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ABSTRACTPulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.

The Biomedical Advanced Research and Development Authority, part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, recognizes that the evaluation of medical countermeasures under the Animal Rule requires well-characterized and reproducible animal models that are likely to be predictive of clinical benefit. Marburg virus (MARV), one of two members of the genus Marburgvirus, is characterized by a hemorrhagic fever and a high case fatality rate for which there are no licensed vaccines or therapeutics available. This natural history study consisted of twelve cynomolgus macaques challenged with 1000 PFU of MARV Angola and observed for body weight, temperature, viremia, hematology, clinical chemistry, and coagulation at multiple time points. All animals succumbed to disease within 8 days and exhibited signs consistent with those observed in human cases, including viremia, fever, systemic inflammation, coagulopathy, and lymphocytolysis, among others. Additionally, this study determined the time from exposure to onset of disease manifestations and the time course, frequency, and magnitude of the manifestations. This study will be instrumental in the design and development of medical countermeasures to Marburg virus disease.

The cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) is widely used for filovirus vaccine testing. To use limited BSL-4 resources efficiently and minimize NHP usage, Simon’s two-stage design was adapted to screen candidate Ebola virus (EBOV) vaccines in up to six NHPs with two (optimal), three, or four NHPs in Stage 1. Using the optimal design, two NHPs were tested in Stage 1. If neither survived, the candidate was rejected. Otherwise, it was eligible for Stage 2 testing in four NHPs. Candidates advanced if four or more NHPs were protected over both stages. An 80% efficacious candidate vaccine had 88.5% probability of advancing, and a 40% efficacious candidate vaccine had 83% probability of rejection. Simon’s two-stage design was used to screen 27 EBOV vaccine candidates in 43 candidate regimens that varied in dose, adjuvant, formulation, or schedule. Of the 30 candidate regimens tested using two NHPs in Stage 1, 15 were rejected, nine were withdrawn, and six were tested in Stage 2. All six tested in Stage 2 qualified to advance in the product development pipeline. Multiple regimens for the EBOV vaccines approved by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) in 2019 were tested in this program. This approach may also prove useful for screening Sudan virus (SUDV) and Marburg virus (MARV) vaccine candidates.

Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.

The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for Zaire ebolavirus. However, effective medical countermeasures, such as vaccines for other filoviruses such as Sudan ebolavirus and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.S. Food and Drug Administration (FDA) Animal Rule. The selection of an appropriate animal model and virus challenge isolates for nonclinical studies are critical aspects of the development program. Here, we have focused on the recommendation of challenge isolates for Sudan ebolavirus and Marburg virus. Based on analyses led by the Filovirus Animal and Nonclinical Group (FANG) and considerations for strain selection under the FDA Guidance for the Animal Rule, we propose prototype virus isolates for use in nonclinical challenge studies.

Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein’s poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations.

Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selection to VEEV IAB and IC epizootic varieties was based on the DOD objective for vaccine protection in a bioterrorism event. The subsequent down-selection of VEEV IAB and IC isolates was based on isolate availability, origin, virulence, culture and animal passage history, known disease progression in animal models, relevancy to human disease, and ability to generate sufficient challenge material. Methods for the propagation of viral stocks (use of uncloned (wild-type), plaque-cloned, versus cDNA-cloned virus) to minimize variability in the potency of the resulting challenge materials were also reviewed. The presented processes for VEEV strain selection and the propagation of viral stocks may serve as a template for animal model development product testing under the Animal Rule to other viral vaccine programs. This manuscript is based on the culmination of work presented at the “Alphavirus Workshop” organized and hosted by the Joint Vaccine Acquisition Program (JVAP) on 15 December 2014 at Fort Detrick, Maryland, USA.

The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the devastating 2014-2016 outbreak of Ebola virus (EBOV) disease (EVD) in Guinea, Sierra Leone, and Liberia that resulted in over 28,000 cases and over 11,300 deaths. In addition, the 2018-2020 outbreak in the Democratic Republic of the Congo currently has over 3,400 cases and over 2,200 deaths. A fully licensed vaccine and at least one other investigational vaccine are being deployed to combat this EVD outbreak. To support vaccine development and pre-clinical/clinical testing a Filovirus Animal Nonclinical Group (FANG) human anti-EBOV GP IgG ELISA was developed to measure anti-EBOV GP IgG antibodies. This ELISA is currently being used in multiple laboratories. Reported here is a characterization of an interlaboratory statistical analysis of the human anti-EBOV GP IgG ELISA as part of a collaborative study between five participating laboratories. Each laboratory used similar method protocols and reagents to measure anti-EBOV GP IgG levels in human serum samples from a proficiency panel consisting of ten serum samples created by the differential dilution of a serum sample positive for anti-GP IgG antibodies (BMIZAIRE105) with negative serum (BMI529). The total assay variability (inter- and intra-assay variability) %CVs observed at each laboratory ranged from 12.2 to 30.6. Intermediate precision (inter-assay variability) for the laboratory runs ranged from 8.9 to 21.7%CV and repeatability (intra-assay variability) %CVs ranged from 7.2 to 23.7. The estimated slope for the relationship between log10(Target Concentration) and the log10(Observed Concentration) across all five laboratories was 0.95 with a 90% confidence interval of (0.93, 0.97). Equivalence test results showed that the 90% confidence interval for the ratios for the sample-specific mean concentrations at the five individual labs to the overall laboratory consensus value were within the equivalence bounds of 0.80 to 1.25 for each laboratory and test sample, except for six test samples from Lab D, two samples from Lab B1, and one sample from Lab B2. The mean laboratory concentrations for Lab D were less than those from the other laboratories by 20% on average across the serum samples. The evaluation of the proficiency panel at these laboratories provides a limited assessment of assay precision (intermediate precision, repeatability, and total assay variability), dilutional linearity, and accuracy. This evaluation suggests that the within-laboratory performance of the anti-EBOV GP IgG ELISA as implemented at the five laboratories is consistent with the intended use of the assay based on the acceptance criteria used by laboratories that have validated the assay. However, the assessment of between-laboratory performance revealed lower observed concentrations at Lab D and greater variability in assay results at Lab B1 relative to other laboratories.

An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes.

The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the recent and devastating 2014-2016 outbreak of Ebola virus (EBOV) disease in Guinea, Sierra Leone, and Liberia that resulted in more than 10,000 casualties. Such a vaccine would need to be vetted through a U.S. Food and Drug Administration (FDA) traditional, accelerated, or Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal EBOV challenge will need to be bridged to human immune response distributions in clinical trials. When possible, species-neutral methods are ideal for detection and bridging of these immune responses, such as methods to quantify anti-EBOV glycoprotein (GP) immunoglobulin G (IgG) antibodies. Further, any method that will be used to support advanced clinical and non-clinical trials will most likely require formal validation to assess suitability prior to use. Reported here is the development, qualification, and validation of a Filovirus Animal Nonclinical Group anti-EBOV GP IgG Enzyme-Linked Immunosorbent Assay (FANG anti-EBOV GP IgG ELISA) for testing human serum samples.