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Background:  Few studies have characterized virus-specific CD8⁺ T cells in infants, particularly during the first few months of life. An important question is whether the CD8⁺ T cell responses generated by young infants are capable of controlling viral replication or exerting selective pressure in vivo over the first year of life.

Methods:  RNA was extracted from plasma obtained from 5 untreated, HIV-1-infected infants at 1 to 3, 4 to 6, and 12 to 15 months of age. Gag and nef cDNA were amplified and 10 clones per gene per patient were sequenced. Molecular HLA class I typing was performed. A modified peptide-based IFN-g ELISpot assay was used to detect and measure the frequencies of epitope-specific CD8⁺ T cells.

Results:  Intrapatient genetic distances and the ratios of nonsynonymous:synonymous (dN/dS) amino acid substitutions increased over time, suggesting diversification of infant sequences. DN/dS ratios were highest in nef and gag p15. Of all amino acid (aa) substitutions, 75% were observed in the nef gene and 19% of amino acid substitutions in gag p15 were either within epitopes corresponding to infant HLA alleles or within regions previously described as targets for HIV-1-specific CD8⁺ T-cell responses. Analysis of sequences within potential epitopes restricted by infant HLA alleles revealed evidence of CD8⁺ T-cell escape as early as 2 to 3 months of age. For example, in an HLA A24⁺ infant, Q192H substitution was detected within an A24-restricted nef epitope (aa 186 to 194; DSRLAFQHM) by 2 months. In another HLA A24⁺ infant, F135Y substitution was detected in an A*24-restricted nef epitope (aa 134 to 143; RFPLTFGWCF) as early as 3 months of age. Reduced recognition of the variant sequences compared with wild type sequences was demonstrated by ELISpot.

Conclusions:  Our findings demonstrate that young infants are capable of generating CD8⁺ T-cell responses that exert potent selective pressures in vivo and that these CD8⁺ T-cell responses shape viral evolution over the first year of life.

Keywords: CD8+ T cells; HIV-1 evolution; selective pressure