Identifying all tissues in which HIV persists during ART is a prerequisite to the design of efficient eradication strategies. Extensive characterization of HIV reservoirs in multiple anatomical sites is only possible by collecting post-mortem tissues within 6 hours following death from people living with HIV who generously gave their body for research on HIV cure.
Two men living with HIV (Participant #1 & #2: 67 and 68 years old, on continuous ART for 11 and 8 years; with neuropathy, and non-Hodgkin lymphoma, respectively) underwent rapid deep-tissue sampling after death. HIV DNA and cell-associated gag RNA were quantified by qPCR in snap-frozen tissues (multiple lymph nodes, gut, liver, spleen, brain and testes). Near-full length (NFL) proviral sequences were obtained by a modified FLIPS assay followed by PacBio sequencing.
HIV-infected-cells were detected in all tissues examined from both participants. Frequencies of infected cells varied across tissues and between participants, with lymph nodes, liver, lungs and spleen displaying the highest levels of HIV DNA/RNA. A total of 300 (participant #1; 14 tissues) and 141 (participant #2; 8 tissues) NFL genomes were isolated (Figure 1). Participant #2 proviruses often contained large deletions (88% of all defects), whereas viral genomes from participant #1 displayed equally hypermutations (39%) or large deletions (42%). Intact HIV genomes represented 2% and 25% of all proviruses in participants #1 and #2, respectively, and were mainly found in spleen and lymph nodes. In both participants, a large fraction of HIV genomes were found in mutiple copies (49% and 47% in participants #1 and #2, respectively), indicating that clonal expansions are not only common in blood, but also in tissues. The vast majority of these expanded clones were shared accross multiple tissues.
This study is the first to report NFL sequencing in multiple deep tissues. Proviral sequencing in samples from these two participants highlights interindividual differences in reservoir integrity and location. Half the viral genomes retrieved from tissues were found to be clonally expanded in both participants, suggesting that persistently infected cells harbouring either intact or defective proviruses expand and recirculate between anatomical sites during long-term ART.