Stoichiometry for entry and binding properties of the Env protein of R5 T cell-tropic HIV-1 and its evolutionary variant of macrophage-tropic HIV-1.

Human immunodeficiency virus type 1 typically requires a high density of CD4 for efficient entry as a mechanism to target CD4+ T cells (T-tropic), with CCR5 being used most often as the coreceptor. When target T cells are limiting, the virus can evolve to infect cells with a low density of CD4 such as macrophages (M-tropic). The entry phenotype is known to be encoded in the viral Env protein on the surface of the virus particle. Using data showing a dose response for infectivity based on CD4 surface density, we built a model consistent with T-tropic viruses requiring multiple CD4 molecules to mediate infection, whereas M-tropic viruses can infect cells using a single CD4 receptor molecule interaction. We also found that T-tropic viruses bound to the surface of cells with a low density of CD4 are released more slowly than M-tropic viruses which we modeled to be due to multiple interactions of the T-tropic virus with multiple CD4 molecules to allow the initial stable binding. Finally, we found that some M-tropic Env proteins, as the gp120 subunit, possess an enhanced affinity for CD4 compared with their T-tropic pair, indicating that the evolution of macrophage tropism can be reflected both in the closed Env trimer conformation on the virion surface and, in some cases, also in the open confirmation of gp120 Env. Collectively, these studies reveal differences in the stoichiometry of interaction of T-tropic and M-tropic viruses with CD4 and start to identify the basis of binding differences at the biochemical level. IMPORTANCE: Human immunodeficiency virus type 1 normally targets CD4+ T cells for viral replication. When T cells are limiting, the virus can evolve to infect myeloid cells. The evolutionary step involves a change from requiring a high surface density of CD4 for entry to being able to infect cells with a low density of CD4, as is found on myeloid lineage cells such as macrophage and microglia. Viruses able to infect macrophages efficiently are most often found in the CNS late in the disease course, and such viruses may contribute to neurocognitive impairment. Here, we examine the CD4 binding properties of the viral Env protein to explore these two different entry phenotypes.

The timing of HIV-1 infection of cells that persist on therapy is not strongly influenced by replication competency or cellular tropism of the provirus.

People with HIV-1 (PWH) on antiretroviral therapy (ART) can maintain undetectable virus levels, but a small pool of infected cells persists. This pool is largely comprised of defective proviruses that may produce HIV-1 proteins but are incapable of making infectious virus, with only a fraction (~10%) of these cells harboring intact viral genomes, some of which produce infectious virus following ex vivo stimulation (i.e. inducible intact proviruses). A majority of the inducible proviruses that persist on ART are formed near the time of therapy initiation. Here we compared proviral DNA (assessed here as 3' half genomes amplified from total cellular DNA) and inducible replication competent viruses in the pool of infected cells that persists during ART to determine if the original infection of these cells occurred at comparable times prior to therapy initiation. Overall, the average percent of proviruses that formed late (i.e. around the time of ART initiation, 60%) did not differ from the average percent of replication competent inducible viruses that formed late (69%), and this was also true for proviral DNA that was hypermutated (57%). Further, there was no evidence that entry into the long-lived infected cell pool was impeded by the ability to use the CXCR4 coreceptor, nor was the formation of long-lived infected cells enhanced during primary infection, when viral loads are exceptionally high. We observed that infection of cells that transitioned to be long-lived was enhanced among people with a lower nadir CD4+ T cell count. Together these data suggest that the timing of infection of cells that become long-lived is impacted more by biological processes associated with immunodeficiency before ART than the replication competency and/or cellular tropism of the infecting virus or the intactness of the provirus. Further research is needed to determine the mechanistic link between immunodeficiency and the timing of infected cells transitioning to the long-lived pool, particularly whether this is due to differences in infected cell clearance, turnover rates and/or homeostatic proliferation before and after ART.

Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy.

Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.

Nonsuppressible viremia during HIV-1 therapy meets molecular virology.

HIV-1 replication can be suppressed with antiretroviral therapy (ART), but individuals who stop taking ART soon become viremic again. Some people experience extended times of detectable viremia despite optimal adherence to ART. In this issue of the JCI, White, Wu, and coauthors elucidate a source of nonsuppressible viremia (NSV) in treatment-adherent patients - clonally expanded T cells harboring HIV-1 proviruses with small deletions or mutations in the 5'-leader, the UTR that includes the major splice donor site of viral RNA. These mutations altered viral RNA-splicing efficiency and RNA dimerization and packaging, yet still allowed production of detectable levels of noninfectious virus particles. These particles lacked the HIV-1 Env surface protein required for cell entry and failed to form the mature capsid cone required for infectivity. These studies improve our understanding of NSV and the regulation of viral functions in the 5'-leader with implications for rationalized care in individuals with NSV.

Biotypes of Central Nervous System Complications in People With Human Immunodeficiency Virus: Virology, Immunology, and Neuropathology.

Despite viral suppression with antiretroviral therapy (ART), people with human immunodeficiency virus (HIV) continue to experience central nervous system (CNS) complications, primarily in the form of mild cognitive impairment and mental health disorders (eg, depression, anxiety, other neuropsychiatric problems). The multifactorial pathogenesis and heterogeneity of mechanisms likely underlying CNS complications must be addressed in the development of preventive interventions and effective treatments. The biotyping approach has previously been useful to define phenotypes of other CNS diseases based on underlying mechanisms and could be translated to the field of neuroHIV. The purpose of the Biotype Workshop series, and the Virology, Immunology and Neuropathology Working Group in particular, is to capitalize on current and new technologies and guide future research efforts using the wealth of available immunological, virologic, and neuropathological data collected from people with HIV on and off ART.

Cardiovascular Disease and Thrombosis in HIV Infection.

HIV infection has transitioned from an acute, fatal disease to a chronic one managed by antiretroviral therapy. Thus, the aging population of people living with HIV (PLWH) continues to expand. HIV infection results in a dysregulated immune system, wherein CD4+ T cells are depleted, particularly in the gastrointestinal tract, disrupting the gut epithelial barrier. Long-term HIV infection is associated with chronic inflammation through potentially direct mechanisms caused by viral replication or exposure to viral proteins and indirect mechanisms resulting from increased translocation of microbial products from the intestine or exposure to antiretroviral therapy. Chronic inflammation (as marked by IL [interleukin]-6 and CRP [C-reactive protein]) in PLWH promotes endothelial cell dysfunction and atherosclerosis. PLWH show significantly increased rates of cardiovascular disease, such as myocardial infarction (risk ratio, 1.79 [95% CI, 1.54-2.08]) and stroke (risk ratio, 2.56 [95% CI, 1.43-4.61]). In addition, PLWH have increased levels of the coagulation biomarker D-dimer and have a two to ten-fold increased risk of venous thromboembolism compared with the general population. Several small clinical trials analyzed the effect of different antithrombotic agents on platelet activation, coagulation, inflammation, and immune cell activation. Although some markers for coagulation were reduced, most agents failed to reduce inflammatory markers in PLWH. More studies are needed to understand the underlying mechanisms driving inflammation in PLWH to create better therapies for lowering chronic inflammation in PLWH. Such therapies can potentially reduce atherosclerosis, cardiovascular disease, and thrombosis rates in PLWH and thus overall mortality in this population.

Characterization of Macrophage-Tropic HIV-1 Infection of Central Nervous System Cells and the Influence of Inflammation.

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

Stable Latent HIV Infection and Low-level Viremia Despite Treatment With the Broadly Neutralizing Antibody VRC07-523LS and the Latency Reversal Agent Vorinostat.

We tested the combination of a broadly neutralizing HIV antibody with the latency reversal agent vorinostat (VOR). Eight participants received 2 month-long cycles of VRC07-523LS with VOR. Low-level viremia, resting CD4+ T-cell-associated HIV RNA (rca-RNA) was measured, and intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) were performed at baseline and posttreatment. In 3 participants, IPDA and QVOA declines were accompanied by significant declines of rca-RNA. However, no IPDA or QVOA declines clearly exceeded assay variance or natural decay. Increased resistance to VRC07-523LS was not observed. This combination therapy did not reduce viremia or the HIV reservoir. Clinical Trials Registration. NCT03803605.

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.

Immunological Correlates of the HIV-1 Replication-Competent Reservoir Size.

Understanding what shapes the latent human immunodeficiency virus type 1 (HIV-1) reservoir is critical for developing strategies for cure. We measured frequency of persistent HIV-1 infection after 5 years of suppressive antiretroviral therapy initiated during chronic infection. Pretreatment CD8+ T-cell activation, nadir CD4 count, and CD4:CD8 ratio predicted reservoir size.

Macrophage Tropism in Pathogenic HIV-1 and SIV Infections.

Most myeloid lineage cells express the receptor and coreceptors that make them susceptible to infection by primate lentiviruses (SIVs and HIVs). However, macrophages are the only myeloid lineage cell commonly infected by SIVs and/or HIVs. The frequency of infected macrophages varies greatly across specific host and virus combinations as well as disease states, with infection rates being greatest in pathogenic SIV infections of non-natural hosts (i.e., Asian nonhuman primates (Asian NHPs)) and late in untreated HIV-1 infection. In contrast, macrophages from natural SIV hosts (i.e., African NHPs) are largely resistant to infection due to entry and/or post-entry restriction mechanisms. These highly variable rates of macrophage infection may stem from differences in the host immune environment, entry and post-entry restriction mechanisms, the ability of a virus to adapt to efficiently infect macrophages, and the pleiotropic effects of macrophage-tropism including the ability to infect cells lacking CD4 and increased neutralization sensitivity. Questions remain about the relationship between rates of macrophage infection and viral pathogenesis, with some evidence suggesting that elevated levels of macrophage infection may contribute to greater pathogenesis in non-natural SIV hosts. Alternatively, extensive infection of macrophages may only emerge in the context of high viral loads and immunodeficiency, making it a symptom of highly pathogenic infections, not a primary driver of pathogenesis.

Curing HIV: Seeking to Target and Clear Persistent Infection.

Human immunodeficiency virus type 1 (HIV-1) infection persists despite years of antiretroviral therapy (ART). To remove the stigma and burden of chronic infection, approaches to eradicate or cure HIV infection are desired. Attempts to augment ART with therapies that reverse viral latency, paired with immunotherapies to clear infection, have advanced into the clinic, but the field is still in its infancy. We review foundational studies and highlight new insights in HIV cure research. Together with advances in ART delivery and HIV prevention strategies, future therapies that clear HIV infection may relieve society of the affliction of the HIV pandemic.

Measuring the contribution of γδ T cells to the persistent HIV reservoir.

OBJECTIVE: To study the contribution of γδ T cells to the persistent HIV reservoir. DESIGN: Fifteen HIV-seropositive individuals on suppressive ART were included. We performed parallel quantitative viral outgrowth assays (QVOA) of resting CD4 T (rCD4) cells in the presence or absence of γδ T cells. METHODS: Resting αß+CD4 T cells were magnetically isolated from PBMCs using two different custom cocktails, only one kit contained antibodies to deplete γδ T cells, resulting in two populations: rCD4 cells and rCD4 cells depleted of γδ cells. Frequency of infection was analyzed by QVOA and DNA measurements. RESULTS: Recovery of replication-competent HIV from cultures of rCD4 cells was similar in 11 individuals despite the presence of γδ T cells. In four donors, HIV recovery was lower when γδ T cells were present. Expression of the cytotoxic marker CD16 on Vδ2 cells was the only variable associated with the lower HIV recovery. Our results highlight the potency of those responses since a mean of 10 000 γδ T cells were present within 2.5 million rCD4 cells. However, despite the low frequency of γδ T cells, the presence of cytotoxic Vδ2 cells correlated with lower HIV recovery from cultures of rCD4 cells. CONCLUSION: Results of this study show that quantification of the contribution of γδ T cells to the reservoir is challenging because of their low numbers compared with conventional rCD4 cells and highlights the potent antiviral function of γδ T cells and the impact of their presence on the frequency of latent HIV infection.

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.

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.

Blocking Formation of the Stable HIV Reservoir: A New Perspective for HIV-1 Cure.

Recent studies demonstrate that the stable HIV-1 reservoir in resting CD4+ T cells is mostly formed from viruses circulating when combination antiretroviral therapy (ART) is initiated. Here we explore the immunological basis for these observations. Untreated HIV-1 infection is characterized by a progressive depletion of memory CD4+ T cells which mostly express CD127, the α chain of the IL-7 receptor (IL-7R). Depletion results from both direct infection and bystander loss of memory CD4+ T cells in part attributed to dysregulated IL-7/IL-7R signaling. While IL-7/IL7R signaling is not essential for the generation of effector CD4+ T cells from naïve cells, it is essential for the further transition of effectors to memory CD4+ T cells and their subsequent homeostatic maintenance. HIV-1 infection therefore limits the transition of CD4+ T cells from an effector to long-lived memory state. With the onset of ART, virus load (VL) levels rapidly decrease and the frequency of CD127+ CD4+ memory T cells increases, indicating restoration of effector to memory transition in CD4+ T cells. Collectively these data suggest that following ART initiation, HIV-1 infected effector CD4+ T cells transition to long-lived, CD127+ CD4+ T cells forming the majority of the stable HIV-1 reservoir. We propose that combining ART initiation with inhibition of IL-7/IL-7R signaling to block CD4+ T cell memory formation will limit the generation of long-lived HIV-infected CD4+ T cells and reduce the overall size of the stable HIV-1 reservoir.

Longitudinal HIV sequencing reveals reservoir expression leading to decay which is obscured by clonal expansion.

After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.

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.

The evolution of HIV-1 entry phenotypes as a guide to changing target cells.

Through a twist of fate the most common form of HIV-1, as defined by entry phenotype, was not appreciated until recently. The entry phenotype is closely linked to the target cell and thus to virus-host interactions and pathogenesis. The most abundant form of HIV-1 uses CCR5 as the coreceptor and requires a high density of CD4 for efficient entry, defining its target cell as the CD4+ memory T cell. This is the transmitted form of the virus, the form that is found in the blood, and the form that rebounds from the latent reservoir. When CD4+/CCR5+ T cells become limiting the virus evolves to use alternative target cells to support viral replication. In the CNS, the virus can evolve to use a cell that displays only a low density of CD4, while maintaining the use of CCR5 as the coreceptor. When this evolutionary variant evolves, it must be sustaining its replication in either macrophages or microglial cells, which display only a low density of CD4 relative to that on T cells. In the blood and lymphoid system, the major switch late in disease is from T cells expressing CD4 and CCR5 to T cells expressing CD4 and CXCR4, with a change in coreceptor specificity. Thus the virus responds in two different ways to different environments when its preferred target cell becomes limiting.