The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies.

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. Because rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting

Development and Testing of a Method for Validating Chemical Inactivation of Ebola Virus

Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods

Transboundary Animal Diseases, an Overview of 17 Diseases with Potential for Global Spread and Serious Consequences

Animals provide food and other critical resources to most of the global population. As such, diseases of animals can cause dire consequences, especially disease with high rates of morbidity or mortality. Transboundary animal diseases (TADs) are highly contagious or transmissible, epidemic diseases, with the potential to spread rapidly across the globe and the potential to cause substantial socioeconomic and public health consequences. Transboundary animal diseases can threaten the global food supply, reduce the availability of non-food animal products, or cause the loss of human productivity or life. Further, TADs result in socioeconomic consequences from costs of control or preventative measures, and from trade restrictions. A greater understanding of the transmission, spread, and pathogenesis of these diseases is required. Further work is also needed to improve the efficacy and cost of both diagnostics and vaccines. This review aims to give a broad overview of 17 TADs, providing researchers and veterinarians with a current, succinct resource of salient details regarding these significant diseases. For each disease, we provide a synopsis of the disease and its status, species and geographic areas affected, a summary of in vitro or in vivo research models, and when available, information regarding prevention or treatment

Development of a Lethal Intranasal Exposure Model of Ebola Virus in the Cynomolgus Macaque

Ebola virus (EBOV) is a filovirus that can cause Ebola virus disease (EVD). No approved vaccines or therapies exist for filovirus infections, despite an urgent need. The development and testing of effective countermeasures against EBOV requires use of animal models and a thorough understanding of how the model aligns with EVD in humans. The majority of published studies report outcomes of parenteral exposures for emulating needle stick transmission. However, based on data from EVD outbreaks, close contact exposures to infected bodily fluid seems to be one of the primary routes of EBOV transmission. Thus, further work is needed to develop models that represent mucosal exposure. To characterize the outcome of mucosal exposure to EBOV, cynomolgus macaques were exposed to EBOV via intranasal (IN) route using the LMA® mucosal atomization device (LMA® MAD). For comparison, four non-human primates (NHPs) were exposed to EBOV via intramuscular (IM) route. This IN exposure model was uniformly lethal and correlated with a statistically significant delay in time to death when compared to exposure via the IM route. This more closely reflects the timeframes observed in human infections. An IN model of exposure offers an attractive alternative to other models as it can offer insight into the consequences of exposure via a mucosal surface and allows for screening countermeasures via a different exposure route

Intramuscular Exposure of Macaca fascicularis to Low Doses of Low Passage- or Cell Culture-Adapted Sudan Virus or Ebola Virus

The filoviruses Ebola virus (EBOV) and Sudan virus (SUDV) can cause severe diseases, and there are currently no licensed countermeasures available for use against them. Transmission occurs frequently via contact with bodily fluids from infected individuals. However, it can be difficult to determine when or how someone became infected, or the quantity of infectious virus to which they were exposed. Evidence suggests the infectious dose is low, but the majority of published studies use high exposure doses. This study characterized the outcome of exposure to a low dose of EBOV or SUDV, using a Macaca fascicularis model. Further, because the effect of virus passage in cell culture may be more pronounced when lower exposure doses are used, viruses that possessed either the characteristics of wild type viruses (possessing predominantly 7-uridine (7U) genotype and a high particle-to-plaque forming unit (PFU) ratio) or cell culture-passaged viruses (predominantly 8-uridine (8U) genotype, a lower particle-to-PFU ratio) were used. The time to death after a low dose exposure was delayed in comparison to higher exposure doses. These data demonstrated that an extremely low dose of EBOV or SUDV is sufficient to cause lethal disease. A low dose exposure model can help inform studies on pathogenesis, transmission, and optimization of prevention strategies

A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

ABSTRACT Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA’s Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo. These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV resulted in a single mutation in the region encoding the glycoprotein (GP). This is a region where mutations can have important consequences on outbreaks and human disease [S. Mahanty and M. Bray, Lancet Infect Dis 4:487–498, 2004, https://doi.org/10.1016/S1473-3099(04)01103-X]. We thus investigated whether this mutation impacted disease by using a cynomolgus macaque model of MARV infection. Monkeys exposed to virus containing the mutation had better clinical outcomes than monkeys exposed to virus without the mutation. We also observed that a remarkably low number of MARV particles was sufficient to cause death. Our results could have a significant impact on how future studies are designed to model MARV disease and test vaccines and therapeutics

Immune Correlates of Protection from Filovirus Efficacy Studies in Non-Human Primates

Non-human primate (NHP) efficacy data for several Ebola virus (EBOV) vaccine candidates exist, but definitive correlates of protection (CoP) have not been demonstrated, although antibodies to the filovirus glycoprotein (GP) antigen and other immunological endpoints have been proposed as potential CoPs. Accordingly, studies that could elucidate biomarker(s) that statistically correlate, whether mechanistically or not, with protection are warranted. The primary objective of this study was to evaluate potential CoP for Novavax EBOV GP vaccine candidate administered at different doses to cynomolgus macaques using the combined data from two separate, related studies containing a total of 44 cynomolgus macaques. Neutralizing antibodies measured by pseudovirion neutralization assay (PsVNA) and anti-GP IgG binding antibodies were evaluated as potential CoP using logistic regression models. The predictive ability of these models was assessed using the area under the receiver operating characteristic (ROC) curve (AUC). Fitted models indicated a statistically significant relationship between survival and log base 10 (log10) transformed anti-GP IgG antibodies, with good predictive ability of the model. Neither (log10 transformed) PsVNT50 nor PsVNT80 titers were statistically significant predictors of survival, though predictive ability of both models was good. Predictive ability was not statistically different between any pair of models. Models that included immunization dose in addition to anti-GP IgG antibodies failed to detect statistically significant effects of immunization dose. These results support anti-GP IgG antibodies as a correlate of protection. Total assay variabilities and geometric coefficients of variation (GCVs) based on the study data appeared to be greater for both PsVNA readouts, suggesting the increased assay variability may account for non-significant model results for PsVNA despite the good predictive ability of the models. The statistical approach to evaluating CoP for this EBOV vaccine may prove useful for advancing research for Sudan virus (SUDV) and Marburg virus (MARV) candidate vaccines

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

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Deactivation of SARS-CoV-2 with pulsed-xenon ultraviolet light: Implications for environmental COVID-19 control.

ObjectivesProlonged survival of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on environmental surfaces and personal protective equipment may lead to these surfaces transmitting this pathogen to others. We sought to determine the effectiveness of a pulsed-xenon ultraviolet (PX-UV) disinfection system in reducing the load of SARS-CoV-2 on hard surfaces and N95 respirators.MethodsChamber slides and N95 respirator material were directly inoculated with SARS-CoV-2 and were exposed to different durations of PX-UV.ResultsFor hard surfaces, disinfection for 1, 2, and 5 minutes resulted in 3.53 log10, >4.54 log10, and >4.12 log10 reductions in viral load, respectively. For N95 respirators, disinfection for 5 minutes resulted in >4.79 log10 reduction in viral load. PX-UV significantly reduced SARS-CoV-2 on hard surfaces and N95 respirators.ConclusionWith the potential to rapidly disinfectant environmental surfaces and N95 respirators, PX-UV devices are a promising technology to reduce environmental and personal protective equipment bioburden and to enhance both healthcare worker and patient safety by reducing the risk of exposure to SARS-CoV-2

Development of a Well-Characterized Rhesus Macaque Model of Ebola Virus Disease for Support of Product Development

Ebola virus (EBOV) is a negative-sense RNA virus that can infect humans and nonhuman primates with severe health consequences. Development of countermeasures requires a thorough understanding of the interaction between host and pathogen, and the course of disease. The goal of this study was to further characterize EBOV disease in a uniformly lethal rhesus macaque model, in order to support development of a well-characterized model following rigorous quality standards. Rhesus macaques were intramuscularly exposed to EBOV and one group was euthanized at predetermined time points to characterize progression of disease. A second group was not scheduled for euthanasia in order to analyze survival, changes in physiology, clinical pathology, terminal pathology, and telemetry kinetics. On day 3, sporadic viremia was observed and pathological evidence was noted in lymph nodes. By day 5, viremia was detected in all EBOV exposed animals and pathological evidence was noted in the liver, spleen, and gastrointestinal tissues. These data support the notion that EBOV infection in rhesus macaques is a rapid systemic disease similar to infection in humans, under a compressed time scale. Biomarkers that correlated with disease progression at the earliest stages of infection were observed thereby identifying potential “trigger-to-treat” for use in therapeutic studies