Human brain biorepositories supporting NeuroHIV research

Several biobanks and research programs have substantially advanced our understanding of HIV persistence in the brain. Two have contributed substantially to the study of HIV in the brain: The National NeuroHIV Tissue Consortium (NNTC) and Last Gift (LG). The NNTC is a federated system of five biorepositories that together are distinguished by a large participant cohort and extensive, longitudinal in-life clinical characterization that provides data associated with most tissues collected. Thus far, 3,567 participants, including 3,094 PWH, have been enrolled in the NNTC, and 1,100 brains (along with other vital organs) have been donated to the biobank, alongside tens of thousands of longitudinal plasma and CSF samples. The NNTC collects extensive baseline and longitudinal data to inform analyses, and biofluids are available for requestors (S1 Table). Participants or legal proxies consent to autopsy, and the NNTC collects post-mortem brain and peripheral tissue, performs pathological assessments, and banks frozen and fixed tissue, providing investigators with a wide-ranging framework for examining the effects of HIV on the CNS over time (S2 Table). The NNTC collects neurological, neurocognitive, psychiatric, and clinical laboratory data during life. Because the time and frequency of viral load measurements vary across participants, two related but distinct operational definitions of viral suppression were instituted in the NNTC cohort (S3 Table). Forty-three participants met Criterion 1, requiring ≥5 years of observation, with ≥4 undetectable viral loads in the last 5 years, and either one undetectable value within 12 months of death or two within 24 months. Seventy-five participants met Criterion 2, requiring ≥2 years of observation and at least one undetectable viral load within 6 months of death. Since these criteria are overlapping, 33 participants met both criteria, for a total of 85 participants in the NNTC suppressed autopsy cohort. Information on these NNTC participants with brain autopsy specimens is presented in S4 Table. Given the effectiveness of contemporary ART regimens, this suppressed autopsy cohort is expected to grow, as currently 71% of active NNTC participants currently have viral loads that are below the limits of detection of current standard assays (<20 copies/mL).

In addition to the site-collected central data, experimental data generated by investigators are returned to the consortium and entered into a case-linked database [19]. These attributes established NNTC as one of the most widely utilized and valuable resources for the study of the effect of HIV on the CNS, including studies on HIV persistence in the brain. Investigators can request specimens and data from the NNTC upon approval of their request, which can be made at http://nntc.org/. To date, the NNTC has received 1,038 requests, resulting in the shipment of 29,461 specimens, along with linked clinical data and metadata.

A complementary effort closely integrated with the NNTC is the LG study, which works in collaboration with studies on PWH at the San Diego NNTC site, the California NeuroHIV Tissue Consortium (CNTN). Complementing the NNTC, the LG program, a rapid-autopsy cohort designed to investigate HIV persistence at the end of life, has provided unique insights into CNS reservoirs through near-death sampling of individuals maintained on suppressive ART until death. In addition to achieving short postmortem intervals, participants undergo intensive prospective clinical and virologic follow-up, with plasma HIV-1 RNA measurements frequently obtained within days, and sometimes hours, of death. The average time from last plasma viral load measurement to death for LG participants is 20 days, with a median of just 6 days. Intracardiac fluid is collected at autopsy to enable additional virologic characterization. Together, these features provide an unusually detailed view of reservoir biology in the context of virologic status immediately preceding death. Upon death, the LG team conducts a rapid research autopsy enabling the collection of high-quality brain and peripheral tissues with minimal post-mortem interval. The time from participant death to completion of the autopsy by the LG team averages 6.8 hours (with a median of 6.05 hours). This approach preserves labile viral and host signals, maximizing RNA integrity and cell viability, thereby providing unique opportunities to study HIV persistence, transcriptional activity, and reservoir dynamics that are not possible with standard autopsy protocols. Because the rapid-autopsy model is highly resource-intensive, the LG cohort is necessarily smaller than the broader NNTC; however, the complementary strengths of these programs, combining deep longitudinal characterization with near-death assessments and rapid tissue collection, provide a powerful framework for studying CNS HIV reservoirs.

Both of these cohorts rely on participants who consent to brain and organ donation at death for research purposes. When the NNTC was established, the program drew on a substantial literature documenting barriers to organ donation within minority communities, and great consideration was given to these issues within the broader context of palliative care. Particular attention was given to developing ethically appropriate approaches for engaging seriously ill individuals in discussions about end-of-life tissue donation. The LG initiative has further expanded this by examining the ethical, social, and emotional dimensions of end-of-life HIV research through a deeply participant-centered model. This work incorporates the perspectives of healthcare workers and research staff, highlighting participant altruism, emotional challenges, and the central role of community engagement in building trust and supporting participants and families with dignity, consent, and respect [20,21]. Together, this has demonstrated a broad willingness among PWH and their families to participate in observational and interventional cure-related research at the end of life and underscores the value participants place on contributing to scientific progress [22–24].

Although many earlier studies examined brain tissue from PWH before the widespread availability of highly effective suppressive ART, several reports in the past five years have focused specifically on individuals who were virally suppressed at the time of death. In the sections below, we indicate whether samples originated from an NNTC participant, an LG participant, or other sources.

As described above, data from investigator-performed studies generated from specimens from NNTC participants must be provided back to the consortium. We have now compiled all available data on quantitative reservoir studies on brains from NNTC’s virally-suppressed cohorts in S5 Table. Information on quantitative proviral loads from brain and lymphoid tissues from NNTC and LG participants are given in Table 1.

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Table 1. Proviral HIV loads from brain and lymphoid tissues in suppressed participants.

https://doi.org/10.1371/journal.ppat.1014446.t001

The goal of this review is to synthesize the published literature that utilizes autopsy brain tissue to examine the CNS as a potential reservoir. We summarize current findings and state of knowledge regarding HIV persistence in the brain, drawing on data from multiple cohorts and tissue sources. We also highlight important directions for future research to fill key gaps in the field to advance our understanding of CNS HIV reservoirs.

Ethics statement

Studies on post-mortem samples are not considered human subjects research. The NNTC and LG operate under IRB approval at their respective institutions.

Identifying HIV-positive cells and characterizing viral persistence

Detection and quantification of cells harboring HIV nucleic acids, together with characterization of viral genomes and transcriptional activity, are key to determining whether HIV persists in the CNS, expresses viral gene products, and retains the potential to produce replication-competent virus. A range of complementary laboratory techniques has been developed for HIV reservoir studies, each with distinct strengths and limitations [25–28]. Importantly, detection of HIV nucleic acids or proteins does not by itself establish ongoing replication or pathogenic relevance, underscoring the need to integrate multiple complementary assays. We note that for many of the techniques, the lower limit of detection may not be known or may differ between labs performing the assay. In the studies described below, the criteria used to define the populations under study, the criteria for suppression (which often differ from the above NNTC criteria), and their findings are summarized in Table 2.

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Table 2. Summary of brain viral reservoir studies discussed in this manuscript.

https://doi.org/10.1371/journal.ppat.1014446.t002

Collectively, the studies summarized in Table 2 support an overarching conclusion that the CNS constitutes a durable HIV sanctuary during suppressive ART, with HIV DNA, RNA, and in several cases intact or replication-competent virus detected across multiple brain regions, most prominently frontal cortex, white matter, and basal ganglia [29–33]. CNS myeloid cells, microglia and macrophages, emerge as the dominant cellular reservoirs, while astrocytes can harbor proviral DNA and may facilitate viral persistence without robust productive infection [34,35]. However, reported variability in reservoir size, proviral integrity, transcriptional activity, and CNS vs peripheral compartmentalization reflects substantial differences in the participants’ conditions, including ART duration, comorbid conditions, peri-death ART interruption, and inconsistent definitions of viral suppression [31,36,37]. Methodological heterogeneity further contributes to divergent conclusions. Bulk ddPCR and qPCR maximize sensitivity for total HIV DNA but lack resolution of proviral integrity, whereas intact proviral DNA assay (IPDA) and near-full-length sequencing are limited by DNA fragmentation. In situ and single-cell approaches provide cellular localization and transcriptional context but often underestimate low-abundance infection due to RNA degradation and limited tissue depth [38–40]. Given the size of the brain and small regions taken for study, sampling bias exists for all methods. Together, these technical and biological constraints underscore that apparent discrepancies across studies largely reflect differences in sampling, assays, and clinical context rather than fundamental disagreement, reinforcing the CNS as a distinct HIV reservoir during ART.

Quantification of HIV in brains from virally suppressed PWH

Many studies using autopsy brain tissues from PWH have focused on detecting and quantifying HIV DNA and RNA in the CNS using PCR-based amplification methods. By amplifying, and in some cases sequencing, regions of HIV DNA and/or RNA, several groups have quantified proviral burden in the brain using the ddPCR, a highly sensitive technique capable of detecting low-level HIV nucleic acids, to evaluate the presence and distribution of HIV in brain tissue from virally suppressed individuals.

Mohammadzadeh and colleagues [41] analyzed autopsy brain samples from NNTC participants and other sources, assessing both HIV DNA and RNA. They found detectable HIV DNA in the cerebral cortex of all four virally suppressed individuals, as well as in all eight viremic PWH, at comparable levels between the two groups. HIV RNA was also detected in all four virally suppressed individuals and in seven of the eight viremic individuals, again at similar levels. Notably, virally suppressed PWH exhibited significantly higher levels of integrated HIV proviral DNA compared with viremic PWH.

Chung and colleagues [42] examined brain tissue from 27 NNTC participants, 16 who were virally suppressed and 11 viremic donors with HIV encephalitis (HIVE). Three CNS regions were examined for the presence of HIV DNA: the basal ganglia, frontal white matter, and corpus callosum. HIV DNA was detected in 5 of the 16 suppressed participants. In contrast, HIV DNA was detected in brain specimens from all 11 non-suppressed participants with HIVE.

Paired autopsy brain and lymphoid tissues from 63 virally suppressed NNTC participants were studied by Oliveira and colleagues [37]. Detection frequencies of HIV DNA were 70% (44 of 63) in the frontal cortex, 70% (41 of 59) in the basal ganglia, and 55% (31 of 56) in the occipital cortex, with significantly lower levels observed in the occipital cortex compared with the other regions. HIV DNA was detected in lymphoid tissue from all participants, underscoring the persistence of viral reservoirs outside the brain.

Quantification of HIV DNA has also been performed on brain regions from six LG participants, four of whom had undetectable plasma viral loads at, or immediately prior to death, in studies by Chaillon and colleagues [31] and Tang and colleagues [35]. HIV was detected in brain tissues from all four LG participants with undetectable plasma viral loads, as well as one of the two with viremia.

Intact versus defective proviruses in the brain

The development of the IPDA, which identifies proviral sequences that are genetically intact and therefore more likely to be replication competent, enables discrimination between intact and defective HIV genomes in tissue-based reservoir studies [43]. Although IPDA provides an improved estimate of intact proviral burden, it remains a surrogate for replication competence and can generate false positive classifications [44].

Four studies from the Churchill group have applied IPDA and related quantitative approaches to brain tissue obtained from NNTC participants [38,45–47]. In the first, Cochrane and colleagues [45] detected HIV DNA in frontal lobe tissue from 12 virally suppressed and 18 viremic PWH, with no significant difference in total HIV DNA levels between groups. IPDA revealed that intact proviruses were present in 6 of 9 virallysuppressed and 8 of 10 viremic individuals, demonstrating that both groups harbored intact potentially replication-competent proviruses in the frontal lobe.

The second study, by Angelovich and colleagues [46], examined regional variation in intact proviral genomes across multiple brain regions. HIV pol DNA was detected in the frontal white matter, basal ganglia, and cerebellum in 16 virally suppressed and 21 viremic PWH. HIV pol DNA was detectable in all three brain regions, with lower levels in the cerebellum and basal ganglia compared with frontal white matter in both groups. Among those assessed for intact proviruses (8 of the 16 virally suppressed and 8 of the 21 viremic PWH), the frontal white matter contained intact proviruses in 6 of 8 virally suppressed and 6 of 8 viremic individuals. The basal ganglia contained intact proviruses in 2 of 8 virally suppressed and 4 of 6 viremic individuals. The cerebellum contained intact proviruses in 2 of 8 virally suppressed and 3 of 8 viremic individuals. Overall, the frontal white matter contained the highest levels of intact proviral DNA in both suppressed and viremic groups.

In a third study, Jamal Eddine and colleagues [47] evaluated paired autopsy brain and lymphoid tissues from 63 virally suppressed NNTC participants. The presence of HIV DNA was examined in the frontal cortex, basal ganglia, and occipital cortex. Detection frequencies were 70% (44 of 63) in the frontal cortex, 70% (41 of 59) in the basal ganglia, and 55% (31 of 56) in the occipital cortex, with significantly lower levels observed in the occipital cortex compared with the other regions. In contrast to the CNS findings, HIV DNA was detected in lymphoid tissue from all participants, underscoring the persistence of viral reservoirs outside the brain. Importantly, HIV RNA levels were strongly correlated with total HIV DNA, suggesting that transcriptional activity is tightly linked to the size of the brain reservoir.

In the most recent study from this group, Byrne and colleagues [38] assessed IPDA-defined measurements of intact and total proviruses in brain tissues from 18 virally suppressed and 17 viremic NNTC participants. As in the earlier work, no significant difference was observed in total HIV DNA between the two groups. This study then shifted focus to evaluating the relationship to neuroinflammation (discussed in the following section). Together, these studies indicate that intact and transcriptionally active proviruses are a persistent feature of the brain, even under long-term ART.

Other investigators have also applied IPDA to examine the HIV reservoir in autopsy brains from virally suppressed PWH. Gabuzda and colleagues [34] detected intact proviral genomes in 18 of 28 NNTC participants using IPDA. These researchers also assessed HIV DNA and RNA using gag-based PCR assays. They found that HIV DNA was detected in all 28 participants and HIV RNA in 26 of 28, confirming persistent viral genomes and transcriptional activity in brain tissue despite plasma viral suppression. While such IPDA studies have not been performed in the LG cohort, the study by Chaillon [31] found that 94% of env proviral sequences derived from the brains of 5 participants were intact.

Beyond IPDA, direct viral sequencing also provides strong evidence for the presence of intact proviruses in the brain. In a recent study examining postmortem brain tissue from three virallysuppressed individuals outside the NNTC and LG cohorts, Sun and colleagues [32] performed near-full-length proviral sequencing and identified intact, clonally related HIV genomes within the basal ganglia and periventricular white matter. These brain-derived sequences were phylogenetically linked to peripheral viral genomes, suggesting trafficking of infected cells or shared cellular ancestry or proviral trafficking mediated by cellular migration between brain and systemic reservoirs, both complementing and contrasting with the prior studies of viral compartmentalization. Overall, these findings demonstrate that the CNS can harbor genome-intact proviruses, clonally related to those in peripheral tissues, and support the possibility that infected cells migrate to and expand within the brain microenvironment.

Taken together, the above studies demonstrate that, despite effective systemic viral suppression, HIV can persist and show varying degrees of compartmentalization across brain regions and other tissues. This complexity underscores the difficulty of defining the boundaries and behavior of HIV reservoirs within the CNS.

Brain cell types harboring HIV

Historically, in individuals with active viremia, numerous studies have demonstrated that cells of myeloid lineage, including microglia and perivascular macrophages, can express HIV RNA and protein within the CNS [48,49]. However, some studies have identified astrocyte infection in this pre-efficacious ART era. Churchill et al, using immunohistochemistry, in situ hybridization, and viral protein detection, reported extensive astrocyte infection in brains from NNTC participants with HIV‑associated dementia [50]. Agbey et al [51] have reported expression of HIV env RNA in the human CNS, including in astrocytes, in the brains of PWH who had never taken ART.

More recently, similar approaches have been applied to brains from virally suppressed individuals. Ko et al [30] examined postmortem brain tissues from virally suppressed PWH, using DNAscope in situ hybridization coupled with cell-type-specific immunostaining to localize HIV DNA that single‑cell resolution. Their central finding was that detectable HIV DNA was confined almost exclusively to CNS myeloid cells, including microglia and perivascular macrophages, with no convincing evidence of HIV DNA within GFAP‑positive astrocytes.

In contrast, Valdebenito et al [52] advanced the concept of astrocytes as CNS HIV reservoirs by demonstrating that astrocytes, although poorly supportive of productive viral replication, can harbor HIV DNA and mediate efficient cell‑to‑cell viral transfer to neighboring cells in vitro through gap junctions and other intercellular mechanisms. Much of this work integrated human brain tissues with in vitro and ex vivo astrocyte models, emphasizing functional consequences of viral presence rather than durable, integrated proviral burden. Compared with Ko et al, which focused on the detection of HIV DNA with cellular co-localization,Valdebenito et al placed greater weight on low‑level or restricted infection and viral protein/RNA detection, raising the possibility that astrocytes contribute to CNS persistence through non‑canonical or indirect mechanisms rather than acting as a major source of long‑lived, integrated proviruses under suppressive ART.

Cell-type assignment was also addressed in two of the studies from the Churchill group. Cochrane and colleagues [45] performed DNAscope combined with immunofluorescence for the myeloid marker CD68, along with laser-capture microdissection of CD68+ cells, confirming that HIV DNA was present in myeloid cells in brains from virally suppressed donors. Jamal Eddine and colleagues [47] used immunofluorescence detection of HIV p24 (Gag) together with markers for myeloid cells (CD68) and T cells (CD3) to demonstrate HIV Gag protein expression predominantly in myeloid cells in the brains of suppressed individuals. Rare HIV p24-positive T cells were detected, but these were restricted to perivascular or intravascular compartments, consistent with transient trafficking rather than stable parenchymal infection.

Taken together, these studies support a unifying model in which CNS myeloid cells constitute the principal stable HIV reservoir in virally suppressed individuals, while astrocyte involvement is variable and context‑dependent, becoming more apparent in settings of uncontrolled viremia, severe neuroinflammation, or advanced neurocognitive disease. This distinction has important implications for HIV cure strategies, suggesting that eradication efforts must primarily target long‑lived CNS myeloid populations, while also accounting for astrocyte‑mediated viral signaling or protein expression that may sustain neuroinflammation independent of replication‑competent virus.

Studies on isolated viable brain cells

Given the consistent detection of HIV within cells of the myeloid lineage within the CNS in brain tissue sections, several investigators have isolated myeloid cells (primarily microglia) from PWH to directly assess viral persistence within these long-lived CNS-resident cells. One of the most compelling studies used LG brain tissues to demonstrate that brain-resident microglia can serve as durable reservoirs for replication-competent HIV despite durable systemic plasma viral suppression. Tang and colleagues [35] isolated viable microglia from the parietal cortex of LG donors. They detected both total and integrated HIV DNA, as well as replication-competent virus that could be reactivated ex vivo, which was macrophage tropic. These findings provided strong evidence that microglia harbor inducible proviruses even when plasma viremia is undetectable.

Building upon this work, Schlachetzki and colleagues [33] isolated microglia from fresh prefrontal cortical tissue of LG donors and performed single-cell transcriptomic and chromatin accessibility profiling (scRNA-seq and scATAC-seq). HIV RNA and DNA were detected in microglia from 2 of the 3 participants, with an infection frequency of ~0.5% among microglia in the virally suppressed participants, demonstrating the durability of the microglial reservoir at the single-cell level. Complementary findings were reported in a study from five NNTC participants by Nuhn and colleagues [40], including two who were virally suppressed, in which microglia, freshly isolated from the frontal lobe, the subventricular zone, and the occipital lobe regions of brain, were assessed using IPDA. In one suppressed participant, both proviral DNA and intact genomes were detected across all three brain regions examined, whereas in the second suppressed participant, proviral DNA and intact genomes were detected in the frontal and lobes. Another suppressed participant had both proviral DNA and intact genomes only in the subventricular zone. Among the remaining viremic participants, HIV DNA was detected sporadically, and intact proviruses were not observed. Collectively, these studies indicate that microglia can harbor genetically intact, inducible HIV proviruses that persist despite long-term viral suppression, supporting the durability of the CNS reservoir. These findings highlight the challenge posed by CNS-resident myeloid cells, which are long-lived and may be less responsive to systemic ART compared with peripheral immune cell populations.

The possibility of T-cell reservoirs in the CNS remains incompletely defined. Several recent studies demonstrate that HIV-infected CD4⁺ T cells can be detected in the CSF. Single-cell analyses detect HIV RNA+ central memory CD4⁺ T cells in CSF of PWH on ART [53]. Both productive and replication-inactive infection in CSF CD4⁺ T cells occur during acute infection in humans and macaques, but the overall frequency of infected CD4⁺ T cells in the CNS appears low relative to myeloid-lineage cells, and the extent to which these cells constitute a stable reservoir remains uncertain [54]. Technical challenges in isolating rare lymphocyte populations from CSF and brain tissue have limited the number of studies capable of rigorously assessing T-cell infection or persistence in CNS compartments.

Compartmentalization of HIV sequences in the brain

Studies of HIV env gene sequences from suppressed NNTC participants by Oliveira and colleagues [37] revealed compartmentalization of viral sequences between the brain and paired lymphoid tissues; an earlier study by Rose and colleagues [29] found similar compartmentalization between brain and lymphoid as well as other organ tissues in suppressed NNTC participants. Chaillon and colleagues [31] studied longitudinal blood samples and postmortem tissues from six suppressed LG participants, examining HIV env gene sequences to investigate viral phylogenetics across multiple anatomical compartments. This work revealed persistent viral nucleic acids and evidence of inter-compartmental exchange among lymphoid, gastrointestinal, and CNS tissues. These data support a model of continuous, low-level replication or migration between peripheral and CNS reservoirs that presents additional challenges to HIV cure strategies.

Earlier studies utilizing viral phylogenetics demonstrated that HIV within the CNS can evolve independently from peripheral viral populations, supporting the concept of CNS compartmentalization [29,37]. Phylogenetic analyses have shown that CSF HIV variants often form genetically distinct lineages relative to contemporaneous plasma virus, reflecting localized replication and independent evolutionary trajectories within the CNS. These compartmentalized CSF populations may exhibit unique tropism, most notably macrophage-tropic or highly adapted T-cell–tropic phenotypes, indicating that the CNS can support selective pressures and viral diversification not observed in peripheral blood [55,56]. More recent tissue-based studies extend these findings by providing direct spatial characterization of viral persistence within specific brain regions and CNS cell populations [31,32,37]. These approaches allow for improved localization of HIV reservoirs within microglia, macrophages, and other CNS-associated cells, thereby offering greater resolution than CSF-based analyses alone. Together, these findings strengthen evidence that the CNS functions as a distinct viral reservoir with compartment-specific viral dynamics.

Summary of brain viral reservoir studies

Collectively, findings from the NNTC, the LG program, and independent cohorts provide evidence demonstrating that HIV persistence in the brain is a consistent and reproducible phenomenon, even among individuals with long-term plasma viral suppression. Across multiple methodological approaches, including ddPCR, IPDA, in situ hybridization, near-full-length sequencing, and viable microglia isolation, researchers have repeatedly detected HIV DNA, HIV RNA, and in many cases intact and inducible proviruses within CNS tissues. The consistent detection of viral DNA, RNA, and intact proviruses in virally suppressed individuals indicates that the CNS reservoir is not inert but capable of maintaining and, in some cases, transcriptionally and functionally active competent HIV genomes. Interestingly, other than in brains with HIVE, the level of HIV proviral DNA does not appreciably differ in those participants whose viral load was suppressed [38,41,45]. This parallels observations in the simian immunodeficiency virus (SIV)/nonhuman primate model, where viremia suppression with ART lowered brain SIV RNA levels but not SIV proviral DNA levels [57]. In addition, measures of neuroinflammation in the brains of the treated animals in this study were also decreased. This recognition naturally raises the next critical questions regarding how persistent HIV within the brain contributes to local immune activation and neuroinflammatory processes. The following section focuses on this emerging area of investigation.

Although most CNS HIV reservoir studies focus primarily on viral nucleic acid detection, a limited number have evaluated viral protein expression within brain tissue. Using a multiplex imaging platform, the Eugen’s group demonstrated that HIV-infected cells in the brains of virally suppressed PWH continue to produce detectable viral proteins, including p24, gp120, nef, vpr, and tat, providing direct evidence of persistent translational activity within the CNS reservoir [39]. Jamal Eddine and colleagues demonstrated HIV Gag (p24) expression predominantly within myeloid-lineage cells in brain tissue, supporting persistent viral activity within CNS reservoirs despite ART [47]. Earlier work by Churchill and colleagues reported extensive astrocyte-associated HIV protein detection in individuals with HIV-associated dementia [50]. However, relatively few studies of brains from suppressed PWH have examined HIV-encoded proteins in CNS tissue compared to nucleic acid-based approaches. This discrepancy may partially reflect technical challenges associated with detecting low-abundance viral proteins in postmortem tissue, as well as the greater sensitivity and broader availability of molecular techniques such as PCR-based assays and sequencing approaches [25–28]. Given evidence suggesting viral proteins may contribute to persistent inflammation despite ART, further investigation of HIV protein expression within the CNS remains an important area for future research.

Neuroinflammation and neurocognitive impairment

Despite effective ART, HIV persistence within the CNS has been repeatedly linked to markers of neuroinflammation and immune activation, several studies have examined this relationship using both bulk and single-cell approaches. Using virally suppressed NNTC donors, Gabuzda et al [34] combined IPDA with differential gene-expression and observed that while intact proviral levels did not differ significantly between individuals with and without neurocognitive impairment, brains showing histological evidence of neuroinflammation exhibited a trend toward higher intact-proviral burden. Among the 78 genes examined that are associated with inflammation, stress responses, and white-matter integrity, most were upregulated in brain tissue from PWH compared with that from people without HIV infection, with notable downregulation of genes related to oxidative phosphorylation and oligodendrocyte function.

Using multiplex immunofluorescence, Byrnes and colleagues [38] demonstrated increased expression of interferon-inducible myxovirus resistance protein 1 (Mx-1) and Tumor Necrosis Factor (TNF) in astrocytes and myeloid cells within frontal cortex tissue from both virally suppressed and viremic PWH. Importantly, the frequency of Mx-1–positive myeloid cells correlated with levels of total, intact, and 5′-defective HIV proviral DNA in virally-suppressed participants, supporting a role for persistent proviral burden with local immune activation of HIV in the CNS.

In a study of participants in the LG cohort who had long-term viral suppression, Trufino and colleagues [58] found that despite consistent detection of HIV DNA and RNA across all CNS regions, quantitative measures of reservoir size and transcriptional activity showed no association with cognitive performance. Interestingly, while CNS HIV burden alone did not account for neurocognitive variability, their examination of the T-cell receptor repertoire in different brain regions revealed linkages to specific cognitive function, suggesting neuroimmune factors may drive cognitive deficits in suppressed PWH.

Single-cell omics approaches have extended these observations on neuroinflammation. In the study of Schlachetzki and colleagues [33], the HIV RNA-positive microglia were enriched for interferon-stimulated and pro-inflammatory gene signatures, demonstrating that infected microglia remain transcriptionally active under suppressive ART. Building on these insights, Tang and colleagues [59] first demonstrated of increased type I interferon signaling in neurocognitively impaired NNTC participants (on ART but not all suppressed) using proteomics. Next, they used scRNA-seq to examine two virally-suppressed participants (one from the LG, the other through the National Disease Research Interchange following LG protocols) and found transcriptome evidence of persistent type I interferon signaling in the cells, providing mechanistic evidence that sustained interferon activity may bridge latent infection and neuroinflammation [59].

A recent study by Wilson and colleagues [60] examined the combined effects of HIV infection and substance use disorder (SUD) on neuronal and microglial gene transcriptional programs in the ventral midbrain using tissue from 90 NNTC participants. The cohort included 28 participants without HIV, 30 virally suppressed PWH, and 32 viremic PWH, with each group further stratified by the presence of opioid or cocaine SUD. Virally suppressed PWH with SUD were found to have a dysregulated transcriptional profile in dopaminergic neurons, and an altered microglial transcriptome consistent with increased susceptibility to HIV infection, although detection of HIV was not reported.

Together, these studies highlight a converging theme: even with effective systemic viral suppression, the CNS remains an immunologically distinct environment in which infected microglia and macrophages can sustain low-level chronic inflammation. Persistent interferon signaling, cellular activation, and ongoing proviral transcription all point to neuroinflammation as both a marker and a potential driver of CNS reservoir maintenance. These processes may help explain the cognitive deficits observed in some PWH, providing the foundation for the next section’s focus on neurocognitive outcomes.

Integrated evidence for HIV persistence in the brain

In the modern ART era, neuropathology in PWH, including those with viral suppression, is generally limited to mild, nonspecific findings, such as low-grade gliosis, microglial activation, and sparse perivascular macrophages, in contrast to the severe HIVE, multinucleated giant cells, and extensive white-matter damage that characterized the pre-ART period [34,61,62]. This broader pathological context underscores that the viral signals detected in brain tissue today arise within a landscape of attenuated but not absent neuroinflammation, highlighting the importance of understanding persistent tissue-resident reservoirs rather than the dramatic encephalitic pathology that dominated the pre-ARTera.

These autopsy-based studies reveal that HIV DNA and RNA remain detectable in the brains of virally suppressed PWH. As summarized in S5A Table, brain tissue from 38 of the virally suppressed NNTC participants has been assayed in one or more studies focused on proviral DNA load, and 32 (84%) had at least one brain region in which HIV DNA was detected. The median level of HIV proviral DNA was 2.53 copies per 10⁶ cells. IPDA-based analyses (S5B Table) have been performed on 30 virally suppressed NNTC participants, with HIV DNA detectable in all 30 (100%) samples, with a median of 3.55% being intact. When examining specimens from suppressed NNTC participants in either set of techniques, samples from 43 of the 45 (96%) had HIV proviral DNA detected.

While there are indications that the distribution of HIV DNA varies by region, the limited number of studies assessing multiple regions within the same individuals using uniform methodologies precludes definitive conclusions regarding regional specificity. The field lacks large-scale, region-by-region analyses conducted across a sufficiently large number of participants using consistent assay platforms. As such, it remains unclear whether differences across brain regions reflect true regional specificity of infection or heterogeneous distribution that varies between individuals. Despite potential region-specific patterns, it is important to underscore that the absolute burden of infected cells is exceedingly low, with most assays detecting only rare events near the limits of quantification, such that much of the field necessarily relies on binary detection rather than robust quantitative distributions, reflecting the profound degree of viral suppression achieved in the CNS on long-term ART.

Sequence analyses reveal various patterns of viral compartmentalization in PWH [36,63]. In some individuals, brain-derived env sequences form distinct clusters, suggesting localized evolution within CNS tissues [63]. In others, viral variants are widely distributed across the brain, lymphoid, gastrointestinal, and peripheral tissues, indicating a lack of strict compartmental separation [36]. Studies from both the NNTC and LG cohorts demonstrate that viral genomes can persist, evolve, and traffic between migrate among anatomical compartments even under long-term ART, potentially through infected cell migration, clonal expansion, or low-level replication. These observations blur traditional distinctions between “brain” and “systemic” reservoirs.