Background:  HIV-1 amino acid sequence polymorphisms associated with expression of specific HLA class I alleles suggest sites of CTL-mediated positive selection pressure and immune escape. However, a substantial number of sites under so-called negative selection pressure have been identified, where HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind these negative associations, and their significance in terms of the evolution and immune control of HIV remains unclear.

Methods:  We focused 3 alleles—HLA-B*57, B*5801, and B*51—that impose strong selection pressure on HIV. More than 300 clade C and B viruses were sequenced for Gag, RT, INT, and Nef to examine associations between sequence polymorphism and HLA expression. Vertical and horizontal transmission pairs were used to study the selection, transmission, and stability of escape mutations. The effect of escape on epitope presentation was examined by peptide binding and mRNA transfection assay.

Results:  We identified a negative association between expression of HLA-B*57/B*5801 and conservation of consensus Gly at Nef residue-83. We show that Nef-83-Gly is an AŕG escape mutant within a previously undefined B*57/B*5801-restricted epitope, KF9. This A83G escape mutant is transmitted, does not revert, and hence has spread in the population to replace Ala as the consensus. Peptide-binding and mRNA transfection assays show that the A83G KF9 variant is no longer presented on the cell surface. Analysis of HLA-B*51 epitopes revealed a second negative association between expression of B*51 and conservation of the consensus AA Val at Int 31, within the B*51-restricted epitope LI9. Again the consensus was found to represent an IŕV escape mutation selected by HLA-B*51 that is stable on transmission and thus has spread to replace I as the consensus.

Conclusions:  Negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. If an escape variant reaches fixation in the population, the epitope can be considered extinct, as it has been lost as a potential target to the immune system, and evidence for the mechanism by which it arose will disappear. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

Keywords: CTL escape; Selction; HIV evolution