Contribution of intact viral genomes persisting in blood and tissues during ART to plasma viral rebound in SHIV-infected rhesus macaques

Abstract

Persistent SIV/HIV reservoirs are the primary obstacle to a cure and the source of viral rebound after ART interruption (ATI). However, the anatomical source of viral rebound remains elusive. Here, we characterized the proviral landscape in the blood, inguinal, and axillary lymph nodes and colon biopsies of five SHIV-infected rhesus macaques (RMs), under ART for 28 weeks. From the 144 near full-length (NFL) proviral sequences obtained pre-ATI, 35% were genetically intact and only 2.8% were found in multiple copies. Envelope sequences of plasma rebounding viruses after ATI, more frequently matched pre-ATI intact proviruses retrieved from lymph nodes compared to sequences isolated from the blood or the colon (4, 1, and 1 pair of matched sequences, respectively). Our results suggest that clonal expansion of infected cells rare in this model, and that intact proviruses persisting in the lymph nodes may be a preferential source of viral rebound upon ATI.

Keywords: Genomics; Virology.

Graphical abstract

Figure 1

Study design and quantification of reservoir markers (A) Schematic representation of the study and sampling schedule. Five rhesus macaques were infected intravenously with SHIV-AD8-EO (red arrow) and treated with ART from week 8 to week 37 (orange box). Violet arrows indicate blood, lymph nodes, and colon-sampling time points. Plasma virions env sequences were obtained from blood at week 40 (blue arrow). (B) Longitudinal plasma viral load measures in the 5 animals. The gray area indicates time on ART. (C) Quantification of SHIV DNA. Results are shown for blood, lymph nodes, and colon and represented as SHIV DNA copies per million CD4 T cells (blood and lymph node) or per million total cells (colon). (D) Quantification of SHIV cell-associated RNA (LTR-gag). Results are shown for blood, lymph node, and colon and represented as SHIV RNA copies per million CD4 T cells (blood and lymph node) or per million total cells (colon). Undetectable measurements are represented by open symbols, and limits of detection are plotted. Statistical analysis was performed by a parametric one-way ANOVA test and Dunn’s multiple comparison post-test. ∗p < 0.05; ∗∗p < 0.01.

Figure 2

SHIV proviral landscape in the 5 animals The SHIV-AD8-EO genome diagram at the top illustrates the coverage of the near-full length (NFL) amplification (92%). Each horizontal line represents and individual proviral sequence obtained by NFL amplification from week 34 and 36 (pre-ATI time points). Each block of sequences derived from a given animal is identified with the corresponding animal ID. The type of defect in each sequence is indicated by a specific color, as shown in the diagram on the right. Alignments were performed using the founder strain SHIV-AD8-EO as a reference.

Figure 3

Phylogenetic trees SHIV NFL sequences Phylogenetic trees were built with all sequences obtained from each animal, after excluding the proviral sequences identified as hypermutated. Trees were built using the estimated by maximum likelihood model rooted to the reference sequence of the founder strain SHIV-AD8-EO (pink). Sequences derived from the different compartments are identified by a specific color: red for the blood, green for the lymph node, and blue for the colon. Scale bars refer to the phylogenetic distance, represented by the length of the branch, in nucleotide substitution per site.

Figure 4

Genetic intactness and clonality of SHIV genomes (A) Pie chart summarizing the proportion of genomes retrieved from each compartment displaying a specific type of genetic defect. (B) Pie chart representing the proportion of identical proviral sequences in each compartment. Clones are identified by a different color. Unique sequences are shown in gray. Statistical analysis was performed by a two-way ANOVA test and Tukey’s multiple comparison post-test. ∗p < 0.05.

Figure 5

Phylogenetic relationships between genetically intact sequences isolated from pre-ATI samples and rebounding viruses (A) Left column: phylogenetic trees of genetically intact proviral sequences from pre-ATI samples and sequences of rebounding viruses. Proviral sequences matching rebound virion sequences are indicated by a hash or an asterisk. Right column: diagram representing the fraction of rebounding viruses that are genetically identical to an intact provirus retrieved from a pre-ATI sample. (B) Minimal genetic distance between the sequence of a rebounding plasma virions and a genetically intact provirus was calculated for all compartments and in all animals. (C) Genetic distances between each rebounding plasma virions and the closest intact proviral sequence from each compartment for all compartments and in all animals. Statistical analysis was performed by a one-way ANOVA test and Tukey’s multiple comparison post-test. ∗∗p < 0.01; ∗∗∗p < 0.001.