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Clinical Trial
. 2018 Nov 27;115(48):E11341-E11348.
doi: 10.1073/pnas.1813512115. Epub 2018 Nov 12.

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption

Ching-Lan Lu  1 , Joy A Pai  1 , Lilian Nogueira  1 , Pilar Mendoza  1 , Henning Gruell  2   3   4 , Thiago Y Oliveira  1 , John Barton  5 , Julio C C Lorenzi  1 , Yehuda Z Cohen  1 , Lillian B Cohn  1 , Florian Klein  2   4   6 , Marina Caskey  1 , Michel C Nussenzweig  7   8 , Mila Jankovic  1
Affiliations
Clinical Trial

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption

Ching-Lan Lu et al. Proc Natl Acad Sci U S A. .

Abstract

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.

Keywords: HIV; analytical treatment interruption; latent reservoir; sequencing.

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Conflict of interest statement

Conflict of interest statement: There are patents on 3BNC117 (PTC/US2012/038400) and 10-1074 (PTC/US2013/065696) that list Michel Nussenzweig as an inventor.

Figures

Fig. 1.
Fig. 1.
Quantitative analysis of the latent reservoir during treatment interruption. (A) Study design. Green arrows indicate combination bNAb infusion. Black arrows indicate the time points that were sampled. (B) NFL HIV-1 genome sequencing strategy (11). All viruses that had deletion in env were excluded from further analysis. (C) Comparison of reservoir measurements. Graph shows frequency per million CD4+ T cells at the preinfusion (wk-2) and week-12 (wk12) time points: gag+ proviruses (gag), env+ proviruses (env), near full-size proviruses (near full size) (Left), intact proviruses (intact) (Middle), and inducible proviruses (Q2VOA) (17) (Right). Each dot represents a different participant. Horizontal bars indicate median values. Statistical significance was determined using two-tailed Mann–Whitney U test. (D) Pearson correlation between frequency of intact proviruses and other reservoir measurements at the preinfusion (wk-2) (circles) and wk12 (triangles) time points. Participant 9254 was excluded from the quantitative analysis because of inadequate sample availability.
Fig. 2.
Fig. 2.
Sequence analysis of near full-size proviruses. (A) Pie charts summarize sequence analysis at the preinfusion (wk-2) and week 12 (wk12) time points. The number in the middle of the pie represents the number of near full-size proviruses sequenced. Pie slices depict the proportion of sequences for each participant that were intact or had different lethal defects, including premature stop codons mediated by hypermutation, single nucleotide indels, nonsense mutations, packaging signal (ψ) deletions, and MSD site mutations. (B) Scatterplot showing the frequency and location of stop codons mediated by hypermutation (red circles) or by other mechanisms (green circles) (Upper) and indels (blue squares) (Lower). The location of stop codons and indels was determined using HXB2 genome as reference. Frequency is expressed as percentage of all defective sequences.
Fig. 3.
Fig. 3.
Qualitative analysis of the circulating latent reservoir. Pie charts show the clonal distribution of env sequences derived from Q2VOA (17) or NFL sequencing for each participant at the preinfusion (wk-2) and week 12 (wk12) time points. The number in the middle indicates the total number of env sequences analyzed. White slices represent unique sequences isolated only once across both time points from both Q2VOA and NFL sequencing (singles), and colored slices represent identical sequences that appear more than once (clones). The colors of the slices represent identical sequences found in Q2VOA and in NFL. Blue arrows indicate clones that show significant differences between the time points (17).
Fig. 4.
Fig. 4.
Comparisons of the circulating latent reservoir and rebound viruses. (A) Diagrams show the overlap between env sequences obtained from Q2VOA (blue), NFL sequencing (yellow), and rebound plasma SGA or PBMC outgrowth culture (red) (17). The intersection of the NFL and Q2VOA circles represents the number of identical env sequences belonging to clones obtained by both methods. Sequences obtained from the preinfusion and week 12 were combined. (B) Maximum likelihood phylogenetic trees of env sequences obtained from Q2VOA at preinfusion (green) and week 12 (blue), NFL at the preinfusion (purple) and week 12 (orange), and rebound viruses from SGA or outgrowth cultures (red) from two representative participants. Additional participants are shown in SI Appendix, Fig. S4. (C) Circos plots depicting recombination events between env sequences derived from Q2VOA at preinfusion (blue), intact NFL at preinfusion (green), and rebound plasma SGA (red). Gray lines show the contribution of parent sequences to recombinant sequences. Clonal env sequences were collapsed and represented as one virus. The thickness of the black outer bars represents the number of sequences obtained from that particular clone. Asterisks indicate the same env sequences between intact NFL and Q2VOA sequences.
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