AEGiS-15IAC: Evaluation of HIV-1 neutralization using flow cytometry on green fluorescent protein-based and intracellular p24Ag-based.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Evaluation of HIV-1 neutralization using flow cytometry on green fluorescent protein-based and intracellular p24Ag-based.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10350)

Louisirirotchanakul S, Pathapanyasat K, Auewarakul P, Siritantikorn S, Suthent R, Puthavathana P
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

OBJECTIVE: To measure neutralization by a flow cytometric method as follows 1) HIV isolate-based using GFP-coreceptor reporter cells (GHOST system) 2) Chimeric HIV construct-GFP (Green Fluorescent Protein) reporter-based using MT2 cells 3) HIV isolate-based by intracellular p24 quantitative and extracellular p24 Ag capture using CD8-depleted PBMC Material and

METHOD: Panel plasma from 40 HIV-1 infected Thais who attended at Siriraj Hospital, Bangkok, Thailand was investigated for antibody-mediated neutralization. Neutralizing antibody (NT Ab) to HIV subtype E was analyzed at day 4 by a flow cytometry on GFP-based methods; chimeric HIV DNA-GFP reporter using MT2 cell and a clinical isolate using GHOST cell-based. NT Ab results were compared to NT Ab on 2 culture systems; a clinical isolate by determining extracellular HIV-1 p24Ag-based at day 7 using PBMC and a T-cell line adapted (TCLA) strain by observing syncytia formation-based at day 4 using C8166 cells. In addition, neutralization by measuring intracellular HIV-1 p24 Ag on CD8-depleted PBMC with monoclonal Ab staining using a flow system was compared with extracellular p24Ag capture using a standard culture system.

RESULT: Neutralization of the same HIV subtype E isolate by GHOST-based and C8166-based methods was more sensitive (90% vs 78%) than PBMC-based assay by extracellular HIV p24 Ag expression (47%). Using a flow cytometer, the chimeric HIV constructed -GFP based in this study demonstrated less sensitivity to neutralization compared to the intracellular HIV p24Ag as end point (50% vs 81%). Flow cytometric detection of intracellular HIV-1 p24Ag showed a good agreement with the standard detection of extracellular p24Ag capture (70% vs 83%).

CONCLUSION: A flow cytometric assay for the evaluation of the HIV neutralization to HIV subtype E can be used. However, reproducibility with more viruses should be further investigated.

Keywords: AEGIS, HIV-1, Green Fluorescent Proteins, Luminescent Proteins, Flow Cytometry, HIV Seropositivity, Acquired Immunodeficiency Syndrome, Indicators and Reagents, Fluorescent Antibody Technique, Thailand, methods

040711
A10350