Antiretroviral therapy (ART) is generally able to suppress HIV-1 RNA to very low levels in the plasma, yet HIV-infected cells persist in all ART-treated individuals and generate viral rebound if ART is stopped [1,2]. Understanding the forces maintaining these persistent reservoirs during ART can inform strategies for eliminating them. On the basis of analyses of blood cells, replication-competent viral reservoirs are very stable during ART [3,4], an observation that has prompted speculation that new HIV-infected cells may be generated during ART by low-level viral replication or by the proliferation of infected cells. Examining these hypotheses using blood and lymphocytic tissue has been the major avenue of productive research related to systemic reservoirs. However, as they require viable cells, these approaches have not been readily applicable to studies of potential reservoirs within the CNS. An alternative approach to studying CNS reservoirs is to examine viral RNA in the CNS of individuals on ART.
CSF escape is used to describe the phenomenon in which HIV-1 RNA levels in the CSF exceed those in the plasma of otherwise well suppressed individuals  and may represent production from viral reservoirs. For our studies, we have followed a relatively tolerant definition of CSF escape in which CSF HIV RNA is equal to or greater than that of the plasma within the quantifiable range of standard clinical assays. Because of slower CSF viral decay in some individuals after ART initiation, this definition also assumes stable therapy for at least 6 months (though chronic incomplete adherence may be common within this definition). Thus, CSF HIV-1 RNA of 50 copies/ml in someone with a detectable plasma HIV-1 RNA concentration of less than 50 copies would qualify in this definition. Once this definition is met, a second stage of classification is applied to categorize these individuals further. This includes neurosymptomatic CSF escape in which patients present with new or progressive CNS symptoms and signs; asymptomatic CSF escape when there are no discernable neurological abnormalities associated with the escape; and secondary CSF escape when dissociation of CSF and plasma HIV-1 RNA is found in the context of another CNS infection (e.g. borreliosis, herpes zoster, cryptococcal meningitis).
Clinically, neurosymptomatic escape is the most important of these types as it is associated with CNS morbidity and requires adjustments in therapy [6,7]. It also may be the most interesting because of its relation to independent CNS replication and the local HIV-1 reservoir as discussed below. It is usually discovered in the context of diagnostic evaluation of new neurological presentation, making it difficult to estimate its prevalence, though it is clearly uncommon. To date, there are over a hundred published accounts of symptomatic CSF escape [6–24], which has been associated with low nadir CD4+ cell count [7,25,26] and resistance-associated mutations [7,27]. These associations have been confirmed in two recent case series that reported a high incidence of symptomatic CSF escape in HIV-positive Indian patients, most of which were receiving second-line atazanavir-based cART and their viruses were harboring multiple resistance-associated mutations [17,28]. The clinical and radiological responses upon treatment optimization for individuals with symptomatic CSF escape suggest the role of incomplete antiretroviral penetration and efficacy in the central nervous system in certain individuals .
Asymptomatic escape is usually found when lumbar punctures are part of a clinical study [30,31] or incidentally when performed for an unrelated reason (e.g. unrelated headache). In cross-sectional analyses, asymptomatic CSF escape has been observed in up to 19% of individuals on monotherapy [11,32] and in 5–13% of individuals receiving combination antiretroviral therapy (cART) [30,33–36]. Individuals with asymptomatic CSF escape typically have CSF HIV-1 RNA levels that are lower than neurosymptomatic escape, though they can overlap. Most instances of asymptomatic escape are sporadic and transient, perhaps similar to plasma ‘blips’, though we have encountered individuals with sustained asymptomatic CSF escape (see Table 1 for examples of persistent CSF escape observed at multiple time points). The clinical significance of asymptomatic escape is unclear and it is not associated with reduced neurocognitive performance  or increased CSF levels of neurofilament light chain (NFL), a biomarker of axonal damage , but is associated with elevated levels of a marker of monocyte/macrophage immune activation (CSF neopterin) [30,31].
Finally, secondary CSF escape is the most poorly studied of the three types of CSF escape, but is presumably self-limited after the associated CNS infection is treated. It is thought to arise because of lymphocyte activation, traffic and secondary local HIV-1 replication provoked by the other pathogen. Accurately estimating the frequency of secondary CSF escape is difficult because it is both rare and because some individuals may experience reactivation without symptoms and therefore not receive a diagnostic lumbar punctures.
In this review, we explore what CSF escape might tell us about HIV-1 persistence in the CNS. We will start by discussing what analyses of HIV-1 populations in the CSF of untreated people have taught us about viral replication in the CNS. We will then focus on CSF escape in treated people to address whether CSF escape is produced by persistent viral reservoirs in the CNS. One limitation of this approach is that CSF escape can only identify reservoirs that are actively producing virus and will not detect latent reservoirs within the CNS. Nonetheless, this approach may be able to detect a subset of individuals with replication-competent viral reservoirs in their CNS.
The source of HIV-1 in the cerebrospinal fluid of untreated people
CSF originates in the choroid plexus, but may be altered by contributions from the brain parenchyma, surrounding leptomeninges and local vascular and perivascular tissues . Likewise, HIV-1 virions within the CSF might originate from a variety of cells in these tissue spaces. Macrophages/microglia are the primary HIV-susceptible resident cells within the CNS and can produce HIV-1 that is detected in CSF. However, CD4+ T cells can migrate from the periphery into the CNS where they may release virus if infected or serve as targets for viral replication if uninfected. Thus, virions within the CSF may be produced by cells trafficking into that compartment or from CNS resident cells.
Compartmentalization studies in untreated people
HIV-1 RNA can typically be detected in the CSF throughout untreated infection, usually at about one-tenth of the concentration noted in plasma, though the ratio of CSF to plasma HIV-1 RNA is variable. Independent replication of viral populations in the CNS for many generations produces CSF viral lineages that are genetically distinct from those in the blood plasma (i.e. compartmentalized CNS populations). Thus, the detection of compartmentalized lineages in the CSF can be used as evidence of sustained local viral replication that evolves independent of the replication taking place in the blood or systemic organs. Using this approach, we have found locally replicating populations begin emerging in the CNS within the first 2 years of infection  and have observed high rates of compartmentalization in individuals with severe neurologic disease .
Potential target cells differ in the density of the HIV-1 receptor (CD4) as well as the type, and density, of coreceptors (CCR5 or CXCR4) expressed on their surface. If a viral lineage replicates in a particular cell type for many generations, its entry properties (i.e. receptor and coreceptor usage) may become adapted to that cell type . The vast majority of HIV-1 isolated from the blood of untreated people is well adapted to entering cells that express a high density of CD4 on their surface , like the density expressed on CD4+ T cells . In contrast, we [39,43] and others [44–49] have previously found that some viruses isolated from the CSF (particularly from the CSF of individuals with neurologic disease), have an enhanced ability to infect cells that express a low density of CD4, similar to that found on macrophages . Such differences in CD4 utilization are indicative of viruses adapted to infect macrophages (M-tropic) and distinguish them from the majority of HIV-1 variants (i.e. T-cell-tropic variants) which require high levels of CD4 in order to enter host cells . This is supported by our analyses showing that compartmentalized, M-tropic CSF HIV-1 populations decay very slowly after ART initiation, reflecting their production by long-lived macrophage/microglia in the CNS  and contrasting with the rapid decay of T-tropic CSF HIV-1 that parallels viral decay in plasma. Together these studies suggest that during untreated infection, HIV-1 populations can be established in macrophage/microglia within the CNS and that HIV-1 can replicate in these cells long enough to form divergent lineages and adapt to utilizing their low surface density of CD4. It remains unknown whether these populations produce viral reservoirs in macrophage/microglia that persist during long-term ART.
Surprisingly, we have also detected compartmentalized T-cell-tropic viral populations in the CSF of some untreated individuals , suggesting that these viral populations have been replicating and evolving in CD4+ T cells in the CNS. The possibility that T cells could maintain viral populations in the CNS is also supported by a recent study showing that HIV-1 colonizes and maintains populations in the CNS of humanized mice that contain human T cells, but lack human macrophage/microglia . This raises the question as to whether CD4+ T cells can form long-term CNS populations that support viral persistence in the CNS during ART. The alternative is that compartmentalized T-cell-tropic lineages in the CNS are generated by replication in CD4+ T cells that continuously migrate into the CNS. However, this hypothesis seems unlikely given that the influx of CD4+ T would also bring virus from the periphery that could recombine with virus in the CNS, thus reducing genetic differences between these compartments and eliminating compartmentalization.
The possibility that CD4+ T cells can exist as long-term populations in the CNS (i.e. CNS resident cells) rather than just as transient migrants has gathered support in recent years. Although neuroinflammation and the detection of T cells in the brain has long been associated with CNS disease, it is now appreciated that CD4+ T cells play important roles in maintaining CNS health and function . Much of the neuroprotective effects of CD4+ T cells may play out at the choroid plexus, which serves as a ‘neuro-immunological interface’ between the immune privileged brain and the periphery [52,53]. Interestingly, CD4+ T cells rapidly home to the choroid plexus where they can become activated by antigen-presenting cells and proliferate . Additional work is needed to determine whether long-term (resident) populations of CD4+ T may be maintained by cellular proliferation at the choroid plexus.
Together, studies of HIV-1 populations in untreated people indicate that the CNS may contain many HIV-infected macrophage, microglia and CD4+ T cells. The next question is whether these HIV-infected cells persist during ART. If they can, then it is important to address their ability to generate viral rebound if ART is stopped and whether low-level viral replication continues in the CNS during ART.
Evidence that cerebrospinal fluid escape virus is produced by persistent viral reservoirs in the central nervous system
What can cerebrospinal fluid escape tell us about central nervous system reservoirs?
Analyses of viral reservoirs typically require cells/tissues collected directly from the tissue/compartment of interest. Unfortunately, current techniques require tissue from autopsy or biopsy in order to directly analyze CNS reservoirs. Further, multiple studies have shown that if brain tissue is collected at autopsy from humans that experienced agonal events, the harvested CNS cells often exhibit reduced cell viability and degraded mRNA . Additionally, ART may be discontinued near the time of death, so that autopsy samples may not be representative of ART-suppressed tissue. Thus, the often-uncertain quality of archived human brain tissue hampers the use of QVOA or analyses of HIV-1 RNA concentrations to assess viral persistence during ART. Although DNA analyses are less likely to suffer from issues of cellular viability or nucleic acid integrity, studies using T cells isolated from the blood of ART-treated people indicate that most proviral genomes are defective , and thus DNA analyses may provide little information about replication-competent reservoirs.
Analyses of CSF escape provide special opportunities to examine CNS reservoirs in living patients, though with some limitations. In ART-treated individuals, detection of CSF escape with undetectable levels of virus in the plasma is evidence that HIV-1 is being produced in the CNS. However, additional information is needed to determine whether CSF escape is produced by CNS reservoirs (macrophage/microglia or possibly long-term populations of CD4+ T cells) rather than by CD4+ T cells that are transiently trafficking through that compartment. Such evidence can come in the form of CSF escape populations that persist over long periods of time, are genetically diverse, are macrophage-tropic, or appear to be evolving during ART (all described below). Such populations are unlikely to be produced by cells that transiently migrate into the CNS and release virus but are much more likely to be produced by HIV-infected cells that persist in that compartment for long periods of time.
Persistent cerebrospinal fluid escape
CSF escape is a rare phenomenon, which few studies have examined longitudinally. This is unfortunate because persistent CSF escape (i.e. CSF escape observed over sequential time points) most likely represents HIV-1 production/replication in CNS resident cells, whereas episodic CSF escape (i.e. CSF escape that is brief and quickly resolves) may be produced by infected cells that traffic into the CNS and release virus and/or transiently support viral replication. The rationale that persistent CSF escape is produced by CNS reservoirs is based on the observation that CSF escape is overall rare and the probability that HIV-infected cells from the periphery migrate into the CNS multiple times over the course of months or years and begin producing virus that reaches detectable levels is exceedingly low. Similarly, the likelihood that an HIV-infected cell would migrate into the CNS and continue producing large amounts of virus for many months also seems unlikely. As a result, we favor the interpretation that persistent CSF escape is typically produced by CNS resident cells and represents persistent reservoirs in this compartment.
Despite a paucity of longitudinal studies of CSF escape, there are now a number of examples of persistent CSF escape (Table 1). We have previously performed lumbar punctures in a study of 98 neurologically asymptomatic people enrolled in the THINC study at Yale, UNC or UCSF, and observed 6 with CSF escape . We then performed longitudinal analyses on three participants with CSF escape and found one persistent case (1% of the cohort). Similarly, in Torino (Italy), among 71 HIV-positive patients who received at least two lumbar punctures (median time from the first to the subsequent lumbar puncture was 14 months, IQR: 4–21) because of advanced infection or a variety of symptoms including HAND, white matter abnormalities, lymphoma, neurologic symptoms and CNS infections, we observed 22 cases of episodic CSF escape and 4 persistent CSF escape cases (5.6% persistent overall; 15.4% persistent among individuals with CSF escape; Table 1). Overall, these findings indicate that persistent CSF escape is rare, but does occur and suggests that CNS reservoirs persist in at least a subset of individuals.
Genetically diverse cerebrospinal fluid escape populations
Detecting genetically diverse CSF escape populations is an additional piece of evidence that viral reservoirs persist in the CNS during ART. This assumes that overall systemic replication is well suppressed so that diverse CSF escape populations do not represent poor suppression in general. It also assumes that the number of HIV-infected cells migrating through the CNS is small making it highly unlikely that many of these cells (most likely CD4+ T cells ) will simultaneously release virus into the CSF of ART-treated people. For example, if the number of CSF white blood cells (CSF WBCs) is greatly elevated to 20 cells/μl and there are ∼150 ml of CSF, then the CSF would contain ∼3 × 106 total white blood cells, of which only about a quarter are CD4+ T cells (see a recent analysis of the cellular composition of CSF in HIV-infected people ). Thus, despite highly elevated CSF WBC counts, the CSF would contain fewer than a million CD4+ T cells (≈7.5 × 105). If we assume that these cells harbor inducible proviruses at a rate similar to that of resting CD4+ T cells in the blood (i.e. ≈1 out of a million cells [3,59]) then the probability of multiple HIV-infected cells migrating into the CSF and expressing virus is extremely low. On the basis of this logic, genetically diverse CSF escape populations are most likely produced by CNS resident cells infected with and expressing a diverse population of HIV-1 proviruses. However, the converse is not informative because a low-diversity CSF escape population could be produced either by small number of migratory cells or a small number of CNS resident cells.
We have used deep sequencing (Illumina MiSeq with PrimerID) and single genome analysis with sanger sequencing to generate env sequences from the CSF of four participants with CSF escape (Table 2). Of these, one participant with symptomatic CSF escape (Fig. 1a and ) and one with asymptomatic CSF escape  had highly diverse populations that were likely produced by many HIV-infected cells that persisted in the CNS during extended ART therapy. For the participant featured in Fig. 1 with symptomatic CSF escape, we identified 225 unique CSF escape viruses (Fig. 1b). The majority of these sequences are likely produced by persistent reservoirs in the CNS, however, given the exceptionally high CSF WBC count in this symptomatic individual (44 cells/μl), we cannot rule out the possibility that a few of the CSF escape variants may have been produced by HIV-infected cells that trafficked into the CNS from the periphery. Of the four individuals examined, two had diverse populations (described above) and the remaining two participants (both of whom had asymptomatic CSF escape) had little to no genetic variation in the CSF population (, Table 2). These low-diversity populations may have either been produced by HIV-infected cells that migrated into the CNS from the periphery or from a small number of CNS resident cells.
Macrophage-tropic cerebrospinal fluid escape virus
Given that macrophage-tropic HIV-1 is almost never observed in the blood plasma  and has primarily been found in compartmentalized lineages in the CNS , macrophage-tropic CSF escape virus is almost certainly produced by HIV-infected cells in the CNS. After performing sequence analyses of CSF escape populations for the four participants described above, we then used our established methods for assessing the entry properties of HIV-1 Envs . Specifically, we assessed the ability of these Envs to facilitate entry into cells expressing a low surface density of CD4, similar to levels expressed by monocyte-derived macrophages. Multiple studies have previously shown that viruses adapted to infecting macrophages have an enhanced ability to infect cells that express a low density of CD4 [39,44–49].
In these four participants, only one participant had a CSF escape population that was macrophage-tropic . This participant had a persistent, highly diverse CSF escape population, which provides very strong support for this population being produced by HIV-infected macrophage/microglia in the brain parenchyma (3026; Table 2). The remaining three individuals had CSF escape populations that were T-cell tropic (Table 2), an observation that cannot be used to discriminate between viruses that are being produced by migratory T cells from the periphery or resident T cells in the CNS.
Evidence of ongoing replication in the central nervous system of individuals with cerebrospinal fluid escape
During ART, HIV-1 particles may be released from infected cells in the absence of replication. Alternatively, virus may continue to replicate if they are resistant to the drug regimen and/or drug concentrations are subtherapeutic. CSF escape has been repeatedly associated with drug resistance [7,27,61] a pattern that could only be produced by ongoing replication of drug-resistant variants in the CNS, not just viral production in the absence of replication. This inference is strengthened by studies showing that CSF escape often resolves after drug regimens are optimized [6,7].
Genetic analyses of persistent CSF escape can also be used to determine whether these populations continue to replicate and evolve during therapy. In one case of persistent CSF escape of a macrophage-tropic population, we found patterns of evolving drug resistance during ART indicating that this population continued to replicate during ART .
To our knowledge, we have performed the first genetic and phenotypic analyses of CSF escape (three as part of the THINC study and one enrolled in Torino, Italy). On the basis of a number of observations, we were able to conclude that half of the CSF escape populations were produced by CNS resident cells (Table 2). Surprisingly, our data suggest that in one subject, this resident cell population is most likely macrophage/microglia, but in the other subject, the population was likely CD4+ T cells. This observation, and our previous studies observed that T-cell-tropic HIV-1 lineages can be compartmentalized in the CNS , indicate that populations of CD4+ T cells may support viral persistence in the CNS both before and after ART initiation.
There are, however, at least two caveats to this work. First, we use a variety of methods to infer the cells producing CSF escape virus, but we do not directly observe infected cells to confirm the cell type being infected or its location within the CNS. Directly observing CNS reservoirs in vivo is challenging and requires either tissues from improved animal models that retain many of the features of HIV-1 colonization of the CNS in humans or autopsy brain samples collected from durably suppressed humans. Second, genetic and phenotypic analyses of CSF escape are often only possible when CSF viral RNA concentrations are high. Whether our results are representative of CSF escape populations with lower viral loads (i.e. <100 cp/ml of HIV-1 RNA) remains unknown. Despite these caveats and the small number of CSF escape populations that have been analyzed so far, our results clearly illustrate that HIV-1 populations can persist in the CNS after extended ART.
This work was supported by NIH grants P01MH094177 and R01 NS094067 and by Università di Torinò Ricerca Locale (ex-60%) – 2015”. The work was also supported by the UNC Center For AIDS Research (NIH award P30 AI050410) and the UNC Lineberger Comprehensive Cancer Center (NIH award P30 CA16068).
Conflicts of interest
There are no conflicts of interest.
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