Rebound HIV-1 in cerebrospinal fluid after antiviral therapy interruption is mainly clonally amplified R5 T cell-tropic virus.

HIV-1 persists as a latent reservoir in people receiving suppressive antiretroviral therapy (ART). When ART is interrupted (treatment interruption/TI), rebound virus re-initiates systemic infection in the lymphoid system. During TI, HIV-1 is also detected in cerebrospinal fluid (CSF), although the source of this rebound virus is unknown. To investigate whether there is a distinct HIV-1 reservoir in the central nervous system (CNS), we compared rebound virus after TI in the blood and CSF of 11 participants. Peak rebound CSF viral loads vary and we show that high viral loads and the appearance of clonally amplified viral lineages in the CSF are correlated with the transient influx of white blood cells. We found no evidence of rebound macrophage-tropic virus in the CSF, even in one individual who had macrophage-tropic HIV-1 in the CSF pre-therapy. We propose a model in which R5 T cell-tropic virus is released from infected T cells that enter the CNS from the blood (or are resident in the CNS during therapy), with clonal amplification of infected T cells and virus replication occurring in the CNS during TI.

HIV-1 is Transported into the Central Nervous System by Trafficking Infected Cells.

Background: In this work, we carried out a cross-sectional study examining HIV-1 and HCV free virus concentrations in blood and cerebrospinal fluid (CSF) to determine whether HIV-1 enters the central nervous system (CNS) passively as virus particles or in the context of migrating infected cells. If virions migrate freely across the blood-cerebrospinal fluid barrier (BCSFB) or the blood-brain barrier (BBB) then HCV and HIV-1 would be detectable in the CSF at proportions similar to that in the blood. Alternatively, virus entry as an infected cell would favor selective entry of HIV-1. Methods: We measured HIV-1 and HCV viral loads in the CSF and blood plasma of 4 co-infected participants who were not on antiviral regimens for either infection. We also generated HIV-1 env sequences and performed phylogenetic analyses to determine whether HIV-1 populations in the CSF of these participants were being maintained by local replication. Results: While CSF samples taken from all participants had detectable levels of HIV-1, HCV was not detectable in any of the CSF samples despite participants having HCV concentrations in their blood plasma, which exceeded that of HIV-1. Further, there was no evidence of compartmentalized HIV-1 replication in the CNS (Supplementary Figure 1). These results are consistent with a model where HIV-1 particles cross the BBB or the BCSFB within infected cells. In this scenario, we would expect HIV-1 to reach the CSF more readily because the blood contains a much greater number of HIV-infected cells than HCV-infected cells. Conclusions: HCV entry into the CSF is restricted, indicating that virions do not freely migrate across these barriers and supporting the concept that HIV-1 is transported across the BCSFB and/or BBB by the migration of HIV-infected cells as part of an inflammatory response or normal surveillance.

What can characterization of cerebrospinal fluid escape populations teach us about viral reservoirs in the central nervous system?

OBJECTIVE: To review the evidence that CSF (cerebrospinal fluid) escape populations are produced by viral reservoirs in the central nervous system (CNS). DESIGN: CSF escape is a rare phenomenon in which individuals on suppressive ART have well controlled systemic infections with elevated levels of HIV-1 RNA in their CSF. However, the rarity of CSF escape coupled with relatively low CSF viral loads has impeded detailed analyses of these populations. Here, and in a previous study, we performed genetic and phenotypic assessments of CSF escape populations to determine whether CSF escape is produced by CNS reservoirs or by cells trafficking through the CNS. METHODS: We report HIV-1 viral loads in the CSF and blood plasma of four individuals with CSF escape (one new example and three previously described examples). We performed phylogenetic analyses of the viral env gene to evaluate diversity within the CSF escape populations and performed entry analyses to determine whether Env proteins were adapted to entering macrophage/microglia. RESULTS: Two individuals had CSF escape produced by CNS reservoirs. In contrast, the remaining two cases were likely because of transient viral production from cells migrating into the CNS and releasing virus. CONCLUSION: Together our analyses indicate that replication-competent HIV-1 can persist in the CNS during ART, but that not all cases of CSF escape are produced by CNS reservoirs. Our results also suggest that both CD4 T cells and macrophage/microglia can serve as persistent viral reservoirs in the CNS.

Rationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies.

An array of carbohydrates masks the HIV-1 surface protein Env, contributing to the evasion of humoral immunity. In most HIV-1 isolates 'glycan holes' occur due to natural sequence variation, potentially revealing the underlying protein surface to the immune system. Here we computationally design epitopes that mimic such surface features (carbohydrate-occluded neutralization epitopes or CONE) of Env through 'epitope transplantation', in which the target region is presented on a carrier protein scaffold with preserved structural properties. Scaffolds displaying the four CONEs are examined for structure and immunogenicity. Crystal structures of two designed proteins reflect the computational models and accurately mimic the native conformations of CONEs. The sera from rabbits immunized with several CONE immunogens display Env binding activity. Our method determines essential structural elements for targets of protective antibodies. The ability to design immunogens with high mimicry to viral proteins also makes possible the exploration of new templates for vaccine development.

Predicting Efavirenz Concentrations in the Brain Tissue of HIV-Infected Individuals and Exploring their Relationship to Neurocognitive Impairment.

Sparse data exist on the penetration of antiretrovirals into brain tissue. In this work, we present a framework to use efavirenz (EFV) pharmacokinetic (PK) data in plasma, cerebrospinal fluid (CSF), and brain tissue of eight rhesus macaques to predict brain tissue concentrations in HIV-infected individuals. We then perform exposure-response analysis with the model-predicted EFV area under the concentration-time curve (AUC) and neurocognitive scores collected from a group of 24 HIV-infected participants. Adult rhesus macaques were dosed daily with 200 mg EFV (as part of a four-drug regimen) for 10 days. Plasma was collected at 8 time points over 10 days and at necropsy, whereas CSF and brain tissue were collected at necropsy. In the clinical study, data were obtained from one paired plasma and CSF sample of participants prescribed EFV, and neuropsychological test evaluations were administered across 15 domains. PK modeling was performed using ADAPT version 5.0 Biomedical Simulation Resource, Los Angeles, CA) with the iterative two-stage estimation method. An eight-compartment model best described EFV distribution across the plasma, CSF, and brain tissue of rhesus macaques and humans. Model-predicted median brain tissue concentrations in humans were 31 and 8,000 ng/mL, respectively. Model-predicted brain tissue AUC was highly correlated with plasma AUC (γ = 0.99, P < 0.001) but not CSF AUC (γ = 0.34, P = 0.1) and did not show any relationship with neurocognitive scores (γ < 0.05, P > 0.05). This analysis provides an approach to estimate PK the brain tissue in order to perform PK/pharmacodynamic analyses at the target site.

Human Immunodeficiency Virus Type 1 RNA Detected in the Central Nervous System (CNS) After Years of Suppressive Antiretroviral Therapy Can Originate from a Replicating CNS Reservoir or Clonally Expanded Cells.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) populations are detected in cerebrospinal fluid (CSF) of some people on suppressive antiretroviral therapy (ART). Detailed analysis of these populations may reveal whether they are produced by central nervous system (CNS) reservoirs. METHODS: We performed a study of 101 asymptomatic participants on stable ART. HIV-1 RNA concentrations were cross-sectionally measured in CSF and plasma. In participants with CSF HIV-1 RNA concentrations sufficient for analysis, viral populations were genetically and phenotypically characterized over multiple time points. RESULTS: For 6% of participants (6 of 101), the concentration of HIV-1 RNA in their CSF was ≥0.5 log copies/mL above that of plasma (ie, CSF escape). We generated viral envelope sequences from CSF of 3 participants. One had a persistent CSF escape population that was macrophage-tropic, partially drug resistant, genetically diverse, and closely related to a minor macrophage-tropic lineage present in the blood prior to viral suppression and enriched for after ART. Two participants (1 suppressed and 1 not) had transient CSF escape populations that were R5 T cell-tropic with little genetic diversity. CONCLUSIONS: Extensive analysis of viral populations in 1 participant revealed that CSF escape was from a persistently replicating population, likely in macrophages/microglia, present in the CNS over 3 years of ART. CSF escape in 2 other participants was likely produced by trafficking and transient expansion of infected T cells in the CNS. Our results show that CNS reservoirs can persist during ART and that CSF escape is not exclusively produced by replicating CNS reservoirs.

Phenotypic Correlates of HIV-1 Macrophage Tropism.

UNLABELLED: HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4(+) T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE: HIV-1 typically replicates in CD4(+) T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses evolve and differ from CCR5-using T cell-tropic viruses may provide insights into viral evolution and pathogenesis within the central nervous system. We characterized the HIV-1 env viral entry gene from subject-matched macrophage-tropic and T cell-tropic viruses to identify entry features of macrophage-tropic viruses. We observed several differences between T cell-tropic and macrophage-tropic Env proteins, including functional differences with host CD4 receptor engagement and possible changes in the CD4 binding site and V1/V2 region. We also identified viruses with phenotypes between that of "true" macrophage-tropic and T cell-tropic viruses, which may represent evolutionary intermediates in a multistep process to macrophage tropism.

Ongoing HIV Transmission and the HIV Care Continuum in North Carolina.

OBJECTIVE: HIV transmission is influenced by status awareness and receipt of care and treatment. We analyzed these attributes of named partners of persons with acute HIV infection (index AHI cases) to characterize the transmission landscape in North Carolina (NC). DESIGN: Secondary analysis of programmatic data. METHODS: We used data from the NC Screening and Tracing of Active Transmission Program (2002-2013) to determine HIV status (uninfected, AHI, or chronic HIV infection [CHI]), diagnosis status (new or previously-diagnosed), and care and treatment status (not in care, in care and not on treatment, in care and on treatment) of index AHI cases' named partners. We developed an algorithm identifying the most likely transmission source among known HIV-infected partners to estimate the proportion of transmissions arising from contact with persons at different HIV continuum stages. We conducted a complementary analysis among a subset of index AHI cases and partners with phylogenetically-linked viruses. RESULTS: Overall, 358 index AHI cases named 932 partners, of which 218 were found to be HIV-infected (162 (74.3%) previously-diagnosed, 11 (5.0%) new AHI, 45 (20.6%) new CHI). Most transmission events appeared attributable to previously-diagnosed partners (77.4%, 95% confidence interval 69.4-85.3%). Among these previously-diagnosed partners, 23.2% (14.0-32.3%) were reported as in care and on treatment near the index AHI case diagnosis date. In the subset study of 33 phylogenetically-linked cases and partners, 60.6% of partners were previously diagnosed (43.9-77.3%). CONCLUSIONS: A substantial proportion of HIV transmission in this setting appears attributable to contact with previously-diagnosed partners, reinforcing the need for improved engagement in care after diagnosis.

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).

Comparison of viral Env proteins from acute and chronic infections with subtype C human immunodeficiency virus type 1 identifies differences in glycosylation and CCR5 utilization and suggests a new strategy for immunogen design.

Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.