Ebola and Marburg virus-like particles activate human myeloid dendritic cells

The filoviruses, Ebola (EBOV) and Marburg (MARV), are potential global health threats, which cause deadly hemorrhagic fevers. Although both EBOV and MARV logarithmically replicate in dendritic cells (DCs), these viruses do not elicit DC cytokine secretion and fail to activate and mature infected DCs. Here, we employed virus-like particles (VLPs) of EBOV and MARV to investigate whether these genome-free particles maintain similar immune evasive properties as authentic filoviruses. Confocal microscopy indicated that human myeloid-derived DCs readily took up VLPs. However, unlike EBOV and MARV, VLPs induced maturation of DCs including upregulation of costimulatory molecules (CD40, CD80, CD86), major histocompatibility complex (MHC) class I and II surface antigens, and the late DC maturation marker CD83. The chemokine receptors CCR5 and CCR7 were also modulated on VLP-stimulated DCs, indicating that DC could migrate following VLP exposure. Furthermore, VLPs also elicited DC secretion of the pro-inflammatory cytokines TNF-alpha, IL-8, IL-6, and MIP-1alpha. Most significantly, in stark contrast to DC treated with intact EBOV or MARV, DC stimulated with EBOV or MARV VLPs showed enhanced ability to support human T-cell proliferation in an allogenic mixed lymphocyte response (MLR). Thus, our findings suggest that unlike EBOV and MARV, VLPs are effective stimulators of DCs and have potential in enhancing innate and adaptive immune responses.     

Development of vaccines for Marburg hemorrhagic fever

Marburg (MARV) and Ebola viruses (EBOV) emerged from the rainforests of Central Africa more than 30 years ago causing outbreaks of severe and, usually, fatal hemorrhagic fever. EBOV has garnered the lion's share of the attention, fueled by the higher frequency of EBOV outbreaks, high mortality rates and importation into the USA, documented in such popular works as the best-selling novel 'The Hot Zone'. However, recent large outbreaks of hundreds of cases of MARV infection in the Democratic Republic of the Congo and Angola with case fatalities approaching 90% dramatically highlight its lethal potential. Although no vaccines or antiviral drugs for MARV are currently available, remarkable progress has been made over the last few years in developing potential countermeasures against MARV in nonhuman primate models. In particular, a vaccine based on attenuated recombinant vesicular stomatitis virus was recently shown to have both preventive and postexposure efficacy.     

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses.     

Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2) recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete protection was achieved when the proteins were given alone or in combination with DNA vaccines. These data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea pigs.     

Rapid detection protocol for filoviruses.

BACKGROUND: The incidence of filovirus disease outbreaks has been increasing in recent years. Although there have been advances in the developments of diagnostics, field tests are rare. Apart from family members of infected patients, health care workers are at high risk of being infected during the initial phase of an outbreak. RT-PCR has been shown to be helpful in containing outbreaks. OBJECTIVES: To develop Taqman-RT-PCR for the detection of Ebola-Zaire virus (EBOV-Z), Ebola-Sudan virus (EBOV-S) and Marburg virus (MBGV). STUDY DESIGN: Quantitative Taqman-RT-PCRs for the detection of these viruses were developed and established on a portable Smartcycler TD. RESULTS AND CONCLUSIONS: All three assays were highly sensitive and specific. The mobility of the assay system may help to contain future outbreaks.     

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.     

Filovirus budding

Family Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), is a growing threat to human and non-human primate populations in central Africa. Although many facets of the filovirus life cycle remain to be deciphered, a great deal has been learned in recent years. In particular, a clearer understanding of the roles played by viral, as well as cellular, proteins in the assembly and budding processes has been achieved. This review will discuss the current state of filovirus budding research, with especial emphasis placed on the viral matrix protein VP40 and its relationship with the cellular vesicular sorting pathway. Possible budding functions of the viral glycoprotein (GP), as well as the membrane-associated viral protein 24 (VP24), will also be described, and a model for filovirus budding will be proposed.     

Prospects for immunisation against Marburg and Ebola viruses

For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource‐poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single‐injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication‐defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV‐based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV‐based Ebola vaccine was recently used to manage a potential laboratory exposure. Copyright © 2010 John Wiley & Sons, Ltd.     

Evidence for occurrence of filovirus antibodies in humans and imported monkeys: do subclinical filovirus infections occur worldwide?

In the present serologocal study 120 monkey sera from different species originating from the Philippines, China, Uganda and undetermined sources and several groups of human sera comprising a total of 1288 specimens from people living in Germany were examined for the presence of antibodies directed against filoviruses (Marburg virus, strain Musoke/Ebola virus, subtype Zaire, strain Mayinga/Reston virus). Sera were screened using a filovirus-specific enzyme-linked immunosorbent assay (ELISA). ELISA-positive sera were then confirmed by the indirect immunofluorescence technique, Western blot technique, and a blocking assay, and declared positive when at least one cornfirmation test was reactive. Altogether 43.3% of the monkey sera and 6.9% of the human sera reacted positively with at least one of the three different filovirus antigens. The blocking assays show that antibodies, detected in the sera, are directed to specific filovirus antigens and not caused by antigenic cross-reactivity with hitherto unknown agents. Data presented in this report suggest that subclinical filovirus infections may also occur in humans and in subhuman primates. They further suggest that filoviruses are not restricted to the African continent.     

Development,characterization and use of monoclonal VP40-antibodies for the detection of Ebola virus

Ebola virus (EBOV) causes uncommon but dramatic outbreaks in remote regions of Africa, where diagnostic facilities are limited. In order to develop diagnostic tests, which can be handled and distributed easily, monoclonal antibodies (mAbs) to EBOV, species Zaire, were produced from mice immunized with inactivated viral particles. Nine stable hybridoma cell lines were obtained producing specific mAbs directed against the viral structural protein VP40. These mAbs were characterized by enzyme-linked immunosorbent, immunoblot and immunofluorescence assays. Subsequently, an antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections.     

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation.     

Production of monoclonal antibodies and development of an antigen capture ELISA directed against the envelope glycoprotein GP of Ebola virus

Ebola virus (EBOV) causes severe outbreaks of Ebola hemorrhagic fever in endemic regions of Africa and is considered to be of impact for other parts of the world as an imported viral disease. To develop a new diagnostic test, monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP were characterized by ELISA, Western blot and immunofluorescence analyses. An antigen capture ELISA was established, which is specific for EBOV-Zaire and shows a sensitivity of approximately 10³ plaque-forming units/ml. Since the ELISA is able to detect even SDS-inactivated EBOV in spiked human sera, it could complement the existing diagnostic tools in the field and in routine laboratories where high containment facilities are not available.     

Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges

The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10(7) pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.     

Modulation of virion incorporation of Ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization

The filoviruses Ebolavirus (EBOV) and Marburgvirus (MARV) cause severe hemorrhagic fever in humans and are potential agents of biological warfare. The envelope glycoprotein (GP) of filoviruses mediates viral entry into cells and is an attractive target for therapeutic intervention and vaccine design. Here, we asked if the efficiency of virion incorporation of EBOV-GP impacts attachment and entry into target cells and modulates susceptibility to neutralizing antibodies. In order to control the level of EBOV-GP expression, we generated cell lines expressing the GPs of the four known EBOV subspecies in an inducible fashion. Regulated expression of GP on the cell surface allowed production of reporter viruses harboring different amounts of GP. A pronounced reduction of virion incorporation of EBOV-GP had relatively little effect on virion infectivity, suggesting that only a few copies of GP might be sufficient for efficient engagement of cellular receptors. In contrast, optimal interactions with cellular attachment factors like the DC-SIGN protein required incorporation of high amounts of GP. Antibody-mediated neutralization of virions bearing high amounts of GP was slightly more efficient than neutralization of virions harboring low amounts of GP, suggesting that the efficiency of GP incorporation into virions might modulate susceptibility to neutralizing antibodies. Finally, regulated expression of GP in permissive 293 cells did not reduce EBOV-GP-driven infection but diminished vesicular stomatitis virus GP (VSV-G) and amphotropic murine leukemia virus (A-MLV) GP mediated entry in a dose-dependent manner. Therefore, intracellular GP does not seem to downmodulate expression of its receptor(s) but might alter expression and/or function of molecules involved in VSV-G and A-MLV-GP-dependent entry. Our results suggest that the efficiency of virion incorporation of GP could impact EBOV attachment to target cells and might modulate control of viral spread by the humoral immune response.     

Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1

Since 1998, multiple strains of bluetongue virus (BTV), belonging to six different serotypes (types 1, 2, 4, 8, 9 and 16) have caused outbreaks of disease in Europe, causing one of the largest epizootics of bluetongue ever recorded, with the deaths of >1.8 million animals (mainly sheep). The persistence and continuing spread of BTV in Europe and elsewhere highlights the importance of sensitive and reliable diagnostic assay systems that can be used to rapidly identify infected animals, helping to combat spread of the virus and disease. BTV has a genome composed of 10 linear segments of dsRNA. We describe a real-time RT-PCR assay that targets the highly conserved genome segment 1 (encoding the viral polymerase--VP1) that can be used to detect all of the 24 serotypes, as well as geographic variants (different topotypes) within individual serotypes of BTV. After an initial evaluation using 132 BTV samples including representatives of all 24 BTV serotypes, this assay was used by the European Community Reference Laboratory (CRL) at IAH Pirbright to confirm the negative status of 2,255 animals imported to the UK from regions that were considered to be at risk during the 2006 outbreak of BTV-8 in Northern Europe. All of these animals were also negative by competition ELISA to detect BTV specific antibodies and none of them developed clinical signs of infection. These studies have demonstrated the value of the assay for the rapid screening of field samples.     

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe hemorrhagic fever disease in humans, with case-fatality rates that range up to 90%. Among the Ebolavirus genus, there are four distinct species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV), and Cote d''Ivoire ebolavirus (CIEBOV) (10), with a possible fifth species identified in a recent outbreak in the Bundibugyo region of Western Uganda (34). Of these, ZEBOV and SEBOV are known to cause lethal disease in humans. The persistence of these viruses in nature is not well understood. Sporadic outbreaks due to EBOV have been occurring in Central Africa since the 1970s, but since the mid-1990s, the incidence of outbreaks has increased more than 4-fold (6, 7, 8, 42-45), and EBOV has spread aggressively throughout the great ape sanctuaries of West and Central Africa, decimating wild populations of gorillas and chimpanzees (2). While the filoviruses infect both humans and great apes, due to the high mortality rates of the infection, neither is thought to serve as reservoirs for these viruses but only as accidental hosts (16). Recent findings suggest that African fruit bats may serve as a reservoir host for filoviruses (2, 22); however, little is known about the nature of transmission to humans and nonhuman primates from bats or the likelihood of other reservoir species. In outbreak situations, filoviruses are believed to transmit from person to person mainly through contact with bodily fluids from infected patients. However, recent studies of Ebola outbreaks in wild apes have suggested that there could be other modes of transmission, including aerosol (2, 36). Studies in nonhuman primates have shown that EBOV and MARV can be spread through aerosolized droplets under controlled laboratory conditions (18, 21). So, despite the low incidence of infections globally, the lethality and potential airborne transmission of filoviruses in heavily populated areas makes them a significant biological threat, resulting in their placement on the Centers for Disease Control and Prevention list of Category A Bioterrorism Agents and the Department of Health and Human Services (DHHS) list of select agents and toxins. Concern is further compounded by the potential for these agents to be obtained from the wild (2, 22). In a biological threat scenario, aerosol transmission will likely be the main mode of viral dissemination, and protection against aerosolized EBOV would be of utmost importance (3). However, most previous vaccine candidates have only been evaluated for efficacy against intramuscular or intraperitoneal challenge and not against an aerosol challenge in nonhuman primates.At present, there are no licensed vaccines or specific antiviral treatments available for EBOV or MARV infections. However, significant progress has been made over the past few years in developing vaccine candidates that can protect nonhuman primates (NHPs) from lethal EBOV and MARV challenges (11, 17, 20, 23, 32, 40, 41). Most of the candidates utilize recombinant vaccine approaches that direct the protective immune response toward the surface glycoprotein (GP) of a single species of EBOV. Importantly, each species of EBOV is antigenically distinct, based on the sequences of the viral GP (10), and therefore, vaccines targeted against the GP of one species of the virus will not provide cross-protection against infection by another (19). Unique among the vaccine candidates is the recombinant complex adenovirus vaccine (CAdVax) system, which provides multivalent protection of NHPs against multiple species of filoviruses (33). The CAdVax vaccine platform is based on a complex, replication-defective adenovirus 5 (Ad5) vector (28-30, 37, 38) that allows for the incorporation of multiple gene inserts into the vector''s genome. Using this design, a bivalent vaccine vector (EBO7) was developed that expresses modified GP gene sequences of SEBOV and ZEBOV. When included in a novel pan-filovirus vaccine formulation, this vaccine was 100% protective in NHPs against two species of EBOV (ZEBOV and SEBOV) and two different strains of MARV (Musoke and Ci67) (33).In the study presented here, we further tested the protective efficacy of the CAdVax-based EBO7 vaccine in macaques by comparing aerosol to parenteral challenge. Aerosol challenge is potentially even more lethal than parenteral infection, because it induces hemorrhagic pneumonia. This is particularly true of SEBOV aerosol challenge, against which protection has not previously been demonstrated. In our studies, we have found that for either route of infection, the vaccine-induced bivalent anti-EBOV responses were protective against lethal challenge with either SEBOV or ZEBOV. This is the first report of a vaccine that is capable of protecting against aerosol SEBOV challenge. In addition, we found that EBO7 was also capable of protecting macaques with preexisting immunity to adenovirus against ZEBOV challenge. These results provide further insight into the feasibility of developing a fully protective multivalent EBOV vaccine using the CAdVax vaccine platform.