Approximately 39 million people in the world live with HIV infection. Currently available treatments can reduce the amount of virus to near undetectable levels. But they do not eliminate the virus. A reservoir of HIV-infected cells persists during treatment. If treatment stops, these cells can cause rebounding virus levels and a return of symptoms. As a result, patients living with HIV must remain on treatment their entire lives.

HIV reservoir cells often do not express viral proteins, making them hard for the immune system to find and destroy. Many of these reservoir cells occur in lymph nodes, which makes them difficult for researchers to access for study. Learning more about where these cells hide in the body may enable scientists to develop new treatments to help eliminate them.

Sun et al. show that HIV reservoir cells exist in many body tissues, including the brain. In the experiments, Sun et al. used single HIV genome sequencing to identify HIV genetic sequences in the brain and other body tissues from three recently deceased individuals with HIV. The individuals agreed to donate their tissues for postmortem studies before their deaths. All received antiretroviral therapy until death. The experiments identified functional HIV genetic sequences in lymph nodes and gastrointestinal tissues, known hotspots for HIV-infected cells. Sun et al. also found genetically intact HIV in brain tissue from two of the individuals. The HIV genetic sequences were identical to sequences found in other body tissues. This discovery suggests HIV-infected cells had divided into more HIV-infected cells and spread.

The results suggest that cells harboring intact HIV invade the brain and persist there for extended periods during antiretroviral therapy. To eradicate the virus, interventions targeting HIV reservoir cells must be able to reach the brain. This new information may help researchers developing HIV-reservoir targeting drugs decide which candidates will likely be the most effective. Future studies may also shed light on how HIV reaches the brain and how the infected cells escape destruction by immune cells, which may suggest more treatment strategies.

Combination antiretroviral therapy has extended the life expectancy of people living with HIV (PLWH) to near-normal levels, but it is clear that antiretroviral therapy (ART), in its present form, does not lead to a sustained, drug-free remission of HIV-1 infection; instead, a long-lived reservoir of virally-infected cells persists despite ART (Chun et al., 1997; Wong et al., 1997) and can effectively fuel rebound viremia in case of treatment interruptions. Hence, ART needs to be taken life-long; finding a way to eliminate HIV-1 reservoir cells remains critical to finding a cure for HIV-1 infection.

HIV-1 reservoir cells that circulate in the peripheral blood have been well-described in recent years (Margolis et al., 2020). These circulating reservoir cells mostly consist of memory CD4 T cells that persist life-long and harbor chromosomally-integrated viral DNA, also referred to as a ‘provirus’. The pool size of HIV-1-infected cells during suppressive antiretroviral therapy is frequently maintained or replenished by clonal proliferation of viral reservoir cells, during which an identical viral DNA copy is passed on to daughter cells (Cohn et al., 2015; Hosmane et al., 2017; Bui et al., 2017; Einkauf et al., 2019; Pinzone et al., 2019). Important advances in recent technology development, including single-genome near full-length proviral sequencing, have demonstrated that the vast majority of HIV-1 DNA species encountered in ART-treated persons are defective (Ho et al., 2013; Lee et al., 2017; Hiener et al., 2017), mostly due to errors occurring during reverse transcription of viral RNA into DNA. Such errors can lead to large deletions, premature stop codons, or defects in the viral packaging signal region; moreover, specific host proteins such as APOBEC-3G/3 F can induce lethal hypermutations into the proviral sequence. The ability of viral reservoir cells to persist indefinitely is frequently attributed to very limited or absent proviral gene transcription; this viral latency protects infected cells from host immune responses and reduces possible cytopathic effects associated with viral gene expression. However, recent studies have emphasized that proviral gene expression can be ongoing in some HIV-1-infected cells during antiretroviral therapy (Yukl et al., 2018), typically when proviruses are integrated in immediate proximity to activating chromatin marks (Einkauf et al., 2022; Lian et al., 2023).

Much less is currently known about HIV-1 reservoir cells in other body compartments that are more difficult to access for analytic purposes. Lymph nodes and lymphoid tissues in the gastrointestinal tract harbor the vast majority of all lymphocytes and are likely to represent a prime location for the persistence of virally infected cells (Estes et al., 2017; Baiyegunhi et al., 2022; Kroon et al., 2022; Beckford-Vera et al., 2022); however, studies that interrogate viral reservoir cells in these tissues remain limited. Even less is known about HIV-1 persistence in other organs, and, in particular, in the CNS, although recent studies have started to explore viral reservoir cells in such specific body compartments (Cochrane et al., 2022). In the present study, we used single-genome proviral sequencing to conduct a detailed analysis of HIV-1 proviral sequences in autopsy samples from up to n=18 different organ systems from three study participants.

The lifelong persistence of viral reservoir cells makes HIV-1 infection an incurable disease that necessitates indefinite antiretroviral suppression therapy. However, the location of HIV-1 viral reservoir cells across different tissues has been difficult to assess in the past, due to the limited availability of tissue samples. Recent studies, pioneered by investigators of the ‘Last Gift Cohort’, have catalyzed investigations to characterize HIV-1 sequences in diverse organ systems, specifically in the CNS (Tang et al., 2023; Chaillon et al., 2020). In our study, we used single-genome near full-length proviral sequencing to evaluate the distribution of HIV-1 reservoir cells in multiple anatomical compartments from autopsy samples of three individuals living with HIV-1 and receiving antiretroviral therapy until the time of their decease. Consistent with previous studies (Banga et al., 2016; Kuo et al., 2020), intact proviruses were readily detected in lymph nodes; moreover, we detected relatively high frequencies of intact proviruses in the colon, likely reflecting viral infection of CD4 T cells residing in gut-associated lymphoid tissues (GALT). Of note, our study is among the first investigations to identify near full-length proviral sequences from the central nervous system in two study participants, supporting the hypothesis that CNS cells can serve as reservoirs for long-term HIV-1 persistence despite antiretroviral therapy. Importantly, we noted that large clones of virally infected cells were broadly disseminated across multiple tissues, and, in selected cases, involved cells from the CNS; this suggests that HIV-1 reservoir cells seeded to the brain via hematogenous spread can proliferate in the local tissue microenvironment of the central nervous system. Together, our work suggests that HIV-1 reservoir cells harboring intact proviruses are broadly distributed across multiple anatomical locations, involving lymphoid tissues, gastrointestinal tissues, genitourinary tissues, and, importantly, central nervous system tissues.

HIV-1 can invade the CNS within days after infection, as demonstrated in animal (Chakrabarti et al., 1991) and in human studies (Valcour et al., 2012). Most likely, infection of CNS cells occurs as a result of transmigration of infected CD4 T cells and, possibly, macrophages across the blood-brain-barrier (BBB; Spudich and González-Scarano, 2012; Liu et al., 2000), a process that may be facilitated by the increased permeability of the BBB during the initial, highly replicative stage of HIV-1 infection. Infected cells that successfully enter the CNS may frequently be short-lived, however some infected CD4 T cells in the brain may persist long-term. Moreover, invading infected cells can transmit the virus to resident CNS cells via effective cell-to-cell transmission (Liu et al., 2000). At least three different CNS cell types seem to be susceptible to HIV-1 infection: perivascular macrophages, microglial cells, and astrocytes, although the role of the latter as HIV-1 target cells is more controversial (Churchill et al., 2006; Wallet et al., 2019; Woodburn et al., 2022). Yet, due to the difficulties in accessing brain tissues for analytic purposes, the role of the central nervous system in HIV-1 persistence during antiretroviral therapy remained largely unknown for a long time. Using the intact proviral DNA assay (IPDA), a ddPCR-based technique allowing to identify proviruses with a high probability of being genome-intact, previous investigators identified intact proviruses in 6 out of 9 ART-treated persons (Cochrane et al., 2022), although the precise proviral DNA sequence and their possible clonality was not assessed with this technology. In our study, a total of 13 CNS tissue samples from three study participants were analyzed, including specimens from the basal ganglia, thalamus, occipital lobe, frontal lobe, and periventricular white matter. In two study persons (participants 1 and 3), intact proviral sequences were detected in basal ganglia, suggesting that HIV-1 may preferentially persist in this anatomical compartment in the CNS; an additional intact provirus was detected in periventricular white matter. Notably, the intact provirus from basal ganglia in one of our study persons (participant 1) was clonal with 4 intact proviruses from the lymph node, indicating, to our knowledge for the first time, that CNS tissue can be involved in the multi-compartment dissemination of large clones of HIV-1 proviruses in ART-treated persons. Moreover, in multiple instances, we observed clones of HIV-1-infected cells that were distributed across different autologous CNS tissues, specifically in study participant 3, suggesting local spread of virally infected cells through clonal proliferation within the immune microenvironment of the CNS.

Our study did not allow to determine which cell types were infected by HIV-1 and responsible for clonal expansion of viral reservoir cells in the CNS; however, it is possible that infected microglia are involved. Microglial cells originate from erythromyeloid progenitors in the yolk sac and colonize the developing CNS during embryogenesis (Kierdorf et al., 2013); these cells act as the main innate immune cell population of the CNS. Due to their long half-life (typically several years), their ability to divide and self-renew, and their high cell-intrinsic susceptibility to HIV-1 (Cenker et al., 2017), these cells may represent a primary cellular site for long-term HIV-1 persistence in the CNS. In particular, self-renewal through homeostatic proliferation in microglia (Réu et al., 2017) may support HIV-1 persistence through clonal expansion. A recent study indeed identified HIV-1 DNA and RNA in microglia cells from autopsies from ART-treated PLWH who did not have specific (HIV or non-HIV associated) CNS pathology (Ko et al., 2019). Evidence for HIV-1 persistence in CD68 +myeloid cells, most likely microglia cells, was also described by previous investigators (Cochrane et al., 2022). That said, the presence of clonal proviral sequences shared between the CNS and lymphoid tissues in our study could also suggest that migrating CD4 T cells infected with R5-tropic viruses may infect the brain as ‘Trojan horses’, and then potentially clonally expand in situ in the CNS; this hypothesis is consistent with recent findings from Kincer et al., 2023. In the future, it may be possible to capture the phenotypic characteristics of HIV-1 reservoir cells from the CNS directly with single-cell assays that permit combined assessments of the phenotype and the proviral sequence; an example for such an assay system was recently described (Sun et al., 2023).

In our study, CXCR4-tropic proviruses were exclusively detected in participant 2. Compared to the other two participants who began ART shortly after HIV-1 diagnosis, participant 2 was diagnosed with HIV-1 25 years prior to starting antiretroviral therapy and died 10 months after ART commencement; therefore, viral CXCR4 tropism most likely resulted from ‘coreceptor switch’ frequently occurring during advanced stages of immune deficiency (Connor et al., 1997). Whether the preferential persistence of CXCR4-tropic viruses in study participant 2 was associated with our inability to detect intact proviruses in the CNS in this person is unclear; however, prior studies suggested that CCR5 can act as the principal co-receptor for HIV-1 isolates in the brain (Albright et al., 1999; He et al., 1997). Moreover, rebound viremia in cerebrospinal fluid after ART interruption is mostly fueled by CCR5-tropic virus (Kincer et al., 2023), further supporting the assumption that R5-tropic viruses are better adjusted to persist in the brain. Another notable finding in our study was the high number of HIV-1 proviruses isolated from the prostate, which had the second highest proviral frequency among all analyzed tissues in study participant 2, second only to lymph node samples. Other studies also reported that the prostate can represent a tissue reservoir for HIV-1 (Chaillon et al., 2020). We were unable to identify the precise cell type harboring HIV-1 in the prostate, but it is possible that myeloid cells may harbor HIV-1 in this location. A prior study indeed demonstrated that intact, replication-competent HIV-1 can persist in myeloid cells from the urethra, located in immediate anatomical proximity to the prostate (Ganor et al., 2019).

Our study has several limitations. Importantly, this work includes only 3 participants, and brain tissues were the only samples available from participant 3. Moreover, due to limited tissue sizes available for investigation, very few cells were assayed from the terminal ileum and testes; prior studies suggested high HIV-1 DNA levels in these 2 tissues during suppressive antiretroviral therapy (Horn et al., 2021; Miller et al., 2019). Another limitation was that peripheral blood samples were not available from the 3 participants, which made it impossible to study phylogenetic associations between tissue reservoirs of HIV-1 relative to viral species circulating in peripheral blood. Moreover, we cannot fully exclude contamination of tissue samples with cells from peripheral blood. However, after autopsy, the tissue samples were washed thoroughly with PBS to eliminate blood as much as possible. Notably, we failed to detect intact proviral sequences from over 100 million liver cells of 2 participants, despite the fact that the liver contains about 13% of all human blood supply at any given time point, arguing against contamination of tissues with blood cells.

In sum, this study provides a deep analysis of tissue reservoirs for HIV-1 that includes a detailed assessment of HIV-1 sequences in CNS tissues. Our work supports the persistence of genome-intact HIV-1 in many tissues, including CNS tissues, emphasizing the difficulties in finding strategies to effectively eliminate HIV-1 from the human body in clinical settings.

Proviral sequencing data have been deposited in Genbank (accession numbers: OR660701–OR661197) and may be accessed at https://www.ncbi.nlm.nih.gov/nuccore.

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Author details

1. Weiwei Sun

   Ragon Institute of MGH, MIT and Harvard, Cambridge, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4578-6292

2. Yelizaveta Rassadkina

   Ragon Institute of MGH, MIT and Harvard, Cambridge, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

3. Ce Gao

   Ragon Institute of MGH, MIT and Harvard, Cambridge, United States

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

4. Sarah Isabel Collens

   Department of Neurology, Massachusetts General Hospital, Boston, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. Xiaodong Lian

   Ragon Institute of MGH, MIT and Harvard, Cambridge, United States

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

6. Isaac H Solomon

   Department of Pathology, Brigham and Women’s Hospital, Boston, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Shibani S Mukerji

   Department of Neurology, Massachusetts General Hospital, Boston, United States

   Contribution

   Conceptualization, Resources, Investigation

   Competing interests

   No competing interests declared

8. Xu G Yu

   1. Ragon Institute of MGH, MIT and Harvard, Cambridge, United States
   2. Infectious Disease Division, Brigham and Women’s Hospital, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration

   Competing interests

   No competing interests declared

9. Mathias Lichterfeld

   1. Ragon Institute of MGH, MIT and Harvard, Cambridge, United States
   2. Infectious Disease Division, Brigham and Women’s Hospital, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing - review and editing

   For correspondence

   mlichterfeld@mgh.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9865-8350

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