Objective: Human immunodeficiency virus (HIV) persistence in blood and tissue reservoirs, including the brain, is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the central nervous system (CNS) reservoir is unclear. Here, we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH).Methods: Total, intact, and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n = 18) or virologically suppressed (n = 12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital polymerase chain reaction (ddPCR). HIV‐seronegative individuals were included as controls (n = 6).Results: Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)‐suppressed individuals (median = 22.3 vs 26.2 HIV pol copies/10⁶ cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8 of 10 viremic and 6 of 9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/10⁶ cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir.Interpretation: Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. ANN NEUROL 2022;92:532–544

HIV-associated neurocognitive disorders (HAND) are a leading cause of morbidity in up to 50% of individuals living with HIV, despite effective treatment with antiretroviral therapy (ART). Current evidence suggests that chronic inflammation associated with HIV is especially attributed to the dysregulated production of reactive oxygen species (ROS) that contribute to neurodegeneration and poor clinical outcomes. While ROS have beneficial effects in eliciting immune responses to infection, chronic ROS production causes damage to macromolecules such as DNA and lipids that has been linked to altered redox homeostasis associated with antioxidant dysregulation. As a result, this disruption in the balance between antioxidant-dependent mechanisms of ROS inactivation and ROS production by enzymes such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family, as well as from the electron transport chain of the mitochondria can result in oxidative stress. This is particularly relevant to the brain, which is exquisitely susceptible to oxidative stress due to its inherently high lipid concentration and ROS levels that have been linked to many neurodegenerative diseases that have similar stages of pathogenesis to HAND. In this review, we discuss the possible role and mechanisms of ROS production leading to oxidative stress that underpin HAND pathogenesis even when HIV is suppressed by current gold-standard antiretroviral therapies. Furthermore, we highlight that pathological ROS can serve as biomarkers for HIV-dependent HAND, and how manipulation of oxidative stress and antioxidant-dependent pathways may facilitate novel strategies for HIV cure.

HIV persistence in the brain is a barrier to cure, and potentially contributes to HIV-associated neurocognitive disorders. Whether HIV transcription persists in the brain despite viral suppression with antiretroviral therapy (ART) and is subject to the same blocks to transcription seen in other tissues and blood, is unclear. Here, we quantified the level of HIV transcripts in frontal cortex tissue from virally suppressed or non-virally suppressed people with HIV (PWH). HIV transcriptional profiling of frontal cortex brain tissue (and PBMCs where available) from virally suppressed (n = 11) and non-virally suppressed PWH (n = 13) was performed using digital polymerase chain reaction assays (dPCR). CD68+ myeloid cells or CD3+ T cells expressing HIV p24 protein present in frontal cortex tissue was detected using multiplex immunofluorescence imaging. Frontal cortex brain tissue from PWH had HIV TAR (n = 23/24) and Long-LTR (n = 20/24) transcripts. Completion of HIV transcription was evident in brain tissue from 12/13 non-virally suppressed PWH and from 5/11 virally suppressed PWH, with HIV p24+CD68+ cells detected in these individuals. While a block to proximal elongation was present in frontal cortex tissue from both PWH groups, this block was more extensive in virally suppressed PWH. These findings suggest that the brain is a transcriptionally active HIV reservoir in a subset of virally suppressed PWH.

Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798–802

It is well established that lung pathology and inflammation are more severe during respiratory infections complicated by the presence of both bacteria and viruses. Whilst co-infection can result in invasive pneumococcal disease and systemic inflammation, the neuroinflammatory consequences of co-infection are poorly characterised. In this study, we utilised a mouse co-infection model involving Streptococcus pneumoniae (S. pneumoniae) and influenza A virus (IAV) lung infection, and we also isolated microglia for ex vivo stimulation with pneumococcus or serum amyloid A (SAA). Co-infection but not S. pneumoniae or IAV alone significantly increased the number of amoeboid-shaped microglia and expression of pro-inflammatory cytokines including tumour necrosis factor α (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), and C-C motif chemokine ligand-2 (CCL-2) in the hypothalamus. Pneumococcus was only detected in the hypothalamus of co-infected mice. In addition, the systemic inflammatory cytokines TNFα, IL-1β and IL-6 were not elevated in co-infected mice relative to IAV-infected mice, whereas SAA levels were markedly increased in co-infected mice (p < 0.05). SAA and its functional receptor termed formyl peptide receptor 2 (Fpr2) transcript expression were also increased in the hypothalamus. In mouse primary microglia, recombinant SAA but not S. pneumoniae stimulated TNFα, IL-1β, IL-6 and CCL-2 expression, and this response was completely blocked by the pro-resolving Fpr2 agonist aspirin-triggered resolvin D1 (AT-RvD1). In summary, lung co-infection increased the number of ‘activated’ amoeboid-shaped microglia and inflammatory cytokine expression in the hypothalamus. Whilst persistent pneumococcal brain infection was observed, SAA proved to be a much more potent stimulus of microglia than pneumococci, and this response was potently suppressed by the anti-inflammatory AT-RvD1. Targeting Fpr2 with pro-resolving eicosanoids such as AT-RvD1 may restore microglial homeostasis during severe respiratory infections.

Despite viral suppression with antiretroviral therapy (ART), people with HIV (PWH) continue to exhibit brain pathology, and ~20% of individuals develop HIV-associated neurocognitive disorders. However, the state of cellular activation in the brain of virally suppressed (VS) PWH and the impact of local viral reservoirs on cellular activation are unclear. Using multiplex immunofluorescence imaging, here, we demonstrate that the frontal cortex brain tissue from both non-virally suppressed (nVS; n=17) and VS PWH (n=18) have higher frequencies of astrocytes and myeloid cells expressing interferon-inducible Mx-1 and proinflammatory TNFα relative to HIV-seronegative individuals (p<0.05 for all). The frequency of TGF-β1+ cells were also elevated in the brain tissue from both nVS and VS PWH, which may support active immunoregulatory responses despite ART. Importantly, the frequency of Mx1+ myeloid cells correlated with levels of total HIV DNA and intact and 5′ defective HIV proviral DNA (p<0.05 for all) in the brain of VS PWH. These findings demonstrate that cell activation persists in the brain of VS PWH and is associated with HIV DNA in the brain, which may contribute to neuropathology.