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.     

Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function

The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP's overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion.     

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.     

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.     

Ebola virus uses clathrin-mediated endocytosis as an entry pathway

Ebola virus (EBOV) infects several cell types and while viral entry is known to be pH-dependent, the exact entry pathway(s) remains unknown. To gain insights into EBOV entry, the role of several inhibitors of clathrin-mediated endocytosis in blocking infection mediated by HIV pseudotyped with the EBOV envelope glycoprotein (EbGP) was examined. Wild type HIV and envelope-minus HIV pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSVg) were used as controls to assess cell viability after inhibiting clathrin pathway. Inhibition of clathrin pathway using dominant-negative Eps15, siRNA-mediated knockdown of clathrin heavy chain, chlorpromazine and sucrose blocked EbGP pseudotyped HIV infection. Also, both chlorpromazine and Bafilomycin A1 inhibited entry of infectious EBOV. Sensitivity of EbGP pseudotyped HIV as well as infectious EBOV to inhibitors of clathrin suggests that EBOV uses clathrin-mediated endocytosis as an entry pathway. Furthermore, since chlorpromazine inhibits EBOV infection, novel therapeutic modalities could be designed based on this lead compound.     

Detection of all known filovirus species by reverse transcription-polymerase chain reaction using a primer set specific for the viral nucleoprotein gene

The filoviruses, Marburg virus (MARV) and Ebola virus (EBOV), are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates. Sporadic outbreaks of filovirus infection have occurred in Central Africa and parts of Asia. Identification of the natural reservoir animals that are unknown yet and epidemiological investigations are current challenges to forestall outbreaks of filovirus diseases. The filovirus species identified currently include one in the MARV group and five in the EBOV group, with large genetic variations found among the species. Therefore, it has been difficult to develop a single sensitive assay to detect all filovirus species, which would advance laboratory diagnosis greatly in endemic areas. In this study, a highly sensitive universal RT-PCR assay targeting the nucleoprotein (NP) gene of filoviruses was developed. The genomic RNAs of all known MARV and EBOV species were detected by using an NP-specific primer set. In addition, this RT-PCR procedure was verified further for its application to detect viral RNAs in tissue samples of animals infected experimentally and blood specimens of infected patients. This assay will be a useful method for diagnostics and epidemiological studies of filovirus infections.     

Permeabilization of the plasma membrane by Ebola virus GP2

The glycoprotein (GP) of Ebola virus (EBOV) is a multifunctional protein known to play a role in virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. EBOV GP is synthesized as a precursor which is subsequently cleaved to yield two disulfide-linked subunits: GP1 (surface-exposed [SU] subunit) and GP2 (membrane-anchored [TM] subunit). We sought to determine the effect of membrane-anchored GP2 protein expression on the integrity of host cell lipid membranes. Our findings indicated that: (i) expression of GP2 enhanced membrane permeability to hygromycin-B (hyg-B), (ii) the transmembrane (TM) domain of GP2 was essential for enhanced membrane permeability, (iii) amino acids (aa) 667ALF669 within the TM region of GP2 were important for enhanced membrane permeability, and (iv) EBOV infected cells were more permeable to hyg-B than mock infected cells. Together, these data suggest that the TM region of GP2 modifies the permeability of the plasma membrane. These findings may have important implications for GP-induced cell damage and pathogenesis of EBOV infection.     

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.     

Cathepsins in the kidney of olive flounder,Paralichthys olivaceus,and their responses to bacterial infection

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.     

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.     

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.     

Schistosoma japonicum,S. mansoni,S. haematobium,S. intercalatum,and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms

 Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN₂ only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species. Received: 1 April 1996 / Accepted: 19 June 1996     

Detection of Ebola virus envelope using monoclonal and polyclonal antibodies in ELISA, surface plasmon resonance and a quartz crystal microbalance immunosensor

Ebola virus (EBOV) Zaire, Sudan, as well as Ivory Coast are virulent human EBOV species. Both polyclonal and monoclonal antibodies (MAbs) were developed against soluble EBOV envelope glycoprotein (GP) for the study of EBOV envelope diversity and development of diagnostic reagents. Three EBOV Sudan-Gulu GP peptides, from the N-terminus, mid-GP, and C-terminus regions were used to immunize rabbits for the generation of anti-EBOV polyclonal antibodies. Polyclonal antisera raised against the C-terminus peptide could detect both Sudan-Gulu as well as Zaire GPs, while anti-N and mid-region peptide polyclonal sera recognized only EBOV Sudan-Gulu GP. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan and Ivory Coast), and as well as reacted with the Reston non-human primate EBOV GPs. In addition, MAb 15H10 bound virion-associated GP of all known EBOV species. MAb 17A3 recognized GPs of both EBOV Sudan-Gulu and Zaire, while MAb 6D11 recognized only EBOV Sudan-Gulu GP. To detect EBOV GP, these antibody reagents were used in ELISA, surface plasmon resonance and in a quartz crystal microbalance immunosensor. Thus, polyclonal and monoclonal antibodies can be used in combination to identify and differentiate both human and non-human primate EBOV GPs.     

Mechanisms of regulation of lysosomal thiol protease activity in thyroid gland: Effects of protein synthesis inhibitors

Triiodothyronine administered for 7 days reduces the contents of total cathepsin B and L (but not cathepsin C) in rat thyroid gland. Cycloheximide reduces the contents of cathepsin B, C, and L, while actinomycin D induces a less pronounced decrease in cathepsin C and L activity, cathepsin B activity being unchanged. Against the background of triiodothyronine, both agents equally inhibit these proteases. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 521–523, May, 1997     

Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.     

The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway

Ebola virus (EBOV) has been reported to enter cultured cell lines via a dynamin-2-independent macropinocytic pathway or clathrin-mediated endocytosis. The route(s) of productive EBOV internalization into physiologically relevant cell types remain unexplored, and viral-host requirements for this process are incompletely understood. Here, we use electron microscopy and complementary chemical and genetic approaches to demonstrate that the viral glycoprotein, GP, induces macropinocytic uptake of viral particles into cells. GP's highly-glycosylated mucin domain is dispensable for virus-induced macropinocytosis, arguing that interactions between other sequences in GP and the host cell surface are responsible. Unexpectedly, we also found a requirement for the large GTPase dynamin-2, which is proposed to be dispensable for several types of macropinocytosis. Our results provide evidence that EBOV uses an atypical dynamin-dependent macropinocytosis-like entry pathway to enter Vero cells, adherent human peripheral blood-derived monocytes, and a mouse dendritic cell line.     

Cathepsin L is required for ecotropic murine leukemia virus infection in NIH3T3 cells

Recently it has been reported that a cathepsin B inhibitor, CA-074Me, attenuates ecotropic murine leukemia virus (Eco-MLV) infection in NIH3T3 cells, suggesting that cathepsin B is required for the Eco-MLV infection. However, cathepsin B activity was negative or extremely low in NIH3T3 cells. How did CA-074Me attenuate the Eco-MLV infection? The CA-074Me treatment of NIH3T3 cells inhibited cathepsin L activity, and a cathepsin L specific inhibitor, CLIK148, attenuated the Eco-MLV vector infection. These results indicate that the suppression of cathepsin L activity by CA-074Me induces the inhibition of Eco-MLV infection, suggesting that cathepsin L is required for the Eco-MLV infection in NIH3T3 cells. The CA-074Me treatment inhibited the Eco-MLV infection in human cells expressing the exogenous mouse ecotropic receptor and endogenous cathepsins B and L, but the CLIK148 treatment did not, showing that only the cathepsin L suppression by CLIK148 is not enough to prevent the Eco-MLV infection in cells expressing both of cathepsins B and L, and CA-074Me inhibits the Eco-MLV infection by suppressing both of cathepsins B and L. These results suggest that either cathepsin B or L is sufficient for the Eco-MLV infection.     

Production and characterization of monoclonal antibodies against different epitopes of Ebola virus antigens

Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with up to 90% mortality rate. In this study, Ebola virus like particles (EVLPs) and the aglycosyl subfragment of glycoprotein (GP(1) subfragment D) were used to generate monoclonal antibodies (MAbs) against different epitopes of the viral antigens. Such MAbs could be useful in diagnostics and potential therapeutics of viral infection and its hemorrhagic symptoms. Hybridoma cell fusion technology was used for production of MAbs. The MAbs were characterized using ELISA and Western blot analysis. Furthermore, five recombinant sub-domains of GP(1) subfragment D were produced, which were used as antigen in Western blot analysis for epitope mapping. Seventeen MAbs of different epitope specificities against EBOV antigens [virion protein (VP40), secreted glycoprotein (sGP), and GP(1) subfragment D] were developed. Based on epitope mapping studies, the anti-GP MAbs were categorized into six groups. The binding of the three anti-sGP MAbs with different epitope specificities were mostly between aa 157 and 221. The two anti-VP40 MAbs with the same or overlapping epitopes are potentially good candidates for developing antigen detection assays for early diagnosis of EBOV infection. The anti-GP MAbs with different epitope specificities as an oligoclonal cocktail could be tested for therapy.     

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.