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192a Enhanced Production of Primary Isolates of HIV-1 in CD4-Enriched Cell Substrate
C. Wu*1,2, C. Evans3, and G. Vyas1
1Univ. of California San Francisco Sch. of Med., USA; 2San Francisco State Univ., CA, USA; and 3ARC Northern California Blood Ctr., Oakland, USA
Background: Clonally derived DNA or envelope proteins are immunogenic but do not induce broadly neutralizing antibodies (NAb) for the prevention of HIV infection. Instead of the genetically restricted lab isolates grown in T-cell lines for whole inactivated virion vaccine, our approach includes natural proteins of the prevalent primary isolates of HIV (pHIV). We report here HIV-1 expansion in CD4-enriched PBMC (CD4-E).
Methods: Blood donations were processed with Pall leukocyte filters. Reverse flow of 300 ml of PBS through used filters recovered cells that were made to 50% (V/V) suspension for ficoll-gradient centrifugation to derive viable PBMC. Removal of CD8+ cells with anti-CD8-coated magnetic beads provided CD4-E. After PHA-stimulation for 3 days, PBMC and CD4-E were compared for in vitro propagation of 5 ( 103 TCID50 of pHIV-1 (92BR014) inoculated into 2.0 ml of cultures containing 2 ( 106cells. During 12 days of culturing, half of the amount of cell-free supernates was removed for monitoring p24 antigen as a measure of pHIV-1 synthesis and replaced with fresh medium at 3-day intervals.
Results: Recovery of PBMC from 6 filters averaged 260 ( 106, yielding 182 ( 106 CD4-E after CD8 depletion. In 12-day cultures p24 synthesis peaked at day 6 and cumulative p24 synthesis per million PBMC and CD4-E was 0.125 and 0.35 mug/ml, respectively. Based on the molecular structure of viral envelope and core, total HIV proteins synthesized in culture was considered twice that of p24 quantitation. In a scaled up culture, 182 ( 106 CD4-E from a blood filter would produce at least 63.7 mug of pHIV-1.
Conclusions: An accelerated large-scale production of pHIV-1 in abundantly available CD4-E cell substrate is practical. Further, CD4-E cocultured with dendritic cells that present pHIV-1 through its unique DC-SIGN receptor would yield adequate pHIV-1 for a polyvalent vaccine using viral inactivation by chemical and physical methods that do not alter the protein structure, composition and immunogenicity of the natural HIV proteins that are capable of inducing both CTL and NAb.
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