Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates.

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.

Small animal models of filovirus disease: recent advances and future directions.

INTRODUCTION: Small animal models have played a critical role in understanding the pathogenesis and transmission of disease caused by filoviruses. Notably, small animals have served to identify and validate many different approaches to countering infection with these highly pathogenic viruses. Nonetheless, predictive efficacy between each model does not appear to be equivalent as higher order animals seem to be more prognostic and therefore successful in the evaluation of medical countermeasures (MCM). Areas covered: This review comprehensively details the available small animal models of filovirus infection and discusses the benefits and shortcomings of each model with respect to the development of MCM. An up-to-date evaluation of mouse, hamster, guinea pig, and ferret models is provided. Expert opinion: The recent development of the domestic ferret model for ebolavirus offers a small animal model that faithfully reproduces most features of human disease without the need for viral adaptation or an immunocompromised host. That being said, choosing a small animal model to evaluate a particular MCM must consider potential confounders associated with each model. These confounding issues include incomplete host immune systems or mutations in the challenge virus that enables the disease.

Postexposure Protective Efficacy of T-705 (Favipiravir) Against Sudan Virus Infection in Guinea Pigs.

Filoviruses such as Ebola virus (EBOV), Marburg virus (MARV), and Sudan virus (SUDV) cause deadly viral hemorrhagic fever in humans, with high case-fatality rates; however, no licensed therapeutic agent or vaccine has been clinically approved to treat or prevent infection. T-705 (favipiravir) is a novel antiviral drug that has been approved for the treatment of influenza in Japan. T-705 exhibits broad-spectrum antiviral activity against different viruses, including MARV and EBOV, and here, we are the first to report the in vitro and in vivo antiviral activity of T-705 against SUDV. T-705 treatment reduced SUDV replication in Vero E6 cells. Subcutaneous administration of T-705, beginning 1-4 days after infection and continuing for 7 days, significantly protected SUDV-infected guinea pigs, with a survival rate of 83%-100%. Viral RNA replication and infectious virus production were also significantly reduced in the blood, spleen, liver, lungs, and kidney. Moreover, early administration of low-dose T-705 and late administration (at 5 days after infection) of higher-dose T-705 also showed partial protection. Overall, our study is the first to demonstrate the antiviral activity of T-705 against SUDV, suggesting that T-705 may be a potential drug candidate for use during outbreaks.

A Predictive Spatial Distribution Framework for Filovirus-Infected Bats.

Tools with predictive capabilities in regards of filovirus outbreaks are mainly anthropocentric and have disregarded the ecological dimension of the problem. Here we contribute to shift the current paradigm by studying the dynamics of the putative main zoonotic niche of filoviruses, bats, and its link to environmental drivers. We propose a framework that combines data analysis, modeling, and the evaluation of sources of variability. We implement a regression analysis using factual data to correlate environmental parameters and the presence of bats to find the distribution of resources. The information inferred by the regression is fed into a compartmental model that describes the infection state. We also account for the lack of knowledge of some parameters using a sampling/averaging technique. As a result we estimate the spatio-temporal densities of bats. Importantly, we show that our approach is able to predict where and when an outbreak is likely to appear when tested against recent epidemic data in the context of Ebola. Our framework highlights the importance of considering the feedback between the ecology and the environment in zoonotic models and sheds light on the mechanisms to propagate filoviruses geographically. We expect that our methodology can help to design prevention policies and be used as a predictive tool in the context of zoonotic diseases associated to filoviruses.

Small Animal Models for Evaluating Filovirus Countermeasures.

The development of novel therapeutics and vaccines to treat or prevent disease caused by filoviruses, such as Ebola and Marburg viruses, depends on the availability of animal models that faithfully recapitulate clinical hallmarks of disease as it is observed in humans. In particular, small animal models (such as mice and guinea pigs) are historically and frequently used for the primary evaluation of antiviral countermeasures, prior to testing in nonhuman primates, which represent the gold-standard filovirus animal model. In the past several years, however, the filovirus field has witnessed the continued refinement of the mouse and guinea pig models of disease, as well as the introduction of the hamster and ferret models. We now have small animal models for most human-pathogenic filoviruses, many of which are susceptible to wild type virus and demonstrate key features of disease, including robust virus replication, coagulopathy, and immune system dysfunction. Although none of these small animal model systems perfectly recapitulates Ebola virus disease or Marburg virus disease on its own, collectively they offer a nearly complete set of tools in which to carry out the preclinical development of novel antiviral drugs.

Neglected filoviruses.

Eight viruses are currently assigned to the family Filoviridae Marburg virus, Sudan virus and, in particular, Ebola virus have received the most attention both by researchers and the public from 1967 to 2013. During this period, natural human filovirus disease outbreaks occurred sporadically in Equatorial Africa and, despite high case-fatality rates, never included more than several dozen to a few hundred infections per outbreak. Research emphasis shifted almost exclusively to Ebola virus in 2014, when this virus was identified as the cause of an outbreak that has thus far involved more than 28 646 people and caused more than 11 323 deaths in Western Africa. Consequently, major efforts are currently underway to develop licensed medical countermeasures against Ebola virus infection. However, the ecology of and mechanisms behind Ebola virus emergence are as little understood as they are for all other filoviruses. Consequently, the possibility of the future occurrence of a large disease outbreak caused by other less characterized filoviruses (i.e. Bundibugyo virus, Lloviu virus, Ravn virus, Reston virus and Taï Forest virus) is impossible to rule out. Yet, for many of these viruses, not even rudimentary research tools are available, let alone medical countermeasures. This review summarizes the current knowledge on these less well-characterized filoviruses.

Personal protective equipment for use in a filovirus disease outbreak: rapid advice guideline

Filoviruses (Ebola and Marburg) are highly contagious pathogens, which cause severe and often fatal illness in humans. Health workers are at increased risk of infection with these viruses because of their close and prolonged contact with severely ill patients with a high viral load. The risk of transmission of Ebola virus can be reduced if appropriate measures are taken, including the use of personal protective equipment (PPE). The urgent need for clear standards for PPE use became acutely apparent during the unprecedented outbreak of Ebola virus disease (EVD) in certain western African countries in 2013­16.

Personal Protective Equipment for Filovirus Epidemics: A Call for Better Evidence.

Personal protective equipment (PPE) is an important part of worker protection during filovirus outbreaks. The need to protect against a highly virulent fluid-borne pathogen in the tropical environment imposes a heat stress on the wearer that is itself a safety risk. No evidence supports the choice of PPE employed in recent outbreaks, and standard testing procedures employed by the protective garment industry do not well simulate filovirus exposure. Further research is needed to determine the appropriate PPE for filoviruses and the heat stress that it imposes.

A call to action to enhance filovirus disease outbreak preparedness and response.

The frequency and magnitude of recognized and declared filovirus-disease outbreaks have increased in recent years, while pathogenic filoviruses are potentially ubiquitous throughout sub-Saharan Africa. Meanwhile, the efficiency and effectiveness of filovirus-disease outbreak preparedness and response efforts are currently limited by inherent challenges and persistent shortcomings. This paper delineates some of these challenges and shortcomings and provides a proposal for enhancing future filovirus-disease outbreak preparedness and response. The proposal serves as a call for prompt action by the organizations that comprise filovirus-disease outbreak response teams, namely, Ministries of Health of outbreak-prone countries, the World Health Organization, Médecins Sans Frontières, the Centers for Disease Control and Prevention-Atlanta, and others.

Challenges, progress, and opportunities: proceedings of the filovirus medical countermeasures workshop.

On August 22-23, 2013, agencies within the United States Department of Defense (DoD) and the Department of Health and Human Services (HHS) sponsored the Filovirus Medical Countermeasures (MCMs) Workshop as an extension of the activities of the Filovirus Animal Non-clinical Group (FANG). The FANG is a federally-recognized multi-Agency group established in 2011 to coordinate and facilitate U.S. government (USG) efforts to develop filovirus MCMs. The workshop brought together government, academic and industry experts to consider the needs for filovirus MCMs and evaluate the status of the product development pipeline. This report summarizes speaker presentations and highlights progress and challenges remaining in the field.

Possible leap ahead in filovirus therapeutics.

In a recent study published in Nature, Warren et al. describe the generation of a novel synthetic adenosine analogue, BCX4430, a synthetic drug-like small molecule that provides protection from Ebola and Marburg virus infection in animal models.

Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430.

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.

Characterization of a putative filovirus vaccine: virus-like particles.

Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.

An interagency collaboration to facilitate development of filovirus medical countermeasures.

The Filovirus Animal Non-Clinical Group (FANG) is a US interdepartmental and interagency group established to support and facilitate the advanced development of filovirus Medical Countermeasures (MCM), both vaccines and therapeutics. It is co-led by one representative from the Department of Defense (DoD), the first author, and one from the Department of Health and Human Services (HHS), the second author. The FANG membership includes operational level program staff and Subject Matter Experts (SME) from performing organizations as well as scientific staff and program managers from DoD and HHS funding and regulatory agencies. Focus areas include animal models, assays, reagents, product manufacture and characterization, and other interagency product development issues that will support Food and Drug Administration (FDA) licensure of safe and effective filovirus MCMs. The FANG continues to develop strategies to address broadly applicable and interagency product development challenges relevant to filovirus MCM development. This paper summarizes FANG structure and accomplishments and is meant to heighten community awareness of this government-led collaborative effort.

Designing and testing broadly-protective filoviral vaccines optimized for cytotoxic T-lymphocyte epitope coverage.

We report the rational design and in vivo testing of mosaic proteins for a polyvalent pan-filoviral vaccine using a computational strategy designed for the Human Immunodeficiency Virus type 1 (HIV-1) but also appropriate for Hepatitis C virus (HCV) and potentially other diverse viruses. Mosaics are sets of artificial recombinant proteins that are based on natural proteins. The recombinants are computationally selected using a genetic algorithm to optimize the coverage of potential cytotoxic T lymphocyte (CTL) epitopes. Because evolutionary history differs markedly between HIV-1 and filoviruses, we devised an adapted computational technique that is effective for sparsely sampled taxa; our first significant result is that the mosaic technique is effective in creating high-quality mosaic filovirus proteins. The resulting coverage of potential epitopes across filovirus species is superior to coverage by any natural variants, including current vaccine strains with demonstrated cross-reactivity. The mosaic cocktails are also robust: mosaics substantially outperformed natural strains when computationally tested against poorly sampled species and more variable genes. Furthermore, in a computational comparison of cross-reactive potential a design constructed prior to the Bundibugyo outbreak performed nearly as well against all species as an updated design that included Bundibugyo. These points suggest that the mosaic designs would be more resilient than natural-variant vaccines against future Ebola outbreaks dominated by novel viral variants. We demonstrate in vivo immunogenicity and protection against a heterologous challenge in a mouse model. This design work delineates the likely requirements and limitations on broadly-protective filoviral CTL vaccines.