Primate Cells Research Articles

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry.

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

Transcriptomic architecture of nuclei in the marmoset CNS

To understand the cellular composition and region-specific specialization of white matter — a disease-relevant, glia-rich tissue highly expanded in primates relative to rodents — we profiled transcriptomes of ~500,000 nuclei from 19 tissue types of the central nervous system of healthy common marmoset and mapped 87 subclusters spatially onto a 3D MRI atlas. We performed cross-species comparison, explored regulatory pathways, modeled regional intercellular communication, and surveyed cellular determinants of neurological disorders. Here, we analyze this resource and find strong spatial segregation of microglia, oligodendrocyte progenitor cells, and astrocytes. White matter glia are diverse, enriched with genes involved in stimulus-response and biomolecule modification, and predicted to interact with other resident cells more extensively than their gray matter counterparts. Conversely, gray matter glia preserve the expression of neural tube patterning genes into adulthood and share six transcription factors that restrict transcriptome complexity. A companion Callithrix jacchus Primate Cell Atlas (CjPCA) is available through https://cjpca.ninds.nih.gov.

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family.

Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized β1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnβ1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

Racial and Ethnic Inequities in Use of Preventive Services Among Privately Insured Adults With a Pediatric-Onset Disability.

Cerebral palsy (CP) and spina bifida (SB) are pediatric-onset disabilities. Adults living with CP/SB are in a greater need of preventive care than the general population due to their increased risk for chronic diseases. Our objective was to compare White/Black and White/Hispanic inequities in the use of preventive services. Using 2007-2017 private claims data, we identified a total of 11,635 adults with CP/BS. Of these, 8,935 were White, 1,457 Black, and 1,243 Hispanic. We matched health-related variables (age, sex, comorbid conditions) between White adults and those in each minority subpopulation. Generalized estimating equations were used and all models were adjusted for age, sex, comorbidities, income, education, and US Census divisions. Outcomes of interest were: (1) any office visit; (2) any physical/occupational therapy; (3) wellness visit; (4) bone density screening; (5) cholesterol screening; and (6) diabetes screening. The rate of recommended services for all subpopulations of adults with CP/SB was low. Compared with White adults, Hispanic adults had lower odds of wellness visits (odds ratio [OR] = 0.71, 95% CI, 0.53-0.96) but higher odds of diabetes screening (OR = 1.48, 95% CI, 1.13-1.93). Compared with White adults, Black adults had lower odds of wellness visits (OR = 0.50, 95% CI, 0.24-1.00) and bone density screening (OR = 0.54, 95% CI, 0.31-0.95). Preventive service use among adults with CP/SB was low. Large White-minority disparities in wellness visits were observed. Interventions to address physical accessibility, adoption of telehealth, and increased clinician education may mitigate these disparities, particularly if initiatives target minority populations.

Porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV): A threat for xenotransplantation?

The potential for a donor-derived transmission of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) to the recipient has been recognized since pigs were considered candidate donors for xenotransplantation. This review gives a short description of the viral properties and summarizes the current evidence of the effects of PCMV/PRV transmission in preclinical xenotransplantation. Despite evidence that PCMV/PRV does not infect human and non-human primate cells, activation in the transplanted organ and detrimental systemic complications have been described. As PCMV/PRV is a herpesvirus able to establish latency, the importance of adequate screening of donor pigs is emphasized, as no efficient treatment is available. Furthermore, easy and successful ways of elimination of PCMV/PRV from pig herds are indicated.

Virus Safety of Xenotransplantation.

The practice of xenotransplantation using pig islet cells or organs is under development to alleviate the shortage of human donor islet cells or organs for the treatment of diabetes or organ failure. Multiple genetically modified pigs were generated to prevent rejection. Xenotransplantation may be associated with the transmission of potentially zoonotic porcine viruses. In order to prevent this, we developed highly sensitive PCR-based, immunologicals and other methods for the detection of numerous xenotransplantation-relevant viruses. These methods were used for the screening of donor pigs and xenotransplant recipients. Of special interest are the porcine endogenous retroviruses (PERVs) that are integrated in the genome of all pigs, which are able to infect human cells, and that cannot be eliminated by methods that other viruses can. We showed, using droplet digital PCR, that the number of PERV proviruses is different in different pigs (usually around 60). Furthermore, the copy number is different in different organs of a single pig, indicating that PERVs are active in the living animals. We showed that in the first clinical trials treating diabetic patients with pig islet cells, no porcine viruses were transmitted. However, in preclinical trials transplanting pig hearts orthotopically into baboons, porcine cytomegalovirus (PCMV), a porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), but no PERVs, were transmitted. PCMV/PRV transmission resulted in a significant reduction of the survival time of the xenotransplant. PCMV/PRV was also transmitted in the first pig heart transplantation to a human patient and possibly contributed to the death of the patient. Transmission means that the virus was detected in the recipient, however it remains unclear whether it can infect primate cells, including human cells. We showed previously that PCMV/PRV can be eliminated from donor pigs by early weaning. PERVs were also not transmitted by inoculation of human cell-adapted PERV into small animals, rhesus monkey, baboons and cynomolgus monkeys, even when pharmaceutical immunosuppression was applied. Since PERVs were not transmitted in clinical, preclinical, or infection experiments, it remains unclear whether they should be inactivated in the pig genome by CRISPR/Cas. In summary, by using our sensitive methods, the safety of xenotransplantation can be ensured.

A reference single-cell regulomic and transcriptomic map of cynomolgus monkeys

Non-human primates are attractive laboratory animal models that accurately reflect both developmental and pathological features of humans. Here we present a compendium of cell types across multiple organs in cynomolgus monkeys (Macaca fascicularis) using both single-cell chromatin accessibility and RNA sequencing data. The integrated cell map enables in-depth dissection and comparison of molecular dynamics, cell-type compositions and cellular heterogeneity across multiple tissues and organs. Using single-cell transcriptomic data, we infer pseudotime cell trajectories and cell-cell communications to uncover key molecular signatures underlying their cellular processes. Furthermore, we identify various cell-specific cis-regulatory elements and construct organ-specific gene regulatory networks at the single-cell level. Finally, we perform comparative analyses of single-cell landscapes among mouse, monkey and human. We show that cynomolgus monkey has strikingly higher degree of similarities in terms of immune-associated gene expression patterns and cellular communications to human than mouse. Taken together, our study provides a valuable resource for non-human primate cell biology.

Spatial profiling of early primate gastrulation in utero.

Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells(PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.

TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates.

2563 Background: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe side effects including cytokine release syndrome (CRS) and vascular leak syndrome (VLS). This is believed to be due to activation of IL-2Rα+ endothelial cells and inflammatory eosinophils as well as high Cmax due to the short half-life requiring frequent high dose administrations. Also, potent activation of immunosuppressive IL-2Rα+ regulatory T cells (Tregs) may limit IL-2’s efficacy. TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog designed to optimally address these shortcomings. Methods: Naïve male and female cynomolgus monkeys received 2-3 doses of TransCon IL-2 β/γ intravenously Q2W. Blood samples were taken to assess plasma concentrations of IL-2 β/γ (active drug), serum cytokines, hematology and immunophenotyping. Histopathology was also performed. Results: Administration of TransCon IL-2 β/γ induced robust increases in absolute lymphocyte counts (ALC), with no/minor increases in eosinophil counts. The increase in ALC was primarily driven by potent activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells and Tregs. At doses with maximum pharmacodynamic (PD) responses, TransCon IL-2 β/γ induced on average > 20-fold ALC increases, > 19-fold CD8+ T cell increases, > 50-fold Effector Memory CD8+ T cell increases, > 18-fold NK cell increases and > 400-fold γδ T cell increases compared to pre-dose levels. These changes corresponded with a low Cmax and a long effective half-life ( > 30 hours) for IL-2 β/γ. A clear induction of soluble CD25 and chemokine MCP-1 was seen after dosing, while levels of IL-5, IL-6, IFN-γ and TNF-α remained low. Further, histopathology showed no signs of vascular damage, tissue necrosis, pulmonary edema or eosinophilia. Conclusions: In cynomolgus monkeys, TransCon IL-2 β/γ demonstrated remarkable PD responses in CD8+ T cells, NK cells and γδ T cells over Tregs and eosinophils. No signs of VLS or CRS were observed. These results are likely due to the selective IL-2Rβ/γ bias along with the slow and sustained release of IL-2 β/γ resulting in low Cmax and long circulatory half-life enabled by the TransCon prodrug technology. The massive expansion of γδ T cells, which are known to possess considerable anti-tumor activity, is to our knowledge a unique finding for TransCon IL-2 β/γ among IL-2 variants in clinical development. Altogether, the responses seen in monkeys are suggestive of a potentially substantial improvement in the therapeutic index beyond what has been achieved by aldesleukin or new IL-2 variants to date. TransCon IL-2 β/γ is currently being evaluated in a clinical Phase 1/2 trial as monotherapy and in combination with pembrolizumab (NCT05081609).

HLA-B*57:01 Complexed to a CD8 T-Cell Epitope from the HSV-2 ICP22 Protein Binds NK and T Cells through KIR3DL1.

HLA-B*57:01 is an HLA allelic variant associated with positive outcomes during viral infections through interactions with T cells and NK cells, but severe disease in persons treated with the anti-HIV-1 drug abacavir. The role of HLA-B*57:01 in the context of HSV infection is unknown. We identified an HLA-B*57:01-restricted CD8 T-cell epitope in the ICP22 (US1) protein of HSV-2. CD8 T cells reactive to the HSV-2 ICP22 epitope recognized the orthologous HSV-1 peptide, but not closely related peptides in human IFNL2 or IFNL3. Abacavir did not alter the CD8 T-cell recognition of the HSV or self-derived peptides. Unexpectedly, a tetramer of HSV-2 ICP22 epitope (228–236) and HLA-B*57:01 bound both CD8 T cells and NK cells. Tetramer specificity for KIR3DL1 was confirmed using KIR3DL1 overexpression on non-human primate cells lacking human KIR and studies with blocking anti-KIR3DL1 antibody. Interaction with KIR3DL1 was generalizable to donors lacking the HLA-B*57:01 genotype or HSV seropositivity. These findings suggest a mechanism for the recognition of HSV infection by NK cells or KIR-expressing T cells via KIR3DL1.

Early development of the cochlea of the common marmoset, a non-human primate model.

BackgroundFine-tuned cochlear development is essential for hearing. Owing to the difficulty in using early human fetal samples, most of our knowledge regarding cochlear development has been obtained from rodents. However, several inter-species differences in cochlear development between rodents and humans have been reported. To bridge these differences, we investigated early otic development of a non-human primate model animal, the common marmoset (Callithrix jacchus).MethodsWe examined 20 genes involved in early cochlear development and described the critical developmental steps for morphogenesis, which have been reported to vary between rodents and marmosets.ResultsThe results revealed that several critical genes involved in prosensory epithelium specifications showed higher inter-species differences, suggesting that the molecular process for hair cell lineage acquisition in primates differs considerably from that of rodents. We also observed that the tempo of cochlear development was three times slower in the primate than in rodents.ConclusionsOur data provide new insights into early cochlear development in primates and humans and imply that the procedures used for manipulating rodent cochlear sensory cells cannot be directly used for the research of primate cells due to the intrinsic inter-species differences in the cell fate determination program.

Antibody-dependent enhancement of SARS-CoV-2 spike binding to human ACE2 receptor by virus neutralizing antibodies

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease COVID-19 has caused over 275 million cases of illness and over 5 million deaths as of December 2021. The spike glycoproteins expressed on surface of the SARS-CoV-2 particle are the primary target of both vaccination and monoclonal antibody therapies. Widely reported evidence suggests that a subset of both naturally produced and synthetic antibodies (Abs) enhance infection, a phenomenon known as antibody-dependent enhancement (ADE). Recently, it has been demonstrated that the mechanism by which some Abs may elicit ADE is by enhancing the binding affinity of SARS-CoV-2 spike to its ligand - the human ACE2 receptor. Using in vitro spike-ACE2 binding assays, we identified two Abs which demonstrated ADE for spike/human ACE2 binding. Surprisingly, these binding-enhancing antibodies sufficiently neutralized SARS-CoV-2 pseudo-virus infection in human ACE2-knockin 293T cells, as well as live virus infection in Vero-E6 (non-human primate cell line) and Calu-3 (human cell line) cells in vitro. Furthermore, these receptor-binding enhancing antibodies significantly reduce but not enhance signs of SARS-CoV-2 infection in human ACE2-knockin mice, including viral replication and tissue pathology. Taken together, our data suggest a discrepancy between results of in vitro spike/receptor binding assay and virus neutralization assays for SARS-CoV-2 infection. Enhanced spike binding to host receptors is not a reliable measure of screening for ADE antibodies, but may be a mechanism by which virus neutralization is achieved in nature. This work was supported in part by grants from the National Institutes of Health (R01 AI151139, R56 AI146226 and P20 GM130555). W. Huang also received research support from MegaRobo Technologies Corporation.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

A New Method of Bone Stromal Cell Characterization by Flow Cytometry.

The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.

Non-human primate pluripotent stem cells for the preclinical testing of regenerative therapies.

Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies. Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases. Therefore, the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years. For the preclinical validation of cell replacement therapies in non-human primates, it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts. However, pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems. In recent years, however, relevant progress has also been made with non-human primate pluripotent stem cells. This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies. We focus on the critical domains of (1) reprogramming and embryonic stem cell line derivation, (2) cell line maintenance and characterization and, (3) application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.

BMAL1 moonlighting as a gatekeeper for LINE1 repression and cellular senescence in primates.

Aging in humans is intricately linked with alterations in circadian rhythms concomitant with physiological decline and stem cell exhaustion. However, whether the circadian machinery directly regulates stem cell aging, especially in primates, remains poorly understood. In this study, we found that deficiency of BMAL1, the only non-redundant circadian clock component, results in an accelerated aging phenotype in both human and cynomolgus monkey mesenchymal progenitor cells (MPCs). Unexpectedly, this phenotype was mainly attributed to a transcription-independent role of BMAL1 in stabilizing heterochromatin and thus preventing activation of the LINE1-cGAS-STING pathway. In senescent primate MPCs, we observed decreased capacity of BMAL1 to bind to LINE1 and synergistic activation of LINE1 expression. Likewise, in the skin and muscle tissues from the BMAL1-deficient cynomolgus monkey, we observed destabilized heterochromatin and aberrant LINE1 transcription. Altogether, these findings uncovered a noncanonical role of BMAL1 in stabilizing heterochromatin to inactivate LINE1 that drives aging in primate cells.

The OM-85 bacterial lysate inhibits SARS-CoV-2 infection of epithelial cells by downregulating SARS-CoV-2 receptor expression

BackgroundTreatments for coronavirus disease 2019, which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through interactions of its spike (S) protein with angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. We found that airway OM-85 administration inhibits Ace2 and Tmprss2 transcription in the mouse lung, suggesting that OM-85 might hinder SARS-CoV-2/host cell interactions.ObjectivesWe sought to investigate whether and how OM-85 treatment protects nonhuman primate and human epithelial cells against SARS-CoV-2.MethodsACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein–pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung, and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2-transfected HEK293T cells treated with OM-85 in vitro.ResultsOM-85 significantly downregulated ACE2 and TMPRSS2 transcription and surface ACE2 protein expression in epithelial cell lines and primary bronchial epithelial cells. OM-85 also strongly inhibited SARS-CoV-2 S1 protein binding to, SARS-CoV-2 S protein–pseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on SARS-CoV-2 receptor downregulation.ConclusionsOM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of coronavirus disease 2019.

Roles of antiviral sensing and type I interferon signaling in the restriction of SARS-CoV-2 replication

SummarySevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019. Few studies have compared replication dynamics and host responses to SARS-CoV-2 in cell lines from different tissues and species. Therefore, we investigated the role of tissue type and antiviral genes during SARS-CoV-2 infection in nonhuman primate (kidney) and human (liver, respiratory epithelial, gastric) cell lines. We report different viral growth kinetics and release among the cell lines despite comparable ACE2 expression. Transcriptomics revealed that absence of STAT1 in nonhuman primate cells appeared to enhance inflammatory responses without effecting infectious viral titer. Deletion of RL-6 in respiratory epithelial cells increased viral replication. Impaired infectious virus release was detected in Huh7 but not Huh7.5 cells, suggesting a role for RIG1. Gastric cells MKN45 exhibited robust antiviral gene expression and supported viral replication. Data here provide insight into molecular pathogenesis of and alternative cell lines for studying SARS-CoV-2 infection.

Highly Specific Sigma Receptor Ligands Exhibit Anti-Viral Properties in SARS-CoV-2 Infected Cells.

(1) Background: There is a strong need for prevention and treatment strategies for COVID-19 that are not impacted by SARS-CoV-2 mutations emerging in variants of concern. After virus infection, host ER resident sigma receptors form direct interactions with non-structural SARS-CoV-2 proteins present in the replication complex. (2) Methods: In this work, highly specific sigma receptor ligands were investigated for their ability to inhibit both SARS-CoV-2 genome replication and virus induced cellular toxicity. This study found antiviral activity associated with agonism of the sigma-1 receptor (e.g., SA4503), ligation of the sigma-2 receptor (e.g., CM398), and a combination of the two pathways (e.g., AZ66). (3) Results: Intermolecular contacts between these ligands and sigma receptors were identified by structural modeling. (4) Conclusions: Sigma receptor ligands and drugs with off-target sigma receptor binding characteristics were effective at inhibiting SARS-CoV-2 infection in primate and human cells, representing a potential therapeutic avenue for COVID-19 prevention and treatment.

Conserved Induction of Distinct Antiviral Signalling Kinetics by Primate Interferon Lambda 4 Proteins.

Interferon lambdas (IFNλ) (also known as type III IFNs) are critical cytokines that combat infection predominantly at barrier tissues, such as the lung, liver, and gastrointestinal tract. Humans have four IFNλs (1–4), where IFNλ1–3 show ~80%–95% homology, and IFNλ4 is the most divergent displaying only ~30% sequence identity. Variants in IFNλ4 in humans are associated with the outcome of infection, such as with hepatitis C virus. However, how IFNλ4 variants impact cytokine signalling in other tissues and how well this is conserved is largely unknown. In this study, we address whether differences in antiviral signalling exist between IFNλ4 variants in human hepatocyte and intestinal cells, comparing them to IFNλ3. We demonstrate that compared to IFNλ3, wild-type human IFNλ4 induces a signalling response with distinct magnitudes and kinetics, which is modified by naturally occurring variants P70S and K154E in both cell types. IFNλ4’s distinct antiviral response was more rapid yet transient compared to IFNλ1 and 3. Additionally, divergent antiviral kinetics were also observed using non-human primate IFNλs and cell lines. Furthermore, an IFNλ4-like receptor-interacting interface failed to alter IFNλ1’s kinetics. Together, our data provide further evidence that major functional differences exist within the IFNλ gene family. These results highlight the possible tissue specialisation of IFNλs and encourage further investigation of the divergent, non-redundant activities of IFNλ4 and other IFNλs.