HIV-associated neurocognitive disorders: recent advances in pathogenesis, biomarkers, and treatment

The brain as a viral reservoir

The concept of quiescent viral reservoirs, systemically or in the CNS, has emerged as a barrier to HIV eradication in the post-HAART era³⁶. Monocytes and macrophages, in both the CNS and the periphery, express co-receptors necessary for HIV infection, are infected early in the course of disease, are long-living, do not die as a consequence of HIV infection or immune surveillance, and hence can become viral reservoirs for HIV^37–41. Furthermore, ARV entry into the brain can be variable because of the blood–brain barrier, and ARV efficacy as well as the ability of agents to reverse latency in brain cells such as microglia and particularly astrocytes is at best limited⁴². These factors point to the brain as a sanctuary site, further reinforcing the brain as a likely viral reservoir. Probable latent brain infection in macrophages, astrocytes, and other glial cells has been demonstrated in multiple neuropathologic and in vitro studies^43–50. The issue remains controversial, though, as some consider that the demonstration of such virus reflects only the phagocytic function of those cells. Further, as yet, the virus in such cells has not been demonstrated to be replication competent, with the exception of macrophages, though the study used animal models⁵¹. Nonetheless, one study found that after 10 years of suppressive antiretroviral therapy (ART) in the plasma, low levels of HIV RNA could still be detected in the cerebrospinal fluid (CSF) – that is, viral escape – with associated evidence of immune activation, thereby emphasizing the brain as both a sanctuary site and a viral reservoir⁵².

Further support for the brain as a viral reservoir comes from a recent study of simian immunodeficiency virus (SIV)-infected macaques that were continuously virally suppressed with CNS-penetrant HAART. When latency-reversing agents were administered, there was reactivation of virus in the macaque brain⁵³. This resulted in increases in CSF viral load, 10 times higher than plasma, and increased CNS immune activation and neuronal damage markers, likely contributing to a harmful CNS inflammatory response⁵³. The most prevalent SIV genotype in the CSF was novel compared with peripheral genotypes, suggesting that distinct genomes that persisted in the CNS compartment were amplified despite long-term peripheral viral suppression⁵³. Similarly, in humans, it is relatively common to find different genotypes of HIV in the CNS and periphery, again demonstrating compartmentalization in the CNS and its role as a reservoir^54,55.

Monocyte populations and biomarkers

Direct evidence of infected monocytes entering the brain has not been demonstrated in humans; however, animal models have demonstrated peripheral monocyte trafficking to the CNS in SIV infection⁵⁶. HIV viral load and proviral HIV DNA in plasma mononuclear cells have been linked to disease progression, AIDS development, and HAND in HAART-naïve advanced infection and proviral DNA in virally suppressed individuals^57–59. Interestingly, a recent study identified that levels of HIV DNA in peripheral mononuclear cells were not associated with HAND in virally suppressed HIV-positive individuals on stable HAART regimens. Instead, an increase in monocyte HIV-positive reservoir size was associated with progression of HAND, in particular in the HAD subgroup⁶⁰. These papers raise a crucial issue in HAND pathogenesis: at what point do systemic factors become less important compared to brain-related factors? In other words, when does systemic HIV disease “metastasize” to the brain, at that point making it a truly brain-autonomous problem no longer driven by systemic disease?

In HIV, the phenotypic subpopulations of monocytes are altered, resulting in increased circulating CD16⁺ monocytes instead of the usually prevalent CD14⁺CD16^– populations found in normal controls^61,62. CD16⁺ cells can be infected by HIV, in part due to differences in host restriction factor expression (APOBEC isoforms and SAMDH1) allowing HIV replication^63–68. The proportion of CD16⁺ monocytes in the total population is associated with uncontrolled viremia (>400 copies/mL), reduced by adherence to ART, and linked to disease progression^61,62,69,70. Increased proportions and absolute numbers of CD16⁺ monocytes are more strongly associated with HAND than with the CSF viral load or proportion of HIV-infected cells in the CNS; hence, measurement of CD16⁺ populations, CD14⁺/CD16⁺ ratio, and high HIV DNA levels in CD16⁺ cells may be used as biomarkers of disease and correlate with neurocognitive dysfunction^71–75.

Alternate monocyte biomarkers have also been identified. CD163 is a hemoglobin–haptoglobin scavenger receptor found exclusively on monocytes and macrophages^76–80. When monocytes are activated by lipopolysaccharide, Fcγ receptors crosslink or oxidative stress occurs, CD163 is shed as soluble CD163 (sCD163) to reduce inflammatory cell activation and cytokine release^{77,78,81–83}. Expression of CD163 is higher on CD16⁺ monocytes⁸⁴. Plasma sCD163 is elevated in individuals with HIV duration greater than one year, and HAART therapy reduces sCD163 commensurate with HIV RNA levels; however, sCD163 levels remain significantly higher than in controls, suggesting ongoing low-level monocyte activation⁸³. Follow-up clinical studies demonstrate that higher levels of sCD163 occur in HAND populations, in particular the MND/HAD groups, compared with those who were cognitively unimpaired⁸⁵. These results suggest that sCD163 is a novel marker of macrophage-mediated disease activity in HIV, systemically and in the CNS. However, it is not specific for HIV, nor does it distinguish the duration of infection or HAND subtype.

Another marker of monocyte activation, sCD14, a soluble monocyte lipopolysaccharide receptor, is similar to sCD163 in that levels are higher in early and chronic HIV infection than in HIV-negative controls⁸³. However, sCD14 levels were not reduced by treatment with HAART and were significantly increased after treatment with HAART in early HIV⁸³. Plasma and CSF sCD163 correlates with sCD14^83,85. However, despite this, sCD14 has had variable associations with HAND severity^85,86. Alternate monocyte biomarkers, such as CSF CCL2, correlate with HAND severity and risk of progression as well as markers of inflammation^87,88. The comparative clinical utility of these biomarkers requires further study.

Chronic inflammation and HIV proteins

The inflammatory milieu, as a potential contributor to HAND, has become an interesting area of research. It is increasingly recognized that low-level latent infection in HIV and its comorbidities (e.g. metabolic syndrome, drug use, and hepatitis C) as well as aging can lead to chronic low-grade immune activation and inflammation^89,90. Sustained CNS inflammation occurring during HAART may be due to latent and low-level infection in the CSF meningeal compartment and possibly brain (viral escape), priming of microglia and macrophages due to circulating products translocated from the gut, altered neuronal and synaptic function, contributions from the metabolic syndrome, and possible ART-related toxicity.

The macrophage and microglia biomarker of immune activation, neopterin, is elevated in most HIV seropositive individuals, with levels declining after commencement of HAART. Low viral persistence in the CNS, as measured by CSF HIV RNA, is significantly correlated with CSF neopterin elevation⁹¹. Neopterin correlates with HAND severity in ART-naïve individuals and predicts the development of HAND; however, its utility in the HAART era is not clear^92–94.

Inflammatory cytokine secretion by infected monocytes causes a cascade, with compounding effects due to positive feedback on gene transcription⁹⁵. This chronic inflammation is probably exacerbated by HIV component proteins expressed in infected cells (especially monocytes), such as Vpr, Tat, Nef, and gp120, which further activate inflammatory pathways, leading directly to neural toxicity^96–99. However, chronic inflammation in latent HIV infection with suppressed HIV RNA may be caused by deregulation of inflammatory cascades by transcriptional regulators, such as BCL11B, with resultant silencing of integrated virus⁴³. Conversely, in HIV-encephalitis (HIVE), reduced transcriptional regulation leads to re-emergence of HIV RNA in CSF and brain⁴³. This suggests that HAND with viral suppression may be driven by viral components, such as Tat and Nef, whereas HAND without viral suppression may be fuelled by whole virus, in particular envelope proteins. This is an attractive potential explanation for HAND in the context of viral suppression, as current HAART does not target the immediate post-integration transcription stage of the HIV replication cycle, allowing the continued production of Tat, Nef, and Vpr.

Tat and Nef have been found to affect endothelial activation and dysfunction, leading to cardiovascular disease¹⁰⁰. Tat transcriptionally deregulates genes and induces cytokine activation of IL-17 pathways, leading to a proinflammatory state with direct neurotoxicity¹⁰¹. In addition, it is soluble, able to exit the infected cell, and can affect neighboring cells¹⁰¹. Thus, measurement of Tat is a plausible biomarker for neural damage in HAND. However, CSF Tat does not correlate directly with HAND severity, possibly because it is only intermittently produced, or there may be difficulties with the lower limit of detection in current assays¹⁰². In the future, antisense oligonucleotides and monoclonal antibodies could be utilized to enhance the efficacy of HAART¹⁰³.

CCR5 chemokine receptor neurotropism

Viral envelope glycoproteins can fuse with only those cells expressing both CD4 and an HIV co-receptor, of which CCR5 has been identified as the most important for microglia and CNS macrophages^104,105. CCR5 receptors are upregulated on activated CD4⁺ and CD8⁺ T cells, enhancing antigen-presenting cell interaction, T cell trafficking into tissues, and cytokine production¹⁰⁶. In response to infection or inflammation, monocytes, microglia, astrocytes, and neurons express CCR5 ligands, which increase the migration of CCR5⁺ T cells into the CNS¹⁰⁶. Locally, these effector T cells secrete CCR5 ligands, which amplify the immune response¹⁰⁶. A subset of CCR5-tropic viruses have been found to be highly fusogenic, requiring lower expression levels of CCR5 and CD4 to infect cells, leading to greater apoptosis¹⁰⁷. There is now good evidence that the vast majority of HIV strains in the brain are CCR5 tropic¹⁰⁸. Given the intrinsic role of CCR5 in CNS disease, it is suggested that CCR5 antagonists, such as maraviroc, may be able to reduce the migration and then activation of effector CD8⁺ T cells in the CNS and hence reduce the neurocognitive sequelae of HIV¹⁰⁶. This is now being assessed (see the section titled “Treatment”).

Biomarkers of HIV replication

While quantification of CSF HIV DNA as a biomarker of HAND is both impracticable and insensitive¹⁰⁹, measurement of HIV RNA expression in CSF is more robust. In the pre-HAART era and ARV-naïve population, HIV RNA in blood is independently associated with cognitive impairment¹¹⁰, high levels have been associated with HAD in individuals with advanced immunosuppression, and a decrease in HIV RNA levels has been correlated with improvements in cognitive measures^111–115. While these factors suggest that HIV RNA level can be a useful biomarker in HAND, it is by no means a perfect marker; its utility in the HAART era is less well established. CSF HIV RNA levels may not accurately reflect HIV replication in brain parenchyma, though single copy assay techniques hold some promise.

Biomarkers of neural injury

Biomarkers of neural damage would be expected to correlate most closely with HAND. CSF neurofilament light chain (NFL), a component of myelinated axons, has recently been identified as a sensitive biomarker of neuronal injury in multiple neurodegenerative diseases^116–118. In HIV, CSF NFL is increased in patients with and without HAND, with levels higher in HAND and increasing as HIV disease advances, suggesting early neuronal injury¹¹⁶. CSF NFL concentrations appear to predict the development of HAD, with levels increasing 1–2 years before symptoms develop¹¹⁹. However, NFL levels are not sensitive in the detection of mild disease, in particular when virally suppressed¹¹⁸. Newer ultrasensitive plasma assays for NFL are highly correlated with CSF NFL concentrations¹¹⁷. NFL levels in plasma and CSF increase with reducing CD4 counts and increasing plasma RNA viral loads in asymptomatic individuals, correlate with HAND severity, reaching the highest values in HAD, and are reduced by HAART^117,118,120. NFL is significantly correlated with sCD163 and sCD14, suggesting that CNS monocyte activation is directly linked with neuronal injury in HAND¹¹⁸. Plasma NFL, although not specific, may be an emerging sensitive, non-invasive assay for HAND, especially as the sensitivity of assays increases.

Other biomarkers of neural damage such as tau and CSF/plasma albumin ratios, reflecting blood–brain barrier integrity, correlate with HAD and in some studies CSF neopterin^121–123. These, however, are significantly reduced in the setting of HAART and hence are less useful in the clinical setting of HAND with viral suppression¹²¹. In addition, non-invasive quantitative MRI techniques can reflect neuroinflammation and neuronal injury. A recent study demonstrated early reductions in parenchymal volume, enlargement of the brainstem and third ventricle, diffusion alterations in the caudate, altered white matter integrity in the corpus callosum, and brain metabolite changes reflecting neural injury within 100 days of seroconversion¹²⁴.

An additional emerging area of interest is microRNAs, the non-coding RNA molecules which regulate gene expression. Specific subtypes miR125b and 146a have a role in microglial infection and cell death¹²⁵. In a small study of 10 patients, nine with HAND, these microRNAs correlated with HIVE; however, the interaction with ART and viral suppression has yet to be elucidated¹²⁵.

CNS penetration by HAART

The CNS penetration effectiveness (CPE) score was proposed in order to rank drug penetration and efficacy in the CNS¹²⁶. The CPE score has been supported in most studies, where treatment regimens with a higher CPE score generally have lower rates of HIV RNA detectible in CSF^127,128. However, the issue of the utility of the CPE score remains controversial. Indeed, the CPE score is limited by its categorical scoring, unclear weighting of each criterion (pharmacokinetic, chemical properties, etc.), and lack of consideration of toxic effects or drug interactions. Furthermore, the CPE score relies on CSF drug concentrations, which may not reflect the brain parenchymal pharmacokinetics, and efficacy in glial cells is not considered, primarily due to limited data availability.

To assess this latter point, recent studies assessing the efficacy of current CNS-penetrating ARVs in astrocytes and macrophage-lineage cells have been performed using in vitro models of infection^42,129. All of the agents tested could inhibit HIV infection in macrophages with concentrations typically found in CSF⁴². Similar results were found in astrocytes, except for lamivudine, stavudine, and zidovudine, which required between 12- and 187-fold greater concentrations than are achievable in the CSF⁴². These results indicate that these drugs may not adequately treat astrocyte reservoirs and suggest that these agents should not be utilized in CNS-penetrating (NeuroHAART) regimens.

The interaction between improved NeuroHAART, commonly defined by CPE score, and clinical efficacy has not been clearly elucidated. A recent systematic review found only six methodologically sound and two sufficiently powered studies that attempted to answer this question¹³⁰. Utilizing these more rigorous studies, it was concluded that a regimen with higher CNS penetration probably improved both neurocognitive function and HIV CSF RNA concentrations¹³⁰. Subsequently, a multisite randomized controlled trial has been performed which showed no benefit for a NeuroHAART regimen¹³¹. However, there was an imbalance at entry for CD4 nadir and hepatitis C status, factors which are known to influence neurocognition. Also, it is known that HAND patients may continue to improve on a stable HAART regimen for approximately 1 year from regimen commencement; the trial requirement for stability for only 8 weeks may have been a further confounding factor. As a result, there remain no definitive HAART guidelines for HAND. Further investigations with randomized controlled trials addressing the latter shortcomings are needed¹³⁰.

HAART intensification for HAND in the context of viral suppression

Recent advances suggest that CCR5 receptor interactions with CNS reservoir cells are intrinsically linked with CNS HIV and HAND. Maraviroc, a CCR5 receptor antagonist, is a target for HAART intensification, as it has adequate CSF penetration, has low rates of resistance, inhibits CNS viral replication, including in monocyte/macrophage cells, and has anti-inflammatory properties in the CNS^132–137. Maraviroc acts by blocking HIV entry into cells, hence restricting virion replication and indirectly inhibiting immediate post-integration viral protein production, which probably drives the CNS inflammatory milieu^96–99,138. A recent prospective, open-label pilot randomized controlled trial in individuals with viral suppression and stable ART for 12 months found maraviroc-intensified HAART improved global neurocognitive performance at both 6 and 12 months without significant side effects²⁷. In the future, large randomized controlled studies will be necessary to confirm these findings; however, this suggests that maraviroc intensification of HAART could be an option for the clinical management of HAND occurring in the context of viral suppression.

Early initiation of HAART

Lower HIV DNA levels in monocytes are associated with HAART initiation within the first year of infection, suggesting that the early commencement of HAART may improve outcomes in HAND^52,139. However, the impact of immediate versus deterred HAART on neurocognitive performance in ARV-naïve patients without profound immunosuppression is unclear. The recent INSIGHT START neurology substudy aimed to assess this^140,141. Individuals were randomized to commence HAART either at baseline (CD4 >500 cells/μL) or when CD4⁺ cells were below 350 cells/μL¹⁴⁰. While serious AIDS and non-AIDS events were reduced in the immediate treatment arm, without increased adverse events, the neurocognitive substudy did not demonstrate similar improvements on neuropsychological tests^140,141. However, less than 50% of the study population reached 4 years of follow-up, the potentially neurotoxic ARV, efavirenz, was commonly used¹⁴², and low rates of HAND were found owing to short disease duration and high CD4⁺ counts. As a result, further studies with longer duration of follow-up and adequate power are required to assess the feasibility of CNS protection with early ART initiation.

Future treatment directions

Current studies are investigating intranasal insulin, IL-1 antagonists, paroxetine, and complement inhibitors as well as limiting the replication of viral transcripts by Tat monoclonal antibodies and antisense oligonucleotides.