CD8⁺ T-cell specificity of IFN-γ responses to synthetic peptides. PBMC from donor 00BW1774 were CD8⁺ CD4⁺ depleted or enriched by using MACS CD8 MicroBeads and CD4 MicroBeads followed by Elispot assay with 20-mer Gag p24 peptide MFTALSEGATPQDLNTMLNT and 9-mer Gag p24 peptide TPQDLNTML (G180-TL9). Experiments were performed in triplicate. The extent of standard deviation is shown (error bars).

Profiles of HIV-1C Tat-specific CTL response. (A) Forty-eight subjects were screened in the IFN-γ-Elispot assay using 18 HIV-1C-based 15-mer synthetic peptides overlapping by 10 amino acids (x axis). Cumulative Elispot-based CTL responses were expressed as a sum of the per-patient responses to a particular peptide (y axis). The immunodominant region is delineated with a dashed box. (B) Nucleotide variability. Nucleotide variability was analyzed across the HIV-1C tat alignment of nucleotide sequences from Botswana isolates by using a sliding window of 10 nucleotides and increments of 3 nucleotides. A dashed box corresponds to the identified immunodominant region within HIV-1C Tat. (C) Amino acid diversity across HIV-1C Tat. Fragments of amino acid alignment analyzed by the program PROTDIST from the PHYLIP package corresponded to the overlapping peptides that were used in the Elispot assay. The dashed box corresponds to the identified immunodominant region within HIV-1C Tat. (D) Consensus of immunodominant region. Amino acid consensus of the immunodominant region within HIV-1C Tat is shown as a sequence in the main row. Residues beneath the main row represent amino acid variation at a particular position. Subscript numbers correspond to the percentage of amino acid occurrence within HIV-1C Tat. (E) Tat-specific CTL responses among a subset of 16 sequenced study subjects. The top line of alignment represents contiguous sequence of 15-mer synthetic peptides used in the Elispot assay. Letters above the top line of alignment correspond to the synthetic peptide variants. Dashes throughout the alignment show the identical amino acids within viral isolates. Boxes across the alignment designate identified CTL responses. Boxes longer than 15 amino acids represent CTL responses to the overlapping peptides. Study subjects that did not demonstrate HIV-1C Tat-specific CTL responses in the Elispot assay are circled.

Profiles of HIV-1C Nef-specific CTL responses. (A) Forty-five subjects were screened in the IFN-γ-Elispot assay using 30 HIV-1C-based 15- to 20-mer overlapping synthetic peptides (x axis). Cumulative Elispot-based CTL responses were expressed as a sum of the per-patient responses to a particular peptide (y axis). Immunodominant regions are boxed with dashed lines. (B) nucleotide variability. Nucleotide variability was analyzed across the HIV-1C nef alignment of nucleotide sequences from Botswana isolates by using a sliding window of 20 nucleotides and increments of 3 nucleotides. Dashed boxes correspond to identified immunodominant regions within HIV-1C Nef. (C) Amino acid diversity across HIV-1C Nef. Fragments of amino acid alignment analyzed by the program PROTDIST from the PHYLIP package corresponded to synthetic peptides that were used in the Elispot assay. Dashed boxes correspond to identified immunodominant regions within HIV-1C Nef. (D) dN/dS substitution rates. dN and dS were analyzed using the SNAP program and are shown per codon within identified immunodominant regions. (E) Consensus of immunodominant regions. Amino acid consensus of immunodominant regions within HIV-1C Nef are shown as sequences in the main rows. Residues beneath the main row represent amino acid variation at a particular position. Subscript numbers correspond to the percentage of amino acid occurrence within HIV-1C Nef.

Magnitude of HIV-1C-specific Elispot-based CTL responses in Gag, Tat, Rev, and Nef expressed as cumulative CTL responses normalized by amino acid diversity (per peptide) and number of study subjects screened.

Fine mapping of HIV-1C Tat CTL epitope FQTKGLGISY (T38-FY10) restricted by HLA-B*1503. PBMC from subjects who demonstrated responses to 15-mer peptide VCFQTKGLGISYGRK were analyzed in peptide titration experiments using a set of 22 synthetic peptides from 9- to 12-mer. The frequency of responses is expressed as SFC per million PBMC. Titration curves represent CTL responses to serial dilutions of synthetic peptides. Only responses relative to peptide FQTKGLGISY are shown. Subjects who were negative for HLA-B*1503 did not demonstrate CTL responses to the T38-FY10 epitope (data not shown).

Phylogenetic relationship of 45 near-full-length HIV-1C sequences from Botswana with other available nonrecombinant HIV-1C sequences from around the world. One isolate from Ethiopia (ETH2220), two from Brazil (92BR025 and 98BR004), nine from India (IN11246, IN301999, IN21068, IN301905, IN301904, IN101, 94IN476-104, 98IN012-14, and 98IN022), two from Zambia (96ZM651-8m and 96ZM751-3m), one from Israel (98IS002-5), two from Tanzania (98TZ013-10 and 98TZ017-2), and one from South Africa (97ZA012-1) were included in the analysis. HIV-1O isolate CM.ANT70 (accession number L20587) was used as an outlier. The neighbor-joining method and the Kimura two-parameter model were used. The bootstrap values of 80% or higher are shown at the nodes supporting branching order. Nucleotide diversity among the whole set of HIV-1C sequences was calculated using the DNADIST program.

Profiles of HIV-1C Gag-specific CTL responses. (A) Forty-six subjects were screened in the IFN-γ-Elispot assay using 49 HIV-1C-based 20-mer overlapping synthetic peptides (x axis). Cumulative Elispot-based CTL responses were expressed as a sum of the per-patient responses to a particular peptide (y axis). Immunodominant regions are boxed with dashed lines. HIV-1C Gag p17, p24, and p7/p6 are delineated. (B) Nucleotide variability. Nucleotide variability was analyzed across the HIV-1C gag alignment of nucleotide sequences from Botswana isolates by using a sliding window of 30 nucleotides and increments of 10 nucleotides. Dashed boxes correspond to identified immunodominant regions within HIV-1C Gag p24. (C) Amino acid diversity across HIV-1C Gag. Fragments of amino acid alignment analyzed by the program PROTDIST from the PHYLIP package corresponded to 49 overlapping peptides used in the Elispot assay. Dashed boxes correspond to identified immunodominant regions within HIV-1C Gag p24. (D) dN/dS substitution rates: dN and dS were analyzed using the SNAP program and are shown per codon within identified immunodominant regions. (E) Consensus of immunodominant regions. The main row represents a consensus amino acid sequence of immunodominant regions within HIV-1C Gag. Residues beneath the main row represent amino acid variation at a particular position. Subscript numbers correspond to the percent occurrence of amino acids within HIV-1C Gag.

Profiles of HIV-1C Rev-specific CTL responses. (A) Forty-seven subjects were screened in the IFN-γ-Elispot assay using 19 HIV-1C-based 15-mer synthetic peptides overlapping by 10 amino acids (x axis). Cumulative Elispot-based CTL responses were expressed as a sum of the per-patient responses to a particular peptide (y axis). (B) Nucleotide variability. Nucleotide variability was analyzed across HIV-1C rev alignment of nucleotide sequences from Botswana isolates by using a sliding window of 20 nucleotides and increments of 3 nucleotides. (C) Amino acid diversity across HIV-1C Rev. Fragments of amino acid alignment analyzed by the program PROTDIST from the PHYLIP package corresponded to nineteen synthetic peptides that were used in the Elispot assay.

Comparison of HIV-1C-specific CTL responses with HIV-1B-specific responses in Nef. Forty-five HIV-1C-infected subjects were screened in the IFN-γ-Elispot assay using 30 HIV-1C-based 15- to 20-mer overlapping synthetic peptides and 20 HIV-1B-based 20-mer peptides (x axis). Cumulative Elispot-based CTL responses were measured as a sum of the per-patient responses to a particular peptide and expressed as the number of cumulative SFC/10⁶ PBMC (y axis).

Fine mapping of HIV-1C Gag p24-specific CTL epitopes. (A) Gag p24 epitope TPQDLNTML (G180-TL9) is restricted by HLA-B*4201 or HLA-B*8101. PBMC from seven subjects who expressed HLA-B*4201 or -B*8101 were analyzed in an Elispot assay. The frequency of responses is expressed as SFC per million PBMC. Titration curves represent CTL responses to serial dilutions of synthetic peptides. Subjects who were negative for HLA-B*4201 or -B*8101 did not demonstrate CTL responses to the G180-TL9 epitope (data not shown). (B) Gag p24 epitope YVDRFFKTL (G296-YL9) is restricted by HLA-B*1510. PMBC from four subjects who expressed HLA-B*1510 were analyzed in an Elispot assay. The frequency of responses is expressed as SFC per million PBMC. Titration curves represent CTL responses to serial dilutions of synthetic peptides. Subjects who were negative for HLA-B*1510 did not demonstrate CTL responses to the G296-YV9 epitope (data not shown).