Animal Rule Research Articles

In vitro efficacy of brincidofovir against variola virus.

Brincidofovir (CMX001), a lipid conjugate of the acyclic nucleotide phosphonate cidofovir, is under development for smallpox treatment using "the Animal Rule," established by the FDA in 2002. Brincidofovir reduces mortality caused by orthopoxvirus infection in animal models. Compared to cidofovir, brincidofovir has increased potency, is administered orally, and shows no evidence of nephrotoxicity. Here we report that the brincidofovir half-maximal effective concentration (EC50) against five variola virus strains in vitro averaged 0.11 μM and that brincidofovir was therefore nearly 100-fold more potent than cidofovir.

The potential of liposome-encapsulated ciprofloxacin as a tularemia therapy.

Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.

Remote monitoring of the progression of primary pneumonic plague in Brown Norway rats in high-capacity, high-containment housing

Development of new vaccines, diagnostics, and therapeutics for biodefense or other relatively rare infectious diseases is hindered by the lack of naturally occurring human disease on which to conduct clinical trials of efficacy. To overcome this experimental gap, the U.S. Food and Drug Administration established the Animal Rule, in which efficacy testing in two well-characterized animal models that closely resemble human disease may be accepted in lieu of large-scale clinical trials for diseases with limited natural human incidence. In this report, we evaluated the Brown Norway rat as a model for pneumonic plague and describe the natural history of clinical disease following inhalation exposure to Yersinia pestis. In high-capacity, high-containment housing, we monitored temperature, activity, heart rate, and rhythm by capturing electronic impulses transmitted from abdominal telemeter implants. Using this system, we show that reduced activity and development of fever are sensitive indications of disease progression. Furthermore, we identified heart arrhythmias as contributing factors to the rapid progression to lethality following the fever response. Together, these data validate the Brown Norway rat as an experimental model for human pneumonic plague and provide new insight that may ultimately lead to novel approaches in postexposure treatment of this devastating infection.

Randomized comparison of single dose of recombinant human IL-12 versus placebo for restoration of hematopoiesis and improved survival in rhesus monkeys exposed to lethal radiation

BackgroundThe hematopoietic syndrome of the acute radiation syndrome (HSARS) is a life-threatening condition in humans exposed to total body irradiation (TBI); no drugs are approved for treating this condition. Recombinant human interleukin-12 (rHuIL-12) is being developed for HSARS mitigation under the FDA Animal Rule, where efficacy is proven in an appropriate animal model and safety is demonstrated in humans.MethodsIn this blinded study, rhesus monkeys (9 animals/sex/dose group) were randomized to receive a single subcutaneous injection of placebo (group 1) or rHuIL-12 at doses of 50, 100, 250, or 500 ng/kg (groups 2–5, respectively), without antibiotics, fluids or blood transfusions, 24–25 hours after TBI (700 cGy).ResultsSurvival rates at Day 60 were 11%, 33%, 39%, 39%, and 50% for groups 1–5, respectively (log rank p < 0.05 for each dose vs. control). rHuIL-12 also significantly reduced the incidences of severe neutropenia, severe thrombocytopenia, and sepsis (positive hemoculture). Additionally, bone marrow regeneration following TBI was significantly greater in monkeys treated with rHuIL-12 than in controls.ConclusionsData from this study demonstrate that a single injection of rHuIL-12 delivered one day after TBI can significantly increase survival and reduce radiation-induced hematopoietic toxicity and infections. These data significantly advance development of rHuIL-12 toward approval under the Animal Rule as an effective stand-alone medical countermeasure against the lethal effects of radiation exposure.

Spatio-temporal Binding: An Adaptation of the Peak Interval Procedure to Assess Spatio-temporal Learning in Rats

Time and space are commonly approached as two distinct dimensions, and rarely combined together in a single task. In this project, we designed a new version of the peak interval procedure in which we included a spatial component. The aim of this experiment was to investigate the learning of a spatio-temporal rule in animals. Adult male Sprague-Dawley rats were placed in front of a 5 nose-poke wall where a given hole could be visited after a precise time after a sound onset in a Fixed Interval (FI) schedule. The animals were presented two different contexts, differentiable by the house-light (A-red or B-green), tone frequency (1 or 11kHz), and spatio-temporal combination rule given by lighting a specific hole and associating it to a specific FI value (i.e., Hole 2-FI 15s vs. Hole 4-FI 60s). Non-reinforced probe trials of longer duration than the FI values (45s for FI15 and 120s for FI60) were pseudo-randomly presented to ascertain temporal peak interval functions. After 8 weeks of training, all the animals were able to differentiate the two contexts as shown by the number of nose-pokes in the correct hole peaking at the appropriate time, while virtually no activity was recorded in the other holes. In a single Pavlovian session of 8 trials, we changed the spatio- temporal rule for half of the animals: in the second context, the hole 1 was reinforced at 30seconds. Here we showed that this one session was sufficient for the animals to learn the new spatio-temporal association. Contrary to the control group, the experimental group exhibited a behavioral pattern associated to each hole indicative of a possible spatio-temporal binding. Interestingly, the behavior in the other control Context, in which the spatio-temporal rule had not been changed, was preserved in both groups. The experiment was followed for an extra 10 days after the Pavlovian Shift. At the end of the experiment, all the animals were perfectly and fully adapted to new spatio-temporal rule. In conclusion, the study suggests that rats are able to learn a spatio-temporal association in a single session, and that the two (space, time) dimensions may be learned together and not separately.

A Mechanistic Model of Drug‐Induced Liver Injury Aids the Interpretation of Elevated Liver Transaminase Levels in a Phase I Clinical Trial

Entolimod (CBLB502) is a Toll-like receptor 5 agonist in development as a single-dose countermeasure against total body irradiation. Efficacy can be assessed from animal studies, but the “Animal Rule” does not apply to safety assessment. Marked elevations of serum aminotransferases (exceeding 1,000 IU/l) were observed in some human subjects receiving Entolimod in a safety study, threatening its continued development. The percentage of total hepatocytes undergoing necrosis in these subjects was estimated using a mechanistic, multiscale, mathematical model (DILIsym). The simulations suggested that no subject in the safety study experienced more than a modest loss of hepatocytes (<5%), which was comparable to estimates from a study of healthy volunteers receiving treatment with heparins. The predicted hepatocyte loss with Entolimod was lower than that required to cause liver dysfunction or that is routinely excised from volunteers donating for autologous liver transplantation and did not likely represent a serious health risk.

Efficacy Testing of Orally Administered Antibiotics against an Inhalational Bacillus anthracis Infection in BALB/C Mice

Anthrax is a potentially fatal disease in man, and events of the last decade have highlighted the possibility for Bacillus anthracis to be used as a biological weapon through deliberate release. Whilst outbreaks of anthrax occur in several countries, there are only a few areas in which outbreaks of inhalational anthrax occur, so it is not possible to test therapies using conventional clinical trials. To overcome these problems, the FDA published the ‘Animal Rule’ in 2002; a rule designed to permit licensure of drugs and biologics intended to reduce or prevent serious conditions caused by exposure to biological and other agents. Due to limited proven post-exposure prophylaxis after inhalational exposure for use in humans, it is essential to have a robust animal model of disease. In a series of three studies conducted in BALB/c mice, the efficacy of postexposure, orally administered antibiotics in preventing disease following inhalational exposure to B. anthracis was evaluated. Mice treated with either azithromycin or clarithromycin displayed a near identical disease progression to the control, untreated mice; in contrast, those treated with levofloxacin, amoxicillin and co-amoxiclav were generally protected with survival of 97%, 95% and 79%, respectively. The details of the assessments and future direction of these studies are presented.

Approaches to modelling the human immune response in transition of candidates from research to development.

This review considers the steps required to evaluate a candidate biodefense vaccine or therapy as it emerges from the research phase, in order to transition it to development. The options for preclinical modelling of efficacy are considered in the context of the FDA's Animal Rule.

Linking the human response to unplanned radiation and treatment to the nonhuman primate response to controlled radiation and treatment.

A key difficulty in developing countermeasures against radiation-induced health impairments is the clear lack of controlled clinical studies, due to the relatively low number of radiation victims worldwide. Instead, established and accepted animal models, as well as the recommendations of national and international expert panels and committees, are the main sources of information. Therefore, the development of countermeasures requires comparison of data from many sources and accumulation of information consistent with the U. S. Food and Drug Administration's "Animal Rule." A new approach is the comparative analysis of human data from the SEARCH (System for Evaluation and Archiving of Radiation Accidents based on Case Histories) database and data from nonhuman primate (NHP) animal model studies. The SEARCH database contains 824 clinical cases from 81 radiation accidents in 19 countries. This exceptional collection of clinical data from accidentally radiation-exposed persons is analyzed regarding clinical signs and symptoms of radiation-induced health impairments. To analyze the time course of radiation syndromes, clinical parameters common to the SEARCH and NHP databases have to be assigned into comparable categories of clinical severity for each species. The goal is to establish a method for comparison of human and NHP data, validate the NHP data as a surrogate for human efficacy/clinical studies, and open a way for the extraction of diagnostic and treatment methods for humans after radiation exposure according to relevant regulations.

Efficacy of primate humoral passive transfer in a murine model of pneumonic plague is mouse strain-dependent.

New vaccines against biodefense-related and emerging pathogens are being prepared for licensure using the US Federal Drug Administration's “Animal Rule.” This allows licensure of drugs and vaccines using protection data generated in animal models. A new acellular plague vaccine composed of two separate recombinant proteins (rF1 and rV) has been developed and assessed for immunogenicity in humans. Using serum obtained from human volunteers immunised with various doses of this vaccine and from immunised cynomolgus macaques, we assessed the pharmacokinetic properties of human and cynomolgus macaque IgG in BALB/c and the NIH Swiss derived Hsd:NIHS mice, respectively. Using human and cynomolgus macaque serum with known ELISA antibody titres against both vaccine components, we have shown that passive immunisation of human and nonhuman primate serum provides a reproducible delay in median time to death in mice exposed to a lethal aerosol of plague. In addition, we have shown that Hsd:NIHS mice are a better model for humoral passive transfer studies than BALB/c mice.

The Delayed Pulmonary Syndrome Following Acute High-dose Irradiation

Several radiation dose- and time-dependent tissue sequelae develop following acute high-dose radiation exposure. One of the recognized delayed effects of such exposures is lung injury, characterized by respiratory failure as a result of pneumonitis that may subsequently develop into lung fibrosis. Since this pulmonary subsyndrome may be associated with high morbidity and mortality, comprehensive treatment following high-dose irradiation will ideally include treatments that mitigate both the acute hematologic and gastrointestinal subsyndromes as well as the delayed pulmonary syndrome. Currently, there are no drugs approved by the Food and Drug Administration to counteract the effects of acute radiation exposure. Moreover, there are no relevant large animal models of radiation-induced lung injury that permit efficacy testing of new generation medical countermeasures in combination with medical management protocols under the FDA animal rule criteria. Herein is described a nonhuman primate model of delayed lung injury resulting from whole thorax lung irradiation. Rhesus macaques were exposed to 6 MV photon radiation over a dose range of 9.0-12.0 Gy and medical management administered according to a standardized treatment protocol. The primary endpoint was all-cause mortality at 180 d. A comparative multiparameter analysis is provided, focusing on the lethal dose response relationship characterized by a lethal dose50/180 of 10.27 Gy [9.88, 10.66] and slope of 1.112 probits per linear dose. Latency, incidence, and severity of lung injury were evaluated through clinical and radiographic parameters including respiratory rate, saturation of peripheral oxygen, corticosteroid requirements, and serial computed tomography. Gross anatomical and histological analyses were performed to assess radiation-induced injury. The model defines the dose response relationship and time course of the delayed pulmonary sequelae and consequent morbidity and mortality. Therefore, it may provide an effective platform for the efficacy testing of candidate medical countermeasures against the delayed pulmonary syndrome.

Characterization of the Dose Response Relationship for Lung Injury Following Acute Radiation Exposure in Three Well-established Murine Strains

Approval of radiation countermeasures through the FDA Animal Rule requires pivotal efficacy screening in one or more species that are expected to react with a response similar to humans (21 C.F.R. § 314.610, drugs; § 601.91, biologics). Animal models used in screening studies should reflect the dose response relationship (DRR), clinical presentation, and pathogenesis of lung injury in humans. Over the past 5 y, the authors have characterized systematically the temporal onset, dose-response relationship (DRR), and pathologic outcomes associated with acute, high dose radiation exposure in three diverse mouse strains. In these studies, C57L/J, CBA/J, and C57BL/6J mice received wide field irradiation to the whole thorax with shielding of the head, abdomen, and forelimbs. Doses were delivered at a rate of 69 cGy min using an x-ray source operated at 320 kVp with half-value layer (HVL) of 1 mm Cu. For all strains, radiation dose was associated significantly with 180 d mortality (p < 0.0001). The lethal dose for 50% of animals within the first 180 d (LD50/180) was 11.35 Gy (95% CI 11.1-11.6 Gy) for C57L/J mice, 14.17 Gy (95% CI 13.9-14.5 Gy) for CBA/J mice, and 14.10 Gy (95% CI 12.2-16.4 Gy) for C57BL/6J mice. The LD50/180 in the C57L/J strain was most closely analogous to the DRR for clinical incidence of pneumonitis in non-human primates (10.28 Gy; 95% CI 9.9-10.7 Gy) and humans (10.60 Gy; 95% CI 9.9-12.1 Gy). Furthermore, in the C57L/J strain, there was no gender-specific difference in DRR (p = 0.5578). The reliability of the murine models is demonstrated by the reproducibility of the dose-response and consistency of disease presentation across studies.Health Phys. 106(1):000-000; 2014.

Recent Patents in the Field of Radioprotector Development: Opportunities and Challenges

Ionizing radiation-induced Acute Radiation Syndrome (ARS) can be prevented/treated using radioprotective agents including; radioprotectors, radiomitigators and radio-therapeutics. Most of the radioprotective molecules currently being developed have emerged from classical drug discovery approaches and have certain shortcomings related to efficacy and toxicity. In order to overcome these issues, adoption of new target-based drug development approaches is required. Like other pharmaceutical drug development programs, patents are being used to protect the new radioprotective compounds that provide right of exclusivity to the inventors. In addition to patents, the FDA approval for safety and efficacy is required for the development of radioprotective drugs that is often being done under FDA's animal rule and requires huge amount of funding. However, radiation countermeasure agents considered emergency medicines and not carrying attractive revenue generation opportunities and are often granted 'orphan drug status'. Thus, pharma giants do not take much interest in such molecules that limit the funding options only to the government agencies, which in turn reduces the interest of researchers in this particular area. In addition to their emergency use, these agents could also be employed for clinical radiotherapy purposes and may provide reasonable incentive to the inventor. Thus, encouraging the development of new radioprotective agent with cutting-edge drug discovery approaches needs more public-private partnership ensuring affordable access; that may stimulate researchers to engage in the area of radioprotector development. This article attempts to cover information available so far on the products/patents/major compounds, known in this critical area of development, along with most relevant regulatory issues and future perspectives.

Personhood, ethics, and animal cognition: situating animals in Hare's two level utilitarianism

Abbreviations Part I - Hare's Two-Level Utilitarianism 1: Introduction 2: Hare on the Logic of Moral Discourse 3: The Nature of Intuitive Level System ILS) Rules 4: Assessing Hare's Theory Part II - Sentience, Personhood, and Utilitarianism 5: Which Animals are Sentient? 6: Personhood and Biography 7: Moral Significance and Autonoetic Consciousness (AC) 8: Candidates for Near-Personhood Part III - Formulating ILS Rules for Humans and Animals 9: Replaceability and Population Policy 10: Humane Sustainable Agriculture 11: Conclusion Bibliography

Recommendations to Improve Collaboration and Expedite Acceptance of Animal Models

The contents of this commentary are the sole responsibility of the authors and do not necessarily represent the official views of the Biomedical Advanced Research and Development Authority, the Office of the Assistant Secretary for Preparedness and Response, or the US Department of Health and Human Services. In our review of animal models for diseases caused by Francisella tularensis, Burkholderia mallei, or Burkholderia pseudomallei, we identified several scientific gaps that need to be filled before any one model will stand up to regulatory scrutiny (this issue). These range from a lack of well-characterized challenge material to the absence of data on appropriate treatment triggers, both of which are necessary to support regulatory qualification of a model in accordance with the US Food and Drug Administration’s (FDA’s) ‘‘Animal Rule’’ (21 CFR 314.610 and 21 CFR 601.91). The Animal Rule provides a regulatory approval pathway for candidate medical countermeasures (MCMs) when human efficacy data are impossible to obtain due to ethical reasons. This alternative regulatory pathway is especially critical for scientists and developers of MCMs for chemical, biologic, and radiologic or nuclear threats (CBRN) and emerging infectious diseases. To support the development of novel MCMs and meet the FDA’s Animal Rule requirements, the Biomedical Advanced Research and Development Authority (BARDA), a division within the US Department of Health and Human Services (HHS), established the Nonclinical Development Network (NDN) to support and accelerate animal model research and development. However, in the 2 years since the NDN was established, we and others have observed 3 areas that still need improvement—specifically, data sharing, data integration, and research coordination. Public and private entities must come together to resolve these challenges. In this commentary, we recommend several policies that could improve research coordination and enable resources to be leveraged across government agencies and their academic and industry partners. Under Federal Acquisition Regulations, the US government (USG) retains unlimited rights to data that are generated under the contracts it supports. This unfettered access to data and information should be the basis for a similarly unrestricted flow of information across the USG. Sharing data and information will allow for the timely identification of scientific and regulatory gaps, reduce the probability of redundant USG funding of studies, and ultimately reduce the number of animals that are used for experimentation. Due to the relative immaturity of animal models for Burkholderia, the USG has an opportunity to effectively implement processes for data sharing prior to engaging in a significant body of work. USG agencies that anticipate supporting Burkholderia model development could introduce common provisions in their contracts to ensure that contractors and grantees understand that their data and methods can, and will be, exchanged across agencies. The barriers to implementing such data-sharing processes should be low since this type of research and development work would be conducted independent of product development and therefore would not be deemed confidential or proprietary. BARDA and other agencies within the USG have begun to incorporate these provisions into their nonclinical development contracts. The USG has supported the development of MCMs for the prophylaxis and treatment of anthrax exposure/infection, including vaccines, antibiotics, and antitoxin therapies. Due to the diverse nature of the anthrax MCM portfolio, many animal efficacy experiments have been conducted. As one approach to addressing some of the regulatory uncertainties regarding the development of MCMs, BARDA engaged its contractors and requested the integration of data from control animals challenged with Bacillus anthracis to conduct a meta-analysis of the data. As a result, data from a larger number of animal studies were obtained and model parameters were effectively refined with greater statistical rigor. This allowed for the identification of reproducible markers of disease that have aided in developing and further characterizing an animal model of anthrax. For pathogens where there are limited animal efficacy data being generated in the context of products, it is imperative that policies and a framework be established that allow for a similar integration of experimental data. Optimally, data integration would extend to all USG agencies conducting animal model

HIV Vaccine Development: Strategies for Preclinical and Clinical Investigation

This article discusses HIV vaccine discovery and candidate vaccine testing in the context of current realities of funding and clinical trial practice. Lacking perfect animal models for testing candidate HIV vaccines, clinical investigators have proposed a strategy of iterative exploratory clinical trials in the model of cancer chemotherapy development. Problems with the appropriateness of this model to HIV vaccine development are discussed. Also, the future feasibility of this strategy in the context of increasing clinical trial costs and emerging new, efficacious prevention modalities is questioned. Strategies for making better use of animal models are presented as an alternative to iterative exploratory clinical efficacy testing. Some ways in which better data from preclinical studies can refine clinical product development are described. Finally, development of an HIV vaccine under the FDA's "Animal Rule" pathway to licensure when human efficacy studies are not feasible is discussed as a fall-back approach. Not making a preventive vaccine against HIV infection is simply not an option because eradication of AIDS will require a preventive vaccine.

Animal rule for drug approval creates a jungle of confusion

Animal rule for drug approval creates a jungle of confusion

Pathology and Pathophysiology of Inhalational Anthrax in a Guinea Pig Model

Nonhuman primates (NHPs) and rabbits are the animal models most commonly used to evaluate the efficacy of medical countermeasures against anthrax in support of licensure under the FDA's "Animal Rule." However, a need for an alternative animal model may arise in certain cases. The development of such an alternative model requires a thorough understanding of the course and manifestation of experimental anthrax disease induced under controlled conditions in the proposed animal species. The guinea pig, which has been used extensively for anthrax pathogenesis studies and anthrax vaccine potency testing, is a good candidate for such an alternative model. This study was aimed at determining the median lethal dose (LD50) of the Bacillus anthracis Ames strain in guinea pigs and investigating the natural history, pathophysiology, and pathology of inhalational anthrax in this animal model following nose-only aerosol exposure. The inhaled LD50 of aerosolized Ames strain spores in guinea pigs was determined to be 5.0 × 10(4) spores. Aerosol challenge of guinea pigs resulted in inhalational anthrax with death occurring between 46 and 71 h postchallenge. The first clinical signs appeared as early as 36 h postchallenge. Cardiovascular function declined starting at 20 h postexposure. Hematogenous dissemination of bacteria was observed microscopically in multiple organs and tissues as early as 24 h postchallenge. Other histopathologic findings typical of disseminated anthrax included suppurative (heterophilic) inflammation, edema, fibrin, necrosis, and/or hemorrhage in the spleen, lungs, and regional lymph nodes and lymphocyte depletion and/or lymphocytolysis in the spleen and lymph nodes. This study demonstrated that the course of inhalational anthrax disease and the resulting pathology in guinea pigs are similar to those seen in rabbits and NHPs, as well as in humans.

Pathology of Experimental Aerosol Zaire Ebolavirus Infection in Rhesus Macaques

There is limited knowledge of the pathogenesis of human ebolavirus infections and no reported human cases acquired by the aerosol route. There is a threat of ebolavirus as an aerosolized biological weapon, and this study evaluated the pathogenesis of aerosol infection in 18 rhesus macaques. Important and unique findings include early infection of the respiratory lymphoid tissues, early fibrin deposition in the splenic white pulp, and perivasculitis and vasculitis in superficial dermal blood vessels of haired skin with rash. Initial infection occurred in the respiratory lymphoid tissues, fibroblastic reticular cells, dendritic cells, alveolar macrophages, and blood monocytes. Virus spread to regional lymph nodes, where significant viral replication occurred. Virus secondarily infected many additional blood monocytes and spread from the respiratory tissues to multiple organs, including the liver and spleen. Viremia, increased temperature, lymphocytopenia, neutrophilia, thrombocytopenia, and increased alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, total bilirubin, serum urea nitrogen, creatinine, and hypoalbuminemia were measurable mid to late infection. Infection progressed rapidly with whole-body destruction of lymphoid tissues, hepatic necrosis, vasculitis, hemorrhage, and extravascular fibrin accumulation. Hypothermia and thrombocytopenia were noted in late stages with the development of disseminated intravascular coagulation and shock. This study provides unprecedented insight into pathogenesis of human aerosol Zaire ebolavirus infection and suggests development of a medical countermeasure to aerosol infection will be a great challenge due to massive early infection of respiratory lymphoid tissues. Rhesus macaques may be used as a model of aerosol infection that will allow the development of lifesaving medical countermeasures under the Food and Drug Administration's animal rule.

Standardization of the Filovirus Plaque Assay for Use in Preclinical Studies

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.