Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial

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1 CORRECTION NOTICE Nat. Med. 17, (2011) Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial Morgane Rolland, Sodsai Tovanabutra, Allan C decamp, Nicole Frahm, Peter B Gilbert, Eric Sanders-Buell, Laura Heath, Craig A Magaret, Meera Bose, Andrea Bradfield, Annemarie O Sullivan, Jacqueline Crossler, Teresa Jones, Marty Nau, Kim Wong, Hong Zhao, Dana N Raugi, Stephanie Sorensen, Julia N Stoddard, Brandon S Maust, Wenjie Deng, John Hural, Sheri Dubey, Nelson L Michael, John Shiver, Lawrence Corey, Fusheng Li, Steve G Self, Jerome Kim, Susan Buchbinder, Danilo R Casimiro, Michael N Robertson, Ann Duerr, M Juliana McElrath, Francine E McCutchan & James I Mullins In the version of this supplementary file originally posted online, the references were omitted. The references are now provided as of 8 March nature medicine

2 Supplementary Materials for: Genetic impact of vaccination on breakthrough HIV- 1 sequences from the Step trial Morgane Rolland 1 *, Sodsai Tovanabutra 2 *, Allan C. decamp 3 *, Nicole Frahm 3 *, Peter B. Gilbert 3, Eric Sanders-Buell 2, Laura Heath 1, Craig A. Magaret 3, Meera Bose 2, Andrea Bradfield 2, Annemarie O Sullivan 2, Jacqueline Crossler 2, Teresa Jones 2, Marty Nau 2, Kim Wong 1, Hong Zhao 1, Dana N. Raugi 1, Stephanie Sorensen 1, Julia N. Stoddard 1, Brandon S. Maust 1, Wenjie Deng 1, John Hural 3, Sheri Dubey 5, Nelson L. Michael 2, John Shiver 5, Lawrence Corey 3, Fusheng Li 3, Steve G. Self 3, Jerome Kim 2, Susan Buchbinder 4, Danilo R. Casimiro 5, Michael N. Robertson 5, Ann Duerr 3, M. Juliana McElrath 3, Francine E. McCutchan 2, and James I. Mullins 1 *contributed equally Supplement Page 1

3 Supplementary methods. Laboratory methods Plasma specimens. HIV-1 testing was done at day 1, weeks 12, 30 and 52, and every 6 months thereafter through year 4 of trial participation. Specimens were screened with an immunoassay and HIV-1 infection was confirmed with a Western blot and a plasma viral RNA assay. Earlier samples were tested to validate the timing of HIV-1 infection. After HIV-1 diagnosis, study participants were followed at weeks 1, 2, 8, 12, 26, 52 and 78. Plasma specimens from the first available RNA positive specimen were used for HIV-1 sequence amplification. HIV- 1 near full- length genome (nflg) sequencing. This study was designed to characterize HIV-1 viruses from volunteers infected before the unblinding date of October 17, Plasma specimens collected at the time of HIV-1 diagnosis were obtained from 88 study volunteers. Viral RNA extracted from plasma (QIAamp Viral RNAKit, Qiagen) was the template for cdna synthesis. cdna synthesis was done using SuperScript III Reverse Transcriptase (Invitrogen) at the University of Washington (UW), or ThermoScript RT (Invitrogen) at the Military HIV Research Program (MHRP). Oligo dt was used to prime cdna synthesis in the UW studies and HIV-specific primers - either JL68R (5 -CTTCTTCCTGCCATAGGAGATGCCTAAG-3 ) or UNINEF-7 (5 -GCACTCAAGGCAAGCTTTATTGAGGCTT-3 )- were used to prime cdna synthesis at MHRP. nflg or half genomes were amplified by nested PCR following endpointdilution of cdna templates (Expand polymerase) according to methods described in Rousseau et al. 1. PCR products derived from single amplifiable viral genome templates were gel purified and sequenced directly 2,3. Sequences have been deposited in GenBank under accession numbers XXXX-YYYY. Supplement Page 2

4 IFN- γ ELISpot Assays. IFN-γ ELISpot assays were performed on frozen peripheral blood mononuclear cells (PBMC) obtained 4 weeks after the second vaccination from all 27 vaccine recipients with available cells using overlapping 15-mer peptides matched to the vaccine inserts. Assays were conducted with 10 5 cells/well as previously described 4. Peptides were tested in a minipool format with each minipool containing 5 consecutive peptides; peptides corresponding to positive wells in the minipools were then tested individually. To be scored as positive, a response had to be greater than three times the mean background and at least 50 spot-forming cells per million (SFC/M) after background subtraction. Sequence Analyses. nflg nucleotide sequences were error-corrected in Sequencher (Gene Codes Corporation). All sequences from a given volunteer were then aligned manually using the MacClade program (version 4.08) 5 and each mutation rechecked in Sequencher. Sequences were normally accepted if they contained 5 ambiguous base calls. Each intra-host alignment was screened for phylogenetically informative sites, which are identified by removing all private mutations (mutations occurring only once) from the set of sequences using the InSites algorithm ( The amplification strategy was to obtain 5 to 10 nflg sequences per specimen, depending on intrahost sequence variation assessments: if 5 or more phylogenetically-informative sites were found in the first 5 nflg sequences generated per individual, then ~5 additional nflg were amplified and sequenced. There was no evidence of population stratification among the samples according to the lab where they were processed (Mean distance at MHRP and at UW, p = 0.62), or according to the number of sequences that we obtained from each individual (n = 3 to 14). Three test samples were evaluated in both labs and returned indistinguishable results. We found no evidence that there was a bias due to the time of infection, when categorizing individuals as seronegative or as in early HIV-1 infection (2-tailed Fisher s exact test comparing vaccine and placebo: p = 0.17). Supplement Page 3

5 Coding sequences were extracted from alignments using Gene Cutter ( and multisubject alignments were created using MUSCLE 6, as implemented in Seaview 7. Codonbased alignments of individual genes along with the translated protein sequence output were manually refined as needed in MacClade. Phylogenetic Analyses. Maximum-likelihood phylogenetic trees were reconstructed by estimating the GTR + I + G nucleotide substitution model using PhyML (version 3.0) 8 implemented in DIVEIN 9 ( Gag, Pol and Nef trees included all founder sequence(s) for all volunteers as well as the MRKAd5 HIV-1 Gag/Pol/Nef (CAM-1 gag, IIIB pol, JRFL nef) vaccine, HXB2 and CON_B04 sequences. Based on nucleotide sequences from gag, pol and nef, pairwise diversity and tree-based divergence measures were calculated from the most-recent common ancestor sequence, the Step vaccine insert sequences, HXB2 and the HIV-1 B consensus Based on phylogenetic analyses and inspection of nflg sequences, we estimated the number of founder variants for each subject and deduced the consensus sequences that corresponded to each founder virus (Supplementary Table 1). To identify multiple founders, groups of at least two sequences each must have a number of shared polymorphisms not shared with the remaining group(s). This number ranged from 1-4 in the current study. A gag tree based on all nucleotide sequences (Fig. 1) is shown along with an env nucleotide tree based on all-volunteerderived env nucleotide sequences and circulating sequences from the US, Canada and Peru, where all infections in our dataset occurred (Supplementary Fig. 1). CTL Epitope Prediction. Known HIV-1 epitopes were included and potential CTL epitopes were predicted using 2 methods: NetMHC 10 ( and Epipred 11 ( NetMHC predicts binding of peptides to 4-digit HLA alleles; the software discriminates on the basis of quantitative peptide Supplement Page 4

6 MHC binding data and discerns strong and weak binders. We accepted known epitopes reported at the Los Alamos National Laboratory HIV database (HIVDB) as well as HIVDB-variant epitopes that had identical HXB2 coordinates and were strong or weak binders in the reference and founder sequences based on each individual s HLA. Epipred identifies known and potential HIV-1 CTL epitope motifs using 2-digit HLA information. In addition to the known HLArestricted epitopes previously reported at LANL, we accepted all epitope motifs with a posterior probability of >0.8. HLA-specific epitopes were predicted in all HIV-1 proteins derived from the volunteers sequences and in the corresponding consensus founder sequences, based on each individual s HLA genotype. Protein sequences from 3 volunteers were excluded from this analysis those corresponding to one individual infected with a non-b subtype virus (CRF02_AG recombinant), the sole female infected volunteer in the study group, and one individual for whom HLA-genotyping information was not available. A separate set of analyses were done while excluding one or the other of two subjects with genetically very similar viruses. Results were the same whether or not both sequences were included, so results from analyses in which both subject s sequences were used are reported. Epitopes were also identified in the MRKAd5 HIV-1 Gag/Pol/Nef vaccine sequences and in all proteins from HXB2 and from the subtype B 2004 consensus sequence (CON_B04) (available at the HIVDB, Sieve analyses. Two types of sieve analyses were performed: global sieve analyses are based on summary measures of distances between founder sequences and a reference sequence (e.g., the MRK Ad5 insert) to compare vaccine and placebo-recipients, whereas local sieve analyses scan the proteome and evaluate amino acid (AA) sites individually or as a set of sites (e.g., AA sites spanning an epitope s length) that discriminate whether a founder sequence is from the vaccine or placebo group. Supplement Page 5

7 Statistical methods for global summary distance sieve analysis. Each founder sequence was assigned a value summarizing its distance to a reference sequence. The MRKAd5 HIV-1 Gag/Pol/Nef insert sequences were the main reference sequences. HXB2 and CON_B04 were also used as references, because they enabled analyses for HIV-1 regions outside the Gag, Pol, and Nef proteins represented in the MRKAd5 vaccine construct. For each distance measure defined below, its distribution was compared between infected vaccine and infected placebo recipients. Hypothesis testing for these comparisons was performed using either the consensus founder variant(s) for each subject or all individual sequences. Whole- Protein Tree- based Sieve Analysis. Tree-based distances between all protein sequences were calculated using an HIV-specific substitution model of protein evolution 12. This HIV-1- specific-10% inter-subject similarity scoring matrix (HIV-10) up-weights substitutions depending on the evolutionary cost they incur and is available in PhyML 8 within HyPhy 13. Treebased distances were extracted from the trees using the NewickTermBranch algorithm ( For each individual, the average of the distances between the MRKAd5 HIV-1 sequence and each founder sequence was computed, and these average distances were compared between the vaccine and placebo groups using a Wilcoxon/Mann-Whitney test. To address the issue of controlling false positive errors in the presence of multiplicity of analyses due to multiple insert proteins, we first assessed significance of the results for Gag-Pol-Nef combined, and, if significant, considered the p-values for the component proteins Gag, Pol, and Nef. Global sieve effects restricted to CTL epitopes from founder variants. We considered measures of distance between the consensus founder sequence(s) of a subject and a reference sequence. Genetic distances were calculated using the HIV-10 model of evolution as described above 12. Supplement Page 6

8 Two T cell epitope-based distance measures were used: the CTL epitope and K-mer distances. The CTL epitope distance is defined in three steps. First, T cell epitopes (among linear peptides of length 8, 9, 10, or 11) were identified as described above in volunteers and reference sequences, based on each individual HLA type. Second, we focused on the subset of epitopes that were shared (i.e., had the same HXB2 positions) in the volunteers sequence(s) and the reference sequence (with at most 2 AA differences). Third, we computed all pairwise distances between these epitopes using the HIV-10 evolutionary model in PhyML. The CTL epitope distance was then defined for each subject as the average of the different epitope-specific pairwise distances. If there were no known or highly likely epitopes in either sequence, then the distance could not be defined and the subject's information was not used. A sieve effect on CTL epitope distances is interpreted in terms of greater epitope distances among AA changes that preserve epitopes. Results for these distances are reported in Fig. 2. If an epitope in a founder sequence is mutated relative to the vaccine-insert sequence such that the mutated form is no longer recognized as an epitope, this peptide is not included in the CTL epitope distances. Thus, we developed a second measure to capture effects where founder sequences have mutations that preclude their identification as epitopes: the K-mer distance or percent epitope mismatch distance, which is based on epitopes in the reference sequence, regardless of whether corresponding peptides in the volunteers sequence(s) may be epitopes. Using Epipred, the first step in computing the percent epitope mismatch distance is to compute the nonparametric maximum likelihood estimate (NPMLE) of the number of peptides shared between the reference sequence and the consensus founder sequence(s), defined as the sum of estimated epitope-probabilities across all 8, 9, 10, 11-mers in the reference sequence that are exactly matched in the founder sequence(s). Then, the distance is the NPMLE of the percent of mismatched peptides, defined as one minus the ratio of the NPMLE of the number of shared peptides (computed in the first step) and the NPMLE of the number of peptides in the reference Supplement Page 7

9 sequence. The denominator NPMLE is computed as the sum across all 8, 9, 10, 11-mers in the reference sequence of the estimated epitope-probabilities. Hence, K-mer distances account for the whole distribution of peptides rather than the limited set of epitopes with a cut-off of 0.8 probability. Because the NetMHC software returns results of non-binder, weak binder, or strong binder, we defined the distance as the estimated percent of mismatched epitopes, the latter defined as the number of weak or strong binding 8, 9, 10, or 11-mers in the reference sequence that mismatch the corresponding peptide in the founder sequence(s). For each distance measure, a Wilcoxon rank sum test (equivalently the Mann-Whitney test) with exact 2-sided p-value was used to test for a different distribution in the majority-consensus sequence summary measures between the infected vaccine and placebo groups. Global sieve effects restricted to CTL epitopes in all individual sequences. Parallel to the above distances, we defined two epitope-based distances that account for all individual founder sequences. The CTL epitope distance was defined as above, except that in the first step, T cell epitopes were identified in all of the subject's founder sequences in addition to the reference sequence. The K-mer distance is defined as above, except that the numerator of the ratio is the estimated number of peptides shared between the reference sequence and all of the subject s founder sequences. Hypothesis testing was the same as described above. Local sieve analysis statistical methods. Individual sites and K-mers were evaluated as signatures wherein there are different AA distributions for vaccine versus placebo recipients. As the following analyses did not take HLA or predicted epitope sites into account, they were conducted on the 66 subjects used for the analyses above plus the one individual for whom HLA data was not available. Signature positions in founder variants. For each AA site in Gag, Pol, and Nef, we compared the rate of AA mismatch to the MRKAd5 insert residue found in the infected vaccine group to this Supplement Page 8

10 rate in the infected placebo group. The same analysis was done for Env with HXB2 as reference. We used the t-statistic numerator-type statistic of Gilbert, Wu, and Jobes 14, and their permutation procedure, to compute an unadjusted p-value for each position. Positions with insufficient AA variability to potentially discover a significant result were screened out using Tarone s procedure 15, which reduced the number of sites for analysis to 97 (from 542) for Gag; 76 (from 885) for Pol; 82 (from 236) for Nef; and 322 (from 957) for Env. To account for the multiplicity of hypothesis tests, we applied, separately for each protein, the Holm-Bonferroni multiplicity adjustment procedure with Tarone s 15 modification to improve power. This procedure yields family-wise error rate adjusted p-values, with a value of 0.05 indicating that the probability of any false positive signatures is below In addition, we also estimated q-values, and judged that sites with q-value < 0.20 have significant evidence of being signature sites. Signature positions in all individual sequences. In addition to the signature analyses of founder sequence(s), signature positions were evaluated using all of the individual sequences. For each position we estimated the probability of a mismatch between an individual sequence and the reference sequence (either MRKAd5 or HXB2) for the infected vaccine group and the infected placebo group. We then took the difference of these estimated probabilities as a test statistic. If a position is a signature position, the parameter estimated by this difference will be significantly different from zero. Unadjusted p-values were computed using the nonparametric bootstrap. This method provides valid and robust inferences when there are different numbers of sequences per subject, as is the case for our data. The analyses were performed on all positions for which at least four subjects had a non-consensus AA in at least one of their sequences. Adjusted p-values and q-values were computed in the same way as described above. Supplement Page 9

11 Signature K- mer peptides based on all individual sequences. For each K-mer peptide in the MRKAd5 and HXB2 reference sequences we compared the distribution of similarity scores for all of the individual sequences between the infected vaccine group and the infected placebo group. For each sequence we computed a similarity score to the reference K-mer by selecting the closest similarity score across all K-mers in that sequence. The similarity score between K- mers was computed by summing the appropriate elements of the HIV-10 matrix over the positions of the peptide. The estimated means were computed using generalized estimating equations to fit restricted-moment models (marginal mean models), using a Gaussian model with exchangeable correlation structure. K-mers of length 8, 9, 10, and 11 were evaluated, because these are the most common lengths of CD8+ T cell epitopes. All statistical tests used 2- sided p-values. Analyses were performed with R. Supplement Page 10

12 Supplementary results. Possible linked pair. A possible linked pair was identified between 2 vaccine recipients who attended the same clinic in NYC. Although no epidemiological evidence supported this transmission, these samples were collected five months apart, processed in separate labs, and this linkage was confirmed by later specimens from both individuals, which also showed intermixed viral populations. Importantly, the analyses described below were performed using sequences from both subjects or from only one or the other, with no differences noted in the results. Predicted CTL epitope sieve analysis based on Epipred results. The predicted CTL epitope sieve analyses were repeated based on epitopes identified using Epipred 11. As with NetMHC, average Epipred-derived epitopic distances to the vaccine were significantly larger for vaccine than for placebo recipients (mean vs ; p = 0.02) (Supplementary Fig. 2a). However, although epitopic distances were larger for vaccinees in both Gag (0.027 vs 0.011) and Nef (0.064 vs 0.021), the difference was only statistically significant in Nef (p = 0.03) (Supplementary Fig. 2b-d). In agreement with the NetMHC-based analyses, the signal was specific to the vaccine inserts and absent in the rest of the proteome. Distances to HXB2 and CON_B04 were larger among vaccinees in epitopes derived from Gag-Pol-Nef (p = with HXB2; p = with CON_B04) but not in the remainder of the proteome (p = 0.90 with HXB2; p = 0.60 with CON_B04). Of note, since both programs use different algorithms, the number of potential epitopes was over three times smaller with Epipred than with NetMHC (628 vs for Gag, Pol and Nef epitopes). The above analyses were conducted using consensus founder sequences from each subject. If we used the entire set of sequences, 18 additional unique epitopes (i.e., a total of 646 for Epipred) were predicted and the results were similar to those obtained with the consensus founder Supplement Page 11

13 variants (data not shown), indicating that consensus founder variants accurately reflected the viral population in the hosts due to the homogeneity among sequences in acute/early infection. K- mers sieve analysis. Comparisons based on Epipred-derived epitopes gave comparable results that did not reach significance (except for marginally significant results in Nef (p = 0.06)). Signature K- mer peptides based on all individual sequences using Epipred. First, results are reported based on epitopes that are known or highly likely (Epipred probability cut-off of 0.80) in the MRKAd5 insert sequence. For pre-screening, only K-mers different from the insert and found in at least 4 subjects were included, that is 30, 29, and 19 epitopes for Gag, Pol, and Nef, respectively. All lengths from 8 to 11 AA were tested simultaneously. However, q-values were all above 0.40, indicating that there were no convincing results (SLYNTVATL in Gag had a p-value of 0.04 but a q value of 0.55). Second, results are reported based on all 8-11-mers in the MRKAd5 insert sequence. The prescreening restricts the analysis to K-mers different from the insert and found in at least 4 subjects. This did not substantially cut down on the number of examined K-mers. Accordingly, each length 8-11 was evaluated separately. There were a large number of K-mers with q-values less than 0.20 (Supplementary Table 2). Greater epitope-distances for vaccine recipient sequences were found in K-mers spanning Gag83-90, Gag , and Gag , Nef 20-27, and Nef , while smaller epitope-distances were found in K-mers spanning Gag Supportive analysis of the per- protocol efficacy take population. If there is a sieve effect, it could be present/strongest if the analyses were restricted to infected vaccine recipients who are members of the protocol-specified per-protocol efficacy (PP) population and who had at least one positive CD8+ T cell response (ELISpot testing) at week 8 post-randomization. Based on the protocol definitions, a subject is part of the PP population if: a) he was HIV negative at baseline and until and on the Week 12 visit; b) he received the first two scheduled vaccinations with the Supplement Page 12

14 correct dosage, with the second vaccination within the acceptable window; and c) he received the correct clinical material for all vaccinations. A positive CD8+ T cell response is an ELISpot result 55 SFCs/10 6 PBMC and 4-fold over media-control for any of the four vaccine peptide pools studied (Gag, Pol-1, Pol-2, Nef). The comparative sequence analyses were repeated in the per-protocol efficacy take (PP take) population, which included 19 (of 39) vaccinees and 17 (of 27) placebo recipients. There were no apparent differences in the results for the PP take population compared to the entire population. The rank-based interaction test of Ohrvik 16 was applied, with a permutation procedure to obtain a p-value. This permutation procedure generates a null distribution of 10, test statistics computed on permuted data sets with the PP take membership status randomly shuffled within each of the infected vaccine and infected placebo groups. Interaction tests assessing a different vaccine effect on sequence distances by membership in the PP take population showed no evidence of interactions (p-values all > 0.10). Sieve analysis of distances and stratification of the cohort. There was no significant bias in the sieve analysis due to different timing of sequence sampling in vaccinees versus placebo recipients; the fraction of infected subjects with an acute-phase sample was 8 of 39 in the vaccine group versus 10 of 27 in the placebo group, excluding the one female and the CRF02_AG infected individual (p = 0.17). Considering the genetic distances between vaccine and placebo recipients in acute infection, we found that in this smaller group the distances to the MRKAd5 inserts were not significantly different between vaccine and placebo recipients (mean vs ; p = ). When considering the genetic distances between vaccine and placebo recipients who were not in acute infection, we found that the distances to the MRKAd5 inserts were significantly higher in vaccine than placebo recipients when all epitopes or epitopes from Gag were tested (Gag: mean vs ; p = ), conforming to results reported with the entire dataset. Supplement Page 13

15 Supplementary Figure Legends Supplementary Fig. 1. Maximum- Likelihood phylogenetic tree using env nucleotide sequences. Four hundred and fifty nine HIV-1 env sequences from infected trial participants are shown, with placebo recipient taxa shown in blue and vaccine recipient taxa in red. Also included are contemporary sequences from the US (in black), from Canada (in green) and from Peru (in yellow). Likewise, sequences isolated from trial participants in Canada are highlighted in green, and sequences isolated from individuals in Peru are highlighted in yellow. The inset represents sequences from two individuals with closely related viruses. Supplementary Fig. 2. Epitope- specific protein distances between epitopes from founder sequences and the MRKAd5 insert sequence (Epipred). Comparison of epitopic distances across treatment assignment based on Epipred predictions using Gag, Pol and Nef epitopes combined (a), or separately (b-d). Supplementary Fig. 3. Percent epitope mismatch distances. Comparison of percent mismatch distances across treatment assignments based on Gag, Pol and Nef epitopes combined (a), or separately (b-d) (using MRKAd5 as reference), and on the combined vaccine insert derived peptides (Gag, Pol and Nef) (e) or of the combined non-insert-derived peptides (Env, Rev, Tat, Vif, Vpr, and Vpu epitopes) (f) (using HXB2 as reference). Supplementary Fig. 4. Relationship between protein distances and the number or timg of immunization(s). Epitopic distances (Gag, Pol, Nef combined; NetMHC derived) are plotted for vaccinees and placebos against: (a) the number of immunizations prior to infection, and; (b) the number of days between the last immunization and infection. The solid and dotted lines are regression lines through the vaccinees and placebo recipients, respectively. Supplement Page 14

16 Supplementary Fig. 5. Summary of sequence analyses (Epipred) and IFN- g ELISpot data from Gag (a,b), Nef (c,d) and Pol (e,f). For each protein, the upper panel (a,c,e) represents the location of predicted epitopes based on Epipred in vaccine and placebo groups (light and medium gray columns, respectively), amino acid signature sites (dashed lines), and signature k-mers (horizontal bars). The lower panel (b,d,f) corresponds to IFN-γ ELISpot responses detected in 21 vaccine recipients prior to infection. 15mer peptides overlapping by 11 amino acids were used to assess responses. The region covered by each responsive peptide is shown as the box width, whereas the number of subjects reacting with that peptide is given by the box height. Supplement Page 15

17 Supplementary Table 1. Genetic characteristics of founder variant(s). Supplement Page 16

18 1 2 3 a b c d Number of additional half-genome sequences. Number of consensus founder variant(s). Nucleotide pairwise sequence diversity (Mean and minimum and maximum). Possible transmission linkage with another volunteer. Female volunteer. Recombinant CRF02_AG isolate. No HLA- genotype data available. Supplement Page 17

19 Supplementary Table 2. Signature analysis of K- mers (K=8, 9, 10, or 11) that are known or possible (Epipred posterior probability > 0.0) in the MRKAd5 insert sequence. Gag 8-mers Position1 Estimate p-value q-value Pol 8-mers: None evaluable Nef 8-mers Position1 Estimate p-value q-value Gag 9-mers Position1 Estimate p-value q-value Pol 9-mers: None evaluable Supplement Page 18

20 Nef 9-mers Position1 Estimate p-value q-value Gag 10-mers Position1 Estimate p-value q-value Pol 10-mers: None evaluable Nef 10-mers: Position1 Estimate p-value q-value Gag 11-mers Position1 Estimate p-value q-value Pol 11-mers: None evaluable Nef 11-mers: None evaluable Supplement Page 19

21

22 Supplementary Fig. 2 a 0.30 p=0.02 b 0.30 p=0.19 c 0.30 p=0.83 d 0.30 p= Protein distance Placebo Vaccine n = 26 n = 39 Placebo Vaccine n = 25 n = 39 Placebo Vaccine n = 26 n = 38 Placebo Vaccine n = 22 n = 33

23 Supplementary Fig. 3 a 100 P = 0.01 b 100 P < c 100 P = 0.75 d 100 P = 0.50 Percent epitope mismatch Placebo Vaccine n = 26 n = 39 Placebo Vaccine n = 25 n = 39 Placebo Vaccine n = 25 n = 39 Placebo Vaccine n = 23 n = 38 e 100 P = 0.06 f 100 P = 0.76 Percent epitope mismatch Placebo Vaccine n = 26 n = 39 Placebo Vaccine n = 26 n = 39

24 Supplementary Fig. 4 a b Protein distance 0.12 Placebo Vaccine Immunizations Days

25 Supplementary Fig. 5 a Placebo Vaccine b e Placebo Vaccine f a HXB2 position c d

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