Rapid Emergence and Evolution of SARS-CoV-2 Variants in Advanced HIV Infection.

Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions1-4, but the evolutionary processes underlying these observations are incompletely understood. Here we used high-throughput, single-genome amplification and sequencing (HT-SGS) to obtain up to ~103 SARS-CoV-2 spike gene sequences in each of 184 respiratory samples from 22 people with HIV (PWH) and 25 people without HIV (PWOH). Twelve of 22 PWH had advanced HIV infection, defined by peripheral blood CD4 T cell counts (i.e., CD4 counts) <200 cells/µL. In PWOH and PWH with CD4 counts ≥200 cells/µL, most single-genome spike sequences in each person matched one haplotype that predominated throughout the infection. By contrast, people with advanced HIV showed elevated intra-host spike diversity with a median of 46 haplotypes per person (IQR 14-114). Higher intra-host spike diversity immediately after COVID-19 symptom onset predicted longer SARS-CoV-2 RNA shedding among PWH, and intra-host spike diversity at this timepoint was significantly higher in people with advanced HIV than in PWOH. Composition of spike sequence populations in people with advanced HIV fluctuated rapidly over time, with founder sequences often replaced by groups of new haplotypes. These population-level changes were associated with a high total burden of intra-host mutations and positive selection at functionally important residues. In several cases, delayed emergence of detectable serum binding to spike was associated with positive selection for presumptive antibody-escape mutations. Taken together, our findings show remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern (VOCs).

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo.

Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

Identification of astrocyte regulators by nucleic acid cytometry.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

The role of CD101-expressing CD4 T cells in HIV/SIV pathogenesis and persistence.

Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101+ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4ß7 in the gut mucosa. Notably, during acute SIV infection, CD101+ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101+ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101+ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101+ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Droplet-microfluidics-assisted sequencing of HIV proviruses and their integration sites in cells from people on antiretroviral therapy.

The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV-host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

A SARS-CoV-2 spike ferritin nanoparticle vaccine protects against heterologous challenge with B.1.1.7 and B.1.351 virus variants in Syrian golden hamsters.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 µg) or low (0.2 µg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

High-throughput, single-copy sequencing reveals SARS-CoV-2 spike variants coincident with mounting humoral immunity during acute COVID-19.

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH2-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.

High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19.

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH 2 -terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting. AUTHOR SUMMARY: Mutant sequences of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) arising during any individual case of coronavirus disease 2019 (COVID-19) could theoretically enable the virus to evade immune responses or antiviral therapies that target the predominant infecting virus sequence. However, commonly used sequencing technologies are not optimally designed to detect variant virus sequences within each sample. To address this issue, we developed novel technology for sequencing large numbers of individual SARS-CoV-2 genomic RNA molecules across the region encoding the virus surface proteins. This technology revealed extensive genetic diversity in cultured viruses from a clinical isolate of SARS-CoV-2, but lower diversity in samples from 7 individuals with COVID-19. Importantly, concurrent analysis of paired serum samples in selected individuals revealed relatively low levels of antibody binding to the SARS-CoV-2 spike protein at the time of initial sequencing. With increased serum binding to spike protein, we detected multiple SARS-CoV-2 variants bearing independent mutations in a single epitope, as well as a transient increase in virus burden. These findings suggest that SARS-CoV-2 replication creates sufficient virus genetic diversity to allow immune-mediated selection of variants within the time frame of acute COVID-19. Large-scale studies of SARS-CoV-2 variation and specific immune responses will help define the contributions of intra-individual SARS-CoV-2 evolution to COVID-19 clinical outcomes and antiviral drug susceptibility.

Fc-mediated effector function contributes to the in vivo antiviral effect of an HIV neutralizing antibody.

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

High levels of genetically intact HIV in HLA-DR+ memory T cells indicates their value for reservoir studies.

OBJECTIVE: The contribution of HLA-DR+ memory CD4 T cells to the HIV reservoir during prolonged antiretroviral therapy is unclear as these cells are commonly excluded when assessing for replication-competent HIV. To address this issue, we examined the distribution of genetically intact HIV DNA within HLA-DR- and HLA-DR+ memory CD4 T cells and the RNA transcriptional profile of these cells during antiretroviral therapy. DESIGN/METHODS: Full-length DNA sequencing was used to examine the HIV DNA landscape within HLA-DR+ and HLA-DR- memory CD4 T cells. RNA quantification and sequencing was used to interrogate the relationship between HLA-DR status and HIV RNA transcription. RESULTS: HLA-DR+ CD4 T cells contained a high frequency of genetically intact HIV genomes, contributing over half of the genetically intact viral sequences to the reservoir. Expansions of genetically identical sequences were identified in all T-cell subsets, indicating that cellular proliferation maintains genetically intact and defective viral DNA during therapy. Intracellular HIV RNA levels in HLA-DR+ and HLA-DR- T cells were not statistically different by either long terminal repeat quantitative PCR quantification or single-genome RNA sequencing of the p6-RT region. CONCLUSION: The high proportion of intact viral DNA sequences in the proliferative HLA-DR+ subset suggests they are critical in maintaining HIV infection during effective therapy. As such, these cells should be included in any immune intervention targeting HIV during effective therapy.

Identification of Genetically Intact HIV-1 Proviruses in Specific CD4+ T Cells from Effectively Treated Participants.

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Perspectives on Human Immunodeficiency Virus (HIV) Cure: HIV Persistence in Tissue.

The uneven anatomic distribution of cell subsets that harbor human immunodeficiency virus (HIV) during antiretroviral therapy (ART) complicates investigation of the barriers to HIV cure. Here we propose that while previous studies done largely in blood cells have led to important investigations into HIV latency, other important mechanisms of HIV persistence during ART may not be readily apparent in the bloodstream. We specifically consider as an example the question of ongoing HIV replication during ART. We discuss how growing understanding of key anatomic sanctuaries for the virus can inform future experiments aimed at further clarifying this issue.