Systems-level temporal immune-metabolic profile in Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever (CCHF) caused by CCHF virus (CCHFV) is one of the epidemic-prone diseases prioritized by the World Health Organisation as public health emergency with an urgent need for accelerated research. The trajectory of host response against CCHFV is multifarious and remains unknown. Here, we reported the temporal spectrum of pathogenesis following the CCHFV infection using genome-wide blood transcriptomics analysis followed by advanced systems biology analysis, temporal immune-pathogenic alterations, and context-specific progressive and postinfection genome-scale metabolic models (GSMM) on samples collected during the acute (T0), early convalescent (T1), and convalescent-phase (T2). The interplay between the retinoic acid-inducible gene-I-like/nucleotide-binding oligomerization domain-like receptor and tumor necrosis factor signaling governed the trajectory of antiviral immune responses. The rearrangement of intracellular metabolic fluxes toward the amino acid metabolism and metabolic shift toward oxidative phosphorylation and fatty acid oxidation during acute CCHFV infection determine the pathogenicity. The upregulation of the tricarboxylic acid cycle during CCHFV infection, compared to the noninfected healthy control and between the severity groups, indicated an increased energy demand and cellular stress. The upregulation of glycolysis and pyruvate metabolism potentiated energy generation through alternative pathways associated with the severity of the infection. The downregulation of metabolic processes at the convalescent phase identified by blood cell transcriptomics and single-cell type proteomics of five immune cells (CD4+ and CD8+ T cells, CD14+ monocytes, B cells, and NK cells) potentially leads to metabolic rewiring through the recovery due to hyperactivity during the acute phase leading to post-viral fatigue syndrome.

Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (ß-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

Rural and urban exposures shape early life immune development in South African children with atopic dermatitis and nonallergic children.

BACKGROUND: Immunological traits and functions have been consistently associated with environmental exposures and are thought to shape allergic disease susceptibility and protection. In particular, specific exposures in early life may have more significant effects on the developing immune system, with potentially long-term impacts. METHODS: We performed RNA-Seq on peripheral blood mononuclear cells (PBMCs) from 150 children with atopic dermatitis and healthy nonallergic children in rural and urban settings from the same ethnolinguistic AmaXhosa background in South Africa. We measured environmental exposures using questionnaires. RESULTS: A distinct PBMC gene expression pattern was observed in those children with atopic dermatitis (132 differentially expressed genes [DEGs]). However, the predominant influences on the immune cell transcriptome were related to early life exposures including animals, time outdoors, and types of cooking and heating fuels. Sample clustering revealed two rural groups (Rural_1 and Rural_2) that separated from the urban group (3413 and 2647 DEGs, respectively). The most significantly regulated pathways in Rural_1 children were related to innate activation of the immune system (e.g., TLR and cytokine signaling), changes in lymphocyte polarization (e.g., TH17 cells), and immune cell metabolism (i.e., oxidative phosphorylation). The Rural_2 group displayed evidence for ongoing lymphocyte activation (e.g., T cell receptor signaling), with changes in immune cell survival and proliferation (e.g., mTOR signaling, insulin signaling). CONCLUSIONS: This study highlights the importance of the exposome on immune development in early life and identifies potentially protective (e.g., animal) exposures and potentially detrimental (e.g., pollutant) exposures that impact key immunological pathways.

Association between gut microbiota development and allergy in infants born during pandemic-related social distancing restrictions.

BACKGROUND: Several hypotheses link reduced microbial exposure to increased prevalence of allergies. Here we capitalize on the opportunity to study a cohort of infants (CORAL), raised during COVID-19 associated social distancing measures, to identify the environmental exposures and dietary factors that contribute to early life microbiota development and to examine their associations with allergic outcomes. METHODS: Fecal samples were sequenced from infants at 6 (n = 351) and repeated at 12 (n = 343) months, using 16S sequencing. Published 16S data from pre-pandemic cohorts were included for microbiota comparisons. Online questionnaires collected epidemiological information on home environment, healthcare utilization, infant health, allergic diseases, and diet. Skin prick testing (SPT) was performed at 12 (n = 343) and 24 (n = 320) months of age, accompanied by atopic dermatitis and food allergy assessments. RESULTS: The relative abundance of bifidobacteria was higher, while environmentally transmitted bacteria such as Clostridia was lower in CORAL infants compared to previous cohorts. The abundance of multiple Clostridia taxa correlated with a microbial exposure index. Plant based foods during weaning positively impacted microbiota development. Bifidobacteria levels at 6 months of age, and relative abundance of butyrate producers at 12 months of age, were negatively associated with AD and SPT positivity. The prevalence of allergen sensitization, food allergy, and AD did not increase over pre-pandemic levels. CONCLUSIONS: Environmental exposures and dietary components significantly impact microbiota community assembly. Our results also suggest that vertically transmitted bacteria and appropriate dietary supports may be more important than exposure to environmental microbes alone for protection against allergic diseases in infancy.

Coordinated pyruvate kinase activity is crucial for metabolic adaptation and cell survival during mitochondrial dysfunction.

Deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported long survival of DGUOK knockout (Dguok-/-) mice despite low (<5%) mtDNA content in liver tissue. However, the molecular mechanisms enabling the extended survival remain unknown. Using transcriptomics, proteomics and metabolomics followed by in vitro assays, we aimed to identify the molecular pathways involved in the extended survival of the Dguok-/- mice. At the early stage, the serine synthesis and folate cycle were activated but declined later. Increased activity of the mitochondrial citric acid cycle (TCA cycle) and the urea cycle and degradation of branched chain amino acids were hallmarks of the extended lifespan in DGUOK deficiency. Furthermore, the increased synthesis of TCA cycle intermediates was supported by coordination of two pyruvate kinase genes, PKLR and PKM, indicating a central coordinating role of pyruvate kinases to support the long-term survival in mitochondrial dysfunction.

Nucleoside-Modified mRNA Vaccines Protect IFNAR-/- Mice against Crimean-Congo Hemorrhagic Fever Virus Infection.

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR-/- mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR-/- and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.

Biological Aging in People Living with HIV on Successful Antiretroviral Therapy: Do They Age Faster?

PURPOSE OF REVIEW: In the absence of a prophylactic/therapeutic vaccine or cure, the most amazing achievement in the battle against HIV was the discovery of effective, well-tolerated combination antiretroviral therapy (cART). The primary research question remains whether PLWH on prolonged successful therapy has accelerated, premature, or accentuated biological aging. In this review, we discuss the current understanding of the immunometabolic profile in PLWH, potentially associated with biological aging, and a better understanding of the mechanisms and temporal dynamics of biological aging in PLWH. RECENT FINDINGS: Biological aging, defined by the epigenetic alterations analyzed by the DNA methylation pattern, has been reported in PLWH with cART that points towards epigenetic age acceleration. The hastened development of specific clinical geriatric syndromes like cardiovascular diseases, metabolic syndrome, cancers, liver diseases, neurocognitive diseases, persistent low-grade inflammation, and a shift toward glutamate metabolism in PLWH may potentiate a metabolic profile at-risk for accelerated aging.

Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication.

Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitro infection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+ T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitro lung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.

Fecal Metabolome Signature in the HIV-1 Elite Control Phenotype: Enrichment of Dipeptides Acts as an HIV-1 Antagonist but a Prevotella Agonist.

HIV-1 elite controllers (EC) are a rare group among HIV-1-infected individuals who can naturally control viral replication for a prolonged period. Due to their heterogeneous nature, no universal mechanism could be attributed to the EC status; instead, several host and viral factors have been discussed as playing a role. In this study, we investigated the fecal metabolome and microbiome in a Swedish cohort of EC (n = 14), treatment-naive viremic progressors (VP; n = 16), and HIV-negative individuals (HC; n = 12). Fecal untargeted metabolomics was performed by four ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Molecular docking and biochemical microscale thermophoresis (MST) were used to describe the peptide-metabolite interactions. Single-cycle infectivity assays were performed in TZM-Bl cell lines using CCR5- and CXCR4-tropic HIV-1 strains. The microbiome analysis was performed using 16S rRNA sequencing. Th effects of metabolites on bacterial species viability were determined using several clinical isolates. We observed an enrichment of dipeptides in EC compared to VP and HC (adjusted P < 0.05). In silico analysis by molecular docking, in vitro biochemical assays, and ex vivo infection assays identified anti-HIV-1 properties for two dipeptides (WG and VQ) that could bind to the HIV-1 gp120, of which WG was more potent. The microbiome analysis identified enrichment of the genus Prevotella in EC, and these dipeptides supported bacterial growth of the genus Prevotella in vitro. The enrichments of the dipeptides and higher abundance of Prevotella have a distinct mechanism of elite control status in HIV-1 infection that influences host metabolism. IMPORTANCE HIV-1 elite controllers (EC) are a rare group among HIV-1-infected individuals who can naturally control viral replication for a prolonged period. Due to their heterogeneous nature, no universal mechanism could be attributed to the EC status; instead, several host and viral factors have been discussed as playing a role. In this study, we investigated the fecal metabolome and microbiome in a Swedish cohort of EC, treatment-naive viremic progressors (VP), and HIV-negative individuals (HC). We observed an enrichment of dipeptides in EC compared to the other two study groups. In silico and in vitro analyses identified anti-HIV-1 properties for two dipeptides that could bind to the HIV-1 gp120 and act as an HIV-1 antagonist. Furthermore, these dipeptides supported bacterial growth of the genus Prevotella in vitro that was enriched in EC, which influences host metabolism. Thus, increased levels of both dipeptides and Prevotella could provide beneficial effects for EC.

Increased acquired protease inhibitor drug resistance mutations in minor HIV-1 quasispecies from infected patients suspected of failing on national second-line therapy in South Africa.

BACKGROUND: HIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs. METHODS: During 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient's treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database. RESULTS: Statistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p = 0.042) and integrase inhibitor RAM (4%; 2/56; p = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation (n = 13) in protease and K65R (n = 5), K103N (n = 7) and M184V (n = 5) in reverse transcriptase. CONCLUSIONS: HTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in < 20% of the population. Deep sequencing could be of greater value in detecting acquired resistance mutations early.

Utility of Proteomics in Emerging and Re-Emerging Infectious Diseases Caused by RNA Viruses.

Emerging and re-emerging infectious diseases due to RNA viruses cause major negative consequences for the quality of life, public health, and overall economic development. Most of the RNA viruses causing illnesses in humans are of zoonotic origin. Zoonotic viruses can directly be transferred from animals to humans through adaptation, followed by human-to-human transmission, such as in human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and, more recently, SARS coronavirus 2 (SARS-CoV-2), or they can be transferred through insects or vectors, as in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), Zika virus (ZIKV), and dengue virus (DENV). At the present, there are no vaccines or antiviral compounds against most of these viruses. Because proteins possess a vast array of functions in all known biological systems, proteomics-based strategies can provide important insights into the investigation of disease pathogenesis and the identification of promising antiviral drug targets during an epidemic or pandemic. Mass spectrometry technology has provided the capacity required for the precise identification and the sensitive and high-throughput analysis of proteins on a large scale and has contributed greatly to unravelling key protein-protein interactions, discovering signaling networks, and understanding disease mechanisms. In this Review, we present an account of quantitative proteomics and its application in some prominent recent examples of emerging and re-emerging RNA virus diseases like HIV-1, CCHFV, ZIKV, and DENV, with more detail with respect to coronaviruses (MERS-CoV and SARS-CoV) as well as the recent SARS-CoV-2 pandemic.

HIV-1 Subtype C with PYxE Insertion Has Enhanced Binding of Gag-p6 to Host Cell Protein ALIX and Increased Replication Fitness.

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C-infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when the PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEi viruses and inserted them in a reference HIV-1 sequence. Viral growth was increased, and the sensitivity to the protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to that of wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion indicates the clinical importance of these viral variants.IMPORTANCE Genomic differences within HIV-1 subtypes is associated with various degrees of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1C has a natural deletion of one of these two motifs, resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of the antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses.

Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

A viral genome wide association study and genotypic resistance testing in patients failing first line antiretroviral therapy in the first large countrywide Ethiopian HIV cohort.

BACKGROUND: Antiretroviral therapy (ART) was rolled-out in Ethiopia in 2005, but there are no reports on outcome of ART and human immunodeficiency virus drug resistance (HIVDR) at national level. We described acquired drug resistance mutations in pol gene and performed a viral genome wide association study in virologic treatment failure patients who started first line ART during 2009-2011 in the first large countrywide HIV cohort in Ethiopia. METHODS: The outcome of tenofovir (TDF)- and zidovudine (ZDV)-based ART was defined in 874 ART naïve patients using the on-treatment (OT) and intention-to-treat (ITT) analyses. Genotypic resistance testing was done in patients failing ART (> 1000 copies/ml) at month 6 and 12. Near full-length genome sequencing (NFLG) was used to assess amino acid changes in HIV-1 gag, pol, vif, vpr, tat, vpu, and nef genes between paired baseline and month 6 samples. RESULTS: High failure rates were found in ITT analysis at month 6 and 12 (23.3%; 33.9% respectively). Major nucleoside and non-nucleoside reverse transcriptase (NRTI/NNRTI) drug resistance mutations were detected in most failure patients at month 6 (36/47; 77%) and month 12 (20/30; 67%). A high rate of K65R was identified only in TDF treated patients (35.7%; 50.0%, respectively). No significant difference was found in failure rate or extent of HIVDR between TDF- and ZDV- treated patients. All target regions of interest for HIVDR were described by NFLG in 16 patients tested before initiation of ART and at month 6. CONCLUSION: In this first Ethiopian national cohort, a high degree of HIVDR was seen among ART failure patients, independent on whether TDF- or ZDV was given. However, the major reason to ART failure was lost-to-follow-up rather than virologic failure. Our NFLG assay covered all relevant target genes for antiretrovirals and is an attractive alternative for HIVDR surveillance.

Antiretroviral potency of 4'-ethnyl-2'-fluoro-2'-deoxyadenosine, tenofovir alafenamide and second-generation NNRTIs across diverse HIV-1 subtypes.

Objectives: 4'-Ethnyl-2'-fluoro-2'-deoxyadenosine (EFdA) is a novel translocation-defective reverse transcriptase inhibitor. We investigated the virological and biochemical inhibitory potentials of EFdA against a broad spectrum of subtype-specific chimeric viruses and compared it with tenofovir alafenamide, nevirapine, efavirenz, rilpivirine and etravirine. Methods: pNL4.3 chimeric viruses encoding gag-pol from treatment-naive patients (n = 24) and therapy-failure patients (n = 3) and a panel of reverse transcriptase inhibitor-resistant strains (n = 7) were used to compare the potency of reverse transcriptase inhibitor drugs. The phenotypic drug susceptibility assay was performed using TZM-bl cells. In vitro inhibition assays were done using patient-derived reverse transcriptase. IC50 values of NNRTIs were calculated using a PicoGreen-based spectrophotometric assay. Steady-state kinetics were used to determine the apparent binding affinity (Km.dNTP) of triphosphate form of EFdA (EFdA-TP) and dATP. Results: Among the chimeric treatment-naive viruses, EFdA had an ex vivo antiretroviral activity [median (IQR) EC50 = 1.4 nM (0.6-2.1 nM)] comparable to that of tenofovir alafenamide [1.6 nM (0.5-3.6 nM)]. Subtype-specific differences were found for etravirine (P = 0.004) and rilpivirine (P = 0.017), where HIV-1C had the highest EC50 values. EFdA had a greater comparative efficiency [calculated by dividing the efficiency of monophosphate form of EFdA (EFdA-MP) incorporation (kcat.EFdA-TP/Km.EFdA-TP) over the efficiency of dATP incorporation (kcat.dATP/Km.dATP)] compared with the natural substrate dATP, with a fold change of between 1.6 and 3.2. Ex vivo analysis on reverse transcriptase inhibitor-resistant strains showed EFdA to have a higher potency. Despite the presence of rilpivirine DRMs, some non-B strains showed hypersusceptibility to rilpivirine. Conclusions: Our combined virological and biochemical data suggest that EFdA inhibits both WT and reverse transcriptase inhibitor-resistant viruses efficiently in a subtype-independent manner. In contrast, HIV-1C is least susceptible to etravirine and rilpivirine.

Immune Checkpoints as the Immune System Regulators and Potential Biomarkers in HIV-1 Infection.

Immune checkpoints are several co-stimulatory and inhibitory pathways that regulate T cell immune responses. Most of the discoveries about immune checkpoints were made in cancer research where inhibitory immune checkpoints cause immune exhaustion and down-regulate anti-tumor responses. In addition to cancer, immune checkpoints are exploited in chronic infectious diseases. In human immunodeficiency virus (HIV) infection, the immune checkpoint molecule called programmed cell death protein 1 (PD-1) has been determined as being a major regulatory factor for T cell exhaustion. Recent studies with antibodies blocking either PD-1 ligand 1 (PD-L1) or PD-1 show not only promising results in the enhancement of HIV-specific immune responses but even in reducing the latent HIV reservoir. Apart from the therapeutic target for a functional cure of HIV-1, immune checkpoint molecules might be used as biomarkers for monitoring disease progression and therapeutic response. In this review, we will summarize and discuss the inhibitory immune checkpoint molecules PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), and T cell immunoglobulin and mucin-domain-containing-3 (TIM3) as well as the co-stimulatory molecules CD40L and CD70, including their role in immunity, with a particular focus on HIV infection, and being potential targets for a functional HIV cure.

Increased replication capacity following evolution of PYxE insertion in Gag-p6 is associated with enhanced virulence in HIV-1 subtype C from East Africa.

BACKGROUND: A lower virulence of HIV-1 subtype C (HIV-1C) is suggested to be related to the global dominance of HIV-1C. In this observational study, combining in vivo (clinical monitoring) and in vitro (genotypic, biochemical, and phenotypic assays), we explored whether HIV-1C from East Africa (HIV-1CEA ) is more pathogenic due to the evolution of a PYxE-insertion (CPYxEi ) in the gag-p6 that also could affect the therapy response. METHODS: HIV-1B (n = 112) and HIV-1CEA (n = 128)-infected individuals residing in Sweden were analyzed with regard to Gag-p6 genotype and clinically monitored. Based on the Gag-p6 characteristics, three HIV-1CEA and one HIV-1 B patient-derived p2-INT-recombinant virus (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) were constructed to analyze viral growth kinetics (VGKs) and drug sensitivity assays. Reverse transcriptase (RT) from the same samples was cloned into the heterodimer expression plasmid (pRT6H-PROT) to analyze catalytic efficiency of RT. RESULTS: A higher viral failure rate and lower pre-therapy CD4+ T-cell counts were observed in HIV-1CEA -infected patients compared to HIV-1B-infected patients. In Gag-p6, PTAP-duplication was more common in HIV-1C. HIV-1CEA -infected patients with signature CPYxEi, evidenced very low pre-therapy CD4+ T-cell counts and suboptimal gain in CD4+ T-cells following therapy, as compared to the non-CPYxEi -strains indicating higher virulence. VGKs showed a statistically significant higher replication capacity (RC) for the CPYxEi viruses than the other two non-CPYxEi strains. No statistically significant difference was observed in the catalytic efficiency among HIV-1C RTs. CONCLUSIONS: This is the first evidence of polymerase independent increased virulence and RC in HIV-1CEA following PYxE-insertion that is associated with suboptimal CD4+ T-cell gain following therapy initiation. J. Med. Virol. 89:106-111, 2017. © 2016 Wiley Periodicals, Inc.

Factors influencing the efficacy of rilpivirine in HIV-1 subtype C in low- and middle-income countries.

OBJECTIVES: The use of the NNRTI rilpivirine in low- and middle-income countries (LMICs) is under debate. The main objective of this study was to provide further clinical insights and biochemical evidence on the usefulness of rilpivirine in LMICs. PATIENTS AND METHODS: Rilpivirine resistance was assessed in 5340 therapy-naive and 13,750 first-generation NNRTI-failed patients from Europe and therapy-naive HIV-1 subtype C (HIV-1C)-infected individuals from India (n = 617) and Ethiopia (n = 127). Rilpivirine inhibition and binding affinity assays were performed using patient-derived HIV-1C reverse transcriptases (RTs). RESULTS: Primary rilpivirine resistance was rare, but the proportion of patients with >100,000 HIV-1 RNA copies/mL pre-ART was high in patients from India and Ethiopia, limiting the usefulness of rilpivirine as a first-line drug in LMICs. In patients failing first-line NNRTI treatments, cross-resistance patterns suggested that 73% of the patients could benefit from switching to rilpivirine-based therapy. In vitro inhibition assays showed ∼ 2-fold higher rilpivirine IC50 for HIV-1C RT than HIV-1B RT. Pre-steady-state determination of rilpivirine-binding affinities revealed 3.7-fold lower rilpivirine binding to HIV-1C than HIV-1B RT. Structural analysis indicated that naturally occurring polymorphisms close to the NNRTI-binding pocket may reduce rilpivirine binding, leading to lower susceptibility of HIV-1C to rilpivirine. CONCLUSIONS: Our clinical and biochemical findings indicate that the usefulness of rilpivirine has limitations in HIV-1C-dominated epidemics in LMICs, but the drug could still be beneficial in patients failing first-line therapy if genotypic resistance testing is performed.

Virological efficacy with first-line antiretroviral treatment in India: predictors of viral failure and evidence of viral resuppression.

OBJECTIVE: Combination antiretroviral therapy (ART) has improved in efficacy, durability and tolerability. Virological efficacy studies in India are limited. We determined incidence and predictors of virological failure among patients initiating first-line ART and described virological resuppression after confirmed failure, with the goal of informing national policy. METHODS: Therapy-naïve patients initiated on first-line ART as per national guidelines were monitored every 3 months for adherence and virological response over 2 years. Genotyping on baseline samples was performed to assess primary drug resistance. Multivariate Cox regression analysis was used to assess predictors of virological failure. RESULTS: Virological failure rate among 599 eligible patients was 10.7 failures per 100 person-years. Cumulative failure incidence was 13.2% in the first year and 16.5% over 2 years. Patients initiated on tenofovir had a significantly lower rate of virological failure than those on stavudine or zidovudine (6.7 vs. 11.9 failures per 100 person-years, P = 0.013). Virological failure was independently associated with age <40 years, mean adherence <95%, non-tenofovir-containing regimens and presence of primary drug resistance. In a subset of 311 patients who were reassessed after treatment failure, 19% (11/58) patients resuppressed their viral load to <400 copies/ml after confirmed virological failure. CONCLUSIONS: Our results support the inclusion of tenofovir as first-line ART in resource-limited settings and a role for regular adherence counselling and virological monitoring for enhanced treatment success. Detection of early virological failure should provide an opportunity to augment adherence counselling and repeat viral load testing before therapy switch is considered.