HIV-1 acute infection: evidence for selection? ROLLAND Morgane University of Washington
Cohort & data S6 S5 T4 S4 T2 S2 T1 S1 S7 T3 DPS (days post symptoms) 3 (Fiebig I) 7 (Fiebig I) 13 (Fiebig V) 14 (Fiebig I) 3 (Fiebig I) 8 (Fiebig I) 3 (Fiebig I) Eleven HIV-1 subtype B infected individuals from the Seattle Primary Infection Cohort (SeaPIC) Near full length genomes (~9,1 Kb) were amplified from plasma by singletemplate PCR, cloned and sequenced 495 HIV-1 subtype B genomes Eight individuals were enrolled during acute HIV-1 infection Four transmission pairs 25 (Fiebig V) HXB2 acute infection
A single founder variant typically establishes HIV-1 infection S5 T2 S2 Phylogenetic tree with sequences from the first time point after infection in recipients: A single HIV-1 variant established infection in 8 of the 9 MSM HXB2 S6 T1 Li et al. PLoS Pathogens. 2010. S1 S7 S4 T4 T3 MSM: 31/50 = 62% HSX: 141/175 subjects = 81% (p = 0.008) Other MSM cohorts: 77% of 64 Step vaccine trial volunteers infected with subtype B (Rolland, Stovanabutra, decamp et al., submitted) 86% of 37 MSM evaluated preseroconversion in the MACS cohort (Gottlieb et al., 2008)
Comparison of sequences from transmission pairs HXB2 S2, sampled at 3 dps Sample from T2 on same day, sampled at 25 dps Sample from T3 8 days later S4, sampled at 13 dps Sample from T4 10 days later S1, sampled at 8 dps Sample from T1 17 days later S1 S2 T2, 14 dps S4 T3, 9 yrs T4, 9 yrs T1, 10 yrs Restriction to a single founder not due to lack of variation in transmitter Comparison between the genome sequences from the transmitter and genomes from visit 1 in the recipient No exact match at the genome level; exact match in gag or pol Closest match: : 98.31% identity to a genome from T3 S4: 98.89% identity to a genome from T4 S1: 99.28% identity to a genome from T1 S2: 99.82% identity to a genome from T2
Comparison to the consensus from the transmitter S2, sampled at 3 dps Sample from T2 on same day S2 T2, 14 dps T3, 9 yrs Divergence from transmitter consensus 0.163 0.154 p = 0.84, sampled at 25 dps Sample from T3 8 days later T2 S2 S4, sampled at 13 dps Sample from T4 10 days later S4 0.899 1.360 T3 p = 0.002 T4, 9 yrs S1, sampled at 8 dps Sample from T1 17 days later S1 0.920 T4 0.926 S4 p = 0.68 T1, 10 yrs HXB2 Consensus from transmitter sequences 1.332 T1 1.224 S1 p = 0.58
Comparison to the consensus from the transmitter Divergence from transmitter consensus 0.163 T2 0.899 1.360 T3 p = 0.68 0.920 T4 1.332 T1 p = 0.84 0.154 S2 p = 0.002 0.926 S4 p = 0.58 1.224 S1 The founder variant: did not correspond to the consensus from the transmitter seemed to be a rare variant in the transmitter, that nonetheless was within the distribution of variants found in the transmitter, except in the case of. The genomes from were significantly more divergent than any genome in the transmitter. Lack of exact match can be due to: Insufficient or non-simultaneous sampling Compartmentalization: plasma vs semen Selection for viruses better adapted to the new host
ELISpot assays with transmitter- and recipient-specific peptides Predicted epitopes Forward mutation Decrease in database frequency by >50% between: 1) the AA in the transmitter and the AA in the recipient 2) the consensus AA in the recipient and the mutated AA Reverse mutation Increase in database frequency by >50% ELISpot responses assays Hypothesis: Sites that differ between transmitter and recipient may correspond to sites under CTL pressure in the transmitter, that reverted back to a consensus-like form upon transmission to a new host One example of a CTL response directed against a Nef region in the transmitter
Longitudinal follow-up Follow-up between: S6 3 and 350 dps, 12 visits 123 genomes S5 7 and 181 dps, 8 visits 85 genomes T4, S4 HXB2 T2, S2 T1, S1 S7 T3 3 and 196 dps, 7 visits, 68 genomes 25 and 247 dps, 9 visits, 93 genomes
Diversification over time Pairwise diversity, genomes 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00-50 50 150 250 350 dps Rate of diversification, % per year 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 genome 0.42 0.53 0.65 0.67 S5 S6 S7 gag pol env, C2V5 nef 0.42 0.44 0.26 0.34 0.20 0.37 0.28 0.06 0.80 1.60 0.90 1.13 1.17 0.02 1.40 0.67 The rate of evolution varied across the genome; it was higher in env and nef. The rates seen in C2V5 are comparable to the rates reported in chronic infection (0.92%+/-0.29, Shankarappa et al., 1999)
A dip in nucleotide diversity in acute infection Pairwise diversity, based on genomes, normalized to zero at visit 1 S6 S7 S5 50 100 150 200 250 300 350 dps The pairwise nucleotide diversity decreased in the earliest time points of infection.
A dip in APOBEC3G-mediated mutations APOBEC3G S6 APOBEC3G diversity S7 Pairwise diversity, genomes Control G>A mutations S5 Diversity in S6 Diversity in S5 Diversity in S7 Diversity in APOBEC3G dps Control G>A mutations The dip in APOBEC3G-mediated mutations was parallel to the dip in nucleotide diversity Yet, the dip in APOBEC3G-induced mutations was not the cause of the dip in nucleotide diversity.
Demographic processes dominate in acute infection Viral loads (x100,000) Viral loads S6 S7 Pairwise diversity, genomes Diversity Significant deviations in neutrality tests S5 Diversity in S6 Diversity in S5 Diversity in S7 Diversity in At the genome level, neutrality tests (Tajima s D and Fu and Li s D*) showed negative deviations from the neutral model, suggesting alternative hypotheses, such as selection or demographic history: founder effects, changes in population size or purifying selection can lead to strong negative deviations. Genome-wide and gene-specific tests were congruent, thus showing the predominance of demographic processes in acute infection. The dip in diversity is likely due to the single variant-founder effect and the subsequent delay before the viral population reaches equilibrium dps
Signs of selection appear at 1-2 weeks post symptoms The earliest fixation of an AA substitution occurred at day 21 in Tat (S5) By 6 months post-infection, there were between 9 (in S6) and 18 (in S7) positively selected sites across the genome. Mutually-exclusive substitution patterns in predicted CTL epitopes More evidence of forward ( escape ) than reverse mutations: S7: 37 forward vs. 2 reverse mutations S5: 53 forward vs. 7 reverse mutations S6: 91 forward vs. 7 reverse mutations : 55 forward vs. 10 reverse mutations X X Forward mutation Reverse mutation
Conclusion HIV-1 acute infection is characterized by a founder effect with single variants establishing infection in 81 % of individuals (based on 110 MSM) The variants establishing infection in the recipient did not correspond to the consensus variant in the transmitter. Demographic processes dominated in the earliest weeks of infection Founder effect (short branch lengths to the MRCA) and multiplicity of variable sites (star-like phylogeny) led to significant negative deviations from the neutral model. Across transmission pairs and over time in the recipients, we found more evidence of forward ( escape ) than reverse mutations Mutually-exclusive substitution patterns in predicted CTL epitopes
Acknowledgments James I. Mullins Joshua T. Herbeck Seattle Primary Infection Cohort Yi Liu Sherry McLaughlin Hong Zhao Kim Wong Julia N. Stoddard Dana Raugi Stephanie Sorensen Indira Genowati Brian Birditt Angela McKay Brandon S. Maust Wenjie Deng M. Juliana McElrath John McNevin Nicole Frahm Justine E. Brown Kurt Diem Joanne D. Stekler Ann C. Collier