A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates

Background: Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variabili ty in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralizati on of HIV-1 primary isolates. Methods: A modified neutralization assay was performed using CD8-depleted p eripheral blood mononuclear cells (PBMC). The cells were fixed, permeabiliz ed, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and a nalyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tes ted using pooled sera or plasma from subtype B' or E infected patients. Results: Primary isolate cultures (without neutralizing antibody) showed fr om 18% to 42% p24(+) cells, depending on the virus. Less than 0.2% p24(+) c ells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. Conclusions: Flow cytometric detection of intracellular HIV-1 p24 can be us ed as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It al so provides an alternative quantitative endpoint for HIV neutralization ass ays. Cytometry 40:141-150, 2000. (C) 2000 Wiley-Liss, Inc.