Overlap between predicted CD8 and CD4 epitopes in Env-V2 and -V3 in sequences from HIV-1-infected RV144 participants.

<p>The x-axis corresponds to the V2 and V3 sequence, and the y-axis corresponds to the number of predicted epitopes starting at each position. The epitope predictions correspond to all unique HLA/peptide combinations, hence the number of epitope predictions starting at a specific location can surpass the number of subjects in the cohort because a given peptide can be predicted as an epitope for multiple HLA alleles. The amino acids in red correspond to sites that were identified as genetic signatures that distinguished breakthrough sequences from vaccine and placebo recipients.</p

Number of CD8 epitopes predicted for each subject depending on his HLA type.

<p>Predictions are given for proteins that corresponded to the vaccine inserts (Gag, Pro, Env), and for proteins that were not part of the vaccine (RT-IN, and Nef).</p><p>Number of CD8 epitopes predicted for each subject depending on his HLA type.</p

Flowchart diagram of HIV-1 breakthrough infections in RV144.

<p>Flowchart diagram of HIV-1 breakthrough infections in RV144.</p

Subject-specific epitopes matched to vaccine-derived epitopes.

<p>Epitopes were considered matched when the subject- and vaccine-derived peptides had at least 67% AA identity. Number and percentages of matched epitopes are given for all of the 110 subjects in the cohort; there was no difference between the vaccine and placebo groups.</p><p>Subject-specific epitopes matched to vaccine-derived epitopes.</p

CD8 and CD4 epitopes predicted to be strong binders matched against different vaccine inserts.

<p>Epitopes were predicted in all HIV-1 proteome sequences derived from RV144 breakthrough infections. The epitopes were matched against epitopes derived from the RV144 vaccine inserts of subtype B (MN, LAI) or CRF01_AE (CM244, 92TH023); two epitope characteristics were used to compare epitopes from the breakthrough to the vaccine: the predicted binding affinity for each epitope and the protein distance between the epitope sequences. One summary measure was computed for each protein and each subject, and comparisons were done between the vaccine (V) and placebo (P) groups (the number of vaccine and placebo recipients included in each group is in parenthesis) with Mann-Whitney tests for proteins corresponding to those included in the RV144 vaccine insert (Gag, Pro, gp120 (including V2)) and those not part of the RV144 vaccine (RT-IN, Nef).</p><p>CD8 and CD4 epitopes predicted to be strong binders matched against different vaccine inserts.</p

Schematic representation of epitope predictions in RV144 HIV-1 breakthrough sequences, and their comparison to RV144 vaccine inserts.

<p>A. Each line represents the Env-gp120 sequence from a subject and each circle a CD8 epitope prediction (different colors for different HLA alleles). The figure represents epitopes predicted based on each subject’s HLA class I genotype for two subjects who were infected with a nearly identical virus (AA100: HLA-A*02∶03, HLA-A*24∶10, HLA-B*18∶01, HLA-B*18∶02, HLA-C*07∶04; AA118: HLA-A*11∶01, HLA-A*24∶07, HLA-B*44∶03, HLA-C*01∶02, HLA-C*07∶01). B. Epitope repertoires from a given subject are compared to the epitope predictions for the vaccine insert sequences (CM244 and MN) based on that subject’s HLA class I genotype. Empty circles represent epitopes predicted in the sequence from a subject that could not be matched to a corresponding epitope prediction based on the vaccine insert sequence and the subject’s HLA class I genotype. More subject-derived epitopes were matched against the vaccine insert CM244 than against MN; both subjects were infected by a CRF01-AE virus like CM244, while MN is a subtype B virus.</p

Vaccine efficacy at the signature sites in the vaccine proteins.

<p>¹HXB2 numbering</p><p>²The sieve effect was detected for the indicated vaccine sequence; for Env positions, these are 92TH023, CM244, both of the CRF01_AE sequences, the subtype B sequence MN, or all three. The corresponding vaccine amino acids are given after the colon. They are in parentheses if the effect was “vMismatch” (with greater divergence from vaccine AA among placebo recipients).</p><p>³Symmetrized estimated vaccine efficacies (for hazard ratio (HR) above 1, symmetrized VE = [1 − hazard ratio (HR)]×100%; for HR below 1, symmetrized VE = − [1 − (1/(HR))]×100%) to prevent infection with specific HIV-1 genotypes.</p><p>⁴The p-value for Env 379 was not calculable for the DVE method because one of the treatment groups (the vaccine-recipient group) exhibited no variation at the site.</p><p>Vaccine efficacy at the signature sites in the vaccine proteins.</p

Signature sites in vaccine proteins: 2-sided unadjusted p-values calculated by five methods.

<p>Signature sites in vaccine proteins: 2-sided unadjusted p-values calculated by five methods.</p

Analysis methods.

<p>Methods evaluate (1) differential deviation (vaccine versus placebo) from the immunogen sequences at specific loci or in peptide regions that are relevant to antibody binding; (2) differential codon selection, and differences in physico-chemical properties across vaccine and placebo; (3) differential vaccine efficacy versus HIV-1 sequences that do not match immunogen sequences at individual sites and in each of several pre-specified antibody-relevant protein regions; (4) greater or more rapid viral escape (vaccine versus placebo) at predicted class I and class II HLA-restricted T cell epitopes; and (5) differences in phylogenetic diversity of the breakthrough amino acid sequences (vaccine versus placebo) or differential evolutionary divergence from the vaccine immunogen sequences. T cell and tree images are from <a href="http://openclipart.org" target="_blank">openclipart.org</a>.</p

Tests for enrichment of signature sites in biologically-defined subsets compared to all other sites.

<p>For each of the six pre-specified site sets, we compared the number of the 12 vaccine immunogen signature sites in Env that are in the set to the number that are outside of the set, to test whether membership in the set is independent of the “signature site” designation. For example, 7 of the 12 signature sites are in the <i>Hotpots</i> site set, while 89 of the 441 tested non-signature sites are in that set.</p><p>Tests for enrichment of signature sites in biologically-defined subsets compared to all other sites.</p