Measurement of HIV Neutralization Using Flow Cytometric & Reporter Virus Assays

诱导广泛交叉反应的中和抗体是艾滋病疫苗开发的一个高度优先事项，但已证明难以实现。虽然大多数免疫原产生的抗体可以中和T细胞系适应的HIV-1毒株的一个亚群，但迄今为止还没有一种免疫原产生针对HIV-1病毒原代分离株的有效的、广泛的交叉反应性反应。即使免疫原改善的微小增量导致中和抗体滴度和交叉中和活性的增加，也会加速疫苗的开发；然而，不同的研究人员在评估交叉中和作用时使用的目标菌株缺乏一致性，这使得比较疫苗诱导的抗体反应变得困难。因此，迫切需要建立广泛分布的HIV-1参考菌株的标准组。为了促进这一点，从急性和早期B亚型感染中克隆了全长gp160基因，并将其作为评估B支HIV-1中和抗体的参考试剂。在JC53-BL （TZM-bl）细胞中进行荧光素酶报告基因检测，筛选gp160克隆个体作为env型病毒的感染性。对功能环境克隆进行了测序，并利用可溶性CD4、单克隆抗体和重组gp120疫苗感染者和非感染者的血清样本对其中和表型进行了表征。从12株R5原代HIV-1分离株中选择环境克隆，这些克隆对中和没有异常敏感或抗性，并且具有广泛的遗传、抗原和地理多样性。这些参考试剂将促进能力测试和其他旨在提高各实验室测定性能的验证工作，并可用于疫苗诱导的中和抗体的标准化评估。

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has been proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T cell line adapted strains of HIV-1, none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies and serum samples from infected individuals and non-infected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine elicited neutralizing antibodies.