HIV-induced RSAD2/Viperin supports sustained infection of monocyte-derived macrophages.

HIV establishes long-term latent infection in memory CD4+ T cells and also establishes sustained long-term productive infection in macrophages, especially in the central nervous system (CNS). To better understand how HIV sustains infection in macrophages, we performed RNAseq analysis after infection of human monocyte-derived macrophages (MDMs) with the brain-derived HIV-1 strain YU2 and compared this with acute infection of CD4+ T cells. HIV infection in MDM and CD4+ T cells altered many gene transcripts, but with few overlaps between these different cell types. We found interferon pathways upregulated in both MDM and CD4+ T cells, but with different gene signatures. The interferon-stimulated gene RSAD2/Viperin was among the most upregulated genes following HIV infection in MDMs, but not in CD4+ T cells. RSAD2/Viperin was induced early after infection with various HIV strains, was sustained over time, and remained elevated in established MDM infection even if new rounds of infection were blocked by antiretroviral treatment. Immunofluorescence microscopy revealed that RSAD2/Viperin was induced in HIV-infected cells, as well as in some uninfected neighboring cells. Knockdown of RSAD2/Viperin following the establishment of infection in MDMs reduced the production of HIV transcripts and viral p24 antigen. This correlated with the reduction in the number of multinucleated giant cells, and changes in the HIV DNA and chromatin structure, including an increased DNA copy number and loss of nucleosomes and histone modifications at the long terminal repeat (LTR). RNAseq transcriptomic analysis of RSAD2/Viperin knockdown during HIV infection of MDMs revealed the activation of interferon alpha/beta and gamma pathways and the inactivation of Rho GTPase pathways. Taken together, these results suggest that RSAD2/Viperin supports the sustained infection in macrophages, potentially through mechanisms involving the alteration of the LTR chromatin structure and the interferon response. IMPORTANCE: HIV infection of macrophages is a barrier to HIV cure and a source of neurocognitive pathology. We found that HIV induces RSAD2/Viperin during sustained infection of macrophages. While RSAD2/Viperin is an interferon-stimulated gene with known antiviral activity, we find RSAD2/Viperin promotes HIV infection in macrophages through multiple mechanisms, including interferon signaling. Therefore, RSAD2/Viperin may be a therapeutic target for the treatment of HIV-infected macrophages.

A high-throughput screening assay for silencing established HIV-1 macrophage infection identifies nucleoside analogs that perturb H3K9me3 on proviral genomes.

HIV-infected macrophages are long-lived cells that represent a barrier to functional cure. Additionally, low-level viral expression by central nervous system (CNS) macrophages contributes to neurocognitive deficits that develop despite antiretroviral therapy (ART). We recently identified H3K9me3 as an atypical epigenetic mark associated with chronic HIV infection in macrophages. Thus, strategies are needed to suppress HIV-1 expression in macrophages, but the unique myeloid environment and the responsible macrophage/CNS-tropic strains require cell/strain-specific approaches. Here, we generated an HIV-1 reporter virus from a CNS-derived strain with intact auxiliary genes expressing destabilized luciferase. We employed this reporter virus in polyclonal infection of primary human monocyte-derived macrophages (MDM) for a high-throughput screen (HTS) to identify compounds that suppress virus expression from established macrophage infection. Screening ~6,000 known drugs and compounds yielded 214 hits. A secondary screen with 10-dose titration identified 24 meeting criteria for HIV-selective activity. Using three replication-competent CNS-derived macrophage-tropic HIV-1 isolates and viral gene expression readout in MDM, we confirmed the effect of three purine analogs, nelarabine, fludarabine, and entecavir, showing the suppression of HIV-1 expression from established macrophage infection. Nelarabine inhibited the formation of H3K9me3 on HIV genomes in macrophages. Thus, this novel HTS assay can identify suppressors of HIV-1 transcription in established macrophage infection, such as nucleoside analogs and HDAC inhibitors, which may be linked to H3K9me3 modification. This screen may be useful to identify new metabolic and epigenetic agents that ameliorate HIV-driven neuroinflammation in people on ART or prevent viral recrudescence from macrophage reservoirs in strategies to achieve ART-free remission. IMPORTANCE Macrophages infected by HIV-1 are a long-lived reservoir and a barrier in current efforts to achieve HIV cure and also contribute to neurocognitive complications in people despite antiretroviral therapy (ART). Silencing HIV expression in these cells would be of great value, but the regulation of HIV-1 in macrophages differs from T cells. We developed a novel high-throughput screen for compounds that can silence established infection of primary macrophages, and identified agents that downregulate virus expression and alter provirus epigenetic profiles. The significance of this assay is the potential to identify new drugs that act in the unique macrophage environment on relevant viral strains, which may contribute to adjunctive treatment for HIV-associated neurocognitive disorders and/or prevent viral rebound in efforts to achieve ART-free remission or cure.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Effects of Mask Reuse on the Oropharyngeal, Skin, and Mask Microbiome.

BACKGROUND: Face masks have been critical in the coronavirus disease 2019 (COVID-19) pandemic, but supplies were sometimes limited and disposable masks contribute greatly to environmental waste. Studies suggest that filtration capacity is retained with repeated use, and surveys indicate many people reuse surgical masks. However, the impact of mask reuse on the host is understudied. METHODS: We applied 16S rRNA gene sequencing to investigate the bacterial microbiome of the facial skin and oropharynx of individuals randomized to wearing fresh surgical masks daily versus masks reused for 1 week. RESULTS: Compared to daily fresh masks, reuse was associated with increased richness (number of taxa) of the skin microbiome and trend towards greater diversity, but no difference in the oropharyngeal microbiome. Used masks had either skin-dominant or oropharynx-dominant bacterial sequences, and reused masks had >100-fold higher bacterial content but no change in composition compared to those used for 1 day. CONCLUSIONS: One week of mask reuse increased the number of low-abundance taxa on the face but did not impact the upper respiratory microbiome. Thus, face mask reuse has little impact on the host microbiome, although whether minor changes to the skin microbiome might relate to reported skin sequelae of masking (maskne) remains to be determined.

Epigenetic Landscape of HIV-1 Infection in Primary Human Macrophage.

Human immunodeficiency virus (HIV)-infected macrophages are long-lived cells that sustain persistent virus expression, which is both a barrier to viral eradication and contributor to neurological complications in patients despite antiretroviral therapy (ART). To better understand the regulation of HIV-1 in macrophages, we compared HIV-infected primary human monocyte-derived macrophages (MDM) to acutely infected primary CD4 T cells and Jurkat cells latently infected with HIV (JLAT 8.4). HIV genomes in MDM were actively transcribed despite enrichment with heterochromatin-associated H3K9me3 across the complete HIV genome in combination with elevated activation marks of H3K9ac and H3K27ac at the long terminal repeat (LTR). Macrophage patterns contrasted with JLAT cells, which showed conventional bivalent H3K4me3/H3K27me3, and acutely infected CD4 T cells, which showed an intermediate epigenotype. 5'-Methylcytosine (5mC) was enriched across the HIV genome in latently infected JLAT cells, while 5'-hydroxymethylcytosine (5hmC) was enriched in CD4 cells and MDMs. HIV infection induced multinucleation of MDMs along with DNA damage-associated p53 phosphorylation, as well as loss of TET2 and the nuclear redistribution of 5-hydoxymethylation. Taken together, our findings suggest that HIV induces a unique macrophage nuclear and transcriptional profile, and viral genomes are maintained in a noncanonical bivalent epigenetic state. IMPORTANCE Macrophages serve as a reservoir for long-term persistence and chronic production of HIV. We found an atypical epigenetic control of HIV in macrophages marked by heterochromatic H3K9me3 despite active viral transcription. HIV infection induced changes in macrophage nuclear morphology and epigenetic regulatory factors. These findings may identify new mechanisms to control chronic HIV expression in infected macrophages.

Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus.

Innate lymphoid cells (ILCs), a heterogeneous cell population, are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor a-chain (CD25), IL-7 receptor a-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus.

Debulking SARS-CoV-2 in saliva using angiotensin converting enzyme 2 in chewing gum to decrease oral virus transmission and infection.

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 ΔRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others.

Label-free detection of synthetic, full genomic length HIV-1 RNA at the few-copy level.

Oligonucleotide-functionalized graphene biosensors show immense promise for use as label-free point of care devices for detection of nucleic acid biomarkers at clinically relevant levels. Graphene-based nucleic acid sensors can be fabricated at low cost and have been shown to reach limits of detection in the attomolar range. Here we demonstrate devices functionalized with 22mer or 8omer DNA probes are capable of detecting full length genomic HIV-1 subtype B RNA, with a limit of detection below 1 aM in nuclease free water. We also show that these sensors are suitable for detection directly in Qiazol lysis reagent, again with a limit of detection below 1 aM for both 22mer and 8omer probes.

The Lung Allograft Microbiome Associates with Pepsin, Inflammation, and Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the principal cause of early morbidity and mortality after lung transplantation. The lung microbiome has been implicated in later transplantation outcomes but has not been investigated in PGD. Objectives: To define the peritransplant bacterial lung microbiome and relationship to host response and PGD. Methods: This was a single-center prospective cohort study. Airway lavage samples from donor lungs before organ procurement and recipient allografts immediately after implantation underwent bacterial 16S ribosomal ribonucleic acid gene sequencing. Recipient allograft samples were analyzed for cytokines by multiplex array and pepsin by ELISA. Measurements and Main Results: We enrolled 139 transplant subjects and obtained donor lung (n = 109) and recipient allograft (n = 136) samples. Severe PGD (persistent grade 3) developed in 15 subjects over the first 72 hours, and 40 remained without PGD (persistent grade 0). The microbiome of donor lungs differed from healthy lungs, and recipient allograft microbiomes differed from donor lungs. Development of severe PGD was associated with enrichment in the immediate postimplantation lung of oropharyngeal anaerobic taxa, particularly Prevotella. Elevated pepsin, a gastric biomarker, and a hyperinflammatory cytokine profile were present in recipient allografts in severe PGD and strongly correlated with microbiome composition. Together, immediate postimplantation allograft Prevotella/Streptococcus ratio, pepsin, and indicator cytokines were associated with development of severe PGD during the 72-hour post-transplantation period (area under the curve = 0.81). Conclusions: Lung allografts that develop PGD have a microbiome enriched in anaerobic oropharyngeal taxa, elevated gastric pepsin, and hyperinflammatory phenotype. These findings suggest a possible role for peritransplant aspiration in PGD, a potentially actionable mechanism that warrants further investigation.

Cytomegalovirus Latent Infection is Associated with an Increased Risk of COVID-19-Related Hospitalization.

Some risk factors for severe coronavirus disease 2019 (COVID-19) have been identified, including age, race, and obesity. However, 20%-50% of severe cases occur in the absence of these factors. Cytomegalovirus (CMV) is a herpesvirus that infects about 50% of all individuals worldwide and is among the most significant nongenetic determinants of immune system. We hypothesized that latent CMV infection might influence the severity of COVID-19. Our analyses demonstrate that CMV seropositivity is associated with more than twice the risk of hospitalization due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immune profiling of blood and CMV DNA quantitative polymerase chain reaction in a subset of patients for whom respiratory tract samples were available revealed altered T-cell activation profiles in absence of extensive CMV replication in the upper respiratory tract. These data suggest a potential role for CMV-driven immune perturbations in affecting the outcome of SARS-CoV-2 infection and may have implications for the discrepancies in COVID-19 severity between different human populations.

Redondovirus Diversity and Evolution on Global, Individual, and Molecular Scales.

Redondoviridae is a newly established family of circular Rep-encoding single-stranded (CRESS) DNA viruses found in the human ororespiratory tract. Redondoviruses were previously found in ∼15% of respiratory specimens from U.S. urban subjects; levels were elevated in individuals with periodontitis or critical illness. Here, we report higher redondovirus prevalence in saliva samples: four rural African populations showed 61 to 82% prevalence, and an urban U.S. population showed 32% prevalence. Longitudinal, limiting-dilution single-genome sequencing revealed diverse strains of both redondovirus species (Brisavirus and Vientovirus) in single individuals, persistence over time, and evidence of intergenomic recombination. Computational analysis of viral genomes identified a recombination hot spot associated with a conserved potential DNA stem-loop structure. To assess the possible role of this site in recombination, we carried out in vitro studies which showed that this potential stem-loop was cleaved by the virus-encoded Rep protein. In addition, in reconstructed reactions, a Rep-DNA covalent intermediate was shown to mediate DNA strand transfer at this site. Thus, redondoviruses are highly prevalent in humans, found in individuals on multiple continents, heterogeneous even within individuals and encode a Rep protein implicated in facilitating recombination. IMPORTANCERedondoviridae is a recently established family of DNA viruses predominantly found in the human respiratory tract and associated with multiple clinical conditions. In this study, we found high redondovirus prevalence in saliva from urban North American individuals and nonindustrialized African populations in Botswana, Cameroon, Ethiopia, and Tanzania. Individuals on both continents harbored both known redondovirus species. Global prevalence of both species suggests that redondoviruses have long been associated with humans but have remained undetected until recently due to their divergent genomes. By sequencing single redondovirus genomes in longitudinally sampled humans, we found that redondoviruses persisted over time within subjects and likely evolve by recombination. The Rep protein encoded by redondoviruses catalyzes multiple reactions in vitro, consistent with a role in mediating DNA replication and recombination. In summary, we identify high redondovirus prevalence in humans across multiple continents, longitudinal heterogeneity and persistence, and potential mechanisms of redondovirus evolution by recombination.

Viruses, periodontitis, and comorbidities.

Seminal studies published in the 1990s and 2000s explored connections between periodontal diseases and systemic conditions, revealing potential contributions of periodontal diseases in the initiation or worsening of systemic conditions. The resulting field of periodontal medicine led to the publication of studies indicating that periodontal diseases can influence the risk of systemic conditions, including adverse pregnancy outcomes, cardiovascular and respiratory diseases, as well as Alzheimer disease and cancers. In general, these studies hypothesized that the periodontal bacterial insult and/or the associated proinflammatory cascade could contribute to the pathogenesis of these systemic diseases. While investigations of the biological basis of the connections between periodontal diseases and systemic conditions generally emphasized the bacteriome, it is also biologically plausible, under an analogous hypothesis, that other types of organisms may have a similar role. Human viruses would be logical "suspects" in this role, given their ubiquity in the oral cavity, association with periodontal diseases, and ability to elicit strong inflammatory response, compromise immune responses, and synergize with bacteria in favor of a more pathogenic microbial consortium. In this review, the current knowledge of the role of viruses in connecting periodontal diseases and systemic conditions is examined. We will also delve into the mechanistic basis for such connections and highlight the importance of those relationships in the management and treatment of patients.

CD4 receptor diversity in chimpanzees protects against SIV infection.

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

ICTV Virus Taxonomy Profile: Redondoviridae.

Viruses in the family Redondoviridae have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Redondoviridae, which is available at ictv.global/report/redondoviridae.

Gene coexpression networks reveal novel molecular endotypes in alpha-1 antitrypsin deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition that causes early onset pulmonary emphysema and airways obstruction. The complete mechanisms via which AATD causes lung disease are not fully understood. To improve our understanding of the pathogenesis of AATD, we investigated gene expression profiles of bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMCs) in AATD individuals. METHODS: We performed RNA-Seq on RNA extracted from matched BAL and PBMC samples isolated from 89 subjects enrolled in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Subjects were stratified by genotype and augmentation therapy. Supervised and unsupervised differential gene expression analyses were performed using Weighted Gene Co-expression Network Analysis (WGCNA) to identify gene profiles associated with subjects' clinical variables. The genes in the most significant WGCNA module were used to cluster AATD individuals. Gene validation was performed by NanoString nCounter Gene Expression Assay. RESULT: We observed modest effects of AATD genotype and augmentation therapy on gene expression. When WGCNA was applied to BAL transcriptome, one gene module, ME31 (2312 genes), correlated with the highest number of clinical variables and was functionally enriched with numerous immune T-lymphocyte related pathways. This gene module identified two distinct clusters of AATD individuals with different disease severity and distinct PBMC gene expression patterns. CONCLUSIONS: We successfully identified novel clusters of AATD individuals where severity correlated with increased immune response independent of individuals' genotype and augmentation therapy. These findings may suggest the presence of previously unrecognised disease endotypes in AATD that associate with T-lymphocyte immunity and disease severity.

Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2.

Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.

Transcriptomics of bronchoalveolar lavage cells identifies new molecular endotypes of sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits. METHODS: RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well-characterised patients with pulmonary sarcoidosis enrolled in the multicentre Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted gene co-expression network analysis and nonparametric statistics were used to analyse genome-wide BAL transcriptome. Validation of results was performed using a microarray expression dataset of an independent sarcoidosis cohort (Freiburg, Germany; n=50). RESULTS: Our supervised analysis found associations between distinct transcriptional programmes and major pulmonary phenotypic manifestations of sarcoidosis including T-helper type 1 (Th1) and Th17 pathways associated with hilar lymphadenopathy, transforming growth factor-ß1 (TGFB1) and mechanistic target of rapamycin (MTOR) signalling with parenchymal involvement, and interleukin (IL)-7 and IL-2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T-cell immune response; extraocular organ involvement with PI3K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan involvement, with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg. CONCLUSION: Taken together, our results identify BAL gene expression programmes that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

An integrated self-powered 3D printed sample concentrator for highly sensitive molecular detection of HIV in whole blood at the point of care.

Rapid and efficient biological sample preparation and pretreatment are crucial for highly sensitive, reliable and reproducible molecular detection of infectious diseases. Herein, we report a self-powered, integrated sample concentrator (SPISC) for rapid plasma separation, pathogen lysis, nucleic acid trapping and enrichment at the point of care. The proposed sample concentrator uses a combination of gravitational sedimentation of blood cells and capillary force for rapid, self-powered plasma separation. The pathogens (e.g., HIV virus) in separated plasma were directly lysed and pathogen nucleic acid was enriched by an integrated, flow-through FTA® membrane in the concentrator, enabling highly efficient nucleic acid preparation. The FTA® membrane of the SPISC is easy to store and transport at room temperature without need for uninterrupted cold chain, which is crucial for point of care sampling in resource-limited settings. The platform has been successfully applied to detect HIV virus in blood samples. Our experiments show that the sample concentrator can achieve a plasma separation efficiency as high as 95% and a detection sensitivity as low as 10 copies per 200 µL blood (∼100 copies per mL plasma) with variability less than 7%. The sample concentrator described is fully compatible with downstream nucleic acid detection and has great potential for early diagnostics, monitoring and management of infectious diseases at the point of care.

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses.

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences.