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35 Development of a Sensitive Flow Cytometric Assay for Measuring HIV-1 Neutralizing Antibodies in Seropositive and Vaccinated Subjects
J. Darden*1, A. Sambor1, S. Presswala1, R. McLinden1, J. Kim1, D. Birx1, V. Polonis1, and the TAVEG2
1The US Military HIV Res. Prgm., Rockville, MD, USA and 2The Thai AIDS Vaccine Evaluation Grp., Thailand, USA, and France
Background: The induction of neutralizing antibody (NAb) by candidate HIV vaccines should be evaluated by sensitive assays that accurately reflect virus, antibody, and host cell interactions. We have previously shown that flow cytometry can be used as an endpoint for NAb assays using HIV-1 subtypes A(G. In many standard neutralization assays, virus and NAb are washed out after infection to accurately measure extracellular p24 as an endpoint for the assay. In this study, we evaluated HIV neutralization using an assay in which NAb is maintained in the cultures and flow cytometric detection of HIV infection using intracellular p24 is the endpoint.
Methods: Cell lines or peripheral blood mononuclear cells (PBMC) were infected with HIV-1 subtype B and CRF01_A/E (E) viruses in the presence or absence of HIV seropositive or vaccinee sera (from 2 ongoing prime-boost trials in Thailand). Virus and NAb were washed out, and NAb was then added (flow cytometry endpoint) or not added (extracellular p24 endpoint) for the culture period. At day 4 to 6 post-infection, cells were stained for intracellular p24 and surface CD4 or CD3 and then analyzed on a flow cytometer. Culture fluids were harvested at day 4 to 8 for extracellular p24 measurement.
Results: The flow cytometry NAb assay was shown to be reproducible and vaccinee sera (at 1:10 dilution) showed 0 to 97% reduction of infected cells by this assay. Greater than 50% reduction of infected cells was observed at 4- to 10-fold higher dilutions (1:2,560(1:10,240) of seropositive sera when the flow cytometry endpoint was used. In a comparative study using sera from 24 subtype B NAb-positive vaccinees in groups receiving protein subunit boosts, the flow cytometry-based assay detected subtype E virus NAb in twice as many post sera, as compared with the extracellular p24 assay.
Conclusions: The maintenance of NAb throughout the assay period and the assessment of reduction in infected cell number may be a more accurate reflection of in vivo conditions and a more sensitive method for evaluating sera from vaccine trials.
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