HIV Diversity and Genetic Compartmentalization in Blood and Testes during Suppressive Antiretroviral Therapy

Abstract

HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune-privileged tissues, such as the testes, may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed the proviral burden and genetics in the blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by quantitative PCR, and subgenomic proviral sequences (nef region) were characterized from single templates. HIV diversity, compartmentalization, and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from the blood (396 sequences; 354 nef-intact sequences) and testes (326 sequences; 309 nef-intact sequences) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and the immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testis genetic compartmentalization, but none did so when the evaluation was restricted to unique sequences per site, suggesting that compartmentalization, when present, is attributable to the clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testis reservoirs. Furthermore, while the testis microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication.IMPORTANCE Two key questions in HIV reservoir biology are whether immune-privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy and, if so, by what mechanism. While our results indicated that blood-testis HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood-tissue data set retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testis reservoirs differ is not via seeding of divergent HIV sequences therein but, rather, via differential clonal expansion of latently infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of a broadly similar magnitude across the blood and testes, clonal expansion represents a challenge. The results support individualized analysis of within-host reservoir diversity to inform curative approaches.

Keywords: HIV; clonal expansion; diversity; genetic compartmentalization; reservoir; testes.

FIG 1

Quantifying and characterizing the HIV reservoir in blood and testes. (A) Representative immunostaining of a frozen testis section (participant 5) showing cells expressing CD3 (red) or CD4 (green). Nuclei were counterstained with DAPI (blue). CD4 T cells (CD3⁺ CD4⁺) appear in yellow in the merged image. (B) HIV DNA loads, expressed in number of copies per million cells in whole-tissue lysate, in left and right testes. ND, not determined; trace, HIV DNA was detected but not quantifiable. The asterisk denotes the fact that HIV DNA loads in testes are measured as copies per million total cells (not per million CD4⁺ T-cells, as for blood). (C) Marginal positive correlation between HIV DNA loads in blood and testes (where the latter is expressed as the average measurements for the left and right testes). (D) Unique HIV sequence distribution in blood and testes, matched by participant. (E) Significant correlation between the HIV DNA load and unique HIV sequence frequency in blood. (F) Lack of a significant correlation between the HIV DNA load and unique HIV sequence frequency in testes.

FIG 2

Relationship between the total number of unique HIV sequences collected per participant as a function of the total number of sequences collected. The dashed line denotes a hypothetical data set where every sampled sequence is unique.

FIG 3

Between-host HIV phylogeny. The numbers on the branches indicate bootstrap values supporting within-host monophyletic clades. The scale is the estimated number of substitutions per nucleotide site. NL4-3 and MJ4 are subtype B and C reference sequences, respectively.

FIG 4

Within-host HIV reservoir diversity in blood and testes. Phylogenies, inferred from nucleotide sequence alignments (HIV nef), are midpoint rooted, with scales denoting the estimated number of substitutions per nucleotide site. Matched highlighter plots, made from amino acid sequence alignments, show substitutions relative to the master sequence (the top sequence in the phylogeny). Symbols denote sampling location: blood (filled circles), right testis (open squares), and left testis (open diamonds). The red A and black B markers for participants 1, 3, 4, 5, 7, 8, and 9 identify sequences collected from independent sections of the same testis (the HIV genetic compartmentalization results for these within-tissue comparisons are summarized in Table 3). Bootstrap values of between 70 and 90% are reported to the left of their respective nodes; those of >90% are marked with asterisks.

FIG 4

Within-host HIV reservoir diversity in blood and testes. Phylogenies, inferred from nucleotide sequence alignments (HIV nef), are midpoint rooted, with scales denoting the estimated number of substitutions per nucleotide site. Matched highlighter plots, made from amino acid sequence alignments, show substitutions relative to the master sequence (the top sequence in the phylogeny). Symbols denote sampling location: blood (filled circles), right testis (open squares), and left testis (open diamonds). The red A and black B markers for participants 1, 3, 4, 5, 7, 8, and 9 identify sequences collected from independent sections of the same testis (the HIV genetic compartmentalization results for these within-tissue comparisons are summarized in Table 3). Bootstrap values of between 70 and 90% are reported to the left of their respective nodes; those of >90% are marked with asterisks.

FIG 4

Within-host HIV reservoir diversity in blood and testes. Phylogenies, inferred from nucleotide sequence alignments (HIV nef), are midpoint rooted, with scales denoting the estimated number of substitutions per nucleotide site. Matched highlighter plots, made from amino acid sequence alignments, show substitutions relative to the master sequence (the top sequence in the phylogeny). Symbols denote sampling location: blood (filled circles), right testis (open squares), and left testis (open diamonds). The red A and black B markers for participants 1, 3, 4, 5, 7, 8, and 9 identify sequences collected from independent sections of the same testis (the HIV genetic compartmentalization results for these within-tissue comparisons are summarized in Table 3). Bootstrap values of between 70 and 90% are reported to the left of their respective nodes; those of >90% are marked with asterisks.

FIG 5

Significant positive correlation between blood and testis proviral diversity. Values represent average within-host patristic (tip-to-tip phylogenetic) distances between all pairs of sequences sampled from each site, after collapsing identical nef sequences to a single copy per compartment.

FIG 6

Genetic compartmentalization results for each participant. “Distance” denotes the results of the Hudson, Boos, and Kaplan nonparametric test for population structure, “Tree” denotes the results of the Slatkin-Maddison test (57), and “Consensus” denotes the final result (compartmentalization was declared only if the results of both tests agreed). Blue denotes compartmentalization; gray denotes no compartmentalization. (A) Overall results when comparing all nef sequences from blood to all those from testes; (B) unique results with identical nef sequences collapsed to a single copy per compartment.

FIG 7

Immune escape landscape in blood and testes. (A) Example of escape burden analysis using participant 7's HIV Nef amino acid sequence alignments in blood and testes. Red, orange, and blue represent HLA-adapted, possibly adapted, and susceptible forms, respectively. For each sequence, the sum of HLA-adapted and possibly adapted sequences is divided by the total number of HLA-associated sites to yield the overall percentage of adapted sites (values shown at the end of each sequence). These values are then averaged to yield site- and participant-specific averages. For viral sites associated with more than one HLA allele (e.g., Nef codon 105), the inferred escape profile is shown for the underlined allele. (B) Correlation between escape burden in blood and testes. (C) Correlation between epitope complexity, defined here as the percentage of known or predicted HLA-restricted CTL epitopes exhibiting amino acid variation, between blood and testes. (D) Examples of escaped and susceptible forms coexisting within known or predicted CTL epitopes within the participants' reservoirs, where the height of the residue represents its frequency.