Anti-Nipah Virus Enzyme-Linked Immunosorbent Assays with Non-human Primate and Hamster Serum.

Enzyme-linked Immunosorbent assays or ELISAs are a versatile method for detecting various immunological ligands of interest. As the name suggests, ELISAs rely on the interaction between a ligand and an antibody to produce results. In the study of infectious disease, ELISAs are commonly used to determine if a pathogen-specific immune response has occurred in a host organism. These assays can be performed in serosurveys as part of epidemiological investigations during, or following, an infectious disease outbreak. In the research environment, ELISAs are used to quantify the humoral immune response following infection or vaccination of a host organism. Data from these assays can be used to determine the type of immune response elicited (e.g. IgG1 vs IgG2) and the robustness of the response. Here, we describe ELISAs that were developed for the study of either hamsters or non-human primates vaccinated against Nipah virus infection, or infected with Nipah virus. The ELISAs described include assays for both IgG and IgM in the hamster and non-human primate models for Nipah virus-induced disease. An assay was also developed for the detection of IgA in bronchoalveolar lavage from non-human primates.

Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Longitudinal analyses using 18F-Fluorodeoxyglucose positron emission tomography with computed tomography as a measure of COVID-19 severity in the aged, young, and humanized ACE2 SARS-CoV-2 hamster models.

This study compared disease progression of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in three different models of golden hamsters: aged (≈60 weeks old) wild-type (WT), young (6 weeks old) WT, and adult (14-22 weeks old) hamsters expressing the human-angiotensin-converting enzyme 2 (hACE2) receptor. After intranasal (IN) exposure to the SARS-CoV-2 Washington isolate (WA01/2020), 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography with computed tomography (18F-FDG PET/CT) was used to monitor disease progression in near real time and animals were euthanized at pre-determined time points to directly compare imaging findings with other disease parameters associated with coronavirus disease 2019 (COVID-19). Consistent with histopathology, 18F-FDG-PET/CT demonstrated that aged WT hamsters exposed to 105 plaque forming units (PFU) developed more severe and protracted pneumonia than young WT hamsters exposed to the same (or lower) dose or hACE2 hamsters exposed to a uniformly lethal dose of virus. Specifically, aged WT hamsters presented with a severe interstitial pneumonia through 8 d post-exposure (PE), while pulmonary regeneration was observed in young WT hamsters at that time. hACE2 hamsters exposed to 100 or 10 PFU virus presented with a minimal to mild hemorrhagic pneumonia but succumbed to SARS-CoV-2-related meningoencephalitis by 6 d PE, suggesting that this model might allow assessment of SARS-CoV-2 infection on the central nervous system (CNS). Our group is the first to use (18F-FDG) PET/CT to differentiate respiratory disease severity ranging from mild to severe in three COVID-19 hamster models. The non-invasive, serial measure of disease progression provided by PET/CT makes it a valuable tool for animal model characterization.

Multivalent viral particles elicit safe and efficient immunoprotection against Nipah Hendra and Ebola viruses.

Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses have focused on targeting individual viruses, although their geographical and bat reservoir host overlaps warrant creation of multivalent vaccines. Here we explored whether replication-incompetent pseudotyped vesicular stomatitis virus (VSV) virions or NiV-based virus-like particles (VLPs) were suitable multivalent vaccine platforms by co-incorporating multiple surface glycoproteins from NiV, HeV, and EBOV onto these virions. We then enhanced the vaccines' thermotolerance using carbohydrates to enhance applicability in global regions that lack cold-chain infrastructure. Excitingly, in a Syrian hamster model of disease, the VSV multivalent vaccine elicited safe, strong, and protective neutralizing antibody responses against challenge with NiV, HeV, or EBOV. Our study provides proof-of-principle evidence that replication-incompetent multivalent viral particle vaccines are sufficient to provide protection against multiple zoonotic deadly viruses with high pandemic potential.

Antiviral innate immunity is diminished in the upper respiratory tract of severe COVID-19 patients.

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-κB signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-κB response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-κB responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-κB responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Sangivamycin is highly effective against SARS-CoV-2 in vitro and has favorable drug properties.

Sangivamycin is a nucleoside analog that is well tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin's potential for clinical administration, pharmacokinetic; absorption, distribution, metabolism, and excretion (ADME); and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 therapeutic.

Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain­RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Naturally Acquired SARS-CoV-2 Immunity Persists for Up to 11 Months Following Infection.

BACKGROUND: Characterizing the kinetics of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of critical importance to developing strategies that may mitigate the public health burden of coronavirus disease 2019 (COVID-19). We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma donors at multiple time points over an 11-month period to determine how circulating antibody levels change over time following natural infection. METHODS: From April 2020 to February 2021, we enrolled 228 donors. At each study visit, subjects either donated plasma or had study samples drawn only. Anti-SARS-CoV-2 donor testing was performed using the VITROS Anti-SARS-CoV-2 Total and IgG assays and an in-house fluorescence reduction neutralization assay. RESULTS: Anti-SARS-CoV-2 antibodies were identified in 97% of COVID-19 convalescent donors at initial presentation. In follow-up analyses, of 116 donors presenting at repeat time points, 91.4% had detectable IgG levels up to 11 months after symptom recovery, while 63% had detectable neutralizing titers; however, 25% of donors had neutralizing levels that dropped to an undetectable titer over time. CONCLUSIONS: Our data suggest that immunological memory is acquired in most individuals infected with SARS-CoV-2 and is sustained in a majority of patients for up to 11 months after recovery. Clinical Trials Registration. NCT04360278.

Formulation, Stability, Pharmacokinetic, and Modeling Studies for Tests of Synergistic Combinations of Orally Available Approved Drugs against Ebola Virus In Vivo.

Outbreaks of Ebola ebolavirus (EBOV) have been associated with high morbidity and mortality. Milestones have been reached recently in the management of EBOV disease (EVD) with licensure of an EBOV vaccine and two monoclonal antibody therapies. However, neither vaccines nor therapies are available for other disease-causing filoviruses. In preparation for such outbreaks, and for more facile and cost-effective management of EVD, we seek a cocktail containing orally available and room temperature stable drugs with strong activity against multiple filoviruses. We previously showed that (bepridil + sertraline) and (sertraline + toremifene) synergistically suppress EBOV in cell cultures. Here, we describe steps towards testing these combinations in a mouse model of EVD. We identified a vehicle suitable for oral delivery of the component drugs and determined that, thus formulated the drugs are equally active against EBOV as preparations in DMSO, and they maintain activity upon storage in solution for up to seven days. Pharmacokinetic (PK) studies indicated that the drugs in the oral delivery vehicle are well tolerated in mice at the highest doses tested. Collectively the data support advancement of these combinations to tests for synergy in a mouse model of EVD. Moreover, mathematical modeling based on human oral PK projects that the combinations would be more active in humans than their component single drugs.

Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants that threaten the efficacy of existing vaccines and therapeutic antibodies underscores the urgent need for new antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells of COVID-19 patients. The three most potent antibodies targeted distinct regions of the RBD, and all three neutralized the SARS-CoV-2 variants B.1.1.7 and B.1.351. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the ACE2 receptor, and has limited contact with key variant residues K417, E484 and N501. We designed bispecific antibodies by combining non-overlapping specificities and identified five ultrapotent bispecific antibodies that inhibit authentic SARS-CoV-2 infection at concentrations of <1 ng/mL. Through a novel mode of action three bispecific antibodies cross-linked adjacent spike proteins using dual NTD/RBD specificities. One bispecific antibody was >100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a 2.5 mg/kg dose. Notably, six of nine bispecific antibodies neutralized B.1.1.7, B.1.351 and the wild-type virus with comparable potency, despite partial or complete loss of activity of at least one parent monoclonal antibody against B.1.351. Furthermore, a bispecific antibody that neutralized B.1.351 protected against SARS-CoV-2 expressing the crucial E484K mutation in the hamster model. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Anti-SARS-CoV-2 Serology persistence over time in COVID-19 Convalescent Plasma Donors.

BACKGROUND: Characterizing the kinetics of the antibody response to SARS□CoV□2 is of critical importance to developing strategies that may mitigate the public health burden of COVID-19. We sought to determine how circulating antibody levels change over time following natural infection. METHODS/MATERIALS: We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma (CCP) donors at multiple time points over a 9-month period. At each study visit, subjects either donated plasma or only had study samples drawn. In all cases, anti-SARS-CoV-2 donor testing was performed using semi-quantitative chemiluminescent immunoassays (ChLIA) targeting subunit 1 (S1) of the SARS-CoV-2 spike (S) protein, and an in-house fluorescence reduction neutralization assay (FRNA). RESULTS: From April to November 2020 we enrolled 202 donors, mean age 47.3 ±14.7 years, 55% female, 75% Caucasian. Most donors reported a mild clinical course (91%, n=171) without hospitalization. One hundred and five (105) (52%) donors presented for repeat visits with a median 42 (12-163) days between visits. The final visit occurred at a median 160 (53-273) days post-symptom resolution. Total anti-SARS-CoV-2 antibodies (Ab), SARS-CoV-2 specific IgG and neutralizing antibodies were detected in 97.5%, 91.1%, and 74% of donors respectively at initial presentation. Neutralizing Ab titers based on FRNA 50 were positively associated with mean IgG levels (p = <0.0001). Mean IgG levels and neutralizing titers were positively associated with COVID-19 severity, increased donor age and BMI (p=0.0006 and p=0.0028, p=0.0083 and p=0.0363, (p=0.0008 and p=0.0018, respectively). Over the course of repeat visits, IgG decreased in 74.1% of donors; FRNA 50 decreased in 44.4% and remained unchanged in 33.3% of repeat donors. A weak negative correlation was observed between total Ab levels and number of days post-symptom recovery (r = 0.09). CONCLUSION: Anti-SARS-CoV-2 antibodies were identified in 97% of convalescent donors at initial presentation. In a cohort that largely did not require hospitalization. IgG and neutralizing antibodies were positively correlated with age, BMI and clinical severity, and persisted for up to 9 months post-recovery from natural infection. On repeat presentation, IgG anti-SARS-CoV-2 levels decreased in 56% of repeat donors. Overall, these data suggest that CP donors possess a wide range of IgG and neutralizing antibody levels that are proportionally distributed across demographics, with the exception of age, BMI and clinical severity.

Scalable, Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was expanding, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

Clinical, laboratory, and temporal predictors of neutralizing antibodies against SARS-CoV-2 among COVID-19 convalescent plasma donor candidates.

BACKGROUNDSARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2-neutralizing antibodies (nAbs) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood.METHODSPotential convalescent plasma donors with virologically documented SARS-CoV-2 infection were tested for serum IgG against SARS-CoV-2 spike protein S1 domain and against nucleoprotein (NP), and for nAb.RESULTSAmong 250 consecutive persons, including 27 (11%) requiring hospitalization, who were studied a median of 67 days since symptom onset, 97% were seropositive on 1 or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titers included older age (adjusted OR [AOR] 1.03 per year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. nAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range 77-120) apart (P < 0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses.CONCLUSIONnAb titers correlated with COVID-19 severity, age, and sex. SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels declined, and a small proportion of convalescent individuals lacked adaptive immune responses.FUNDINGThe project was supported by the Frederick National Laboratory for Cancer Research with support from the NIAID under contract number 75N91019D00024, and was supported by the Fred Hutchinson Joel Meyers Endowment, Fast-Grants, a New Investigator award from the American Society for Transplantation and Cellular Therapy, and NIH contracts 75N93019C0063, 75N91019D00024, and HHSN272201800013C, and NIH grants T32-AI118690, T32-AI007044, K08-AI119142, and K23-AI140918.

The SKI complex is a broad-spectrum, host-directed antiviral drug target for coronaviruses, influenza, and filoviruses.

The SARS-CoV-2 pandemic has made it clear that we have a desperate need for antivirals. We present work that the mammalian SKI complex is a broad-spectrum, host-directed, antiviral drug target. Yeast suppressor screening was utilized to find a functional genetic interaction between proteins from influenza A virus (IAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) with eukaryotic proteins that may be potential host factors involved in replication. This screening identified the SKI complex as a potential host factor for both viruses. In mammalian systems siRNA-mediated knockdown of SKI genes inhibited replication of IAV and MERS-CoV. In silico modeling and database screening identified a binding pocket on the SKI complex and compounds predicted to bind. Experimental assays of those compounds identified three chemical structures that were antiviral against IAV and MERS-CoV along with the filoviruses Ebola and Marburg and two further coronaviruses, SARS-CoV and SARS-CoV-2. The mechanism of antiviral activity is through inhibition of viral RNA production. This work defines the mammalian SKI complex as a broad-spectrum antiviral drug target and identifies lead compounds for further development.

Clinical, laboratory, and temporal predictors of neutralizing antibodies to SARS-CoV-2 after COVID-19.

BACKGROUND: SARS-CoV-2-specific antibodies may protect from reinfection and disease, providing the rationale for administration of plasma containing SARS-CoV-2 neutralizing antibodies (nAb) as a treatment for COVID-19. The clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood. METHODS: Adults with virologically-documented SARS-CoV-2 infection in a convalescent plasma donor screening program were tested for serum IgG to SARS-CoV-2 spike protein S1 domain, nucleoprotein (NP), and for nAb. RESULTS: Amongst 250 consecutive persons studied a median of 67 days since symptom onset, 243/250 (97%) were seropositive on one or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titer included older age (adjusted OR [AOR] 1.03/year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during acute illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic (ROC) analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. NAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range, 77-120) apart (P<0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses. CONCLUSIONS: Nab titers correlated with COVID-19 severity, age, and sex. Standard commercially available SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels were found to decline and a small proportion of COVID-19 survivors lack adaptive immune responses.

A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging human pathogen, endemic in areas of China, Japan, and the Korea (KOR). It is primarily transmitted through infected ticks and can cause a severe hemorrhagic fever disease with case fatality rates as high as 30%. Despite its high virulence and increasing prevalence, molecular and functional studies in situ are scarce due to the limited availability of high-titer SFTSV exposure stocks. During the course of field virologic surveillance in 2017, we detected SFTSV in ticks and in a symptomatic soldier in a KOR Army training area. SFTSV was isolated from the ticks producing a high-titer viral exposure stock. Through the use of advanced genomic tools, we present here a complete, in-depth characterization of this viral stock, including a comparison with both the virus in its arthropod source and in the human case, and an in vivo study of its pathogenicity. Thanks to this detailed characterization, this SFTSV viral exposure stock constitutes a quality biological tool for the study of this viral agent and for the development of medical countermeasures, fulfilling the requirements of the main regulatory agencies.

Repurposing Pyramax®, quinacrine and tilorone as treatments for Ebola virus disease.

We have recently identified three molecules (tilorone, quinacrine and pyronaridine tetraphosphate) which all demonstrated efficacy in the mouse model of infection with mouse-adapted Ebola virus (EBOV) model of disease and had similar in vitro inhibition of an Ebola pseudovirus (VSV-EBOV-GP), suggesting they interfere with viral entry. Using a machine learning model to predict lysosomotropism these compounds were evaluated for their ability to possess a lysosomotropic mechanism in vitro. We now demonstrate in vitro that pyronaridine tetraphosphate is an inhibitor of Lysotracker accumulation in lysosomes (IC50 = 0.56 µM). Further, we evaluated antiviral synergy between pyronaridine and artesunate (Pyramax®), which are used in combination to treat malaria. Artesunate was not found to have lysosomotropic activity in vitro and the combination effect on EBOV inhibition was shown to be additive. Pyramax® may represent a unique example of the repurposing of a combination product for another disease.

Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6-12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

A Novel Ebola Virus VP40 Matrix Protein-Based Screening for Identification of Novel Candidate Medical Countermeasures.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.