In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection

Abstract

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.

FIG. 1.

Recombinant structure of the initial strain. A virtually complete HIV-1 genome sequence from participant 123 was analyzed by bootscanning with subtype A, C, D, and J reference sequences. The top panel shows the maximum parsimony bootstrap values for subtype associations across the genome. Subregions of different subtypes (I through XIII) were excised and analyzed with reference sequences of all subtypes using neighbor joining with the bootstrap. The position of the 123 strain (open circle) and bootstrap support for subtype assignments are indicated. The boundaries of each segment are delineated according to positions in reference strain HXB2. The diagram in the center summarizes the recombinant structure.

FIG. 2.

Envelope sequences before and after superinfection. (A) Plasmas from visits 1 and 3 were the source of complete envelope sequences, represented by open and filled circles, respectively. The open circle indicated by the arrow is the corresponding sequence from the complete genome shown in Fig. 1. For visit 3, two related but distinct variants were found, i.e., variants 1 and 2. The tree was constructed by neighbor joining with the bootstrap, and the scale bar represents a 10% difference. Bootstrap values at important nodes are indicated. A subtype J sequence was designated as the outgroup root. (B) Recombinant structures of the envelopes present at visit 1 and 3 are shown, with breakpoints numbered according to positions in strain HXB2. The center panel compares regions of the same subtype in the two recombinants, using selected clones from visits 1 and 3 (open and filled circles, respectively). Visit 1 and visit 3 strains clustered separately within their subtype, indicating different lineages, which are represented by shading or crosshatching, respectively.

FIG. 3.

Temporal evolution of gp41/nef recombinant forms in plasma and PBMC. (A) The source of gp41/nef recombinant sequences was RNA extracted from plasma. The phylogenetic tree was constructed by neighbor joining, including gp41/nef sequences from visits 0, 1, 2, and 3 (open triangles, open circles, open squares, and closed circles, respectively) and reference sequences of relevant subtypes. Bar, 10% difference. Bootstrap values at important nodes are indicated. The different molecular forms are indicated by Roman numerals. (B) Structure of recombinant forms with breakpoints indicated (HXB2 numbering). For the subtype A segments, shading indicates regions from the initial strain; crosshatched regions are from the superinfecting strain. (C) The source of gp41/nef recombinant sequences was DNA extracted from PBMC. The phylogenetic tree was constructed by neighbor joining, including gp41/nef sequences from visits 1, 2, 3, 4, 7, and 10 (open circles, open squares, closed circles, closed triangles, closed squares, and closed diamonds, respectively) and reference sequences of relevant subtypes. Bar, 10% difference. Bootstrap values at important nodes are indicated. Stars represent sequences from plasma (panel A) representing forms I, III, IV, V, and VI, respectively. Form II was found only in PBMC.

FIG. 4.

Consistency of sampling and temporal fluctuation of molecular forms in plasma and PBMC. The compositions of the viral quasispecies in plasma and PBMC from visit 0 through visit 10 are shown, comparing sequences obtained from plasma RNA with those obtained from PBMC DNA. Each pie chart represents approximately 20 sequences. The legend indicates the colors used to represent the various molecular forms. Form I is the initial form, and form II is the superinfecting strain; all other forms are recombinants of these. At visit 3, the PBMC compartment was independently sampled (S1 and S2) to compare the proportions of forms recovered.

FIG. 5.

Evolution of the viral quasispecies: the gag gene. (A) Phylogenetic tree for sequences from DNAs extracted from PBMC. The phylogenetic tree was constructed by neighbor joining, including gag sequences from visits 1, 2, 3, 4, and 7 (open circles, open squares, closed circles, closed triangles, and closed squares, respectively) and reference sequences of relevant subtypes. Bar, 10% difference. Bootstrap values at important nodes are indicated, and molecular forms are identified by Roman numerals. (B) Structure of recombinant forms. The subtype A portions are from two lineages, indicated by shading (initial strain) or crosshatching (superinfecting strain). The numbering of breakpoints is according to positions in HXB2. The diagram at the bottom indicates the portion of gag that was sequenced, from codon 43 of p17 up to 15 codons before the end of p6.

FIG. 6.

Detection of initial and superinfecting strains in serial PBMC and plasma samples from participant 123 using strain-specific PCRs. (Left) DNAs from PBMC from the indicated visits were amplified in the first round with universal primers and in the second round with strain-specific primers, either for gag or for gp41. PCR products were separated in agarose gels and stained with ethidium bromide. (Right) Similarly, nested PCR was applied to RNAs from plasma samples by incorporation of an initial reverse transcription step. Boxes highlight the absence of the superinfecting strain before visit 3.

FIG. 7.

Contrasting dynamics in gag and env of viral quasispecies. (A) Fluctuating proportions of molecular forms of gag and gp41/nef sampled from PBMC after superinfection. Each pie chart represents approximately 20 sequences. I, initial strain; S, deduced superinfecting strain; R, recombinant between initial and superinfecting strains. (B) Percentages of sequences that represented initial, superinfecting, and new recombinant strains (blue, magenta, and light blue, respectively) at each visit in gag and gp41/nef analyses. (C) Relative timing of initial infection, seroconversion, attainment of viral load set point, superinfection, and detection of new recombinant forms from the seronegative window period through 30 months of follow-up of participant 123.