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84 Improved Measurement of Antibody-Mediated Neutralization of HIV-1 by Flow Cytometric Enumeration of Infected T Cells
J. Mascola1*, M. Louder1, C. Winter1, D. Gabuzda2, and M. Roederer1
1Vaccine Res. Ctr., NIAID, NIH, Bethesda, MD, USA and 2Dana-Farber Cancer Inst., Boston, MA, USA
Background: Accurate measurement of neutralization of primary HIV-1 strains is important for the evaluation of immune responses to candidate vaccines. The variability inherent in our current assay, which quantifies infection of PBMC indirectly by measurement of secreted p24-antigen, prompted development of an assay to directly enumerate infected T cells by flow cytometry.
Methods: Primary stocks of HIV-1 were grown in PBMC and concentrated up to 10-fold through a 100-kd Millipore filter. Mitogen-stimulated PBMC were exposed to HIV-1 in 96-well microtiter plates. A protease inhibitor (indinavir 1 mum) was added to cultures to prevent secondary rounds of viral replication. Infection of individual cells was evaluated by intracellular staining for p24-antigen using a directly conjugated anti-p24 MAb (Coulter KC57). Some assays were done with env-pseudotyped GFP reporter viruses, and both GFP and p24-Ag were measured by 2-color flow cytometry.
Results: Upon exposure to virus, p24-positive T cells could be readily distinguished beginning 1 day post infection. Without indinavir present, secondary rounds of virus replication resulted in infection of the large majority of CD4+ T cells by 4 to 6 days post infection. With indinavir, first round infection measured by flow cytometry on day 2 was seen in 1 to 5% of PBMC. Single-round replication env-pseudotyped GFP reporter viruses gave similar levels of infection of PBMC, and the large majority of GFP+ cells were also p24-antigen positive. Serial dilution of viral stocks demonstrated that the number of target cells infected varied proportionally with the viral input. Whereas the level of neutralization measured in this assay appears to be similar to that observed using the secreted p24-antigen capture assay, the improved assay reproducibility allows more accurate measurement of modest (i.e., 50%) neutralization effects.
Conclusions: This flow cytometric assay measures single-round infection of PBMC after exposure to HIV-1. The direct enumeration of infected target cells allows more accurate and reproducible measurement of antibody-mediated neutralization.
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