HIV-1 Replication in the Central Nervous System Occurs in Two Distinct Cell Types

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to the development of HIV-1-associated dementia (HAD). We examined the virological characteristics of HIV-1 in the cerebrospinal fluid (CSF) of HAD subjects to explore the association between independent viral replication in the CNS and the development of overt dementia. We found that genetically compartmentalized CCR5-tropic (R5) T cell-tropic and macrophage-tropic HIV-1 populations were independently detected in the CSF of subjects diagnosed with HIV-1-associated dementia. Macrophage-tropic HIV-1 populations were genetically diverse, representing established CNS infections, while R5 T cell-tropic HIV-1 populations were clonally amplified and associated with pleocytosis. R5 T cell-tropic viruses required high levels of surface CD4 to enter cells, and their presence was correlated with rapid decay of virus in the CSF with therapy initiation (similar to virus in the blood that is replicating in activated T cells). Macrophage-tropic viruses could enter cells with low levels of CD4, and their presence was correlated with slow decay of virus in the CSF, demonstrating a separate long-lived cell as the source of the virus. These studies demonstrate two distinct virological states inferred from the CSF virus in subjects diagnosed with HAD. Finally, macrophage-tropic viruses were largely restricted to the CNS/CSF compartment and not the blood, and in one case we were able to identify the macrophage-tropic lineage as a minor variant nearly two years before its expansion in the CNS. These results suggest that HIV-1 variants in CSF can provide information about viral replication and evolution in the CNS, events that are likely to play an important role in HIV-associated neurocognitive disorders

Compartmentalization, Viral Evolution, and Viral Latency of HIV in the CNS

Human immunodeficiency virus type 1 (HIV-1) infection occurs throughout the body, and can have dramatic physical effects, such as neurocognitive impairment in the central nervous system (CNS). Furthermore, examining the virus that resides in the CNS is challenging due to its location and can only be done using samples collected either at autopsy, indirectly form the cerebral spinal fluid (CSF), or through the use of animal models. The unique milieu of the CNS fosters viral compartmentalization as well as evolution of viral sequences, allowing for new cell types, such as macrophages and microglia, to be infected. Treatment must also cross the blood brain barrier adding additional obstacles in eliminating viral populations in the CNS. These long-lived infected cell types and treatment barriers may affect functional cure strategies in people on highly active antiretroviral therapy (HAART)

Viral characteristics associated with human immunodeficiency virus type 1 infection of the central nervous system early following transmission

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can occur shortly after transmission and compartmentalized HIV-1 variants, genetically distinct from virus in the blood, can be detected in the cerebrospinal fluid (CSF) in some individuals throughout the course of infection. HIV-1 replication can induce neuropathogenesis and may also represent a distinct reservoir from that found in CD4+ T cells in the blood and lymphoid tissue. We do not know the time course of the virologic events that lead to neurological dysfunction and the potential establishment of a CNS reservoir, or the extent to which these long-term outcomes are predicted by the initial aspects of virus-host interaction. In this dissertation, I examined the viral genetic and phenotypic determinants associated with CNS compartmentalization early following both vertical and horizontal transmission. Additionally, I examined CNS compartmentalization over the full course of disease in simian immunodeficiency virus (SIV)-infected rhesus macaques. Using single genome amplification (SGA), sequencing and phylogenetic analysis, I detected significant compartmentalization in both the pediatric and adult cohorts, predominantly associated with a longer time since HIV-1 infection. Compartmentalization was also shown to persist and evolve over a period of up to two years in the adult cohort. Through the use of Bayesian Evolutionary Analysis by Sampling Trees (BEAST), I identified and examined multiple variant transmission events and defined two distinct pathways to compartmentalization: the early sequestration of a transmitted virus in the CNS; and the later establishment of an independently replicating population within the CNS that originated from the periphery. I also concluded via entry tropism analyses that in most children and adults, replication in the CNS is sustained by growth in CD4+ T cells, while in some young children the virus in the CNS evolves to replicate in cells with low CD4 surface expression, potentially macrophages and/or microglia. The observation of significant compartmentalization early following both vertical and horizontal transmission, evidence of adaptation of virus to the CNS, and the potential for early CNS involvement to result in long-term neurocognitive damage and reservoir establishment adds further justification to the policy of prompt initiation of antiretroviral treatment.Doctor of Philosoph

A Novel Role for and Potential Therapeutic Targeting of Heme Oxygenase-1 in HIV Neuropathogenesis

HIV-associated neurocognitive disorders (HAND) affect up to 50% of HIV-infected adults despite potent viral suppression with antiretroviral therapy (ART) and are associated with persistent neuronal damage, monocyte/macrophage activation, chronic inflammation, and oxidative stress. Heme oxygenase-1 (HO-1) is a highly inducible, detoxifying enzyme critical for limiting oxidative stress, inflammation, and cellular injury within the central nervous system (CNS) and other tissues. Our analysis of HO-1 expression in the brain prefrontal cortex from HIV-infected individuals demonstrated a significant HO-1 protein deficiency, even in HIV-infected subjects treated with ART. This HO-1 deficiency associated with a diagnosis of HAND and HIV-encephalitis (HIVE) as well as with elevated CNS HIV replication, type I interferon responses, and macrophage activation. Within this cohort longer variants of a HO-1 promoter region (GT)n microsatellite polymorphism, which cause reduced HO-1 gene expression, associated with increased risk of HIVE and elevated CNS macrophage activation. HIV replication in macrophages, a primary CNS HIV reservoir, selectively reduced HO-1 protein and RNA expression and induced production of neurotoxic levels of glutamate. This HO-1 deficiency and associated neurotoxin production was a conserved feature of infection with macrophage-tropic HIV-1 and HIV-2 strains that correlated closely with the extent of replication. ART regimens applied to macrophages after HIV infection was established failed to prevent this HO-1 loss and associated neurotoxin production. HO-1 siRNA knockdown and enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. These findings identify HO-1 as a host factor that is deficient in the brains of HIV-infected individuals and suggest that loss of HO-1 and its protective functions may contribute to HIV neuropathogenesis. Moreover, this work defines a predictable and conserved relationship between HIV replication, HO-1 loss, and neurotoxin production in macrophages that likely reflects processes in place in the HIV-infected brain of individuals receiving ART. Correcting this HO-1 deficiency could provide a novel approach for neuroprotection in individuals with or at risk for developing HAND above that provided by current ART

HIV-1 Compartmentalization and Reservoir Formation

With over 37 million people living with HIV-1, and over one million new infections occurring each year, HIV-1 continues to be a significant public health burden. Current therapies that eliminate viral replication are not curative and require life-long adherence, placing substantial economic, social, and physical burdens on people living with HIV-1. The implementation of an HIV-1 cure is hampered by mechanisms of HIV-1 persistence. In this dissertation, I examine two such mechanisms of persistence – compartmentalization and reservoir formation – through the analysis of genetic diversity. The genotypic and phenotypic properties of HIV-1 in the male genital tract (Chapter 2) and the central nervous system (Chapter 3) are examined using sensitive methodologies such as next generation sequencing with Primer ID and ddPCR-based single genome amplification. The results obtained further elucidate the factors influencing the establishment of compartmentalized lineages and shed light on the sources of HIV-1 observed in the cerebrospinal fluid. Currently a cure is not possible due to the presence of a long-lived reservoir of latently infected cells. Understanding the mechanisms behind reservoir formation could facilitate the design of therapeutic approaches to limit the size of the reservoir. To this end, we analyzed the formation of the long-lived HIV-1 DNA reservoir and the replication-competent reservoir in the same set of ART-treated participants. Results demonstrated that the majority of both the largely defective HIV-1 DNA and replication-competent reservoirs were seeded in the year before ART initiation, suggesting a common mechanism of formation. Furthermore, the results suggest that strategies to block reservoir formation employed near the time of ART initiation would substantially reduce the size of the reservoir, thereby lowering the barrier to an HIV-1 cure. Overall, the results presented here advance our understanding of HIV-1 compartmentalization and reservoir formation. This work examines HIV-1 diversity in multiple anatomic regions, introduces tissue-derived insights into HIV-1 replication in the central nervous system and further elucidates the mechanisms behind the establishment of the long-lived reservoir. By advancing our understanding of mechanisms of HIV-1 persistence, we continue to make progress toward a cure.Doctor of Philosoph

Shock and kill within the CNS: A promising HIV eradication approach?

The most studied HIV eradication approach is the “shock and kill” strategy, which aims to reactivate the latent reservoir by latency reversing agents (LRAs) and allowing elimination of these cells by immune-mediated clearance or viral cytopathic effects. The CNS is an anatomic compartment in which (persistent) HIV plays an important role in HIV-associated neurocognitive disorder. Restriction of the CNS by the blood–brain barrier is important for maintenance of homeostasis of the CNS microenvironment, which includes CNS-specific cell types, expression of transcription factors, and altered immune surveillance. Within the CNS predominantly myeloid cells such as microglia and perivascular macrophages are thought to be a reservoir of persistent HIV infection. Nevertheless, infection of T cells and astrocytes might also impact HIV infection in the CNS. Genetic adaptation to this microenvironment results in genetically distinct, compartmentalized viral populations with differences in transcription profiles. Because of these differences in transcription profiles, LRAs might have different effects within the CNS as compared with the periphery. Moreover, reactivation of HIV in the brain and elimination of cells within the CNS might be complex and could have detrimental consequences. Finally, independent of activity on latent HIV, LRAs themselves can have adverse neurologic effects. We provide an extensive overview of the current knowledge on compartmentalized (persistent) HIV infection in the CNS and on the “shock and kill” strategy. Subsequently, we reflect on the impact and promise of the “shock and kill” strategy on the elimination of persistent HIV in the CNS

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication

Compartmentalized Replication of R5 T Cell-Tropic HIV-1 in the Central Nervous System Early in the Course of Infection

Compartmentalized HIV-1 replication within the central nervous system (CNS) likely provides a foundation for neurocognitive impairment and a potentially important tissue reservoir. The timing of emergence and character of this local CNS replication has not been defined in a population of subjects. We examined the frequency of elevated cerebrospinal fluid (CSF) HIV-1 RNA concentration, the nature of CSF viral populations compared to the blood, and the presence of a cellular inflammatory response (with the potential to bring infected cells into the CNS) using paired CSF and blood samples obtained over the first two years of infection from 72 ART-naïve subjects. Using single genome amplification (SGA) and phylodynamics analysis of full-length env sequences, we compared CSF and blood viral populations in 33 of the 72 subjects. Independent HIV-1 replication in the CNS (compartmentalization) was detected in 20% of sample pairs analyzed by SGA, or 7% of all sample pairs, and was exclusively observed after four months of infection. In subjects with longitudinal sampling, 30% showed evidence of CNS viral replication or pleocytosis/inflammation in at least one time point, and in approximately 16% of subjects we observed evolving CSF/CNS compartmentalized viral replication and/or a marked CSF inflammatory response at multiple time points suggesting an ongoing or recurrent impact of the infection in the CNS. Two subjects had one of two transmitted lineages (or their recombinant) largely sequestered within the CNS shortly after transmission, indicating an additional mechanism for establishing early CNS replication. Transmitted variants were R5 T cell-tropic. Overall, examination of the relationships between CSF viral populations, blood and CSF HIV-1 RNA concentrations, and inflammatory responses suggested four distinct states of viral population dynamics, with associated mechanisms of local viral replication and the early influx of virus into the CNS. This study considerably enhances the generalizability of our results and greatly expands our knowledge of the early interactions of HIV-1 in the CNS

HIV-1 subtype B determinants of neuropathogenesis: viral characteristics associated with dementia

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is a severe neurological disease resulting from HIV-1 infection of cells in the central nervous system (CNS). Significant genetic compartmentalization has been detected between virus in the periphery and virus in the cerebrospinal fluid (CSF)/CNS in subjects with dementia. Although progress has been made over the past thirty years in understanding HIV-1-associated dementia, the mechanisms leading to the development of neurological disease during HIV-1 infection remain unclear. In this dissertation, I examine the neuropathogenesis of HIV-1 over the course of infection by determining the viral characteristics associated with the development of dementia in HIV-1-infected adults. Compartmentalization between the periphery and the CNS has not been previously described for subjects with primary HIV-1 infection. I detected compartmentalized HIV-1 variants in the CSF of a subset of primary infection subjects, and using longitudinal analyses I found that compartmentalization in the CSF can be resolved during primary infection. Compartmentalized HIV-1 variants in the CNS/CSF of subjects with dementia are thought to replicate in long-lived perivascular macrophages and/or microglia in the CNS. I examined the source of compartmentalized HIV-1 in the CSF of subjects with neurological disease and in neurologically-asymptomatic subjects who were initiating antiretroviral therapy. In subjects with neurological disease, I found that rapid decay of CSF-compartmentalized variants was associated with high CSF pleocytosis, whereas slow decay measured for CSF-compartmentalized variants in subjects with neurological disease was correlated with low peripheral CD4 cell count and reduced CSF pleocytosis. The longer half-lives I detected suggest that compartmentalized HIV-1 in the CSF of some HAD subjects may be originating from a long-lived cell type in the brain. I also examined the viral genotypes and phenotypes associated with the CSF-compartmentalized variants with differential decay rates. I detected significant compartmentalization in the CSF HIV-1 population for subjects with neurological disease, and the envelope phenotype characterization revealed two distinct classes of viral encephalitis associated with extensive genetic compartmentalization and the clinical diagnosis of dementia. These results will form the basis of future studies to decipher the biology underlying viral evolution and enhanced HIV-1 replication in the CNS