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HIV cure strategists: ignore the central nervous system at your patients' peril
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AIDS Jan2 2017 Spector, Stephen A.; Rappaport, Jay
aDepartment of Pediatrics, University of California San Diego, La Jolla bRady Children's Hospital San Diego, California
Early in the AIDS epidemic, numerous investigators determined that central nervous system (CNS) disorders including dementia were common among persons infected with HIV. Moreover, it has been established that infected persons without clearly identified dementia can have mild HIV associated neurological disorders [1]. HIV can be identified within cerebrospinal fluid (CSF) of almost all HIV-infected persons not receiving antiretroviral therapy (ART) and post-mortem brain tissues have readily detectable viral expression primarily within resident microglial cells and macrophages [2,3]. Furthermore, in patients with undetectable viral load in both plasma and CSF, HIV DNA was detectable in all subjects in brain autopsy tissue [4]. Further, experimental animal models in which CD4+ T cells are depleted or absent emphasize the importance of the macrophage reservoir [5,6]. Thus, it is somewhat surprising that the CNS as an important reservoir in persons receiving treatment remains hotly debated.
Numerous publications in the era of effective ART have continued to identify subtle neurological deficits in HIV-infected persons despite what appears to be effective long-term viral suppression. This could be due to immune activation rather than infection of the CNS per-se. However, low-level replication leading to chronic inflammation remains possible. The existence of areas of persistent virus is supported by the multiple pattern decay kinetics of virus in the CNS in patients with neurological disease suggesting differences in the cellular reservoirs in these patients [7]. The association of slower decaying virus in CSF, with low CD4+ T-cell tropism, is suggestive of viral reservoirs within macrophages/microglia, rather than CD4+ T cells. If the slow decline of virus within the CNS is indeed even only partially due to macrophage infection, then curative strategies that ignore this reservoir will fail.
The article by Gama et al.[8] in this issue of AIDS provides additional support in a macaque model for the persistence of virus [in this case simian immunodeficiency virus (SIV)] within the CNS despite what appears to be effective ART suppression of virus for more than a year. In their study, one of three macaques showed increases in activation markers within CSF, and SIV transcripts were identified within the occipital cortex of resident CD68+ macrophages (likely microglia cells). Virus could be reactivated from plasma and tissues including brain. Notably, reactivation in peripheral tissues and the CNS occurred independently and viral genotypes could be distinguished between the CNS and the periphery based on phylogeny. The investigators could also show increasing levels of SIV RNA in the brain resulting from what appears to have been focal viral reactivation. It is important to note that only one of three animals showed this pattern of select CNS latency and reactivation, and it is possible that not all macaques (or humans) will harbor viral reservoirs within the CNS.
The data from the study by Gama et al.[8] are consistent with recent findings from other groups, strongly suggesting that the CNS can serve as an isolated and independent reservoir of HIV during ART. Several lines of reasoning argue that the CNS should not be ignored when developing a cure strategy for HIV including the CNS is seeded early following initial HIV infection [9,10]; ART penetration into the CNS is known to be poor for many antiretrovirals, thus establishing an environment with inadequate drug levels to fully suppress virus [11,12]; despite prolonged viral suppression on ART, markers of immune activation persist within CSF [13]; neuroimaging studies provide additional evidence for persistent inflammation in patients fully suppressed on ART [14,15]; viral RNA can be detected in the CSF of some patients but not in blood when using the same sensitive assays (CSF escape) [16-18]; and discrete viral sequences can be identified within CSF and plasma supporting the notion that compartmentalized HIV infection occurs independently within the CNS [7,19].
It is interesting to note that only a single patient has been cured after receiving a stem cell transplant from a CCR5-Δ32 homozygous donor [20]. In other patients receiving similar transplants, the time to rebound appears to be elongated, suggestive of a smaller reservoir of infection. It is important to fully characterize this reservoir. Long-lived cells, such as macrophages with slow turnover kinetics, may be particularly resistant to eradication strategies. Moreover, cure strategies should be more inclusive when approaching HIV reservoirs rather than exclusive in devising approaches to eradicate virus. The study by Gama et al.[8] adds additional information in the rhesus macaque model to the increasing accumulated data supporting the CNS, as well as the macrophage/microglia as an occult reservoir of HIV. For those HIV cure strategists, we warn that you ignore the CNS as an HIV reservoir at your patients' peril.
Reactivation of simian immunodeficiency virus reservoirs in the brain of virally suppressed macaques
Gama, Lucio; Abreu, Celina M.; Shirk, Erin N.; Price, Sarah L.; Li, Ming; Laird, Greg M.; Pate, Kelly A. Metcalf; Wietgrefe, Stephen W.; O'Connor, Shelby L.; Pianowski, Luiz; Haase, Ashley T.; Van Lint, Carine; Siliciano, Robert F.; The LRA-SIV Study Group; Clements, Janice E.
Objective: Resting CD4+ T cells have been recognized as the major cell reservoir of latent HIV-1 during antiretroviral therapy (ART). Using an simian immunodeficiency virus (SIV)/macaque model for AIDS and HIV-related neurocognitive disorders we assessed the contribution of the brain to viral latency and reactivation.
Design: Pigtailed macaques were dual inoculated with SIVDeltaB670 and SIV17E-Fr and treated with an efficacious central nervous system-penetrant ART. After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined.
Methods: Longitudinal plasma and cerebrospinal fluid (CSF) viral loads were analyzed by quantitative and digital droplet PCR. After necropsy, viral transcripts in organs were analyzed by PCR, in-situ hybridization, and phylogenetic genotyping based on env V1 loop sequences. Markers for neuronal damage and CSF activation were measured by ELISA. Results: Increases in activation markers and plasma and CSF viral loads were observed in one animal treated with latency reversing agents, despite ongoing ART. SIV transcripts were identified in occipital cortex macrophages by in-situ hybridization and CD68+ staining. The most abundant SIV genotype in CSF was unique and expanded independent from viruses found in the periphery.
Conclusion: The central nervous system harbors latent SIV genomes after long-term viral suppression by ART, indicating that the brain represents a potential viral reservoir and should be seriously considered during AIDS cure strategies.
Despite abundant evidence of HIV-1 infection in the central nervous system (CNS) during the AIDS epidemic [1,2], neuroAIDS is no longer a critical concern in the era of antiretroviral therapy (ART). HIV-1 encephalitis and dementia have declined significantly in patients whose virus replication is well suppressed by ART in peripheral blood. However, there is evidence that 30-50% of HIV-1-infected individuals on long-term ART have mild to moderate HIV-associated neurocognitive disorder (HAND) [3-5] associated with ongoing low-level inflammation in the brain, which can be directly or indirectly related to viral presence in the CNS [6-8]. ART suppresses viral replication and improves survival in HIV-1-infected patients, but does not eliminate replication-competent viral reservoirs [9-11]. Current research on ART intensification and HIV eradication focus on resting CD4+ T cells (rCD4s) with little consideration for the potential of latent reservoirs in other target cells, such as brain macrophages [12,13]. Two patients in an eradication trial in Boston were removed from ART and both developed encephalitis [14], suggesting that viral reservoirs in brain contribute to virus rebound and cause CNS disease. In addition, initial trials of HIV eradication examine only viral load in peripheral blood as an indication of HIV reactivation or change in the latent reservoir, although novel data indicate that many latent HIV-1 genomes are in tissues and may respond differently to latency reversing agents (LRA) [15].
To evaluate the contribution of the brain in virus latency and reactivation during ART we used a well characterized and consistent macaque model for AIDS and HAND in which more than 80% of infected macaques develop simian immunodeficiency virus (SIV)-associated neurological symptoms in 90 days [16]. This model has been previously evaluated under fully suppressed ART and achieved levels of viral suppression similar to those described in ART-treated HIV-1-infected patients [17]. For in-vivo activation of latent reservoirs, we tested a combination of two synergistic LRAs: the protein kinase C (PKC) activator ingenol-B and the histone deacetylase (HDAC) inhibitor vorinostat. Our results show that LRA administration lead to an increase in viral load in cerebrospinal fluid (CSF), indicating that the CNS harbors latent SIV genomes despite long-term ART suppression. Although a small number of animals were assessed, we provide for the first time in-vivo proof of concept that the brain represents a consequential viral reservoir and should be seriously considered during AIDS cure strategies.
We have previously established a reproducible and accelerated SIV macaque model for AIDS and HAND [16,26]. The model was also used to successfully demonstrate the efficacy of ART in suppressing SIV and in decreasing the number of infected rCD4s to levels similar to those in ART-treated HIV-1 infected individuals [17,22,26]. To rigorously develop an ART suppression model to evaluate latent reservoirs in the brain that closely resembles long-term treatment in virally suppressed patients, three SIV-infected macaques started receiving a CNS-penetrant ART regimen (tenofovir, darunavir, ritonavir, and the integrase inhibitor L-870812) [27] at 12 days post inoculation (p.i.). After 400 days of viral suppression, defined as fewer than 30 SIV copies/ml in plasma measured by qPCR and confirmed by digital droplet PCR, one animal (Mn0) was maintained as procedural control and two macaques (Mn1 and Mn2) were treated with latency reversing agents. Because no Food and Drug Administration-approved compound has been successfully used to reactivate HIV-1 latent reservoirs in virally suppressed humans or nonhuman primates [28-31], we used the novel PKC activator ingenol-B, which had been previously evaluated in vitro and was efficient in activating HIV-1 long-term repeats in reporter cell lines [32-34]. Before testing the compound in our SIV-macaque model, however, we evaluated whether ingenol-B activates viral genomes in ex-vivo primary cells. We initially tested the LRA in two distinct HIV-1 latency models with successful results (Fig. 1a and b). Then, to corroborate the HIV-1 data, we evaluated ingenol-B ex vivo in rCD4s isolated from SIV-infected macaques that have been virally suppressed for more than 180 days, which is the minimum time in humans for blood rCD4 viral reservoirs to stabilize [35]. Similarly to what was observed in the HIV-1 models, the compound significantly increased viral transcription, demonstrating that ingenol-B also activates SIV in macaque latently infected rCD4s (Fig. 1c). Based on these results, and also on previous studies done on dogs, it shows that the compound can be administered orally to mammals for more than 14 days without causing apparent side-effects (Fig. S1, http://links.lww.com/QAD/A981), we started the in-vivo experiments using our SIV macaque model [17,36].
For the first round of ingenol-B treatment, we chose to administer 0.4 mg/kg per day, which is 10% of the nonobserved adverse effect level established in toxicology studies (Fig. S1, http://links.lww.com/QAD/A981). After 30 days of treatment we did not observe increase in plasma viral load and therefore treated the animals for 10 more days with an increased dose of 0.6 mg/kg per day. Again, no changes in viral load were observed and ingenol-B treatment was withdrawn for 2 weeks. Treatment was reinitiated and maintained for 10 days at 1 mg/kg per day in combination with the HDAC inhibitor vorinostat (6 mg/kg subcutaneous infusion four times), which is known to synergize with PKC agonists and does not appear to successfully impact viral reservoirs as monotherapy [18,37]. Animals were kept on CNS-penetrant ART until the end of the experiment and suppressive levels of antiretroviral drugs were detected in blood, brain, spleen, and liver of all three macaques (Table S3, http://links.lww.com/QAD/A981). By the end of the dual-LRA treatment, macaque Mn2 showed detectable plasma viral load, which increased for several days after LRAs were withdrawn (Fig. 2a). No change in viral load was observed in the other LRA-treated animal Mn1 or the untreated Mn0.
The increase of plasma viral load in macaque Mn2 was concomitant with an increase of SIV viral RNA in CSF (Fig. 2b). In addition, we observed a simultaneous increase in CNS immune activation markers (CCL2 and neopterin) and in the neuronal damage marker neurofilament light chain [38], which began before the cotreatment with vorinostat (Fig. 2c). CSF viral load was 10 times higher than plasma despite the CNS-penetrant suppressive ART and the animal was euthanized 18 days after LRA interruption because of neurological symptoms (lethargy and lack of appetite).
To assess the origin of the virus detected in CSF, postmortem RNA samples from different brain regions were analyzed by ddPCR. A significant level of SIV RNA was observed in the occipital cortex of Mn2 (1700 copies/μg RNA) when compared with Mn0 and Mn1 (3 and < 1 copies/μg RNA, respectively; Table 1). These values, however, were considerably lower than RNA levels historically observed in our model in brain of animals with symptoms associated with SIV encephalitis [22]. Viral reactivation appeared to be focal as SIV RNA levels in other parts of brain (basal ganglia and parietal cortex) were as low as those in the LRA untreated animal. ISH results corroborated the PCR findings showing focal, that is,. individual cells expressing viral RNA in occipital cortex parenchyma, in and near microglial nodules (Fig. 3a, Table S4, http://links.lww.com/QAD/A981). No SIV RNA was detected by ISH in other brain regions. SIV RNA colocalized with CD68+ cells, indicating infection and reactivation in brain macrophages (Fig. 3b). To compare LRA-induced viral reactivation to viral recrudescence after ART withdrawal, we analyzed brain tissue of two virally suppressed macaques (Mn3 and Mn4, Fig. S2, http://links.lww.com/QAD/A981) who were euthanized when plasma viral load was first detectable after cessation of ART (3-4 days). Although macaque Mn3 showed lower levels of SIV RNA in brain and CSF when compared with Mn2 (164 versus 1700copies/μg brain RNA and 149 versus 18 000copies/ml of CSF), a greater number of clustered foci with varied intensities were observed in its occipital cortex (Fig. 3a), suggesting viral spread. In the LRA-untreated macaque Mn0, the LRA-treated macaque Mn1, and ART-withdrawn macaque Mn4, brain tissues were negative for SIV RNA by ISH and ddPCR.
Genotypic analyses done on the V1 region in Env of SIV RNA in plasma and CSF by RNA sequencing demonstrated that the most abundant variant in CSF (Mn2-Seq5) in macaque Mn2 was unique and had only 81.9-83.3% homology to those in the plasma (Fig. 4a-d). This suggests that LRA treatment activated distinct genomes that persisted in the CNS compartment despite long-term viral suppression. In the ART-withdrawn macaques Mn3 and Mn4, in which viral reactivation was LRA-independent, sequences from both plasma and CSF were indistinguishable from DNA sequences found in PBMCs (Fig. 4e). In contrast, in LRA-treated Mn2 macaque, the Mn2-Seq5 variant was unique to the CSF and was not represented in viral DNA samples isolated from PBMCs and peripheral tissues at necropsy.
Before the advent of ART, cognitive impairment caused by HIV-1 affected up to 50% of patients [39], indicating that, at that time, at least half of HIV-1-infected patients harbored viral genomes in the brain. In this era of efficacious ART, similar number of patients is affected by neurologic dysfunctions, the majority of which are milder forms. In addition, some groups have reported the presence of viral RNA in the CSF of treated patients [40]. Our macaque model for AIDS and HAND has been successfully used as an ART model as well, with full viral suppression in both periphery and CNS [17,26]. Here we demonstrate that, after 400 days of suppression, LRA treatment reactivated latent virus that could be detected in plasma and tissues, including brain. Reactivation in peripheral tissues and CNS occurred independently, as demonstrated by distinct SIV genotypes isolated from each compartment. Lack of cell-to-cell viral spread in the brain during LRA induction suggests that the observed levels of antiretroviral drugs in the CNS were effective and prevented de novo viral replication. The increasing levels of viral RNA in the CSF after LRA interruption appeared to be unrelated to viral spread, as indicated by the focal reactivation observed in brain by ISH. In contrast, two macaques released from suppressive ART demonstrated cell-to-cell viral spread in brain parenchyma and genotypically identical SIV genotypes in CSF and plasma. In addition, the identification of at least four distinct circulating SIV genotypes substantiates the LRA-activation hypothesis over the possibility of the development of ART-resistant strains. Yet, regardless of whether viral increase in CSF was directly caused by LRA activation, we demonstrate that macrophages in brain harbor replicative competent SIV after long-term ART treatment. Longitudinal surveillance of CSF showed an increase in viral load concomitant with detectable plasma viremia during the dual LRA induction. Inflammatory markers in this animal were upregulated in CSF by the end of the first ingenol-B cycle, prior to the addition of vorinostat, suggesting that immune activation in the CNS occurred independently of the HDAC inhibitor. Further, viral RNA in CSF was 10-fold greater than in plasma and presented unique phenotypically distinct genotypes that were not present in the periphery. Because of the focal nature of SIV infection [16], extensive sampling of brain sections would be necessary to precisely determine the CNS origin of Mn2-Seq5. These results suggest that LRA treatment in one of the macaques led to compartmentalized activation in the CNS, which may represent an obstacle for a complete and well tolerated viral eradication in HIV+ patients.
Other organs apart from brain showed increase in viral RNA expression after LRA treatment. Current reports point to follicular helper T cells as the major CD4+ T-cell compartment for HIV-1 infection and latent reservoirs [41]. A potential mechanism for the effect of LRA treatment is that the compounds reactivated latently infected follicular T cells, triggering increase in leukocyte trafficking into lymphoid tissues. Specifically, in one of the macaques, significant viral reactivation was observed in the liver, an organ that is not commonly associated with HIV-1 reservoirs.
Thus, our data suggest that the presence of virus-producing genomes in the CNS should be a cause of concern during AIDS cure strategies aimed at reactivating latent viral genomes. Increased immune activation in the brain, as we observed, may lead to reactivation of latent reservoirs followed by an exacerbated and harmful inflammatory response even in the presence of ART. As the CNS harbors macrophages with persistent replication-competent virus, monitoring CSF for viral activation or residual viremia should be seriously considered during eradication strategies.

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