The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Mechanism of human antibody-mediated neutralization of Marburg virus.

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

Potent immunogenicity and protective efficacy of a multi-pathogen vaccination targeting Ebola, Sudan, Marburg and Lassa viruse.

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.

Macaque antibodies targeting Marburg virus glycoprotein induced by multivalent immunization.

Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.

Contrasting effects of filamin A and B proteins in modulating filovirus entry.

Ebola (EBOV) and Marburg viruses (MARV) cause severe hemorrhagic fever associated with high mortality rates in humans. A better understanding of filovirus-host interactions that regulate the EBOV and MARV lifecycles can provide biological and mechanistic insight critical for therapeutic development. EBOV glycoprotein (eGP) and MARV glycoprotein (mGP) mediate entry into host cells primarily by actin-dependent macropinocytosis. Here, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry. We found that entry of pseudotype psVSV-RFP-eGP, infectious recombinant rVSV-eGP-mCherry, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their specific effects on EBOV and MARV entry. This study is the first to identify the filamin family of proteins as regulators of EBOV and MARV entry. These findings may provide insight into the development of new countermeasures to prevent EBOV and MARV infections.

ICTV Virus Taxonomy Profile: Filoviridae 2024.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.

Safety, tolerability, and immunogenicity of the chimpanzee adenovirus type 3-vectored Marburg virus (cAd3-Marburg) vaccine in healthy adults in the USA: a first-in-human, phase 1, open-label, dose-escalation trial.

BACKGROUND: WHO has identified Marburg virus as an emerging virus requiring urgent vaccine research and development, particularly due to its recent emergence in Ghana. We report results from a first-in-human clinical trial evaluating a replication-deficient recombinant chimpanzee adenovirus type 3 (cAd3)-vectored vaccine encoding a wild-type Marburg virus Angola glycoprotein (cAd3-Marburg) in healthy adults. METHODS: We did a first-in-human, phase 1, open-label, dose-escalation trial of the cAd3-Marburg vaccine at the Walter Reed Army Institute of Research Clinical Trials Center in the USA. Healthy adults aged 18-50 years were assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 1010 or 1 × 1011 particle units (pu). Primary safety endpoints included reactogenicity assessed for the first 7 days and all adverse events assessed for 28 days after vaccination. Secondary immunogenicity endpoints were assessment of binding antibody responses and T-cell responses against the Marburg virus glycoprotein insert, and assessment of neutralising antibody responses against the cAd3 vector 4 weeks after vaccination. This study is registered with ClinicalTrials.gov, NCT03475056. FINDINGS: Between Oct 9, 2018, and Jan 31, 2019, 40 healthy adults were enrolled and assigned to receive a single intramuscular dose of cAd3-Marburg vaccine at either 1 × 1010 pu (n=20) or 1 × 1011 pu (n=20). The cAd3-Marburg vaccine was safe, well tolerated, and immunogenic. All enrolled participants received cAd3-Marburg vaccine, with 37 (93%) participants completing follow-up visits; two (5%) participants moved from the area and one (3%) was lost to follow-up. No serious adverse events related to vaccination occurred. Mild to moderate reactogenicity was observed after vaccination, with symptoms of injection site pain and tenderness (27 [68%] of 40 participants), malaise (18 [45%] of 40 participants), headache (17 [43%] of 40 participants), and myalgia (14 [35%] of 40 participants) most commonly reported. Glycoprotein-specific antibodies were induced in 38 (95%) of 40 participants 4 weeks after vaccination, with geometric mean titres of 421 [95% CI 209-846] in the 1 × 1010 pu group and 545 [276-1078] in the 1 × 1011 pu group, and remained significantly elevated at 48 weeks compared with baseline titres (39 [95% CI 13-119] in the 1 ×1010 pu group and 27 [95-156] in the 1 ×1011 pu group; both p<0·0001). T-cell responses to the glycoprotein insert and neutralising responses against the cAd3 vector were also increased at 4 weeks after vaccination. INTERPRETATION: This first-in-human trial of this cAd3-Marburg vaccine showed the agent is safe and immunogenic, with a safety profile similar to previously tested cAd3-vectored filovirus vaccines. 95% of participants produced a glycoprotein-specific antibody response at 4 weeks after a single vaccination, which remained in 70% of participants at 48 weeks. These findings represent a crucial step in the development of a vaccine for emergency deployment against a re-emerging pathogen that has recently expanded its reach to new regions. FUNDING: National Institutes of Health.

Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies.

Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (ß-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.

An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

Assembly and transport of filovirus nucleocapsids.

Filovirus-infected cells are characterized by typical cytoplasmic inclusion bodies (IBs) located in the perinuclear region. The formation of these IBs is induced mainly by the accumulation of the filoviral nucleoprotein NP, which recruits the other nucleocapsid proteins, the polymerase co-factor VP35, the polymerase L, the transcription factor VP30 and VP24 via direct or indirect protein-protein interactions. Replication of the negative-strand RNA genomes by the viral polymerase L and VP35 occurs in the IBs, resulting in the synthesis of positive-strand genomes, which are encapsidated by NP, thus forming ribonucleoprotein complexes (antigenomic RNPs). These newly formed antigenomic RNPs in turn serve as templates for the synthesis of negative-strand RNA genomes that are also encapsidated by NP (genomic RNPs). Still in the IBs, genomic RNPs mature into tightly packed transport-competent nucleocapsids (NCs) by the recruitment of the viral protein VP24. NCs are tightly coiled left-handed helices whose structure is mainly determined by the multimerization of NP at its N-terminus, and these helices form the inner layer of the NCs. The RNA genome is fixed by 2 lobes of the NP N-terminus and is thus guided by individual NP molecules along the turns of the helix. Direct interaction of the NP C-terminus with the VP35 and VP24 molecules forms the outer layer of the NCs. Once formed, NCs that are located at the border of the IBs recruit actin polymerization machinery to one of their ends to drive their transport to budding sites for their envelopment and final release. Here, we review the current knowledge on the structure, assembly, and transport of filovirus NCs.

Reemergence of Marburgvirus disease: Update on current control and prevention measures and review of the literature.

In 1967, the very first case of the Marburgvirus disease (MVD) was detected in Germany and Serbia sequentially. Since then, MVD has been considered one of the most serious and deadly infectious diseases in the world with a case-fatality rate between 23% and 90% and a substantial number of recorded deaths. Marburgvirus belongs to the family of Filoviridae (filoviruses), which causes severe viral hemorrhagic fever (VHF). Some major risk factors for human infections are close contact with African fruit bats, MVD-infected non-human primates, and MVD-infected individuals. Currently, there is no vaccine or specific treatment for MVD, which emphasizes the seriousness of this disease. In July 2022, the World Health Organization reported outbreaks of MVD in Ghana after two suspected VHF cases were detected. This was followed in February and March 2023 with the emergence of the virus in two countries new to the virus: Equatorial Guinea and Tanzania, respectively. In this review, we aim to highlight the characteristics, etiology, epidemiology, and clinical symptoms of MVD, along with the current prevention measures and the possible treatments to control this virus.

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection.

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

Modelling Marburg Virus Disease in Syrian Golden Hamsters: Contrasted Virulence Between Angola and Ci67 Strains.

BACKGROUND: Marburg virus (MARV) has caused numerous sporadic outbreaks of severe hemorrhagic fever in humans. Human case fatality rates of Marburg virus disease (MVD) outbreaks range from 20% to 90%. Viral genotypes of MARV can differ by over 20%, suggesting variable virulence between lineages may accompany this genetic divergence. Comparison of existing animal models of MVD employing different strains of MARV support differences in virulence across MARV genetic lineages; however, there are few systematic comparisons in models that recapitulate human disease available. METHODS: We compared features of disease pathogenesis in uniformly lethal hamster models of MVD made possible through serial adaptation in rodents. RESULTS: No further adaptation from a previously reported guinea pig-adapted (GPA) isolate of MARV-Angola was necessary to achieve uniform lethality in hamsters. Three passages of GPA MARV-Ci67 resulted in uniform lethality, where 4 passages of a GPA Ravn virus was 75% lethal. Hamster-adapted MARV-Ci67 demonstrated delayed time to death, protracted weight loss, lower viral burden, and slower histologic alteration compared to GPA MARV-Angola. CONCLUSIONS: These data suggest isolate-dependent virulence differences are maintained even after serial adaptation in rodents and may serve to guide choice of variant and model used for development of vaccines or therapeutics for MVD.

AAV-Vectored Expression of Marburg Virus-Neutralizing Antibody MR191 Provides Complete Protection From Challenge in a Guinea Pig Model.

Although there are no approved countermeasures available to prevent or treat disease caused by Marburg virus (MARV), potently neutralizing monoclonal antibodies (mAbs) derived from B cells of human survivors have been identified. One such mAb, MR191, has been shown to provide complete protection against MARV in nonhuman primates. We previously demonstrated that prophylactic administration of an adeno-associated virus (AAV) expressing MR191 protected mice from MARV. Here, we modified the AAV-MR191 coding sequence to enhance efficacy and reevaluated protection in a guinea pig model. Remarkably, 4 different variants of AAV-MR191 provided complete protection against MARV, despite administration 90 days prior to challenge. Based on superior expression kinetics, AAV-MR191-io2, was selected for evaluation in a dose-reduction experiment. The highest dose provided 100% protection, while a lower dose provided ∼88% protection. These data confirm the efficacy of AAV-mediated expression of MR191 and support the further development of this promising MARV countermeasure.

Long-term Prophylaxis Against Aerosolized Marburg Virus in Nonhuman Primates With an Afucosylated Monoclonal Antibody.

Marburg virus (MARV) causes a hemorrhagic fever disease in human and nonhuman primates with high levels of morbidity and mortality. Concerns about weaponization of aerosolized MARV have spurred the development of nonhuman primate (NHP) models of aerosol exposure. To address the potential threat of aerosol exposure, a monoclonal antibody that binds MARV glycoprotein was tested, MR186YTE, for its efficacy as a prophylactic. MR186YTE was administered intramuscularly to NHPs at 15 or 5 mg/kg 1 month prior to MARV aerosol challenge. Seventy-five percent (3/4) of the 15 mg/kg dose group and 50% (2/4) of the 5 mg/kg dose group survived. Serum analyses showed that the NHP dosed with 15 mg/kg that succumbed to infection developed an antidrug antibody response and therefore had no detectable MR186YTE at the time of challenge. These results suggest that intramuscular dosing of mAbs may be a clinically useful prophylaxis for MARV aerosol exposure.

The Inability of Marburg Virus to Cause Disease in Ferrets Is Not Solely Linked to the Virus Glycoprotein.

Ebola virus (EBOV) causes lethal disease in ferrets, whereas Marburg virus (MARV) does not. To investigate this difference, we first evaluated viral entry by infecting ferret spleen cells with vesicular stomatitis viruses pseudotyped with either MARV or EBOV glycoprotein (GP). Both viruses were capable of infecting ferret spleen cells, suggesting that lack of disease is not due to a block in MARV entry. Next, we evaluated replication kinetics of authentic MARV and EBOV in ferret cell lines and demonstrated that, unlike EBOV, MARV was only capable of low levels of replication. Finally, we inoculated ferrets with a recombinant EBOV expressing MARV GP in place of EBOV GP. Infection resulted in uniformly lethal disease within 7-9 days postinfection, while MARV-inoculated animals survived until study endpoint. Together these data suggest that the inability of MARV to cause disease in ferrets is not entirely linked to GP.

Structural and Energetic Basis for Differential Binding of Ebola and Marburg Virus Glycoproteins to a Bat-Derived Niemann-Pick C1 Protein.

BACKGROUND: Our previous study demonstrated that the fruit bat (Yaeyama flying fox)-derived cell line FBKT1 showed preferential susceptibility to Ebola virus (EBOV), whereas the human cell line HEK293T was similarly susceptible to EBOV and Marburg virus (MARV). This was due to 3 amino acid differences of the endosomal receptor Niemann-Pick C1 (NPC1) between FBKT1 and HEK293T (ie, TET and SGA, respectively, at positions 425-427), as well as 2 amino acid differences at positions 87 and 142 of the viral glycoprotein (GP) between EBOV and MARV. METHODS/RESULTS: To understand the contribution of these amino acid differences to interactions between NPC1 and GP, we performed molecular dynamics simulations and binding free energy calculations. The average binding free energies of human NPC1 (hNPC1) and its mutant having TET at positions 425-427 (hNPC1/TET) were similar for the interaction with EBOV GP. In contrast, hNPC1/TET had a weaker interaction with MARV GP than wild-type hNPC1. As expected, substitutions of amino acid residues at 87 or 142 in EBOV and MARV GPs converted the binding affinity to hNPC1/TET. CONCLUSIONS: Our data provide structural and energetic insights for understanding potential differences in the GP-NPC1 interaction, which could influence the host tropism of EBOV and MARV.

Preexisting Immunity Does Not Prevent Efficacy of Vesicular Stomatitis Virus-Based Filovirus Vaccines in Nonhuman Primates.

Ebola virus (EBOV) and Marburg virus (MARV) made headlines in the past decade, causing outbreaks of human disease in previously nonendemic yet overlapping areas. While EBOV outbreaks can be mitigated with licensed vaccines and treatments, there is not yet a licensed countermeasure for MARV. Here, we used nonhuman primates (NHPs) previously vaccinated with vesicular stomatitis virus (VSV)-MARV and protected against lethal MARV challenge. After a resting period of 9 months, these NHPs were revaccinated with VSV-EBOV and challenged with EBOV, resulting in 75% survival. Surviving NHPs developed EBOV glycoprotein (GP)-specific antibody titers and no viremia or clinical signs of disease. The single vaccinated NHP succumbing to challenge showed the lowest EBOV GP-specific antibody response after challenge, supporting previous findings with VSV-EBOV that antigen-specific antibodies are critical in mediating protection. This study again demonstrates that VSVΔG-based filovirus vaccine can be successfully used in individuals with preexisting VSV vector immunity, highlighting the platform's applicability for consecutive outbreak response.