价，包括假型病毒检测中的同源中和效价(p lt 0.001)。 末次免疫后4周，经直肠接种1800只50%组织培养感染量的SHIVSF162 P4。SHIVSF162 P4在猪尾猕猴中的感染很强，平均血浆病毒峰值GT为10.7拷贝/毫升，GT 1/3的动物出现持续性病毒血症。所有免疫组的平均血浆病毒载量均显著降低(第2组和第6组与对照组相比，Plt为0.01；所有其他组为Plt 0.05)。我们还观察到攻击当天同源中和抗体效价与攻击后血浆病毒载量峰值呈显著负相关(Spearman‘s r=-0.819，p lt 0.0001)，这与NtAb对感染的保护作用一致。在攻击前，在流式细胞仪分析中，由HIV特异性干扰素-γ和肿瘤坏死因子-α分泌细胞测量的T细胞反应很弱或检测不到。 综上所述，这些结果表明，使用CCR5的灵长类慢病毒对黏膜感染的保护性免疫可以通过系统的初始加强免疫来激发，在重组痘苗病毒或DNA免疫的动物中，用蛋白免疫原加强免疫似乎优于DNA或病毒载体来产生抗原特异性抗体反应。

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. Heterologous "prime-boost" immunization has been shown to elicit protective immunity against multiple primate lentiviruses, including SIVmne, SHIVIIIB and SHIV89.6P. However, there have been relatively few studies comparing the protective efficacy of different prime-boost combinations. The goal of this project is to examine "prime-boost" immunization strategies to identify combinations that induce antibody and CD8+ T cell-mediated immune responses against primate lentiviruses. In the first two stages of this study, we compared recombinant vaccinia virus or DNA vaccine for priming, followed by DNA, protein, or alphavirus boosting, for their ability to protect against mucosal challenge with a CCR5-using SHIVSF162 P4. Twenty-four pig-tailed macaques were primed either with recombinant vaccinia viruses expressing HIV-1 SF162 full length env and SIVmac239 gag/pol (Groups 1-3), or DNA plasmids expressing the same antigens (Groups 5-7). After a resting period, six animals per group were boosted twice (3 mos apart) with one of the following immunogens: Groups 1 and 5, DNA plasmids expressing codon-optimized SF162 env gp140, or SIVmac239 gag-pol, formulated in PBS; Groups 2 and 6, protein immunogens SIVmac239 Gag-Pol particles and SF162 Env gp140 protein formulated in MF59; or recombinant viral vectors expressing the same antigens (Group 3 with alpha virus replicons and Group 7 with vaccinia virus recombinants). Control animals received parental vaccinia virus for prime and empty vector or adjuvant only for boost. After the last immunization, animals in all experimental groups generated SIV- and HIV-specific antibody responses. Compared to animals in other experimental groups, animals boosted with proteins (Groups 2 and 6) had significantly higher SIV-specific and HIV-1-specific gp120 antibody titers, including homologous neutralization titers in a pseudotyped virus assay (p lt 0.001). Four weeks after the last immunization, all animals were challenged with 1,800 50% tissue culture infectious dose of SHIVSF162 P4 by intrarectal inoculation. SHIVSF162 P4 infection in pig-tailed macaques was robust, as shown by mean peak plasma viral of gt 10^7 copies/ml and persistent viremia in gt 1/3 of the animals. Significant reduction of mean plasma viral load was observed in all immunization groups (p lt 0.01 for Groups 2 and 6 vs controls; p lt 0.05 for all other groups). We also observed a significant inverse correlation (Spearman's r = -0.819, p lt 0.0001) between homologous neutralizing antibody (NtAb) titer on day of challenge and peak plasma viral load after challenge, consistent with the role of NtAb to protect against infection. T-cell responses as measured by HIV-specific interferon-gamma and TNF-alpha secreting cells in flow cytometric analysis were weak or undetectable before challenge. Together, these results indicate that protective immunity against mucosal infection with a CCR5-using primate lentivirus can be elicited by systemic prime-boost immunization and that boosting with protein immunogens appears to be superior than DNA or viral vectors in recombinant vaccinia virus or DNA primed animals in the generation of antigen-specific antibody responses.