Ebolavirus: a brief review of novel therapeutic targets
2011; Future Medicine; Volume: 7; Issue: 1 Linguagem: Inglês
10.2217/fmb.11.110
ISSN1746-0921
AutoresAndrew S. Kondratowicz, Wendy Maury,
Tópico(s)Hepatitis B Virus Studies
ResumoFuture MicrobiologyVol. 7, No. 1 EditorialFree AccessEbolavirus: a brief review of novel therapeutic targetsAndrew S Kondratowicz & Wendy J MauryAndrew S KondratowiczDepartment of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA & Wendy J Maury* Author for correspondencePublished Online:22 Dec 2011https://doi.org/10.2217/fmb.11.110AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: antiviralebolavirusfilovirusmarburgvirusvaccineThe filovirus genome & infectionEbolavirus (EBOV) and Marburgvirus (MARV) are members of the family Filoviridae and contain a single-stranded, negative sense ∼19 kb RNA genome. There is a single species of MARV, whereas there are five known species of EBOV: Zaire EBOV, Reston EBOV, Ivory Coast EBOV, Sudan EBOV and Bundibugyo EBOV. While Reston EBOV does not cause disease in humans, the other four species cause Ebola hemorrhagic fever (EHF), with human mortality rates between 40–90% [1] and no vaccines or antivirals are currently available. As disease symptoms are thought to be caused by both replication of the virus and host immune responses [1], promising future therapies logically would focus on reducing virus load and/or enhancing productive immune responses.The EBOV genome encodes seven genes that produce ten mature viral proteins. These proteins include a nucleoprotein, viral proteins 35 and 40 (VP35 and VP40), a glycoprotein (GP) that is processed by the cellular protease furin into GP1 and GP2, with the GP1 gene producing two additional soluble glycoproteins (sGP and ssGP), viral proteins 30 and 24 (VP30 and VP24), and the viral RNA-dependent RNA polymerase (L). The ribonucleoprotein nucleoprotein encapsulates the genome and forms a complex with VP30, VP35 and L, which is required for both genome replication and the transcription of viral genes [2]. VP40 is the matrix protein and the main determinate of viral budding through the host cell plasma membrane [2].Filovirus introduced to the gastrointestinal tract, eye, skin, and through intravenous and intramuscular injections, can produce productive infections in a wide array of laboratory animals [3]. Additionally, the spread of infection through aerosolized droplets has been observed in nonhuman primates (NHPs). Macrophages and dendritic cells (DCs) are an early and consistently identified target of EBOV infection [4]. At later times during infection, numerous additional cell types become viral antigen positive, with lymphocytes being the only cells believed to be resistant to EBOV infection [1]. By 3 days following experimental infection of primate infections, the virus can be detected in the gastrointestinal tract, kidneys, liver, thymus, spleen, lymph nodes, adrenal glands, brain, eye and skin [1]. Death from EHF typically occurs approximately 9 days after the onset of symptoms, resulting from a cytokine storm, multiorgan failure, and the breakdown of the vascular epithelium [1,2,5].Current palliative care of EHFGiven that EHF is rare event in the endemic area of sub-Saharan Africa and many of the early symptoms mimic other more common infectious diseases, such as malaria, early cases of an EBOV outbreak are usually misdiagnosed [6]. Treatment of EHF in rural African hospitals is generally limited to fluid and blood volume maintenance, pain management and antipyrogenics [2,3]. Conventional antiviral strategies, such as ribavirin, recombinant IFN-α, and treatment with convalescent antisera have proven ineffective in animal trials [2]. The most effective palliative strategies identified thus far target the abnormal coagulation characteristic of EHF. Daily, postexposure injections of recombinant nematode anticoagulant protein c2, a known inhibitor of tissue factor (TF)-initiated blood coagulation, resulted in 33% of rhesus monkeys surviving a fatal challenge [2]. In a similar strategy, continuous intravenous drip of recombinant human-activated protein C, a major component of the blood anticoagulation and currently licensed treatment of sepsis, protected 18% of rhesus monkeys from a fatal challenge [7]. These therapeutic approaches have all been shown to delay EHF fatalities by several days, which would give additional time for a potential postexposure treatment to be effective if such a treatment was identified.Dysregulated host immune responses: can we alter the outcome?As with other viral infections, initial induction of type 1 IFNs are likely to elicit subsequent productive immune responses against filoviruses. However, filoviruses encode two different proteins that block induction of this cytokine. Ebolavirus VP24 directly blocks IFN-α/β and IFN-γ signaling by preventing STAT1 localization to the nucleus. In addition, dsRNA-binding protein, VP35, inhibits type 1 IFN induction by several independent mechanisms [8]. Firstly, VP35 directly binds to dsRNA, competing with RIG-I-like receptor binding. VP35 also induces sumolyation of IRF7, decreasing IRF7 transcriptional activity and antagonizing PKR activity. This global inhibition of type I interferon is strongly associated with increased virulence [8]. In addition, EBOV directly inhibits the antiviral activities of the RNAi pathway through the actions of VP35, VP30 and VP40. VP35 inhibits the RNAi pathway by both binding dsRNA and, along with VP30, direct interaction with various components of the pathway [7].The absence of type 1 interferons in filovirus-infected macrophages and DCs is also associated with the dysregulation of other immune responses in fatal infections. Filovirus infections of macrophages trigger the prodigious release of cytokines and other cell modulators including IL-1β, IL-6, IL-8, IL-15, IL-16, MIP-1α and -β, IP-10 and nitrous oxide with an upregulation of CD95 on lymphocytes [5]. This pronounced and aberrant cytokine profile is thought to be responsible for an inappropriately stimulated and quickly depleted immune response characteristic of fatal cases [5]. NHP infectivity studies suggest that elevated TF on macrophages or macrophage microparticles leads to breakdown of the vasculature, resulting in the characteristic disseminated intravascular coagulation [2]. In contrast to the robust activation of cytokine production by filovirus-infected macrophages, activation of infected DCs appears to be minimal and an absence of activation of this important APC may critically play into the nonproductive immune responses observed during filovirus infection [2]. Consistent with this possibility, APCs from fatally infected individuals do not upregulate MHC class I and II complexes, preventing T-cell activation [2] and, consequently, despite the significant production of cytokines in infected macrophages, the cytokine release is ineffective at stimulating effective adaptive immune responses and production of T-cell specific cytokines is limited [5]. Macrophages and DCs likely undergo cell death as a direct result of viral replication, while T cells are killed by a poorly understood process called 'bystander apoptosis' that is characteristic of fatal filovirus infection.Survival in humans is correlated with lower levels of proinflammatory cytokines, T-cell survival, production of antifilovirus antibodies and lower viremia [5]. Interestingly, a recent study on human peripheral blood mononuclear cells infected with the least pathogenic EBOV Bundibugyo demonstrated 2–10-fold lower levels of proinflammatory cytokines, reduced macrophage activation and decreased cell death, compared with cells infected with the pathogenic Zaire strain [4]. Therefore, therapies that appropriately 'prime' the immune response and prevent the subversion of the immune system should provide protection against EHF.While no vaccine is currently available, vaccine development shows great promise in NHP models and could be used to protect researchers, physicians and first responders, as well serving as an effective means of containing outbreaks through 'ring vaccination' [2]. Several recombinant filovirus vaccines, which express GP antigen alone or in combination with nucleoprotein and/or VP40 in an nonpathogenic virus or virus vector, have shown pre-exposure efficacy against filovirus challenge. These include adenovirus (serotype 5, 26 and 35), Venezuelan equine encephalitis virus replicons, human parainfluenza virus type 3, vesicular stomatitis virus, and EBOV VLP-based vaccines ([6], see [9] for a review on vaccine development). Intriguingly, the Ad5 vector was recently shown to be safe and immunogenic in humans and the VSV vector was shown to be safe when administered following a presumptive exposure event [9]. To date, vaccine-elicited immune responses that lead to protection remain unclear and appear to differ between vaccine candidates.Virus loads: can they be controlled?Fatal filovirus infections of humans and NHPs are associated with higher virus loads than those infections where individuals survive. Studies suggest that the difference between a fatal outcome and recovery is a 2–3 log difference in viral titer [1]. Thus, antivirals that reduce virus load, even transiently, may be effective.Animal studies have strongly implicated prophylactic use of the vaccines discussed above as efficacious at reducing virus load. However, eliciting productive host immune responses to EHF as a postexposure therapy may not serve as a predictably effective strategy, as most cases are not identified until well after the onset of symptoms.Recently, researchers have begun to focus on inhibiting the virus directly. Perhaps the most promising antifilovirus therapy targets the mRNA of different viral genes directly through steric hindrance or degradation of the target sequence. Phosphorodiamidate morpholino oligomers are cell permeable, highly specific nucleotide analogs that can be targeted to precise sequences of mRNA and cause a steric block in the translation of that sequence. Pre- and post-exposure administration of phosphorodiamidate morpholino oligomers targeting VP24, VP35 or L provided protection to rodents and rhesus monkeys against a lethal challenge and a postexposure, optimized phosphorodiamidate morpholino oligomer vector protected 62 and 100% of NHPs from lethal challenge with EBOV or MARV, respectively [2,10]. RNAi-based therapeutics have shown similar promise with seven postexposure treatments of siRNA targeted against EBOV polymerase completely protecting rhesus monkeys from lethal challenge [2]. It should be noted that these protective treatments were administered soon after exposure and the useful window of efficacy remains to be determined. This is especially true of the RNAi therapies due to the possible interference of the aforementioned EBOV-encoded suppressors of RNA silencing.An additional approach to blocking virus replication may be to interfere with the ability of filoviruses to enter the body. The cell surface protein T-cell immunoglobulin mucin-1 (TIM-1) was recently demonstrated to serve as a receptor for the filoviruses on epithelia, including a number of mucosal surfaces that are important for virus transmission [11]. Antibodies or small molecules that interfere with EBOV GP/TIM-1 interactions may prove to be effective therapies for blocking person-to-person transmission. Alternatively, small molecules that directly target EBOV GP may be efficacious. Compound 7 is a benzodiazepine compound that binds directly to the EBOV entry glycoprotein, blocking infectivity [12]. Compound 8a also inhibited EBOV entry, although the mechanism has yet to be determined [13]. Both of these compounds displayed potent inhibition at low concentrations, but have yet to be evaluated for in vivo efficacy.GP-mediated fusion between the viral envelope and host endosomal membranes may also be an excellent therapeutic target. Two recent studies targeting fusion show promising in vitro results. Miller et al. demonstrated that an endosome-targeted peptide analog of the GP C-terminal heptad repeat, a region critical in the formation of the 6-helix bundle and subsequent membrane fusion, significantly inhibited infection of both EBOV and MARV [14]. Although this study was performed in tissue culture, a similar approach provided some protection to Nipah (NiV) virus in hamsters, with survival rates dependent on the timing of the initiation of treatment. In a second set of studies, Wolf et al. recently identified a potent inhibitor of EBOV entry during an attempt to identify NiV entry inhibitors using a high-throughput screen [15]. LJ001 irreversibly intercalated into lipid membranes, inhibiting viral-cell fusion of a wide range of enveloped viruses. Unfortunately, postexposure treatment failed to protect mice from a lethal challenge with EBOV, likely due to the short half-life of the compound in the host. Given the broad antiviral range, low cytotoxicity, and a probable inability of the virus to develop resistance, a pharmacokinetically optimized form of LJ001 could potentially serve as an EBOV therapeutic. Additionally, compounds targeting the lysosomal protein Niemann–Pick C1 that is required for EBOV GP-dependent entry may also have future clinical efficacy [16,17].Small molecules that target other portions of the filovirus life cycle are currently limited and in vivo testing of these is only being initiated. However, through high throughput screens with compound libraries, several promising candidates have been identified [18–20]. High throughput screens of compounds containing heterocyclic aromatic structures identified FGI-103 and FGI-106 as potent EBOV inhibitors [19,20]. Both provided complete postexposure protection in mice with little or no cytotoxicity. A more focused high throughput screens containing compounds predicted to inhibit viral ligand/TSG-101 interaction identified FGI-104 [18]. This compound, along with FGI-106, inhibited replication of a broad range of viruses and provided protection against EBOV challenge in mice. FGI-106 and FGI-103, and potentially FGI-104, target events late in the replication cycle.Conclusion & future perspectiveThe last few years have seen great advances in the development of filovirus therapeutics, most notably with vaccine development and potential treatments to target viral mRNA. However, given the questionable financial incentive of developing EBOV-specific antivirals or vaccines, future cures for EBOV might stem from broad-efficacy antivirals. Ongoing studies to understand the EBOV replication cycle – as well as that of other RNA viruses – will help to identify novel therapeutic targets. These targets possibly include required host factors that have been identified by large-scale screens. This, along with recent crystallization of several EBOV proteins, will allow for more rational, structure-based development of antivirals. One of the most significant outstanding questions is the timing of EBOV therapeutics. All of the strategies described in this article that provided protection were administered either before or shortly after a lethal challenge. This strategy would be useful for a healthcare worker or researcher, but is of questionable value for a patient already displaying signs of EHF. Strategies that provide cheap, easily administered, and potent virus inhibition in a patient displaying EHF remain elusive.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Hartman AL, Towner JS, Nichol ST. Ebola and marburg hemorrhagic fever. Clin. Lab. Med.30,161–177 (2010).Crossref, Medline, Google Scholar2 Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet377,849–862 (2011).Crossref, Medline, Google Scholar3 Sullivan NJ, Martin JE, Graham BS, Nabel GJ. Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. Nat. Rev. Microbiol.7,393–400 (2009).Crossref, Medline, CAS, Google Scholar4 Gupta M, Goldsmith CS, Metcalfe MG, Spiropoulou CF, Rollin PE. Reduced virus replication, proinflammatory cytokine production, and delayed macrophage cell death in human PBMCs infected with the newly discovered Bundibugyo ebolavirus relative to Zaire ebolavirus. 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This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download
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