Design and characterization of protective pan-ebolavirus and pan-filovirus bispecific antibodies.

Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.

Serological Evidence of Filovirus Infection in Nonhuman Primates in Zambia.

Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.

Filovirus-reactive antibodies in humans and bats in Northeast India imply zoonotic spillover.

Bats are reservoirs for several zoonotic pathogens, including filoviruses. Recent work highlights the diversity of bat borne filoviruses in Asia. High risk activities at the bat-human interface pose the threat of zoonotic virus transmission. We present evidence for prior exposure of bat harvesters and two resident fruit bat species to filovirus surface glycoproteins by screening sera in a multiplexed serological assay. Antibodies reactive to two antigenically distinct filoviruses were detected in human sera and to three individual filoviruses in bats in remote Northeast India. Sera obtained from Eonycteris spelaea bats showed similar patterns of cross-reactivity as human samples, suggesting them as the species responsible for the spillover. In contrast, sera from Rousettus leschenaultii bats reacted to two different virus glycoproteins. Our results indicate circulation of several filoviruses in bats and the possibility for filovirus transmission from bats to humans.

Cytokine Effects on the Entry of Filovirus Envelope Pseudotyped Virus-Like Particles into Primary Human Macrophages.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

Comparative analysis of serologic cross-reactivity using convalescent sera from filovirus-experimentally infected fruit bats.

With the exception of Reston and Bombali viruses, the marburgviruses and ebolaviruses (family Filoviridae) cause outbreaks of viral hemorrhagic fever in sub-Saharan Africa. The Egyptian rousette bat (ERB) is a natural reservoir host for the marburgviruses and evidence suggests that bats are also natural reservoirs for the ebolaviruses. Although the search for the natural reservoirs of the ebolaviruses has largely involved serosurveillance of the bat population, there are no validated serological assays to screen bat sera for ebolavirus-specific IgG antibodies. Here, we generate filovirus-specific antisera by prime-boost immunization of groups of captive ERBs with all seven known culturable filoviruses. After validating a system of filovirus-specific indirect ELISAs utilizing infectious-based virus antigens for detection of virus-specific IgG antibodies from bat sera, we assess the level of serological cross-reactivity between the virus-specific antisera and heterologous filovirus antigens. This data is then used to generate a filovirus antibody fingerprint that can predict which of the filovirus species in the system is most antigenically similar to the species responsible for past infection. Our filovirus IgG indirect ELISA system will be a critical tool for identifying bat species with high ebolavirus seroprevalence rates to target for longitudinal studies aimed at establishing natural reservoir host-ebolavirus relationships.

First Evidence of Antibodies Against Lloviu Virus in Schreiber's Bent-Winged Insectivorous Bats Demonstrate a Wide Circulation of the Virus in Spain.

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber's Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber's Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber's Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber's Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

Generation and Characterization of Anti-Filovirus Nucleoprotein Monoclonal Antibodies.

Filoviruses cause lethal hemorrhagic fever in humans. The filovirus nucleoprotein (NP) is expressed in high abundance in infected cells and is essential for virus replication. To generate anti-filovirus monoclonal antibodies (mAbs) against the NP, mice were immunized with peptides known as B-cell epitopes corresponding to different filovirus NPs, and hybridomas were screened using FLAG-tagged filovirus NP constructs. Numerous mAbs were identified, isotyped, and characterized. The anti-NP mAbs demonstrated different ranges of binding affinities to various filovirus NPs. Most of the clones specifically detected both recombinant and wild-type NPs from different filoviruses, including Ebola (EBOV), Sudan (SUDV), Bundibugyo (BDBV), Marburg (MARV), Tai Forest (TAFV), and Reston (RESTV) viruses in western blot analysis. The mAbs were also able to detect native NPs within the cytoplasm of infected cells by immunofluorescence confocal microscopy. Thus, this panel of mAbs represents an important set of tools that may be potentially useful for diagnosing filovirus infection, characterizing virus replication, and detecting NP⁻host protein interactions.

Post-exposure immunotherapy for two ebolaviruses and Marburg virus in nonhuman primates.

The 2013-2016 Ebola virus (EBOV) disease epidemic demonstrated the grave consequences of filovirus epidemics in the absence of effective therapeutics. Besides EBOV, two additional ebolaviruses, Sudan (SUDV) and Bundibugyo (BDBV) viruses, as well as multiple variants of Marburg virus (MARV), have also caused high fatality epidemics. Current experimental EBOV monoclonal antibodies (mAbs) are ineffective against SUDV, BDBV, or MARV. Here, we report that a cocktail of two broadly neutralizing ebolavirus mAbs, FVM04 and CA45, protects nonhuman primates (NHPs) against EBOV and SUDV infection when delivered four days post infection. This cocktail when supplemented by the anti-MARV mAb MR191 exhibited 100% efficacy in MARV-infected NHPs. These findings provide a solid foundation for clinical development of broadly protective immunotherapeutics for use in future filovirus epidemics.

A Two-Antibody Pan-Ebolavirus Cocktail Confers Broad Therapeutic Protection in Ferrets and Nonhuman Primates.

Recent and ongoing outbreaks of Ebola virus disease (EVD) underscore the unpredictable nature of ebolavirus reemergence and the urgent need for antiviral treatments. Unfortunately, available experimental vaccines and immunotherapeutics are specific for a single member of the Ebolavirus genus, Ebola virus (EBOV), and ineffective against other ebolaviruses associated with EVD, including Sudan virus (SUDV) and Bundibugyo virus (BDBV). Here we show that MBP134AF, a pan-ebolavirus therapeutic comprising two broadly neutralizing human antibodies (bNAbs), affords unprecedented effectiveness and potency as a therapeutic countermeasure to antigenically diverse ebolaviruses. MBP134AF could fully protect ferrets against lethal EBOV, SUDV, and BDBV infection, and a single 25-mg/kg dose was sufficient to protect NHPs against all three viruses. The development of MBP134AF provides a successful model for the rapid discovery and translational advancement of immunotherapeutics targeting emerging infectious diseases.

[Innate immunity evasion mechanisms of filoviruses]. / Mécanismes d'échappement des filovirus à l'immunité innée.

Ebola virus is an important pathogen that emerged in Central Africa where it was responsible of numerous outbreaks of haemorrhagic fevers associated with a extremely high mortality rate (up to 90%). The filovirus pathogenicity is related to an inappropriate antiviral response. Indeed, this family of viruses has developed evasion strategies from early innate immunity mechanisms. As a result, a massive viral replication induces an unsuitable immune response causing an acute inflammatory reaction associated with the haemorrhagic syndrome. In this review, we describe the mechanisms adopted by filoviruses like Ebola virus to escape innate immunity response.

Suspected Exposure to Filoviruses Among People Contacting Wildlife in Southwestern Uganda.

Background: Human and filovirus host interactions remain poorly understood in areas where Ebola hemorrhagic fever outbreaks are likely to occur. In the Bwindi region of Uganda, a hot spot of mammalian biodiversity in Africa, human livelihoods are intimately connected with wildlife, creating potential for exposure to filoviruses. Methods: We tested samples from 331 febrile patients presenting to healthcare facilities near Bwindi Impenetrable Forest, Uganda, by polymerase chain reaction (PCR) analysis and Western blot, using recombinant glycoprotein antigens for Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus. Behavioral data on contact with wildlife were collected to examine risk factors for filovirus seropositivity. Results: All patients were negative for active filovirus infection, by PCR analysis. However, patients were seroreactive to SUDV (4.7%), EBOV (5.3%), and BDBV (8.9%), indicating previous exposure. Touching duikers was the most significant risk factor associated with EBOV seropositivity, while hunting primates and touching and/or eating cane rats were significant risk factors for SUDV seropositivity. Conclusions: People in southwestern Uganda have suspected previous exposure to filoviruses, particularly those with a history of wildlife contact. Circulation of filoviruses in wild animals and subsequent spillover into humans could be more common than previously reported.

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats in Zambia.

Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.

The structural basis for filovirus neutralization by monoclonal antibodies.

Filoviruses, including ebolaviruses and marburgviruses, are the causative agents of highly lethal disease outbreaks. The 2013-2016 Ebola virus outbreak was responsible for >28000 infections and >11000 deaths. Although there are currently no licensed vaccines or therapeutics for any filovirus-induced disease, monoclonal antibodies (mAbs) are among the most promising options for therapeutic development. Hundreds of mAbs have been isolated from human survivors of filovirus infections that target the viral spike glycoprotein (GP). The binding, neutralization, and cross-reactivity of many of these mAbs has been determined. Several mAbs have been characterized structurally, and this information has been crucial for strategizing therapeutic and vaccine design. Here we present an overview of the structural features of the neutralizing/protective epitopes on filovirus glycoproteins.

Epigraph: A Vaccine Design Tool Applied to an HIV Therapeutic Vaccine and a Pan-Filovirus Vaccine.

Epigraph is an efficient graph-based algorithm for designing vaccine antigens to optimize potential T-cell epitope (PTE) coverage. Epigraph vaccine antigens are functionally similar to Mosaic vaccines, which have demonstrated effectiveness in preliminary HIV non-human primate studies. In contrast to the Mosaic algorithm, Epigraph is substantially faster, and in restricted cases, provides a mathematically optimal solution. Epigraph furthermore has new features that enable enhanced vaccine design flexibility. These features include the ability to exclude rare epitopes from a design, to optimize population coverage based on inexact epitope matches, and to apply the code to both aligned and unaligned input sequences. Epigraph was developed to provide practical design solutions for two outstanding vaccine problems. The first of these is a personalized approach to a therapeutic T-cell HIV vaccine that would provide antigens with an excellent match to an individual's infecting strain, intended to contain or clear a chronic infection. The second is a pan-filovirus vaccine, with the potential to protect against all known viruses in the Filoviradae family, including ebolaviruses. A web-based interface to run the Epigraph tool suite is available (http://www.hiv.lanl.gov/content/sequence/EPIGRAPH/epigraph.html).

Integrated sequence and immunology filovirus database at Los Alamos.

The Ebola outbreak of 2013-15 infected more than 28 000 people and claimed more lives than all previous filovirus outbreaks combined. Governmental agencies, clinical teams, and the world scientific community pulled together in a multifaceted response ranging from prevention and disease control, to evaluating vaccines and therapeutics in human trials. As this epidemic is finally coming to a close, refocusing on long-term prevention strategies becomes paramount. Given the very real threat of future filovirus outbreaks, and the inherent uncertainty of the next outbreak virus and geographic location, it is prudent to consider the extent and implications of known natural diversity in advancing vaccines and therapeutic approaches. To facilitate such consideration, we have updated and enhanced the content of the filovirus portion of Los Alamos Hemorrhagic Fever Viruses Database. We have integrated and performed baseline analysis of all family ITALIC! Filoviridaesequences deposited into GenBank, with associated immune response data, and metadata, and we have added new computational tools with web-interfaces to assist users with analysis. Here, we (i) describe the main features of updated database, (ii) provide integrated views and some basic analyses summarizing evolutionary patterns as they relate to geo-temporal data captured in the database and (iii) highlight the most conserved regions in the proteome that may be useful for a T cell vaccine strategy.Database URL:www.hfv.lanl.gov.

The Role of Cytokines and Chemokines in Filovirus Infection.

Ebola- and marburgviruses are highly pathogenic filoviruses and causative agents of viral hemorrhagic fever. Filovirus disease is characterized by a dysregulated immune response, severe organ damage, and coagulation abnormalities. This includes modulation of cytokines, signaling mediators that regulate various components of the immune system as well as other biological processes. Here we examine the role of cytokines in filovirus infection, with an emphasis on understanding how these molecules affect development of the antiviral immune response and influence pathology. These proteins may present targets for immune modulation by therapeutic agents and vaccines in an effort to boost the natural immune response to infection and/or reduce immunopathology.

Filoviruses: One of These Things is (not) Like the Other.

The family Filoviridae contains several of the most deadly pathogens known to date and the current Ebola virus disease (EVD) outbreak in Western Africa, due to Ebola virus (EBOV) infection, highlights the need for active and broad research into filovirus pathogenesis. However, in comparison, the seven other known filovirus family members are significantly understudied. Many of these, including Marburgviruses and Ebolaviruses other than EBOV, are also highly virulent and fully capable of causing widespread epidemics. This review places the focus on these non-EBOV filoviruses, including known immunological and pathological data. The available animal models, research tools and currently available therapeutics will also be discussed along with an emphasis in the large number of current gaps in knowledge of these less highlighted filoviruses. It is evident that much research is yet to be done in order to bring the non-EBOV filovirus field to the forefront of current research and, importantly, to the development of more effective vaccines and therapeutics to combat potential future outbreaks.

Stat1-Deficient Mice Are Not an Appropriate Model for Efficacy Testing of Recombinant Vesicular Stomatitis Virus-Based Filovirus Vaccines.

Stat1(-/-) mice lack a response to interferon α, ß, and γ, allowing for replication of nonadapted wild-type (wt) Ebolavirus and Marburgvirus. We sought to establish a mouse model for efficacy testing of live attenuated recombinant vesicular stomatitis virus (rVSV)-based filovirus vaccine vectors using wt Ebolavirus and Marburgvirus challenge strains. While infection of immunocompetent mice with different rVSV-based filovirus vectors did not cause disease, infection of Stat1(-/-) mice with the same vectors resulted in systemic infection and lethal outcome for the majority of tested rVSVs. Despite differences in viral loads, organ tropism was remarkably similar between rVSV filovirus vaccine vectors and rVSVwt, with the exception of the brain. In conclusion, Stat1(-/-) mice are not an appropriate immunocompromised mouse model for efficacy testing of live attenuated, replication-competent rVSV vaccine vectors.

A Single-Vector, Single-Injection Trivalent Filovirus Vaccine: Proof of Concept Study in Outbred Guinea Pigs.

The filoviruses, Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SEBOV), cause severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Monovalent recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode a filovirus glycoprotein (GP) in place of the VSV glycoprotein, have shown 100% efficacy against homologous filovirus challenge in rodent and NHP studies. Here, we examined the utility of a single-vector, single-injection trivalent rVSV vector expressing MARV, ZEBOV, and SEBOV GPs to protect against MARV-, ZEBOV-, and SEBOV-induced disease in outbred Hartley guinea pigs where we observed protection from effects of all 3 filoviruses.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa.

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.