High Multiplicity Infection by HIV-1 in Men Who Have Sex with Men

Abstract

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5' and 3' half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3-6 days before symptom onset and 14-17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized.

Figure 1. Neighbor-joining (NJ) tree of full-length HIV-1 gp160 env sequences from 28 acutely infected subjects and 2 chronically infected sexual partners.

Two chronic-to-acute (LACU9000 to HOBR0961 and AD18 to AD17) and two acute-to-acute (AD77 to AD75 and AD83 to 04013240) transmissions were documented, with donor sequences shown in blue and recipient sequences shown in green. Individual sequences with APOBEC G-to-A hypermutation were excluded from the analysis. Bootstrap values (≥70%) are shown for intra-subject clusters, partner pairs, and additional sequences with evidence of epidemiologic linkage. The horizontal scale bar represents 1.0% genetic distance.

Figure 2. NJ trees and Highlighter plots of HIV-1 env diversity.

Full-length gp160 env sequences from four subjects are depicted by NJ tree phylogenies and by Highlighter, a sequence visualization tool that allows tracing of common ancestry between sequences based on individual nucleotide polymorphisms . Sequences from subject 04013440 (A) showed productive infection by a single virus, from subject 04013211 (B) infection by two closely related viruses, from subject 04013383 (C) infection by two distantly related viruses, and from subject 04013448 (D) infection by four viruses with inter-lineage recombinants denoted by orange symbols in the NJ tree. The horizontal scale bar represents genetic distance.

Figure 3. Time course of HIV-1 exposure, symptom onset, viral kinetics, and initiation of antiretroviral therapy in subject AD17.

ARS, acute retroviral syndrome. HAART, highly active antiretroviral therapy. For purposes of mathematical modeling, a plasma virus load of 10 RNA molecules per milliliter was estimated at day 6.

Figure 4. NJ trees and Highlighter plots of HIV-1 diversity in 5′ and 3′ half genomes in subject AD17.

Sequences in (A) and (B) were derived from overlapping 5′ and 3′ SGA amplicons spanning the complete viral genome. Blue symbols represent sequences from day 14 and green symbols day 17 as depicted in Fig. 3 . Solid ovals represent sequences derived from plasma vRNA and solid triangles represent sequences derived from peripheral blood mononuclear cell DNA.

Figure 5. Molecular cloning and biological analysis of the transmitted/founder virus from subject AD17.

(A) Cloning strategy and genome organization of the transmitted/founder HIV-1 provirus pAD17.1. (B) Replication of pAD17.1 virus in activated primary human CD4+ lymphocytes (left panel) and monocyte-derived macrophages (right panel) from the same normal blood donor. Results were replicated three times in cells from different donors, each time showing efficient replication of pAD17.1 virus in CD4+ T cells but not in macrophages. (C) pAD17.1 virus infection of JC53BL-13 cells assessed by luciferase expression in the absence or presence of the CXCR4 inhibitor AMD3100 (1.2 uM) or the CCR5 inhibitor TAK779 (10 uM) or both. Results from four experiments are expressed as infectivity (mean ±1 S.D.) relative to control wells lacking coreceptor inhibitor: NL4.3 is X4 tropic, YU2 is R5 tropic, WEAU1.60 is dual R5/X4 tropic, and pAD17.1 is R5 tropic.

Figure 6. NJ tree and Highlighter plot of HIV-1 env diversity in subject 04013171.

Sequences emanating from ten transmitted/founder viruses are color-coded and identified as variants 1–10. Inter-lineage recombinants are depicted in orange. The horizontal scale bar represents genetic distance.

Figure 7. NJ trees and Highlighter plots of HIV-1 diversity in env gp41, env gp160 and 3′ half genomes in subject 7010100068.

Seventy-two 3′ half genomes were amplified and sequenced and segments of each are represented in panels A (env gp41), B (env gp160) and C (3′ half genome). The progeny of transmitted/founder viruses are color-coded and identifiable as discrete ‘rakes’ of identical or nearly identical sequences (variants 1–7) in the env gp41 segments shown in panel A. The relatedness of sequences emanating from the seven transmitted/founder viruses is progressively obscured in panels B and C as longer segments are compared due to inter-lineage recombination. The horizontal scale bar represents genetic distance.