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Cure Studies/ART Interruption & Brain Affects, Vorinostat
 
 
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"......nine participants presented a compartmentalized HIV RNA rebound within the CSF after interruption of ART, even when sampled within 2 weeks from viral rebound......our study suggests that the HIV RNA population often rebounds independently within the CSF and that the HIV DNA reservoirs in anatomic compartments might present additional obstacles to eradication and need to be actively targeted to achieve a complete cure. Our data suggest that failing to perform lumbar punctures during structured ART interruption in the setting of cure trials might overlook important events emanating from the CNS, which could be contributing to overall rebound. Future prospective studies with more frequent sampling should determine if the virus originated within the CNS meaningfully contributes to reconstituting the HIV RNA population in blood......The most likely source of compartmentalized HIV RNA is a CNS reservoir that would need to be targeted to achieve complete HIV eradication.
 
This is important since compartmentalized HIV reservoirs within the CNS will be particularly difficult to target as part of viral eradication strategies, as a consequence of limited drug penetration, compartmentalization, tissue-specific viral adaptation and the presence of unique cellular targets"
 
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"Reactivation of simian immunodeficiency virus reservoirs in the brain of virally suppressed macaques"....tested HDAC vorinostat on brain
AIDS Jan 2017 - 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.....After 500 days of viral suppression animals were treated with two cycles of latency reversing agents and increases in viral transcripts were examined..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.....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.
 
Pdf attached here

 
from Jules: what about reseeding HIV-DNA after interruption

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HIV cure strategists: ignore the central nervous system at your patients' peril - (12/21/16) we would like to highlight additional potential perils facing HIV cure strategists with respect to the CNS; namely adverse CNS outcomes that may include toxicities of HIV cure therapies, direct immune-mediated CNS pathogenesis or the impact of viral reactivation on the brain. Mechanisms of negative outcomes on the CNS and neuronal tissue due to cure strategies could include, first, adverse effects on brain function secondary to the removal or elimination of latently infected neuronal cells with crucial function for brain health, such as microglial cells and astrocytes [4]. Second, neuronal damage from either drug utilized during cure research strategies or neuronal damage from viral proteins, the expression of which may be upregulated during cure treatments. An example being the gene upregulation resulting from histone deacetylase (HDAC) inhibitor use. Finally, a further adverse outcome, which could be a catastrophic event, is immune reconstitution inflammatory syndromes occurring in the CNS compartment. This could occur due to cytokine storms caused by immunotherapeutic agents modifying neuroinflammatory responses, or immune activation following viral rebound and blips caused by HDAC inhibitors (and similar agents) or viral rebounds associated with antiretroviral treatment interruptions.
 
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ART Interruption Associated with HIV in the Brain: "Reseeding CNS during ART Interruption Leading to Intrathecal Immune Activation" - "HIV-1 Viral Escape in Cerebrospinal Fluid of Subjects on Suppressive Antiretroviral Treatment" - (09/18/17)
 
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Compartmentalized HIV rebound in the central nervous system after interruption of antiretroviral therapy
 
Pdf attached

 
Virus Evolution 2016
 
Sara Gianella,1,*,† Sergei L. Kosakovsky Pond,3,*,† Michelli F. Oliveira,1 Konrad Scheffler,1 Matt C. Strain,1 Antonio De la Torre,2 Scott Letendre1, Davey M. Smith1,4,‡, and Ronald J. Ellis2 1Department of Medicine, University of California, San Diego, La Jolla, CA, USA, 2Departments of Neurosciencesand Psychiatry, University of California, San Diego, La Jolla, CA, USA, 3Department of Biology, Temple University, Philadelphia, PA, USA and 4Veterans Affairs San Diego Healthcare System, San Diego, CA, USA
 
Abstract
 
To design effective eradication strategies, it may be necessary to target HIV reservoirs in anatomic compartments other than blood. This study examined HIV RNA rebound following interruption of antiretroviral therapy (ART) in blood and cerebrospinal fluid (CSF) to determine whether the central nervous system (CNS) might serve as an independent source of resurgent viral replication.
 
Paired blood and CSF samples were collected longitudinally from 14 chronically HIV-infected individuals undergoing ART interruption. HIV env (C2-V3), gag (p24) and pol (reverse transcriptase) were sequenced from cell-free HIV RNA and cell-associated HIV DNA in blood and CSF using the Roche 454 FLX Titanium platform. Comprehensive sequence and phylogenetic analyses were performed to search for evidence of unique or differentially represented viral subpopulations emerging in CSF supernatant as compared with blood plasma.
 
Using a conservative definition of compartmentalization based on four distinct statistical tests, nine participants presented a compartmentalized HIV RNA rebound within the CSF after interruption of ART, even when sampled within 2 weeks from viral rebound. The degree and duration of viral compartmentalization varied considerably between subjects and between time-points within a subject. In 10 cases, we identified viral populations within the CSF supernatant at the first sampled time-point after ART interruption, which were phylogenetically distinct from those present in the paired blood plasma and mostly persisted over time (when longitudinal time-points were available). Our data suggest that an independent source of HIV RNA contributes to viral rebound within the CSF after treatment interruption. The most likely source of compartmentalized HIV RNA is a CNS reservoir that would need to be targeted to achieve complete HIV eradication.
 
This is important since compartmentalized HIV reservoirs within the CNS will be particularly difficult to target as part of viral eradication strategies, as a consequence of limited drug penetration, compartmentalization, tissue-specific viral adaptation and the presence of unique cellular targets

 
Introduction
 
Antiretroviral therapy (ART) suppresses viral replication to undetectable levels in most HIV-infected individuals (Palella et al. 2006). However, ART cannot eradicate latently infected cells (Wong et al. 1997; Finzi et al. 1999; Siliciano et al. 2003), and robust viral replication resumes following treatment interruption (Hoen et al. 2005). Discouragingly, this pattern holds in 80-90% of cases even when ART is initiated during the earliest phase of HIV infection (2-10 weeks), specifically to limit the size of the HIV reservoir and improve immune reconstitution (Hocqueloux et al. 2010; Saez-Cirion et al. 2013). Among individuals who initiate ART during chronic HIV infection (i.e. at least 3 months post infection) viral rebound is virtually universal (Palmisano et al. 2007; Saez-Cirion et al. 2013).
 
Rebounding virus might originate from a variety of sources: the DNA compartment in blood, namely peripheral blood mononuclear cells (PBMC), RNA or DNA compartments in lymphoid tissues, and possibly other anatomical compartments that harbor replication-competent HIV-infected cells or viral particles (Lewin et al. 2011; Gray et al. 2014; Margolis, 2014).
 
In most HIV-infected individuals, the establishment of viral reservoirs in multiple tissues and anatomic compartments, including the central nervous system (CNS), occurs within the first few weeks of HIV infection (Clements et al. 2005; Thompson et al. 2011; Gray et al. 2014; Salemi and Rife, 2015; Sturdevant et al. 2015). The HIV population during early HIV infection is typically homogenous (Keele et al. 2008), but compartmentalization can occur as a consequence of tissue-specific genetic differentiation and restricted viral migration between anatomic sites or tissues (Zarate et al. 2007; Blackard, 2012; Svicher et al. 2014). Compartmentalized viral evolution is frequently a consequence of discordant selective pressures (Pillai et al. 2006), and gives rise to tissue-adapted variants, which subsequently contribute to disease pathogenesis (Strain et al. 2005; Pillai et al. 2006). Phylogenetic methods needed to quantify this restriction of gene flow between compartments are numerous and well developed (Zarate et al. 2007). Our group and others have used similar methods to describe the presence of compartmentalized HIV RNA populations in the cerebrospinal fluid (CSF) supernatant, mostly using samples from ART naïve individuals (Harrington et al. 2009; Smith et al. 2009; Choi et al. 2012). While direct assessment of viral variants sampled from brain parenchyma in living subjects is rarely feasible, HIV RNA collected from the CSF can be an informative surrogate.
 
Interestingly, viral rebound in CSF supernatant almost always follows the rebound in blood plasma after ART interruption (Monteiro de Almeida et al. 2005). This lag might indicate that rebounding virus in CSF is simply imported from blood, in which case, the sequences from the two compartments should be genetically indistinguishable early after viral rebound. In contrast, if sequences of the rebounding CSF viral population are demonstrably distinct from blood, they may be derived from separate CNS sources. Additionally, the degree of compartmentalization is expected to vary over time and can be influenced by pleocytosis and other factors (Smith et al. 2009), which are proxies for the extent of cellular trafficking between CNS and blood.
 
In this study, we quantified the reservoir size and characterized the complex dynamics of viral subpopulations in a unique cohort of 14 HIV-infected individuals who had been serially sampled in CSF and blood plasma before and after interruption of ART. Comprehensive sequence and phylogenetic analyses were performed to explore the relative contribution of viral reservoirs within the CNS to the HIV RNA rebound following ART interruption. This is important since compartmentalized HIV reservoirs within the CNS will be particularly difficult to target as part of viral eradication strategies, as a consequence of limited drug penetration, compartmentalization, tissue-specific viral adaptation and the presence of unique cellular targets (Gray et al. 2014).
 
Discussion
 
A successful HIV eradication strategy must block or cripple all known mechanisms of viral persistence and recovery (Richman et al. 2009). One such mechanism is the establishment of genetically distinct viral populations in different tissues and anatomical compartments (Svicher et al. 2014). Completely eradicating viral replication and reservoirs in blood might be insufficient to achieve a cure if the virus is able to reconstitute its population within the CNS compartment. Numerous previous studies estimated that 30-60% of HIV-infected individuals harbor viral populations that are compartmentalized between blood and CNS (Blower et al. 2005; Pillai et al. 2006; Harrington et al. 2009; Sturdevant et al. 2015). Through the use of more sensitive sequencing techniques and high-throughput bioinformatics methods, our study suggests that an even larger proportion of individuals may have some degree of compartmentalization in rebounding HIV RNA populations following treatment interruption. Most of our participants (nine out of 14) presented a compartmentalized HIV RNA rebound within the CNS after ART interruption (conservatively based on 4 different tests), even when sampled a few days after viral rebound.
 
Two processes can affect the extent of differences between viral populations between blood and CSF. The first such process is genetic divergence: standard population genetics results imply that in the absence of mixing, two anatomically separate viral populations will diverge genotypically via neutral drift and/or adaptation. It is possible that a considerable amount of divergence happens before viral suppression, particularly in patients starting ART during chronic infection, as most of treated patients in the USA. Such pre-suppression divergence in turn would lead to distinct populations rebounding in the two compartments, if the rebounding populations arise independently from compartment-specific sources. The second process is trafficking between the CNS and other compartments, most importantly blood, which reduces differences between compartments by mixing and/or recombination between the viral populations (this process has been called "a fundamental evolutionary mechanism that helps to shape HIV-1 env intrahost diversity in natural infection" (Brown et al. 2011)). When significant compartmentalization is observed, as we show here in most cases of viral rebound after treatment interruption, this might suggest that the rate of trafficking is not sufficiently high to completely homogenize the populations. In contrast, the absence of compartmentalization (i.e. high population inter-mixing) could reflect either high trafficking or that the populations were not distinguishable to begin with (due to low divergence rates). We remark that the finding of viral compartmentalization does not imply completely segregated viral populations, but that there is a detectable divergence between some subpopulations in each compartment. Nevertheless, in 12 cases out of 14, we identified unique viral populations within the CSF after ART interruption, which were phylogenetically distinct from those present in the paired blood plasma. These CSF-only clades persist over time and, in two cases (of whom one was suppressed for >1.2 years before interrupting ART), the same pre-ART population reappeared in the CSF (but not in blood) after interrupting therapy. This pattern implies that at least some of the rebounding virus within the CNS originates from a CNS source and might represent a notable barrier to sterilizing cure.
 
To investigate the possible source of rebounding viruses, we also sequenced HIV DNA collected from PBMC (N = 15) and CSF cellular pellets (N = 10) for a subset of individuals. While approximately 40% of the blood populations were not compartmentalized between rebounding HIV RNA and HIV DNA sampled from the corresponding PBMC (e.g., that would occur if viral rebound were seeded by cells circulating in the blood), all but one (80%) of the CSF populations were compartmentalized between HIV RNA and HIV DNA sampled from corresponding CSF cellular pellets. While this analysis is limited by sampling bias as a consequence of low cellular input especially from the CNS cellular pellets, it is also consistent with the hypothesis that HIV RNA rebound within the CNS compartment most likely originates from cells within the brain parenchyma, which cannot be directly sampled using our methods.
 
None of the tested variables were associated with time to viral rebound, which is likely a consequence of small sample size and retrospective study design with uncertain time to rebound for most included individuals.
 
This study has several limitations. First, it only includes 14 individuals (of whom only 10 were certainly suppressed at the time of treatment interruption), thereby limiting the power of subsequent statistical analysis; we note that despite the small sample size, our study delivers the most detailed characterization of a cohort of such size to date, highlighting the amount of clinical, laboratory and informatics effort involved. Second, this is a retrospective study taking advantage of samples collected from people interrupting ART, and the optimal samples (closest to ART interruption) were not always available (except for the five participants belonging to our prospective Group 1). Consequently, there are considerable differences in timing of sampling time-points after treatment interruption. While cohort characteristics are relatively heterogeneous, our results were robust to choosing individuals sampled early or late during rebound, and independent of HIV RNA levels, time on ART or any other clinical variables. Nevertheless, the HIV RNA populations were evaluated several days after viral rebound in most participants and we cannot exclude the possibility that HIV RNA did cross from the blood to the CNS very early and diversified fast enough to confound our analysis.
 
Third, low template input into the NGS reaction and sequencing errors could cause biases or skewing in the sampling of the original viral population, and negatively impact our ability to perform accurate analyses on these samples. We have, therefore, significantly elevated the threshold of compartmentalization detection and specifically included computational tests for robustness against significant errors in frequency estimation. Template input was particularly low in some (but not all) CSF samples, which could negatively impact our capacity to find unique clades within the CSF: assuming we are simply resampling the most common variants, we are more likely to find that CSF sequences fall within better sampled blood clades. In contrast, despite the possible sampling bias in CSF, we were still able to observe CSF-unique clades in all compartmentalized time points and some were repeated across longitudinal samples further confirming the validity of our methods.
 
Despite these limitations, our study suggests that the HIV RNA population often rebounds independently within the CSF and that the HIV DNA reservoirs in anatomic compartments might present additional obstacles to eradication and need to be actively targeted to achieve a complete cure. Our data suggest that failing to perform lumbar punctures during structured ART interruption in the setting of cure trials might overlook important events emanating from the CNS, which could be contributing to overall rebound. Future prospective studies with more frequent sampling should determine if the virus originated within the CNS meaningfully contributes to reconstituting the HIV RNA population in blood.
 
Results
 
Participants, samples and clinical laboratory tests

 
Study participants (N = 14) were HIV-infected individuals with a median age of 41 years (interquartile range [IQR]: [37-45]), who had been receiving ART for a median of 4 years (IQR: 1.7-8.5 years) and voluntarily interrupted therapy between February 1999 and December 2005. Characteristics of the study participants are summarized in Table 1.
 
Ten participants had documented undetectable HIV RNA levels for at least 2 years (prospective Group 1) or 6 months (retrospective Group 2) before ART interruption. For the remaining four participants (convenience cohort), duration of HIV RNA suppression before treatment interruption was uncertain. Rebound dynamics of HIV RNA in blood plasma and CSF supernatant for five participants belonging to Group 1 are showed in Fig. 1.

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The median time from ART interruption to viral rebound in blood plasma was 19 days (IQR: 8-31 days), while the median time from ART interruption to viral rebound in CSF supernatant was 30 days (IQR: 16-44 days). The median time from viral rebound to HIV RNA sampling in blood plasma (referred as Time point 1) was 24 days (IQR: 20-87 days), while the median time from viral rebound (within the CSF compartment) to HIV RNA sampling in CSF supernatant (Time point 1) was 14 days (IQR: 4-87 days). Since our study was primarily interested in analyzing viral rebound in CSF, the earliest available time points at which HIV RNA levels were sufficient for sequence analysis were included as Time point 1. Because of the characteristic delay between rebound in blood and CSF, HIV RNA levels in blood had plateaued and were no longer substantially increasing during this time period (see Fig. 1). At Time point 1, median CD4 T cell count was 412 cells/μl [IQR: 397-476] and HIV RNA levels in blood and CSF supernatant were 4.7 log10 (copies/ml) [IQR: 4.2-5.1] and 3.3 log10 (copies/ml) [IQR: 2.7-3.8], respectively. With the exception of participant 34535, we sampled HIV RNA from blood and CSF supernatant for one or more additional time points (referred as Time points 2-5) after a median of 55 days from Time point 1 (IQR: 9-364 days). For participants 34535, 26919 and 25839, plasma and CSF supernatant were available from a time point preceding the initiation of ART.

 
 
 
 
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