Table 1.
HIV-1 epitopes and CTL clones.
Fig 1.
Production of HIV-1 epitope mutant virus libraries.
Double-stranded DNA encoding an HIV-1 genomic region including an epitope of interest was synthesized to represent every possible single and double amino acid variant within the epitope and its immediately flanking amino acids, using degenerate codons (NNK, where N is any nucleotide and K is guanine or thymidine, encoding every amino acid while reducing stop codons). This DNA was then substituted into the full length HIV-1 NL4-3 genome to create a plasmid HIV-1 library for virus production.
Table 2.
Epitope variants contained in plasmid and virus libraries.
Fig 2.
CTL selective pressure results in altered HIV-1 epitope variant frequencies.
A. The frequencies of each SL9 epitope variant are plotted for the plasmid library, initial virus library, and virus populations after one or two weeks of passage (in the absence of CTL selection). All sequences below a frequency threshold of 2.5x10-5 in the plasmid library (inadequately represented for carry-through to the virus library) are represented in black. The frequencies of the consensus variant are significantly different between time points (p<0.00001). The subtype B consensus variant of the SL9 epitope is indicated by the bottom light blue bars in all bar graphs, and color coding of each variant is consistent across panels; variants not achieving the frequency cutoff of 1x10-4 in both replicates of the starting virus library are labeled “other sequences” and indicated by hatched gray bars. B. The frequencies of epitope variants after one and two weeks of passaging in the presence of the SL9-specific CTL clone 3.23T are indicated. Again, the consensus variant differs significantly between time points (p<0.00001), and additionally significantly different (p<0.00001) between libraries cultured without versus with 3.23T after 1 and 2 weeks. C. Epitope variant frequencies are plotted after one week of passaging in the presence of a control CTL clone 68A62 recognizing an A*02-restricted epitope in reverse transcriptase, or different effector to target (E:T) ratios of clone 3.23T. The frequencies of the consensus variant are significantly different (p<0.00001) between culture with 68A62 versus 3.23T. D. The formula to calculate relative enrichment (RE) of each variant compared to the subtype B consensus epitope variant using the frequency of each variant compared to the consensus epitope is shown. E and F. The REs between two biological replicate experiments with the SL9 variant library passaged in the absence (E) or presence (F) of clone 3.23T (RE-CTL values without CTLs and RE+CTL values with CTLs respectively) are plotted. Red dots in both panels E and F indicate variants with high RE+CTL values, demonstrating their locations in the RE-CTL plot. All panels are representative of individual replicate experiments.
Fig 3.
Single amino acid epitope variants display distinct patterns of selection in the absence or presence of CTLs (SL9 epitope).
The RE values of all single amino acid variants with or without addition of the indicated CTL clones are displayed as color-scaled boxes. The horizontal axis indicates each subtype B consensus amino acid of each epitope and its immediately flanking amino acids, and the vertical axis indicates substituting amino acids. Hatched boxes indicate consensus amino acids. Additionally, the mean REs for substitutions of amino acids that are hydrophobic (A, V, I, L, M, F, Y, W, G, and P), polar-noncharged (C, S, T, N, and Q), basic (R, H, K), or acidic (D and E) are indicated below each plot. Variants that were detected above threshold in the plasmid library but not in the virus library were considered non-replicating and assigned RE-CTL = -2.0 for these analyses.
Fig 4.
Single amino acid epitope variants display distinct patterns of selection in the absence or presence of CTLs (KF11 epitope).
The RE values of all single amino acid variants with or without addition of the indicated CTL clones are displayed as color-scaled boxes. The horizontal axis indicates each subtype B consensus amino acid of each epitope and its immediately flanking amino acids, and the vertical axis indicates substituting amino acids. Hatched boxes indicate consensus amino acids. Additionally, the mean REs for substitutions of amino acids that are hydrophobic (A, V, I, L, M, F, Y, W, G, and P), polar-noncharged (C, S, T, N, and Q), basic (R, H, K), or acidic (D and E) are indicated below each plot. Variants that were detected above threshold in the plasmid library but not in the virus library were considered non-replicating and assigned RE-CTL = -2.0 for these analyses.
Fig 5.
Single amino acid epitope variants display distinct patterns of selection in the absence or presence of CTLs (KK10 epitope).
The RE values of all single amino acid variants with or without addition of the indicated CTL clones are displayed as color-scaled boxes. The horizontal axis indicates each subtype B consensus amino acid of each epitope and its immediately flanking amino acids, and the vertical axis indicates substituting amino acids. Hatched boxes indicate consensus amino acids. Additionally, the mean REs for substitutions of amino acids that are hydrophobic (A, V, I, L, M, F, Y, W, G, and P), polar-noncharged (C, S, T, N, and Q), basic (R, H, K), or acidic (D and E) are indicated below each plot. Variants that were detected above threshold in the plasmid library but not in the virus library were considered non-replicating and assigned RE-CTL = -2.0 for these analyses.
Fig 6.
Identification of single amino acid epitope variants that potentially escape from CTLs.
The net changes in RE values due to CTL selection (ΔRE = RE+CTL—RE-CTL) are displayed as color-scaled boxes for all single amino acid variants of epitopes SL9 (A), KF11 (B), and KK10 (C). The horizontal axis indicates each subtype B consensus amino acid and its immediately flanking amino acids, and the vertical axis indicates substituting amino acids. Variants that detected above threshold in the plasmid library but not in the virus library are considered non-replicating and coded by dark grey. Consensus amino acids are coded by hatched boxes.
Fig 7.
Confirmation that ΔRE values reflect potential for escape from CTLs.
Clonal HIV-1NL4-3 with five SL9 epitope variants with increased (1.39–1.66 and 1.37–2.32 for CTL clones 3.23T and 10.11T respectively), one variant with decreased (-1.42 and -1.43 with CTL clones 3.23T and 10.11T respectively) ΔRE, and the subtype B consensus epitope were individually tested for susceptibility for suppression by CTLs. T1 cells were acutely infected and cultured in the presence or absence of CTL clones 3.23T or 10.11T; supernatant p24 antigen values after 7 days are plotted for each indicated variant. Each value is the mean of triplicates, and error bars represent standard deviations.
Fig 8.
CTL selection of all library epitope variants.
The RE values of all epitope variants with or without addition of the indicated CTL clones are displayed as color-scaled boxes for epitopes SL9, KF11, and KK10. The horizontal axis indicates each consensus amino acid and its immediately flanking amino acids, and the vertical axis indicates substituting amino acids. First columns indicate relative enrichment versus consensus variant without CTLs (RE-CTL), second columns indicate relative enrichment versus consensus variant with added CTLs (RE+CTL), third columns indicate the difference between those values (ΔRE = RE+CTL-RE-CTL), and fourth columns indicate variants with ΔRE ≥ 0.7 = 5-fold (white boxes) or < 0.7 (gray boxes). Variants with RE-CTL < -1.5 and stop codons are omitted.
Fig 9.
Distributions of epitope variant viabilities.
Top panel: The mean RE-CTL values of all variants in the virus library are plotted for each epitope. The numbers achieving thresholds of RE-CTL≥0 and RE-CTL≥-0.5 and their frequencies (over all variants adequately represented in the plasmid library) are indicated for each epitope. Variants with RE-CTL<-2 are not plotted. Bottom panel: The numbers of variants in different ranges of mean RE-CTL values are indicated.
Fig 10.
CTL promiscuity for epitope variant recognition.
Top panel: The mean ΔRE values of all variants in the virus library are plotted for each epitope, excluding those with mutations in the epitope flanking residues. The numbers and frequencies (over all variants adequately represented in the virus library) of variants achieving a threshold of ΔRE≥0.7 are indicated. Bottom panel: The numbers of variants in different ranges of mean ΔRE values are indicated.
Fig 11.
Comparisons of viabilities of potential escape variant epitopes for SL9, KF11, and KK10.
Top panel: RE-CTL values are plotted for all SL9, KF11, and KK10 epitope variants (excluding those with substitutions in flanking residues) in the virus libraries. Potential escape variants (ΔRE≥0.7) are indicated by filled symbols, and the remainder are open symbols. Bottom panel: RE-CTL values are plotted for all SL9, KF11, and KK10 epitope variants as above, where variants with mean ΔRE≥0.7 for all tested CTLs are indicated by filled symbols. Numbers of potential escape variants with RE-CTL>-0.5 and their frequencies among variants in the virus libraries are indicated.
Fig 12.
Schematic representation of fitness landscapes and CTL escape options for SL9, KF11, and KK10.
Epitope variants (including the immediate flanking residues) with mean ΔRE≥0.7 were considered potential escape variants, and RE-CTL≥-0.5 were considered viable. The percentages of viability and susceptibility to CTLs for double amino acid variants that were missing in our plasmid library were inferred to be the same as those that were present and tested in this study. Top row: Venn diagrams indicate total numbers of possible single and double amino acid variants (white circles), numbers of viable variants (red circles), and numbers of viable variants susceptible to CTLs (green circles). The surface areas of each circle are approximately proportional to the numbers of variants contained. Bottom panel: Bar graphs indicate the same data, where gray bars indicate non-viable variants, green bars indicate viable variants that are recognized by CTLs, and red bars indicate viable variants that escape CTLs.
Fig 13.
Correlations of epitope variant susceptibilities to different CTLs targeting SL9, KF11, or KK10 epitopes.
The ΔRE values are compared between CTLs targeting targeting each epitope. Shaded areas indicate ΔRE≥0.7 for one or both CTLs, and red filled symbols indicate those with RE-CTL≥-0.5. Epitope variants that were not detected in the virus libraries (non-replicating) were excluded.