Targeting the virus-host interface for the development of therapeutics against filoviruses.

Analyzing The Ebola ViruAnalyzing The Impact That The Ebola Virus Disease Has on Children

The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Infection with Marburg and Ebola viruses cause haemorrhagic fevers that are characterized by organ malfunction, bleeding complications, and high mortality. The viruses are members of the family Filoviridae, a group of membrane-enveloped filamentous RNA viruses. Five distinct species of the genus Ebolavirus have been reported; the genus Marburgvirus contains only one species. Both Marburg and Ebola virus diseases are zoonotic infections whose primary hosts are thought to be bats. The initial human infection is acquired from wildlife and subsequent person-to-person spread propagates the outbreak until it is brought under control. Ebola and Marburg viruses are classified as hazard or risk group 4 pathogens because of the very high case fatality rates observed for Ebola and Marburg virus diseases, the frequency of person-to-person transmission and community spread, and the lack of an approved vaccine or antiviral therapy. This mandates that infectious materials are handled and studied in maximum containment laboratory facilities. Epidemics have occurred sporadically since the discovery of Marburg in 1967 and Ebola virus in 1976. While some of these outbreaks have been relatively large, infecting a few hundreds of individuals, they have generally occurred in rural settings and have been controlled relatively easily. However, the 2013–2016 epidemic of Ebola virus disease in West Africa was different, representing the first emergence of the Zaire species of Ebola in a high-density urban location. Consequently, this has been the largest recorded filovirus outbreak in both the number of people infected and the range of geographical spread. Many of the reported and confirmed cases were among people living in high-density and impoverished urban environments. The chapter summarizes the most up-to-date taxonomic status of the family Filoviridae. It focuses on Marburg and Ebola viruses in a historical context, culminating in the 2013–2016 outbreak of Ebola virus in West Africa. Virus biology of the most well-studied member is described, with details of the viral genome and the protein machinery necessary to propagate viruses at the molecular and cellular level. This information is used to build a wider-scale virus–host perspective with detail on the pathology and pathogenesis of Ebola virus disease. The consequences of cell infection are examined, together with our current understanding of the immune response to Ebola virus, leading to a broader description of the clinical features of disease. The chapter closes by drawing information together in a section on diagnosis, ecology, prevention, and control.

Peter L Collins & Alexander Bukreyev† †Author for correspondence Laboratory of Infectious Disease, National Institute of Allergy & Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6505, Bethesda, MA 20892-8007, USA Tel.: +1 301 594 1854; Fax: +1 301 496 8312; ab176v@nih.gov ‘The development of vaccines against MARV and EBOV ... is a high priority because of their high virulence, ability to spread from person to person and the possibility of their use in bioterrorism.’

developed 6 weeks later (6).Blood culture grew B. longum and B. infantis, which were probiotic strains.Apart from 1 case of sepsis caused by B. longum associated with acupuncture in a 19-year-old healthy patient (7), we did not find other reports of invasive Bifidobacterium spp.infections.Because neutropenic episodes, even with bowel involvement, are common during treatment for cancer (8), no reason to promote therapeutic use of probiotics has been proven.Probiotics can cause substantial bacterial overgrowth when stimulating factors are present.In our opinion, avoiding fecal impaction is crucial for preventing colonic bacterial overgrowth and minimizes the chance that bacteria will translocate and cause invasive infection.Nutritional recommendations for a neutropenic diet for children are still debated.The problem is not probiotic therapy but rather fermented food products to which small amounts of probiotics are added.After we reviewed the literature, we did not find enough data to safely recommend the use of these products in children receiving chemotherapy (9).Nevertheless, probiotic therapy is recommended for many immunocompromised patients, such as preterm infants and persons with chronic inflammatory bowel disease (10).We believe that this case of B. breve sepsis in an oncology patient underscores the invasive potential of probiotics.

Ebola virus disease (EVD) is a complex zoonosis that is highly virulent in humans. The largest recorded outbreak of EVD is ongoing in West Africa, outside of its previously reported and predicted niche. We assembled location data on all recorded zoonotic transmission to humans and Ebola virus infection in bats and primates (1976–2014). Using species distribution models, these occurrence data were paired with environmental covariates to predict a zoonotic transmission niche covering 22 countries across Central and West Africa. Vegetation, elevation, temperature, evapotranspiration, and suspected reservoir bat distributions define this relationship. At-risk areas are inhabited by 22 million people; however, the rarity of human outbreaks emphasises the very low probability of transmission to humans. Increasing population sizes and international connectivity by air since the first detection of EVD in 1976 suggest that the dynamics of human-to-human secondary transmission in contemporary outbreaks will be very different to those of the past.DOI: http://dx.doi.org/10.7554/eLife.04395.001

Dear Editor, In humans and great apes, Ebola viruses are pathogenic agents linked to severe, life-threatening organ dysfunction. Equatorial or West Africa is home to four different Ebola virus species. The spread of the most virulent species into the world population happens mostly through interaction with contaminated biological fluids and can cause severe illness outbreaks in places with few resources1. Widespread transcription and replication, immunological suppression, aberrant inflammatory reactions, significant fluid and electrolyte losses, and high mortality mark a sickness brought on by these viruses. Although the latest innovations in vaccine technology and the lack of a licensed prophylaxis or therapeutic, counseling for multiple severe organ failures brought on by immune-mediated cellular damage primarily consists of supportive care. The WHO categorized the 2013–2016 outbreak as a Public Health Emergency of International Concern, highlighting the difficulties associated with treating Ebola virus infections and raising concerns about society's readiness to explore the potential epidemics on a scientific, clinical, and sociological level2. This pathogen creates serious public health concerns and immediate illness with a high fatality rate, primarily spread by living person exposure to infected bodily fluids and corpses. The 2013–2016 West African outbreaks demonstrated the significant pandemic potential of Ebola viruses. This pandemic was unparalleled, with almost 28 000 cases reported and 11 000 fatalities3. The Sudan ebolavirus, which causes Ebola, broke out in the Western and Central Regions of Uganda in the 2022 Uganda Ebola outbreak. Over 160 persons were affected, and 77 of them passed away. The Sudan ebolavirus, which causes Ebola, broke out in the Western and Central Regions of Uganda in the 2022 Uganda Ebola outbreak. Over 160 persons were affected, and 77 of them passed away. It was the fifth epidemic of Sudan ebolavirus in Uganda4. The Ugandan Ministry of Health reported an occurrence of the illnesses on 20 September 20225. There were 44 fatalities and 90 recognized or likely circumstances as of 24 October 2022. The first fatality in the country's capital of Kampala was reported on 12 October. Twelve days later, on 24 October, there had been 14 infections over the previous 2 days6. On 11 January 2023, the epidemic was deemed to be over after 42 days with no suspected cases7. Ebolaviruses are viruses with a singular, negatively polarized strand of RNA as their genome. They are members of the genus Ebolavirus in the group Filoviridae of the order Mononegaviruses. Bundibugyo ebolavirus (Bundibugyo virus), Reston ebolavirus (Reston virus), Sudan ebolavirus (Sudan virus), Ta Forest ebolavirus (Ta Forest virus), and Zaire ebolavirus are the five species that make up the genus Ebolavirus (Ebola virus). It is important to emphasize this taxonomy because virtually identical names have different meanings: Ebolavirus and Zaire ebolavirus relate to taxonomic classifications, whereas Ebola is a virus. This taxonomy was amended in 20118. The Ebola and Sudan viruses are mostly to blame for these outbreaks. Since the discovery of ebolaviruses in 1976, more than 20 countries of the virus have been documented in Sub-Saharan Africa, particularly in Sudan, Uganda, the Democratic Republic of the Congo, and Gabon. The infection in Gulu, Uganda, in 2000 was in a semi-urban region, in contrast to most of these outbreaks, which took place in remote rural areas. It is possible that minor epidemics may not have been recognized as such. The biggest Ebola outbreak occurred in West Africa from 2013 to 2016, primarily affecting Guinea, Sierra Leone, and Liberia9. It spread to numerous nations in rural and urban regions and had a very high incidence and fatality rate (more than 28 000 cases and more than 11 000 fatalities). The actual impact, meanwhile, may have been far higher due to underrepresentation. The total average mortality rate in this epidemic was 62.9% (95% CI: 61.9–64.0%) for known cases with documented health outcomes10. If someone has been exposed to the virus and is showing symptoms, public health officials should be contacted, and the patient should be quarantined. The patient's infection status may be confirmed by testing blood samples taken from the patient. After the beginning of manifestations, the Ebola virus may be identified in the blood. For the virus to be detected, it may take up to 3 days following the onset of symptoms. Because of its sensitivity to identifying even trace amounts of the Ebola virus, polymerase chain reaction (PCR) is one of the most widely utilized diagnostic tools. Even while PCR techniques are sensitive enough to identify individual virus particles in low blood concentrations, their sensitivity improves as the viral load rises in an ongoing infection. Once there are not enough virus particles in a patient's blood, PCR tests will no longer be able to detect the disease. Patients may be confirmed to have been exposed to and infected with the Ebola virus using other means, such as identifying antibodies an Ebola virus disease (EVD) case develops in response to an infection11. Still, if this pathogen infects someone, they should administer supportive clinical treatment, which includes rehydration, nourishment, analgesics, and blood transfusions, when necessary, which is currently a pillar of EVD patient management, despite the lack of conclusive evidence for its usefulness. Maintaining intravascular volume with oral rehydration solution or intravenous fluids that provide proper electrolyte exchange is a crucial component of supportive treatment. Individuals with recurrent diarrhea and vomiting may also benefit from antiemetics and antidiarrheal medications12. To improve the prognosis of infected individuals and lower the infection rate and hence the danger of future infections, an appropriate therapy would be required for epidemic control. Several possible therapeutic compounds for antiviral medications have shown promise, and they each have unique mechanisms13. Ethics approval Not applicable. Sources of funding Not applicable. Conflicts of interest disclosure The authors declare no conflicts of interest, financial or otherwise. Data availability statement All data used to support the findings of this study are included in the article.

More than 25 years have passed since the discovery of a filovirus, Marburg virus, which caused an epidemic of fever among laboratory workers in Marburg, Germany, in 1967. The persons affected had contact with the blood or tissues of monkeys or with other infected persons. Marburg virus has reappeared only three times since its discovery, with the largest and most recent outbreak occurring in 1999 in Durba, Democratic Republic of the Congo. Ebola virus, another filovirus, was first described in 1976 during two fever epidemics in Zaire and Sudan. Since then, Ebola virus has caused large hospital outbreaks of fever in Kikwit, Zaire, in 1995, and Gulu, Uganda, in 2000. Ebola virus has also been implicated in small chains of transmission among persons with direct contact with intermediary hosts, mostly nonhuman primates in the central African countries of Gabon and Republic of the Congo. The reservoirs for both viruses are still unknown, and the rarity of outbreaks and the remote location of human outbreaks make it difficult, if not impossible, to study the pathogenesis of the human disease. Thus, animal models have been the best, and often only, approach available for studying the progression of disease caused by Marburg and Ebola viruses. Dr. Ryabchikova, the principal author of this book, and her laboratory group have studied the pathogenesis of filoviruses for several decades by using animal models and electron microscopy, a unique approach that has made her one of the few filovirus experts in the world. This book is a compilation not only of her work but of all the information available on Marburg and Ebola viruses. The first three chapters of the book provide a general review of filovirus history, laboratory methods (with an emphasis on electron microscopy), viral structure, morphology, and replication. Chapters 4 and 5 provide more specific details on infection of the target cells (macrophages and reticuloendothelial system) in different organs and during the course of filoviral infection. In chapter 6, the authors deal with the hemorrhagic side of Ebola and Marburg virus infection. Not all patients infected with these viruses bleed, and when bleeding disorders do occur, no correlated infection of endothelial and hematopoietic cells occurs. Dr. Ryabchikova has found that changes in the microcirculation system, such as the appearance of hemorrhages, clotting, and fibrin deposits, vary by virus and by animal species. Chapter 7, which describes pathologic changes in the organs during the course of filoviral infection, could have been combined with chapter 5. Likewise, the last chapter, which covers immunopathology, appears more like a discussion of the previous chapters. Much of the data have already been published in the Russian or Western literature. However, this book provides one source for all information available on Marburg and Ebola viruses and has a great advantage over other sources. The number and the quality of the illustrations are impressive, and a comprehensive index is provided. The book will prove useful to clinicians and researchers interested in understanding the pathogenesis of fevers, and it will provide researchers working with other viruses a lesson in the benefits of using electron microscopy technology.

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

Ebola‐ and marburgviruses belong to the family of filoviruses and cause severe haemorrhagic fevers in humans and nonhuman primates. Since their discovery in 1967, during outbreaks in Germany and former Yugoslavia originating with nonhuman primates imported from Uganda, they have been responsible for numerous disease outbreaks in Africa, including that of unprecedented size, which is currently ongoing in West Africa. Filoviruses have also been implicated in massive die‐offs of great apes, and in recent years there have been several cases of imported infections into Europe and North America. Filoviral haemorrhagic fevers are severe diseases with case fatality rates of up to 90%. Currently, there is neither a licensed vaccine nor a specific therapy available, although experimental vaccines and treatments have shown promise in nonhuman primates, and are now being vigorously pursued in human clinical trials. Key Concepts Human pathogenic filoviruses (Ebola virus (EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV), Bundibugyo virus (BDBV) and Marburg virus (MARV)) are found in Africa, primarily in bats and nonhuman primates. In contrast, Reston virus (RESTV), which is nonpathogenic in humans, originates in the Philippines, where it is found in both nonhuman primates and pigs. Imported nonhuman primates have been the cause of outbreaks of filoviral haemorrhagic fever in Europe and North America, both among humans and nonhuman primates. Tourism and the return of medical aid workers have also resulted in several imported infections during the last years. Bats represent the most likely reservoir for filoviruses. The recent outbreak in West Africa has been responsible for over 27 000 cases (as of 27 May 2015) and is still ongoing; highlighting the threat that filovirus infection continues to pose to human health. While the absolute number of filoviral haemorrhagic fever cases remain low compared to other diseases, the severe disease picture and high case fatality rates have led to a high public profile of filoviral haemorrhagic fevers. The pathophysiology of filoviral haemorrhagic fevers involves vascular dysfunction, impairment of the immune system and massive dysregulation of cytokine production. Death is caused by multiple organ failure as result of a syndrome resembling septic shock. Currently, the only measures to combat filovirus infections are supportive therapy and patient isolation. However, basic public health measures are very effective at controlling outbreaks, when consistently and rigorously implemented. Experimental treatments and vaccines in nonhuman primates exist, but are not yet licensed for use in humans. However, public interest in the current West African EBOV outbreak has accelerated efforts to see several of the more advanced therapeutic and vaccination approaches licenced.

Ebola and Marburg viruses belong to the family of filoviruses and cause severe haemorrhagic fevers in humans and nonhuman primates. Since their discovery in 1967, during outbreaks in Germany and former Yugoslavia originating with nonhuman primates imported from Uganda, they have been responsible for numerous disease outbreaks in Africa. Ebola has also been implicated in massive die‐offs of great apes and in recent years there have been several cases of imported Marburg infections into Europe and North America. Filoviral haemorrhagic fevers (FHF) are severe diseases with case fatality rates of up to 90%. Currently, there is neither a licensed vaccine nor a specific therapy available. Although experimental vaccines and treatments have shown promise in nonhuman primates, at the present time supportive therapy and prevention of disease transmission through rigorous case management and patient isolation are the only means available to combat FHF. Key Concepts: Human pathogenic filoviruses (Ebola and Marburg viruses) are found in Africa, whereas Reston ebolavirus , which is nonpathogenic to humans, originates in the Philippines, where it is found in both nonhuman primates and pigs. Although the absolute number of filoviral haemorrhagic fever cases is low when compared to other diseases, the severe disease picture and high case fatality rates have led to a high public profile of filoviral haemorrhagic fevers. Imported nonhuman primates have been the cause of outbreaks of filoviral haemorrhagic fever in Europe and North America, both among humans and nonhuman primates. Tourism has also resulted in several imported infections during the last years. Bats represent the most likely reservoir for filoviruses. The pathophysiology of filoviral haemorrhagic fevers involves vascular dysfunction, impairment of the immune system and massive dysregulation of cytokine production. Death is caused by multiple organ failure as result of a syndrome resembling septic shock. Experimental treatments and vaccines in nonhuman primates exist, but are not licensed for use in humans. Currently the only measures to combat filovirus infections are supportive therapy and patient isolation.

Cholesterol-conjugated stapled peptides inhibit Ebola and Marburg viruses in vitro and in vivo

Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan virus resulted in 164 cases with 55 deaths. In 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea and Tanzania resulting in over 49 confirmed or suspected cases; 41 of which were fatal. There are no clearly defined correlates of protection against these VHF, impeding targeted vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Ebola virus, Sudan virus, Marburg virus) and an arenavirus (Lassa virus). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19/AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Ebola virus, Sudan virus and Marburg virus challenges in a small animal model.

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Filoviruses: closing the gap on a killer: Marburg and Ebola Viruses (Current Topics in Microbiology and Immunology series) edited by H-D. Klenk