Filovirus VP24 Proteins Differentially Regulate RIG-I and MDA5-Dependent Type I and III Interferon Promoter Activation.

Filovirus family consists of highly pathogenic viruses that have caused fatal outbreaks especially in many African countries. Previously, research focus has been on Ebola, Sudan and Marburg viruses leaving other filoviruses less well studied. Filoviruses, in general, pose a significant global threat since they are highly virulent and potentially transmissible between humans causing sporadic infections and local or widespread epidemics. Filoviruses have the ability to downregulate innate immunity, and especially viral protein 24 (VP24), VP35 and VP40 have variably been shown to interfere with interferon (IFN) gene expression and signaling. Here we systematically analyzed the ability of VP24 proteins of nine filovirus family members to interfere with retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA5) induced IFN-ß and IFN-λ1 promoter activation. All VP24 proteins were localized both in the cell cytoplasm and nucleus in variable amounts. VP24 proteins of Zaire and Sudan ebolaviruses, Lloviu, Taï Forest, Reston, Marburg and Bundibugyo viruses (EBOV, SUDV, LLOV, TAFV, RESTV, MARV and BDBV, respectively) were found to inhibit both RIG-I and MDA5 stimulated IFN-ß and IFN-λ1 promoter activation. The inhibition takes place downstream of interferon regulatory factor 3 phosphorylation suggesting the inhibition to occur in the nucleus. VP24 proteins of Mengla (MLAV) or Bombali viruses (BOMV) did not inhibit IFN-ß or IFN-λ1 promoter activation. Six ebolavirus VP24s and Lloviu VP24 bound tightly, whereas MARV and MLAV VP24s bound weakly, to importin α5, the subtype that regulates the nuclear import of STAT complexes. MARV and MLAV VP24 binding to importin α5 was very weak. Our data provides new information on the innate immune inhibitory mechanisms of filovirus VP24 proteins, which may contribute to the pathogenesis of filovirus infections.

Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Menglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus.

We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb-APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb-APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Menglà virus genome. Antibody-antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Menglà NP C-terminal domain. The sdAb-APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb-APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.

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.

Complete protection of the BALB/c and C57BL/6J mice against Ebola and Marburg virus lethal challenges by pan-filovirus T-cell epigraph vaccine.

There are a number of vaccine candidates under development against a small number of the most common outbreak filoviruses all employing the virus glycoprotein (GP) as the vaccine immunogen. However, antibodies induced by such GP vaccines are typically autologous and limited to the other members of the same species. In contrast, T-cell vaccines offer a possibility to design a single pan-filovirus vaccine protecting against all known and even likely existing, but as yet unencountered members of the family. Here, we used a cross-filovirus immunogen based on conserved regions of the filovirus nucleoprotein, matrix and polymerase to construct simian adenovirus- and poxvirus MVA-vectored vaccines, and in a proof-of-concept study demonstrated a protection of the BALB/c and C57BL/6J mice against high, lethal challenges with Ebola and Marburg viruses, two distant members of the family, by vaccine-elicited T cells in the absence of GP antibodies.

Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies.

UNLABELLED: Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection. IMPORTANCE: The study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.

Ad35 and ad26 vaccine vectors induce potent and cross-reactive antibody and T-cell responses to multiple filovirus species.

Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26-Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.

Filovirus vaccines.

Filoviruses can cause severe and often fatal hemorrhagic fever in humans and non-human primates (NHPs). Although there are currently no clinically proven treatments for filovirus disease, much progress has been made in recent years in the discovery of therapeutics and vaccines against these viruses. A variety of vaccine platforms have been shown to be effective against filovirus infection. This review summarizes the literature in this field, focusing on vaccines that have been shown to protect NHPs from infection. Furthermore, the uses of rodent models in vaccine development, as well as correlates of immunity, are discussed.

Filovirus emergence and vaccine development: a perspective for health care practitioners in travel medicine.

Recent case reports of viral hemorrhagic fever in Europe and the United States have raised concerns about the possibility for increased importation of filoviruses to non-endemic areas. This emerging threat is concerning because of the increase in global air travel and the rise of tourism in central and eastern Africa and the greater dispersion of military troops to areas of infectious disease outbreaks. Marburg viruses (MARV) and Ebola viruses (EBOV) have been associated with outbreaks of severe hemorrhagic fever involving high mortality (25-90% case fatality rates). First recognized in 1967 and 1976 respectively, subtypes of MARV and EBOV are the only known viruses of the Filoviridae family, and are among the world's most virulent pathogens. This article focuses on information relevant for health care practitioners in travel medicine to include, the epidemiology and clinical features of filovirus infection and efforts toward development of a filovirus vaccine.

Vaccine to confer to nonhuman primates complete protection against multistrain Ebola and Marburg virus infections.

Filoviruses (Ebola and Marburg viruses) are among the deadliest viruses known to mankind, with mortality rates nearing 90%. These pathogens are highly infectious through contact with infected body fluids and can be easily aerosolized. Additionally, there are currently no licensed vaccines available to prevent filovirus outbreaks. Their high mortality rates and infectious capabilities when aerosolized and the lack of licensed vaccines available to prevent such infectious make Ebola and Marburg viruses serious bioterrorism threats, placing them both on the category A list of bioterrorism agents. Here we describe a panfilovirus vaccine based on a complex adenovirus (CAdVax) technology that expresses multiple antigens from five different filoviruses de novo. Vaccination of nonhuman primates demonstrated 100% protection against infection by two species of Ebola virus and three Marburg virus subtypes, each administered at 1,000 times the lethal dose. This study indicates the feasibility of vaccination against all current filovirus threats in the event of natural hemorrhagic fever outbreak or biological attack.

Vaccine research efforts for filoviruses.

Ebola and Marburg viruses belong to the family Filoviridae, and cause acute, frequently fatal, haemorrhagic fever in humans and non-human primates. No vaccines are available for human use. This review describes the status of research efforts to develop vaccines for these viruses and to identify the immune mechanisms of protection. The vaccine approaches discussed include DNA-based vaccines, and subunit vaccines vectored by adenovirus, alphavirus replicons, and vaccinia virus.

The role of the Type I interferon response in the resistance of mice to filovirus infection.

Adult immunocompetent mice inoculated with Ebola (EBO) or Marburg (MBG) virus do not become ill. A suckling-mouse-passaged variant of EBO Zaire '76 ('mouse-adapted EBO-Z') causes rapidly lethal infection in adult mice after intraperitoneal (i.p.) inoculation, but does not cause apparent disease when inoculated subcutaneously (s.c.). A series of experiments showed that both forms of resistance to infection are mediated by the Type I interferon response. Mice lacking the cell-surface IFN-alpha/beta receptor died within a week after inoculation of EBO-Z '76, EBO Sudan, MBG Musoke or MBG Ravn, or after s.c. challenge with mouse-adapted EBO-Z. EBO Reston and EBO Ivory Coast did not cause illness, but immunized the mice against subsequent challenge with mouse-adapted EBO-Z. Normal adult mice treated with antibodies against murine IFN-alpha/beta could also be lethally infected with i.p.-inoculated EBO-Z '76 or EBO Sudan and with s.c.-inoculated mouse-adapted EBO-Z. Severe combined immunodeficient (SCID) mice became ill 3-4 weeks after inoculation with EBO-Z '76, EBO Sudan or MBG Ravn, but not the other viruses. Treatment with anti-IFN-alpha/beta antibodies markedly accelerated the course of EBO-Z '76 infection. Antibody treatment blocked the effect of a potent antiviral drug, 3-deazaneplanocin A, indicating that successful filovirus therapy may require the active participation of the Type I IFN response. Mice lacking an IFN-alpha/beta response resemble primates in their susceptibility to rapidly progressive, overwhelming filovirus infection. The outcome of filovirus transfer between animal species appears to be determined by interactions between the virus and the innate immune response.

Retrovirus and filovirus "immunosuppressive motif" and the evolution of virus pathogenicity in HIV-1, HIV-2, and Ebola viruses.

The "immunosuppressive motif" was found to be present in the glycoproteins of retroviruses and filoviruses. This sequence is also conserved in the pathogenic lentiviruses, HIV-1 and SIV, and is absent from HIV-2 gp41 and from an apathogenic simian retrovirus. The present analysis deals with the possible involvement of the "immunosuppresessive motif" in the pathogenicity of retroviruses and filoviruses, and the reasons for the conservation of this motif. The ancestral gene from which the "immunosuppressive motif" originated is not known.

Improved specificity of testing methods for filovirus antibodies.

An epizootic among monkeys imported into the United States created an immediate need for detection of antibodies to filoviruses. Thousands of samples were submitted to the Centers for Disease Control and Prevention for testing. Problems of sensitivity and specificity existed in the methods available for these assays. The experiments described in this report resulted in improved methods for the detection of antibodies to filoviruses, both for indirect fluorescent antibody assays (IFA) by standardizing methods and the Western blot (WB) by minimizing antigen load and by incorporating skim milk in diluents.