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80 Rapid Screening of Subtype B and C Neutralizing Antibody Response against PBMC-Grown HIV-1 Isolates in Engineered CEM Cell Line
Y. Sun*1, N. Laudau2, T. Brown, and S. Susan1
1Chiron Corp., Emeryville, CA, USA and 2Aaron Diamond AIDS Res. Ctr., New York, NY, USA
Background: The PBMC-based assays are usually time consuming and costly. Our purpose is to establish a high throughput neutralization assay using an engineered CEM cell line that can detect neutralizing antibody activity against a number of HIV-1 subtype B and C isolates.
Methods: A new 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/coreceptors such as the CD4, CCR5, CXCR4, and BONZO. The GFP-positive cells after infection can then be detected by flow cytometry. This assay was compared with 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 mul of titrated virus was incubated with 50 mul for 1 hour. This mixture was added to 104/ml cells and incubated at 37(C 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.
Results: The results demonstrate the 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 serum against subtype B and C isolates. TV2 was more susceptible to neutralization than TV1.
Conclusions: The development of this reproducible high throughput 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.
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