If strategies currently in development succeed in eradicating HIV reservoirs in peripheral blood and lymphoid tissues, residual sources of virus may remain in anatomic compartments. Paired blood and semen samples were collected from 12 individuals enrolled in a randomized, double-blind, placebo-controlled therapeutic vaccine clinical trial in people with HIV (PWH) who began antiretroviral therapy (ART) during acute or early infection (ClinicalTrials registration no. NCT01859325). After the week 56 visit (postintervention), all participants interrupted ART. At the first available time points after viral rebound, we sequenced HIV-1 env (C2-V3), gag (p24), and pol (reverse transcriptase) regions amplified from cell-free HIV RNA in blood and seminal plasma using the MiSeq Illumina platform. Comprehensive sequence and phylogenetic analyses were performed to evaluate viral population structure, compartmentalization, and viral diversity in blood and seminal plasma. Compared to that in blood, HIV RNA rebound in semen occurred significantly later (median of 66 versus 42 days post-ART interruption, P < 0.01) and reached lower levels (median 164 versus 16,090 copies/ml, P < 0.01). Three of five participants with available sequencing data presented compartmentalized viral rebound between blood and semen in one HIV coding region. Despite early ART initiation, HIV RNA molecular diversity was higher in semen than in blood in all three coding regions for most participants. Higher HIV RNA molecular diversity in the genital tract (compared to that in blood plasma) and evidence of compartmentalization illustrate the distinct evolutionary dynamics between these two compartments after ART interruption. Future research should evaluate whether the genital compartment might contribute to viral rebound in some PWH interrupting ART.IMPORTANCE To cure HIV, we likely need to target the reservoirs in all anatomic compartments. Here, we used sophisticated statistical and phylogenetic methods to analyze blood and semen samples collected from 12 persons with HIV who began antiretroviral therapy (ART) during very early HIV infection and who interrupted their ART as part of a clinical trial. First, we found that HIV RNA rebound in semen occurred significantly later and reached lower levels than in blood. Second, we found that the virus in semen was genetically different in some participants compared to that in blood. Finally, we found increased HIV RNA molecular diversity in semen compared to that in blood in almost all study participants. These data suggest that the HIV RNA populations emerging from the genital compartment after ART interruption might not be the same as those emerging from blood plasma. Future research should evaluate whether the genital compartment might contribute to viral rebound in some people with HIV (PWH) interrupting ART.

If strategies currently in development succeed in eradicating HIV reservoirs in peripheral blood and lymphoid tissues, residual sources of virus may remain in anatomic compartments. Paired blood and semen samples were collected from 12 individuals enrolled in a randomized, double-blind, placebo-controlled therapeutic vaccine clinical trial in people with HIV (PWH) who began antiretroviral therapy (ART) during acute or early infection (ClinicalTrials registration no. NCT01859325). After the week 56 visit (postintervention), all participants interrupted ART. At the first available time points after viral rebound, we sequenced HIV-1 env (C2-V3), gag (p24), and pol (reverse transcriptase) regions amplified from cell-free HIV RNA in blood and seminal plasma using the MiSeq Illumina platform. Comprehensive sequence and phylogenetic analyses were performed to evaluate viral population structure, compartmentalization, and viral diversity in blood and seminal plasma. Compared to that in blood, HIV RNA rebound in semen occurred significantly later (median of 66 versus 42 days post-ART interruption, P < 0.01) and reached lower levels (median 164 versus 16,090 copies/ml, P < 0.01). Three of five participants with available sequencing data presented compartmentalized viral rebound between blood and semen in one HIV coding region. Despite early ART initiation, HIV RNA molecular diversity was higher in semen than in blood in all three coding regions for most participants. Higher HIV RNA molecular diversity in the genital tract (compared to that in blood plasma) and evidence of compartmentalization illustrate the distinct evolutionary dynamics between these two compartments after ART interruption. Future research should evaluate whether the genital compartment might contribute to viral rebound in some PWH interrupting ART.IMPORTANCE To cure HIV, we likely need to target the reservoirs in all anatomic compartments. Here, we used sophisticated statistical and phylogenetic methods to analyze blood and semen samples collected from 12 persons with HIV who began antiretroviral therapy (ART) during very early HIV infection and who interrupted their ART as part of a clinical trial. First, we found that HIV RNA rebound in semen occurred significantly later and reached lower levels than in blood. Second, we found that the virus in semen was genetically different in some participants compared to that in blood. Finally, we found increased HIV RNA molecular diversity in semen compared to that in blood in almost all study participants. These data suggest that the HIV RNA populations emerging from the genital compartment after ART interruption might not be the same as those emerging from blood plasma. Future research should evaluate whether the genital compartment might contribute to viral rebound in some people with HIV (PWH) interrupting ART.

HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.

HIV cure research requires interrogating latent HIV reservoirs in deep tissues, which necessitates autopsies to avoid risks to participants. An HIV autopsy biobank would facilitate this research, but such research raises ethical issues and requires participant engagement. This study explores the willingness to participate in HIV cure research at the end of life. Participants include Canadians with HIV [people with HIV (PWHIV)] aged 55 years or older. Following a mixed-method study design, all participants completed a phone or online survey, and a subset of participants participated in in-depth phone or videoconference interviews. We produced descriptive statistics of quantitative data and a thematic analysis of qualitative data. Barriers and facilitators were categorized under domains of the Theoretical Domains Framework. From April 2020 to August 2021, 37 participants completed the survey (mean age = 69.9 years old; mean duration of HIV infection = 28.5 years), including 15 interviewed participants. About three quarters of participants indicated being willing to participate in hypothetical medical studies toward the end of life (n = 30; 81.1%), in HIV biobanking (n = 30; 81.1%), and in a research autopsy (n = 28; 75.7%) to advance HIV cure research, mainly for altruistic benefits. The main perceived risks had to do with physical pain and confidentiality. Barriers and facilitators were distributed across five domains: social/professional role and identity, environmental context and resources, social influences, beliefs about consequences, and capabilities. Participants wanted more information about study objectives and procedures, possible accommodations with their last will, and rationale for studies or financial interests funding studies. Our results indicate that older PWHIV would be willing to participate in HIV cure research toward the end of life, HIV biobanking, and research autopsy. However, a dialogue should be initiated to inform participants thoroughly about HIV cure studies, address concerns, and accommodate their needs and preferences. Additional work is required, likely through increased community engagement, to address educational needs.