Christine R. Fisher

The average cost to bring a new drug from its initial discovery to a patient's bedside is estimated to surpass $2 billion and requires over a decade of research and development. There is a need for new drug screening technologies that can parse drug candidates with increased likelihood of clinical utility early in development in order to increase the cost‐effectiveness of this pipeline. For example, during the COVID‐19 pandemic, resources were rapidly mobilized to identify effective therapeutic treatments but many lead antiviral compounds failed to demonstrate efficacy when progressed to human trials. To address the lack of predictive preclinical drug screening tools, PREDICT96‐ALI, a high‐throughput (n = 96) microphysiological system (MPS) that recapitulates primary human tracheobronchial tissue,is adapted for the evaluation of differential antiviral efficacy of native SARS‐CoV‐2 variants of concern. Here, PREDICT96‐ALI resolves both the differential viral kinetics between variants and the efficacy of antiviral compounds over a range of drug doses. PREDICT96‐ALI is able to distinguish clinically efficacious antiviral therapies like remdesivir and nirmatrelvir from promising lead compounds that do not show clinical efficacy. Importantly, results from this proof‐of‐concept study track with known clinical outcomes, demonstrate the feasibility of this technology as a prognostic drug discovery tool.

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system.

The COVID-19 pandemic necessitated a rapid mobilization of resources toward the development of safe and efficacious vaccines and therapeutics. Finding effective treatments to stem the wave of infected individuals needing hospitalization and reduce the risk of adverse events was paramount. For scientists and healthcare professionals addressing this challenge, the need to rapidly identify medical countermeasures became urgent, and many compounds in clinical use for other indications were repurposed for COVID-19 clinical trials after preliminary preclinical data demonstrated antiviral activity against SARS-CoV-2. Two repurposed compounds, fluvoxamine and amodiaquine, showed efficacy in reducing SARS-CoV-2 viral loads in preclinical experiments, but ultimately failed in clinical trials, highlighting the need for improved predictive preclinical tools that can be rapidly deployed for events such as pandemic emerging infectious diseases. The PREDICT96-ALI platform is a high-throughput, high-fidelity microphysiological system (MPS) that recapitulates primary human tracheobronchial tissue and supports highly robust and reproducible viral titers of SARS-CoV-2 variants Delta and Omicron. When amodiaquine and fluvoxamine were tested in PREDICT96-ALI, neither compound demonstrated an antiviral response, consistent with clinical outcomes and in contrast with prior reports assessing the efficacy of these compounds in other human cell-based in vitro platforms. These results highlight the unique prognostic capability of the PREDICT96-ALI proximal airway MPS to assess the potential antiviral response of lead compounds.

Despite the relatively common observation of therapeutic efficacy in discovery screens with immortalized cell lines, the vast majority of drug candidates do not reach clinical development. Candidates that do move forward often fail to demonstrate efficacy when progressed from animal models to humans. This dilemma highlights the need for new drug screening technologies that can parse drug candidates early in development with regard to predicted relevance for clinical use. PREDICT96-ALI is a high-throughput organ-on-chip platform incorporating human primary airway epithelial cells in a dynamic tissue microenvironment. Here we demonstrate the utility of PREDICT96-ALI as an antiviral screening tool for SARS-CoV-2, combining the high-throughput functionality of a 96-well plate format in a high containment laboratory with the relevant biology of primary human tissue. PREDICT96-ALI resolved differential efficacy in five antiviral compounds over a range of drug doses. Complementary viral genome quantification and immunofluorescence microscopy readouts achieved high repeatability between devices and replicate plates. Importantly, results from testing the three antiviral drugs currently available to patients (nirmatrelvir, molnupiravir, and remdesivir) tracked with clinical outcomes, demonstrating the value of this technology as a prognostic drug discovery tool.

Background: Ebola virus (EBOV) is a highly lethal member of the Filoviridae family associated with human hemorrhagic disease. Despite being a sporadic disease, it caused a large outbreak in 2014–2016 in West Africa and another outbreak recently in the Democratic Republic of Congo. Several vaccine candidates are currently in preclinical and clinical studies but none are stable without cold chain storage.Methods: We used preservation by vaporization (PBV), a novel processing technology to heat-stabilize FiloRab1 (inactivated rabies-based Ebola vaccine), a candidate Ebola vaccine, and stored the vials at temperatures ranging from 4°C to 50°C for 10 days to 12 months. We immunized Syrian hamsters with the best long-term stable FiloRab1 PBV vaccines and challenged them with rabies virus (RABV).Results: Syrian hamsters immunized with FiloRab1 PBV–processed vaccines stored at temperatures of 4°C and 37°C for 6 months, and at 50°C for 2 weeks, seroconverted against both RABV-G and EBOV-GP. Notably, all of the FiloRab1 PBV vaccines proved to be 100% effective in a RABV challenge model.Conclusions: We successfully demonstrated that the FiloRab1 PBV vaccines are stable and efficacious for up to 6 months when stored at temperatures ranging from 4°C to 37°C and for up to 2 weeks at 50°C.

Current rabies vaccines are safe and, when administered properly, they are highly effective. In addition, they elicit long-lasting immunity, with virus-neutralising antibody titres persisting for years after vaccination. However, current regimens require multiple doses to achieve high neutralising titres and they are costly, which means that it is difficult for developing countries, where rabies deaths are highest, to implement widespread vaccination. New innovations are the only way to reduce rabies disease to acceptable rates. Numerous preclinical and clinical studies are under way, testing novel vaccines, adjuvants and injection methods. Research into the use of live vaccines and alternative vaccine vectors is ongoing, while attempts to develop DNA vaccines have so far failed to match the immunogenicity and neutralising capability of traditional vaccines. The development of molecular adjuvants that induce faster, stronger immune responses with less antigen has yielded exciting preclinical results and appears to edge us closer to a better rabies vaccine. However, steep challenges remain: molecular adjuvants require administration with live vaccines, and differences in species specificity of immune molecules complicate development. Over all, the array of research undertaken over the past decade is impressive and encouraging, but most new vaccines have yet to be tested in clinical trials, and the viability of such experimental vaccines in the global market remains to be seen. Only a vaccine that outperforms currently available vaccines in every area will have a chance at widespread adoption. Nevertheless, the authors are confident that some vaccine candidates will meet these criteria. Las actuales vacunas antirrábicas son seguras y, si se administran debidamente, muy eficaces. Además, inducen inmunidad duradera, con títulos de anticuerpos neutralizantes que subsisten años después de la vacunación. Sin embargo, los regímenes actuales resultan costosos y exigen dosis múltiples para lograr títulos de neutralización elevados, lo que dificulta a los países en desarrollo, que son los más golpeados por la rabia, la implantación generalizada de la vacunación. El único camino para reducir la rabia a niveles aceptables pasa por la innovación. Están en marcha numerosos estudios preclínicos y clínicos en los que se ensayan vacunas, adyuvantes y métodos de inyección novedosos. También sigue adelante la investigación sobre el uso de vacunas vivas y vectores vacunales alternativos, mientras que ninguna de las tentativas realizadas hasta la fecha con vacunas de ADN ha deparado niveles de inmunogenicidad y capacidad de neutralización equiparables a los de las vacunas tradicionales. La obtención de adyuvantes moleculares que inducen una respuesta inmunitaria más rápida y vigorosa en presencia de menos cantidad de antígeno ha dado resultados preclínicos muy interesantes y poco a poco parece acercarnos al logro de una mejor vacuna antirrábica. Subsisten, empero, arduas dificultades: los adyuvantes moleculares solo funcionan si se administran con vacunas vivas, y las diferencias existentes entre las especies en cuanto a la especificidad de las moléculas inmunitarias complican las labores de desarrollo. Globalmente, el conjunto de investigaciones emprendidas en el último decenio es impresionante y alentador, pero la mayoría de las nuevas vacunas aún deben pasar por la fase de ensayo clínico, y está por ver qué viabilidad tienen estas vacunas experimentales en el mercado mundial. Solo una vacuna que supere a las actuales en todos los aspectos tiene posibilidades de ser adoptada a gran escala. Pese a todo, los autores expresan su confianza en que algunas de las vacunas candidatas cumplan estos criterios. Les vaccins actuels contre la rage sont sûrs et très efficaces lorsqu’ils sont administrés correctement. En outre, ils confèrent une immunité durable, avec le maintien de titres neutralisants d’anticorps plusieurs années après la vaccination. Néanmoins, les régimes actuels nécessitent l’administration de plusieurs doses pour obtenir des titres élevés d’anticorps neutralisants et ils sont onéreux, de sorte que la vaccination à grande échelle est difficile à mettre en oeuvre dans les pays en développement, pourtant les plus touchés par la mortalité par rage. Seule l’adoption de solutions innovantes permettra de ramener l’incidence de la rage à un niveau acceptable. De nombreuses études précliniques et cliniques sont en cours, visant à tester les innovations en matière de vaccins, de modes d’injection et d’adjuvants. La recherche sur l’utilisation de vaccins à virus vivant et sur de nouveaux vecteurs vaccinaux se poursuit, alors que les tentatives de développement de vaccins à ADN n’ont pas réussi jusqu’à présent à obtenir un effet immunogène ou des capacités de neutralisation virale équivalents à ceux des vaccins traditionnels. Les résultats d’essais précliniques sur de nouveaux adjuvants moléculaires induisant une réponse immune plus rapide et plus puissante avec moins d’antigène sont extrêmement prometteurs et semblent annoncer l’imminence de meilleurs vaccins contre la rage. Il subsiste toutefois d’importantes difficultés : les adjuvants moléculaires ne peuvent être administrés qu’avec des vaccins vivants et les différences de spécificité d’espèce des molécules immunes rendent le développement plus complexe. Globalement, les efforts déployés depuis une...

Significance: Generating mAbs against the native extracellular epitopes of multispanning membrane proteins is challenging, and as a result, few nonpeptidic mAbs against transporters have ever been isolated. Our approach here using virus-like particles and divergent host species for immunizations provides a means to overcome these challenges. The specific mAbs isolated here recognize native GLUT4 on the cell surface and can distinguish its different conformational states, thus representing some of the only state-specific mAbs ever isolated against any transporter. Epitope mapping of these mAbs revealed their binding sites as well as the mechanisms by which amino acids control the inward-open and outward-open states of GLUT4. Our studies demonstrate a valuable platform to isolate functional mAbs against important multispanning membrane proteins.

With much of the molecular virology characterized, we are now gaining an appreciation of how nuanced rabies virus infection dynamics are and how immune status relates to transmission. The growing number of recognized rabies virus-related lyssaviruses highlights shortcomings in our discriminatory diagnostics and raises questions about their impact on human health. The lack of therapeutics for some of these lyssaviruses is a major concern. Enzootic maintenance of rabies virus within a population depends on transmission within narrow windows. The virus avoids extinction by both general and reservoir host-specific mechanisms with remarkable epidemiological plasticity. Features of rabies virus biology, ecology and evolution have made the virus a model pathogen for disease ecology and evolution. Recent work using mathematical models and phylodynamic analyses have enabled reconstruction and forecasting of epizootic spread of the virus at the landscape level. At an evolutionary level, there is evidence that strong barriers prevent rabies virus from establishing in new host species, and both ecological opportunities and viral adaptation are needed to overcome these barriers. Studies of rabies virus molecular evolutionary dynamics have revealed the role of purifying selection and the importance of viral and host genetic backgrounds in host shifts. Further study of contemporary host shifts will likely shed light on the unknown origin of rabies. We suggest that efforts be focused on developing strategies and technologies to manage rabies viruses within bat reservoirs as previously done with carnivores, integrating diagnostics that can rapidly differentiate strains into surveillance efforts, honing our predictive power to detect outbreaks and coordinating local resources to halt the spread of this lethal zoonosis.

The 2013–2016 West African Ebola virus (EBOV) disease outbreak was the largest filovirus outbreak to date. Over 28 000 suspected, probable, or confirmed cases have been reported, with a 53% case-fatality rate. The magnitude and international impact of this EBOV outbreak has highlighted the urgent need for a safe and efficient EBOV vaccine. To this end, we demonstrate the immunogenicity and protective efficacy of FILORAB1, a recombinant, bivalent, inactivated rabies virus–based EBOV vaccine, in rhesus and cynomolgus monkeys. Our results demonstrate that the use of the synthetic Toll-like receptor 4 agonist glucopyranosyl lipid A in stable emulsion (GLA-SE) as an adjuvant increased the efficacy of FILORAB1 to 100% protection against lethal EBOV challenge, with no to mild clinical signs of disease. Furthermore, all vaccinated subjects developed protective anti–rabies virus antibody titers. Taken together, these results support further development of FILORAB1/GLA-SE as an effective preexposure EBOV vaccine.