Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption

Basiel Cole, Laurens Lambrechts, Zoe Boyer, Ytse Noppe, Marie-Angélique De Scheerder, John-Sebastian Eden, Bram Vrancken, Timothy E Schlub, Sherry McLaughlin, Lisa M Frenkel, Sarah Palmer, Linos Vandekerckhove, Basiel Cole, Laurens Lambrechts, Zoe Boyer, Ytse Noppe, Marie-Angélique De Scheerder, John-Sebastian Eden, Bram Vrancken, Timothy E Schlub, Sherry McLaughlin, Lisa M Frenkel, Sarah Palmer, Linos Vandekerckhove

Abstract

The HIV-1 reservoir is composed of cells harboring latent proviruses that have the potential to contribute to viremia upon antiretroviral treatment (ART) interruption. While this reservoir is known to be maintained by clonal expansion of infected cells, the contribution of these cell clones to residual viremia and viral rebound remains underexplored. Here, we conducted an extensive analysis on four ART-treated individuals who underwent an analytical treatment interruption (ATI), characterizing the proviral genomes and associated integration sites of large infected clones and phylogenetically linking these to plasma viremia. We show discrepancies between different assays in their ability to assess clonal expansion. Furthermore, we demonstrate that proviruses could phylogenetically be linked to plasma virus obtained before or during an ATI. This study highlights a role for HIV-infected cell clones in the maintenance of the replication-competent reservoir and suggests that infected cell clones can directly contribute to rebound viremia upon ATI.

Trial registration: ClinicalTrials.gov NCT02641756.

Keywords: CP: Microbiology; HIV; analytical treatment interruption; antiretroviral therapy; cellular proliferation; clonal expansion; full-length HIV sequencing; integration site sequencing; latency; rebound; replication competency.

Conflict of interest statement

Declaration of interests The authors declare that no conflict of interest exists.

Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Overview of the workflow for…
Figure 1.. Overview of the workflow for HIV-1 reservoir characterization and viral loads at each time point of sample collection
(A) Workflow of HIV-1 reservoir characterization by single-genome sequencing (SGS), full-length individual proviral sequencing (FLIPS), integration site loop amplification (ISLA), and multiple displacement amplification (MDA). In a first step, potentially clonal HIV-1-infected cells were identified by SGS, FLIPS, and ISLA on lysed sorted CD4 T cell subsets. In a second step, MDA with subsequent SGS and ISLA was performed on selected sorted cell lysates. In the final step, MDA reactions containing a potentially clonal provirus were identified, and the near full-length (NFL) genome of the according provirus was amplified and sequenced. (B) Viral load (copies/mL) at each time point of sample collection for all participants. The day of analytical treatment interruption (ATI) initiation is indicated with a vertical red line. The plasma was sampled during ART (time point 1, T1), 8 to 14 days after ATI (time point 2, T2), at the first detectable viral load (time point 3, T3), and at later rebound (time point 4, T4). Note that T1 is not shown to scale. The horizontal dashed lines indicate the limit of detection at 20 copies/mL. See also Figure S4 and Tables S1, S6, and S7.
Figure 2.. Classification of HIV-1 integration sites…
Figure 2.. Classification of HIV-1 integration sites and proviral near full-length genome sequences across different cell subsets before ATI
(A) Proportions of integration sites (IS) retrieved by integration site loop amplification (ISLA) for participants STAR 9, STAR 10, and STAR 11 from TCM/TTM and TEM subsets from peripheral blood. IS found more than once are defined as “clonal” and are shown in color as proportion of all IS. Identical IS found in both subsets are linked with dashed lines. p values test was used for a difference in the proportion of unique IS between TCM/TTM and TEM by “prop.test” in R. (B) Proportions of intact and defective near full-length sequences from full-length individual proviral sequencing (FLIPS) within TCM/TTM and TEM peripheral blood for each participant. See Figures S1 and S2A for figures including all FLIPS data. (C) Proportions of expansions of identical sequences (EIS) found in TCM/TTM and TEM peripheral blood subsets based on FLIPS data for each participant. EIS consisting of a detective and intact provirus are shown in shades of red and green, respectively, while unique proviruses are represented in white. The “prop.test” in R was used to test for differences in the proportion of unique proviruses between TCM/TTM and TEM. TCM/TTM, central/transitional memory CD4 T cell; TEM, effector memory CD4 T cell; PSI, packaging signal; MSD, major splice donor. See also Figures S1 and S2 and Tables S2 and S4.
Figure 3.. Near full-length proviral HIV-1 genomes…
Figure 3.. Near full-length proviral HIV-1 genomes and associated integration sites recovered from the peripheral blood by MDA
(A) For each participant, the recovered proviral genome structures are shown aligned to the HXB2 reference genome, and corresponding integration sites are listed on the right. For more information, see Table S3. Each provirus is colored according to their structural category. (B–D) For each participant, the number of integration site (IS) and near full-length (NFL) proviruses generated via full-length individual proviral sequencing (FLIPS) linked to clones characterized by multiple displacement amplification (MDA) is shown together with their corresponding cellular subset. TCM/TTM, central/transitional memory CD4 T cell; TEM, effector memory CD4 T cell; PSI, packaging signal; MSD, major splice donor. See also Table S3.
Figure 4.. Comparison of assays to identify…
Figure 4.. Comparison of assays to identify potentially clonal HIV-1-infected cell populations
Clonality assessments are shown for integration site loop amplification (ISLA), single-genome sequencing (SGS), and full-length individual proviral sequencing (FLIPS). Identical sequences found multiple times within the same assay are shown as light gray slices. The total number of examined integration sites (IS), V1–V3 env sequences, and near full-length (NFL) proviral sequences is noted in the middle of each donut plot. When an IS, V1–V3 env SGS, or NFL FLIPS sequence could be unambiguously linked to data from previously identified infected cell clones by multiple displacement amplification (MDA), they were given a distinct standout color and chromosome designation as indicated in the legend. Dark gray slices show identical sequences that did not allow for overlap detection as (1) not all IS could be linked via MDA to a V1–V3 env or NFL sequence and (2) not all NFL genomes contained the V1–V3 env region. Arrows are used to indicate discrepancies between the different assays. EIS, expansion of identical sequences. See also Table S5.
Figure 5.. Maximum-likelihood phylogenetic trees based on…
Figure 5.. Maximum-likelihood phylogenetic trees based on the V1–V3 env region from proviral and plasma sequences before and during ATI
Proviral sequences collected before ATI from full-length individual proviral sequencing (FLIPS) and multiple displacement amplification (MDA) are shown as squares and circles, respectively. The integration sites (IS) associated with MDA-derived proviruses are noted if available. Plasma sequences are shown as triangles (V1–V3 env) or diamonds (3′-half genome) where the color indicates the time point during analytical treatment interruption (ATI). Arrows indicate identical matches between proviral and plasma V1–V3 env sequences. All trees are rooted to the HXB2 reference sequence. (A) In participant STAR 10, three identical matches between defective proviral and plasma rebound sequences were found. For two, the corresponding IS ZBTB20 and Chr8:100792121 could be recovered. (B) In participant STAR 4, only one match between a unique major splice donor (MSD) deleted provirus and plasma sequences was observed. (C) In STAR 9, a match between a unique intact provirus and multiple plasma sequences from different time points were found. (D) In STAR 11, a rebounding plasma sequence could be linked to an expansion of identical intact near full-length (NFL) genomes located in the ZNF141 gene. One unique intact provirus can be linked to a residual plasma sequence from T1. SGS, single-genome sequencing; PSI, packaging signal. See also Figure S3.

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