Background: Assessing HIV integration site location and frequency in latently infected cells can inform how HIV persists on antiretroviral therapy (ART). We aimed to characterise HIV integration sites in CD4 T-cells from blood and tissue from HIV-infected individuals on suppressive ART.
Methods: Genomic DNA from 1 million CD4 T-cells obtained from blood, lymph node (LN) or rectal tissue from virologically suppressed participants on ART was enzymatically cut to random sized fragments, tagged and amplified by nested PCR with barcoded primers before Miseq sequencing. Chromosomal alignment was determined using Blat-UCSC Genome Browser (GRCH38/hg38). Clonal expansion of HIV integration sites was defined if identical HIV integration sites differed >2 base pairs in PCR-product length and ≥3 length polymorphisms were present.
Results: We assessed 1794 integration sites from 7 participants receiving ART for a median [range] of 14 [7-19] years with an undetectable viral load for 6 [4-16] years. Integration was enriched in genes vs non-genes (78 [58-87]% vs 23 [13-42]%, p< 0.001). The majority of integration sites into genes were intronic (median 86 [72-97]% vs 13 [3-18]% exonic, p< 0.001) and demonstrated preferential insertion in the opposite orientation relative to gene transcription (median 60 [47-68]% vs 40 [32-52]% same orientation, p=0.007). In blood we observed a median of 54 [25-70]% clonally expanded integration sites. Interestingly, the frequency of clonal expansion was different in blood and tissue. In matched samples of 2 participants, the frequency of clonal expansion in LN was similar to blood, whereas rectal tissue showed the least amount of clonal expansion (table). Integration sites in expanded compared to non-expanded clones were enriched in genes (84% vs 69% respectively, p=0.04) but there was no enrichment in oncogenes (median 22% vs 25% respectively, p=0.74).

 CD4 countHIV DNA /10^6 CD4 cells% Clonal expansion in blood% Clonal expansion in LN% Clonal expansion Rectal tissue
Participant 65781500323817
Participant 752149425-10
[Clonal expansion of HIV integration sites]

Conclusions: Expanded clones of infected cells in blood and tissues are common. HIV integration is preferentially detected in intronic regions but not oncogenes with orientations that are usually in opposite direction. The relative contribution of expanded clones to HIV persistence may differ in different tissue sites but analysis of further tissue samples are needed.