P39.07
Background: RV217/ECHO study presents a unique opportunity to identify subjects very early in HIV infection, with a median time to last negative nucleic acid test (NAT) of 4 days. Two transmitted/founder (T/F) variants were isolated from a Thai volunteer, with a representation of 99% and 1% for the major (Mj) and the minor (mn) variants respectively at the peak of viral load. Using next-generation, targeted deep sequencing, we confirmed the presence of that mn variant on the day of the first positive NAT. After peak viremia the mn variant grew exponentially, reaching>30% by day 31. Six months post infection, the mn variant became the predominant quasispecies. We hypothesized that viral fitness is responsible for this viral dynamic profile.
Methods: Full-length infectious molecular clones (FLIMC) of the Mj and mn variants were generated. Each Mj and mn FLIMC was engineered to express the fluorescent proteins, GFP and mCherry. The four constructs Mj.C2, Mj.G2, mn.C2 and mn.G2 were tested for infectivity in cell lines (TZMbl & A3R5) and primary cells (PBMC, macrophages, dendritic cells) and monitored by flow cytometry, fluorescent microscopy and p24 capture assay; cells were singly infected and co-infected. Cell tropism was also assessed using CXCR4 and CCR5 Ghost cells.
Results: Both mn and Mj FLIMCs showed productive infection in cell lines and primary cells and used the co-receptor CCR5 exclusively. Neither the GFP nor mCherry proteins modified in vitro infection kinetics of Mj and mn FLIMCs. Cell lines and primary cells were co-infected with different ratios of Mj/mn. Contrary to their representation at the time of initial infection, we found that the mn variant was more fit, as measured by replication kinetics, than the majority transimitted Mj variant. Interestingly, dual infection of single cells was a very rare event.
Conclusions: We successfully applied a new fitness assay to evaluate multiple T/F in acute infection and found fitness didn't account for dominance of the Mj variant during acute infection.
