Background:  Most HIV-1 infections result from transmission of single infectious units, yet the genetic and evolutionary pathways that HIV-1 undergoes to establish productive infection in the new host remain largely unclear. Our aim was to identify the earliest HLA-restricted cytotoxic T lymphocyte (CTL) escape mutations in full-length transmitted HIV-1 genomes.

Methods:  Single genome amplification (SGA) was used to derive genomic (9 Kb) and env (2.5 Kb) amplicons from plasma virion RNA of subjects acutely infected with HIV-1 subtype B. Direct DNA sequencing and phylogenetic analysis allowed the identification of the transmitted/early virus which, together with human HLA phenotyping, was used to identify early escape mutations.

Results: Nucleotide sequences were determined for 5 full-length transmitted/early founder HIV-1 genomes and 102 transmitted env genes. For a subset of patients, env sequences were obtained from multiple sequential time-points. Of 102 transmitted env genes, 14 showed mutations suggestive of CTL recognition and escape which fell within 3 categories: those that matched a known HLA-restricted CTL epitope (from the LANL database); those that matched a known CTL epitope and were experimentally shown to be recognized by autologous CTL; and those that did not match any known CTL epitope. Analyses of sequential samples revealed a marked increase in the proportion of mutant viruses over time, most of which encoded single amino acid substitutions at various positions within a 9-mer region. In one acutely infected individual a set of identical or near identical env sequences seen at peak viremia was replaced 14 days later by a new swarm of viral mutants having mutations within a 9-mer and accounting for 93% of the virus population. Coupled with this response, 18 other CTL epitopes were mapped throughout the proteome using enzyme-linked immunosorbent spot (ELISpot) assays, while the viral load progressively declined to <2000 copies/mL. Functional assays are underway to determine the immunological (or other) pressures responsible for the rapid selection in this and other patients.

Conclusions: These findings provide a unique description of early genetic diversification of the transmitted virus and a method for identifying the earliest biological selection pressures in vivo.