Patent application title: Recombinant proteins from filoviruses and their use
Inventors:
Axel Thomas Lehrer (Honolulu, HI, US)
David E. Clements (Honolulu, HI, US)
Tom Humphreys (Honolulu, HI, US)
Assignees:
PanThera Biopharma LLC
IPC8 Class: AC07K1408FI
USPC Class:
530350
Class name: Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof proteins, i.e., more than 100 amino acid residues
Publication date: 2012-03-15
Patent application number: 20120065371
Abstract:
Filovirus subunit protein immunogens are produced using a recombinant
expression system and combined with one or more adjuvants in immunogenic
formulations. The subunit proteins include GP95, GP-FL, VP40, VP24, and
NP derived from Ebola Virus and Marburg Virus. Adjuvants include
saponins, emulsions, alum, and dipeptidyl peptidase inhibitors. The
disclosed immunogenic formulations are effective in inducing strong
antibody responses directed against individual Filovirus proteins and
intact Filovirus particles as well as stimulating cell-mediated immune
responses to the Filoviruses.Claims:
1.-22. (canceled)
23. An immunogenic composition comprising a recombinant Filovirus VP40 protein subunit, wherein the protein subunit is expressed and secreted as a soluble protein from stably transformed insect cells.
24. An immunogenic composition comprising a recombinant Filovirus VP24 protein subunit, wherein the protein subunit is expressed and secreted as a soluble protein from stably transformed insect cells.
25. An immunogenic composition comprising a recombinant Filovirus NP protein subunit, wherein the protein subunit is expressed and secreted as a soluble protein from stably transformed insect cells.
26.-49. (canceled)
Description:
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/724,747, filed Oct. 7, 2005, which prior application is hereby incorporated by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] A sequence listing file in ST.25 format on CD-ROM is appended to this application and fully incorporated herein by reference. The sequence listing information recorded in computer readable form is identical to the written sequence listing herein (per WIPO ST.25 para. 39, the information recorded on the form is identical to the written sequence listing). With respect to the appended CD-ROM, the format is ISO 9660; the operating system compatibility is MS-Windows; the single file contained on the CD-ROM is named "EBO.ADJ.03.ST25.txt" and is a text file produced by PatentIn 3.3 software; the file size in bytes is 63 KB; and the date of file creation is 2 Oct. 2006.
BACKGROUND OF THE INVENTION
[0003] 1. Technical Field
[0004] The invention relates to protection against Filovirus induced hemorrhagic fevers using recombinant proteins. The invention, more specifically, concerns several recombinant Filovirus structural proteins produced from eukaryotic cells. In the invention, proteins are post-translationally modified and are purified so that native structure is retained, thereby providing material for use as immunogens to elicit protection against virally induced disease.
[0005] "Subunit protein" is defined here as any protein derived or expressed independently from the complete organism that it is derived from. Furthermore, a subunit protein may represent a full length native protein sequence or any fraction of the full length native protein sequence. Additionally, a subunit protein may contain in addition to the full length or partial protein sequence, one or more sequences, which may contain sequences that are homologous or heterologous to the organism from which the primary sequence was derived. This definition is significantly broader than the concept of a subunit protein as a single protein molecule that co-assembles with other protein molecules to form a multimeric or oligomeric protein. The subunit proteins of the invention are produced in a cellular production system by means of recombinant DNA methods and, after purification, are formulated in a vaccine.
[0006] 2. Related Art
[0007] Ebola and Marburg viruses are the only two members of the Filovirus family. They are enveloped, negative strand RNA viruses. The viral RNAs have a length of approximately 19 kb and their genome structure is 3'-NP-VP35-VP40-GP-VP30-VP24-L-5'. Upon entry into the cytoplasm of host cells, individual subgenomic, viral mRNA species are transcribed by viral RNA polymerase (RdRp). While the host cell synthesizes viral proteins in the cytoplasm, RNA dependent RNA polymerase (L) in complex with VP35 replicates the complete Filovirus RNA. Viral RNA together with nucleoprotein NP and VP30 forms the nuclear core complex. The nuclear core is inserted into Filovirus envelopes formed by the viral matrix protein VP40 in combination with VP24 and mature viral surface glycoprotein (GP) anchored into the host cell membrane. A schematic diagram of a mature Filovirus particle is shown in FIG. 1.
[0008] Marburg virus was first identified in an outbreak that occurred during 1967 in Marburg, Germany. The first human cases were animal handlers, and it was established that the source of infection were monkeys imported from Africa. For the next 9 years there were no reported outbreaks of Filovirus induced hemorrhagic fevers. In 1976, the Zaire subtype of Ebola virus first appeared in an outbreak in the Democratic Republic of Congo (known then as Zaire). A second outbreak occurred in the same year in Sudan caused by the Sudan subtype which showed lower fatality rates. Later two more subtypes were identified, Ebola Ivory-Coast and Ebola Reston. To date a total of 18 Ebola virus outbreaks have caused 1860 cases of human disease and resulted in 1296 deaths. Six Marburg virus outbreaks have to date caused 613 human infections and were fatal in 494 cases (source: WHO). Case fatality rates are dependent on the specific subtype of Filovirus causing the disease and generally vary between 50-90%, with Ebola Zaire and Marburg viruses showing the highest mortality rates. Primates are the only confirmed natural hosts for Filoviruses and human outbreaks can usually be traced back to contact with infected primates. Even though bats could serve as a potential Marburg and Ebola virus reservoir based on observations and laboratory experiments (Swanepoel et al. 1996, Leroy et al. 2005), no natural reservoir has been confirmed to date (Feldmann et al 2004).
[0009] Despite relatively few outbreaks, Ebola and Marburg viruses are well known due to their extreme virulence. Both viruses cause fulminant hemorrhagic fevers and death in up to 90% of human infections depending on the infecting strain and route of infection. Although the viruses are endemic only to certain parts of central Africa and the Philippines, the threat of a bioterrorist attack using Ebola virus has raised concern about the virus worldwide. Intentional exposure of non-human primates with aerosolized Ebola virus (Johnson et al. 1995) has demonstrated that a bioterrorist attack using Ebola or Marburg virus is feasible and could affect thousands of people. In outbreaks, it is believed that human-to-human spread only occurs upon direct contact with bodily fluids of an infected patient. However, it has been documented that an unintentional animal to animal infection was possible even without direct body contact of the individually caged rhesus monkeys (Jaax et al. 1995). In addition, recent revelations related to the efforts to produce weaponized Marburg virus in the former Soviet Union (Alibek 1999) reinforced the need to prioritize Ebola/Marburg viruses as a biodefense target. While state of the art medical treatment increases the chances of survival for patients, there are currently no antiviral therapies available to cure the disease, nor a vaccine that protects healthy individuals from infection. Supportive treatment is costly and resource demanding due to the need for strict containment. An Ebola or Marburg virus outbreak, especially in a densely populated urban area, whether caused by natural transmission or terrorist attack, could lead to many casualties and have a dramatic impact on public health systems. A safe and effective vaccine or antiviral treatment that could be used to protect healthcare workers, other at-risk persons, and travelers is therefore highly desirable.
[0010] The nucleotide sequence analysis of gene 4 of Marburg virus revealed the basic features of Filovirus surface glycoproteins (singular, "GP"; plural, "GPs") (Will et al. 1993). Filovirus GPs typically possess a hydrophobic signal peptide at the N-terminus, a hydrophilic external domain, a hydrophobic transmembrane anchor and a small hydrophilic cytoplasmic tail. The signal peptide is removed during the maturation of the glycoproteins and the external domain is heavily glycosylated, with carbohydrates accounting for approximately 50% of the molecular weight of the mature protein. Most research on the biology of Filoviruses focuses on GP, a type I transmembrane glycoprotein. GP is involved in cell entry and is therefore presumed to be a key target for virus neutralization. The Filovirus glycoproteins, on the genomic as well as on the protein level, show strain specific features; Filoviruses can therefore be differentiated and identified based on GP structure. Further analysis of the proteins expressed in Ebola and Marburg virus-infected cells revealed that in addition to the mature GP, Ebola, but not Marburg, viruses express a soluble glycoprotein (sGP). This product results from early translation termination and lacks the majority of the C-terminal region of mature GP (Volchkov et al. 1998). Since it lacks the transmembrane domain it is secreted as dimers into the extracellular space. GP and sGP are synthesized from the same gene. While sGP is synthesized from the direct mRNA transcript, it was shown that the expression of mature GP requires post-transcriptional editing of the mRNA. Expression of the edited GP gene in mammalian cells results in trimerization and the formation of characteristic spikes on the cell surface. In contrast, sGP is secreted as antiparallel-oriented homodimers stabilized by intermolecular disulfide bonds (Volchkova et al. 1998). An Ebola virus mutant was generated from cDNA that included the edited form of the GP gene (expressing mature GP only). This mutant showed a much higher cytotoxicity in comparison to wild-type Ebola virus, which is most likely linked to the much higher concentration of GP present in infected cells (Volchkov et al. 2001). The focus of the current application is on expression of the native mature GP which is expected to form trimeric structures. Immune responses targeting the native structure are more likely to protect individuals from disease than responses targeting non-native (e.g., sGP) structures.
[0011] After translation of the post-transcriptionally edited mRNA into the native (non-mutant) polypeptide, preGPer, in the endoplasmatic reticulum, the preGPer polypeptide is heavily glycosylated to form preGP. Furin cleavage separates preGP into two mature subunits, GP1 and GP2, which are linked by an intermolecular disulfide bond (Volchkov et al. 1998). Mutational analysis revealed that inter- and intramolecular disulfide bonding is highly conserved and important for virus entry. Jeffers et al. (2002) suggest that disulfide bonds play a more important role in formation of functional glycoprotein than the presence of N-linked glycosylations. A similar post-translational cleavage into GP1 and GP2 is also observed on Marburg virus GP (Volchkov et al. 2000). A summary of the different pathways of Filovirus GP synthesis can be found in Feldmann et al. 2001.
[0012] The surface glycoproteins, i.e., the GPs, may contribute to the cytopathic effects observed in Filovirus infections via a variety of direct and indirect mechanisms. It has been shown that mature Ebola GP alone induces cellular detachment in cell cultures without inducing cell death (Chan et al. 2000). This is in contrast to sGP or GP1 which do not induce cellular detachment. Interestingly, Marburg virus GP does not induce similar detachment. Further characterization of the cytopathicity of GP identified that cell detachment was dependent on dynamin (Sullivan et al., 2005). It has also been shown that native mature GP can trigger activation of primary human macrophages resulting in production of proinflammatory cytokines (Wahl-Jensen et al. 2005). As Filovirus disease is often associated with overproduction of cytokines, this represents a possible mechanism for direct cytopathic effect (see further discussion of pathogenesis of Filovirus disease below). Again, this activity was associated with native, mature GP (trimeric form) and not GP1 and sGP. Furthermore, GP may contribute to the severe pathogenicity of Filovirus infection via indirect mechanisms (e.g., by inhibiting a potent protective immune response to the virus). It has been demonstrated that GP can decrease cell surface expression of MHC class I molecules (Sullivan et al., 2005). This may result in decreased immune responses upon infection. In addition, secretion of sGP as well as shedding of GP1 from virus infected cells (Dolnik et al. 2004) may diminish the activity of neutralizing antibodies.
[0013] The Filovirus surface glycoprotein is the only viral protein that is exclusively localized on the surface of the virus particles and possesses structural features to facilitate cell binding and fusion with the cell membrane. It was demonstrated that Ebola GP binds to the cell surface exposed lectins DC-SIGN and DC-SIGNR (Simmons et al. 2003). These lectins are exposed on early infection target cells such as dendritic and other antigen presenting cells and binding of these molecules may facilitate fusion efficiency by changing GP's conformation. Work by Takada et al. (2004) further confirmed the enhancement of Filovirus cell entry caused by galactose- and N-acetylgalactosamine specific C-type lectins (hMGL) present on these early target cells. The role of GP1 in cell surface receptor-binding has been suggested. Extensive mutational analysis has identified the N-terminal third of GP1 as the region most important for this first step of viral entry (Manicassamy et al. 2005). Sequence analysis of GP2 revealed the presence of a putative fusion domain (Volchkov et al. 1992). Ruiz-Arguello et al. (1998) demonstrated in vitro that the putative fusion peptide possesses liposome-binding capability. Mutational analysis using pseudotyped vesicular stomatitis viruses confirmed these findings, suggesting that the fusion process of Ebola virus might mirror that of influenza or human immunodeficiency viruses (Ito et al. 1999). A model was developed based on the hydrophobicity of the fusion peptide and tested in vitro; the testing confirmed that the fusion peptide spans residues 25 to 35 of the GP2 protein (Adam et al. 2004). Gomara et al. (2004) demonstrated further that an internal proline residue in the fusion peptide is responsible for the necessary membrane destabilization to initiate the fusion process. GP2 assembles into a rod-like trimeric structure which resembles that of other viral membrane-fusion proteins such as HIV gp41 or influenza HA2 (Weissenhorn et al. 1998). The crystal structure revealed that an internal GP2 fragment forms a trimer with a central three-stranded coiled coil structure surrounded by shorter C-terminal helices packed in an antiparallel conformation into hydrophobic grooves on the surface of the coiled coils (Malashkevich et al. 1999). Further functional analysis of the coiled-coil interactions revealed their importance in facilitating the entry of Ebola virus into host cells and demonstrated that inhibitors of this interaction could act as efficient antiviral compounds. Thus, in a mature, wild-type GP, the GP2 portion of GP is likely involved in trimerization and fusion, while the GP1 portion is likely involved in binding to the cellular receptors and facilitating entry into target cells.
[0014] To date no effective antiviral treatment against Filoviruses is available. Bray (2003) provides a comprehensive review of methods to defend against the use of Filoviruses as biological weapons. In another publication, Geisbert and Jahrling (2004) report the current status of progress towards protection against viral hemorrhagic fevers in general. In addition to immunotherapeutics and antivirals, the publication discusses several potentially supportive interventions targeting the host immune system. Potent antiviral compounds could either target virus proteins or host proteins required for appropriate virus protein processing during the viral lifecycle. The surface glycoprotein GP is a prime antiviral target because of its involvement in host cell entry. DC-SIGN binding can effectively be inhibited with hyperbranched dendritic polymers functionalized with mannose (Rojo and Delgado 2004). Another potential antiviral drug, cyanovirin-N, also exploits the DC-SIGN binding activity of GP and shows activity inhibiting GP-pseudotyped lentivirus entry into HeLa cells (Barrientos et al. 2004c). Another study demonstrated that monomeric as well as dimeric cyanovirin-N show approximately the same antiviral activity (Barrientos et al. 2004b). The importance of endosomal proteolysis by cathepsin B and L for Ebola virus entry was demonstrated by Chandran et al. (2005) and further confirmed by Schornberg et al. (2006). This suggests that cathepsin inhibitors may show activity as anti-Ebola virus drugs.
[0015] Filovirus matrix proteins (VP40) possess the general characteristic functionality of virus matrix proteins, although they show only 2-7% sequence identity to matrix proteins of other virus families (Paramyxoviridae, Rhabdoviridae, Bornaviridae). The late domain sequences important for virus budding are some of the few structurally conserved features present on the proteins of different virus families (Timmins et al. 2004). The crystal structure of a truncated Ebola virus VP40 protein (Dessen et al. 2000) revealed that it consists of two domains with unique folds connected by a flexible linker. At the beginning of the second domain is a trypsin-cleavage site after amino acid 212. The N-terminal cleavage fragment shows spontaneous hexamerization in vitro (Ruigrok et al. 2000). The N-terminal domain of VP40 is involved in the protein-protein interactions required for oligomerization while the C-terminal part is responsible for membrane-binding properties. Denaturing conditions (such as 4M urea) or membrane association induce hexamer formation of full-length VP40 (Scianimanico et al. 2000). VP40-hexamers formed from three antiparallel homodimers line the inside of Filovirus particles. On electron micrographs of Ebola virus VP40 oligomers hexameric and octameric structures were identified (Timmins et al. 2003). The disc-shaped octamers are formed in association with RNA by four antiparallel homodimers of Ebola VP40. The crystal structure revealed that the interaction with RNA induces two conformational changes of the protein (Gomis-Ruth et al. 2003). In contrast to Ebola virus VP40, the Marburg virus matrix protein forms oligomers that stack up to form complex polymeric structures. Therefore the oligomeric status could not be determined by electron microscopy.
[0016] In addition to the in vitro characterization of VP40, the cellular mechanisms involved in Filovirus budding from mammalian cells have been studied. Timmins et al. (2001) demonstrated that full-length Ebola VP40, but not the C-terminally truncated protein, is released into the culture supernatant of mammalian cells and that the matrix protein seems to be released inside vesicles. The expression of VP40 alone leads to secretion of few Filovirus-like particles, but co-expression with GP increases the formation of these structures (Noda et al. 2002, Kolesnikova et al. 2004b). Panchal et al. 2003 demonstrated in vivo that oligomers of full length Ebola VP40 are formed only in association with membranes. These oligomers seem to be transported via lipid rafts to the site of assembly. It was also shown that VP40 recruits the cellular protein Tsg101 via specific interaction with an intrinsic late budding domain (PTAP) (Licata et al. 2003). Tsg101 is involved in the vacuolar sorting pathway of mammalian cells and is important for efficient budding of numerous viruses. In addition, hexamers of VP40 can bind to the WW domain of human Nedd4, another protein involved in the late endosomal pathway necessary for efficient virus budding (Timmins et al. 2003). It was demonstrated by Yasuda et al. (2003) that Nedd4 regulates the egress of Ebola virus-like particles from mammalian cells. Despite only 29% sequence homology to the Ebola matrix protein, Marburg virus VP40 protein also associates with membranes of the late endosomal compartment both in virus-infected cells as well as in recombinant expression in mammalian cells (Kolesnikova et al. 2002, Kolesnikova et al. 2004a). While the Tsg101-binding PTAP motif is absent from Marburg virus VP40, the PPXY motif for binding of Nedd4 (Yasuda et al., 2003) is found in Marburg virus and may also to be important for efficient budding. Ebola virus VP40 shows the properties of a microtubule-associate protein (MAP) because of its binding to tubulin. It enhances tubulin polymerization in vitro (Ruthel et al. 2005). It further stabilizes microtubules against depolymerization and could therefore play a role in directed transport of virus particles to the site of budding
[0017] Reviews describing the current knowledge of the impact of VP40 on molecular mechanisms involved in Filovirus cellular trafficking (Aman et al 2003) and the current understanding of the process of Filovirus budding (Jasenosky and Kawaoka 2004) have been published.
[0018] The nucleoprotein (NP) of Marburg virus self-assembles tubule-like structures when recombinantly expressed in mammalian cells. These aggregates resemble structures observed in sections of viral inclusions of Marburg virus infected cells (Kolesnikova et al. 2000). In association with cellular RNA (when expressed in Sf21 cells), Marburg virus NP forms regularly structured loose coils (Mavrakis et al. 2002). High salt treatment tightens the coiling. Ebola virus NP expressed recombinantly in mammalian cells forms intact nucleocapsids only if expressed in combination with Ebola VP35 and VP24 (Huang et al. 2002). Other virus proteins are not necessary for nucleocapsid formation, but post-translational O-glycosylation of NP is required to facilitate VP35-binding. NP increases the release of Ebola virus-like particles if co-expressed with VP40 in mammalian cells. Further addition of Ebola GP potentiates this effect (Licata et al. 2004).
[0019] The structure and function of the minor matrix protein VP24 are not fully understood. While co-expression of VP24 and VP40 in mammalian cells does not increase VLP release, co-expression of VP40, NP and VP24 in mammalian cells yields a higher amount of Ebola VLPs than co-expression of VP40 and NP alone, suggesting that VP24 may interact with NP (Licata et al. 2004). In cells infected with Ebola virus, as well as upon recombinant expression, VP24 appears to be concentrated in the perinuclear region (Han et al. 2003). VP24 from virus infected mammalian cells is not glycosylated via N-linked sugar residues and appears to tetramerize. Studies have further shown that VP24 strongly interacts with lipid bilayers and is potentially located within lipid rafts. Recent studies have shown that VP24 is essential for the formation of a functional ribonucleoprotein complex (Hoenen et al. 2006). In addition, it has been shown that VP24 seems to block proper IFN signaling giving another potential explanation of how Filoviruses evade the host immune responses (Reid et al. 2006).
[0020] The two small proteins contained in the nucleocapsid complex have different functions. VP35 is an essential part of the Filovirus nucleocapsid (Huang et al. 2002, Muhlberger et al. 1998) but the exact role in capsid assembly has not been shown yet. VP35 is a type I IFN antagonist and therefore a major contributor to Filovirus pathogenicity (Basler et al. 2003, Hartman et al. 2004). The role of the phosphoprotein VP30 is linked to transcription activation of the nucleocapsid complex. It was shown that phosphorylation on two serine residues (40 and 42) is essential for binding to NP and therefore inclusion into the nuclear core complex (Modrof et al. 2001). Phosphorylation negatively regulates transcription activation mediated by VP30 and phosphorylase inhibition can therefore inhibit Ebola virus growth (Modrof et al. 2002). It was further shown that the effect of VP30 seems to be directed at a very early step in transcription initiation (Weik et al. 2002). VP30 oligomerizes due to an internal cluster of hydrophobic residues (containing four leucine residues). Mutation of the leucine residues or application of a peptide binding to this region abolishes VP30-dependent Ebola virus transcription activation (Hartlieb et al. 2003).
[0021] The pathogenesis of Ebola virus infection is still not fully understood, even though progress towards understanding the mechanisms has been made in recent years. One finding with most hemorrhagic fever cases caused by Filoviruses is that lesions caused by the viral infection are not severe enough to account for terminal shock and death of the host. Yet this is the most common cause of death in viral hemorrhagic fevers. This suggests the involvement of inflammatory mediators as an important part in the pathogenic pathways. A simplified primate model (Sullivan et al. 2003b) suggests that initial host immune responses as well as cell damage due to infection of monocytes and macrophages causes the release of pro-inflammatory cytokines. Infection of endothelial cells causes damage to the endothelial barrier which in combination with effects of the cytokines leads to loss in vascular integrity resulting in hemorrhage and vasomotor collapse (primary cause of death in Ebola patients). Another model further elaborates on the detailed chain of events (Geisbert and Jahrling. 2004). The authors suggest that cytokines released by initially infected monocytes and macrophages recruit further macrophages to the sites of infection. This makes more target cells available for viral exploitation and further amplifies the dysregulated host response, leading to loss of lymphocytes by apoptosis. Ebola virus enhances expression of tissue factor, resulting in activation of the clotting pathway and formation of fibrin in the vasculature. These coagulation disorders are complemented by infection of hepatocytes and adrenal cortical cells resulting in impaired synthesis of important clotting factors. Studies of human Ebola Sudan patients showed leucopenia and unresponsive PBMC's, high viral loads in infected monocytes and elevated nitric oxide levels to be associated with a fatal outcome of the disease (Sanchez et al. 2004).
[0022] Primates are the only animals known to develop disease following natural infection with Filoviruses. However, in order to research virus biology, several animal models of disease have been developed, including a non-human primate model, guinea pig models, and a mouse model for Ebola virus infection using a mouse-adapted Ebola Zaire virus. Fisher-Hoch and McCormick (1999) describe several of these animal models and a publication by Ryabchikova et al. (1999) reviews animal pathology of Filoviral infections in more detail. Animal models have been used to develop the current pathogenesis model based on in vitro experiments using primate monocyte/macrophage cultures (Feldmann et al. 1996, Stroher et al. 2001) as well as in vivo experiments comparing the coagulation changes in mice, guinea pigs, and monkeys (Bray et al. 2001). While small animal models can provide insight into the virus life cycle and pathogenicity and help as an economical choice for vaccine and antiviral drug development, only non-human primates show a disease progression similar to human disease (Geisbert et al. 2002). Pathogenesis in the cynomolgus macaque model of Ebola Hemorrhagic Fever has been described in detail by Geisbert et al. (2003).
[0023] Since there is no treatment to cure Filovirus induced hemorrhagic fevers, the majority of patients succumb to the disease. However, between 10 to 50% (depending on virus strain and medical support available) of infected humans survive the disease. Following the large 1995 outbreak of Ebola Zaire in the Democratic Republic of Congo, sera of patients were analyzed. It was found that while IgM and IgG antibodies appeared at approximately the same time after onset of disease, high IgM titers persisted for a much shorter time in survivors in comparison to fatal cases (Ksiazek et al. 1999). IgG titers in survivors were detectable for at least two years following recovery. Ebola virus patients receiving convalescent sera during their illness have been reported to show a higher survival rate (Mupapa et al. 1999). Based on this observation, immune mouse sera from mice receiving sub-lethal doses of Ebola virus were transferred to naive immunodeficient mice. Dose-dependent protection against virus challenge was observed in recipients of immune sera (Gupta et al 2001). Recombinant human monoclonal antibodies were constructed via phage display from bone marrow RNA of two Ebola survivors (Maruyama et al. 1999) and in vitro virus neutralization was demonstrated with one of these antibodies (Maruyama et al. 1999b). The same human monoclonal antibody was able to provide pre- and post-exposure prophylaxis in the guinea pig model of Ebola hemorrhagic fever (Parren et al. 2002). Similarly, mouse monoclonal antibodies to Ebola GP applied pre- or post-exposure were successful to protect mice against lethal Ebola virus challenge (Wilson et al. 2000). Another route to provide antibody-based protection against viral diseases is the preparation of hyperimmune serum in non-susceptible animals. Sera prepared in sheep and goats tested successfully for protection in the guinea pig model of Ebola virus (Kudoyarova-Zubavichene et al. 1999). A similarly prepared equine anti-Ebola Zaire serum was tested for protection in baboons, mice and guinea pigs. Results were inconsistent and showed low levels of protection in mice and monkeys, while all guinea pigs were protected against a lethal dose of Ebola virus. While viremia and disease onset in cynomolgus monkeys treated with the equine hyperimmune serum challenged with Ebola virus was delayed, all animals succumbed to the disease when protective antibodies had been depleted (Jahrling et al. 1996, Jahrling et al. 1999). One potential reason for the failure of passive immunizations in non-human primates could be antibody-dependent enhancement of infection (as demonstrated in vitro by Takada et al. 2003b). A further hurdle for the prophylactic or therapeutic use of neutralizing antibodies could be strain specificity. Out of five different neutralizing epitopes that have been identified on Ebola Zaire GP, none seems to have neutralizing activity against glycoproteins from the Sudan, Ivory Coast or Reston strains of Ebola virus or against the glycoprotein of Marburg virus (Takada et al. 2003).
[0024] In contrast to the mostly dramatic symptomatic cases of Filovirus infections, asymptomatic cases have been reported (Leroy et al. 2000). In only a few of these cases, specific antibody responses against Ebola virus proteins could be detected. Interestingly, the responses were directed against NP as well as VP40, and not against Filovirus glycoprotein, which is thought to be the main protective antigen and the target for virus neutralizing antibodies. The asymptomatic individuals furthermore showed early and strong inflammatory responses with high levels of circulating cytokines. These clinical parameters emphasize the importance of proper humoral and cell-mediated immunity in protection against Filovirus infection. Induction of cytotoxic T cells directed against GP was observed upon immunization of mice with liposome-encapsulated irradiated Ebola virus particles (Rao et al. 1999). Responses seem to be dependent on proper delivery of antigen to antigen presenting cells (APC). It was further demonstrated that the liposome-encapsulated virus particles can induce T cell help necessary to protect mice against lethal challenge (Rao et al. 2002). However, the same vaccine did not provide good protection in a primate model. Another study reports the generation of protective cytotoxic T lymphocytes after 2 or 3 immunizations with recombinant VEE replicons expressing Ebola NP (Wilson and Hart 2001). Even though the cellular response achieved did not yield 100% protection in the mouse model, it provides evidence that proper cellular responses will aid in protection. Identification of CTL epitopes on the nucleoprotein may lead to the discovery of potential defense mechanisms (Simmons et al. 2004). Another experiment in the mouse model demonstrated that cytotoxic memory T cells alone can protect against death, but that a persistent infection can develop if mice are deficient in helper T-cells or mount an insufficient humoral response (Gupta et al. 2004). This suggests a potential mechanism for development of a natural reservoir host animal for Filoviruses.
[0025] One of the characteristic features of Filovirus disease in infected primates is the rapid decline in the number of circulating lymphocytes. It was shown that Ebola virus induces apoptosis in natural killer cell populations by day two post-infection (Reed et al. 2004). It may therefore inhibit activation of lymphocytes by eliminating the subsets that are most likely to be capable of mounting an effective response to the virus. The viral component responsible for this has not been identified yet, although Ebola virus immune suppression seems to be linked to a domain of the surface glycoprotein and/or the VP35 protein. Studies with Ebola and Marburg virus-like particles containing GP and VP40 (Bosio et al. 2004) demonstrated activation of human myeloid dendritic cells. In contrast, administration of live or inactivated Ebola virus does not induce dendritic cell maturation. Other virus proteins not contained in the recombinant particles may be responsible for preventing this early immune response. Particles containing only the viral matrix protein VP40 were able to specifically activate natural killer cells and induce an early protective immunity against Ebola virus infection 1-3 days after immunization (Warfield et al. 2004). It was shown that the protection was mediated by perforin-mediated lysis of target cells by NK cells. This highlights another pathway that may be critical to provide successful protection against Filovirus infection.
[0026] Conventional vaccine approaches against Filovirus infection, such as attenuated or killed Filovirus particles are not generally considered feasible due to the high safety risks involved. Demonstration of proper attenuation of live viral strains in humans would be much too risky to consider and the risk of reversion would further complicate this approach. For example, a serendipitously acquired virus strain which had proven to be nonlethal to strain 13 guinea pigs proved to be lethal to Hartley guinea pigs (Hevey et al. 2002). Production of large quantities of virus to generate a killed vaccine would likewise be unacceptable from a safety point of view. Instead, several modern vaccine approaches have been the major area of research. Approaches evaluated or under evaluation include DNA vaccines, adeno-, alpha- or vesicular stomatitis virus vectored vaccines, and recombinantly produced proteins. Summarizing overviews of the advances in developing vaccines to protect against Filovirus induced hemorrhagic fevers were published in several review papers (Enserink 2003, Hart 2003, Geisbert and Jahrling 2004).
[0027] Xu et al. (1998) reported successful protection of guinea pigs against Ebola virus infection after injecting animals with 3 doses of DNA vaccines expressing either Ebola surface glycoprotein, the soluble glycoprotein sGP, or the nucleoprotein. A similar approach (Vanderzanden et al. 1998) reported successful protection of mice against Ebola virus infection after gene gun application of 4 or 5 doses of DNA vaccines encoding Ebola GP or NP. Protection of non-human primates against Ebola virus infection has been demonstrated after immunization with three doses of a combination of DNA vaccines followed by a boost utilizing a recombinant adenovirus vector expressing Ebola Zaire GP (Sullivan et al. 2000). The Vaccine Research Center at NIH is currently developing a prime-boost vaccine for human use based on this approach. It has further been shown that a single dose of the recombinant adenovirus can elicit a protective response against Ebola virus challenge four weeks after immunization (Sullivan et al. 2003). Data provided by these immunological studies strongly suggest the importance of Ebola GP in development and persistence of vaccine protection (Sullivan et al. 2003b). DNA vaccines need to be applied many times in order to raise effective immune responses. They could potentially lead to the development of autoimmune diseases and the use of strong promoters contained in the used plasmids may present a safety risk in human application. Recombinant adenoviruses may not be efficacious as vaccines on individuals with pre-existing immunity to adenoviruses and can for the same reason only be applied once. Due to safety concerns, development of adenoviral vectors for gene therapy and vaccine development has slowed in past years. In a clinical trial using adenovirus vectors for gene therapy, a patient died due to complications associated with the use of Ad5 virus (Raper et al. 2003).
[0028] Another approach for vaccine development focused on the use of alphavirus (VEE) replicons. Using vectors expressing several Marburg virus structural proteins, Hevey et al. (1998) were able to demonstrate that guinea pigs developed high specific serum antibody titers and could be protected if Marburg GP, NP or VP35 proteins were applied. Marburg virus VP40, VP30 or VP24 did not protect against viral challenge. Analogous vectors expressing Ebola virus GP, NP, VP24, VP30 or VP40 (Wilson et al 2001, Pushko et al. 2001) showed a significant level of protection against homologous challenge in vaccinated mice and guinea pigs. Passive transfer of immune sera was unsuccessful in providing protection against disease.
[0029] Recently a new vaccine approach has been tested utilizing replication-competent vesicular stomatitis virus vectors expressing surface and secreted glycoproteins of Ebola Zaire and Marburg viruses. A VSV vector expressing Ebola surface GP protected mice against lethal challenge (Garbutt et al. 2004). Further studies showed protection against homologous (but not heterologous) Filovirus challenge in guinea pigs and cynomolgus monkeys (Jones et al. 2005). Daddario-DiCaprio et al. (2006) showed that this vaccine may even provide post-exposure protection if administered at a high dose within 20-30 minutes after infection.
[0030] Virus-like particles based on recombinant proteins expressed in mammalian cell lines have been evaluated for their potential as protective vaccines against Filovirus infections. Particles containing Ebola GP and VP40 have shown to be more successful in protecting mice against lethal Ebola virus challenge than inactivated virus particles (Warfield et al. 2003). This indicates that virus inactivation may destroy necessary native structural features on the contained virus proteins. It was further demonstrated that Marburg virus-like particles constructed using Marburg virus GP and VP40 successfully induced protection against Marburg virus infection in guinea pigs, while Ebola VLPs were not protective against Marburg virus challenge (Warfield et al. 2004). Only a vaccine containing both Marburg and Ebola virus-like particles was able elicit protection against challenge with both types of Filoviruses in the guinea pig model (Swenson et al. 2005) demonstrating a lack of cross-protection between Filoviruses.
[0031] Marburg virus GP was expressed by baculovirus recombinants in Sf9 and Trichoplusia ni cells as full-length or truncated version without the transmembrane region. Culture supernatants formulated with Ribi adjuvant (Jennings, V.M. Review of Selected Adjuvants Used in Antibody Production. ILARJ 37:119-125. 1995) were utilized to immunize guinea pigs. Protection was achieved only against the homologous Marburg virus strain (Hevey et al. 1997). It was furthermore demonstrated that passive transfer of immune sera conferred protection to naive guinea pigs. Baculovirus expressed full length or truncated Ebola GP was administered to guinea pigs alone (2 doses) or in a prime-boost scheme following one dose of a DNA vaccine. Prime-boost and the protein-only vaccines (applied with adjuvant) induced protective immunity in less than 50% of the animals (Mellquist-Riemenschneider et al. 2003).
[0032] An article published by Hevey et al. 2002 compared the protection in guinea pigs provided by several Marburg virus GP-based vaccine strategies. Approaches included a DNA vaccine, VEE-replicons, baculovirus expressed recombinant GP or a prime-boost approach combining DNA vaccine and recombinant protein. The study suggested that DNA vaccines and replicon-based vaccines were particularly interesting. Hart (2003) cautions that it may be dangerous to focus a vaccine approach solely on the surface glycoproteins. Certain individuals may not develop the desired T-cell responses to GP and may therefore not be protected.
[0033] Adjuvants are materials that increase the immune response to a given antigen. Since the first report of such an enhanced immunogenic effect by materials added to an antigen (Ramon, G., Bull. Soc. Centr. Med. Vet. (1925) 101:227-234), a large number of adjuvants have been developed, but only calcium and aluminum salts are currently licensed in the United States for use in human vaccine products. Numerous studies have demonstrated that other adjuvants are significantly more efficacious for inducing both humoral and cellular immune responses. However, most of these have significant toxicities or side-effects which make them unacceptable for human and veterinary vaccines. In fact, even aluminum hydroxide has recently been associated with the development of injection site granulomas in animals, raising safety concerns about its use. Because of these problems, significant efforts have been invested in developing highly potent, but relatively non-toxic adjuvants. A number of such adjuvant formulations have been developed and show significant promise (Cox, J. C. and Coulter, A. R., Vaccine (1997) 15:248-256; Gupta, R. K. and Siber, G. R., Vaccine (1995) 13:1263-1276), especially in combination with recombinant products. Several of these modern adjuvants are being tested in preclinical and clinical trials designed to examine both efficacy and safety.
[0034] The main modes of action of adjuvants include (i) a depot effect, (ii) direct immunomodulation through interaction with receptors, etc., on the surface of immune cells and (iii) targeting antigens for delivery into specific antigen-presenting cell populations (e.g., through the formation of liposomes or virosomes). The depot effect results from either the adsorption of protein antigens onto aluminum gels or the emulsification of aqueous antigens in emulsions. In either case this results in the subsequent slow release of these antigens into the circulation from local sites of deposition. This prevents the rapid loss of most of the antigen that would occur by passage of the circulating antigen through the liver. Immunomodulation involves stimulation of the "innate" immune system through interaction of particular adjuvants with cells such as monocytes/macrophages or natural killer (NK) cells. These cells become activated and elaborate proinflammatory cytokines such as TNF-α and IFN-γ, which in turn stimulate T lymphocytes and activate the "adaptive" immune system. Bacterial cell products, such as lipopolysaccharides, cell wall derived material, DNA, or oligonucleotides often function in this manner (Krieg, A. M. et al., Nature (1995) 374:546; Ballas, Z, J, et al., J. of Immunology (2001) 167:4878-4886; Chu, R. S., et al., J. Exp. Med. (1997) 186:1623; Hartmann, G. and Krieg, A., J. Immunol. (2000) 164:944-952; Hartmann, G., et al., J. of Immunol (2000) 164:1617-1624; Weeratna, R. D. et al., Vaccine (2000) 18:1755-1762; U.S. Pat. Nos. 5,663,153; 5,723,335; 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; 6,429,199). Targeting of antigens to (and within) antigen presenting cells is accomplished through the delivery and fusion of antigen bearing vehicles (e.g., liposomes or virosomes) with antigen presenting cells, thereby delivering the antigen into the intracellular pathways necessary for presentation of antigen in the context of MHC Class I and/or II molecules (Leserman, L., J. Liposome Res. (2004) 14:175-89; Bungener et al., Vaccine (2005) 23:1232-41).
[0035] In addition to these more traditional adjuvants, various new technologies are under development. One of them includes the use of dipeptidyl peptidases, which have shown promising results in human cancer therapy. The biological activity of dipeptidyl peptidases may reflect adjuvant-like properties (e.g. generating cytokine and chemokine production), as shown in preclinical animal models in response to their administration. These responses are believed to enhance both antigen-presentation to naive T-cells and the co-stimulation of antigen-specific T-cells. The use of adjuvants in combination with recombinant antigens is widely known in the art and can result in vaccines with better efficacy.
[0036] Thus, there is an unmet need for vaccines against Filoviruses. A key technical problem to be solved is the efficient production of conformationally relevant Filovirus surface glycoproteins, as well as additional structural proteins, that serve as potent immunogens in vaccinated subjects. Related technical problems are the production and formulation of effective vaccines that have improved costs of production and distribution.
SUMMARY OF THE INVENTION
[0037] The inventors have identified unique combinations of antigens and adjuvants that induce relevant protective immune responses and have shown an acceptable safety profile in vaccinated individuals. These unique formulations depend upon several novel, properly folded recombinant subunit proteins (Filovirus GP, VP40, VP24 or NP) combined with one or more adjuvants, such as saponins, emulsions, and alum-based formulations. These antigen/adjuvant combinations (1) induce or enhance relevant, protective immune responses, such as protein specific antibody and cell mediated responses, and (2) maintain an acceptable safety profile. The disclosed invention provides immunogenic compositions containing as active ingredients recombinantly-produced Filovirus glycoprotein(s), and optionally, additional structural virus proteins. A preferred embodiment of the disclosed invention comprises the recombinant truncated surface glycoprotein of one or more Ebola or Marburg viruses as active ingredients. A preferred embodiment of the disclosed invention alternatively includes a full-length Filovirus surface glycoprotein and/or several other structural Filovirus proteins. A preferred embodiment of the disclosed invention also includes an adjuvant, such as a saponin or a saponin-like material (e.g., GPI-0100, ISCOMATRIX®), alum-based formulations (e.g., Alhydrogel®), or emulsion-based formulations (e.g., ISA-51, Co-Vaccine HT, Ribi R-700), either alone or in combination with other immunostimulants and adjuvants, as a component of the immunogenic formulations described herein. Typically, the disclosed immunogenic formulations are capable of eliciting the production of antibodies against Filoviruses, for example Ebola Zaire, and stimulating cell-mediated immune responses.
[0038] Other aspects of this invention include use of a therapeutically effective amount of the immunogenic composition in an acceptable carrier for use as an immunoprophylactic against Filovirus infection and a therapeutically effective amount of the immunogenic composition in an acceptable carrier as a pharmaceutical composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows a schematic structure of Ebola virus particle.
[0040] FIG. 2 shows a Sodium Dodecyl Sulfate Polyacrylamide Gel electrophoresis (SDS-PAGE) panel demonstrating expression and secretion of Ebola Zaire GP95, VP40 and VP24 proteins. Loading of gel: Lanes 1, 6 and 11--prestained broad range protein marker showing molecular weights in kDa (NEB); Lanes 2-4--each loaded with 10 μl of 3 different culture supernatants from a VP40 expression line (reducing sample buffer); Lanes 7-9--each loaded with 10 μl of 3 different culture supernatants from a VP24 expression line (non reducing); lanes 13-15--each loaded with 10 μl of 3 different culture supernatants from GP95 expression line (non reducing conditions).
[0041] FIG. 3 shows an SDS-PAGE panel demonstrating Immunoaffinity Chromatography (IAC) purified Ebola Zaire GP95 under reducing and non-reducing conditions. Sample loading on 10% Coomassie stained SDS-PAGE gel: (1) prestained broad range molecular weight marker with molecular weights in kDa (NEB; www.neb.com); (2) 1 μg GP95, reducing conditions; (3+9) 1 μg GP95, PNGase treated to remove N-linked glycosylations, reducing conditions; (4+10) 1 μg GP95, fully deglycosylated, reducing conditions; (5 and 6) fetuin as control to verify proper enzymatic deglycosylation; (8) 1 μg GP95, non-reducing conditions. A: identifies band of GP95, B: shows GP1 region, C: identifies location of deglycosylated GP1, D: GP2 (2 or more bands), E: deglycosylated GP2.
[0042] FIG. 4 shows an SDS-PAGE panel demonstrating IAC purified Ebola Zaire VP40 under reducing and non-reducing Conditions. Sample loading on silver-stained 12% SDS-PAGE gel (1 μg VP40 loaded per lane): (1) Mark12 (Invitrogen Corp.)--molecular weights in kDa, (2) VP40 (lot #588-161) reduced, (3) VP40 (lot #609-73) reduced, (4) VP40 (lot #619-23) reduced, (5) VP40 (lot #588-161) not reduced, (6) VP40 (lot #609-73) not reduced, (7) VP40 (lot #619-23) not reduced.
[0043] FIG. 5 shows an SDS-PAGE panel demonstrating IAC purified Ebola Zaire VP24 under reducing and non-reducing conditions. VP24 is expressed by S2 cells in three glycoforms (mono-, di- and tri-glycosylated) which co-purify on the IAC column. The lanes have been loaded with Mark12 (protein marker from Invitrogen Corp.)--molecular weights in kDa, (1)=1.0 μg final purified VP24 and (2)=0.1 μg final purified VP24.
[0044] FIG. 6 shows an SDS-PAGE panel demonstrating reactivity of individual recombinant Ebola Zaire virus proteins with Ebola specific hyperimmune mouse ascites fluid (HMAF). FIG. 6 is a Western blot of 12% SDS-PAGE gel with following loading pattern: Lane 1--prestained broad range protein marker (NEB)--molecular weights in kDa; Lanes 2, 4, 5--each loaded with 10 μl of 3 different culture supernatants from VP40 expression line (reducing conditions), lane 3: PNGase treated supernatant from VP40 expression line (reducing conditions); Lane 6: loaded with 10 μl of culture supernatant from GP95 expression line (non reducing conditions); Lane 7: loaded with 10 μl of PNGase treated supernatant from GP95 expression line (reducing conditions); Lanes 8, 9, 10: each loaded with 10 μl of 3 different culture supernatants from GP95 expression line (non reducing), lanes 11, 13 and 14--each loaded with 10 μl of 3 different culture supernatants from VP24 expression line, lane 12: PNGase treated supernatant from VP24 expression line (non reducing conditions). Western blot was probed with anti-Ebola HMAF (reactive against ZEBOV, SEBOV, REBOV).
[0045] FIG. 7 shows an SDS-PAGE panel demonstrating the reactivity of individual recombinant proteins with a VP40-specific monoclonal antibody. FIG. 7 is a Western blot of 12% SDS-PAGE gel with identical loading to FIG. 6, but probed with A 10A (binds to ZEBOV VP40)--which antibody binds to glycosylated and non-glycosylated proteins, and also reacts with VP40 dimers.
[0046] FIG. 8 shows an SDS-PAGE panel demonstrating the reactivity of individual recombinant proteins with a GP-specific monoclonal antibody. FIG. 8 is a Western blot of 12% SDS-PAGE gel with identical loading to FIG. 6, but probed with the conformationally sensitive antibody 13C6 (reactive against ZEBOV GP). The antibody does not bind to the deglycosylated GP95 protein (see lane 7).
[0047] FIG. 9 shows an SDS-PAGE panel demonstrating the reactivity of individual recombinant proteins with a VP24-specific monoclonal antibody. FIG. 9 is a Western blot of 12% SDS-PAGE gel with identical loading to FIG. 6, but probed with BG11 (reactive against ZEBOV VP24)--which antibody binds to the three glycoforms as well as deglycosylated forms.
[0048] FIG. 10 is a histograph showing induction of B Cell Memory in Balb/c Mice with recombinant Ebola Zaire GP95 and VP40.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] The invention described herein provides subunit Filovirus immunogenic formulations that are produced using a recombinant expression system; the formulations typically include one or more adjuvants. The disclosed immunogenic formulations are effective in inducing strong antibody responses directed against individual Filovirus proteins and intact Filovirus particles as well as stimulating cell-mediated immune responses to the viruses (see Examples 3 and 4).
[0050] The inventors and their colleagues have successfully developed a method of expression in insect cell culture systems that produce recombinant structural proteins from Filoviruses such as Ebola Zaire, Ebola Sudan or Marburg virus. The surface glycoprotein is either expressed full length or in a truncated form, removing about 5% of the mature protein. The scope of the truncated proteins used in the invention includes any surface glycoprotein subunit secretable by the expression system. The preferred truncation deletes the membrane spanning portion (situated within the GP2 region, entailing approximately the carboxy-terminal 30 amino acids, thus allowing it to efficiently be secreted into the extracellular medium, facilitating recovery). "Expression" and "to express" are synonymous with "secretion" and "to secrete" as used herein. Cloning and expressing full length Filovirus GP is possible, but secretion is less efficient due to the membrane anchor. However, the membrane spanning domain does not contain any known antigenic epitopes and inclusion of the membrane spanning domain reduces yields; therefore the membrane spanning domain and the cytosolic tail portions of GP are not included in the preferred antigen, a subunit, surface glycoprotein of Filoviruses described below and called "GP95". "Secretable" means able to be secreted, and typically secreted, from the transformed cells in the expression system. Furthermore, the expressed proteins have been shown to be properly glycosylated and achieve native conformation as determined by reactivity with the conformationally sensitive monoclonal antibody, 13C6-1-1 ("13C6"; Wilson et al., 2000) (Example 2). The proteins are potent immunogens when administered in combination with modern adjuvants (Examples 3 and 4) and have been shown to induce protective efficacy in a small animal model for Ebola Hemorrhagic Fever (see Example 5 below). Thus the inventors have found a novel solution to a key technical problem: the efficient production of conformationally relevant Filovirus surface glycoproteins that serve as potent immunogens in vaccinated subjects.
[0051] The invention also provides for the use of adjuvants as components in an immunogenic composition compatible with the purified proteins to boost the immune response resulting from vaccination. One or more preferred adjuvants are selected from the group comprising saponins (e.g., GPI-0100 (Hawaii Biotech, Inc., Aiea, Hawaii), ISCOMATRIX® (CSL Limited, Parkville, Victoria, Australia)) (saponin, saponin-derivative, and saponin-like substances are collectively referred to herein as "saponin-based"), alum-based adjuvants (e.g., Alhydrogel (Superfos Export Co., 15 Amaliegade, Copenhagen, Denmark), or emulsion-based adjuvants (e.g., Co-Vaccine HT (Co-Vaccine BV, Lelystad, Flevoland, Netherlands), Ribi R-700 (GlaxoSmithKline, Philadelphia, Pa.), Montanide ISA-51 (Seppic, Paris, France). Aluminum-based adjuvants (collectively, "alum" or "alum-based adjuvants") are aluminum hydroxide, aluminum phosphate, or a mixture thereof. Aluminum hydroxide (commercially available as "Alhydrogel®") was used as alum in the Examples.
[0052] The antigens used in the disclosed immunogenic compositions typically comprise one or more truncated Filovirus surface glycoprotein subunits and may further contain additional structural virus protein subunits. For example, a preferred immunogenic composition comprises one or more surface glycoprotein subunits (preferably GP95) expressed and secreted from insect cells, preferably Drosophila S2 cells. The surface glycoprotein subunit (i.e., truncated, secretable GP95 protein) from the Ebola Zaire virus ("ZEBOV") is used in the prototype vaccine composition. Surface glycoproteins subunits from other Filoviruses, such as Marburg virus ("MARV"), e.g. of the Musoke, Angola or Popp strains, Ebola Sudan Virus ("SEBOV"), Ebola Ivory-Coast Virus ("ICEBOV") or Ebola Reston Virus ("REBOV") can be used as replacement or additional antigens in the disclosed invention.
[0053] One or more optional recombinant Filovirus structural protein subunits can be included in the disclosed immunogenic composition. For example, one or more insect cell, preferably Drosophila Schneider 2 cell-expressed, structural protein subunits (preferably VP40, VP24, or NP), preferably from the homologous Filovirus, are included in some embodiments of the disclosed immunogenic compositions. Inclusion of such structural protein subunits typically results in exceptionally potent vaccine formulations.
[0054] The combination of one or more viral subunit GP95, with or without other structural protein subunits, and optionally with one or more adjuvants, induces very high titer antibodies in mice. For example, certain combinations of a saponin-based adjuvant, preferably GPI-0100, with a given recombinant antigen, yield a high titer of antibodies and cell-mediated immune responses. Examples illustrating the immunogenicity and protective efficacy of such novel combinations are disclosed below (Examples 3, 4 and 5).
[0055] For C-terminally truncated protein subunits of the invention, the C-terminal truncation point can be varied up to plus or minus 2% of the length of the specified sequence length of a given protein so long as such variation does not affect secretion of the protein and conformation of the epitopes of the secreted subunit protein. For instance, the specified length of the ZEBOV GP95 protein subunit is 617 residues, but the actual truncation point may range from approximately 605 residues to 629 residues so long as such variation does not affect secretion of ZEBOV GP95 and conformation of the epitopes of the secreted ZEBOV GP95.
Surface Glycoprotein Subunits (GP95)
[0056] In a preferred embodiment of the invention, the recombinant protein subunits of the Filovirus vaccine formulations described herein are produced by a eukaryotic expression system, Drosophila melanogaster Schneider 2 ("Drosophila S2" or simply "S2") cells (Johansen, H. et al., Genes Dev. (1989) 3:882-889; Ivey-Hoyle, M., Curr. Opin. Biotechnol. (1991) 2:704-707; Culp, J. S., et al., Biotechnology (NY) (1991) 9:173-177). This method of expression successfully produces recombinant surface glycoproteins from Filoviruses, such as Ebola Zaire (ZEBOV). These proteins are truncated at the C-terminus, leaving approximately 95% of the mature surface glycoprotein (GP95) sequence. The truncation deletes the membrane spanning region of the protein, thus allowing it to be efficiently secreted into the extracellular medium using a homologous or heterologous secretion signal, facilitating recovery. Alternatively, full-length GP (GP-FL) may be expressed using a homologous or heterologous secretion signal. The expressed proteins (GP95 and GP-FL) have been shown to be properly glycosylated and to maintain native conformation as determined by reactivity with a conformationally sensitive monoclonal antibody (13C6, see Example 2). The amino acid sequence listing of ZEBOV GP95 is SEQ ID: 1. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes ZEBOV GP95 is SEQ ID: 2. The nucleotide sequence listing, without "bookends" used in cloning, that encodes ZEBOV GP95 is SEQ ID: 3. The amino acid sequence listing of SEBOV GP95 is SEQ ID: 4. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes SEBOV GP95 is SEQ ID: 5. The nucleotide sequence listing, without "bookends" used in cloning, that encodes SEBOV GP95 is SEQ ID: 6. The amino acid sequence listing of MARV GP95 is SEQ ID: 7. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes MARV GP95 is SEQ ID: 8. The nucleotide sequence listing, without "bookends" used in cloning, that encodes MARV GP95 is SEQ ID: 9. The amino acid sequence listing of ZEBOV GP-FL is SEQ ID: 10. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes ZEBOV GP-FL is SEQ ID: 11. The nucleotide sequence listing, without "bookends" used in cloning, that encodes ZEBOV GP-FL is SEQ ID: 12.
[0057] In another embodiment of the invention, GP95 is defined more broadly as a truncated surface glycoprotein that comprises two subunits (GP1 and GP2-95) that are linked via a disulfide bridge, wherein the polypeptide has been secreted as a recombinant protein from Drosophila cells, and wherein the polypeptide generates antibody responses to a homologous strain of a species of Filovirus.
[0058] In another preferred embodiment, the surface glycoprotein subunit further comprises a hydrophilicity profile characteristic of at least a homologous 95% portion of a surface glycoprotein (GP) starting from the first amino acid at the N-terminus of the mature surface glycoprotein of a strain of a species of Filovirus. In other words, amino acids can be substituted in the sequence comprising GP-FL or GP95 so long as the hydrophilicity profile and immunogenicity are unchanged.
[0059] The immunogenicity and protective efficacy of such GP proteins have also been demonstrated in an animal model (see Examples 3-5).
[0060] "GP95" in one instance refers to a polypeptide that spans a Filovirus surface glycoprotein, preferably one starting from the N-terminal amino acid of the mature surface glycoprotein and ending at an amino acid in the range of the 647th to 648th amino acid, for example, such GP95 can be the polypeptide comprising amino acids 33 to 647 of ZEBOV or SEBOV or amino acids 19 to 648 of MARV.
[0061] "GP-FL" in one instance refers to a polypeptide that spans a Filovirus surface glycoprotein, preferably one starting from the N-terminal amino acid of the mature surface glycoprotein and ending at an amino acid in the range of the 676th to 681st amino acid, for example, such GP-FL can be the polypeptide comprising amino acids 33 to 676 of ZEBOV or SEBOV or amino acids 19 to 681 of MARV.
[0062] Preferably, the ZEBOV surface glycoprotein subunit is a portion of the ZEBOV surface glycoprotein that comprises approximately 95% of its length starting from amino acid residue 1 at its N-terminus and which portion has been recombinantly produced and secreted from Drosophila cells. In another embodiment, GP95 is at least 80%, or 85%, or 90%, or 95% homologous over the entire sequence relative to native Filovirus GP, or alternatively relative to the sequence listings disclosed in SEQ ID NOS:1, 4, and 7, as the case may be, so long as such variation from native Filovirus GP, or SEQ ID NOS:1, 4, and 7, respectively, does not affect secretion of the GP95 subunit and conformation of the epitopes of the secreted GP95 subunit.
[0063] In another embodiment, GP-FL is at least 80%, or 85%, or 90%, or 95% homologous over the entire sequence relative to native Filovirus GP, or alternatively relative to the sequence listing disclosed in SEQ ID NO:10, so long as such variation from native Filovirus GP, or SEQ ID NO:10, respectively, does not affect secretion of the GP-FL subunit and conformation of the epitopes of the secreted GP-FL subunit.
[0064] More preferably, GP is derived from homologs or variants as described above, e.g., all Ebola Zaire strains as well as any serotypes of: Ebola Sudan virus (SEBOV), Ebola Ivory-Coast and Marburg virus (MARV). The GP95 and GP-FL proteins preferably are produced from vectors containing the DNA encoding the Filovirus GP fused to a heterologous secretion signal (e.g. BiP (Kirkpatrick et al., 1995) or TPA (Culp et al., 1991)). The proteins are processed by cellular signalases to release the mature GP95 or GP-FL proteins.
[0065] In one embodiment, the immunogenic composition comprises the surface glycoprotein subunit derived from Ebola Zaire virus. Preferably, the GP95 subunit from Ebola Zaire virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody (e.g. 13C6; Wilson et al., 2000) as described in Example 2. Alternatively, the GP95 subunit from Ebola Zaire virus is purified by conventional chromatography methods or alternative affinity purification methods (e.g., wheat germ lectin chromatography (Dolnik et al. 2004)), which are known to one skilled in the art.
[0066] In one embodiment, the immunogenic composition comprises the surface glycoprotein subunit derived from Ebola Sudan virus. Preferably, the GP95 subunit from Ebola Sudan virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody (e.g., 13C6) as described in Example 2. Alternatively, the GP95 subunit from Ebola Sudan virus is purified by conventional chromatography methods or alternative affinity purification methods (e.g., wheat germ lectin chromatography (Dolnik et al. 2004)) which are known to one skilled in the art.
[0067] In one embodiment, the immunogenic composition comprises the surface glycoprotein subunit derived from Marburg virus. Preferably, the GP95 subunit from Marburg virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody as described for Ebola Zaire GP95 in Example 2. Alternatively, the GP95 subunit from Marburg virus is purified by conventional chromatography methods or alternative affinity purification methods (e.g., wheat germ lectin chromatography (Dolnik et al. 2004)), which are known to one skilled in the art.
Trimeric GP95
[0068] Numerous studies have demonstrated that immunogenicity is directly related both to the size of the immunogen and to the antigenic complexity of the immunogen. Thus, in general, larger antigens make better immunogens. The native form of GP found on the surface of the Filovirus virion is a homotrimer (Volchkov et al. 1998b). The majority of the recombinant GP95 protein prepared using the method of production disclosed herein is present in solution as trimers and dimers in addition to a smaller portion of monomer. Trimers of GP may be stabilized by protein cross-linking agents (e.g., formaldehyde, EGS) after purification. In a preferred embodiment, the surface glycoproteins from ZEBOV, SEBOV or MARV are prepared as trimeric antigens.
Matrix Protein (VP40)
[0069] In addition to the Filovirus surface glycoproteins discussed above, the immunogenic formulations of the described invention optionally include the Filovirus matrix protein VP40. The recombinant protein components of the Filovirus vaccine formulations described herein are produced by a eukaryotic expression system, Drosophila melanogaster Schneider 2 (S2) cells (see references above). This method of expression successfully produces recombinant matrix proteins from Filoviruses, such as Ebola Zaire (ZEBOV). These proteins are expressed full length and may be expressed as a fusion with a eukaryotic secretion signal (e.g., from BiP or TPA) thus allowing it to be efficiently secreted into the extracellular medium, facilitating recovery. Furthermore, the secreted protein has been shown to be glycosylated, while the non-secreted protein is not. The secreted V40 was shown to maintain native conformation as determined by reactivity with a VP40-specific monoclonal antibody M-HD06-A10A ("A10A") produced by United States Army Medical Research Institute for Infectious Diseases ("USAMRIID"), as shown in Example 2. The amino acid sequence listing of ZEBOV VP40 is SEQ ID: 13. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes ZEBOV VP40 is SEQ ID: 14. The nucleotide sequence listing, without "bookends" used in cloning, that encodes ZEBOV VP40 is SEQ ID: 15.
[0070] In another embodiment of the invention, VP40 is defined more broadly as a full length matrix protein that comprises one or more N-linked glycosylations, wherein the polypeptide has been secreted with or without the use of an eukaryotic secretion signal as a recombinant protein from Drosophila cells, and wherein the polypeptide generates antibody responses to a homologous or heterologous strain of a species of Filovirus.
[0071] In another preferred embodiment, the matrix protein further comprises a hydrophilicity profile characteristic of a homologous matrix protein (VP40) starting from the first amino acid at the N-terminus of the matrix protein of a strain of a species of Filovirus. In other words, amino acids can be substituted in the sequence comprising VP40 so long as the hydrophilicity profile and immunogenicity are unchanged.
[0072] The immunogenicity of such VP40 proteins have also been demonstrated in an animal model (see Examples 3-5).
[0073] "VP40" in one instance refers to a polypeptide that spans the entire Filovirus matrix protein, preferably one starting from the N-terminal amino acid of the mature matrix protein and ending at an amino acid in the range of the 303rd to 326th amino acid, for example, such VP40 can be the polypeptide comprising amino acids 1 to 326 of the ZEBOV protein.
[0074] Preferably, the ZEBOV matrix protein is a portion of the ZEBOV matrix protein that comprises up to 100% of its length starting from amino acid residue 1 at its N-terminus and which portion has been recombinantly produced and secreted with or without a heterologous secretion signal from Drosophila cells. In another embodiment, VP40 is at least 80%, or 85%, or 90% or 95% homologous over the entire sequence relative to native Filovirus VP40, or alternatively relative to the sequence listing disclosed in SEQ ID NO:13, so long as such variation from native Filovirus, or SEQ ID NO:13, respectively, VP40 does not affect secretion of the VP40 subunit and conformation of the epitopes of the secreted VP40 subunit. More preferably, VP40 is derived from homologs or variants as described above, e.g., all Ebola virus variants as well as any serotypes of Marburg virus (MARV). The VP40 proteins preferably are produced from vectors containing the DNA encoding the VP40 proteins fused at the N-terminus to a heterologous secretion signal (e.g., BiP or TPA). The proteins are processed by cellular signalases to release the mature and glycosylated VP40 proteins. The VP40 proteins alternatively are produced without secretion signal from vectors containing the DNA encoding the VP40 proteins. The proteins are released at similar expression levels via alternative pathways into the culture supernatant mimicking the protein shedding observed from Filovirus infected cells.
[0075] In one embodiment, the immunogenic composition comprises the VP40 matrix protein derived from Ebola Zaire virus. Preferably, recombinant VP40 from Ebola Zaire virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody (e.g., A10A) as described in Example 2. Alternatively, the recombinant VP40 from Ebola Zaire virus is purified by conventional chromatography methods which are known to one skilled in the art.
Minor Matrix Protein (VP24)
[0076] In addition to the Filovirus surface glycoproteins discussedabove, the immunogenic formulations of the described invention optionally include the Filovirus minor matrix protein VP24. The Filovirus vaccine formulations described herein are produced by a eukaryotic expression system, Drosophila melanogaster Schneider 2 (S2) cells (see references above). This method of expression successfully produces recombinant minor matrix proteins from Filoviruses, such as Ebola Zaire (ZEBOV). The proteins are expressed full length and are expressed as a fusion with a eukaryotic secretion signal (e.g., BiP or TPA) thus allowing it to be efficiently secreted into the extracellular medium, facilitating recovery. Furthermore, the secreted protein has been shown to be glycosylated (Example 2). It was shown to maintain native conformation as determined by reactivity with a VP24-specific monoclonal antibody (BG11, see Example 2). The amino acid sequence listing of ZEBOV VP24 is SEQ ID: 16. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes ZEBOV VP24 is SEQ ID: 17. The nucleotide sequence listing, without "bookends" used in cloning, that encodes ZEBOV VP24 is SEQ ID: 18.
[0077] In another embodiment of the invention, VP24 is defined more broadly as a full-length, minor matrix protein, wherein the polypeptide has been secreted as a recombinant protein from Drosophila cells which may or may not be glycosylated, and wherein the polypeptide generates antibody responses to a homologous or heterologous strain of a species of Filovirus.
[0078] In another preferred embodiment, the minor matrix protein further comprises a hydrophilicity profile characteristic of a homologous portion of a minor matrix protein (VP24) starting from the first amino acid at the N-terminus of the minor matrix protein of a strain of a species of Filovirus. In other words, amino acids can be substituted in the sequence comprising VP24 so long as the hydrophilicity profile and immunogenicity are unchanged.
[0079] The immunogenicity and protective efficacy of such glycosylated VP24 proteins have also been demonstrated in an animal model (see Examples 3-5).
[0080] "VP24" in one instance refers to a polypeptide that spans a Filovirus minor matrix protein, preferably one starting from the N-terminal amino acid of the mature minor matrix protein and ending at an amino acid in the range of the 251st to 253rd amino acid, for example, such VP24 can be the polypeptide comprising amino acids 1 to 251 of ZEBOV virus.
[0081] Preferably, the ZEBOV minor matrix protein VP24 is a portion of the ZEBOV minor matrix protein that comprises up to 100% of its length starting from amino acid residue 1 at its N-terminus and which protein has been recombinantly produced and secreted with a heterologous secretion signal from Drosophila cells. In another embodiment, VP24 is at least 80%, or 85%, or 90% or 95% homologous over the entire sequence relative to native Filovirus VP24, or alternatively relative to the sequence listing disclosed in SEQ ID NO:16, so long as such variation from native Filovirus VP24 does not affect secretion of the VP24 subunit and conformation of the epitopes of the secreted VP24 subunit. More preferably, VP24 is derived from homologs or variants as described above, e.g., all Ebola virus variants as well as any serotypes of Marburg virus (MARV). The VP24 proteins preferably are produced from vectors containing the DNA encoding the VP24 proteins fused at the N-terminus to a heterologous secretion signal (e.g., BiP or TPA). The proteins are processed by cellular signalases to release the mature and glycosylated VP24 proteins.
[0082] In one embodiment, the immunogenic composition comprises the minor matrix protein derived from Ebola Zaire virus. Preferably, recombinant VP24 from Ebola Zaire virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody, for example Z-AC01-BG11-01 ("BG11"; Wilson et al., 2001), as described in Example 2. Alternatively, the recombinant VP24 from Ebola Zaire virus is purified by conventional chromatography methods, which are known to one skilled in the art.
[0083] Nucleoprotein Subunits (NP)
[0084] In addition to the Filovirus glycoproteins discussed above, the immunogenic formulations of the described invention optionally include the Filovirus nucleoprotein NP. The Filovirus vaccine formulations described herein are produced by a eukaryotic expression system, Drosophila melanogaster Schneider 2 (S2) cells (see references above). This method of expression successfully produces recombinant nucleoproteins from Filoviruses, such as Ebola Zaire (ZEBOV). These proteins are expressed full length or as truncated subunits and may be expressed as a fusion with a eukaryotic secretion signal (e.g., from BiP or TPA) thus allowing it to be secreted into the extracellular medium, facilitating recovery. Furthermore, the expressed protein has been shown to be glycosylated. It was shown to maintain native conformation as determined by reactivity with two monoclonal antibodies (D04-AE8 and FB03-BE08, see Example 2). The amino acid sequence listing of ZEBOV NP is SEQ ID: 19. The nucleotide sequence listing, including leading and trailing nucleotides (collectively, "bookends") used in cloning, that encodes ZEBOV NP is SEQ ID: 20. The nucleotide sequence listing, without "bookends" used in cloning, that encodes ZEBOV NP is SEQ ID: 21.
[0085] In another embodiment of the invention, NP is defined more broadly as a full length nucleoprotein that may comprise one or more N-linked glycosylations, wherein the polypeptide may have been secreted with the use of an eukaryotic secretion signal as a recombinant protein from Drosophila cells, and wherein the polypeptide generates antibody responses to a homologous or heterologous strain of a species of Filovirus.
[0086] In another preferred embodiment, the nucleoprotein further comprises a hydrophilicity profile characteristic of a homologous nucleoprotein (NP) starting from the first amino acid at the N-terminus of the nucleoprotein of a strain of a species of Filovirus. In other words, amino acids can be substituted in the sequence comprising NP so long as the hydrophilicity profile and immunogenicity are unchanged.
[0087] "NP" in one instance refers to a polypeptide that spans the nucleoprotein, preferably one starting from the N-terminal amino acid of the mature nucleoprotein and ending at an amino acid in the range of the 692nd to 739th amino acid, for example, such NP can be the polypeptide comprising amino acids 1 to 739 of ZEBOV virus.
[0088] Preferably, the ZEBOV nucleoprotein NP is a portion of the ZEBOV nucleoprotein that comprises up to 100% of its length starting from amino acid residue 1 at its N-terminus and which portion has been recombinantly produced with or without a heterologous secretion signal from Drosophila cells. In another embodiment, NP is at least 80%, or 85%, or 90% or 95% homologous over the entire sequence relative to native Filovirus NP, or alternatively relative to the sequence listing disclosed in SEQ ID NO:19, so long as such variation from native Filovirus NP or SEQ ID NO:19, respectively, does not affect secretion of the NP subunit and conformation of the epitopes of the secreted NP subunit. More preferably, NP is derived from homologs or variants as described above, e.g., all Ebola virus variants as well as any serotypes of Marburg virus (MARV). The NP proteins preferably are produced from vectors containing the DNA encoding the NP proteins. The NP proteins alternatively are produced from vectors containing the DNA encoding the NP proteins fused at the N-terminus to a heterologous secretion signal (e.g., BiP or TPA). The proteins are then processed by cellular signalases to release the mature and glycosylated NP proteins.
[0089] In one embodiment, the immunogenic composition comprises the nucleoprotein derived from Ebola Zaire virus. Preferably, recombinant NP from Ebola Zaire virus is purified by immunoaffinity chromatography (IAC) using a monoclonal antibody (e.g., Z-D04-AE8 produced by the United States Army Medical Research Institute for Infectious Diseases) as described in Example 2. Alternatively, the recombinant NP from Ebola Zaire virus is purified by conventional chromatography methods, which are known to one skilled in the art.
[0090] Adjuvants
[0091] In addition to the antigenic components described above, the invention optionally contains an adjuvant which aids in inducing a potent, protective immune response to the conformationally relevant antigen,
[0092] Saponin or Saponin-Based Adjuvants
[0093] In an especially preferred embodiment of the invention, a saponin or saponin-based adjuvant such as ISCOMATRIX® or GPI-0100 are added to the recombinant subunit surface glycoprotein, with or without additional structural proteins in the composition. Targeting specific antigen-presenting cell (APC) populations may involve a particular receptor on the surface of the APC, which could bind the adjuvant/antigen complex and thereby result in more efficient uptake and antigen processing as discussed above. For example, a carbohydrate-specific receptor on an APC may bind the sugar moieties of a saponin such as ISCOMATRIX® or GPI-0100 (Kensil, C. R. et al., J. Immunol. (1991) 146:431-437; Newman M. J. et al. J. Immunol. (1992) 148:2357-2362; U.S. Pat. Nos. 5,057,540; 5,583,112; 6,231,859). Although the validity of the invention is not bound by this theory, a possible mechanism of action may be that if the saponin is also bound to an antigen, this antigen would thus be brought into close proximity of the APC and more readily taken up and processed. Similarly, if the adjuvant forms micellar or liposomal complexes with antigen and the adjuvant can interact or fuse with the APC membrane, this may allow the antigen access to the cytosolic or endogenous pathway of antigen processing. As a result, peptide epitopes of the antigen may be presented in the context of MI-IC class I molecules on the APC, thereby inducing the generation of CD8+ cytotoxic T lymphocytes ("CTL"; Newman et al., supra; Oxenius, A., et al., J. Virol. (1999) 73: 4120).
[0094] A saponin is any plant glycoside with soapy action that can be digested to yield a sugar and a sapogenin aglycone. Sapogenin is the nonsugar portion of a saponin. It is usually obtained by hydrolysis, and it has either a complex terpenoid or a steroid structure that forms a practicable starting point in the synthesis of steroid hormones. The saponins of the invention can be any saponin as described above or saponin-like derivative with hydrophobic regions, especially the strongly polar saponins, primarily the polar triterpensaponins such as the polar acidic bisdesmosides, e.g. saponin extract from Quillajabark Araloside A, Chikosetsusaponin IV, Calendula-Glycoside C, chikosetsusaponin V, Achyranthes-Saponin B. Calendula-Glycoside A, Araloside B, Araloside C, Putranjia-Saponin III, Bersamasaponiside, Putrajia-Saponin IV, Trichoside A, Trichoside B, Saponaside A, Trichoside C, Gypsoside. Nutanoside, Dianthoside C, Saponaside D, aescine from Aesculus hippocastanum or sapoalbin from Gyposophilla struthium, preferably, saponin extract Quillaja saponaria Molina and Quil A. In addition, saponin may include glycosylated triterpenoid saponins derived from Quillaja Saponaria Molina of Beta Amytin type with 8-11 carbohydrate moieties as described in U.S. Pat. No. 5,679,354. Saponins as defined herein include saponins that may be combined with other materials, such as in an immune stimulating complex ("ISCOM")-like structure as described in U.S. Pat. No. 5,679,354. Saponins also include saponin-like molecules derived from any of the above structures, such as GPI-0100, such as described in U.S. Pat. No. 6,262,029.
[0095] Preferably, the saponins of the invention are amphiphilic natural products derived from the bark of the tree, Quillaia saponaria. Preferably, they consist of mixtures of triterpene glycosides with an average molecular weight (Mw) of 2000. A particularly preferred embodiment of the invention is a purified fraction of this mixture.
[0096] The most preferred embodiment of the invention is one or more GP95 proteins combined with ISCOMATRIX® or GPI-0100 to produce a vaccine formulation able to induce potent, safe, protective immune responses in vaccinated subjects, including members of the immunodeficient population.
Emulsion and Emulsion-Based Adjuvants
[0097] In another preferred embodiment, an emulsion or emulsion-based adjuvant, such as Montanide ISA-51, Co-Vaccine HT, or Ribi R-700, is added to the recombinant subunit GP protein, with or without additional structural proteins in the composition. Emulsions and emulsion-based vaccines such as those formulated with Montanide ISA-51 (see Example 3) are known in the art (Podda A. and G. DelGiudice, Expert Rev. Vaccines (2003) 2:197-203; Banzhoff A. et al., Gerontology (2003) 49:177-84) and are believed to function primarily through a depot effect. CoVaccine HT does not function in this manner. Rather it most likely functions as an immune modulator, since it contains carbohydrate moieties bound to micro-droplets of vegetable oil, which is thought to mimic the bacterial cell surface. Thus, while this adjuvant is physically an emulsion, its mode of action as an adjuvant is that of an immunomodulator. Ribi R-700 (or other adjuvants of the Ribi Adjuvant System, (GlaxoSmithKline, Philadelphia, Pa.) contains highly purified microbial cell wall constituents such as monophosphoryl lipid A (MPL) and Synthetic Trehalose Dicorynomycolate (TDM) in an emulsion of metabolizable oil. Like CoVaccine HT, Ribi R-700 also demonstrates a immunomodulatory effect in addition to the depot and antigen presentation advantages of an emulsion.
[0098] While reports in the literature (Podda and DelGiudice; Banzhoff et al.) have suggested that addition of emulsions such as MF59 to subunit vaccines may overcome some of the immune difficulties in the elderly, side by side evaluation with another unadjuvanted, subunit-like (split influenza) vaccine formulation failed to show an added benefit (Ruf et al.), resulting in a lack of medical endorsement for the use of the MF59 adjuvanted vaccine (Prescrire Int.). Addition of carbohydrates or immunostimulatory molecules to an emulsion (e.g., Co-Vaccine HT) is believed to further enhance the adjuvant effect through more effective stimulation of antigen presenting cells as described above. Thus, the combination of an emulsion-based adjuvant containing additional immunostimulatory molecules with a conformationally relevant recombinant Filovirus surface glycoprotein produces a particularly potent vaccine composition. In a highly preferred embodiment, ZEBOV GP95 is combined with Co-Vaccine HT to produce a vaccine formulation able to induce potent, protective immune responses in vaccinated subjects.
[0099] Alum
[0100] In a preferred embodiment, the recombinant Filovirus surface glycoprotein, with or without additional structural proteins in the composition, is formulated with aluminum-based adjuvants (collectively, "alum" or "alum-based adjuvants") such as aluminum hydroxide, aluminum phosphate, or a mixture thereof. Aluminum-based adjuvants remain the only adjuvants currently registered for human use in the United States and their effectiveness is widely recognized. Aluminum-based adjuvants are believed to function via a depot mechanism and the combination of the conformationally relevant Filovirus surface glycoprotein antigen with the depot effect is sufficient to induce a potent immune response in vaccinated individuals.
Dipeptidyl Peptidase Inhibitors
[0101] Dipeptidyl Peptidase Inhibitors are synthetic molecules that inhibit the dipeptidyl peptidase (DPP) family of serine proteases. These enzymes digest proteins found naturally in the body and regulate tumor growth and immune responses. Inhibition of certain DPPs appears to stimulate the immune system to mount an attack against infectious agents or tumors. Inhibition of DPP 8/9 in monocytes has been shown to induce the release of IL-1β, a key molecule stimulating immune responses, which then stimulates cytokine and chemokine production (www.pther.com).
[0102] The cytokines and chemokines involved in the response to orally administered DPP inhibitors are known to promote the body's own anti-tumor defenses, including the activity of T-cells, neutrophils, monocytes/macrophages and natural killer cells. Based on the same mechanism, the responses to externally administered antigens can be enhanced.
Administration and Use
[0103] The described invention thus concerns and provides a means for preventing or attenuating infection by Filovirus. As used herein, a vaccine is said to prevent or attenuate a disease if administration of the vaccine to an individual results either in the total or partial immunity of the individual to the disease, or in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease.
[0104] A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
[0105] The active vaccines of the invention can be used alone or in combination with other active vaccines such as those containing other active subunits or formulations containing or expressing homologous or heterologous immunogens. Corresponding or different subunits from one or several serotypes may be included in a particular formulation.
[0106] The therapeutic compositions of the described invention can be administered parenterally by subcutaneous, intramuscular, intradermal, or transdermal (e.g., topical) application.
[0107] Many different techniques exist for the timing of the immunizations when a multiple administration regimen is utilized. It is preferable to use the compositions of the invention more than once to increase the levels and diversities of expression of the immunoglobulin repertoire as well as the population size of mature T-cells in the immunized subject. Typically, if multiple immunizations are given, they will be given one to two months apart.
[0108] To immunize subjects against Filovirus-induced disease for example, the vaccines containing the subunit(s) are administered to the subject in conventional immunization protocols involving, usually, multiple administrations of the vaccine. Administration is typically by injection, typically intramuscular or subcutaneous injection; however, other modes of administration may also be employed.
[0109] According to the described invention, an "effective amount" of a therapeutic composition is one which is sufficient to achieve a desired biological effect. Generally, the dosage needed to provide an effective amount of the composition will vary depending upon such factors as the subject's age, condition, sex, and extent of disease, if any, and other variables which can be adjusted by one of ordinary skill in the art. The antigenic preparations of the invention can be administered by either single or multiple dosages of an effective amount. Effective amounts of the compositions of the invention can vary from 0.01-500 μg per dose, more preferably from 0.1-20 μg per dose, and most preferably 1-5 μg per dose.
EXAMPLES
Example 1
Cloning of Filovirus Structural Genes and Expression in Insect Cells
[0110] Cloning of Required Genes into Vectors for Expression in Insect Cells
[0111] Sequences for expression of four selected Ebola Zaire virus proteins nucleoprotein NP, glycoprotein GP and the small viral proteins VP24 and VP40 were accessed from Genbank (www.ncbi.nlm.nih.gov/Genbank/index.html) Ebola Zaire, Maying a strain; accession number NC--002549. PCR amplification used specially designed DNA primers and cDNA plasmids to prepare the properly positioned inserts containing required restriction sites and stop codons. This allowed directional cloning into the selected expression vectors (pMT/BiPN5-His A (with BiP secretion signal) and pMTN5-His A (no secretion signal), Invitrogen Corp., www.invitrogen.com). All plasmids contained the Drosophila metallothionein promoter to allow inducible protein expression in insect cells.
[0112] The endogenous secretion signal (first 32 amino acids) of the surface glycoprotein GP gene was removed to allow for standardized expression conditions in all constructs using the well characterized BiP secretion signal. Native full-length GP contains a membrane anchor at the C-terminal end. In one construct the full-length GP was cloned. In another construct, the membrane anchor was removed in order to achieve maximum secretion of the expressed protein (last 29 amino acids deleted) with the resulting product comprising 95% of the full-length GP protein and being referred to as GP95. As NP and VP40 are not normally secreted proteins, expression was evaluated from both a secreted and non-secreted construct. Table 1 summarizes the cloned coding sequences.
[0113] For details about the preparation of the expression plasmids and use in the Drosophila expression system, see commonly assigned U.S. Pat. Nos. 6,165,477; 6,416,763; 6,432,411; and 6,749,857, the contents of which are fully incorporated herein by reference. Unless otherwise defined herein, the definitions of terms used in such commonly assigned patents and related to the Drosophila expression system shall apply herein. The DNA sequences cloned into the plasmids in such commonly assigned patents are, of course, different from, and superseded by, the cloned Filovirus sequences disclosed herein.
TABLE-US-00001 TABLE 1 Summary of cloned genomic sequences for expression of Ebola subunit proteins Cloned genomic Expressed subunit Ebola subunit information protein (based on protein Plasmid (position in bp)* wildtype protein) Glycoprotein pMT/BiP-EboZ-GP95 6135-7979 amino acids 33-647 (GP95) (secreted) (edited) Nucleoprotein (NP) pMT/BiP-EboZ-NP 470-2689 Full length (secreted) VP24 (secreted) pMT/BiP-EboZ-VP24 10346-11101 Full length VP40 (secreted) pMT/BiP-EboZ-VP40 4479-5459 Full length Glycoprotein Full- pMT/BiP-EboZ-GP-FL 6135-8069 amino acids 33-676 length (GP-FL) (edited) (secreted) Nucleoprotein (NP pMT-EboZ-NP-NS 470-2689 Full length NS) (not secreted) VP40 NS (not pMT-EboZ-VP40-NS 4479-5459 Full length secreted) *Basepair numbers listed are based on Genbank accession number NC_002549)
[0114] Stable Transformation of Drosophila S2 Cells and Analysis of Protein Expression
[0115] Drosophila S2 cells adapted to ExCell 420 (SAFC; www.sigmaaldrich.com) were co-transformed with one of the expression plasmids for Ebola Zaire VP40 (secreted or non-secreted), VP24, NP (secreted or non-secreted) or GP (95% or full-length) and one of the selectable marker plasmids pCoHygro or pCoBlast (Invitrogen Corp.; www.invitrogen.com) using the calcium phosphate coprecipitation method. Stable expression of each individual subunit was verified in at least 1-2 duplicate cell lines. Samples of culture medium following induction with 200 μM CuSO4 were evaluated by polyacrylamide gel electrophoresis followed by Coomassie staining and Western Blot. Blots were probed with anti-Ebola HMAF (polyvalent hyperimmune mouse ascitic fluid) or individual monoclonal antibodies reactive against Ebola Zaire virus proteins. HMAF (raised in ICR mice immunized with irradiated SEBOV, ZEBOV and REBOV) reacted with all expressed Ebola subunit proteins. Some background reactions were observed when HMAF was used, whereas monoclonal antibodies detected only the appropriate subunits.
[0116] A summary of expression yields and product properties can be found in Table 2.
TABLE-US-00002 TABLE 2 Summary of Expression for Recombinant Ebola Virus Proteins Ebola Aminoacid Predicted Estimated Subunit sequence molecular expression Protein contained* weight level Comments on expressed protein VP40 1-326 (full 35.4 kDa 10-20 mg/L Uniform expression pattern of major (secreted) length) (>90%) and minor (<10%) band of singly glycosylated product. Potentially differentially folded products. VP24 1-251 (full 28.5 kDa 10-20 mg/L Product secreted as three glycoforms. (secreted) length) NP 1-739 (full 83.6 kDa 1-2 mg/L Majority of the expression product is (secreted) length) secreted, various product sizes in supernatant, single band product in cell fraction. GP95 33-647 67.6 kDa 10 mg/L Single band suggests uniform (secreted) glycosylation status of the product. VP40 NS 1-326 (full 35.2 kDa 1-5 mg/L Approximately half of the protein is (Non- length) retained within cells, the rest can be secreted) found in culture supernatant. No glycosylation and much lower expression level compared to secreted VP40. NP NS 1-739 (full 83.3 kDa 1-5 mg/L >90% retained inside the cells, one (Non- length) uniform size of product of around 100 Secreted) kDa. Higher yield than when expressed in secreted form. GP-FL 33-676 70.9 kDa 1-5 mg/L Majority of protein is secreted as a single size product. Expression level is lower than for truncated GP. *Position based on native Ebola virus proteins as contained in Genbank NC_002549
[0117] Production of Soluble Secreted Ebola VP40, VP24, GP95 and NP Proteins
[0118] The cell lines expressing secreted forms of VP40, VP24, GP95 or NP were scaled up and several 400 ml spinner flask cultures or a stirred-tank Bioreactor were inoculated at 1-2×106 cells/ml. Expression in each culture flask was induced after 48 hours by adding CuSO4 to the media. Culture supernatants were harvested on day 7. Expression was verified by polyacrylamide gel electrophoresis and Western Blot. FIG. 2 shows a Coomassie stained PAGE gel containing expression products from selected spinner flask culture supernatants.
Example 2
Purification of Recombinant Filovirus Proteins
[0119] Monoclonal antibodies specific to individual Ebola virus proteins obtained from United States Army Medical Research Institute for Infectious Diseases ("USAMRIID") (Table 3) were coupled to NHS-sepharose (Amersham; www.amershambiosciences.com) at ratios of 10 mg to 1 ml of matrix. Culture medium was loaded directly onto the immunoaffinity chromatography (IAC) columns and bound protein was eluted with 20 mM glycine buffer at pH 2.5 after a PBS wash. Eluates were neutralized and buffer exchanged into PBS using ultrafiltration devices (Centriplus, Millipore; www.millipore.com). Final products were filtered using 0.2 μm syringe filters and protein content determined by UV absorption and gel-electrophoresis. Silver staining of final gels was used to determine the approximate purity of proteins (FIGS. 3, 4 and 5). Yields and purity of each protein are summarized in Table 4.
TABLE-US-00003 TABLE 3 Monoclonal antibodies used for Ebola subunit purification Antibody specificity Antibody name VP24 Z-AC01-BG11-01 VP40 M-HD06-A10A GP 13C6-1-1 NP ZD04-AE8 NP FB03-BE-08
TABLE-US-00004 TABLE 4 Summary of purified soluble Ebola proteins Soluble Ebola protein Estimated Purity Amount purified VP24 >95% 13.9 mg GP95 (batch 1) >90% 10.5 mg GP95 (batch 2) >90% 12.1 mg GP95 (batch sw) >90% 37.9 mg VP40 (batch 1) 85-90% 3.55 mg VP40 (batch 2) 90% 2.8 mg VP40 (batch 3) 85-90% 3.8 mg
[0120] The glycosylation status of purified soluble VP40, VP24, NP and GP95 proteins was determined using PNGase (New England Biolabs, www.neb.com) or an enzymatic deglycosylation kit (E-DEGLY, Sigma, www.sigma-aldrich.com) to remove N- or O-linked sugars from the tested proteins. Molecular weight analysis of glycosylated versus non-glycosylated proteins was conducted on SDS-PAGE gels using Coomassie staining. This was correlated with results from gels probed with a glycoprotein in-gel stain kit (Invitrogen; www.probes.invitrogen.com) and Western blots probed with antibodies reactive to individual proteins (see FIGS. 6-9).
GP
[0121] For recombinant GP95 protein, a 10 ml immunoaffinity column (prepared with 107 mg of EGP13C6 MAb) was used to purify three batches from 800 ml (batch I), 1200 ml (batch 2) or 4000 ml (batch sw) culture supernatant as described above. Coomassie stained SDS-PAGE gels were used to analyze the product (FIG. 3). One major product can consistently be observed (>90% purity) in purified material of all batches. The size of the secreted product is estimated to be approximately 110 kDa (full length--see lane 8). In a reduced sample (lane 2), GP1 can be observed around 90 kDa and GP2 at approximately 20 kDa, indicating efficient post-translational processing by furin. Native protein gels as well as FPLC data (size exclusion chromatography) suggest that the majority of GP95 in solution is present as dimers and trimers. A Western blot probed with the conformationally sensitive antibody 13C6 is shown in FIG. 8.
[0122] Purified GP95 was also analyzed by MALDI-ToF. The molecular weight of the GP95 aglycon (monomer) is 89 kDa and the N-linked glycosylation adds an additional 21.4 kDa per monomer.
[0123] GP contains 7 potential N-linked glycosylation sites, 5 in the GP1 region and two in the GP2 region. N-linked glycosylations were confirmed in both regions by PNGase treatment. Analysis showed no evidence for O-linked glycosylation of the up to 21 potential O-glycosylation sites.
VP40
[0124] For the purification of 3 batches (lots #588-161, #609-73 and #619-23) of the recombinant VP40 protein a 2 ml immunoaffinity column (prepared with 19 mg of M-HD06-A10A MAb) was used. 800 to 1600 ml of culture supernatant were loaded directly onto the IAC column and VP40 eluted after a PBS wash. Silver stained gels were used to analyze the products of all three lots (FIG. 4). VP40 is secreted by S2 cells as two products that co-purify on the IAC column. The apparent sizes of the two VP40 bands on protein gels are 44 and 40 kDa. A Western blot probed with the Ebola Zaire VP40-specific monoclonal antibody A10A is shown in FIG. 7.
[0125] Both forms of secreted VP40 show a single N-linked sugar moiety as determined by PNGase digestion and consistently appear as a major and a minor band product on SDS-PAGE gels. Dimers and higher oligomers are observed as a "smear" above the major protein band under non-reducing conditions but disappear under reducing conditions (FIG. 4). Based on enzymatic deglycosylation, there is no indication of O-linked glycosylation in VP40 expressed and secreted from insect cells.
[0126] N-terminal sequencing of both of the VP40 bands showed efficient processing of the BiP secretion signal and the amino acid sequence of the first 10 amino acids was identical. This rules out that the alternate translation initiation site at M14 is being used by our insect cell line. This alternate expression product was confirmed when VP40 was expressed in mammalian cells (Jasenosky et al. 2001).
[0127] Purified VP40 (lot #588-161) was also analyzed by MALDI-ToF (Autoflex, Bruker Daltonics, www.bdal.com). It showed a molecular weight of 36.8 kDa for the secreted VP40 with one confirmed N-linked glycosylation. The theoretical weight of the aglyco-form is 35.4 kDa. Peptide mass fingerprint data suggests that the glycosylation site at Asn25 is processed. The second site at Asn222 is apparently not glycosylated.
VP24
[0128] For recombinant VP24 protein, a 10 ml immunoaffinity column (prepared with 93 mg of Z-AC1-BG11 MAb) was used to purify VP24 from 800 ml of culture supernatant. The purification scheme was as described above. Silver stained gels were used to analyze the product (FIG. 5). The apparent molecular weights for the three bands on this silver stained PAGE gel are 32, 29 and 26 kDa which can be attributed to the presence of single, double and triple glycosylated products. PNGase treatment results in reduction to one band of lower molecular weight (˜25 kDa) on SDS-PAGE. Purity of the product is estimated at >95%. A Western blot of purified VP24 probed with ZEBOV specific monoclonal antibody reactive against VP24 is shown in FIG. 9.
[0129] Purified VP24 (predicted molecular weight of polypeptide: 28.4 kDa) shows the following molecular weights by MALDI-ToF: 28.9, 29.9 and 30.9 kDa. The peptide mass fingerprint suggests that the protein is partially glycosylated at the predicted sites Asn86, Asn187 and Asn248.
NP
[0130] Both available NP reactive monoclonal antibodies were coupled to NHS-sepharose (approx. 100 mg of monoclonal antibody bound to 10 ml sepharose). Both columns were tested for their ability to purify NP recombinant protein. The binding efficiency of both NP columns was low compared to GP or VP24 columns. The Z-D04-AE8 column in comparison to the FB03-BE-08 column showed higher yields. The purification of recombinant NP proteins from S2 culture medium by this method resulted in product that was estimated to be below 50% in purity.
Example 3
Immunogenicity of Recombinant Ebola Virus Proteins in Balb/c Mice
[0131] The immunogenicity of the purified recombinant VP24, VP40, and GP95 proteins was tested in Balb/c mice. The candidate vaccines were formulated using two adjuvants with different modes of action. The saponin-based GPI-0100 (Hawaii Biotech, Inc.) provides a direct immuno-stimulatory and -modulatory effect, while Montanide ISA-51 (Seppic, www.seppic.com) is an oil-in-water emulsion that provides a depot effect. Mice were immunized by subcutaneous injection three times at 4-week intervals with the test vaccine formulations. Details of the experimental groups are summarized in Table 5 below. One week after the third dose, 5 mice from each group were splenectomized. The rest were terminally bled two weeks after the third dose. All mice were also tail-bled at weeks 2 and 6. Splenocytes were stimulated in vitro with antigen and analyzed for proliferative capacity and secretion of cytokines post-stimulation (by ELISA). Antigen specific antibody titers in the tail- and terminal bleeds were determined by ELISA using immunizing antigen or irradiated Ebola virus as coating antigen.
TABLE-US-00005 TABLE 5 Mouse immunogenicity study using soluble recombinant Ebola virus proteins Group No. of no. Adjuvant Immunogen mice Formulation Route 1 GPI-0100 10 μg GP95 15 100 μg of GPI-0100 is mixed with s.c. 10 μg antigen and PBS 2 ISA-51 10 μg GP95 15 50% ISA-51 is mixed into adequate s.c. volume of immunogen in PBS 3 GPI-0100 10 μg VP-40 10 100 μg of GPI-0100 is mixed with s.c. 10 μg antigen and PBS 4 ISA-51 10 μg VP-40 10 50% ISA-51 is mixed into adequate s.c. volume of immunogen in PBS 5 GPI-0100 10 μg VP-24 10 100 μg of GPI-0100 is mixed with s.c. 10 μg antigen and PBS 6 ISA-51 10 μg VP-24 10 50% ISA-51 is mixed into adequate s.c. volume of immunogen in PBS 7 GPI-0100 NONE 10 100 μg of GPI-0100 is mixed with s.c. PBS 8 ISA-51 NONE 10 50% ISA-51 is mixed into adequate s.c. volume of PBS
[0132] The results of the immunogenicity study are summarized in Table 6. The Stimulation Indices represent the fold-increase of tritiated thymidine uptake in antigen-stimulated cells divided by unstimulated control cells of the same animal. To allow comparison, data from adjuvant-only control groups stimulated with respective antigens has also been included. The ELISA titers are expressed as the GMT of individual EC50 titers based on sigmoid curve fitting on a logarithmic scale (using Origin 7.5 software, www.originlab.com).
[0133] The lymphoproliferation data reveals strong immune responses to all three recombinant Ebola proteins. The specific stimulation indices (SI) ranged from 2.2 up to 15.1 where an S13 is generally considered significant. Relatively high levels of cytokines (IL-4, IL-5, IL-10) were secreted by stimulated splenocytes, particularly in the groups receiving the GPI-0100 adjuvant.
[0134] Interferon gamma (IFN-γ) levels rose to levels clearly above control values and suggest that Th1 type responses have been induced with all three immunogens. Formulations with both adjuvants seem to induce approximately equal IFN-γ responses with all three antigens.
[0135] ELISA titers obtained with all three immunogens show that relatively high serum titers against the antigens were induced following 2 vaccine doses with further increase following administration of a 3rd dose.
TABLE-US-00006 TABLE 6 Summary of results of mouse immunogenicity study ELISA ELISA ELISA titer titer titer Vaccine Stimulation IL-4 IL-5 IL-10 IFN-γ (post (post (post formulation Index [ng/ml] [ng/ml] [ng/ml] [ng/ml] dose 1) dose 2) dose 3) GP95 15.1 ± 3.5 5.0 ± 0.5 10.9 ± 1.8 6.6 ± 1.0 5.8 ± 1.0 <250 7325 9949 (GPI-0100) GP95 10.6 ± 4.9 1.4 ± 0.1 2.1 ± 0.5 1.6 ± 0.4 5.5 ± 2.1 <250 934 3384 (ISA51) GPI-0100 1.0 ± 0.1 0.02 ± 0.00 0.06 ± 0.00 0.04 ± 0.00 0.15 ± 0.01 <250 <250 <250 control (GP stimulated) ISA51 control 1.1 ± 0.2 0.01 ± 0.01 0.06 ± 0.00 0.04 ± 0.00 0.14 ± 0.01 <250 <250 <250 (GP stimulated) VP40 3.0 ± 0.3 7.0 ± 1.3 20.2 ± 1.0 11.8 ± 1.4 6.3 ± 2.0 441 18699 21767 (GPI-0100) VP40 4.4 ± 1.0 2.3 ± 0.2 12.4 ± 2.5 8.8 ± 1.0 5.1 ± 0.4 275 5538 25866 (ISA51) GPI-0100 1.1 ± 0.2 0.01 ± 0.00 0.05 ± 0.00 0.04 ± 0.00 0.09 ± 0.03 <250 <250 <250 control (VP40 stimulated) ISA51 control 0.9 ± 0.1 0.00 ± 0.00 0.05 ± 0.00 0.04 ± 0.01 0.22 ± 0.14 <250 <250 <250 (VP40stimulated) VP24 6.2 ± 2.1 6.4 ± 0.9 23.8 ± 1.7 14.8 ± 1.7 5.4 ± 1.4 <250 3764 7780 (GPI-0100) VP24 2.2 ± 0.7 1.2 ± 0.3 9.5 ± 2.6 5.9 ± 1.4 6.6 ± 0.4 <250 1521 7662 (ISA51) GPI-0100 1.0 ± 0.1 0.02 ± 0.00 0.08 ± 0.02 0.05 ± 0.01 0.33 ± 0.14 <250 <250 <250 control (VP24 stimulated) ISAS1 control 0.9 ± 0.1 0.01 ± 0.01 0.06 ± 0.01 0.04 ± 0.00 0.16 ± 0.03 <250 <250 <250 (VP24 stimulated)
[0136] As described above, immune mouse sera from the first immunogenicity study were tested by ELISA using the recombinant proteins as coating antigens. In addition, the same sera were titered using irradiated Ebola virus particles as coating antigens. Table 7 compares the geometric mean titers of the highest dilution yielding an absorption value of 0.2 above background after color development.
TABLE-US-00007 TABLE 7 Comparison of ELISA titers in the same immune mouse sera obtained using different coating antigens: Recombinant protein antigens irradiated Ebola virus post post post post post post dose 1 dose 2 dose 3 dose 1 dose 2 dose 3 GP95 (GPI-0100) 250 27858 64000 <100 (33) 1397 3363 GP95 (ISA-51) <250 6964 16000 <100 (33) 155 580 VP24 (GPI-0100) <250 16000 36758 <100 (33) <100 (33) 51 VP24 (ISA-51) <250 9190 36758 <100 (33) 124 33 VP40 (GPI-0100) 2297 147033 256000 41 37710 30271 VP40 (ISA-51) 758 48503 256000 64 15659 30271 GPI-0100 control <250 <250 <250 <100 (33) <100 (33) 41 ISA-51 control <250 <250 <250 <100 (33) <100 (33) <100 (33)
[0137] The two methods yielded different relative titer values likely due to the different sources and composition of coating antigen. 0.375 μg of each individual recombinant protein (either GP95, VP40 or VP24) was applied per well resulting in a controlled amount of protein. In contrast, when irradiated virus was used as coating antigen, the relative amounts of each viral protein reflected the composition of the virus particles. VP40 is the major protein in virus particles. High titers in the assay can be attributed to its abundance of about 40% of the total virus protein. In contrast, GP is the major surface protein, but represents only 2-5% of the particle mass. Therefore, relatively less GP protein is contained in each virus-coated well, perhaps resulting in lower overall titers. Similarly, VP24 is only a minor component (2-5%) of virions and in addition resides exclusively inside the particle. It therefore would not be unexpected that anti-VP24 ELISA titers measured against whole virus would be lower than the anti-VP40 and anti-GP titers. The results show that immunization with Ebola proteins expressed in Drosophila S2 cells results in production of high titer antibodies that are reactive with intact viral particles.
Example 4
Optimization of Ebola Vaccine Formulations Based on Recombinant Proteins
[0138] The major vaccine candidate protein (GP95) was tested at 1, 3 or 9 pg formulated with three adjuvants (GPI-0100 (Hawaii Biotech, Inc.), CoVaccine HT® (CoVaccine, www.covaccine.com) and Ribi-700 (GlaxoSmithKline, Philadelphia, Pa.) while VP40 was tested only at 10 μg per dose using the same three adjuvants. 10 Balb/c mice per group were vaccinated three times at four-week intervals (Table 8). Tail bleeds were collected 2 weeks after the first and second immunizations. Three mice were splenectomized 4 days following the third dose, three on day seven. The remaining four mice from each group were terminally bled 14 days post third immunization.
TABLE-US-00008 TABLE 8 Dose/adjuvant formulation study groups: Group no. Adjuvant Immunogen No. of mice Formulation Adm. 1 GPI-0100 1 μg GP95 10 250 μg of GPI-0100 is mixed with 2 sites s.c. antigen and PBS 2 CoVaccine 1 μg GP95 10 0.1 ml of CoVaccine HT is mixed 2 sites s.c. HT with 0.1 ml of immunogen in PBS 3 RIBI R-700 1 μg GP95 10 Prepare formulation according to 2 sites s.c. manufacturer's instructions 4 GPI-0100 3 μg GP95 10 250 μg of GPI-0100 is mixed with 2 sites s.c. antigen and PBS 5 CoVaccine 3 μg GP95 10 0.1 ml of CoVaccine HT is mixed 2 sites s.c. HT with 0.1 ml of immunogen in PBS 6 RIBI R-700 3 μg GP95 10 Prepare formulation according to 2 sites s.c. manufacturer's instructions 7 GPI-0100 9 μg GP95 10 250 μg of GPI-0100 is mixed with 2 sites s.c. antigen and PBS 8 CoVaccine 9 μg GP95 10 0.1 ml of CoVaccine HT is mixed 2 sites s.c. HT with 0.1 ml of immunogen in PBS 9 RIBI R-700 9 μg GP95 10 Prepare formulation according to 2 sites s.c. manufacturer's instructions 10 GPI-0100 10 μg VP-40 10 250 μg of GPI-0100 is mixed with 2 sites s.c. antigen and PBS 11 CoVaccine 10 μg VP-40 10 0.1 ml of CoVaccine HT is mixed 2 sites s.c. HT with 0.1 ml of immunogen in PBS 12 RIBI R-700 10 μg VP-40 10 Prepare formulation according to 2 sites s.c. manufacturer's instructions 13 GPI-0100 NONE 10 250 μg of GPI-0100 is mixed with 2 sites s.c. PBS 14 CoVaccine NONE 10 0.1 ml of CoVaccine HT is mixed 2 sites s.c. HT with 0.1 ml of PBS 15 RIBI R-700 NONE 10 Prepare formulation according to 2 sites s.c. manufacturer's instructions
[0139] Analysis of antibodies raised (ELISA titers) of the combined dose/adjuvant study are shown in Table 9. Titers are expressed as the dilution yielding the half maximal absorption value (determined by using a sigmoidal curve fitting algorithm). Homologous antigen preparations (GP95 or VP40) from the same lots used for immunizations were used as coating antigens.
TABLE-US-00009 TABLE 9 Summary of ELISA titers from the dose/adjuvant study Recombinant GP95 as Recombinant VP40 as coating antigen coating antigen Post Post Post Post Post Post dose dose dose dose dose dose Vaccine formulation 1 2 3 1 2 3 1 μg GP95 <250 4097 24644 ND ND ND (250 μg GPI-0100) 3 μg GP95 <250 6215 16577 ND ND ND (250 μg GPI-0100) 9 μg GP95 <250 10403 18627 ND ND ND (250 μg GPI-0100) 1 μg GP95 <250 <250 1179 ND ND ND (CoVaccine HT) 3 μg GP95 <250 581 7588 ND ND ND (CoVaccine HT) 9 μg GP95 <250 1345 7297 ND ND ND (CoVaccine HT) 1 μg GP95 <250 <250 <250 ND ND ND (Ribi R-700) 3 μg GP95 <250 <250 696 ND ND ND (Ribi R-700) 9 μg GP95 <250 <250 2913 ND ND ND (Ribi R-700) 10 μg VP40 (250 ND ND ND 906 22732 57638 μg GPI-0100) 10 μg VP40 ND ND ND 697 9023 22689 (CoVaccine HT) 10 μg VP40 (Ribi ND ND ND 157 9429 24045 R-700) Control (250 μg <250 <250 <250 <250 <250 <250 GPI-0100) Control <250 <250 <250 <250 <250 <250 (CoVaccine HT) Control <250 <250 <250 <250 <250 <250 (Ribi R-700) *(ND: titer not determined)
[0140] Table 10 shows the cytokine secretion data obtained from antigen-stimulated immune splenocytes. It also lists the Stimulation Index expressed as the fold-increase of tritiated thymidine uptake in antigen-stimulated cells divided by unstimulated control cells of the same animal. Data from adjuvant-only control groups is included, stimulated with the antigen shown. Results shown are the mean values with standard deviation obtained using splenocyte preparations from three individual mice per group harvested on day 4 (upper value) and 7 (lower value) post 3rd dose of vaccine.
TABLE-US-00010 TABLE 10 Summary of Cellular Responses Elicited During the Dose/Adjuvant Study Stimulation IL-4 IL-5 TNF-α IFN-γ Vaccine formulation index [ng/ml] [ng/ml] [ng/ml] [ng/ml] 1 μg GP95 4.3 ± 3.6 0.9 ± 1.2 8.3 ± 1.2 1.0 ± 0.3 3.8 ± 2.2 (250 μg GPI-0100) 3 μg GP95 6.5 ± 3.6 1.7 ± 1.8 7.4 ± 1.3 1.3 ± 0.7 4.6 ± 0.4 (250 μg GPI-0100) 9 μg GP95 5.7 ± 3.3 1.3 ± 1.3 3.3 ± 2.9 1.2 ± 1.6 2.6 ± 2.5 (250 μg GPI-0100) 1 μg GP95 9.7 ± 5.6 0.0 ± 0.0 2.8 ± 0.5 1.4 ± 0.1 3.4 ± 0.3 (CoVaccine HT) 3 μg GP95 (CoVaccine 5.7 ± 0.8 0.0 ± 0.0 3.3 ± 1.2 1.4 ± 0.3 3.5 ± 0.7 HT) 9 μg GP95 (CoVaccine 6.1 ± 1.7 2.3 ± 3.4 3.9 ± 3.0 1.4 ± 1.0 3.4 ± 1.5 HT) 1 μg GP95 1.8 ± 0.7 0.0 ± 0.0 0.3 ± 0.5 1.4 ± 1.0 2.8 ± 2.2 (Ribi R-700) 3 μg GP95 33.0 ± 34.0 0.0 ± 0.0 0.3 ± 0.4 1.0 ± 1.6 1.7 ± 2.8 (Ribi R-700) 9 μg GP95 18.7 ± 6.9 0.0 ± 0.0 0.1 ± 0.1 0.3 ± 0.3 0.3 ± 0.3 (Ribi R-700) Control (250 μg GPI- 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0100) GP95 stim. Control (CoVaccine 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 HT) GP95 stimulated Control (Ribi R-700) 1.1 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 GP95 stimulated 10 μg VP40 (250 μg 2.9 ± 0.8 4.2 ± 3.6 6.2 ± 2.1 0.9 ± 0.7 1.9 ± 2.2 GPI-0100) 10 μg VP40 20.9 ± 7.9 3.7 ± 3.6 4.8 ± 2.6 0.7 ± 0.4 1.6 ± 0.4 (CoVaccine HT) 10 μg VP40 26.5 ± 15.0 0.0 ± 0.0 0.8 ± 1.5 0.0 ± 0.0 0.0 ± 0.0 (Ribi R-700) Control (GPI-0100) 1.2 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 VP40 stim. Control (CoVaccine 1.6 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 HT) VP40 stim. Control (Ribi R-700) 1.4 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 VP40 stimulated
[0141] Overall the GPI-0100 adjuvant shows a strong advantage with both antigens compared to other formulations for both humoral and cellular responses. Based on the same immunological markers, CoVaccine HT appears to have a moderate effect. The responses elicited by formulations containing Ribi R-700 (especially based on cytokine responses) are the lowest. As seen in the previous immunogenicity study, the kinetics of the response to VP40 are more rapid compared to the other antigens.
[0142] Splenocytes from immunized mice receiving the leading vaccine formulations (GP95 and VP40 formulated with GPI-0100) harvested on day 4 post third dose were cultured at a final concentration of 2×106/ml in the presence (or absence) of either GP95 or VP40 antigens or Pokeweed mitogen (PWM) at final concentrations of 5 μg/ml for 18 hrs. Cells were then harvested, washed, and stained with fluorochrome-conjugated antibodies to stain for cell surface (CD3 or CD19) and activation markers (CD69), and then analyzed by flow cytometry.
[0143] The data shown in FIG. 10 demonstrates that the total percentage of CD19+ B-cells stayed approximately the same regardless of the treatment (none, PWM, or antigen). Addition of Pokeweed mitogen activated (CD69+) about 40% of the cells in all mice tested. In contrast to the control groups (GPI-0100 only), the cells of the four mice immunized with recombinant Ebola antigens showed a substantial increase (five fold above background or more) in expression of surface activation marker CD69 in response to in vitro antigen stimulation. This suggests that the tested vaccine formulations induce strong cellular memory in vaccinated Balb/c mice.
Example 5
Protective Efficacy of Ebola Vaccine Candidates in the Mouse Challenge Model for Ebola Zaire
[0144] Vaccine formulations containing 10 μg of either GP95, VP40, or VP24 as individual antigens or the mix of all three, unadjuvanted or in combination with the adjuvants GPI-0100, CoVaccine HT, Talabostat or PT-510 (both from Point Therapeutics, Inc., Boston, Mass.) were tested at USAMRIID in the mouse challenge model for protection against Ebola Zaire virus. GPI-0100 as well as CoVaccine HT were co-administered to animals in injectable formulations, while Talabostat (10 μg doses) and PT-510 (80 μg doses) were given by oral gavage on days -1, 0 and +1 relative to injection of antigen preparation (these animals received antigen preparations in PBS). Three doses of vaccine were administered subcutaneously in 4-week intervals and yielded high serum antibody titers in all vaccinated mice (table 11). VP40 showed excellent immunogenicity which can be seen in the ELISA titers raised by unadjuvanted formulations in groups 11 (even after one dose). GP95 alone (group 1) showed a similar immunogenicity as VP40 while VP24 without adjuvant is raising moderate ELISA antibody titers. The efficacy against virus challenge by i.p. injection of 100 pfu of mouse adapted Ebola virus approximately two weeks after the last dose is also shown in table 11. The column showing survivors lists animals that remained healthy for up to 20 days after challenge (when the experiment was terminated) and all surviving mice in groups 3, 4, 5, 16, 17, 18, 19 and 20 remained healthy throughout the study. In contrast, survivors in groups 1, 2, 8, 10 and 22 developed signs of disease for at least one day during the experiment. The vaccine formulation containing Ebola Zaire GP95 with CoVaccine HT as well as Talabostat or PT-510 completely protected all of the animals from both morbidity and mortality. Vaccine formulations containing all three antigens (GP95, VP40 and VP24) and either of the adjuvants showed the same level of protection. Furthermore, the combination of the three proteins without adjuvant, completely protected 9/10 animals. GP95 alone or formulated with GPI-0100 partially protected the animals against death but not morbidity. Interestingly, 6 of 10 animals in the group vaccinated with VP24 in formulation with CoVaccine HT as well as two animals receiving the antigens and oral doses of PT-510 survived the challenge. In contrast, all animals receiving VP40 alone with or without adjuvant succumbed to the infection despite very high levels of serum antibodies.
[0145] In combination, these results demonstrate that vaccine formulations containing recombinant Ebola subunit antigens produced from stably transformed insect cell lines have the potential to be used as efficacious vaccine products, even without adjuvant.
TABLE-US-00011 TABLE 11 Vaccine formulations and results of the mouse challenge study No. Day 20 (Jul. 05, 2005) Group of ELISA titers Percent no. Adjuvant Immunogen mice first second third dead healthy alive 1 NONE 10 μg GP95 10 155 520 14030 3 7 70% all were sick 2 GPI-0100 10 μg GP95 10 100 1251 19507 1 9 90% all were sick 3 CoVaccine 10 μg GP95 10 300 58520 65315 0 10 100% HT 4 Talabostat 10 μg GP95 10 90 14030 72900 0 9 100% 5 PT510 10 μg GP95 10 100 14913 72900 0 9 100% 6 NONE 10 μg VP24 10 72 139 580 10 0 0% 7 GPI-0100 10 μg VP24 10 46 89 579 10 0 0% 8 CoVaccine 10 μg VP24 10 155 27,122 37,710 4 6 60% HT all were sick 9 Talabostat 10 μg VP24 10 193 1251 12570 10 0 0% 10 PT510 10 μg VP24 10 193 335 2419 8 2 (were 20% sick) 11 NONE 10 μg VP40 10 100 139 24,300 10 0 0% 12 GPI-0100 10 μg VP40 10 80 33,786 101,359 10 0 0% 13 CoVaccine 10 μg VP40 10 3,754 1,415,647 1,968,300 10 0 0% HT 14 Talabostat 10 μg VP40 10 269 647 244096 10 0 0% 15 PT510 10 μg VP40 10 193 580 339389 10 0 0% 16 NONE mix 10 155 900 21,772 1 9 90% 17 GPI-0100 mix 10 112 33,786 101,359 0 10 100% 18 CoVaccine mix 10 1,740 378,800 656,100 0 10 100% HT 19 Talabostat mix 10 235 8100 134213 0 9 100% 20 PT510 mix 10 128 4971 93058 0 9 100% 21 NONE NONE 9 64 72 72 9 0 0% 22 GPI-0100 NONE 10 51 46 57 9 1 10% Was sick 23 CoVaccine NONE 9 128 208 266 9 0 0% HT 24 Talabostat NONE 10 100 111 139 10 0 0% 25 PT510 NONE 10 125 100 112 10 0 0%
Sequence CWU
1
211617PRTEbola Zaire Virus 1Arg Ser Ile Pro Leu Gly Val Ile His Asn Ser
Thr Leu Gln Val Ser1 5 10
15Asp Val Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln
20 25 30Leu Arg Ser Val Gly Leu Asn
Leu Glu Gly Asn Gly Val Ala Thr Asp 35 40
45Val Pro Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro
Pro 50 55 60Lys Val Val Asn Tyr Glu
Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn65 70
75 80Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu Cys
Leu Pro Ala Ala Pro 85 90
95Asp Gly Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser
100 105 110Gly Thr Gly Pro Cys Ala
Gly Asp Phe Ala Phe His Lys Glu Gly Ala 115 120
125Phe Phe Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg
Gly Thr 130 135 140Thr Phe Ala Glu Gly
Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys145 150
155 160Lys Asp Phe Phe Ser Ser His Pro Leu Arg
Glu Pro Val Asn Ala Thr 165 170
175Glu Asp Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala
180 185 190Thr Gly Phe Gly Thr
Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn 195
200 205Leu Thr Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro
Gln Phe Leu Leu 210 215 220Gln Leu Asn
Glu Thr Ile Tyr Thr Ser Gly Lys Arg Ser Asn Thr Thr225
230 235 240Gly Lys Leu Ile Trp Lys Val
Asn Pro Glu Ile Asp Thr Thr Ile Gly 245
250 255Glu Trp Ala Phe Trp Glu Thr Lys Lys Asn Leu Thr
Arg Lys Ile Arg 260 265 270Ser
Glu Glu Leu Ser Phe Thr Val Val Ser Asn Gly Ala Lys Asn Ile 275
280 285Ser Gly Gln Ser Pro Ala Arg Thr Ser
Ser Asp Pro Gly Thr Asn Thr 290 295
300Thr Thr Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser Ala Met305
310 315 320Val Gln Val His
Ser Gln Gly Arg Glu Ala Ala Val Ser His Leu Thr 325
330 335Thr Leu Ala Thr Ile Ser Thr Ser Pro Gln
Ser Leu Thr Thr Lys Pro 340 345
350Gly Pro Asp Asn Ser Thr His Asn Thr Pro Val Tyr Lys Leu Asp Ile
355 360 365Ser Glu Ala Thr Gln Val Glu
Gln His His Arg Arg Thr Asp Asn Asp 370 375
380Ser Thr Ala Ser Asp Thr Pro Ser Ala Thr Thr Ala Ala Gly Pro
Pro385 390 395 400Lys Ala
Glu Asn Thr Asn Thr Ser Lys Ser Thr Asp Phe Leu Asp Pro
405 410 415Ala Thr Thr Thr Ser Pro Gln
Asn His Ser Glu Thr Ala Gly Asn Asn 420 425
430Asn Thr His His Gln Asp Thr Gly Glu Glu Ser Ala Ser Ser
Gly Lys 435 440 445Leu Gly Leu Ile
Thr Asn Thr Ile Ala Gly Val Ala Gly Leu Ile Thr 450
455 460Gly Gly Arg Arg Thr Arg Arg Glu Ala Ile Val Asn
Ala Gln Pro Lys465 470 475
480Cys Asn Pro Asn Leu His Tyr Trp Thr Thr Gln Asp Glu Gly Ala Ala
485 490 495Ile Gly Leu Ala Trp
Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile 500
505 510Tyr Ile Glu Gly Leu Met His Asn Gln Asp Gly Leu
Ile Cys Gly Leu 515 520 525Arg Gln
Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg 530
535 540Ala Thr Thr Glu Leu Arg Thr Phe Ser Ile Leu
Asn Arg Lys Ala Ile545 550 555
560Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro
565 570 575Asp Cys Cys Ile
Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys 580
585 590Ile Asp Gln Ile Ile His Asp Phe Val Asp Lys
Thr Leu Pro Asp Gln 595 600 605Gly
Asp Asn Asp Asn Trp Trp Thr Gly 610 61521875DNAEbola
Zaire Virus 2acgtacagat ctatcccact tggagtcatc cacaatagca cattacaggt
tagtgatgtc 60gacaaactag tttgtcgtga caaactgtca tccacaaatc aattgagatc
agttggactg 120aatctcgaag ggaatggagt ggcaactgac gtgccatctg caactaaaag
atggggcttc 180aggtccggtg tcccaccaaa ggtggtcaat tatgaagctg gtgaatgggc
tgaaaactgc 240tacaatcttg aaatcaaaaa acctgacggg agtgagtgtc taccagcagc
gccagacggg 300attcggggct tcccccggtg ccggtatgtg cacaaagtat caggaacggg
accgtgtgcc 360ggagactttg ccttccataa agagggtgct ttcttcctgt atgatcgact
tgcttccaca 420gttatctacc gaggaacgac tttcgctgaa ggtgtcgttg catttctgat
actgccccaa 480gctaagaagg acttcttcag ctcacacccc ttgagagagc cggtcaatgc
aacggaggac 540ccgtctagtg gctactattc taccacaatt agatatcagg ctaccggttt
tggaaccaat 600gagacagagt acttgttcga ggttgacaat ttgacctacg tccaacttga
atcaagattc 660acaccacagt ttctgctcca gctgaatgag acaatatata caagtgggaa
aaggagcaat 720accacgggaa aactaatttg gaaggtcaac cccgaaattg atacaacaat
cggggagtgg 780gccttctggg aaactaaaaa aaacctcact agaaaaattc gcagtgaaga
gttgtctttc 840acagttgtat caaacggagc caaaaacatc agtggtcaga gtccggcgcg
aacttcttcc 900gacccaggga ccaacacaac aactgaagac cacaaaatca tggcttcaga
aaattcctct 960gcaatggttc aagtgcacag tcaaggaagg gaagctgcag tgtcgcatct
aacaaccctt 1020gccacaatct ccacgagtcc ccaatccctc acaaccaaac caggtccgga
caacagcacc 1080cataatacac ccgtgtataa acttgacatc tctgaggcaa ctcaagttga
acaacatcac 1140cgcagaacag acaacgacag cacagcctcc gacactccct ctgccacgac
cgcagccgga 1200cccccaaaag cagagaacac caacacgagc aagagcactg acttcctgga
ccccgccacc 1260acaacaagtc cccaaaacca cagcgagacc gctggcaaca acaacactca
tcaccaagat 1320accggagaag agagtgccag cagcgggaag ctaggcttaa ttaccaatac
tattgctgga 1380gtcgcaggac tgatcacagg cgggagaaga actcgaagag aagcaattgt
caatgctcaa 1440cccaaatgca accctaattt acattactgg actactcagg atgaaggtgc
tgcaatcgga 1500ctggcctgga taccatattt cgggccagca gccgagggaa tttacataga
ggggctaatg 1560cacaatcaag atggtttaat ctgtgggttg agacagctgg ccaacgagac
gactcaagct 1620cttcaactgt tcctgagagc cacaactgag ctacgcacct tttcaatcct
caaccgtaag 1680gcaattgatt tcttgctgca gcgatggggc ggcacatgcc acattctggg
accggactgc 1740tgtatcgaac cacatgattg gaccaagaac ataacagaca aaattgatca
gattattcat 1800gattttgttg ataaaaccct tccggaccag ggggacaatg acaattggtg
gacaggatag 1860taactcgagg tacgt
187531851DNAEbola Zaire Virus 3atcccacttg gagtcatcca
caatagcaca ttacaggtta gtgatgtcga caaactagtt 60tgtcgtgaca aactgtcatc
cacaaatcaa ttgagatcag ttggactgaa tctcgaaggg 120aatggagtgg caactgacgt
gccatctgca actaaaagat ggggcttcag gtccggtgtc 180ccaccaaagg tggtcaatta
tgaagctggt gaatgggctg aaaactgcta caatcttgaa 240atcaaaaaac ctgacgggag
tgagtgtcta ccagcagcgc cagacgggat tcggggcttc 300ccccggtgcc ggtatgtgca
caaagtatca ggaacgggac cgtgtgccgg agactttgcc 360ttccataaag agggtgcttt
cttcctgtat gatcgacttg cttccacagt tatctaccga 420ggaacgactt tcgctgaagg
tgtcgttgca tttctgatac tgccccaagc taagaaggac 480ttcttcagct cacacccctt
gagagagccg gtcaatgcaa cggaggaccc gtctagtggc 540tactattcta ccacaattag
atatcaggct accggttttg gaaccaatga gacagagtac 600ttgttcgagg ttgacaattt
gacctacgtc caacttgaat caagattcac accacagttt 660ctgctccagc tgaatgagac
aatatataca agtgggaaaa ggagcaatac cacgggaaaa 720ctaatttgga aggtcaaccc
cgaaattgat acaacaatcg gggagtgggc cttctgggaa 780actaaaaaaa acctcactag
aaaaattcgc agtgaagagt tgtctttcac agttgtatca 840aacggagcca aaaacatcag
tggtcagagt ccggcgcgaa cttcttccga cccagggacc 900aacacaacaa ctgaagacca
caaaatcatg gcttcagaaa attcctctgc aatggttcaa 960gtgcacagtc aaggaaggga
agctgcagtg tcgcatctaa caacccttgc cacaatctcc 1020acgagtcccc aatccctcac
aaccaaacca ggtccggaca acagcaccca taatacaccc 1080gtgtataaac ttgacatctc
tgaggcaact caagttgaac aacatcaccg cagaacagac 1140aacgacagca cagcctccga
cactccctct gccacgaccg cagccggacc cccaaaagca 1200gagaacacca acacgagcaa
gagcactgac ttcctggacc ccgccaccac aacaagtccc 1260caaaaccaca gcgagaccgc
tggcaacaac aacactcatc accaagatac cggagaagag 1320agtgccagca gcgggaagct
aggcttaatt accaatacta ttgctggagt cgcaggactg 1380atcacaggcg ggagaagaac
tcgaagagaa gcaattgtca atgctcaacc caaatgcaac 1440cctaatttac attactggac
tactcaggat gaaggtgctg caatcggact ggcctggata 1500ccatatttcg ggccagcagc
cgagggaatt tacatagagg ggctaatgca caatcaagat 1560ggtttaatct gtgggttgag
acagctggcc aacgagacga ctcaagctct tcaactgttc 1620ctgagagcca caactgagct
acgcaccttt tcaatcctca accgtaaggc aattgatttc 1680ttgctgcagc gatggggcgg
cacatgccac attctgggac cggactgctg tatcgaacca 1740catgattgga ccaagaacat
aacagacaaa attgatcaga ttattcatga ttttgttgat 1800aaaacccttc cggaccaggg
ggacaatgac aattggtgga caggatagta a 18514619PRTEbola Sudan
Virus 4Arg Ser Pro Trp Met Pro Leu Gly Val Val Thr Asn Ser Thr Leu Glu1
5 10 15Val Thr Glu Ile Asp
Gln Leu Val Cys Lys Asp His Leu Ala Ser Thr 20
25 30Asp Gln Leu Lys Ser Val Gly Leu Asn Leu Glu Gly
Ser Gly Val Ser 35 40 45Thr Asp
Ile Pro Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val 50
55 60Pro Pro Lys Val Val Ser Tyr Glu Ala Gly Glu
Trp Ala Glu Asn Cys65 70 75
80Tyr Asn Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Pro
85 90 95Pro Pro Asp Gly Val
Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys 100
105 110Ala Gln Gly Thr Gly Pro Cys Pro Gly Asp Tyr Ala
Phe His Lys Asp 115 120 125Gly Ala
Phe Phe Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg 130
135 140Gly Val Asn Phe Ala Glu Gly Val Ile Ala Phe
Leu Ile Leu Ala Lys145 150 155
160Pro Lys Glu Thr Phe Leu Gln Ser Pro Pro Ile Arg Glu Ala Val Asn
165 170 175Tyr Thr Glu Asn
Thr Ser Ser Tyr Tyr Ala Thr Ser Tyr Leu Glu Tyr 180
185 190Glu Ile Glu Asn Phe Gly Ala Gln His Ser Thr
Thr Leu Phe Lys Ile 195 200 205Asp
Asn Asn Thr Phe Val Arg Leu Asp Arg Pro His Thr Pro Gln Phe 210
215 220Leu Phe Gln Leu Asn Asp Thr Ile His Leu
His Gln Gln Leu Ser Asn225 230 235
240Thr Thr Gly Arg Leu Ile Trp Thr Leu Asp Ala Asn Ile Asn Ala
Asp 245 250 255Ile Gly Glu
Trp Ala Phe Trp Glu Asn Lys Lys Asn Leu Ser Glu Gln 260
265 270Leu Arg Gly Glu Glu Leu Ser Phe Glu Ala
Leu Ser Leu Asn Glu Thr 275 280
285Glu Asp Asp Asp Ala Ala Ser Ser Arg Ile Thr Lys Gly Arg Ile Ser 290
295 300Asp Arg Ala Thr Arg Lys Tyr Ser
Asp Leu Val Pro Lys Asn Ser Pro305 310
315 320Gly Met Val Pro Leu His Ile Pro Glu Gly Glu Thr
Thr Leu Pro Ser 325 330
335Gln Asn Ser Thr Glu Gly Arg Arg Val Gly Val Asn Thr Gln Glu Thr
340 345 350Ile Thr Glu Thr Ala Ala
Thr Ile Ile Gly Thr Asn Gly Asn His Met 355 360
365Gln Ile Ser Thr Ile Gly Ile Arg Pro Ser Ser Ser Gln Ile
Pro Ser 370 375 380Ser Ser Pro Thr Thr
Ala Pro Ser Pro Glu Ala Gln Thr Pro Thr Thr385 390
395 400His Thr Ser Gly Pro Ser Val Met Ala Thr
Glu Glu Pro Thr Thr Pro 405 410
415Pro Gly Ser Ser Pro Gly Pro Thr Thr Glu Ala Pro Thr Leu Thr Thr
420 425 430Pro Glu Asn Ile Thr
Thr Ala Val Lys Thr Val Leu Pro Gln Glu Ser 435
440 445Thr Ser Asn Gly Leu Ile Thr Ser Thr Val Thr Gly
Ile Leu Gly Ser 450 455 460Leu Gly Leu
Arg Lys Arg Ser Arg Arg Gln Thr Asn Thr Lys Ala Thr465
470 475 480Gly Lys Cys Asn Pro Asn Leu
His Tyr Trp Thr Ala Gln Glu Gln His 485
490 495Asn Ala Ala Gly Ile Ala Trp Ile Pro Tyr Phe Gly
Pro Gly Ala Glu 500 505 510Gly
Ile Tyr Thr Glu Gly Leu Met His Asn Gln Asn Ala Leu Val Cys 515
520 525Gly Leu Arg Gln Leu Ala Asn Glu Thr
Thr Gln Ala Leu Gln Leu Phe 530 535
540Leu Arg Ala Thr Thr Glu Leu Arg Thr Tyr Thr Ile Leu Asn Arg Lys545
550 555 560Ala Ile Asp Phe
Leu Leu Arg Arg Trp Gly Gly Thr Cys Arg Ile Leu 565
570 575Gly Pro Asp Cys Cys Ile Glu Pro His Asp
Trp Thr Lys Asn Ile Thr 580 585
590Asp Lys Ile Asn Gln Ile Ile His Asp Phe Ile Asp Asn Pro Leu Pro
595 600 605Asn Gln Asp Asn Asp Asp Asn
Trp Trp Thr Gly 610 61551873DNAEbola Sudan Virus
5aggttccatg gatgcctttg ggtgttgtga ctaacagcac tttagaagta acagagattg
60accagctagt ctgcaaggat catcttgcat ctactgacca gctgaaatca gttggtctca
120acctcgaggg gagcggagta tctactgata tcccatctgc aacaaagcgt tggggcttca
180gatctggtgt tcctcccaag gtggtcagct atgaagcggg agaatgggct gaaaattgct
240acaatcttga aataaagaag ccggacggga gcgaatgctt acccccaccg ccagatggtg
300tcagaggctt tccaaggtgc cgctatgttc acaaagccca aggaaccggg ccctgcccag
360gtgactacgc ctttcacaag gatggagctt tcttcctcta tgacaggctg gcttcaactg
420taatttacag aggagtcaat tttgctgagg gggtaattgc attcttgata ttggctaaac
480caaaagaaac gttccttcag tcacccccca ttcgagaggc agtaaactac actgaaaata
540catcaagtta ttatgccaca tcctacttgg agtatgaaat cgaaaatttt ggtgctcaac
600actccacgac ccttttcaaa attgacaata atacttttgt tcgtctggac aggccccaca
660cgcctcagtt ccttttccag ctgaatgata ccattcacct tcaccaacag ttgagtaata
720caactgggag actaatttgg acactagatg ctaatatcaa tgctgatatt ggtgaatggg
780ctttttggga aaataaaaaa aatctctccg aacaactacg tggagaagag ctgtctttcg
840aagctttatc gctcaacgag acagaagacg atgatgcggc atcgtcgaga attacaaagg
900gaagaatctc cgaccgggcc accaggaagt attcggacct ggttccaaag aattcccctg
960ggatggttcc attgcacata ccagaagggg aaacaacatt gccgtctcag aattcgacag
1020aaggtcgaag agtaggtgtg aacactcagg agaccattac agagacagct gcaacaatta
1080taggcactaa cggcaaccat atgcagatct ccaccatcgg gataagaccg agctccagcc
1140aaatcccgag ttcctcaccg accacggcac caagccctga ggctcagacc cccacaaccc
1200acacatcagg tccatcagtg atggccaccg aggaaccaac aacaccaccg ggaagctccc
1260ccggcccaac aacagaagca cccactctca ccaccccaga aaatataaca acagcggtta
1320aaactgtcct gccacaggag tccacaagca acggtctaat aacttcaaca gtaacaggga
1380ttcttgggag tcttgggctt cgaaaacgca gcagaagaca aactaacacc aaagccacgg
1440gtaagtgcaa tcccaactta cactactgga ctgcacaaga acaacataat gctgctggga
1500ttgcctggat cccgtacttt ggaccgggtg cggaaggcat atacactgaa ggcctgatgc
1560ataaccaaaa tgccttagtc tgtggactta ggcaacttgc aaatgaaaca actcaagctc
1620tgcagctttt cttaagagcc acaacggagc tgcggacata taccatactc aataggaagg
1680ccatagattt ccttctgcga cgatggggcg ggacatgcag gatcctggga ccagattgtt
1740gcattgagcc acatgattgg acaaaaaaca tcactgataa aatcaaccaa atcatccatg
1800atttcatcga caacccctta cctaatcagg ataatgatga taattggtgg acgggctagt
1860aagtcgacaa cct
187361845DNAEbola Sudan Virus 6atgcctttgg gtgttgtgac taacagcact
ttagaagtaa cagagattga ccagctagtc 60tgcaaggatc atcttgcatc tactgaccag
ctgaaatcag ttggtctcaa cctcgagggg 120agcggagtat ctactgatat cccatctgca
acaaagcgtt ggggcttcag atctggtgtt 180cctcccaagg tggtcagcta tgaagcggga
gaatgggctg aaaattgcta caatcttgaa 240ataaagaagc cggacgggag cgaatgctta
cccccaccgc cagatggtgt cagaggcttt 300ccaaggtgcc gctatgttca caaagcccaa
ggaaccgggc cctgcccagg tgactacgcc 360tttcacaagg atggagcttt cttcctctat
gacaggctgg cttcaactgt aatttacaga 420ggagtcaatt ttgctgaggg ggtaattgca
ttcttgatat tggctaaacc aaaagaaacg 480ttccttcagt caccccccat tcgagaggca
gtaaactaca ctgaaaatac atcaagttat 540tatgccacat cctacttgga gtatgaaatc
gaaaattttg gtgctcaaca ctccacgacc 600cttttcaaaa ttgacaataa tacttttgtt
cgtctggaca ggccccacac gcctcagttc 660cttttccagc tgaatgatac cattcacctt
caccaacagt tgagtaatac aactgggaga 720ctaatttgga cactagatgc taatatcaat
gctgatattg gtgaatgggc tttttgggaa 780aataaaaaaa atctctccga acaactacgt
ggagaagagc tgtctttcga agctttatcg 840ctcaacgaga cagaagacga tgatgcggca
tcgtcgagaa ttacaaaggg aagaatctcc 900gaccgggcca ccaggaagta ttcggacctg
gttccaaaga attcccctgg gatggttcca 960ttgcacatac cagaagggga aacaacattg
ccgtctcaga attcgacaga aggtcgaaga 1020gtaggtgtga acactcagga gaccattaca
gagacagctg caacaattat aggcactaac 1080ggcaaccata tgcagatctc caccatcggg
ataagaccga gctccagcca aatcccgagt 1140tcctcaccga ccacggcacc aagccctgag
gctcagaccc ccacaaccca cacatcaggt 1200ccatcagtga tggccaccga ggaaccaaca
acaccaccgg gaagctcccc cggcccaaca 1260acagaagcac ccactctcac caccccagaa
aatataacaa cagcggttaa aactgtcctg 1320ccacaggagt ccacaagcaa cggtctaata
acttcaacag taacagggat tcttgggagt 1380cttgggcttc gaaaacgcag cagaagacaa
actaacacca aagccacggg taagtgcaat 1440cccaacttac actactggac tgcacaagaa
caacataatg ctgctgggat tgcctggatc 1500ccgtactttg gaccgggtgc ggaaggcata
tacactgaag gcctgatgca taaccaaaat 1560gccttagtct gtggacttag gcaacttgca
aatgaaacaa ctcaagctct gcagcttttc 1620ttaagagcca caacggagct gcggacatat
accatactca ataggaaggc catagatttc 1680cttctgcgac gatggggcgg gacatgcagg
atcctgggac cagattgttg cattgagcca 1740catgattgga caaaaaacat cactgataaa
atcaaccaaa tcatccatga tttcatcgac 1800aaccccttac ctaatcagga taatgatgat
aattggtgga cgggc 18457631PRTMarburg Virus 7Arg Ser Leu
Pro Ile Leu Glu Ile Ala Ser Asn Asn Gln Pro Gln Asn1 5
10 15Val Asp Ser Val Cys Ser Gly Thr Leu
Gln Lys Thr Glu Asp Val His 20 25
30Leu Met Gly Phe Thr Leu Ser Gly Gln Lys Val Ala Asp Ser Pro Leu
35 40 45Glu Ala Ser Lys Arg Trp Ala
Phe Arg Thr Gly Val Pro Pro Lys Asn 50 55
60Val Glu Tyr Thr Glu Gly Glu Glu Ala Lys Thr Cys Tyr Asn Ile Ser65
70 75 80Val Thr Asp Pro
Ser Gly Lys Ser Leu Leu Leu Asp Pro Pro Thr Asn 85
90 95Ile Arg Asp Tyr Pro Lys Cys Lys Thr Ile
His His Ile Gln Gly Gln 100 105
110Asn Pro His Ala Gln Gly Ile Ala Leu His Leu Trp Gly Ala Phe Phe
115 120 125Leu Tyr Asp Arg Ile Ala Ser
Thr Thr Met Tyr Arg Gly Arg Val Phe 130 135
140Thr Glu Gly Asn Ile Ala Ala Met Ile Val Asn Lys Thr Val His
Lys145 150 155 160Met Ile
Phe Ser Arg Gln Gly Gln Gly Tyr Arg His Met Asn Leu Thr
165 170 175Ser Thr Asn Lys Tyr Trp Thr
Ser Asn Asn Gly Thr Gln Thr Asn Asp 180 185
190Thr Gly Cys Phe Gly Ala Leu Gln Glu Tyr Asn Ser Thr Lys
Asn Gln 195 200 205Thr Cys Ala Pro
Ser Lys Ile Pro Ser Pro Leu Pro Thr Ala Arg Pro 210
215 220Glu Ile Lys Pro Thr Ser Thr Pro Thr Asp Ala Thr
Thr Leu Asn Thr225 230 235
240Thr Asp Pro Asn Asn Asp Asp Glu Asp Leu Ile Thr Ser Gly Ser Gly
245 250 255Ser Gly Glu Gln Glu
Pro Tyr Thr Thr Ser Asp Ala Val Thr Lys Gln 260
265 270Gly Leu Ser Ser Thr Met Pro Pro Thr Pro Ser Pro
Gln Pro Ser Thr 275 280 285Pro Gln
Gln Glu Gly Asn Asn Thr Asp His Ser Gln Gly Thr Val Thr 290
295 300Glu Pro Asn Lys Thr Asn Thr Thr Ala Gln Pro
Ser Met Pro Pro His305 310 315
320Asn Thr Thr Ala Ile Ser Thr Asn Asn Thr Ser Lys Asn Asn Phe Ser
325 330 335Thr Leu Ser Val
Ser Leu Gln Asn Thr Thr Asn Tyr Asp Thr Gln Ser 340
345 350Thr Ala Thr Glu Asn Glu Gln Thr Ser Ala Pro
Ser Lys Thr Thr Leu 355 360 365Pro
Pro Thr Gly Asn Leu Thr Thr Ala Lys Ser Thr Asn Asn Thr Lys 370
375 380Gly Pro Thr Thr Thr Ala Pro Asn Met Thr
Asn Gly His Leu Thr Ser385 390 395
400Pro Ser Pro Thr Pro Asn Pro Thr Thr Gln His Leu Val Tyr Phe
Arg 405 410 415Lys Lys Arg
Ser Ile Leu Trp Arg Glu Gly Asp Met Phe Pro Phe Leu 420
425 430Asp Gly Leu Ile Asn Ala Pro Ile Asp Phe
Asp Pro Val Pro Asn Thr 435 440
445Lys Thr Ile Phe Asp Glu Ser Ser Ser Ser Gly Ala Ser Ala Glu Glu 450
455 460Asp Gln His Ala Ser Pro Asn Ile
Ser Leu Thr Leu Ser Tyr Phe Pro465 470
475 480Asn Ile Asn Glu Asn Thr Ala Tyr Ser Gly Glu Asn
Glu Asn Asp Cys 485 490
495Asp Ala Glu Leu Arg Ile Trp Ser Val Gln Glu Asp Asp Leu Ala Ala
500 505 510Gly Leu Ser Trp Ile Pro
Phe Phe Gly Pro Gly Ile Glu Gly Leu Tyr 515 520
525Thr Ala Gly Leu Ile Lys Asn Gln Asn Asn Leu Val Cys Arg
Leu Arg 530 535 540Arg Leu Ala Asn Gln
Thr Ala Lys Ser Leu Glu Leu Leu Leu Arg Val545 550
555 560Thr Thr Glu Glu Arg Thr Phe Ser Leu Ile
Asn Arg His Ala Ile Asp 565 570
575Phe Leu Leu Thr Arg Trp Gly Gly Thr Cys Lys Val Leu Gly Pro Asp
580 585 590Cys Cys Ile Gly Ile
Glu Asp Leu Ser Arg Asn Ile Ser Glu Gln Ile 595
600 605Asp Gln Ile Lys Lys Asp Glu Gln Lys Glu Gly Thr
Gly Trp Gly Leu 610 615 620Gly Gly Lys
Trp Trp Thr Ser625 63081915DNAMarburg Virus 8aggttagatc
tctccctatt ttagagatag ctagtaacaa tcaaccccaa aatgtggatt 60cggtatgctc
cggaactctc cagaagacag aagatgtcca tctgatggga ttcacactga 120gtgggcaaaa
agttgctgat tcccctttgg aggcatccaa gcgatgggct ttcaggacag 180gtgtacctcc
caagaatgtt gagtatacag aaggggagga agccaaaaca tgctacaata 240taagtgtaac
ggatccctct ggaaaatcct tgctgttgga tcctcctacc aacatccgtg 300actatcctaa
atgcaaaact atccatcata ttcaaggtca aaaccctcat gcgcaaggga 360tcgccctcca
tttgtgggga gcatttttcc tgtatgatcg cattgcctcc acaacaatgt 420accgaggcag
agtcttcact gaagggaaca tagcagctat gattgtcaat aagacagtgc 480acaaaatgat
tttctcgagg caaggacagg ggtaccgtca catgaatctg acttctacta 540ataaatattg
gacaagtaac aatggaacac aaacgaatga cactggatgc ttcggtgctc 600ttcaagaata
caactccacg aagaatcaaa catgtgctcc gtccaaaata ccctcaccac 660tgcccacagc
ccgtccagag atcaaaccca caagcacccc aactgatgcc accacactca 720acaccacaga
cccaaacaat gatgatgagg acctcataac atccggttca gggtccggag 780aacaggaacc
ctatacaact tcagatgcgg tcactaagca agggctttca tcaacaatgc 840cacccactcc
ctcaccacaa ccaagcacgc cacagcaaga aggaaacaac acagaccatt 900cccaaggtac
tgtgactgaa cccaacaaaa ccaacacaac ggcacaaccg tccatgcccc 960cccacaacac
cactgcaatc tctactaaca acacctccaa gaacaacttc agcaccctct 1020ctgtatcact
acaaaacacc accaattacg acacacagag cacagccact gaaaatgaac 1080aaaccagtgc
cccctcgaaa acaaccctgc ctccaacagg aaatcttacc acagcaaaga 1140gcactaacaa
cacgaaaggc cccaccacaa cggcaccaaa tatgacaaat gggcatttaa 1200ccagtccctc
ccccaccccc aacccgacca cacaacatct tgtatatttc agaaagaaac 1260gaagtatcct
ctggagggaa ggcgacatgt ttccttttct ggacgggtta ataaatgctc 1320caattgattt
tgatccagtt ccaaatacaa agacgatctt tgatgaatct tctagttctg 1380gtgcttcggc
tgaggaagat caacatgcct cccccaatat cagtttaact ttatcctatt 1440ttcctaatat
aaatgaaaac actgcctact ctggagaaaa tgagaacgat tgtgatgcag 1500agttaagaat
ttggagcgtt caggaggatg acctggcagc agggctcagt tggataccgt 1560tttttggccc
tggaatcgaa ggactttata ctgctggttt aattaaaaac caaaacaatt 1620tggtctgcag
gttgaggcgt ctagccaatc aaactgccaa atccttggaa ctcttattaa 1680gagtcacaac
cgaggaaagg acattttcct taattaatag acatgccatt gactttctac 1740tcacaaggtg
gggaggaaca tgcaaagtgc ttggacctga ttgttgcatt ggaatagaag 1800acttgtccag
gaatatttcg gaacaaattg accaaatcaa aaaagatgaa caaaaagagg 1860ggactggttg
gggtctaggt ggtaaatggt ggacatccta gtaagtcgac aacct
191591898DNAMarburg Virus 9aggttagatc tctccctatt ttagagatag ctagtaacaa
tcaaccccaa aatgtggatt 60cggtatgctc cggaactctc cagaagacag aagatgtcca
tctgatggga ttcacactga 120gtgggcaaaa agttgctgat tcccctttgg aggcatccaa
gcgatgggct ttcaggacag 180gtgtacctcc caagaatgtt gagtatacag aaggggagga
agccaaaaca tgctacaata 240taagtgtaac ggatccctct ggaaaatcct tgctgttgga
tcctcctacc aacatccgtg 300actatcctaa atgcaaaact atccatcata ttcaaggtca
aaaccctcat gcgcaaggga 360tcgccctcca tttgtgggga gcatttttcc tgtatgatcg
cattgcctcc acaacaatgt 420accgaggcag agtcttcact gaagggaaca tagcagctat
gattgtcaat aagacagtgc 480acaaaatgat tttctcgagg caaggacagg ggtaccgtca
catgaatctg acttctacta 540ataaatattg gacaagtaac aatggaacac aaacgaatga
cactggatgc ttcggtgctc 600ttcaagaata caactccacg aagaatcaaa catgtgctcc
gtccaaaata ccctcaccac 660tgcccacagc ccgtccagag atcaaaccca caagcacccc
aactgatgcc accacactca 720acaccacaga cccaaacaat gatgatgagg acctcataac
atccggttca gggtccggag 780aacaggaacc ctatacaact tcagatgcgg tcactaagca
agggctttca tcaacaatgc 840cacccactcc ctcaccacaa ccaagcacgc cacagcaaga
aggaaacaac acagaccatt 900cccaaggtac tgtgactgaa cccaacaaaa ccaacacaac
ggcacaaccg tccatgcccc 960cccacaacac cactgcaatc tctactaaca acacctccaa
gaacaacttc agcaccctct 1020ctgtatcact acaaaacacc accaattacg acacacagag
cacagccact gaaaatgaac 1080aaaccagtgc cccctcgaaa acaaccctgc ctccaacagg
aaatcttacc acagcaaaga 1140gcactaacaa cacgaaaggc cccaccacaa cggcaccaaa
tatgacaaat gggcatttaa 1200ccagtccctc ccccaccccc aacccgacca cacaacatct
tgtatatttc agaaagaaac 1260gaagtatcct ctggagggaa ggcgacatgt ttccttttct
ggacgggtta ataaatgctc 1320caattgattt tgatccagtt ccaaatacaa agacgatctt
tgatgaatct tctagttctg 1380gtgcttcggc tgaggaagat caacatgcct cccccaatat
cagtttaact ttatcctatt 1440ttcctaatat aaatgaaaac actgcctact ctggagaaaa
tgagaacgat tgtgatgcag 1500agttaagaat ttggagcgtt caggaggatg acctggcagc
agggctcagt tggataccgt 1560tttttggccc tggaatcgaa ggactttata ctgctggttt
aattaaaaac caaaacaatt 1620tggtctgcag gttgaggcgt ctagccaatc aaactgccaa
atccttggaa ctcttattaa 1680gagtcacaac cgaggaaagg acattttcct taattaatag
acatgccatt gactttctac 1740tcacaaggtg gggaggaaca tgcaaagtgc ttggacctga
ttgttgcatt ggaatagaag 1800acttgtccag gaatatttcg gaacaaattg accaaatcaa
aaaagatgaa caaaaagagg 1860ggactggttg gggtctaggt ggtaaatggt ggacatcc
189810646PRTEbola Zaire Virus 10Arg Ser Ile Pro Leu
Gly Val Ile His Asn Ser Thr Leu Gln Val Ser1 5
10 15Asp Val Asp Lys Leu Val Cys Arg Asp Lys Leu
Ser Ser Thr Asn Gln 20 25
30Leu Arg Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp
35 40 45Val Pro Ser Ala Thr Lys Arg Trp
Gly Phe Arg Ser Gly Val Pro Pro 50 55
60Lys Val Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn65
70 75 80Leu Glu Ile Lys Lys
Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro 85
90 95Asp Gly Ile Arg Gly Phe Pro Arg Cys Arg Tyr
Val His Lys Val Ser 100 105
110Gly Thr Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala
115 120 125Phe Phe Leu Tyr Asp Arg Leu
Ala Ser Thr Val Ile Tyr Arg Gly Thr 130 135
140Thr Phe Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala
Lys145 150 155 160Lys Asp
Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr
165 170 175Glu Asp Pro Ser Ser Gly Tyr
Tyr Ser Thr Thr Ile Arg Tyr Gln Ala 180 185
190Thr Gly Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val
Asp Asn 195 200 205Leu Thr Tyr Val
Gln Leu Glu Ser Arg Phe Thr Pro Gln Phe Leu Leu 210
215 220Gln Leu Asn Glu Thr Ile Tyr Thr Ser Gly Lys Arg
Ser Asn Thr Thr225 230 235
240Gly Lys Leu Ile Trp Lys Val Asn Pro Glu Ile Asp Thr Thr Ile Gly
245 250 255Glu Trp Ala Phe Trp
Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg 260
265 270Ser Glu Glu Leu Ser Phe Thr Val Val Ser Asn Gly
Ala Lys Asn Ile 275 280 285Ser Gly
Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr 290
295 300Thr Thr Glu Asp His Lys Ile Met Ala Ser Glu
Asn Ser Ser Ala Met305 310 315
320Val Gln Val His Ser Gln Gly Arg Glu Ala Ala Val Ser His Leu Thr
325 330 335Thr Leu Ala Thr
Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr Lys Pro 340
345 350Gly Pro Asp Asn Ser Thr His Asn Thr Pro Val
Tyr Lys Leu Asp Ile 355 360 365Ser
Glu Ala Thr Gln Val Glu Gln His His Arg Arg Thr Asp Asn Asp 370
375 380Ser Thr Ala Ser Asp Thr Pro Ser Ala Thr
Thr Ala Ala Gly Pro Pro385 390 395
400Lys Ala Glu Asn Thr Asn Thr Ser Lys Ser Thr Asp Phe Leu Asp
Pro 405 410 415Ala Thr Thr
Thr Ser Pro Gln Asn His Ser Glu Thr Ala Gly Asn Asn 420
425 430Asn Thr His His Gln Asp Thr Gly Glu Glu
Ser Ala Ser Ser Gly Lys 435 440
445Leu Gly Leu Ile Thr Asn Thr Ile Ala Gly Val Ala Gly Leu Ile Thr 450
455 460Gly Gly Arg Arg Thr Arg Arg Glu
Ala Ile Val Asn Ala Gln Pro Lys465 470
475 480Cys Asn Pro Asn Leu His Tyr Trp Thr Thr Gln Asp
Glu Gly Ala Ala 485 490
495Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile
500 505 510Tyr Thr Glu Gly Leu Met
His Asn Gln Asp Gly Leu Ile Cys Gly Leu 515 520
525Arg Gln Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe
Leu Arg 530 535 540Ala Thr Thr Glu Leu
Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile545 550
555 560Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr
Cys His Ile Leu Gly Pro 565 570
575Asp Cys Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys
580 585 590Ile Asp Gln Ile Ile
His Asp Phe Val Asp Lys Thr Leu Pro Asp Gln 595
600 605Gly Asp Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln
Trp Ile Pro Ala 610 615 620Gly Ile Gly
Val Thr Gly Val Ile Ile Ala Val Ile Ala Leu Phe Cys625
630 635 640Ile Cys Lys Phe Val Phe
645111964DNAEbola Zaire Virus 11acgtacagat ctatcccact tggagtcatc
cacaatagca cattacaggt tagtgatgtc 60gacaaactag tttgtcgtga caaactgtca
tccacaaatc aattgagatc agttggactg 120aatctcgaag ggaatggagt ggcaactgac
gtgccatctg caactaaaag atggggcttc 180aggtccggtg tcccaccaaa ggtggtcaat
tatgaagctg gtgaatgggc tgaaaactgc 240tacaatcttg aaatcaaaaa acctgacggg
agtgagtgtc taccagcagc gccagacggg 300attcggggct tcccccggtg ccggtatgtg
cacaaagtat caggaacggg accgtgtgcc 360ggagactttg ccttccataa agagggtgct
ttcttcctgt atgatcgact tgcttccaca 420gttatctacc gaggaacgac tttcgctgaa
ggtgtcgttg catttctgat actgccccaa 480gctaagaagg acttcttcag ctcacacccc
ttgagagagc cggtcaatgc aacggaggac 540ccgtctagtg gctactattc taccacaatt
agatatcagg ctaccggttt tggaaccaat 600gagacagagt acttgttcga ggttgacaat
ttgacctacg tccaacttga atcaagattc 660acaccacagt ttctgctcca gctgaatgag
acaatatata caagtgggaa aaggagcaat 720accacgggaa aactaatttg gaaggtcaac
cccgaaattg atacaacaat cggggagtgg 780gccttctggg aaactaaaaa aaacctcact
agaaaaattc gcagtgaaga gttgtctttc 840acagttgtat caaacggagc caaaaacatc
agtggtcaga gtccggcgcg aacttcttcc 900gacccaggga ccaacacaac aactgaagac
cacaaaatca tggcttcaga aaattcctct 960gcaatggttc aagtgcacag tcaaggaagg
gaagctgcag tgtcgcatct aacaaccctt 1020gccacaatct ccacgagtcc ccaatccctc
acaaccaaac caggtccgga caacagcacc 1080cataatacac ccgtgtataa acttgacatc
tctgaggcaa ctcaagttga acaacatcac 1140cgcagaacag acaacgacag cacagcctcc
gacactccct ctgccacgac cgcagccgga 1200cccccaaaag cagagaacac caacacgagc
aagagcactg acttcctgga ccccgccacc 1260acaacaagtc cccaaaacca cagcgagacc
gctggcaaca acaacactca tcaccaagat 1320accggagaag agagtgccag cagcgggaag
ctaggcttaa ttaccaatac tattgctgga 1380gtcgcaggac tgatcacagg cgggagaaga
actcgaagag aagcaattgt caatgctcaa 1440cccaaatgca accctaattt acattactgg
actactcagg atgaaggtgc tgcaatcgga 1500ctggcctgga taccatattt cgggccagca
gccgagggaa tttacacaga ggggctaatg 1560cacaatcaag atggtttaat ctgtgggttg
agacagctgg ccaacgagac gactcaagct 1620cttcaactgt tcctgagagc cacaactgag
ctacgcacct tttcaatcct caaccgtaag 1680gcaattgatt tcttgctgca gcgatggggc
ggcacatgcc acattctggg accggactgc 1740tgtatcgaac cacatgattg gaccaagaac
ataacagaca aaattgatca gattattcat 1800gattttgttg ataaaaccct tccggaccag
ggggacaatg acaattggtg gacaggatgg 1860agacaatgga taccggcagg tattggagtt
acaggcgtta taattgcagt tatcgcttta 1920ttctgtatat gcaaatttgt cttttagtaa
ctcgaggtac gtac 1964121932DNAEbola Zaire Virus
12atcccacttg gagtcatcca caatagcaca ttacaggtta gtgatgtcga caaactagtt
60tgtcgtgaca aactgtcatc cacaaatcaa ttgagatcag ttggactgaa tctcgaaggg
120aatggagtgg caactgacgt gccatctgca actaaaagat ggggcttcag gtccggtgtc
180ccaccaaagg tggtcaatta tgaagctggt gaatgggctg aaaactgcta caatcttgaa
240atcaaaaaac ctgacgggag tgagtgtcta ccagcagcgc cagacgggat tcggggcttc
300ccccggtgcc ggtatgtgca caaagtatca ggaacgggac cgtgtgccgg agactttgcc
360ttccataaag agggtgcttt cttcctgtat gatcgacttg cttccacagt tatctaccga
420ggaacgactt tcgctgaagg tgtcgttgca tttctgatac tgccccaagc taagaaggac
480ttcttcagct cacacccctt gagagagccg gtcaatgcaa cggaggaccc gtctagtggc
540tactattcta ccacaattag atatcaggct accggttttg gaaccaatga gacagagtac
600ttgttcgagg ttgacaattt gacctacgtc caacttgaat caagattcac accacagttt
660ctgctccagc tgaatgagac aatatataca agtgggaaaa ggagcaatac cacgggaaaa
720ctaatttgga aggtcaaccc cgaaattgat acaacaatcg gggagtgggc cttctgggaa
780actaaaaaaa acctcactag aaaaattcgc agtgaagagt tgtctttcac agttgtatca
840aacggagcca aaaacatcag tggtcagagt ccggcgcgaa cttcttccga cccagggacc
900aacacaacaa ctgaagacca caaaatcatg gcttcagaaa attcctctgc aatggttcaa
960gtgcacagtc aaggaaggga agctgcagtg tcgcatctaa caacccttgc cacaatctcc
1020acgagtcccc aatccctcac aaccaaacca ggtccggaca acagcaccca taatacaccc
1080gtgtataaac ttgacatctc tgaggcaact caagttgaac aacatcaccg cagaacagac
1140aacgacagca cagcctccga cactccctct gccacgaccg cagccggacc cccaaaagca
1200gagaacacca acacgagcaa gagcactgac ttcctggacc ccgccaccac aacaagtccc
1260caaaaccaca gcgagaccgc tggcaacaac aacactcatc accaagatac cggagaagag
1320agtgccagca gcgggaagct aggcttaatt accaatacta ttgctggagt cgcaggactg
1380atcacaggcg ggagaagaac tcgaagagaa gcaattgtca atgctcaacc caaatgcaac
1440cctaatttac attactggac tactcaggat gaaggtgctg caatcggact ggcctggata
1500ccatatttcg ggccagcagc cgagggaatt tacacagagg ggctaatgca caatcaagat
1560ggtttaatct gtgggttgag acagctggcc aacgagacga ctcaagctct tcaactgttc
1620ctgagagcca caactgagct acgcaccttt tcaatcctca accgtaaggc aattgatttc
1680ttgctgcagc gatggggcgg cacatgccac attctgggac cggactgctg tatcgaacca
1740catgattgga ccaagaacat aacagacaaa attgatcaga ttattcatga ttttgttgat
1800aaaacccttc cggaccaggg ggacaatgac aattggtgga caggatggag acaatggata
1860ccggcaggta ttggagttac aggcgttata attgcagtta tcgctttatt ctgtatatgc
1920aaatttgtct tt
193213328PRTEbola Zaire Virus 13Arg Ser Met Arg Arg Val Ile Leu Pro Thr
Ala Pro Pro Glu Tyr Met1 5 10
15Glu Ala Ile Tyr Pro Val Arg Ser Asn Ser Thr Ile Ala Arg Gly Gly
20 25 30Asn Ser Asn Thr Gly Phe
Leu Thr Pro Glu Ser Val Asn Gly Asp Thr 35 40
45Pro Ser Asn Pro Leu Arg Pro Ile Ala Asp Asp Thr Ile Asp
His Ala 50 55 60Ser His Thr Pro Gly
Ser Val Ser Ser Ala Phe Ile Leu Glu Ala Met65 70
75 80Val Asn Val Ile Ser Gly Pro Lys Val Leu
Met Lys Gln Ile Pro Ile 85 90
95Trp Leu Pro Leu Gly Val Ala Asp Gln Lys Thr Tyr Ser Phe Asp Ser
100 105 110Thr Thr Ala Ala Ile
Met Leu Ala Ser Tyr Thr Ile Thr His Phe Gly 115
120 125Lys Ala Thr Asn Pro Leu Val Arg Val Asn Arg Leu
Gly Pro Gly Ile 130 135 140Pro Asp His
Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu145
150 155 160Gln Glu Phe Val Leu Pro Pro
Val Gln Leu Pro Gln Tyr Phe Thr Phe 165
170 175Asp Leu Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu
Pro Ala Ala Thr 180 185 190Trp
Thr Asp Asp Thr Pro Thr Gly Ser Asn Gly Ala Leu Arg Pro Gly 195
200 205Ile Ser Phe His Pro Lys Leu Arg Pro
Ile Leu Leu Pro Asn Lys Ser 210 215
220Gly Lys Lys Gly Asn Ser Ala Asp Leu Thr Ser Pro Glu Lys Ile Gln225
230 235 240Ala Ile Met Thr
Ser Leu Gln Asp Phe Lys Ile Val Pro Ile Asp Pro 245
250 255Thr Lys Asn Ile Met Gly Ile Glu Val Pro
Glu Thr Leu Val His Lys 260 265
270Leu Thr Gly Lys Lys Val Thr Ser Lys Asn Gly Gln Pro Ile Ile Pro
275 280 285Val Leu Leu Pro Lys Tyr Ile
Gly Leu Asp Pro Val Ala Pro Gly Asp 290 295
300Leu Thr Met Val Ile Thr Gln Asp Cys Asp Thr Cys His Ser Pro
Ala305 310 315 320Ser Leu
Pro Ala Val Ile Glu Lys 325141008DNAEbola Zaire Virus
14acgtacagat ctatgaggcg ggttatattg cctactgctc ctcctgaata tatggaggcc
60atataccctg tcaggtcaaa ttcaacaatt gctagaggtg gcaacagcaa tacaggcttc
120ctgacaccgg agtcagtcaa tggggacact ccatcgaatc cactcaggcc aattgccgat
180gacaccatcg accatgccag ccacacacca ggcagtgtgt catcagcatt catccttgaa
240gctatggtga atgtcatatc gggccccaaa gtgctaatga agcaaattcc aatttggctt
300cctctaggtg tcgctgatca aaagacctac agctttgact caactacggc cgccatcatg
360cttgcttcat acactatcac ccatttcggc aaggcaacca atccacttgt cagagtcaat
420cggctgggtc ctggaatccc ggatcatccc ctcaggctcc tgcgaattgg aaaccaggct
480ttcctccagg agttcgttct tccgccagtc caactacccc agtatttcac ctttgatttg
540acagcactca aactgatcac ccaaccactg cctgctgcaa catggaccga tgacactcca
600acaggatcaa atggagcgtt gcgtccagga atttcatttc atccaaaact tcgccccatt
660cttttaccca acaaaagtgg gaagaagggg aacagtgccg atctaacatc tccggagaaa
720atccaagcaa taatgacttc actccaggac ttcaagatcg ttccaattga tccaaccaaa
780aatatcatgg gaatcgaagt gccagaaact ctggtccaca agctgaccgg taagaaggtg
840acttctaaaa atggacaacc aatcatccct gttcttttgc caaagtacat tgggttggac
900ccggtggctc caggagacct caccatggta atcacacagg attgtgacac gtgtcattct
960cctgcaagtc ttccagctgt gattgagaag taataactcg aggtacgt
100815978DNAEbola Zaire Virus 15atgaggcggg ttatattgcc tactgctcct
cctgaatata tggaggccat ataccctgtc 60aggtcaaatt caacaattgc tagaggtggc
aacagcaata caggcttcct gacaccggag 120tcagtcaatg gggacactcc atcgaatcca
ctcaggccaa ttgccgatga caccatcgac 180catgccagcc acacaccagg cagtgtgtca
tcagcattca tccttgaagc tatggtgaat 240gtcatatcgg gccccaaagt gctaatgaag
caaattccaa tttggcttcc tctaggtgtc 300gctgatcaaa agacctacag ctttgactca
actacggccg ccatcatgct tgcttcatac 360actatcaccc atttcggcaa ggcaaccaat
ccacttgtca gagtcaatcg gctgggtcct 420ggaatcccgg atcatcccct caggctcctg
cgaattggaa accaggcttt cctccaggag 480ttcgttcttc cgccagtcca actaccccag
tatttcacct ttgatttgac agcactcaaa 540ctgatcaccc aaccactgcc tgctgcaaca
tggaccgatg acactccaac aggatcaaat 600ggagcgttgc gtccaggaat ttcatttcat
ccaaaacttc gccccattct tttacccaac 660aaaagtggga agaaggggaa cagtgccgat
ctaacatctc cggagaaaat ccaagcaata 720atgacttcac tccaggactt caagatcgtt
ccaattgatc caaccaaaaa tatcatggga 780atcgaagtgc cagaaactct ggtccacaag
ctgaccggta agaaggtgac ttctaaaaat 840ggacaaccaa tcatccctgt tcttttgcca
aagtacattg ggttggaccc ggtggctcca 900ggagacctca ccatggtaat cacacaggat
tgtgacacgt gtcattctcc tgcaagtctt 960ccagctgtga ttgagaag
97816253PRTEbola Zaire Virus 16Arg Ser
Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Ile Ser Pro Lys1 5
10 15Lys Asp Leu Glu Lys Gly Val Val
Leu Ser Asp Leu Cys Asn Phe Leu 20 25
30Val Ser Gln Thr Ile Gln Gly Trp Lys Val Tyr Trp Ala Gly Ile
Glu 35 40 45Phe Asp Val Thr His
Lys Gly Met Ala Leu Leu His Arg Leu Lys Thr 50 55
60Asn Asp Phe Ala Pro Ala Trp Ser Met Thr Arg Asn Leu Phe
Pro His65 70 75 80Leu
Phe Gln Asn Pro Asn Ser Thr Ile Glu Ser Pro Leu Trp Ala Leu
85 90 95Arg Val Ile Leu Ala Ala Gly
Ile Gln Asp Gln Leu Ile Asp Gln Ser 100 105
110Leu Ile Glu Pro Leu Ala Gly Ala Leu Gly Leu Ile Ser Asp
Trp Leu 115 120 125Leu Thr Thr Asn
Thr Asn His Phe Asn Met Arg Thr Gln Arg Val Lys 130
135 140Glu Gln Leu Ser Leu Lys Met Leu Ser Leu Ile Arg
Ser Asn Ile Leu145 150 155
160Lys Phe Ile Asn Lys Leu Asp Ala Leu His Val Val Asn Tyr Asn Gly
165 170 175Leu Leu Ser Ser Ile
Glu Ile Gly Thr Gln Asn His Thr Ile Ile Ile 180
185 190Thr Arg Thr Asn Met Gly Phe Leu Val Glu Leu Gln
Glu Pro Asp Lys 195 200 205Ser Ala
Met Asn Arg Met Lys Pro Gly Pro Ala Lys Phe Ser Leu Leu 210
215 220His Glu Ser Thr Leu Lys Ala Phe Thr Gln Gly
Ser Ser Thr Arg Met225 230 235
240Gln Ser Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala Ile
245 25017783DNAEbola Zaire Virus 17acgtacagat ctatggctaa
agctacggga cgatacaatc taatatcgcc caaaaaggac 60ctggagaaag gggttgtctt
aagcgacctc tgtaacttct tagttagcca aactattcag 120gggtggaagg tttattgggc
tggtattgag tttgatgtga ctcacaaagg aatggcccta 180ttgcatagac tgaaaactaa
tgactttgcc cctgcatggt caatgacaag gaatctcttt 240cctcatttat ttcaaaatcc
gaattccaca attgaatcac cgctgtgggc attgagagtc 300atccttgcag cagggataca
ggaccagctg attgaccagt ctttgattga acccttagca 360ggagcccttg gtctgatctc
tgattggctg ctaacaacca acactaacca tttcaacatg 420cgaacacaac gtgtcaagga
acaattgagc ctaaaaatgc tgtcgttgat tcgatccaat 480attctcaagt ttattaacaa
attggatgct ctacatgtcg tgaactacaa cggattgttg 540agcagtattg aaattggaac
tcaaaatcat acaatcatca taactcgaac taacatgggt 600tttctggtgg agctccaaga
acccgacaaa tcggcaatga accgcatgaa gcctgggccg 660gcgaaatttt ccctccttca
tgagtccaca ctgaaagcat ttacacaagg atcctcgaca 720cgaatgcaaa gtttgattct
tgaatttaat agctctcttg ctatctaata actcgaggta 780cgt
78318753DNAEbola Zaire Virus
18atggctaaag ctacgggacg atacaatcta atatcgccca aaaaggacct ggagaaaggg
60gttgtcttaa gcgacctctg taacttctta gttagccaaa ctattcaggg gtggaaggtt
120tattgggctg gtattgagtt tgatgtgact cacaaaggaa tggccctatt gcatagactg
180aaaactaatg actttgcccc tgcatggtca atgacaagga atctctttcc tcatttattt
240caaaatccga attccacaat tgaatcaccg ctgtgggcat tgagagtcat ccttgcagca
300gggatacagg accagctgat tgaccagtct ttgattgaac ccttagcagg agcccttggt
360ctgatctctg attggctgct aacaaccaac actaaccatt tcaacatgcg aacacaacgt
420gtcaaggaac aattgagcct aaaaatgctg tcgttgattc gatccaatat tctcaagttt
480attaacaaat tggatgctct acatgtcgtg aactacaacg gattgttgag cagtattgaa
540attggaactc aaaatcatac aatcatcata actcgaacta acatgggttt tctggtggag
600ctccaagaac ccgacaaatc ggcaatgaac cgcatgaagc ctgggccggc gaaattttcc
660ctccttcatg agtccacact gaaagcattt acacaaggat cctcgacacg aatgcaaagt
720ttgattcttg aatttaatag ctctcttgct atc
75319741PRTEbola Zaire Virus 19Arg Ser Met Asp Ser Arg Pro Gln Lys Ile
Trp Met Ala Pro Ser Leu1 5 10
15Thr Glu Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser
20 25 30Val Gln Gln Gly Ile Val
Arg Gln Arg Val Ile Pro Val Tyr Gln Val 35 40
45Asn Asn Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe
Glu Ala 50 55 60Gly Val Asp Phe Gln
Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys65 70
75 80Leu His His Ala Tyr Gln Gly Asp Tyr Lys
Leu Phe Leu Glu Ser Gly 85 90
95Ala Val Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys
100 105 110Arg Asp Gly Val Lys
Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser 115
120 125Gly Lys Asn Ile Lys Arg Thr Leu Ala Ala Met Pro
Glu Glu Glu Thr 130 135 140Thr Glu Ala
Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu145
150 155 160Pro Lys Leu Val Val Gly Glu
Lys Ala Cys Leu Glu Lys Val Gln Arg 165
170 175Gln Ile Gln Val His Ala Glu Gln Gly Leu Ile Gln
Tyr Pro Thr Ala 180 185 190Trp
Gln Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr 195
200 205Asn Phe Leu Ile Lys Phe Leu Leu Ile
His Gln Gly Met His Met Val 210 215
220Ala Gly His Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln225
230 235 240Ala Arg Phe Ser
Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile 245
250 255Leu Gln Lys Thr Glu Arg Gly Val Arg Leu
His Pro Leu Ala Arg Thr 260 265
270Ala Lys Val Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser
275 280 285Leu Ala Lys His Gly Glu Tyr
Ala Pro Phe Ala Arg Leu Leu Asn Leu 290 295
300Ser Gly Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser
Ala305 310 315 320Ile Ala
Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val
325 330 335Asn Val Gly Glu Gln Tyr Gln
Gln Leu Arg Glu Ala Ala Thr Glu Ala 340 345
350Glu Lys Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp
His Leu 355 360 365Gly Leu Asp Asp
Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys 370
375 380Lys Asn Glu Ile Ser Phe Gln Gln Thr Asn Ala Met
Val Thr Leu Arg385 390 395
400Lys Glu Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu
405 410 415Pro Lys Thr Ser Gly
His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro 420
425 430Gly Pro Ile Asn Asp Asp Asp Asn Pro Gly His Gln
Asp Asp Asp Pro 435 440 445Thr Asp
Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp 450
455 460Asp Gly Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser
Glu Asn Gly Met Asn465 470 475
480Ala Pro Asp Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp
485 490 495Thr Lys Pro Val
Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn 500
505 510Ser Gln Lys Gly Gln His Ile Glu Gly Arg Gln
Thr Gln Ser Arg Pro 515 520 525Ile
Gln Asn Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala 530
535 540Pro Leu Thr Asp Asn Asp Arg Arg Asn Glu
Pro Ser Gly Ser Thr Ser545 550 555
560Pro Arg Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp
Asp 565 570 575Ala Asp Asp
Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu 580
585 590Glu Gln Asp Arg Asp Gly Thr Ser Asn Arg
Thr Pro Thr Val Ala Pro 595 600
605Pro Ala Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln 610
615 620Asp Glu Gln Gln Asp Gln Asp His
Thr Gln Glu Ala Arg Asn Gln Asp625 630
635 640Ser Asp Asn Thr Gln Ser Glu His Ser Phe Glu Glu
Met Tyr Arg His 645 650
655Ile Leu Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met
660 665 670Met Lys Asp Glu Pro Val
Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr 675 680
685Thr Tyr Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu
Thr Glu 690 695 700Lys Glu Ala Met Asn
Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln705 710
715 720Gln Phe Tyr Trp Pro Val Met Asn His Lys
Asn Lys Phe Met Ala Ile 725 730
735Leu Gln His His Gln 740202247DNAEbola Zaire Virus
20atgctaggat ccatggattc tcgtcctcag aaaatctgga tggcgccgag tctcactgaa
60tctgacatgg attaccacaa gatcttgaca gcaggtctgt ccgttcaaca ggggattgtt
120cggcaaagag tcatcccagt gtatcaagta aacaatcttg aagaaatttg ccaacttatc
180atacaggcct ttgaagcagg tgttgatttt caagagagtg cggacagttt ccttctcatg
240ctttgtcttc atcatgcgta ccagggagat tacaaacttt tcttggaaag tggcgcagtc
300aagtatttgg aagggcacgg gttccgtttt gaagtcaaga agcgtgatgg agtgaagcgc
360cttgaggaat tgctgccagc agtatctagt ggaaaaaaca ttaagagaac acttgctgcc
420atgccggaag aggagacaac tgaagctaat gccggtcagt ttctctcctt tgcaagtcta
480ttccttccga aattggtagt aggagaaaag gcttgccttg agaaggttca aaggcaaatt
540caagtacatg cagagcaagg actgatacaa tatccaacag cttggcaatc agtaggacac
600atgatggtga ttttccgttt gatgcgaaca aattttctga tcaaatttct cctaatacac
660caagggatgc acatggttgc cgggcatgat gccaacgatg ctgtgatttc aaattcagtg
720gctcaagctc gtttttcagg cttattgatt gtcaaaacag tacttgatca tatcctacaa
780aagacagaac gaggagttcg tctccatcct cttgcaagga ccgccaaggt aaaaaatgag
840gtgaactcct ttaaggctgc actcagctcc ctggccaagc atggagagta tgctcctttc
900gcccgacttt tgaacctttc tggagtaaat aatcttgagc atggtctttt ccctcaacta
960tcggcaattg cactcggagt cgccacagca cacgggagta ccctcgcagg agtaaatgtt
1020ggagaacagt atcaacaact cagagaggct gccactgagg ctgagaagca actccaacaa
1080tatgcagagt ctcgcgaact tgaccatctt ggacttgatg atcaggaaaa gaaaattctt
1140atgaacttcc atcagaaaaa gaacgaaatc agcttccagc aaacaaacgc tatggtaact
1200ctaagaaaag agcgcctggc caagctgaca gaagctatca ctgctgcgtc actgcccaaa
1260acaagtggac attacgatga tgatgacgac attccctttc caggacccat caatgatgac
1320gacaatcctg gccatcaaga tgatgatccg actgactcac aggatacgac cattcccgat
1380gtggtggttg atcccgatga tggaagctac ggcgaatacc agagttactc ggaaaacggc
1440atgaatgcac cagatgactt ggtcctattc gatctagacg aggacgacga ggacactaag
1500ccagtgccta atagatcgac caagggtgga caacagaaga acagtcaaaa gggccagcat
1560atagagggca gacagacaca atccaggcca attcaaaatg tcccaggccc tcacagaaca
1620atccaccacg ccagtgcgcc actcacggac aatgacagaa gaaatgaacc ctccggctca
1680accagccctc gcatgctgac accaattaac gaagaggcag acccactgga cgatgccgac
1740gacgagacgt ctagccttcc gcccttggag tcagatgatg aagagcagga cagggacgga
1800acttccaacc gcacacccac tgtcgcccca ccggctcccg tatacagaga tcactctgaa
1860aagaaagaac tcccgcaaga cgagcaacaa gatcaggacc acactcaaga ggccaggaac
1920caggacagtg acaacaccca gtcagaacac tcttttgagg agatgtatcg ccacattcta
1980agatcacagg ggccatttga tgctgttttg tattatcata tgatgaagga tgagcctgta
2040gttttcagta ccagtgatgg caaagagtac acgtatccag actcccttga agaggaatat
2100ccaccatggc tcactgaaaa agaggctatg aatgaagaga atagatttgt tacattggat
2160ggtcaacaat tttattggcc ggtgatgaat cacaagaata aattcatggc aatcctgcaa
2220catcatcagt gataactcga ggtacgt
2247212217DNAEbola Zaire Virus 21atggattctc gtcctcagaa aatctggatg
gcgccgagtc tcactgaatc tgacatggat 60taccacaaga tcttgacagc aggtctgtcc
gttcaacagg ggattgttcg gcaaagagtc 120atcccagtgt atcaagtaaa caatcttgaa
gaaatttgcc aacttatcat acaggccttt 180gaagcaggtg ttgattttca agagagtgcg
gacagtttcc ttctcatgct ttgtcttcat 240catgcgtacc agggagatta caaacttttc
ttggaaagtg gcgcagtcaa gtatttggaa 300gggcacgggt tccgttttga agtcaagaag
cgtgatggag tgaagcgcct tgaggaattg 360ctgccagcag tatctagtgg aaaaaacatt
aagagaacac ttgctgccat gccggaagag 420gagacaactg aagctaatgc cggtcagttt
ctctcctttg caagtctatt ccttccgaaa 480ttggtagtag gagaaaaggc ttgccttgag
aaggttcaaa ggcaaattca agtacatgca 540gagcaaggac tgatacaata tccaacagct
tggcaatcag taggacacat gatggtgatt 600ttccgtttga tgcgaacaaa ttttctgatc
aaatttctcc taatacacca agggatgcac 660atggttgccg ggcatgatgc caacgatgct
gtgatttcaa attcagtggc tcaagctcgt 720ttttcaggct tattgattgt caaaacagta
cttgatcata tcctacaaaa gacagaacga 780ggagttcgtc tccatcctct tgcaaggacc
gccaaggtaa aaaatgaggt gaactccttt 840aaggctgcac tcagctccct ggccaagcat
ggagagtatg ctcctttcgc ccgacttttg 900aacctttctg gagtaaataa tcttgagcat
ggtcttttcc ctcaactatc ggcaattgca 960ctcggagtcg ccacagcaca cgggagtacc
ctcgcaggag taaatgttgg agaacagtat 1020caacaactca gagaggctgc cactgaggct
gagaagcaac tccaacaata tgcagagtct 1080cgcgaacttg accatcttgg acttgatgat
caggaaaaga aaattcttat gaacttccat 1140cagaaaaaga acgaaatcag cttccagcaa
acaaacgcta tggtaactct aagaaaagag 1200cgcctggcca agctgacaga agctatcact
gctgcgtcac tgcccaaaac aagtggacat 1260tacgatgatg atgacgacat tccctttcca
ggacccatca atgatgacga caatcctggc 1320catcaagatg atgatccgac tgactcacag
gatacgacca ttcccgatgt ggtggttgat 1380cccgatgatg gaagctacgg cgaataccag
agttactcgg aaaacggcat gaatgcacca 1440gatgacttgg tcctattcga tctagacgag
gacgacgagg acactaagcc agtgcctaat 1500agatcgacca agggtggaca acagaagaac
agtcaaaagg gccagcatat agagggcaga 1560cagacacaat ccaggccaat tcaaaatgtc
ccaggccctc acagaacaat ccaccacgcc 1620agtgcgccac tcacggacaa tgacagaaga
aatgaaccct ccggctcaac cagccctcgc 1680atgctgacac caattaacga agaggcagac
ccactggacg atgccgacga cgagacgtct 1740agccttccgc ccttggagtc agatgatgaa
gagcaggaca gggacggaac ttccaaccgc 1800acacccactg tcgccccacc ggctcccgta
tacagagatc actctgaaaa gaaagaactc 1860ccgcaagacg agcaacaaga tcaggaccac
actcaagagg ccaggaacca ggacagtgac 1920aacacccagt cagaacactc ttttgaggag
atgtatcgcc acattctaag atcacagggg 1980ccatttgatg ctgttttgta ttatcatatg
atgaaggatg agcctgtagt tttcagtacc 2040agtgatggca aagagtacac gtatccagac
tcccttgaag aggaatatcc accatggctc 2100actgaaaaag aggctatgaa tgaagagaat
agatttgtta cattggatgg tcaacaattt 2160tattggccgg tgatgaatca caagaataaa
ttcatggcaa tcctgcaaca tcatcag 2217
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