RESUMEN
RNA editing has been discovered as an essential mechanism for the transcription of the glycoprotein (GP) gene of Ebola virus but not Marburg virus. We developed a rapid transcript quantification assay (RTQA) to analyze RNA transcripts generated through RNA editing and used immunoblotting with a pan-ebolavirus monoclonal antibody to confirm different GP gene-derived products. RTQA successfully quantified GP gene transcripts during infection with representative members of 5 ebolavirus species. Immunoblotting verified expression of the soluble GP and the transmembrane GP. Our results defined RNA editing as a general trait of ebolaviruses. The degree of editing, however, varies among ebolaviruses with Reston virus showing the lowest and Bundibugyo virus the highest degree of editing.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/genética , Edición de ARN , Glicoproteínas , Anticuerpos Antivirales , Anticuerpos Monoclonales , Fiebre Hemorrágica Ebola/genéticaRESUMEN
BACKGROUND: The filovirus Bundibugyo virus (BDBV) causes severe disease with a mortality rate of approximately 20%-51%. The only licensed filovirus vaccine in the United States, Ervebo, consists of a recombinant vesicular stomatitis virus (rVSV) vector that expresses Ebola virus (EBOV) glycoprotein (GP). Ervebo was shown to rapidly protect against fatal Ebola disease in clinical trials; however, the vaccine is only indicated against EBOV. Recent outbreaks of other filoviruses underscore the need for additional vaccine candidates, particularly for BDBV infections. METHODS: To examine whether the rVSV vaccine candidate rVSVΔG/BDBV-GP could provide therapeutic protection against BDBV, we inoculated seven cynomolgus macaques with 1000 plaque-forming units of BDBV, administering rVSVΔG/BDBV-GP vaccine to 6 of them 20-23 minutes after infection. RESULTS: Five of the treated animals survived infection (83%) compared to an expected natural survival rate of 21% in this macaque model. All treated animals showed an early circulating immune response, while the untreated animal did not. Surviving animals showed evidence of both GP-specific IgM and IgG production, while animals that succumbed did not produce significant IgG. CONCLUSIONS: This small, proof-of-concept study demonstrated early treatment with rVSVΔG/BDBV-GP provides a survival benefit in this nonhuman primate model of BDBV infection, perhaps through earlier initiation of adaptive immunity.
Asunto(s)
Vacunas contra el Virus del Ébola , Ebolavirus , Fiebre Hemorrágica Ebola , Estomatitis Vesicular , Vacunas Virales , Animales , Estomatitis Vesicular/prevención & control , Anticuerpos Antivirales , Vesiculovirus/genética , Glicoproteínas/genética , Macaca fascicularis , Inmunoglobulina GRESUMEN
Ebolaviruses are at the forefront of emerging viruses and present a very perceptible threat to global peace and harmony. In the last decade, Ebola virus disease has claimed more than 90% of total lives since its inception in 1976. Owing to multiple host immune evasion methods employed by the virus and the limitations of traditional vaccine development approaches, finding a globally effective and reliable counter measure against Ebola virus remains a challenge. Highly conserved peptide fragments belonging to critical viral proteins and containing multiple epitopes which have the capacity to interact with a wide array of HLA molecules present a viable solution. Immunoinformatics or computational immunology enables rapid screening and shortlisting of plausible epitopes with a high immunogenic potential, thus, supporting expeditious elucidation of efficacious vaccine candidates. In light of above facts, we describe a computational methodology in this chapter for identification of potent peptide vaccine candidates against human infecting viruses. By applying this stringent methodology, we were able to identify multiple, immunogenic ebolavirus peptide fragments which, after verification in animal models, might be considered as part of future synthetic Ebola vaccine.
Asunto(s)
Vacunas contra el Virus del Ébola , Ebolavirus , Fiebre Hemorrágica Ebola , Animales , Biología Computacional/métodos , Epítopos de Linfocito T/química , Fiebre Hemorrágica Ebola/prevención & control , Péptidos , Vacunas de SubunidadRESUMEN
Mali had 2 reported introductions of Ebola virus (EBOV) during the 2013-2016 West Africa epidemic. Previously, no evidence for EBOV circulation was reported in Mali. We performed an EBOV serosurvey study in southern Mali. We found low seroprevalence in the population, indicating local exposure to EBOV or closely related ebola viruses.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Anticuerpos Antivirales , Humanos , Inmunoglobulina G , Malí , Estudios SeroepidemiológicosRESUMEN
Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form ß-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Ebolavirus/efectos de los fármacos , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/química , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión , Microscopía por Crioelectrón , Ebolavirus/crecimiento & desarrollo , Ebolavirus/inmunología , Ebolavirus/patogenicidad , Epítopos/química , Epítopos/inmunología , Femenino , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Humanos , Células Jurkat , Ratones , Modelos Moleculares , Polisacáridos/química , Polisacáridos/inmunología , Unión Proteica , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Ebola virus disease (EVD) remains among the biggest public health threats in Africa, even though recently a vaccine was approved for human use. However, in outbreak situations treatment strategies are needed in combination with vaccination campaigns to impact and stop the spread of the disease. Here, we discuss the development of the immunotherapeutics against EDV both targeting the virus itself and bolstering the immunological environment of the host at both the pre-clinical and clinical level. The early development of antibody therapy in preclinical settings and the early pitfalls in the implementation of this therapeutic strategy are discussed. We also consider the advancement of the production, modulation, and specificity of the antibody treatment that garnered increased success in preclinical studies to the point that it was warranted to test them in a clinical setting. Initial clinical trials in an outbreak scenario proved difficult to definitively confirm the efficacy of the implemented treatment. Upon further modification and with the experiences from the challenging outbreak conditions in mind, the PALM clinical trial demonstrated efficacy of an antibody cocktail which recently received approval for human use.
RESUMEN
Introduction. The consistent emergence/reemergence of filoviruses into a world that previously lacked an approved pharmaceutical intervention parallels an experience repeatedly played-out for most other emerging pathogenic zoonotic viruses. Investment to preemptively develop effective and low-cost prophylactic and therapeutic interventions against viruses that have high potential for emergence and societal impact should be a priority.Areas covered. Candidate drugs can be characterized into those that interfere with cellular processes required for Ebola virus (EBOV) replication (host-directed), and those that directly target virally encoded functions (direct-acting). We discuss strategies to identify pharmaceutical interventions for EBOV infections. PubMed/Web of Science databases were searched to establish a detailed catalog of these interventions.Expert opinion. Many drug candidates show promising in vitro inhibitory activity, but experience with EBOV shows the general lack of translation to in vivo efficacy for host-directed repurposed drugs. Better translation is seen for direct-acting antivirals, in particular monoclonal antibodies. The FDA-approved monoclonal antibody treatment, Inmazeb™ is a success story that could be improved in terms of impact on EBOV-associated disease and mortality, possibly by combination with other direct-acting agents targeting distinct aspects of the viral replication cycle. Costs need to be addressed given EBOV emergence primarily in under-resourced countries.
Asunto(s)
Antivirales/farmacología , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Desarrollo de Medicamentos , Reposicionamiento de Medicamentos , Ebolavirus/efectos de los fármacos , Fiebre Hemorrágica Ebola/virología , HumanosRESUMEN
In 2008, an outbreak of Reston ebolavirus (RESTV) in pigs in the Philippines expanded our understanding of the host range of ebolaviruses. Subsequent experimental infections with the human-pathogenic species Zaire ebolavirus (EBOV) confirmed that pigs are susceptible to African species of ebolaviruses. Pig keeping has become an increasingly important livelihood strategy throughout parts of sub-Saharan Africa, driven by increasing demand for pork. The growth in pig keeping is particularly rapid in Uganda, which has the highest per capita pork consumption in East Africa and a history of sporadic human outbreaks of Ebola virus disease (EVD). Using a systematic sampling protocol, we collected sera from 658 pigs presented for slaughter in Uganda between December 2015 and October 2016. Forty-six pigs (7%) were seropositive based on ELISA tests at two different institutions. Seropositive pigs had antibodies that bound to Sudan NP (n = 27), Zaire NP (Kikwit; n = 8) or both NPs (n = 11). Sera from 4 of the ELISA-positive pigs reacted in Western blot (EBOV NP = 1; RESTV NP = 2; both NPs = 2), and one sample had full neutralizing antibody against Sudan ebolavirus (SUDV) in virus neutralization tests. Pigs sampled in June 2016 were significantly more likely to be seropositive than pigs sampled in October 2016 (p = .03). Seropositive pigs were sourced from all regions except Western region. These observed temporal and spatial variations are suggestive of multiple introductions of ebolaviruses into the pig population in Uganda. This is the first report of exposure of pigs in Uganda to ebolaviruses and the first to employ systematic abattoir sampling for ebolavirus surveillance during a non-outbreak period. Future studies will be necessary to further define the role pigs play (if any) in ebolavirus maintenance and transmission so that potential risks can be mitigated.
Asunto(s)
Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/veterinaria , Enfermedades de los Porcinos/epidemiología , Mataderos , Animales , Femenino , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Masculino , Prevalencia , Factores de Riesgo , Estudios Seroepidemiológicos , Análisis Espacio-Temporal , Sus scrofa , Porcinos , Enfermedades de los Porcinos/virología , Uganda/epidemiologíaRESUMEN
BACKGROUND: Uganda has experienced seven Ebola Virus Disease (EVD) outbreaks and four Marburg Virus Disease (MVD) outbreaks between 2000 and 2019. We investigated the seroprevalence and risk factors for Marburg virus and ebolaviruses in gold mining communities around Kitaka gold mine in Western Uganda and compared them to non-mining communities in Central Uganda. METHODS: A questionnaire was administered and human blood samples were collected from three exposure groups in Western Uganda (gold miners, household members of miners, non-miners living within 50 km of Kitaka mine). The unexposed controls group sampled was community members in Central Uganda far away from any gold mining activity which we considered as low-risk for filovirus infection. ELISA serology was used to analyse samples, detecting IgG antibodies against Marburg virus and ebolaviruses (filoviruses). Data were analysed in STATA software using risk ratios and odds ratios. RESULTS: Miners in western Uganda were 5.4 times more likely to be filovirus seropositive compared to the control group in central Uganda (RR = 5.4; 95% CI 1.5-19.7) whereas people living in high-risk areas in Ibanda and Kamwenge districts were 3.6 more likely to be seropositive compared to control group in Luweeero district (RR = 3.6; 95% CI 1.1-12.2). Among all participants, filovirus seropositivity was 2.6% (19/724) of which 2.3% (17/724) were reactive to Sudan virus only and 0.1% (1/724) to Marburg virus. One individual seropositive for Sudan virus also had IgG antibodies reactive to Bundibugyo virus. The risk factors for filovirus seropositivity identified included mining (AOR = 3.4; 95% CI 1.3-8.5), male sex (AOR = 3.1; 95% CI 1.01-9.5), going inside mines (AOR = 3.1; 95% CI 1.2-8.2), cleaning corpses (AOR = 3.1; 95% CI 1.04-9.1) and contact with suspect filovirus cases (AOR = 3.9, 95% CI 1.04-14.5). CONCLUSIONS: These findings indicate that filovirus outbreaks may go undetected in Uganda and people involved in artisan gold mining are more likely to be exposed to infection with either Marburg virus or ebolaviruses, likely due to increased risk of exposure to bats. This calls for active surveillance in known high-risk areas for early detection and response to prevent filovirus epidemics.
Asunto(s)
Brotes de Enfermedades , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/epidemiología , Enfermedad del Virus de Marburg/diagnóstico , Enfermedad del Virus de Marburg/epidemiología , Marburgvirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Quirópteros/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fiebre Hemorrágica Ebola/sangre , Humanos , Masculino , Enfermedad del Virus de Marburg/sangre , Persona de Mediana Edad , Mineros , Estudios Retrospectivos , Estudios Seroepidemiológicos , Uganda/epidemiología , Adulto JovenRESUMEN
The genus Ebolavirus comprises several virus species with zoonotic potential and varying pathogenicity for humans. Ebolaviruses are considered to circulate in wildlife with occasional spillover events into the human population which then often leads to severe disease outbreaks. Several studies indicate a significant role of bats as reservoir hosts in the ebolavirus ecology. However, pigs from the Philippines have been found to be naturally infected with Reston virus (RESTV), an ebolavirus that is thought to only cause asymptomatic infections in humans. The recent report of ebolavirus-specific antibodies in pigs from Sierra Leone further supports natural infection of pigs with ebolaviruses. However, susceptibility of pigs to highly pathogenic Ebola virus (EBOV) was only shown under experimental settings and evidence for natural infection of pigs with EBOV is currently lacking. Between October and December 2017, we collected 308 serum samples from pigs in Guinea, West Africa, and tested for the presence of ebolavirus-specific antibodies with different serological assays. Besides reactivity to EBOV nucleoproteins in ELISA and Western blot for 19 (6.2%) and 13 (4.2%) samples, respectively, four sera recognized Sudan virus (SUDV) NP in Western blot. Furthermore, four samples specifically detected EBOV or SUDV glycoprotein (GP) in an indirect immunofluorescence assay under native conditions. Virus neutralization assay based on EBOV (Mayinga isolate) revealed five weakly neutralizing sera. The finding of (cross-) reactive and weakly neutralizing antibodies suggests the exposure of pigs from Guinea to ebolaviruses or ebola-like viruses with their pathogenicity as well as their zoonotic potential remaining unknown. Future studies should investigate whether pigs can act as an amplifying host for ebolaviruses and whether there is a risk for spillover events.
Asunto(s)
Anticuerpos Antivirales/sangre , Brotes de Enfermedades/veterinaria , Reservorios de Enfermedades/veterinaria , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/veterinaria , Inmunoglobulina G/sangre , Enfermedades de los Porcinos/epidemiología , Animales , Anticuerpos Neutralizantes/sangre , Reacciones Cruzadas , Ebolavirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/veterinaria , Granjas , Femenino , Guinea/epidemiología , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Humanos , Masculino , Nucleoproteínas/inmunología , Sus scrofa , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
Filoviruses cause outbreaks which lead to high fatality in humans and non-human primates, thus tagging them as major threats to public health and species conservation. In this review, we give account of index cases responsible for filovirus disease outbreaks that have occurred over the past 52 years in a chronological fashion, by describing the circumstances that led to the outbreaks, and how each of the outbreaks broke out. Since the discovery of Marburg virus and Ebola virus in 1967 and 1976, respectively, more than 40 filovirus disease outbreaks have been reported; majority of which have occurred in Africa. The chronological presentation of this review is to provide a concise overview of filovirus disease outbreaks since the discovery of the viruses, and highlight the patterns in the occurrence of the outbreaks. This review will help researchers to better appreciate the need for surveillance, especially in areas where there have been no filovirus disease outbreaks. We conclude by summarizing some recommendations that have been proposed by health and policy decision makers over the years.
RESUMEN
Filoviruses are among the most pathogenic viruses known to man, and work with live viruses is restricted to maximum containment laboratories. In order to study individual aspects of the virus life cycle outside of maximum containment laboratories, life cycle modeling systems have been established, which use reporter-encoding miniature versions of the viral genome called minigenomes. With basic minigenome systems viral genome replication and transcription can be studied, whereas more advanced systems also allow us to model other aspects of the virus life cycle outside of a maximum containment laboratory. These systems, therefore, represent powerful tools to study the biology of filoviruses, and for the screening and development of antivirals.
Asunto(s)
Genoma Viral/genética , Ebolavirus/genética , Transcripción Genética , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Ebola virus (EBOV) infection is highly lethal and results in severe febrile bleeding disorders that affect humans and non-human primates. One of the therapeutic approaches for treating EBOV infection focus largely on cocktails of monoclonal antibodies (mAbs) that bind to specific regions of the EBOV glycoprotein (GP) and neutralize the virus. Recent structural studies using cryo-electron microscopy have identified key epitopes for several EBOV mAbs. While such information has yielded deep insights into antibody binding, limitations on resolution of these structures often preclude a residue-level analysis of EBOV epitopes. In this study, we performed combinatorial peptide-based epitope mapping of EBOV GP against a broad panel of mAbs and polyclonal sera derived from several animal species vaccinated with EBOV DNA and replicon vaccines and/or exposed to EBOV infection to identify residue-level determinants of antibody binding. The peptide-based epitope mapping obtained from a wide range of serum and mAb samples, combined with available cryo-EM structure reconstructions revealed fine details of antibody-virus interactions, allowing for a more precise and comprehensive mapping of antibody epitopes on EBOV GP. We show how these residue-level epitope definitions can be used to characterize antigenic variation across different filoviruses, and provide a theoretical basis for predicting immunity and cross-neutralization in potential future outbreaks.
Asunto(s)
Anticuerpos Antivirales/inmunología , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Mapeo Epitopo , Vacunas de ADN/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Unión ProteicaRESUMEN
Ebolaviruses are the causative agent of a severe hemorrhagic fever with high case fatality rates, for which no approved specific therapy is available. As biosafety level 4 (BSL4) agents, work with live ebolaviruses is restricted to maximum containment laboratories. Transcription and replication-competent viruslike particle (trVLP) systems are reverse genetics-based life cycle modeling systems that allow researchers to model virtually the entire ebolavirus life cycle outside of a maximum containment laboratory. These systems can be used to dissect the virus life cycle, and thus increase our understanding of virus biology, as well as for more applied uses such as the screening and development of novel antivirals, and thus represent powerful tools for work on ebolaviruses.
Asunto(s)
Ebolavirus/genética , Genoma Viral/efectos de los fármacos , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Transcripción Genética , Antivirales/uso terapéutico , Ebolavirus/efectos de los fármacos , Ebolavirus/patogenicidad , Genoma Viral/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Estadios del Ciclo de Vida/efectos de los fármacos , Virión/efectos de los fármacos , Virión/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Ebolaviruses cause severe hemorrhagic fever with high case fatality rates. Despite recent progress, there is a continued need for the development of antivirals against these viruses. Reporter-expressing ebolaviruses, which can be generated using reverse genetics systems, are powerful tools for antiviral screening. While viruses expressing fluorescent reporters are amenable for this purpose and can be used for high-content imaging-type screens, as an alternative, luciferase-expressing reporter viruses have recently been developed and have the advantages of being extremely easy to use and having short assay times. Here we provide a detailed protocol for the use of such a luciferase-expressing reporter virus for antiviral screening in a 96-well format, with parallel assessment of cytotoxicity of the screened compounds.
Asunto(s)
Antivirales/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Ebolavirus/efectos de los fármacos , Antivirales/química , Antivirales/farmacología , Ebolavirus/química , Ebolavirus/patogenicidad , Humanos , Luciferasas/química , Luciferasas/genética , Replicación Viral/efectos de los fármacosRESUMEN
Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6-16h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology.