Michael G. Kurilla, M.D., Ph.D.
National Institute of Allergy and Infectious Diseases
The Ebola outbreak in West Africa has represented a unique public health emergency with global impact. The National Institute of Allergy and Infectious Diseases (NIAID) has been engaged with a full spectrum of research activities on Ebola virus and other related filoviruses predating this outbreak by several decades. Activities have included basic virology, the development of Ebola-specific vaccines, therapeutics, and diagnostic tests, culminating in multiple candidate products undergoing clinical evaluation for the first time during an ongoing Ebola virus outbreak.
Since December 2013, when the first case was encountered, the West African Ebola outbreak has exceeded all previous Ebola virus outbreaks combined by more than an order of magnitude (Victory et al., 2015). Because of a combination of limited regional medical and public health infrastructure, lack of prior medical and public health experience with this virus, spread to large urban centers and prevailing cultural practices, Ebola demonstrated a previously unrecognized capability to spark a large, widespread outbreak with global impact (Alexander et al., 2015). In addition, beyond the direct effect of Ebola virus disease (EVD) on individuals, the impact on and disruption of routine delivery of medical care has had both immediate and long-term consequences for the region.
While Ebola virus has been recognized since 1976 (WHO, 1978a,b), its sporadic emergence, along with the highest level of biocontainment mandated for research, has resulted in a slow accumulation of even basic virological and
medical information about the virus. While Ebola virus possesses the capacity to cause significant disease, human-to-human transmission has been viewed as limited because of the nonrespiratory mode of transmission requiring direct contact. Since previous Ebola outbreaks were successfully stopped with infection control procedures and contact tracing, specific medical countermeasures such as vaccines and therapeutics had not proceeded to advanced development.
Throughout this outbreak and extending decades prior, NIAID has supported research with Ebola virus as well as other members of the filovirus family, including basic virology, vector identification, development of vaccines, therapeutics, diagnostics, and clinical research. Ebola virus as a public health threat represents a salient exemplar of overall preparedness efforts for emerging infectious diseases. A robust research response to such threats requires preexisting research infrastructure, coupled with trained, knowledgeable, and experienced investigators. In addition, a comprehensive response requires a diverse array of supported research and development activities operating before, during, and extending well after outbreaks occur. This article will present a high-level overview of Ebola virus research efforts, many of which NIAID has supported, such as basic virological aspects of Ebola virus as well as product development efforts that have culminated in the evaluation of multiple Ebola virus–specific interventions during an ongoing outbreak for the first time.
EBOLA AS A PATHOGEN: CLINICAL MANIFESTATIONS
Since its original recognition in 1976 (WHO, 1978a,b), multiple independent, relatively small, isolated outbreaks have occurred, mostly in the Central African region (Mahanty and Bray, 2004; Marzi and Feldmann, 2014). A definitive reservoir for the virus has not been identified, although primates and bats have been identified as potential sources for initial human infections (Changula et al., 2014; Groseth et al., 2007). Once established in a human population, transmission progresses through close contact involving bodily fluids with either skin breaks or mucous membranes. Despite limited evidence of aerosol transmission in animals (Zumbrun et al., 2012), the aerosol route among humans has not been observed. After a typical incubation period averaging around 8–10 days, but lasting up to 3 weeks (during which time, transmission is infrequent), initial symptoms are largely nonspecific, such as fever and malaise (Bah et al., 2015; Chertow et al., 2014; Park et al., 2015). Given the myriad of other potential etiologies eliciting a fever, in the absence of a specific molecular diagnostic test, recognition of Ebola virus infection may be delayed, resulting in additional transmission and disease progression in the absence of appropriate medical care. Historically, EVD has been regarded as a hemorrhagic fever disease (Kortepeter et al., 2011); progression in this outbreak has involved gastrointestinal symptoms more commonly, such as nausea, vomiting, and diarrhea, while bleeding was an infrequent finding. Careful attention to fluid and electrolyte management along with recognition of
late-stage complications, such as secondary infections and meningoencephalitis, are critical for successful resolution of disease (Bah et al., 2015; Schieffelin et al., 2014). Typically, perhaps owing to prior occurrences in remote, low-population density regions, infection control practices, including contact tracing and quarantining, outbreaks have been brought under control without the involvement of specific EVD interventions.
EBOLA AS A VIRUS
Within the category of nonsegmented negative-strand RNA viruses, Ebola virus is a member of the Filoviridae family. Five genetically related Ebola viruses have been described, of which Zaire (the cause of the West Africa outbreak) is the most pathogenic (Ascenzi et al., 2008). Virion structure, while uniquely filamentous, is nevertheless typical for enveloped viruses with a nucleocapsid structure comprising a lipid membrane studded with a single type of glycoprotein. Rather than a unique cell surface receptor, the heavily glycosylated glycoprotein appears to interact with cell surface lectins followed by membrane fusion. Cytoplasmic entry occurs from an endosomal compartment after processing by host proteases (Bhattacharyya et al., 2010; Chandran et al., 2005; Hunt et al., 2012). The mode of viral entry allows Ebola to infect a wide array of cell types, contributing to pathogenicity. Following release into the cytoplasm, the virus undergoes typical viral RNA polymerase-directed transcription of its genes, followed by genome replication, viral assembly, and, finally, release from the cell. More detailed descriptions can be found in several excellent reviews. One intriguing aspect of note is the functional diversity that Ebola virus can achieve with a sparse genome of only seven genes. The Ebola virus has evolved multifunctional activities using the same proteomic sequences. For example, viral protein 40 (VP40) can adopt three distinct, folded structures mediating three distinct functional activities expressed during the viral life cycle, including membrane trafficking, viral budding, and transcriptional regulation (Bornholdt et al., 2013).
Ebola as a Species-Specific Infectious Agent with Immune Evasion Strategies
The Ebola virus possesses specific gene products that mediate immune evasion, especially early responses by the innate immune system, which is partly responsible for the severe virulence and systemic pathology that are observed with Ebola virus infection (Audet and Kobinger, 2015; Ramanan et al., 2011). Selected aspects of Ebola virus immune evasion strategies are highlighted below.
VP35 confers species specificity to Ebola virus and has been implicated as a mediator of Type 1 interferon suppression (Basler et al., 2000). Normal mice are relatively resistant to Ebola virus due to a Type 1 interferon response when infected with wild-type Ebola virus. Treatment with antibodies to murine interferon
results in mice sensitive to wild-type Ebola virus, while a mouse-adapted Ebola virus strain that displays enhanced virulence suppresses murine interferon production upon infection. In addition to interferon suppression, VP35 has also been demonstrated to suppress dendritic cell maturation (Yen et al., 2014).
Type 1 interferon represents an early response to viral infection whereby uninfected, bystander cells can be alerted to infection and prepare an antiviral state in surrounding uninfected cells to limit further viral replication (Levy and Garcia-Sastre, 2001). Inhibition of this innate response allows for faster and greater viral dissemination. Exogenous interferon would perhaps overcome inhibition of interferon production in infected cells; however, Ebola virus possesses a second immune evasion gene, VP24, which interferes with interferon signaling (Xu et al., 2014). This effect likely accounts for the lack of rescue in nonhuman primates when interferon is administered postexposure, although survival is prolonged (Smith et al., 2013).
In addition to the evasion of the innate immune response, Ebola virus also has a strategy to circumvent the adaptive immune response (beyond suppression of antigen presentation). Specifically, the Ebola virus glycoprotein (GP) is assembled as a homotrimer on the virion surface. An alternative, edited version of GP produces a soluble homodimer form (sGP) that is secreted in copious quantities during infection. Initially, sGP was regarded as a mere decoy to overwhelm GP-specific antibodies that would otherwise participate in virion clearance and cell surface–mediated killing of infected cells. More recently however, an additional function of sGP has been described (Mohan et al., 2012). Antibody responses directed toward GP typically consist of two populations: (1) a cross-reactive set recognizing both trimer GP and sGP, and (2) a more targeted trimer GP-specific response. In the presence of both trimer GP and sGP, the antibody response skews in favor of the cross-reactive set, and the adaptive humoral immune response is compromised because sGP can function as a decoy. In the presence of only trimer GP, the cross-reactive antibody responses are reduced, while enhancing trimer GP reactivity, thus focusing the humoral response toward antibodies more likely to participate in viral clearance. Termed antigenic subversion, this suggests that vaccination strategies that skew in favor of a trimer-specific response will likely be more effective.
In conjunction with the various cell types that Ebola virus can infect, suppression of host immune defenses leads to its propensity to cause severe disease with multiple organ failure. Adequate, intensive medical support is crucial while host defenses slowly respond. At the same time, the targeted nature of these attacks on various host defenses is responsible for some degree of species-specific pathogenesis of these effects and introduces additional challenges and limitations for researchers attempting to model viral disease in animals.
General Restrictions and Limitations in Ebola Virus Countermeasure Development
In the nearly 40 years since Ebola virus was first described (WHO, 1978a,b), and nearly 50 years since the filovirus family of viruses has been recognized (Malherbe and Strickland-Cholmley, 1968; Martini et al., 1968), significant challenges have hampered the analysis and characterization of this infectious agent, slowing development of appropriate countermeasures. First and foremost, the substantial initial virulence and pathogenesis of cases resulting in fatality rates approaching 90 percent have restricted laboratory investigations with live virus to specialized facilities offering biosafety level-4 (BSL-4) containment. Second, the species specificity of viral pathogenesis described above, renders traditional small, rodent animal models less informative compared to other infectious agents, necessitating studies with nonhuman primates (NHPs) that have constraints owing to both cost and numbers of animals available at any time. Finally, despite previous outbreaks (Mahanty and Bray, 2004), the small number of observed human cases (prior to 2013) and sparse medical investigation into human pathology further limit the understanding and appreciation of animal pathogenesis for relevance to human disease. The requirement for NHP studies conducted under BSL-4 containment to evaluate various vaccine and therapeutic approaches severely limits the number of candidates that can be examined. A lack of information of what constitutes a human infectious dose and typical routes of inoculation leads to questions regarding the level of stringency of NHP models with regard to challenge studies evaluating product candidates.
In the absence of sufficient human cases of EVD (the situation prior to 2013), the capacity for clinical evaluation of candidate vaccines and therapeutics has been viewed as a major obstacle. The sporadic and unpredictable nature of outbreaks, along with their remote locations and small number of potential available cases for study during an outbreak given the lag time for recognizing Ebola virus as the etiologic agent of disease and alerting international aid organizations for response, severely hampered advancing early-stage promising candidates into clinical evaluation. With an extreme paucity of human cases of EVD and their periodic and unpredictable nature, a viable regulatory pathway for product development was uncertain. Only since 2002 when the U.S. Food and Drug Administration introduced the Animal Rule (Snoy, 2010) did a viable licensure pathway become available. As mentioned previously, this Ebola virus outbreak represents the first time Ebola virus–specific interventions have been used in patients (except for a single report of convalescent whole blood transfusion). While multiple trials of vaccines and therapeutics have been initiated, descriptions and outcomes of these trials (many of which are ongoing) are beyond the scope of this article.
Initial efforts shortly after identification of Ebola virus with inactivated virions as a vaccine demonstrated efficacy in rodent models but failed to protect in primate models (Lupton et al., 1980). With limited survival from infection with both humans and primates, characterizing and defining sterilizing immunity has been a major challenge. Curiously, isolation of a human monoclonal antibody from an EVD survivor led to a paradoxical result of in vitro viral neutralization, but lack of protection in vivo for primates from challenge (Oswald et al., 2007). Cellular depletion studies have identified T cells, specifically CD8+ T cells, as capable of mediating protection to challenge following vaccination (Sullivan et al., 2011). In addition, immunoglobulin from vaccinated primates that are protected from subsequent challenge fails to mediate protection by passive transfer, but passive transfer of immunoglobulin derived following vaccination and after survival from challenge can mediate protection (Dye et al., 2012; Parren et al., 2002). Thus, the nature of a protective immune response appears to involve both humoral and cell-mediated effector functions (Warfield et al., 2005). Finally, the durability and long-term immunity to EVD remains to be characterized. Because of the infrequent and sporadic nature of outbreaks, there has been little opportunity for human observation. Primate studies suggest that immunity can and does wane following vaccination, but no data exist for natural infections (Stanley et al., 2014).
While various subunit and vectored approaches have been pursued, the first Ebola vaccine candidate to reach human clinical testing as a proof of concept was a DNA-based vaccine in 2006, using glycoprotein and nucleocapsid inserts (Martin et al., 2006). The vaccine was well tolerated without significant adverse event, and both antibody and T cell responses were described. Ebola virus–specific neutralizing antibodies were not observed. Because this candidate required a three-dose regimen, it was not considered to be a viable option in the field during an outbreak and thus was not pursued further. Since that time, additional candidates have advanced, including human and chimp adenoviral vectored candidates, along with recombinant vesicular stomatitis virus (rVSV) and modified vaccinia Ankara (MVA) systems (Rampling et al., 2016; Regules et al., 2015; Zahn et al., 2012). All of these vaccines target the Ebola glycoprotein (GP). NIAID’s support for vaccine development has spanned all stages of development, from early discovery to animal challenges, preclinical investigational new drug-enabling studies, and clinical evaluation from phase I through currently ongoing phase II/III trials. In addition, NIAID supports a vaccine candidate primate screening service for qualified vaccine developers who require access to primate testing under BSL-4 containment. Since 2010, NIAID has evaluated more than 50 vaccine candidates through this service.1
1 See http://www.niaid.nih.gov/labsandresources/resources/dmid/animalmodels/Pages/default.aspx (accessed November 3, 2016).
Four broad categories of treatment strategies have been pursued with variable results:
- Immunotherapeutic approaches
- Small molecule direct-acting antivirals targeting either viral proteins or nucleic acid
- Host-based targeting that results in an antiviral effect
- Host targeting to intervene in pathogenic complications of EVD, such as particular organ dysfunction
The usefulness of convalescent plasma or whole blood remains inconclusive (Mupapa et al., 1999). Because a single monoclonal antibody (MAb) fails to protect primates to Ebola virus challenge (Oswald et al., 2007), subsequent work has focused on MAb cocktails, with progressively improving efficacy. The most advanced product, ZMapp, consists of three MAbs and demonstrates 100 percent recovery at 5 days postinfection (Qiu et al., 2014). Human clinical evaluation is currently under way.
Small-molecule antiviral compounds have been extensively evaluated for in vitro viral inhibition. A few of the currently licensed RNA virus-specific drugs have demonstrated in vitro inhibitory activity against Ebola virus, including known RNA virus inhibitors, ribavirin, and lamivudine (Hensley et al., 2015; Huggins, 1989). Of note is T-705, an RNA polymerase inhibitor that has activity against influenza, which has some activity (Oestereich et al., 2014). One recent candidate—BCX4430, a nucleoside analogue—has demonstrated high potency in vitro and NHP postinfection survival for both Marburg and Ebola viruses (Warren et al., 2014). This agent is currently undergoing initial human testing. Oligonucleotide-based therapies, such as TKM-Ebola and AVI-7537, have been developed that display postexposure survival and have advanced to early-stage clinical evaluation (Geisbert et al., 2010; Iversen et al., 2012; Kraft et al., 2015).
Other classes of small molecules that target specific host functions necessary for the viral life cycle have been described, such as cathepsin (endosomal proteases) inhibitors that block viral entry, or amiodarone, an ion channel blocker that interferes with viral egress (Chandran et al., 2005; Gehring et al., 2014). Animal model data for both are less promising than in vitro data. Additionally, several classes of licensed drugs have been described with in vitro potency; however, the specific hosts targets or mechanism of action have not been delineated (Gehring et al., 2014; Johansen et al., 2013). Given the species specificity of Ebola virus along with potential species-specific mechanisms of the drugs themselves, caution is warranted with interpretation of animal model results, as well as drawing conclusions regarding human dosing. Additionally, toxicity concerns with these agents may be distinct from their routine clinical usage in the setting of EVD. Finally, approaches to treatment specifically addressing reversal of organ and
metabolic dysfunction have been attempted with limited success, such as recombinant nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation (Geisbert et al., 2003).
A final note regarding antiviral intervention: as is typical for drug screening activities, the vast majority of studies are negative; however, negative data are glaringly absent from the published literature, unless buried within dense tables as part of other more positive results. As a result, previous negative experiments are routinely repeated or advocated for additional support, especially during an outbreak. To partly address this concern of unproductive efforts, NIAID, in conjunction with the World Health Organization, established a database2 to aggregate negative datasets from screens, in vitro assessment, and any available animal model results to assist the scientific community in a more focused application of effort and avoidance of duplication of effort.
Because initial symptoms such as fever and malaise are nonspecific and, more often than not, likely to indicate another infectious agent, accurate diagnostic tests are crucial to identify and isolate EVD patients, not only for prompt treatment for the patient but also to interrupt active transmission links. Currently, polymerase chain reaction (PCR) testing remains the gold standard for diagnostic evaluation, with the caveat that positive results are not obtained until after the onset of symptoms. In addition, PCR can also provide prognostic assessment as well as identify a noninfectious state in survivors (Fitzpatrick et al., 2015). Unfortunately, PCR testing also requires sophisticated laboratory infrastructure with highly trained lab personnel and suffers from limited deployability, which is critical in an evolving and expanding outbreak.
PCR relies on the availability of genomic information, and the recent advances in genomic technologies offer new opportunities for outbreak assessment, including whole viral genome sequencing. During this outbreak, Ebola virus dynamics were directly observed based on sequencing viral isolates obtained during the outbreak (Gire et al., 2014; Park et al., 2015). In the future, interventions that rely on specific genomic sequences, including vaccines and therapeutics, can be evaluated for suitability as an outbreak develops and as it evolves.
NIAID has supported the early development of both nucleic acid–based tests and antigenic tests that potentially offer greater flexibility in terms of rapid turnaround owing to greater mobility as well as less technical support needed for operation (Broadhurst et al., 2015; Leski et al., 2015). The role of these newer testing modalities remains to be fully evaluated, but the introduction of point-of-care testing should improve future outbreak responses.
2 See http://www.who.int/medicines/ebola-treatment/test-database/en (accessed November 3, 2016).
LESSONS FOR OTHER RARE, BUT HIGHLY VIRULENT, INFECTIOUS AGENTS
The Ebola outbreak in West Africa has highlighted a number of pertinent issues that are relevant for emerging infectious diseases. The complex interplay of a susceptible population, their past social and immunological experience with this infectious agent, the level of existing medical and public health infrastructures, as well as unique cultural practices, all contributed to creating a vastly different scenario than had been observed in prior Ebola outbreaks. In this regard, past behavior may not always predict future events, which should be borne in mind when new outbreaks emerge.
One unique situation created with this outbreak is the large number of EVD survivors that are available for ongoing evaluation. Unanswered questions regarding long-term immunity as well as the significance of immunologically privileged sites will directly affect medical care as well as inform approaches to future outbreaks (Christie et al., 2015; Varkey et al., 2015). Survivor cohorts will provide a unique opportunity to characterize human immunity to Ebola virus as well as document and address longer-term sequelae (Clark et al., 2015). Every infectious disease outbreak offers potential for novel discovery that will assist in the current outbreak, inform our approach to the next one, and fundamentally refine and alter our understanding of the complex interplay between infectious agents and human host defenses.
And so, while evolving technology has allowed medical science to identify newly emerging infectious diseases with ever increasing speed and precision, vigilance is required to avoid surprise from a reemerging infectious disease that professional experience and textbooks assert has been addressed, whether that be a novel clinical presentation, enhanced transmission, or emerging drug resistance. The ability of the biomedical research enterprise to respond expeditiously and effectively to future outbreaks of Ebola virus as well as other emerging infectious diseases depends on comprehensive, sustained research efforts that span the full range of scientific and medical investigations from basic research through translational science to clinical research and testing prior to an outbreak, during the outbreak, and extending well after any particular outbreak has ended.
The critical review and editorial commentary provided by Drs. Anthony Fauci and Hilary Marston are greatly appreciated.
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