Evaluation of Archival HIV DNA in Brain and Lymphoid Tissues.

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-env) sequences from a subset of 14 participants. We detected HIV DNA (gag) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain (P < 0.01). Levels of HIV gag between BG and FC were similar (P > 0.2), while OCC had the lowest levels (P = 0.01). Females had higher HIV DNA levels in tissues than males (gag, P = 0.03; 2-LTR, P = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV-env sequences (n = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available.

Early Antiretroviral Therapy Is Associated with Lower HIV DNA Molecular Diversity and Lower Inflammation in Cerebrospinal Fluid but Does Not Prevent the Establishment of Compartmentalized HIV DNA Populations.

Even when antiretroviral therapy (ART) is started early after infection, HIV DNA might persist in the central nervous system (CNS), possibly contributing to inflammation, brain damage and neurocognitive impairment. Paired blood and cerebrospinal fluid (CSF) were collected from 16 HIV-infected individuals on suppressive ART: 9 participants started ART <4 months of the estimated date of infection (EDI) ("early ART"), and 7 participants started ART >14 months after EDI ("late ART"). For each participant, neurocognitive functioning was measured by Global Deficit Score (GDS). HIV DNA levels were measured in peripheral blood mononuclear cells (PBMCs) and CSF cell pellets by droplet digital (dd)PCR. Soluble markers of inflammation (sCD163, IL-6, MCP-1, TNF-α) and neuronal damage (neurofilament light [NFL]) were measured in blood and CSF supernatant by immunoassays. HIV-1 partial C2V3 env deep sequencing data (Roche 454) were obtained for 8 paired PBMC and CSF specimens and used for phylogenetic and compartmentalization analysis. Median exposure to ART at the time of sampling was 2.6 years (IQR: 2.2-3.7) and did not differ between groups. We observed that early ART was significantly associated with lower molecular diversity of HIV DNA in CSF (p<0.05), and lower IL-6 levels in CSF (p = 0.02), but no difference for GDS, NFL, or HIV DNA detectability compared to late ART. Compartmentalization of HIV DNA populations between CSF and blood was detected in 6 out of 8 participants with available paired HIV DNA sequences (2 from early and 4 from late ART group). Phylogenetic analysis confirmed the presence of monophyletic HIV DNA populations within the CSF in 7 participants, and the same population was repeatedly sampled over a 5 months period in one participant with longitudinal sampling. Such compartmentalized provirus in the CNS needs to be considered for the design of future eradication strategies and might contribute to the neuropathogenesis of HIV.

Transient and asymptomatic meningitis in human immunodeficiency virus-1 subtype C: a case study of genetic compartmentalization and biomarker dynamics.

Human immunodeficiency virus (HIV) genetic compartmentalization is defined as genetic differences in HIV in different tissue compartments or subcompartments that characterize viral quasispecies. This descriptive, longitudinal study assessed the dynamics of inflammation, humoral immune response, blood-brain barrier, blood-cerebrospinal fluid (CSF) barrier, as well as neuronal injury biomarkers in serially obtained CSF and serum samples from an antiretroviral (ARV) therapy-naïve patient with HIV-1 subtype C with CSF HIV genetic compartmentalization that resolved spontaneously without ARV treatment. The first CSF sample showed an increase in white blood cell (WBC) count (382 cells/mm3) and a marked increase in the levels of inflammatory cytokines and chemokines, including tumor necrosis factor (TNF)α, interleukin (IL)-10, IP-10, and regulated on activation, normal T cell expressed and secreted (RANTES), which raise the suspicion of dual infection. Serum sample analysis showed all cytokine levels to be normal, with only IP-10 slightly increased. These results corroborate the hypothesis that the CNS immunologic response in a patient with HIV infection was independent of the systemic immunologic response. The patient also had persistently elevated levels of sCD14, neopterin, and ß2M, which were strongly suggestive of persistent CNS immunologic stimulation. This report describes a patient with HIV subtype C who developed a transient episode of asymptomatic HIV meningitis with compartmentalization of HIV in the CSF that resolved independently of ARV therapy. Extensive CSF studies were performed as part of an ongoing longitudinal study, which revealed CNS immune abnormalities. This case presents evidence of HIV-1 subtype C neurotropism and compartmentalization.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection.

UNLABELLED: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4(+)count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P= 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P< 0.001) and increased unspliced cellular HIV RNA transcription (P= 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE: Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4(+)T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

Increased cell-free mitochondrial DNA is a marker of ongoing inflammation and better neurocognitive function in virologically suppressed HIV-infected individuals.

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (<50 copies/mL) by droplet digital PCR. We measured soluble CD14, soluble CD163, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), neopterin, and neurofilament light chain (NFL) by immunoassays in CSF supernatant or blood plasma. Higher levels of mtDNA in CSF were associated with higher levels of MCP-1 (r = 0.56, p < 0.01) in CSF and TNF-α (r = 0.43, p < 0.01) and IL-8 (r = 0.44, p < 0.01) in blood plasma. Subjects with a previous diagnosis of AIDS showed significantly higher levels of mtDNA (p < 0.01) than subjects without AIDS. The associations between mtDNA and MCP-1 in CSF and TNF-α in blood remained significant after adjusting for previous diagnosis of AIDS (p < 0.01). Additionally, higher levels of mtDNA were associated with a lower CD4 nadir (r = -0.41, p < 0.01) and lower current CD4% (r = -0.34, p = 0.03). Paradoxically, higher levels of mtDNA in CSF were significantly associated with better neurocognitive performance (r = 0.43, p = 0.02) and with less neuronal damage (i.e. lower NFL). Higher cell-free mtDNA is associated with inflammation during treated HIV infection, but the impact on neurocognitive functioning and neuronal damage remains unclear and may differ in the setting of suppressive ART.

Dynamic of CSF and serum biomarkers in HIV-1 subtype C encephalitis with CNS genetic compartmentalization-case study.

Despite the effective suppression of viremia with antiretroviral therapy, HIV can still replicate in the central nervous system (CNS). This was a longitudinal study of the cerebrospinal fluid (CSF) and serum dynamics of several biomarkers related to inflammation, the blood-brain barrier, neuronal injury, and IgG intrathecal synthesis in serial samples of CSF and serum from a patient infected with HIV-1 subtype C with CNS compartmentalization.The phylogenetic analyses of plasma and CSF samples in an acute phase using next-generation sequencing and F-statistics analysis of C2-V3 haplotypes revealed distinct compartmentalized CSF viruses in paired CSF and peripheral blood mononuclear cell samples. The CSF biomarker analysis in this patient showed that symptomatic CSF escape is accompanied by CNS inflammation, high levels of cell and humoral immune biomarkers, CNS barrier dysfunction, and an increase in neuronal injury biomarkers with demyelization. Independent and isolated HIV replication can occur in the CNS, even in HIV-1 subtype C, leading to compartmentalization and development of quasispecies distinct from the peripheral plasma. These immunological aspects of the HIV CNS escape have not been described previously. To our knowledge, this is the first report of CNS HIV escape and compartmentalization in HIV-1 subtype C.

Asymptomatic CMV Replication During Early Human Immunodeficiency Virus (HIV) Infection Is Associated With Lower CD4/CD8 Ratio During HIV Treatment.

BACKGROUND: A low CD4/CD8 ratio in human immunodeficiency virus (HIV)-infected individuals is associated with inflammation and higher risk of non-AIDS morbidity and mortality. In this study, we investigated the effect of subclinical cytomegalovirus (CMV) and Epstein-Barr virus (EBV) replication on CD4+ and CD8+ T-cell dynamics when antiretroviral therapy (ART) is started during early infection. METHODS: We investigated 604 peripheral blood mononuclear cell samples from 108 CMV- and EBV-seropositive HIV-infected men who have sex with men, who started ART within a median of 4 months from their estimated date of infection and were followed for a median of 29.1 months thereafter. Levels of CMV and EBV DNA were measured at each timepoint. Mixed-effects asymptotic regression models were applied to characterize CD4+ and CD8+ T-cell dynamics, and Bayesian hierarchical models were used to quantify individual differences in CMV and EBV DNA replication. RESULTS: Higher levels of subclinical CMV replication were associated with lower predicted maximum levels of CD4/CD8 ratio (P < .05), which was driven by higher levels of CD8+ T-cell counts (P < .05), without affecting CD4+ T-cell counts (P > .1). Age was negatively associated with CD4/CD8 levels (P < .05), and this effect was independent of the CMV association (P < .05 for both CMV and age in a multivariate model). CONCLUSIONS: Subclinical CMV replication in blood cells during early HIV infection and younger age were associated with lower CD4/CD8 ratios during suppressive ART. These findings suggest that active CMV infection in the setting of treated HIV may represent an attractive potential target for therapeutic intervention.

Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.