Modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice

The emergence of highly pathogenic human coronaviruses (hCoVs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. Global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. Limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hCoV infections. Therefore, understanding how hCoV-host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hCoVs in humans. Pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hCoV-host interactions that govern pathogenesis. Notably, tractable mouse genetics combined with hCoV reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus-host interaction networks in disease. In addition to assessing etiologies of known hCoVs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent CoVs. Knowledge of CoV pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the World Health Organization. Although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hCoV mouse models, their use as tractable complex genetic organisms for untangling complex hCoV-host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions

Increasing the translation of mouse models of MERS coronavirus pathogenesis through kinetic hematological analysis

Newly emerging viral pathogens pose a constant and unpredictable threat to human and animal health. Coronaviruses (CoVs) have a penchant for sudden emergence, as evidenced by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and most recently, swine acute diarrhea syndrome coronavirus (SADS-CoV). Small animal models of emerging viral pathogenesis are crucial to better understand the virus and host factors driving disease progression. However, rodent models are often criticized for their limited translatability to humans. The complete blood count is the most ordered clinical test in the United States serving as the cornerstone of clinical medicine and differential diagnosis. We recently generated a mouse model for MERS-CoV pathogenesis through the humanization of the orthologous entry receptor dipeptidyl peptidase 4 (DPP4). To increase the translatability of this model, we validated and established the use of an automated veterinary hematology analyzer (VetScan HM5) at biosafety level 3 for analysis of peripheral blood. MERS-CoV lung titer peaked 2 days post infection concurrent with lymphopenia and neutrophilia in peripheral blood, two phenomena also observed in MERS-CoV infection of humans. The fluctuations in leukocyte populations measured by Vetscan HM5 were corroborated by standard flow cytometry, thus confirming the utility of this approach. Comparing a sublethal and lethal dose of MERS-CoV in mice, analysis of daily blood draws demonstrates a dose dependent modulation of leukocytes. Major leukocyte populations were modulated before weight loss was observed. Importantly, neutrophil counts on 1dpi were predictive of disease severity with a lethal dose of MERS-CoV highlighting the predictive value of hematology in this model. Taken together, the inclusion of hematological measures in mouse models of emerging viral pathogenesis increases their translatability and should elevate the preclinical evaluation of MERS-CoV therapeutics and vaccines to better mirror the complexity of the human condition

Artificial intelligence for dementia prevention

INTRODUCTION: A wide range of modifiable risk factors for dementia have been identified. Considerable debate remains about these risk factors, possible interactions between them or with genetic risk, and causality, and how they can help in clinical trial recruitment and drug development. Artificial intelligence (AI) and machine learning (ML) may refine understanding.// METHODS: ML approaches are being developed in dementia prevention. We discuss exemplar uses and evaluate the current applications and limitations in the dementia prevention field.// RESULTS: Risk-profiling tools may help identify high-risk populations for clinical trials; however, their performance needs improvement. New risk-profiling and trial-recruitment tools underpinned by ML models may be effective in reducing costs and improving future trials. ML can inform drug-repurposing efforts and prioritization of disease-modifying therapeutics.// DISCUSSION: ML is not yet widely used but has considerable potential to enhance precision in dementia prevention

Efficacy of host cell serine protease inhibitor MM3122 against SARS-CoV-2 for treatment and prevention of COVID-19

We developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases, including transmembrane protease serine 2 (TMPRSS2), matriptase, and hepsin. TMPRSS2 is a membrane-associated protease that is highly expressed in the upper and lower respiratory tracts and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell entry, replication, and dissemination of new virus particles. We have previously shown that compound MM3122 exhibited subnanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Furthermore, we evaluated MM3122 in a mouse model of COVID-19 and demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in proinflammatory cytokine and chemokine production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2-infected mice. Therefore, MM3122 is a promising lead candidate small-molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses

Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2′O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2′O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2′O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2′O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2′O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2′O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines

Mouse Adapted SARS-CoV-2 (MA10) Viral Infection Induces Neuroinflammation in Standard Laboratory Mice

Increasing evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection impacts neurological function both acutely and chronically, even in the absence of pronounced respiratory distress. Developing clinically relevant laboratory mouse models of the neuropathogenesis of SARS-CoV-2 infection is an important step toward elucidating the underlying mechanisms of SARS-CoV-2-induced neurological dysfunction. Although various transgenic models and viral delivery methods have been used to study the infection potential of SARS-CoV-2 in mice, the use of commonly available laboratory mice would facilitate the study of SARS-CoV-2 neuropathology. Herein we show neuroinflammatory profiles of immunologically intact mice, C57BL/6J and BALB/c, as well as immunodeficient (Rag2−/−) mice, to a mouse-adapted strain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 (MA10)). Our findings indicate that brain IL-6 levels are significantly higher in BALB/c male mice infected with SARS-CoV-2 MA10. Additionally, blood-brain barrier integrity, as measured by the vascular tight junction protein claudin-5, was reduced by SARS-CoV-2 MA10 infection in all three strains. Brain glial fibrillary acidic protein (GFAP) mRNA was also elevated in male C57BL/6J infected mice compared with the mock group. Lastly, immune-vascular effects of SARS-CoV-2 (MA10), as measured by H&E scores, demonstrate an increase in perivascular lymphocyte cuffing (PLC) at 30 days post-infection among infected female BALB/c mice with a significant increase in PLC over time only in SARS-CoV-2 MA10) infected mice. Our study is the first to demonstrate that SARS-CoV-2 (MA10) infection induces neuroinflammation in laboratory mice and could be used as a novel model to study SARS-CoV-2-mediated cerebrovascular pathology

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Herein, we show that the ribonucleoside analog β-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (β-D-N4-hydroxycytidine-5′-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple coronaviruses and oral bioavailability highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses

Swine acute diarrhea syndrome coronavirus replication in primary human cells reveals potential susceptibility to infection

Zoonotic coronaviruses represent an ongoing threat, yet the myriads of circulating animal viruses complicate the identification of higher-risk isolates that threaten human health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered, highly pathogenic virus that likely evolved from closely related HKU2 bat coronaviruses, circulating in Rhinolophus spp. bats in China and elsewhere. As coronaviruses cause severe economic losses in the pork industry and swine are key intermediate hosts of human disease outbreaks, we synthetically resurrected a recombinant virus (rSADS-CoV) as well as a derivative encoding tomato red fluorescent protein (tRFP) in place of ORF3. rSADS-CoV replicated efficiently in a variety of continuous animal and primate cell lines, including human liver and rectal carcinoma cell lines. Of concern, rSADS-CoV also replicated efficiently in several different primary human lung cell types, as well as primary human intestinal cells. rSADS-CoV did not use human coronavirus ACE-2, DPP4, or CD13 receptors for docking and entry. Contemporary human donor sera neutralized the group I human coronavirus NL63, but not rSADS-CoV, suggesting limited human group I coronavirus cross protective herd immunity. Importantly, remdesivir, a broad-spectrum nucleoside analog that is effective against other group 1 and 2 coronaviruses, efficiently blocked rSADS-CoV replication in vitro. rSADS-CoV demonstrated little, if any, replicative capacity in either immune-competent or immunodeficient mice, indicating a critical need for improved animal models. Efficient growth in primary human lung and intestinal cells implicate SADS-CoV as a potential higher-risk emerging coronavirus pathogen that could negatively impact the global economy and human health

Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice

Improving clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small-molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAbs) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). It is not known whether combination RDV/mAb will improve outcomes over single-agent therapies or whether antibody therapies will remain efficacious against variants. Here, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 h after infection and have therapeutic efficacy in vivo against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared with single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants

High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VHFc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic