From 1984 through November 2004, a total of 6973 men were enrolled in the MACS, including 615 seroconverters, of whom 57 were identified as having a positive plasma HIV-1 RNA load at their last seronegative visit by systematic testing of the last seronegative visit of all seroconverters who had specimens available. Forty-five of the 57 subjects had RNA-positive and antibody-negative (RNA+Ab−) blood samples available for further analyses. We confirmed viral RNA positivity at the RNA+Ab− visit for 38 of the 45 subjects (). In the 7 subjects for whom we could not confirm the presence of viral RNA (with a sensitivity of ~ 1–10 copies/PCR, or less than ~40–80 copies/mL of plasma; see Methods), the plasma viral loads determined previously by the Amplicor HIV-1 RNA assay (versions 1.0 and 1.5; cutoff of <400 copies/mL) were between 423 and 1029 copies/mL (whether these represent false-positive results or subsequent sample degradation occurring before our analysis could not be determined). These subjects were excluded from subsequent analyses. Fourteen (36.8%) of the 38 subjects had plasma viral loads >500,000 copies/mL at the RNA+Ab− visit, suggesting that samples were obtained from them near the time of peak viremia of primary infection.
Characteristics of the Multicenter AIDS Cohort Study HIV RNA–positive and antibody-negative cohort.
Each of the 38 confirmed RNA+Ab− subjects was shown by viral phylogenetic analysis to be infected with HIV-1 subtype B. Each subject’s sequence population was monophyletic; hence, there was no evidence of dual infection (). Nor was there any evidence of clustering of intersubject sequences; thus, there was no evidence of close epidemiologic linkages between subjects (). Viral population heterogeneity of the env V1–V5 region was examined in the 37 subjects for whom env V1–V5 sequences were available (mean, 15.2 sequences/subject; range, 11–19). A wide distribution of intrasubject diversities was observed at both the amino acid (median, 0.86%; range, 0.2%–5.6%) and nucleotide levels ( and ). There was no significant correlation between viral load at the seronegative visit and envelope V1–V5 region diversity (nucleotide or amino acid; P = .70 and P = .47, respectively).
Figure 1 A, Maximum-likelihood phylogenetic tree (gap stripped; implemented in PhyML) of the envelope V1–V5 region (567 independent clones from 37 subjects) at the HIV RNA-positive and antibody-negative visit. Median pairwise nucleotide diversity was 0.4% (more ...)
Distributions of intrasubject mean amino acid diversity.
The number of unique variants replicating in, and potentially transmitted to, each subject was estimated by examining phylogenetically informative sites (nucleotide changes shared by 2 or more sequences). Six subjects (16%) had clonal populations (1 variant; i.e., no informative sites) and 17 (46%) had 0 or 1 informative site (1 or 2 unique variants), clearly suggesting outgrowth from a single unique variant. In contrast, 9 (24%) subjects had sequences with 7 or more informative sites (4–13 unique variants, counting an insertion or deletion of any length as an informative site), suggesting that multiple variants were likely to have been transmitted in these cases. In addition, 12 subjects (32%) had evidence of recombination between unique viral variants ( and ).
Unique envelope gp120 viral variants in 37 subjects.
One (0.17%) of 587 V3 loop sequences from the 38 subjects had a genotype consistent with SI/X4 tropism (this sequence had a positive SI PSSM [syncytium-inducing position-specific scoring matrix] score but no canonical 11/25 mutation); thus, nearly all transmitted viruses were CCR5 tropic ().
Envelope V3 loop amino acid variation.
We evaluated V1–V2 loop length variation and PNLGS in the subset of 487 sequences with open reading frames (ORFs) from 37 subjects (). The median length was 66 aa (range, 58–93 aa), with length variation detected in 7 subjects. Thirty-three subjects (89%) had variation in the number of PNLGS in this region. A mean of 5.6 PNLGS (range, 2–9) were found, which was strongly correlated with loop length (adjusted R2 = 0.59; P < .0001).
Histogram of V1–V2 amino acid loop length vs. potential N-linked glycosylation sites (PNLGS).
Analysis of PNLGS in the 447 env V1–V5 sequences with ORFs also demonstrated a wide range of variation (data not shown). Only 4 (11%) of 37 subjects had the same numbers of PNLGS in all clones. However, there was no significant correlation between PNLGS and envelope V1–V5 nucleotide or amino acid diversity (P = .4 and P = .8, respectively).
We found no significant correlations between any of the aforementioned early viral genetic parameters (i.e., diversity, number of unique viral variants, divergence from the estimated ANC, V1–V2 loop length, PNLGS) and any HIV disease outcome measure or surrogate marker evaluated (i.e., set-point viral load and time from seroconversion to clinical AIDS, death, or CD4 cell count <200 cells/μL), self-reported risk factors for mode of transmission, or history of sexually transmitted infections in the 6 months preceding their visit (P > .05, for all comparisons). However, as expected, there was a significant correlation between set-point viral load at ~1 year after seroconversion and time from seroconversion to AIDS (adjusted R2 = 0.3; P = .009)