Measurement of HIV Neutralization Using Flow Cytometric & Reporter Virus Assays

广泛交叉反应性中和抗体的诱导是艾滋病疫苗开发的高度优先事项，但已被证明难以实现。 虽然大多数免疫原产生中和HIV-1的T细胞系适应株的一个子集的抗体，但迄今为止没有一种免疫原产生针对病毒的主要分离株的有效的、广泛的交叉反应性应答。 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增加，也会加速疫苗开发;然而，不同研究者用于评估交叉中和的靶菌株缺乏一致性，使得难以比较疫苗诱导的抗体应答。 因此，迫切需要建立广泛分布的HIV-1参考毒株标准组。 为了促进这一点，从急性和早期亚型B感染中克隆全长gp 160基因，并将其表征为用作参考试剂，以评估针对进化枝B HIV-1的中和抗体。 在JC 53-BL（TZM-bl）细胞中的荧光素酶报告基因测定中，筛选单个gp 160克隆作为Env假型病毒的感染性。 对功能性env克隆进行测序，并通过使用可溶性CD 4、单克隆抗体和来自重组gp 120疫苗的感染个体和非感染接受者的血清样品来表征其中和表型。 选择来自12个R5主要HIV-1分离株的Env克隆，这些克隆对中和反应不异常敏感或具有抗性，并且包含广泛的遗传、抗原和地理多样性。 这些参比试剂将促进能力验证和其他验证工作，旨在提高各实验室的检测性能，并可用于疫苗引起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.