417 A Single-round, Recombinant-based Viral Infectivity Assay Allows for the Sensitive and Specific Detection of Biologically Relevant Neutralizing Antibodies to HIV-1 J. Decker*1, X. Wei1, S. Wang1, X. Wu2, B. Hahn2, J. Kappes2, G. Shaw1 1Howard Hughes Med Inst, Univ of Alabama at Birmingham and 2Univ of Alabama at Birmingham
Background: Infection of human cells by R5 and X4 HIV-1 viruses is a complicated, multi-step process that lies at the heart of viral persistence and pathogenesis. Furthermore, inhibition of virus infection represents a primary objective in HIV-1 vaccine design. Assay methods are needed which measure biologically-relevant neutralizing antibodies (Nab) in a sensitive and specific manner.
Methods: A virus entry assay was developed based on Hela cells (JC53BL) genetically engineered to constitutively express CD4, CCR5, and CXCR4, as well as beta galactosidase (b-gal) and firefly luciferase (luc) under tight regulatory control of the HIV-1 LTR.
Results: JC53BL cells were found to be equally sensitive to R5 and X4 primary viruses compared with PHA lymphoblasts; b-gal and luc expression were linearly related to each other and to input virus. By carefully controlling for virus input, human plasma concentration, and duration of virus-cell interaction (2-6 hrs; terminated by the virus entry inhibitors T-20 or T-649), we were able to detect autologous and heterologous Nab with high sensitivity and specificity. Compared with plasma or serum from uninfected subjects which had no effect on virus entry in dilutions ranging from 1:20-1:5120, inhibition by autologous (and heterologous) plasma reached IC50 titers as high as 1:2500, with autologous titers generally higher by 6-40 fold. Virus entry was inhibited by as much as 99.9% by plasmas from HIV-1 infected subjects at 1:20 dilutions.
Conclusions: By utilizing a virus infectivity assay that limits variables to the entry event and controls for non-specific viral inhibitory effects in normal serum or plasma, we could specifically detect HIV-1 Nab titers as high as 1:2,500 and show that autologous and heterologous serum (or plasma) frequently reduces virus infectivity by greater than 99%. The utility of this assay in evaluating correlates of immune protection afforded by candidate vaccines holds substantial promise.