HIV neutralization assay using polymerase chain reaction-derived molecular signals

Abstract

Characterization of the capacity of human polyclonal antibody to neutralize wild-type patient isolates has important implications for vaccine development. We report the development of a polymerase chain reaction-based neutralization assay that quantitatively measures each infection using HIV proviral formation. These molecular end points identified the absence or quantitative diminution of DNA provirus formation as well as a delay in the kinetics of HIV DNA provirus formation. Using both laboratory strain prototype isolates (HIV-1-MN, HIV-IIIb) and primary wild-type patients' isolates, neutralization end points were reproducibly determined. End points were reached within 72 h, thereby minimizing the impact of subsequent rounds of infection on interpretation of results. Although the neutralization titer of polyclonal sera was usually comparable using standard technology, this assay did find isolate-dependent variation in the relationship between p24 production and HIV proviral DNA formation. Finally, we noted the disparity between the ability of human sera to neutralize prototype and wild-type isolates in primary peripheral blood mononuclear cell targets. We believe this assay provides unique opportunities to characterize the initial events of virus-antibody interaction and will help to elucidate clinically relevant neutralization immunoregulatory mechanisms.