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.

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.

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.

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.

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.

Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination.

Recently approved vaccines have shown remarkable efficacy in limiting SARS-CoV-2-associated disease. However, with the variety of vaccines, immunization strategies, and waning antibody titers, defining the correlates of immunity across a spectrum of antibody titers is urgently required. Thus, we profiled the humoral immune response in a cohort of non-human primates immunized with a recombinant SARS-CoV-2 spike glycoprotein (NVX-CoV2373) at two doses, administered as a single- or two-dose regimen. Both antigen dose and boosting significantly altered neutralization titers and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were associated with distinct levels of protection in the upper and lower respiratory tract. Moreover, NVX-CoV2373 elicited antibodies that functionally targeted emerging SARS-CoV-2 variants. Collectively, the data presented here suggest that a single dose may prevent disease via combined Fc/Fab functions but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.

Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 in nonhuman primates following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination.

Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants. HIGHLIGHTS: NVX-CoV2373 subunit vaccine elicits receptor blocking, virus neutralizing antibodies, and Fc-effector functional antibodies.The vaccine protects against respiratory tract infection and virus shedding in non-human primates (NHPs).Both neutralizing and Fc-effector functions contribute to protection, potentially through different mechanisms in the upper and lower respiratory tract.Both macaque and human vaccine-induced antibodies exhibit altered Fc-receptor binding to emerging mutants.

Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M™ vaccination.

Recently approved vaccines have already shown remarkable protection in limiting SARS-CoV-2 associated disease. However, immunologic mechanism(s) of protection, as well as how boosting alters immunity to wildtype and newly emerging strains, remain incompletely understood. Here we deeply profiled the humoral immune response in a cohort of non-human primates immunized with a stable recombinant full-length SARS-CoV-2 spike (S) glycoprotein (NVX-CoV2373) at two dose levels, administered as a single or two-dose regimen with a saponin-based adjuvant Matrix-M™. While antigen dose had some effect on Fc-effector profiles, both antigen dose and boosting significantly altered overall titers, neutralization and Fc-effector profiles, driving unique vaccine-induced antibody fingerprints. Combined differences in antibody effector functions and neutralization were strongly associated with distinct levels of protection in the upper and lower respiratory tract, pointing to the presence of combined, but distinct, compartment-specific neutralization and Fc-mechanisms as key determinants of protective immunity against infection. Moreover, NVX-CoV2373 elicited antibodies functionally target emerging SARS-CoV-2 variants, collectively pointing to the critical collaborative role for Fab and Fc in driving maximal protection against SARS-CoV-2. Collectively, the data presented here suggest that a single dose may prevent disease, but that two doses may be essential to block further transmission of SARS-CoV-2 and emerging variants.