Single-Shot ChAd3-MARV Vaccine in Modified Formulation Buffer Shows 100% Protection of NHPs.

Marburg virus (MARV) is a virus of high human consequence with a case fatality rate of 24-88%. The global health and national security risks posed by Marburg virus disease (MVD) underscore the compelling need for a prophylactic vaccine, but no candidate has yet reached regulatory approval. Here, we evaluate a replication-defective chimpanzee adenovirus type 3 (ChAd3)-vectored MARV Angola glycoprotein (GP)-expressing vaccine against lethal MARV challenge in macaques. The ChAd3 platform has previously been reported to protect against the MARV-related viruses, Ebola virus (EBOV) and Sudan virus (SUDV), and MARV itself in macaques, with immunogenicity demonstrated in macaques and humans. In this study, we present data showing 100% protection against MARV Angola challenge (versus 0% control survival) and associated production of GP-specific IgGs generated by the ChAd3-MARV vaccine following a single dose of 1 × 1011 virus particles prepared in a new clinical formulation buffer designed to enhance product stability. These results are consistent with previously described data using the same vaccine in a different formulation and laboratory, demonstrating the reproducible and robust protective efficacy elicited by this promising vaccine for the prevention of MVD. Additionally, a qualified anti-GP MARV IgG ELISA was developed as a critical pre-requisite for clinical advancement and regulatory approval.

Development of a Well-Characterized Cynomolgus Macaque Model of Sudan Virus Disease for Support of Product Development.

The primary objective of this study was to characterize the disease course in cynomolgus macaques exposed to Sudan virus (SUDV), to determine if infection in this species is an appropriate model for the evaluation of filovirus countermeasures under the FDA Animal Rule. Sudan virus causes Sudan virus disease (SVD), with an average case fatality rate of approximately 50%, and while research is ongoing, presently there are no approved SUDV vaccines or therapies. Well characterized animal models are crucial for further developing and evaluating countermeasures for SUDV. Twenty (20) cynomolgus macaques were exposed intramuscularly to either SUDV or sterile phosphate-buffered saline; 10 SUDV-exposed animals were euthanized on schedule to characterize pathology at defined durations post-exposure and 8 SUDV-exposed animals were not part of the scheduled euthanasia cohort. Survival was assessed, along with clinical observations, body weights, body temperatures, hematology, clinical chemistry, coagulation, viral load (serum and tissues), macroscopic observations, and histopathology. There were statistically significant differences between SUDV-exposed animals and mock-exposed animals for 26 parameters, including telemetry body temperature, clinical chemistry parameters, hematology parameters, activated partial thromboplastin time, serum viremia, and biomarkers that characterize the disease course of SUDV in cynomolgus macaques.

Development of a Well-Characterized Cynomolgus Macaque Model of Marburg Virus Disease for Support of Vaccine and Therapy Development.

Marburg virus (MARV) is a filovirus that can infect humans and nonhuman primates (NHPs), causing severe disease and death. Of the filoviruses, Ebola virus (EBOV) has been the primary target for vaccine and therapeutic development. However, MARV has an average case fatality rate of approximately 50%, the infectious dose is low, and there are currently no approved vaccines or therapies targeted at infection with MARV. The purpose of this study was to characterize disease course in cynomolgus macaques intramuscularly exposed to MARV Angola variant. There were several biomarkers that reliably correlated with MARV-induced disease, including: viral load; elevated total clinical scores; temperature changes; elevated ALT, ALP, BA, TBIL, CRP and decreased ALB values; decreased lymphocytes and platelets; and prolonged PTT. A scheduled euthanasia component also provided the opportunity to study the earliest stages of the disease. This study provides evidence for the application of this model to evaluate potential vaccines and therapies against MARV and will be valuable in improving existing models.

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.

Animal Models of COVID-19: Nonhuman Primates.

With the advent of the novel SARS-CoV-2, the entire world has been thrown into chaos with severe disruptions from a normal life. While the entire world was going chaotic, the researchers throughout the world were struggling to contribute to the best of their capabilities to advance the understanding of this new pandemic and fast track the development of novel therapeutics and vaccines. While various animal models have helped a lot to understand the basic physiology, nonhman primates have been promising and much more successful in modelling human diseases compared to other available clinical models. Here we describe the different aspects of modelling the SARS-CoV-2 infection in NHPs along with the associated methods used in NHP immunology.

A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 µg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

Skin Vaccination with Ebola Virus Glycoprotein Using a Polyphosphazene-Based Microneedle Patch Protects Mice against Lethal Challenge.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

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.

Natural history of disease in cynomolgus monkeys exposed to Ebola virus Kikwit strain demonstrates the reliability of this non-human primate model for Ebola virus disease.

Filoviruses (Family Filoviridae genera Ebolavirus and Marburgvirus) are negative-stranded RNA viruses that cause severe health effects in humans and non-human primates, including death. Except in outbreak settings, vaccines and other medical countermeasures against Ebola virus (EBOV) will require testing under the FDA Animal Rule. Multiple vaccine candidates have been evaluated using cynomolgus monkeys (CM) exposed to EBOV Kikwit strain. To the best of our knowledge, however, animal model development data supporting the use of CM in vaccine research have not been submitted to the FDA. This study describes a large CM database (122 CM, 62 female and 60 male, age 2 to 9 years) and demonstrates the consistency of the CM model through time to death models and descriptive statistics. CMs were exposed to EBOV doses of 0.1 to 100,000 PFU in 33 studies conducted at three Animal Biosafety Level 4 facilities, by three exposure routes. Time to death was modeled using Cox proportional hazards models with a frailty term that incorporated study-to-study variability. Despite significant differences attributed to exposure variables, all CMs exposed to the 100 to 1,000 pfu doses commonly used in vaccine studies died or met euthanasia criteria within 21 days of exposure, median 7 days, 93% between 5 and 12 days of exposure. Moderate clinical signs were observed 4 to 5 days after exposure and preceded death or euthanasia by approximately one day. Viremia was detected within a few days of infection. Hematology indices were indicative of viremia and the propensity for hemorrhage with progression of Ebola viremia. Changes associated with coagulation parameters and platelets were consistent with coagulation disruption. Changes in leukocyte profiles were indicative of an acute inflammatory response. Increased liver enzymes were observed shortly after exposure. Taken together, these factors suggest that the cynomolgus monkey is a reliable animal model for human disease.

Identification and Characterization of Defective Viral Genomes in Ebola Virus-Infected Rhesus Macaques.

Ebola virus (EBOV), of the family Filoviridae, is an RNA virus that can cause a hemorrhagic fever with a high mortality rate. Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including in vitro EBOV infection, and have previously been associated with viral persistence and immunostimulatory activity. As DVGs have been detected in cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in filovirus-infected hosts. Therefore, we interrogated sequence data from serum and tissue samples using a bioinformatics tool in order to identify the presence of DVGs in nonhuman primates (NHPs) infected with EBOV, Sudan virus (SUDV), or Marburg virus (MARV). Multiple 5' copy-back DVGs (cbDVGs) were detected in NHP serum during the acute phase of filovirus infection. While the relative abundance of total DVGs in most animals was low, serum collected during acute EBOV and SUDV infections, but not MARV infections, contained a higher proportion of short trailer sequence cbDVGs than the challenge stock. This indicated an accumulation of these DVGs throughout infection, potentially due to the preferential replication of short DVGs over the longer viral genome. Using reverse transcriptase PCR (RT-PCR) and deep sequencing, we also confirmed the presence of 5' cbDVGs in EBOV-infected NHP testes, which is of interest due to EBOV persistence in semen of male survivors of infection. This work suggests that DVGs play a role in EBOV infection in vivo and that further study will lead to a better understanding of EBOV pathogenesis. IMPORTANCE The study of filovirus pathogenesis is critical for understanding the consequences of infection and for the development of strategies to ameliorate future outbreaks. Defective viral genomes (DVGs) have been detected during EBOV infections in vitro; however, their presence in in vivo infections remains unknown. In this study, DVGs were detected in samples collected from EBOV- and SUDV-infected nonhuman primates (NHPs). The accumulation of these DVGs in the trailer region of the genome during infection indicates a potential role in EBOV and SUDV pathogenesis. In particular, the presence of DVGs in the testes of infected NHPs requires further investigation as it may be linked to the establishment of persistence.

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.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 µg RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only ∼2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. SIGNIFICANCE STATEMENT: The emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 µ g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFN's potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. ONE-SENTENCE SUMMARY: A SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

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.

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

OBJECTIVES: Prolonged 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. METHODS: Chamber slides and N95 respirator material were directly inoculated with SARS-CoV-2 and were exposed to different durations of PX-UV. RESULTS: For 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. CONCLUSION: With 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.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.