Background:  PBMC based assays are usually time consuming and costly. The purpose is to establish a high throughput neutralization assay using an engineered CEM cell line that can detect neutralizing antibody activity and also establish a correlation between the antibody binding profile and neutralizing activity against a number of HIV-1 subtype B and C isolates.

Methods:  A cell line, CEMx174.R5.LTR-GFP; CLONE 5.25 (5.25), was selected for neutralization assays using PBMC grown HIV-1 isolates. This cell line expresses multiple receptor/co-receptors such as the CD4, CCR5, CXCR4 and BONZO. GFP positive cells after infection can then be detected by flow cytometry. This assay was compared to a traditional PBMC-based neutralization assay by using various HIV-1 positive B and C patient sera to test against SF162 (B), SF2 (B), TV1(C), and TV2 (C). For a single 1-mL culture well, 50 mL of titrated virus was incubated with 50 mL for 1 hour. This mixture was added to 10⁴/mL cells, and incubated at 37 ⁰C for 5 to 7 days. The quantitative percentage neutralization is calculated based on the number of GFP-positive cells as follows:   % Inhibition = (virus control-experimental)/(virus control -cell control)*100. In addition, we have also performed Biacore assays to measure the relative affinities of antibodies induced by different HIV structures and their ability to neutralize respective viruses. Results:  The results demonstrate neutralizing antibody activity of subtype B and C patient sera against above isolates. The SF2 isolate appears to be more sensitive to neutralization than the SF162 isolate. Cross-neutralizing antibody responses were detected in subtype B and C sera against subtype B and C isolates. TV2 was more susceptible to neutralization than TV1. Relative binding affinities of antibodies induced by different structures will be presented.

Conclusions:  The development of this reproducible high through put reporter gene based assay using engineered CEM cell line will facilitate the rapid evaluation of immune sera for the presence of neutralizing antibody activity against diverse HIV-1 primary isolates. A positive correlation between the binding and neutralizing antibodies will help to understand the neutralizing aspects of the immune responses.

Keywords: Neutralizing Antibodies; CEM cells; Biacore, Affinity