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

Abstract

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 variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV‐1 primary isolates. Methods A modified neutralization assay was performed using CD8‐depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV‐1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV‐1 subtype B′ and E primary isolates were tested using pooled sera or plasma from subtype B′ or E infected patients. Results Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24+ cells, depending on the virus. Less than 0.2% p24+ cells 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 used 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 also provides an alternative quantitative endpoint for HIV neutralization assays. Cytometry 40:141–150, 2000. © 2000 Wiley‐Liss, Inc.

Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

“…Linear regression analysis showed a very low correlation coeffient (r 2 ϭ 0.147) between these two biological parameters. This phenomenon has been observed in previous studies (17,40) and indicates that viral stock p24 antigen concentration is not always reflective of the level of infectious virus contained in the stock.…”

Human Immunodeficiency Virus Type 1 Neutralization Measured by Flow Cytometric Quantitation of Single-Round Infection of Primary Human T Cells

“…Ideally, the amount of secreted p24-Ag would be directly related to the number of PBMC infected, but this is not necessarily the case. Factors such as multiple rounds of virus replication, cell death and release of p24-Ag, and release of noninfectious virions can each affect the total amount of cell-free p24-Ag measured in culture (7,8,16,21,37,62,69). Also, primary virus isolates have varied replication kinetics in activated PBMC.…”

“…Although this allows comparisons among HIV-1 strains with varied growth kinetics, the preneutralization steps are labor intensive and time-consuming. In addition, the reproducibility of the Ag-capture neutralization assays is limited by factors such as the numerous cell washes required to remove viral antigens and serum anti-p24 antibody, varied viral growth in culture, death of cells and release of p24-Ag, and manipulations required to harvest and measure p24-Ag (7,8,16,21,37,62,69).…”

“…Folghera et al used flow cytometric detection of intracellular p24 to measure neutralization of HIV-IIIB in H9 target cells (22). More recently, Darden and colleagues reported the use of a flow cytometric primary isolate HIV-1 neutralization assay that enumerated the number of HIV-1-infected PBMC after 4 to 7 days in culture (16).…”

The Role of Natural Killer (NK) Cells and NK Cell Receptor Polymorphisms in the Assessment of HIV-1 Neutralization

“…The assay was performed as previously described [36] with some modifications. Antibody and virus were incubated together for 60 min at 37°C then added to PHA stimulated PBMC.…”