672 An Improved Culture Method for Stimulating the Production of HIV from PBMCs C. Jennings*1, D. Brambilla2, D. Huang1, J. Bremer1 1Rush Med Coll, Chicago, IL and 2New England Res Inst Inc, Watertown, MA
Background: A method for the improved recovery of HIV from PBMCs, even in patients (pts) with undetectable plasma viral loads (< 50cp/mL), is described. This new method couples the use of T-cell specific stimulation with the depletion of CD8+ cells in the absence of PHA-stimulated HIV-seronegative indicator cells to reproducibly stimulate the production of virus from PBMCs.
Methods: EDTA blood (2 x 10 mL) from 9 pts was centrifuged, and each buffy coat cell layer was re-suspended in 10 mL of PBS. We added 250 uL of Dynal anti-CD8 magnetic beads to 1 of the diluted buffy coat mixtures and both were rocked at 4°C for 30 min. PBMCs for the standard HIV co-culture and CD8-depleted PBMCs for the enhanced HIV culture were then obtained by density gradient centrifugation (cells rosetted by the Dynal beads were depleted during ficoll centrifugation). For the enhanced culture method, 6 million CD8-depleted cells were cultured in a flask containing 6 mL MoAb media (IL-2/FBS/gentamycin/RPMI containing 0.05 ug/mL of anti-CD3/CD28 monoclonal antibodies [Southern Biotechnology Associates, Inc.]). Half of the culture supernatant was removed every 3-4 days, tested for p24 antigen production, and replenished with fresh IL-2 media (no MoAbs). For the standard co-culture, 6 million PBMCs were cultured as previously described (equal number of pt PBMCs and PHA-stimulated indicator cells in IL-2/FBS/antibiotic containing RPMI). Half the supernatants harvested every 3-4 days were tested for p24 antigen production and replenished with fresh IL-2 media or fresh indicator cells. All cultures were maintained for a minimum of 28 days.
Results: Replication-competent virus was generated in 9/9 (100%) enhanced cultures compared to 4/9 (44%) standard co-cultures. The average number of days to obtain a positive culture (2 consecutive p24 results above 100 pg/mL) was 17 days for the enhanced culture and 15 days for the standard co-culture.
Conclusions: These data demonstrate that the enhanced culture methodology is more effective than standard culturing methods in stimulating the production of HIV from PBMCs in patients with undetectable plasma viral loads. Additionally, the use of this culture method will eliminate the need for the maintenance of PHA-stimulated indicator cells, a procedure that is both costly and time-consuming. Future studies will involve the use of this method for culturing virus out of other HIV reservoirs, including the genital compartment.
