Kendra J. Alfson

Orthoebolavirus causes severe Ebola virus disease (EVD) and deadly outbreaks in humans. This infection occurs through macropinocytosis and trafficking to late endosomes or lysosomes that utilize the receptor Niemann–Pick C1 (NPC1) to enter the cell cytoplasm. We designed peptide inhibitors based on the NPC1 receptor to target the NPC1 binding site to block viral entry. The results indicated that the ligand-based peptide inhibitors showed potent inhibition activities in vitro studies against pseudotyped or replication-competent Orthoebolavirus. Therefore, we further evaluated one of them in a mouse model challenged with mice-adapted Ebola viruses, which showed some protection efficacy compared with the control group. This study suggests that ligand-based peptides are encouraging inhibitors in the development of inhibitors against Ebola virus infection.

In human and non-human primates, filoviruses, e.g., Ebolaviruses, cause severe hemorrhagic fever for which there are few therapeutic options. While there are licensed vaccines and therapeutics for Ebola virus disease, there is no approved vaccine or treatment for other Ebola diseases. There is a need for broad-spectrum antivirals to treat Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV). We have previously demonstrated that probenecid, an FDA-approved drug with a safety profile spanning over 7 decades, is safe and effective in preventing the replication of influenza A viruses, SARS-CoV-2, and other RNA respiratory viruses, such as HMPV and RSV, both in vitro and in vivo. In this study, probenecid was shown to inhibit the replication of infectious EBOV, SUDV, and MARV in Vero E6 cells, with IC50 Values of 3 μM, 8 μM, and 13 μM, respectively. It also reduced plaque size in infected Vero cell lawns, suggesting reduced virus spread. These studies show that probenecid is an effective, broad-spectrum, host-directed antiviral drug.

Algal lectin Griffithsin (GRFT) is a well-known mannose-binding protein which has broad-spectrum antiviral activity against several important infectious viruses including HIV, HCV, and SARS-CoV-2. Therefore, GRFT has been brought great attention to antiviral therapeutic development. In this report, we have tested GRFT’s activity against the lethal Ebola virus in vitro and in vivo. Our data have shown that the IC₅₀ value is about 42 nM for inhibiting Zaire Ebola virus (EBOV) infection in vitro. The preliminary in vivo mice model using mouse-adapted EBOV has also shown a certain efficacy for delayed mortality compared to the control animals. A GRFT pull-down experiment using viral particles demonstrates that GRFT can bind to N-glycans of EBOV. Thus, it can be concluded that GRFT, through binding to viral glycans, may block Ebola virus infection and has potential for the treatment of Ebola virus disease (EVD).

Algal lectin Griffithsin (GRFT) is a well-known mannose-binding protein which has a broad-spectrum antiviral activity against several important infectious viruses including HIV, HCV, and SARS-CoV-2. Therefore, GRFT has been brought great attention to antiviral therapeutic development. In this report, we have tested GRFT’s activity against the lethal Ebola virus in vitro and in vivo. Our data has shown that the IC50 value is about 42 nM for inhibiting Zair Ebola virus (EBOV) infection in vitro. The preliminary in vivo mice model using mouse-adapted EBOV has also shown a certain efficacy for delayed mortality compared to the control animals. GRFT pulldown experiment by viral particles demonstrates that GRFT can bind to N-glycans of EBOV. Thus, it can be concluded that GRFT through binding to the viral glycans for blocking Ebola virus infection and has potential for treating Ebola virus disease (EVD).

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.

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.

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.

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.

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.

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. 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 T helper cell 1 (T _H 1)–biased CD4 T _H 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 after high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates support the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.