Filovirus consensus antigens, nucleic acid constructs and vaccines made therefrom, and methods of using the same

Abstract

To provide vaccines for inducing immune responses and preventing filovirus infection and / or treating individuals infected with filovirus.SOLUTION: The present invention provides nucleic acid molecules and compositions comprising one or more nucleic acid sequences that encode a consensus filovirus immunogen, including a consensus Marburgvirus filovirus glycoprotein MARV GP immunogen, a consensus Ebolavirus Sudan filovirus glycoprotein SEBOV GP immunogen and a consensus Ebolavirus Zaire glycoprotein ZEBOV GP immunogen.SELECTED DRAWING: Figure 1C

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to vaccines for inducing an immune response and for preventing filovirus infection and / or treating individuals infected with filoviruses, specifically infection by Marburg virus, Ebola virus Sudan, and Ebola virus Zaire. The present invention relates to consensus filovirus proteins, specifically Marburg virus, Ebola virus Sudan, and Ebola virus Zaire filovirus proteins, and nucleic acid molecules encoding them. [Background technology]

[0002] 2. Background of the Invention The Filoviridae family is a group of non-segmented, single-stranded RNA viruses that contains two divergent genera, Marburg virus (MARV) and Ebola virus (EBOV). Members of each of these can cause severe and highly fatal hemorrhagic fever diseases for which there are no treatments or licensed vaccines (Bradfute SB,et al.(2011)Filovirus vaccines.Hum Vaccin 7:701-711; Falzarano D.,et al.(2011)Progress in filovirus vaccine development:evaluating the potential for clinical use.Expert Rev Vaccines 10:63-77; Fields BN,et al.(2007)Fields'virology.Philadelphia:Lippincott Williams&Wilkins.2v.(xix,3091,I-3086p.); Richardson JS,et al.(2009)Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.PLoS One 4:e5308; and Towner JS,et al.(2006)Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola.J Virol 80:6497-6516).

[0003] With a fatality rate of up to 90%, they are listed by the World Health Organization as "one of the most virulent known viral diseases to mankind". The US Centers for Disease Control and Prevention classifies them as "Category A bioterrorism agents", in part due to their potential threat to national security if used as a weapon (Burki TK (2011) USA focuses on Ebola vaccine but research gaps remain. Lancet 378:389). These "high priority" agents could, in theory, be easily transmitted, cause high fatality rates, cause major public health shock and panic, and require special action on public health preparedness (CDC (2011) Bioterrorism Agents / Diseases. Atlanta: Centers for Disease Control and Prevention).

[0004] Hemorrhagic fever is acutely infectious without a carrier state, but is readily transmissible to humans and non-human primates by direct contact with contaminated body fluids, blood, and tissues (Feldmann H., et al. (2003) Ebola virus: from discovery to vaccine. Nat Rev Immunol 3:677-685). In outbreak situations, reuse of medical equipment, resource-limited medical facilities, and delayed prevention measures increase disease transmission and amplify infections in health care facilities.

[0005] The natural hosts of these zoonotic pathogens are likely African bats and pigs (Kobinger GP, et al. (2011) Replication, pathogenicity, shedding, and transmission of Zaire ebolavirus in pigs. J Infect Dis 204:200-208), the latter being a more prolific host, and the initial mode of emergence of the virus at the onset of the outbreak is thought to be through humans in contact with infected animals. The unpredictable epidemic emergence of the disease in the Philippines, potentially Europe, and mainly Africa further constitutes a major public health problem (Outbreak news. (2009) Ebola Reston in pigs and humans, Philippines. Wkly Epidemiol Rec 84:49-50).

[0006] Experiments are being conducted to determine protective efficacy and the ability of the vaccine to induce broad CTLs, including experiments in preclinical rodent studies. Fenimore PW, et al. (2012). Designing and testing broadly-protective filoviral vaccines optimized for cytotoxic T-lymphocyte epitope coverage. PLoS ONE 7:e44769, Hensley LE, et al. (2010). Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus Species. PLoS Pathog 6:el000904, Zahn R,et al(2012).Ad35 and ad26 vaccine vectors induce potent and cross-reactive antibody and T-cell responses to multiple filovirus species.PLoS ONE 7:e44115, Geisbert TW,Feldmann H(2011).Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections.J Infect Dis 204 Suppl 3:S1075-1081, and Grant-Klein RJ, Van Deusen NM, Badger CV, Hannaman D, Dupuy LC, Schmaljohn CS (2012). A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.Hum Vaccin Immunother 8, Grant-Klein RJ, Altamura. L.A., Schmaljohn C.S. (2011).Progress in recombinant DNA-derived vaccines for Lassa virus and filoviruses.Virus Res 162: 148-161)。.

[0007] Vaccine-induced adaptive immune responses have been described in a number of preclinical animal models (Blaney JE,et al.(2011).Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses.J Virol 85:10605-10616, Dowling W,et al.(2007).Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines.J Virol 81:1821-1837, Jones SM,et al.(2005).Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.Nat Med 11:786-790, Kalina WV,Warfield KL,Olinger GG,Bavari S(2009).Discovery of common marburgvirus protective epitopes in a BALB / c mouse model.Virol J 6:132, Kobinger GP, et al. (2006).Chimpanzee adenovirus vaccine protects against Zaire Ebola virus.Virology 346:394-401, Olinger GG, et al. (2005).Protective cytotoxic T-cell responses induced by Venezuelan equine encephalitis virus replicons expressing Ebola virus proteins.J Virol 79:14189-14196, Rao M, Bray M, Alving CR, Jahrling P, Matyas GR (2002).Induction of immune responses in mice and monkeys to Ebola virus after immunization with liposome-encapsulated irradiated Ebola virus: protection in mice requires CD4(+) T cells.J Virol 76:9176-9185、Rao M,Matyas GR,Grieder F,Anderson K,Jahrling PB,Alving CR(1999).Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A.Vaccine 17:2991-2998、Richardson JS,et al.(2009).Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.PLoS One 4:e5308、Vanderzanden L,et al(1998).DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge.Virology 246:134-144、Warfield KL,et al.(2005).Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection.J Immunol 175:1184-1191、Jones SM,et al.(2007).Assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of Ebola virus hemorrhagic fever.J Infect Dis 196 Suppl2:S404-412 Grant-Klein RJ, Van Deusen NM, Badger CV, Hannaman D, Dupuy LC, Schmaljohn CS(2012).A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.Hum Vaccin Immunother 8., Geisbert TW,et al.(2010).Vector choice determines immunogenicity and potency of genetic vaccines against Angola Marburg virus in nonhuman primates.J Virol 84:10386-10394). Viral vaccines are promising and include primarily recombinant adenoviruses and vesicular stomatitis viruses. Noninfectious strategies such as recombinant DNA and Ag-conjugated virus-like particle (VLP) vaccines also have demonstrated levels of preclinical efficacy and are generally considered safer than virus-based platforms. Virus-specific Abs can be protective when applied passively, either before or immediately after infection (Gupta M, Mahanty S, Bray M, Ahmed R, Rollin PE (2001). Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication. J Virol 75:4649-4654, Marzi A, et al. (2012).Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.PLoS ONE 7:e36192、Parren PW,Geisbert TW,Maruyama T,Jahrling PB,Burton DR(2002).Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody.J Virol 76:6408-6412、Qiu X,et al.(2012).Ebola GP-Specific Monoclonal Antibodies Protect Mice and Guinea Pigs from Lethal Ebola Virus Infection.PLoSNegl Trop Dis 6: el575、Wilson JA,et al.(2000).Epitopes involved in antibody-mediated protection from Ebola virus.Science 287:1664-1666、Sullivan NJ,et al.(2011).CD8(+)cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates.Nat Med 17:1128-1131、Bradfute SB,Warfield KL,Bavari S(2008).Functional CD8+ T cell responses in lethal Ebola virus infection.J Immunol 180:4058-4066、Warfield KL,Olinger GG(2011).Protective role of cytotoxic T lymphocytes in filovirus hemorrhagic fever.J Biomed Biotechnol 2011:984241). T cells have also been shown to provide protection based on studies performed in knockout mice, depletion studies in NHPs, and mouse adoptive transfer studies in which efficacy was largely related to the lytic function of adoptively transferred CD8+ T cells. However, relevant analyses of this response as driven by a protective vaccine have rarely been reported.

[0008] Development of countermeasures will ultimately require an improved understanding of protective immune correlates and how they are regulated during infection. This proves challenging when infected individuals suffering from filoviral diseases are unable to mount an early immune response. These fast-moving hemorrhagic fever diseases result in immune dysregulation as indicated by the loss of virus-specific Ab responses, and a marked reduction in total T cell numbers, leading to uncontrolled viral replication and multi-organ infection and failure. Conversely, survivors of Ebola virus (EBOV) disease display an early and transient IgM response, followed shortly by increased levels of virus-specific IgG and CTL. These observations suggest that humoral and cell-mediated immune responses contribute to conferring protection to disease. These data are also supported by numerous preclinical efficacy studies demonstrating the contribution of vaccine-induced adaptive immunity to protection against lethal challenge. However, a mountain of evidence demonstrates a critical role for T cells in providing protection, with efficacy being highly linked to the functional phenotype of CD8+ T cells. Although these recent studies highlight the importance of T cells in providing protection, their precise contribution remains uncharacterized and controversial, and detailed analyses of this response driven by protective vaccines have rarely been reported. Summary of the Invention

[0009] Summary of the Invention Compositions are provided that include a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen. The amino acid sequence of the consensus Zaire Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1, an amino acid sequence homologous to SEQ ID NO:1, or a fragment of an amino acid sequence homologous to SEQ ID NO:1. An amino acid sequence homologous to SEQ ID NO:1 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:1. A fragment of SEQ ID NO:1 or a fragment of an amino acid sequence homologous to SEQ ID NO:1 is typically 600 or more, 630 or more, or 660 or more amino acids long. The amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2, an amino acid sequence homologous to SEQ ID NO:2, or a fragment of an amino acid sequence homologous to SEQ ID NO:2. An amino acid sequence homologous to SEQ ID NO:1 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:2. A fragment of SEQ ID NO:2 or a fragment of an amino acid sequence homologous to SEQ ID NO:2 is typically 600 or more, 630 or more, or 660 or more amino acids. The amino acid sequence of the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen may be SEQ ID NO:3 (MARV ANG), a fragment of SEQ ID NO:3, an amino acid sequence homologous to SEQ ID NO:3, or a fragment of an amino acid sequence homologous to SEQ ID NO:3. An amino acid sequence homologous to SEQ ID NO: 3 is typically at least 95%, at least 96%, at least 97%, at least 99%, or at least 99% homologous to SEQ ID NO: 3. Fragments of SEQ ID NO: 3 or of an amino acid sequence homologous to SEQ ID NO: 3 are typically at least 600, at least 637, or at least 670 amino acids. The amino acid sequence may optionally include a leader sequence, such as an IgE leader.

[0010] Also provided are compositions comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a Marburg-Marburg virus first consensus envelope glycoprotein immunogen, a nucleic acid sequence encoding a Marburg-Marburg virus second consensus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a Marburg-Marburg virus third consensus envelope glycoprotein immunogen. The amino acid sequence of the consensus Zaire Ebola virus envelope glycoprotein immunogen may be SEQ ID NO: 1 (ZEBOV CON), a fragment of SEQ ID NO: 1, an amino acid sequence homologous to SEQ ID NO: 1, or a fragment of an amino acid sequence homologous to SEQ ID NO: 1. An amino acid sequence homologous to SEQ ID NO: 1 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO: 1. A fragment of SEQ ID NO:1 or a fragment of an amino acid sequence homologous to SEQ ID NO:1 is typically 600 or more, 630 or more, or 660 or more amino acids. The amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2, an amino acid sequence homologous to SEQ ID NO:2, or a fragment of an amino acid sequence homologous to SEQ ID NO:2. An amino acid sequence homologous to SEQ ID NO:1 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:2. A fragment of SEQ ID NO:2 or a fragment of an amino acid sequence homologous to SEQ ID NO:2 is typically 600 or more, 630 or more, or 660 or more amino acids. The amino acid sequence of the Marburg Marburg virus 1 consensus envelope glycoprotein immunogen may be SEQ ID NO:4 (MARV RAV), a fragment of SEQ ID NO:4, an amino acid sequence homologous to SEQ ID NO:4, or a fragment of an amino acid sequence homologous to SEQ ID NO:4. An amino acid sequence homologous to SEQ ID NO:4 is typically greater than 95%, greater than 96%, greater than 97%, greater than 99%, or greater than 99% homologous to SEQ ID NO:4.A fragment of SEQ ID NO:4 or a fragment of an amino acid sequence homologous to SEQ ID NO:4 is typically 600 or more, 637 or more, or 670 or more amino acids long. The amino acid sequence of the Marburg virus second consensus envelope glycoprotein immunogen may be SEQ ID NO:5 (MARV OZO), a fragment of SEQ ID NO:5, an amino acid sequence homologous to SEQ ID NO:5, or a fragment of an amino acid sequence homologous to SEQ ID NO:5. An amino acid sequence homologous to SEQ ID NO:5 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:4. A fragment of SEQ ID NO:5 or a fragment of an amino acid sequence homologous to SEQ ID NO:5 is typically 600 or more, 637 or more, or 670 or more amino acids long. The amino acid sequence of the Marburg virus third consensus envelope glycoprotein immunogen may be SEQ ID NO:6 (MARV MUS), a fragment of SEQ ID NO:6, an amino acid sequence homologous to SEQ ID NO:6, or a fragment of an amino acid sequence homologous to SEQ ID NO:6. An amino acid sequence homologous to SEQ ID NO:6 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:6. A SEQ ID NO:6 fragment or a fragment of an amino acid sequence homologous to SEQ ID NO:6 is typically 600 or more, 637 or more, or 670 or more amino acids. The amino acid sequence may optionally include a leader sequence, such as an IgE leader. In some embodiments, the composition further comprises a nucleic acid sequence encoding a Marburg virus Angola 2005 envelope glycoprotein immunogen. The amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen may be SEQ ID NO:3 (MARV ANG), a fragment of SEQ ID NO:3, an amino acid sequence homologous to SEQ ID NO:3, or a fragment of an amino acid sequence homologous to SEQ ID NO:3. An amino acid sequence homologous to SEQ ID NO:3 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:3. A fragment of SEQ ID NO:3 or a fragment of an amino acid sequence homologous to SEQ ID NO:3 is typically 600 or more, 637 or more, or 670 or more amino acids long.The amino acid sequence may optionally include a leader sequence, such as the IgE leader.

[0011] Also provided are compositions comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen and a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen. The amino acid sequence of the consensus Zaire Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1, an amino acid sequence homologous to SEQ ID NO:1, or a fragment of an amino acid sequence homologous to SEQ ID NO:1. An amino acid sequence homologous to SEQ ID NO:1 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:1. A fragment of SEQ ID NO:1 or a fragment of an amino acid sequence homologous to SEQ ID NO:1 is typically 600 or more, 630 or more, or 660 or more amino acids long. The amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2, an amino acid sequence homologous to SEQ ID NO:2, or a fragment of an amino acid sequence homologous to SEQ ID NO:2. An amino acid sequence homologous to SEQ ID NO: 1 is typically at least 95%, at least 96%, at least 97%, at least 99%, or at least 99% homologous to SEQ ID NO: 2. Fragments of SEQ ID NO: 2 or of an amino acid sequence homologous to SEQ ID NO: 2 are typically at least 600, at least 630, or at least 660 amino acids. The amino acid sequence may optionally include a leader sequence, such as an IgE leader.

[0012] Compositions are provided that include a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a Marburg Marburg Virus Angola 2005 envelope glycoprotein immunogen. The nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:64, a fragment of SEQ ID NO:64, a nucleic acid sequence homologous to SEQ ID NO:64, or a fragment of a nucleotide sequence homologous to SEQ ID NO:64. A nucleic acid sequence homologous to SEQ ID NO:64 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:64. A fragment of SEQ ID NO:64 or a fragment of an amino acid sequence homologous to SEQ ID NO:64 typically encodes 600 or more, 630 or more, or 660 or more amino acids of the consensus Zaire Ebola virus envelope glycoprotein immunogen encoded by SEQ ID NO:64. The nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen may be SEQ ID NO:65, a fragment of SEQ ID NO:65, a nucleic acid sequence homologous to SEQ ID NO:65, or a fragment of a nucleotide sequence homologous to SEQ ID NO:65. A nucleic acid sequence homologous to SEQ ID NO:65 is typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:65. A fragment of SEQ ID NO:65 or a fragment of an amino acid sequence homologous to SEQ ID NO:65 typically encodes 600 or more, 630 or more, or 660 or more amino acids of the consensus Sudan Ebola virus envelope glycoprotein immunogen encoded by SEQ ID NO:65. A nucleic acid sequence encoding the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen may be SEQ ID NO:66, a fragment of SEQ ID NO:66, a nucleic acid sequence homologous to SEQ ID NO:66, or a fragment of a nucleotide sequence homologous to SEQ ID NO:66. Nucleic acid sequences homologous to SEQ ID NO:66 are typically 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:66.Fragments of SEQ ID NO: 66 or fragments of amino acid sequences homologous to SEQ ID NO: 66 typically encode 600 or more, 630 or more, or 670 or more amino acids of the Marburg virus Angola 2005 envelope glycoprotein immunogen encoded by SEQ ID NO: 66. The nucleic acid sequence may optionally include a sequence encoding a leader sequence, such as an IgE leader, linked to the sequence encoding the immunogen.

[0013] Each of the different nucleic acid sequences may be on a single nucleic acid molecule, each on a separate nucleic acid molecule, or in various permutations. The nucleic acid molecule may be a plasmid.

[0014] The composition may be formulated for delivery to an individual using electroporation.

[0015] The composition may further comprise a nucleic acid sequence encoding one or more proteins selected from the group consisting of IL-12, IL-15, and IL-28.

[0016] The composition may be used in a method of inducing an immune response against a filovirus, the filovirus may be selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire.

[0017] Methods are provided for treating an individual diagnosed with a filovirus comprising administering to the individual a therapeutically effective amount of the composition, the filovirus may be selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire.

[0018] Methods for preventing filovirus infection in an individual are provided, comprising administering to the individual a prophylactically effective amount of a composition, the filovirus being selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire.

[0019] Compositions are provided that include two or more proteins selected from the group consisting of a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a Marburg MARburg virus Angola 2005 envelope glycoprotein immunogen, a first consensus Marburg MARburg virus envelope glycoprotein immunogen, a second consensus Marburg MARburg virus envelope glycoprotein immunogen, and a third consensus Marburg MARburg virus envelope glycoprotein immunogen. [Brief description of the drawings]

[0020] [Figure 1A]Figures 1A-1C describe the multivalent vaccine construction strategy and expression experiments in Example 1. Figure 1A shows the phylogenetic tree for MGP (top), SGP (bottom right), and ZGP (bottom left). Significant support values ​​were verified by bootstrap analysis and are indicated by (*). The consensus strategy was applied to ZGP and SGP immunogens (CON vaccine). The scale bar represents the distance of amino acids per site, and the analysis was performed using MEGA version 5 software. GP transgenes were commercially synthesized, genetically optimized, and subcloned into a modified pVAX1 mammalian expression vector. Antigen expression was analyzed by Western immunoblotting and FACS following transfection of HEK293T cells. The results of Western immunoblotting are shown in Figure 1B, and the results of FACS are shown in Figure 1C. For comparative control, rVSV expressing MGP, SGP, or ZGP were run simultaneously with each GP specimen, and species-specific anti-GP1 mAb was used for detection. Sizes are indicated (kDa). For FACS, transfected cells were indirectly stained with mouse-derived GP-specific serum reagents, then washed extensively, followed by goat anti-mouse IgG and MHC class I. Western immunoblotting and FACS experiments were repeated at least three times with similar results. Significance for unrooted phylogenetic trees was determined by maximum likelihood method and verified by bootstrap analysis, and significant support values ​​(≥80%; 1,000 bootstrap replicates) were determined by MEGA version 5 software. [Figure 1B]Figures 1A-1C describe the multivalent vaccine construction strategy and expression experiments in Example 1. Figure 1A shows the phylogenetic tree for MGP (top), SGP (bottom right), and ZGP (bottom left). Significant support values ​​were verified by bootstrap analysis and are indicated by (*). The consensus strategy was applied to ZGP and SGP immunogens (CON vaccine). The scale bar represents the distance of amino acids per site, and the analysis was performed using MEGA version 5 software. GP transgenes were commercially synthesized, genetically optimized, and subcloned into a modified pVAX1 mammalian expression vector. Antigen expression was analyzed by Western immunoblotting and FACS following transfection of HEK293T cells. The results of Western immunoblotting are shown in Figure 1B, and the results of FACS are shown in Figure 1C. For comparative control, rVSV expressing MGP, SGP, or ZGP were run simultaneously with each GP specimen, and species-specific anti-GP1 mAb was used for detection. Sizes are indicated (kDa). For FACS, transfected cells were indirectly stained with mouse-derived GP-specific serum reagents, then washed extensively, followed by goat anti-mouse IgG and MHC class I. Western immunoblotting and FACS experiments were repeated at least three times with similar results. Significance for unrooted phylogenetic trees was determined by maximum likelihood method and verified by bootstrap analysis, and significant support values ​​(≥80%; 1,000 bootstrap replicates) were determined by MEGA version 5 software. [Figure 1C]Figures 1A-1C describe the multivalent vaccine construction strategy and expression experiments in Example 1. Figure 1A shows the phylogenetic tree for MGP (top), SGP (bottom right), and ZGP (bottom left). Significant support values ​​were verified by bootstrap analysis and are indicated by (*). The consensus strategy was applied to ZGP and SGP immunogens (CON vaccine). The scale bar represents the distance of amino acids per site, and the analysis was performed using MEGA version 5 software. GP transgenes were commercially synthesized, genetically optimized, and subcloned into a modified pVAX1 mammalian expression vector. Antigen expression was analyzed by Western immunoblotting and FACS following transfection of HEK293T cells. The results of Western immunoblotting are shown in Figure 1B, and the results of FACS are shown in Figure 1C. For comparative control, rVSV expressing MGP, SGP, or ZGP were run simultaneously with each GP specimen, and species-specific anti-GP1 mAb was used for detection. Sizes are indicated (kDa). For FACS, transfected cells were indirectly stained with mouse-derived GP-specific serum reagents, then washed extensively, followed by goat anti-mouse IgG and MHC class I. Western immunoblotting and FACS experiments were repeated at least three times with similar results. Significance for unrooted phylogenetic trees was determined by maximum likelihood method and verified by bootstrap analysis, and significant support values ​​(≥80%; 1,000 bootstrap replicates) were determined by MEGA version 5 software. [Figure 2A]2A-2H show the experimental results in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in FIG. 2A and FIG. 2E. FIG. 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. FIG. 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in FIG. 2B. The y-axis shows the change in body weight as shown in FIG. 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2B]2A-2H show the experimental results in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in FIG. 2A and FIG. 2E. FIG. 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. FIG. 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in FIG. 2B. The y-axis shows the change in body weight as shown in FIG. 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2C]2A-2H show the experimental results in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in FIG. 2A and FIG. 2E. FIG. 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. FIG. 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in FIG. 2B. The y-axis shows the change in body weight as shown in FIG. 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2D]Figures 2A-2H show the results of the experiment in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. Figure 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in Figure 2B. The y-axis shows the change in body weight as shown in Figure 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2E]Figures 2A-2H show the results of the experiment in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. Figure 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in Figure 2B. The y-axis shows the change in body weight as shown in Figure 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2F]Figures 2A-2H show the results of the experiment in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. Figure 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in Figure 2B. The y-axis shows the change in body weight as shown in Figure 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2G]Figures 2A-2H show the results of the experiment in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. Figure 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in Figure 2B. The y-axis shows the change in body weight as shown in Figure 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 2H]Figures 2A-2H show the results of the experiment in Example 1, where complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that animals receiving the trivalent vaccine survived MARV challenge, while control animals had all died by day 10. Figure 2E shows that animals receiving the trivalent vaccine survived ZEBOV challenge, while control animals had all died by day 7. Data on the % change in body weight for vaccinated and control animals relative to those challenged with MARV vaccine is shown in Figure 2B. The y-axis shows the change in body weight as shown in Figure 2F. The thin solid line is for animals receiving the trivalent vaccine. The thin dashed line is for TriAVE, the average result of animals receiving the trivalent vaccine. The thick solid line is for control animals. The thick dashed line is for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-9 post-challenge, terminating with a dagger indicating that animals died of disease by day 10. Data for % change in body weight for vaccinated and control animals relative to those challenged with vaccinated ZEBOV are shown in FIG. 2F. The y-axis shows the change in body weight in percent. The thin solid lines are for animals that received the trivalent vaccine. The thin dashed lines are for TriAVE, the average result of animals that received the trivalent vaccine. The thick solid lines are for control animals. The thick dashed lines are for ControlAVE, the average result of control animals. The thin solid and thin dashed lines remain graphically stable on the days post-challenge, indicating no significant weight loss among vaccinated animals. The thick solid and thick dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals died of disease before day 8. (n=3 for gpMARV, n=6 for gpZEBOV).Binding Abs (Figures 2C and 2G) and NAbs (Figures 2D and 2H) were measured in sera from vaccinated animals before (Pre) and after the first (1X) and second (2X) immunization. Analysis was performed on pooled sera (Figure 2H). *p<0.1, ***p<0.001, ****p<0.0001. [Figure 3A] Figures 3A-3C show the results of Example 1 demonstrating the induction of neutralizing Abs. B cell responses were assessed in mice (n=5 / group) for 20 days after each of the two vaccinations, with 3 weeks between 40 μg E-DNA vaccination injections. Figure 3A shows serum GP-specific IgG responses from vaccinated (solid line) or prebleed (dotted line) mice measured by ELISA. Data are summarized in Figure 3B. Total responses from pEBOS- and pEBOZ-immunized animals were measured against sucrose-purified ZGP, as SGP was not available in this study. IgG responses from pMARV-immunized mice were measured against MARV-Ozolin GP or with the negative control sucrose-purified Nipah G protein, and neutralizing activity of serum specimens was measured against ZEBOV-EGFP, SUDV-Boniface, and MARV-Angola in a BSL-4 facility, with NAb titers shown in Figure 3C. NAbs against SUDV-Boniface were analyzed based on cytopathic effect (CPE) in CV-1 cells, and NAbs against MARV-Angola were analyzed using immunofluorescence analysis. Means are shown in Figure 3B and Figure 3C, and error bars represent standard error. Group analysis was completed by matched two-tailed independent t-test. Experiments were repeated at least twice with similar results, *p<0.1, **p<0.01, ***p<0.001. [Figure 3B]Figures 3A-3C show the results of Example 1 demonstrating the induction of neutralizing Abs. B cell responses were assessed in mice (n=5 / group) for 20 days after each of the two vaccinations, with 3 weeks between 40 μg E-DNA vaccination injections. Figure 3A shows serum GP-specific IgG responses from vaccinated (solid line) or prebleed (dotted line) mice measured by ELISA. Data are summarized in Figure 3B. Total responses from pEBOS- and pEBOZ-immunized animals were measured against sucrose-purified ZGP, as SGP was not available in this study. IgG responses from pMARV-immunized mice were measured against MARV-Ozolin GP or with the negative control sucrose-purified Nipah G protein, and neutralizing activity of serum specimens was measured against ZEBOV-EGFP, SUDV-Boniface, and MARV-Angola in a BSL-4 facility, with NAb titers shown in Figure 3C. NAbs against SUDV-Boniface were analyzed based on cytopathic effect (CPE) in CV-1 cells, and NAbs against MARV-Angola were analyzed using immunofluorescence analysis. Means are shown in Figure 3B and Figure 3C, and error bars represent standard error. Group analysis was completed by matched two-tailed independent t-test. Experiments were repeated at least twice with similar results, *p<0.1, **p<0.01, ***p<0.001. [Figure 3C]Figures 3A-3C show the results of Example 1 demonstrating the induction of neutralizing Abs. B cell responses were assessed in mice (n=5 / group) for 20 days after each of the two vaccinations, with 3 weeks between 40 μg E-DNA vaccination injections. Figure 3A shows serum GP-specific IgG responses from vaccinated (solid line) or prebleed (dotted line) mice measured by ELISA. Data are summarized in Figure 3B. Total responses from pEBOS- and pEBOZ-immunized animals were measured against sucrose-purified ZGP, as SGP was not available in this study. IgG responses from pMARV-immunized mice were measured against MARV-Ozolin GP or with the negative control sucrose-purified Nipah G protein, and neutralizing activity of serum specimens was measured against ZEBOV-EGFP, SUDV-Boniface, and MARV-Angola in a BSL-4 facility, with NAb titers shown in Figure 3C. NAbs against SUDV-Boniface were analyzed based on cytopathic effect (CPE) in CV-1 cells, and NAbs against MARV-Angola were analyzed using immunofluorescence analysis. Means are shown in Figure 3B and Figure 3C, and error bars represent standard error. Group analysis was completed by matched two-tailed independent t-test. Experiments were repeated at least twice with similar results, *p<0.1, **p<0.01, ***p<0.001. [Figure 4A]Figures 4A-4D show the broad T cell responses generated by vaccination. In Figure 4A, H-2b (light bars) and H-2d (dark bars) mice (n=5 / group) were immunized twice with pMARV, pEBOS, or pEBOZ DNA, and IFNγ responses were measured by IFNγ ELISPOT analysis. Splenocytes harvested 8 days after the second immunization were incubated in the presence of individual GP peptides (15mer overlaps of 9 amino acids), and results are shown in stacked bar graphs. Epitope-containing peptides were confirmed (average ≧10 spots and ≧80% response rate), corroborated by flow cytometry, and characterized in populations of total activated IFNγ+ and CD44+CD4+ and / or CD8+ T cells (Tables 1-6), and the number of peptides of positive inducers is shown above the bars. Peptides containing CD4+ epitopes alone, peptides containing CD8+ epitopes alone (*), and peptides containing both CD4+ and CD8+ epitopes (**) are counted. Putative common and / or partial epitopes were searched for in consecutive positive peptide responses (Tables 1-6). [Figure 4B] Figures 4A-4D show the broad T cell responses generated by vaccination. Figure 4B shows an amino acid similarity plot comparing the GP sequences from MARV, SUDV, and ZEBOV viruses shown in Figure 1A. [Figure 4C] Figures 4A-4D show the broad T cell responses generated by vaccination. Figure 4C shows the putative domains within ZEBOV GP (GenBank #VGP_EBOZM). SP, signal peptide; RB, receptor binding; MUC, mucin-like region; FC, furin cleavage site; TM, transmembrane region. [Figure 4D] Figures 4A-4D show the broad range of T cell responses generated by vaccination. In Figure 4D, the total subdominant (dark fill) and immunodominant (light fill) T cell epitope responses are shown as a percentage of the total IFNγ response generated by each vaccine. The experiment was repeated at least twice with similar results. [Figure 5A]Figures 5A-5D show data from an experiment evaluating protective "single dose" vaccination to induce neutralizing Abs and CTLs. H-2k mice (n=10 / group) were vaccinated intramuscularly once with pEBOZ E-DNA in a BSL-4 facility, then received 1,000 LD50 of mZEBOV on day 28 post-challenge. Mice were weighed daily and monitored for disease progression. Animal survival data is in Figure 5A. Vaccinated animals survived challenge, whereas control animals died by day 7. Figure 5B shows data on % change in body weight in challenged animals. Data for immunized animals is shown as a light solid line, and the average data for immunized animals is shown as a light dashed line. Data for control animals is shown as a heavy solid line, and the average data for control animals is shown as a heavy dashed line. The light solid and light dashed lines remain stable within approximately 85%-120% on the graph on the days post-challenge, indicating no significant weight loss among vaccinated animals. The dark solid and dark dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals had died of disease by day 7. NAbs measured prior to challenge, data are shown in Figure 5C. T cell responses following single dose pEBOZ immunization measured by FACS are summarized in Figure 5D as the mean % of total CD44+ / IFNγ+CD4+ (dark) or CD8+ (light) cells. Th1-type effector markers were assessed (TNF and T-bet) and data for CD44+ / IFNγ+CD4+ and CD8+ T cells were compared to total T cell data, which were as follows: for total cells: TNF 2.9 ± 0.8, Tbet 13.0 ± 1.1. for CD4+ / CD44+ / IFNγ+ cells: TNF 61.4 ± 3.1, Tbet 72.6 ± 2.0. For CD8+ / CD44+ / IFNγ+ cells: TNF3 3.0±3.3, Tbet9 2.1±1.4 (*p<0.1, ***p<0.001, ****p<0.0001). Group analysis was completed by matched two-tailed independent t-test and survival curves were analyzed by log-rank (Mantel-Cox) test. Experiments were performed twice with similar results and error bars represent standard error. [Figure 5B] Figures 5A-5D show data from an experiment evaluating protective "single dose" vaccination to induce neutralizing Abs and CTLs. H-2k mice (n=10 / group) were vaccinated intramuscularly once with pEBOZ E-DNA in a BSL-4 facility, then received 1,000 LD50 of mZEBOV on day 28 post-challenge. Mice were weighed daily and monitored for disease progression. Animal survival data is in Figure 5A. Vaccinated animals survived challenge, whereas control animals died by day 7. Figure 5B shows data on % change in body weight in challenged animals. Data for immunized animals is shown as a light solid line, and the average data for immunized animals is shown as a light dashed line. Data for control animals is shown as a heavy solid line, and the average data for control animals is shown as a heavy dashed line. The light solid and light dashed lines remain stable within approximately 85%-120% on the graph on the days post-challenge, indicating no significant weight loss among vaccinated animals. The dark solid and dark dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals had died of disease by day 7. NAbs measured prior to challenge, data are shown in Figure 5C. T cell responses following single dose pEBOZ immunization measured by FACS are summarized in Figure 5D as the mean % of total CD44+ / IFNγ+CD4+ (dark) or CD8+ (light) cells. Th1-type effector markers were assessed (TNF and T-bet) and data for CD44+ / IFNγ+CD4+ and CD8+ T cells were compared to total T cell data, which were as follows: for total cells: TNF 2.9 ± 0.8, Tbet 13.0 ± 1.1. for CD4+ / CD44+ / IFNγ+ cells: TNF 61.4 ± 3.1, Tbet 72.6 ± 2.0. For CD8+ / CD44+ / IFNγ+ cells: TNF3 3.0±3.3, Tbet9 2.1±1.4 (*p<0.1, ***p<0.001, ****p<0.0001). Group analysis was completed by matched two-tailed independent t-test and survival curves were analyzed by log-rank (Mantel-Cox) test. Experiments were performed twice with similar results and error bars represent standard error. [Figure 5C] Figures 5A-5D show data from an experiment evaluating protective "single dose" vaccination to induce neutralizing Abs and CTLs. H-2k mice (n=10 / group) were vaccinated intramuscularly once with pEBOZ E-DNA in a BSL-4 facility, then received 1,000 LD50 of mZEBOV on day 28 post-challenge. Mice were weighed daily and monitored for disease progression. Animal survival data is in Figure 5A. Vaccinated animals survived challenge, whereas control animals died by day 7. Figure 5B shows data on % change in body weight in challenged animals. Data for immunized animals is shown as a light solid line, and the average data for immunized animals is shown as a light dashed line. Data for control animals is shown as a heavy solid line, and the average data for control animals is shown as a heavy dashed line. The light solid and light dashed lines remain stable within approximately 85%-120% on the graph on the days post-challenge, indicating no significant weight loss among vaccinated animals. The dark solid and dark dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals had died of disease by day 7. NAbs measured prior to challenge, data are shown in Figure 5C. T cell responses following single dose pEBOZ immunization measured by FACS are summarized in Figure 5D as the mean % of total CD44+ / IFNγ+CD4+ (dark) or CD8+ (light) cells. Th1-type effector markers were assessed (TNF and T-bet) and data for CD44+ / IFNγ+CD4+ and CD8+ T cells were compared to total T cell data, which were as follows: for total cells: TNF 2.9 ± 0.8, Tbet 13.0 ± 1.1. for CD4+ / CD44+ / IFNγ+ cells: TNF 61.4 ± 3.1, Tbet 72.6 ± 2.0. For CD8+ / CD44+ / IFNγ+ cells: TNF3 3.0±3.3, Tbet9 2.1±1.4 (*p<0.1, ***p<0.001, ****p<0.0001). Group analysis was completed by matched two-tailed independent t-test and survival curves were analyzed by log-rank (Mantel-Cox) test. Experiments were performed twice with similar results and error bars represent standard error. [Figure 5D]Figures 5A-5D show data from an experiment evaluating protective "single dose" vaccination to induce neutralizing Abs and CTLs. H-2k mice (n=10 / group) were vaccinated intramuscularly once with pEBOZ E-DNA in a BSL-4 facility, then received 1,000 LD50 of mZEBOV on day 28 post-challenge. Mice were weighed daily and monitored for disease progression. Animal survival data is in Figure 5A. Vaccinated animals survived challenge, whereas control animals died by day 7. Figure 5B shows data on % change in body weight in challenged animals. Data for immunized animals is shown as a light solid line, and the average data for immunized animals is shown as a light dashed line. Data for control animals is shown as a heavy solid line, and the average data for control animals is shown as a heavy dashed line. The light solid and light dashed lines remain stable within approximately 85%-120% on the graph on the days post-challenge, indicating no significant weight loss among vaccinated animals. The dark solid and dark dashed lines are graphically decreasing from days 0-6 post-challenge, terminating with a dagger indicating that animals had died of disease by day 7. NAbs measured prior to challenge, data are shown in Figure 5C. T cell responses following single dose pEBOZ immunization measured by FACS are summarized in Figure 5D as the mean % of total CD44+ / IFNγ+CD4+ (dark) or CD8+ (light) cells. Th1-type effector markers were assessed (TNF and T-bet) and data for CD44+ / IFNγ+CD4+ and CD8+ T cells were compared to total T cell data, which were as follows: for total cells: TNF 2.9 ± 0.8, Tbet 13.0 ± 1.1. for CD4+ / CD44+ / IFNγ+ cells: TNF 61.4 ± 3.1, Tbet 72.6 ± 2.0. For CD8+ / CD44+ / IFNγ+ cells: TNF3 3.0±3.3, Tbet9 2.1±1.4 (*p<0.1, ***p<0.001, ****p<0.0001). Group analysis was completed by matched two-tailed independent t-test and survival curves were analyzed by log-rank (Mantel-Cox) test. Experiments were performed twice with similar results and error bars represent standard error. [Figure 6] FIG. 1 shows GP-specific T cell gating as disclosed in Example 1. [Figure 7A] FIG. 7A shows that the vaccination experiment in Example 1 generated robust T cells. [Figure 7B] FIG. 7B shows that the vaccination experiment in Example 1 generated robust T cells. [Figure 8A] FIG. 8A shows T cell induction by "single dose" vaccination as disclosed in Example 1. [Figure 8B] FIG. 8B shows T cell induction by "single dose" vaccination as disclosed in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one embodiment of the invention, it is desirable for the consensus antigen to provide improved transcription and translation, including having one or more of the following: a leader sequence with low GC content to increase transcription, mRNA stabilization and codon optimization, elimination of cis-acting sequence motifs (i.e., internal TATA-boxes) wherever possible.

[0022] In some embodiments of the invention, it is desirable to generate consensus antigens that generate a broad immune response across multiple strains, including having at least one of the following: incorporation of all available full-length sequences, computer-generated sequences that utilize the most commonly occurring amino acids at each position, and increased cross-reactivity between strains.

[0023] Diversity in the Filoviridae family is relatively high. Great efforts are being made towards the development of a universal and broadly reactive filovirus vaccine that would ideally provide protection against the multiple species responsible for the highest human mortality. However, this is difficult due to the relatively high level of diversity in the Filoviridae family. EBOV is currently classified into five separate species: Zaire Ebolavirus (ZEBOV), Sudan Ebolavirus (SUDV), Reston Ebolavirus (RESTV), Bundibugyo Ebolavirus (BDBV), and Tai Forest Ebolavirus (TAFV; formerly Côte d'Ivoire Ebolavirus), the first two of which are responsible for the highest mortality rates and are the most likely candidates for weaponization. Diversity is lower in Marburg virus (MARV), which can also have a mortality rate of up to 90%. Currently, there is only one taxonomic species, Marburg Marburg virus (formerly Lake Victoria Marburg virus), but recent revisions have proposed that this contains two viruses, including Rabin virus (RAVV). Adding to the complexity of multivalent vaccine development, MARV and EBOV are highly divergent, with approximately 67% divergence at the nucleotide level. Furthermore, the phylogenetic diversity in filovirus GP is also very high (82% overall). These suggest a potential for filovirus envelope formation, as demonstrated by the recent emergence of BDBV in 2007. Therefore, due to the relative divergence in the Filoviridae family, we hypothesized that the development of an effective multivalent filovirus vaccine will likely require a mixture of immunogenic components.

[0024] A synthetic polyvalent filovirus DNA vaccine against Marburg virus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV) was developed. The novel polyvalent filovirus vaccine applied a multidrug combination approach and contained three DNA plasmids encoding the envelope glycoprotein (GP) genes of Marburg virus (MARV), Sudan ebolavirus (SUDV), or Zaire ebolavirus (ZEBOV). As filovirus vaccine candidates, enhanced DNA (DNA)-based platforms show many advantages due to recent advances in gene optimization and delivery technologies (Bagarazzi ML, et al. (2012). Immunotherapy Against HPV16 / 18 Generates Potent TH1 and Cytotoxic Cellular Immune Responses. Sci Transl Med 4:155ral38; Kee ST, Gehl J, W.LE (2011). Clinical Aspects of Electroporation, Springer, New York, NY.; Hirao LA, et al. (2011). Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge. J Infect Dis 203:95-102). Therefore, each GP was genetically optimized and subcloned into a modified mammalian expression vector and then delivered using in vivo electroporation (EP).

[0025] Preclinical efficacy studies were carried out in guinea pigs and mice using rodent-adapted viruses, and mouse T cell responses were analyzed on a large scale using the novel modified analysis described herein.T cell responses were analyzed on a large scale, including the use of the novel method for epitope identification and characterization described herein.This model provides an important preclinical tool for studying protective immune correlates that can be applied to existing platforms.

[0026] Vaccination in preclinical rodent studies has been shown to be highly potent and elicited robust neutralizing antibodies (NAbs) and T h CTLs expressing type l markers and were fully protected against MARV and ZEBOV challenge. Comprehensive T cell analysis analyzed at scale using the novel modified assay described herein (Shedlock DJ, et al. (2012). Vaccination with synthetic constructs expressing cytomegalovirus immunogens is highly T cell immunogenic in mice. Hum Vaccin Immunother 8:1668-1681) demonstrated large-scale cytotoxic T lymphocytes, epitope breadth, and T h l-type marker expression. Overall, 52 novel T cell epitopes from two different mouse genetic backgrounds were identified (19 of 20 MARV epitopes, 15 of 16 SUDV, and 18 of 22 ZEBOV epitopes), occurring primarily in highly conserved regions of their corresponding glycoproteins (GPs). These data represent the most comprehensive report of preclinical glycoprotein epitopes to date.

[0027] In developing a strategy to provide protection against multiple species responsible for the highest human mortality, we focused on MARV, SUDV, and ZEBOV. Because of their relative divergence, we hypothesized that the development of a multivalent filovirus vaccine would require a mixture of components that could be quickly and easily adapted depending on future emerging strains and / or species. Although the overall diversity of EBOV is about 33%, the amino acid identity increases significantly when SUDV and ZEBOV are analyzed separately (about 94% identity in each species). Thus, as shown in Figure 1A, a two-component strategy, one for SUDV and one for ZEBOV, was designed for coverage against the most lethal EBOV. Because of the relatively low GP diversity in each species (5.6% for SUDV and 7.1% for ZEBOV), we developed a consensus immunogen to increase cross-species coverage, a strategy previously shown to enhance protection in divergent strains of influenza and HIV. These GP sequences were consistent for all reported occurrence sequences (GenBank) as determined by alignment using Vector NTI software (Invitrogen, CA, USA). Non-consensus residues, four amino acids each in SUDV (95, 203, 261, and 472) and ZEBOV (314, 377, 430, and 440), were weighted to Gulu and Mbomo / Mbanza, respectively. Gulu was selected because it is responsible for the highest human mortality of Filoviridae epidemics (n=425), while Mbomo / Mbanza was selected because it is the most recent and deadliest epidemic with published sequence data. The consensus GPs for SUDV (SUDV CON vaccine) and ZEBOV (ZEBOV CON vaccine) were phylogenetically intermediate, parentally aligned strains.

[0028] Identification of proteins in Figure 1A is as follows: MARV Durba (05DRC99)'99: ABE27085; Uganda (01Uga07)'07: ACT79229; Durba (07DRC99)'99: ABE27078; Ozolin'75: VGP_MABVO; Musoke'80: VGP_MABVM; Popp'67: VGP_MABVP; Leiden'08: AEW11937; Angola'05: VGP_MABVA; Ravn'87: VGP_MABVR; Durba (09DRC99)'99; ABE27092; Uganda (02Uga07)'07: ACT79201. SUDV:Boniface'76:VGP_EBOSB;Maleo'79:VGP_EBOSM;Yambio'04:ABY75325;Gulu'00:VGP_EBOSU. ZEBOV:Booue'96:AAL25818;Mayibout'96:AEK25495;Mekouka'94:AAC57989,VGP_EBOG4;Kikwit'95:VGP_EBOZ5;Yambuku(Ekron)'76:VGP_E BOEC;Yambuku(Mayinga)'76:VGP_EBOZM;Kasai'08:AER59712;Kassai'07:AER59718;Etoumbi'05:ABW34742;Mbomo / Mbandza'03:ABW34743.

[0029] The sequence listing provided herein contains a list of 66 sequences, including:

[0030] SEQ ID NO:1 is the amino acid sequence of ZEBOV CON, the consensus Zaire Ebola virus envelope glycoprotein immunogen.

[0031] SEQ ID NO:2 is the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein immunogen, SUDV CON.

[0032] SEQ ID NO:3 is the amino acid sequence of MARV or MARV ANG, the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein and the Marburg virus Angola 2005 envelope glycoprotein immunogen.

[0033] SEQ ID NO:4 is the amino acid sequence of MARV CON1, the first consensus Marburg virus envelope glycoprotein immunogen.

[0034] SEQ ID NO:5 is the amino acid sequence of MARV CON2, a second consensus Marburg virus envelope glycoprotein immunogen.

[0035] SEQ ID NO:6 is the amino acid sequence of MARV CON3, a third consensus Marburg virus envelope glycoprotein immunogen.

[0036] SEQ ID NOs:7 to 25 are peptides derived from MARV ANG.

[0037] SEQ ID NOs: 26 to 41 are peptides derived from SUDV CON.

[0038] SEQ ID NOs: 42 to 62 are peptides derived from ZEBOV CON.

[0039] SEQ ID NO: 63 is the sequence of the IgE signal peptide: MDWTWILFLVAAATRVHS.

[0040] SEQ ID NO:64 is the nucleotide sequence insert within plasmid pEBOZ encoding the consensus Ebola Zaire virus envelope glycoprotein immunogen.

[0041] SEQ ID NO:65 is the nucleotide sequence insert within plasmid pEBOS encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

[0042] SEQ ID NO:66 is the nucleotide sequence insert within plasmid pMARZ ANG encoding the Marburg virus Angola 2005 envelope glycoprotein.

[0043] In some embodiments, the strategy employs coding sequences for three filovirus immunogens: MARV, SUDV, and ZEBOV. The MARV immunogen is the glycoprotein of the Angola 2005 isolate. For SUDV and ZEBOV, consensus glycoprotein sequences were designed.

[0044] In some embodiments, the strategy employs coding sequences for five filovirus immunogens. Three MARV immunogens are provided. Consensus glycoproteins Ozolin, Musoke, or Ravn from three clusters were designed. These three MARV immunogens are together targets for immune response of the designed SUDV and ZEBOV consensus glycoprotein sequences.

[0045] In some embodiments, the strategy employs coding sequences for six filovirus immunogens. Four MARV immunogens are provided. Three consensus glycoproteins from three clusters were designed. These three MARV immunogens are both targets for immune responses to the designed SUDV and ZEBOV consensus glycoprotein sequences, and the MARV immunogen is a glycoprotein of the Angola 2005 isolate.

[0046] As a filovirus vaccine candidate, DNA vaccines show numerous advantages, including fast and inexpensive large-scale production, stability at room temperature, and ease of transportation, all of which further enhance this platform from an economic and geographical perspective. Due to the synthetic nature of the plasmid, the Ag sequence can be quickly and easily modified in response to new emerging species and / or expanded to include additional vaccine components and / or treatment regimens for rapid response during outbreak situations. For example, the MARV strategy herein can be easily expanded to a larger coverage area by co-administration of additional plasmids encoding the consensus MARV GP (MGP) immunogen for other phylogenetic clusters.

[0047] Although "first generation" DNA vaccines have been poorly immunogenic, recent technological advances have dramatically improved their immunogenicity in clinical trials. Optimization of plasmid DNA vectors and their encoded Ag genes has led to increased in vivo immunogenicity. Cellular uptake and subsequent Ag expression are substantially amplified when highly concentrated plasmid vaccine formulations are administered by in vivo electroporation, a technique that uses brief square-wave electric pulses to drive plasmids into temporarily permeabilized cells within the vaccination site. In theory, mixtures of DNA plasmids can be assembled to direct highly specialized immune responses against any number of diverse Ags. Immunity can be further directed by co-delivery of plasmid molecular adjuvants encoding species-specific cytokine genes, and by "consensus engineering" of Ag amino acid sequences to promote biased vaccine-induced immunity against specific strains. This strategy has been shown to enhance protection in divergent strains of influenza virus and HIV. Due in part to these technological advances, immunization regimens involving these DNA vaccines are highly versatile and highly customizable.

[0048] 1. Definition. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0049] In reciting numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision, for example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0050] Adjuvant "Adjuvant," as used herein, can mean any molecule added to the DNA plasmid vaccines described herein to enhance the antigenicity of one or more consensus filovirus immunogens encoded by the DNA plasmids and coding nucleic acid sequences described below.

[0051] b.Antibodies "Antibody" may mean an antibody of the IgG, IgM, IgA, IgD, or IgE class, or a fragment, fragment, or derivative thereof, including Fab, F(ab')2, Fd, as well as single chain antibodies, bispecific antibodies, diabodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0052] C. coding sequence "Coding sequence" or "encoding nucleic acid" as used herein may refer to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. In some embodiments, the coding sequence may further comprise an initiation codon that optionally encodes an N-terminal methionine or a signal peptide, such as an IgE or IgG signal peptide.

[0053] D. Complement "Complement" or "complementary" as used herein means a nucleic acid can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0054] e. consensus or consensus sequence "Consensus" or "consensus sequence," as used herein, can refer to a synthetic nucleic acid sequence, or a corresponding polypeptide sequence, that is constructed based on the analysis of an alignment of multiple subtypes of a particular filovirus antigen and can be used to induce broad immunity against multiple specific filovirus antigen subtypes or serotypes.

[0055] Consensus Zaire Ebola virus envelope glycoprotein immunogen refers to SEQ ID NO:1, fragments of SEQ ID NO:1, variants of SEQ ID NO:1, and fragments of variants of SEQ ID NO:1. (ZEBOV or ZEBOV CON or ZEBOV CON vaccine). Plasmids containing the coding sequence of SEQ ID NO:1 can be referred to as pZEBOV or pEBOZ. Coding sequences for consensus Zaire Ebola virus envelope glycoprotein immunogen include SEQ ID NO:64, fragments of SEQ ID NO:64, variants of SEQ ID NO:64, and fragments of variants of SEQ ID NO:64. Plasmid pEBOZ comprises SEQ ID NO:64.

[0056] Consensus Sudan Ebola virus envelope glycoprotein immunogen refers to SEQ ID NO:2, fragments of SEQ ID NO:2, variants of SEQ ID NO:2, and fragments of variants of SEQ ID NO:2. (SUDV or SUDV CON or SUDV CON vaccine) Plasmids containing the coding sequence of SEQ ID NO:2 can be referred to as pSUDV or pEBOS. Coding sequences for consensus Sudan Ebola virus envelope glycoprotein immunogen include SEQ ID NO:65, fragments of SEQ ID NO:65, variants of SEQ ID NO:65, and fragments of variants of SEQ ID NO:65. Plasmid pEBOS comprises SEQ ID NO:65.

[0057] The Marburg virus Angola 2005 envelope glycoprotein is not a consensus, but is a protein sequence derived from isolates. It has the sequence SEQ ID NO:3. The Marburg virus Angola 2005 envelope glycoprotein immunogen refers to SEQ ID NO:3, fragments of SEQ ID NO:3, variants of SEQ ID NO:3, and fragments of variants of SEQ ID NO:3. (MARV or MARV ANG or MARV ANG or MARV ANG vaccine) A plasmid containing the coding sequence of SEQ ID NO:3 can be referred to as pMARV or pMARV-ANG. Coding sequences for the Marburg virus Angola 2005 envelope glycoprotein immunogen include SEQ ID NO:66, fragments of SEQ ID NO:66, variants of SEQ ID NO:66, and fragments of variants of SEQ ID NO:66. The plasmid pMARV ANG contains SEQ ID NO:66.

[0058] The first consensus Marburg virus envelope glycoprotein immunogen refers to SEQ ID NO:4, fragments of SEQ ID NO:4, variants of SEQ ID NO:4, and fragments of variants of SEQ ID NO:4. SEQ ID NO:4 is the Marburg virus consensus sequence derived from the Rabin Cluster Consensus (Ravn, Durba (09DRC99) and Uganda (02Uga07Y). (MARV CON1 or MARV-RAV CON or MARV-RAV CON vaccine) A plasmid containing the coding sequence of SEQ ID NO:4 can be referred to as pMARV-RAV.

[0059] The second consensus Marburg virus envelope glycoprotein immunogen refers to SEQ ID NO:5, fragments of SEQ ID NO:5, variants of SEQ ID NO:5, and fragments of variants of SEQ ID NO:5. SEQ ID NO:5 is the Marburg virus consensus sequence derived from the Ozolin cluster consensus (Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99)). (MARV CON2 or MARV-OZO CON or MARV-OZO CON vaccine) A plasmid containing the coding sequence of SEQ ID NO:5 may also be referred to as pMARV-OZO.

[0060] The third consensus Marburg virus envelope glycoprotein immunogen refers to SEQ ID NO:6, fragments of SEQ ID NO:6, variants of SEQ ID NO:6, and fragments of variants of SEQ ID NO:6. SEQ ID NO:6 is the Marburg virus consensus sequence derived from the Musoke cluster consensus (Musoke, Popp, and Leiden). (MARV CON1 or MARV-MUS CON or MARV-MUS CON vaccine) A plasmid containing the coding sequence of SEQ ID NO:6 can be referred to as pMARV-MUS.

[0061] f. Constant current "Constant current" as used herein defines the current that a tissue or cells defining said tissue undergo or experience over the duration of an electrical pulse delivered to said tissue. The electrical pulse is delivered from an electroporation device as described herein. This current remains at a constant amperage over the life of the electrical pulse in said tissue because the electroporation device provided herein has a feedback element, preferably with instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) over the duration of the pulse, and cause the electroporation device to change the electrical energy output (e.g., increase the voltage) so that the current remains constant in the same tissue over the entire electrical pulse (on the order of a few microseconds) and from pulse to pulse. In some embodiments, the feedback element includes a control device.

[0062] g. Current feedback or feedback "Current feedback" or "feedback" as used herein may be used interchangeably and may refer to the active response of the electroporation device provided, which includes measuring the current in the tissue between the electrodes and changing the energy output delivered by the EP device accordingly to maintain the current at a constant level. This constant level is preset by the user before starting the pulse sequence or electrical treatment. Feedback may be achieved by the electroporation components of the electroporation device, such as the control device, whose electrical circuitry continuously monitors the current in the tissue between the electrodes and compares the monitored current (or current in the tissue) to preset the current and continuously adjust the energy output to maintain the monitored current at the preset level. The feedback loop may be instantaneous, since it is an analog closed-loop feedback.

[0063] h. Distributed current "Distributed current," as used herein, may refer to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein, which pattern minimizes or eliminates the occurrence of electroporation-related thermal stress in any region of the tissue being electroporated.

[0064] i. Electroporation "Electroporation," "electro-permeabilization," or "electro-kinetic enhancement" ("EP"), as used interchangeably herein, may refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0065] j. Feedback mechanism "Feedback mechanism" as used herein may refer to a process implemented by either software or hardware (or firmware) that receives the desired tissue impedance (before, during, and / or after delivery of an energy pulse), compares it to a current value, preferably the current, and adjusts the delivered pulse of energy to achieve a pre-set value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0066] K. Fragment "Fragment" may refer to a polypeptide fragment of a filovirus immunogen capable of eliciting an immune response in a mammal against a filovirus by recognizing a specific filovirus antigen. Filovirus envelope glycoprotein immunogens may optionally include a signal peptide and / or a methionine at position 1, a protein that is 98% or more homologous to a consensus sequence described herein, a protein that is 99% or more homologous to a consensus sequence described herein, and a protein that is 100% identical to a consensus sequence described herein, in each case with or without a signal peptide and / or a methionine at position 1. Fragments may or may not include a fragment of a filovirus immunogen linked to, for example, a signal peptide, such as, for example, an immunoglobulin signal peptide, such as an IgE signal peptide or an IgG signal peptide.

[0067] A fragment of any of ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, or MARV-MUS CON, or a variant thereof, may comprise a percentage (%) of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, or MARV-MUS CON, or a variant thereof, in each case with or without a signal peptide and / or methionine at position 1. Fragments refer to fragment polypeptides that are 100% identical to the sequence ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, or MARV-MUS CON, in each case with or without a signal peptide and / or methionine at position 1. Fragments also refer to variants, i.e., fragments of polypeptides that are 95% or more, 98% or more, or 99% or more homologous to the sequence ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, or MARV-MUS CON, in each case with or without a signal peptide and / or methionine at position 1. Fragments may include fragments of polypeptides that are 98% or more homologous, 99% or more homologous, or 100% identical to the filovirus immunogens set forth in SEQ ID NOs: 1-6, and may additionally include a signal peptide, such as an immunoglobulin signal peptide, that is not included when calculating the percent homology. In some embodiments, a fragment of SEQ ID NO: 1-6 is linked to a signal peptide, such as an immunoglobulin signal peptide, for example, an IgE signal peptide or an IgG signal peptide. The fragment may include a fragment of SEQ ID NO: 1-6 that includes an N-terminal methionine.Fragments also refer to fragments of polypeptides that are 95% or more, 98% or more, or 99% or more homologous to the sequences disclosed in SEQ ID NOs: 1 to 6. The signal peptide sis, if present, is not included in the calculation of percent homology.

[0068] In some embodiments, a fragment of SEQ ID NO: 1-6 or a variant thereof may comprise 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 or more consecutive amino acids of any of SEQ ID NO: 1-6 or a variant thereof. In some embodiments, a fragment of SEQ ID NO: 1-6 or a variant thereof may comprise 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670, 675 or fewer consecutive amino acids of any of SEQ ID NO: 1-6 or a variant thereof.

[0069] "Fragment" may also refer to a fragment of a nucleic acid sequence encoding a filovirus immunogen, a nucleic acid fragment encoding a fragment of a filovirus immunogen capable of eliciting an immune response in a mammal against a filovirus by recognizing a particular filovirus antigen. A fragment of a nucleic acid fragment encoding a filovirus immunogen or a variant thereof may comprise a percentage (%) of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of the particular full-length nucleic acid sequence encoding a filovirus immunogen or a variant thereof, in each case with or without a signal peptide and / or methionine at position 1. Fragments may include fragments of nucleotide sequences encoding polypeptides that are 98% or more homologous, 99% or more homologous, or 100% identical to the filovirus immunogens set forth in SEQ ID NOs: 1-6, and may additionally include a signal peptide, such as an immunoglobulin signal peptide, which is not included when calculating percent homology. In some embodiments, fragments of nucleotide sequences encoding fragments of SEQ ID NOs: 1-6 are linked to a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE signal peptide or an IgG signal peptide. Although coding sequences for the signal peptide cis are present, they are not included when calculating percent homology. In some embodiments, a fragment of a nucleotide sequence encoding a fragment of SEQ ID NOs:1-6 or a variant thereof may include a sequence encoding 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 or more consecutive amino acids of any of SEQ ID NOs:1-6 or variants thereof.In some embodiments, a fragment of a nucleotide sequence encoding a fragment of SEQ ID NOs:1-6 or a variant thereof may include a sequence encoding no more than 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670, 675 contiguous amino acids of any of SEQ ID NOs:1-6 or variants thereof.

[0070] In some embodiments, the fragment is a fragment of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66. A fragment of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length nucleic acid sequence encoding a filovirus immunogen or variant thereof, in each case with or without a signal peptide and / or methionine at position 1. A fragment of SEQ ID NO:64, SEQ ID NO:64, or SEQ ID NO:66 may include a fragment of a nucleotide sequence encoding a polypeptide that is 98% or more homologous, 99% or more homologous, or 100% identical to a filovirus immunogen encoded by SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, and may additionally include a signal peptide, such as an immunoglobulin signal peptide, which is not included when calculating percent homology. A fragment of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 may include a sequence encoding 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670 or more contiguous amino acids of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, or variants thereof. A fragment of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 may include a sequence encoding no more than 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, 670, 675 consecutive amino acids of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66, or a variant thereof.

[0071] is.Identical "Identical" or "identity", as used herein in the context of two or more nucleic acid or polypeptide sequences, can mean that the sequences have a certain percentage of residues that are the same over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a particular region, determining the number of positions where identical residues occur in both sequences resulting in the number of matched positions, dividing the number of matched positions by the total number of positions in the particular region, and multiplying the result by 100 to produce the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more sticky ends and a particular comparison region contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0072] m. impedance "Impedance", as used herein, can be used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, thus allowing comparison to a preset current.

[0073] Immune response "Immune response," as used herein, can refer to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of one or more filovirus consensus antigens via a provided DNA plasmid vaccine. The immune response can be in the form of a cellular or humoral response, or both.

[0074] o.Nucleic acid "Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein may refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0075] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids, where the nucleic acids can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0076] p. Functionally linked "Operatively linked" as used herein may mean that the expression of a gene is under the control of a promoter that is spatially linked. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The spacing between the promoter and the gene may be approximately the same as the spacing between the promoter and the gene it controls in the gene from which the promoter is derived. As known in the art, variations in this spacing may be accommodated without loss of promoter function.

[0077] q. Promoter "Promoter" as used herein may refer to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may differentially regulate expression of genetic components constitutively, or with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0078] r. Stringent hybridization conditions "Stringent hybridization conditions," as used herein, can refer to the conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions are those that meet the thermal melting point (T) of a particular sequence at a defined ionic strength pH. m ) can be selected to be about 5 to 10°C lower than T m is when 50% of the probes complementary to the target are in equilibrium with the target sequence (when the target sequence is T mStringent conditions can be a temperature (under defined ionic strength, pH, and nuclei concentration) at which probes hybridize to nucleotides (50% of the probes are at equilibrium, since they are present in excess at 20° C.). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion (or other salt), such as a sodium ion concentration of about 0.01-1.0 M at pH 7.0-8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times higher than background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5xSSC, and 1% SDS, incubation at 42°C, or 5xSSC, 1% SDS, incubation at 65°C with a wash in 0.2xSSC and 0.1% SDS at 65°C.

[0079] s. Substantially complementary "Substantially complementary," as used herein, can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0080] t. Substantially identical "Substantially identical" as used herein can mean that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0081] u. Variation "Variant," as used herein with respect to nucleic acids, means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with a reference nucleic acid, its complement or a sequence substantially identical thereto.

[0082] "Variants" refers to peptides or polypeptides that differ in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variants can also refer to proteins with amino acid sequences that are substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with an amino acid that has a different property (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art, typically with minor modifications. These minor modifications can be identified, in part, by considering the hydropathic index of an amino acid as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic index can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in a protein retaining biological activity. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values ​​can result in a peptide retaining biological activity, such as immunogenicity, as is understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are comparable in biological function depend on the relative similarity of the amino acids, particularly the side chains of those amino acids, as evidenced by hydrophobicity, hydrophilicity, charge, size, and other properties. Variants are preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NOs: 1-6.

[0083] "Variants" with respect to nucleic acid sequences that code for the same specific amino acid sequence differ in nucleotide sequence due to different codon usage. Variants of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 that code for the same amino acid sequence as the amino acid sequence encoded by SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 may be of any degree of homology, preferably 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Variants may also be variants of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 that code for proteins that are variants of proteins encoded by SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66 that have an amino acid sequence that is substantially identical to the reference protein with the amino acid sequence retaining at least one biological activity, typically the amino acid sequence is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous.

[0084] v. Vector "Vector" as used herein may refer to a nucleic acid sequence that contains an origin of replication.Vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome.Vector may be a DNA or RNA vector.Vector may be either a self-replicating extrachromosomal vector or a vector that is integrated into a host genome.

[0085] 2. Protein Provided herein are filovirus immunogens that can be used to induce broad immunity against multiple subtypes or serotypes of a particular filovirus antigen. Each consensus filovirus antigen may include the consensus amino acid sequence of the Marburg virus filovirus glycoprotein MARV RAV immunogen, the consensus amino acid sequence of the Marburg virus filovirus glycoprotein MARV OZO immunogen, the consensus amino acid sequence of the Marburg virus filovirus glycoprotein MARV MUS immunogen, the isolated amino acid sequence of the Marburg virus filovirus glycoprotein MARV ANG immunogen, the consensus amino acid sequence of the Zaire Ebola virus glycoprotein ZEBOV immunogen, and the consensus amino acid sequence of the Sudan Ebola virus glycoprotein SUDV immunogen. In some embodiments, the immunogen may include a signal peptide derived from a different protein, such as, for example, an immunoglobulin protein, such as an IgE signal peptide or an IgG signal peptide.

[0086] The amino acid sequences for the immunogens may include SEQ ID NOs: 1-6, variants thereof, and fragments of SEQ ID NOs: 1-6 and variants thereof, and may optionally include a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0087] 3. Coding sequence that encodes a protein The coding sequence encoding the protein described herein can be produced using a defined method. A composition is provided that includes a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a Marburg MARBURG virus Angola 2005 envelope glycoprotein immunogen, and a nucleic acid sequence encoding a first consensus Marburg MARBURG virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a second consensus Marburg MARBURG virus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a third consensus Marburg MARBURG virus envelope glycoprotein immunogen can be produced based on the disclosed amino acid sequence.

[0088] The nucleic acid sequence can encode a full length consensus Zaire Ebola virus envelope glycoprotein immunogen, a full length consensus Sudan Ebola virus envelope glycoprotein immunogen, a full length Marburg Angola 2005 envelope glycoprotein immunogen, a full length first consensus Marburg MARburg virus envelope glycoprotein immunogen, a full length second consensus Marburg MARburg virus envelope glycoprotein immunogen, or a full length third consensus Marburg MARburg virus envelope glycoprotein immunogen. The nucleic acid sequence may comprise a sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The nucleic acid sequence may comprise SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66. The nucleic acid sequence may optionally comprise a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0089] The nucleic acid sequence can encode a fragment of a full-length consensus Zaire Ebola virus envelope glycoprotein immunogen, a fragment of a full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a fragment of a full-length Marburg Angola 2005 envelope glycoprotein immunogen, a fragment of a full-length first consensus Marburg Angola virus envelope glycoprotein immunogen, a fragment of a full-length second consensus Marburg Angola virus envelope glycoprotein immunogen, or a fragment of a full-length third consensus Marburg Angola virus envelope glycoprotein immunogen. The nucleic acid sequence may comprise a sequence encoding a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The nucleic acid sequence may comprise a fragment of SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66. The size of the fragment is disclosed herein as described in the section entitled "Fragments". The acid sequence may optionally include a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0090] The nucleic acid sequence may encode a protein homologous to a full-length consensus Zaire Ebola virus envelope glycoprotein immunogen, a protein homologous to a full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a protein homologous to a full-length Marburg Angola 2005 envelope glycoprotein immunogen, a protein homologous to a full-length first consensus Marburg Angola virus envelope glycoprotein immunogen, a protein homologous to a full-length second consensus Marburg Angola virus envelope glycoprotein immunogen, or a protein homologous to a full-length third consensus Marburg Angola virus envelope glycoprotein immunogen. The nucleic acid sequence may comprise a sequence encoding a protein homologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The nucleic acid sequence may be homologous to SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66. The degree of homology is set forth herein such as in the variants section. The nucleic acid sequence may optionally include a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0091] The nucleic acid sequence may encode a protein homologous to a fragment of a full-length consensus Zaire Ebola virus envelope glycoprotein immunogen, a protein homologous to a fragment of a full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a protein homologous to a fragment of a full-length Marburg Angola 2005 envelope glycoprotein immunogen, a protein homologous to a fragment of a full-length first consensus Marburg Angola virus envelope glycoprotein immunogen, a protein homologous to a fragment of a full-length second consensus Marburg Angola virus envelope glycoprotein immunogen, or a protein homologous to a fragment of a full-length third consensus Marburg Angola virus envelope glycoprotein immunogen. The nucleic acid sequence may comprise a sequence encoding a protein homologous to a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The nucleic acid sequence may comprise a fragment homologous to SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66. Degrees of homology are set forth herein such as in the variants section. The nucleic acid sequence may optionally include a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0092] SEQ ID NO:64 is the nucleotide sequence insert within plasmid pEBOZ encoding the consensus Ebola Zaire virus envelope glycoprotein immunogen.

[0093] SEQ ID NO:65 is the nucleotide sequence insert within plasmid pEBOS encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

[0094] SEQ ID NO:66 is the nucleotide sequence insert within plasmid pMARZ ANG encoding the Marburg virus Angola 2005 envelope glycoprotein.

[0095] 4. Plasmids The plasmid may contain a nucleic acid sequence encoding one or more of the various immunogens disclosed above, including a coding sequence encoding a consensus antigen capable of eliciting an immune response against a synthetic phyloprotein.

[0096] A single plasmid may contain coding sequences for a single phylloprotein immunogen, coding sequences for two phylloprotein immunogens, coding sequences for three phylloprotein immunogens, coding sequences for four phylloprotein immunogens, coding sequences for five phylloprotein immunogens, or coding sequences for six phylloprotein immunogens. A single plasmid may contain coding sequences for a single phylloprotein immunogen that can be formulated together. In some embodiments, the plasmid may include coding sequences encoding IL-12, IL-15, and / or IL-28.

[0097] The plasmid can further comprise a start codon, which can be upstream of the coding sequence, and a stop codon, which can be downstream of the coding sequence. The start and stop codons can be in frame with the antigen coding sequence.

[0098] The plasmid can also include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence can be a human immunodeficiency virus (HIV) promoter, such as a Simian Virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 20040175727, the contents of which are incorporated herein by reference in their entirety.

[0099] The plasmid can also include a polyadenylation signal that can be downstream of the coding sequence. The polyadenylation signal can be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the human β-globulin polyadenylation signal. The SV40 polyadenylation signal can be the polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0100] The plasmid can also contain an enhancer upstream of the coding sequence.The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, FMDV, RSV, or EBV.Enhancement of polynucleotide function is described in U.S. Patent No. 5,939,972, U.S. Patent No. 5,962,428, and International Publication No. WO 94 / 016737, the contents of each of which are fully incorporated by reference.

[0101] The plasmid can also contain a mammalian origin of replication to maintain the plasmid extrachromosomal and produce multiple copies of the plasmid within the cell. The plasmid can be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain an Epstein-Barr virus origin of replication and a nuclear antigen EBNA-1 coding region that can produce high copy episomal replication without integration. The backbone of the plasmid can be pAV0242. The plasmid can be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0102] The plasmid may also contain regulatory sequences that may be sufficient for gene expression in a cell into which the plasmid is administered. The coding sequence may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0103] The coding sequence may also include an Ig leader sequence. The leader sequence may be 5' of the coding sequence. The consensus antigen encoded by this sequence may include an N-terminal Ig leader followed by the consensus antigen protein. The N-terminal Ig leader may be an IGE or an IgG.

[0104] The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The plasmid can be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid can be MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid can be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0105] 5. Composition Compositions are provided that include nucleic acid molecules. The compositions can include multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, the compositions can include a plurality of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different nucleic acid molecules. Such compositions can include a plurality of 2, 3, 4, 5, 6, or more different plasmids.

[0106] The composition may comprise a nucleic acid molecule, e.g., a plasmid, that collectively contains a coding sequence for a single filoprotein immunogen selected from the group consisting of one or more consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen, Marburg MARburg virus Angola 2005 envelope glycoprotein, a first consensus Marburg MARburg virus envelope glycoprotein immunogen, a second consensus Marburg MARburg virus envelope glycoprotein immunogen, and a third consensus Marburg MARburg virus envelope glycoprotein immunogen.

[0107] The composition comprises a consensus Zaire Ebola virus envelope glycoprotein immunogen and a consensus Sudan Ebola virus envelope glycoprotein immunogen; or a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, and a Marburg Marburg virus Angola 2005 envelope glycoprotein; or a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a first consensus Marburg Marburg virus envelope glycoprotein immunogen, a second consensus Marburg Marburg virus envelope glycoprotein immunogen, or a combination of consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen, Marburg Angola 2005 envelope glycoprotein, a first consensus Marburg envelope glycoprotein immunogen, a second consensus Marburg envelope glycoprotein immunogen, and a third consensus Marburg envelope glycoprotein immunogen. Each coding sequence for each filoprotein immunogen is preferably contained on a separate plasmid. Thus, the composition comprising the nucleic acid sequences encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen and the consensus Sudan Ebola virus envelope glycoprotein immunogen can be on a single plasmid, but is preferably on two separate plasmids. The composition comprising nucleic acid sequences encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, the consensus Sudan Ebola virus envelope glycoprotein immunogen, and the Marburg Marburg virus Angola 2005 envelope glycoprotein may be on a single plasmid or on two plasmids in any permutation, but are preferably on three separate plasmids.The composition comprising nucleic acid sequences encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, the consensus Sudan Ebola virus envelope glycoprotein immunogen, the first consensus Marburg virus envelope glycoprotein immunogen, the second consensus Marburg virus envelope glycoprotein immunogen, and the third consensus Marburg virus envelope glycoprotein immunogen may be on a single plasmid, or on two plasmids in any permutation, or on three plasmids in any permutation, or on four plasmids in any permutation, but preferably on five separate plasmids. Similarly, a composition comprising nucleic acid sequences encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a first consensus Marburg virus envelope glycoprotein immunogen, a second consensus Marburg virus envelope glycoprotein immunogen, and a third consensus Marburg virus envelope glycoprotein immunogen may be on a single plasmid, or on two plasmids in any permutation, or on three plasmids in any permutation, or on four plasmids in any permutation, but preferably on five separate plasmids.

[0108] 6. Vaccines Provided herein is a vaccine capable of generating an immune response in a mammal against filoviruses, specifically Marburg virus, Ebola virus Sudan, and / or Ebola virus Zaire. The vaccine can include each of the plasmids discussed above. The vaccine can include multiple plasmids or combinations thereof. The vaccine can be provided to induce a therapeutic or prophylactic immune response.

[0109] The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen and a consensus Sudan Ebola virus envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, and a Marburg Marburg virus Angola 2005 envelope glycoprotein. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a first consensus Marburg Marburg virus envelope glycoprotein immunogen, a second consensus Marburg Marburg virus envelope glycoprotein immunogen, and a third consensus Marburg Marburg virus envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a Marburg Angola 2005 envelope glycoprotein, a first consensus Marburg envelope glycoprotein immunogen, a second consensus Marburg envelope glycoprotein immunogen, and a third consensus Marburg envelope glycoprotein immunogen. The vaccine is preferably a composition comprising a plasmid.

[0110] The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule as a vehicle, adjuvant, carrier, or diluent. The pharmaceutically acceptable excipient may be a transfection facilitator, which may include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, virus particles, polyanions, polycations, or nanoparticles, or other known transfection facilitators.

[0111] The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The transfection facilitating agent is poly-L-glutamic acid, and more preferably, the poly-L-glutamic acid is present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may include detergents such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be used for administration with the genetic construct. In some embodiments, the DNA plasmid vaccine may also include transfection facilitating agents such as liposomes, including other liposomes known in the art such as lipids, lectin liposomes, or DNA liposome mixtures (see, e.g., International Publication No. WO 09324640), calcium ions, viral particles, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0112] The pharma- ceutically acceptable excipient may be one or more adjuvants, which may be other genes in the vaccine that are expressed on the same or alternative plasmids, or delivered as proteins in combination with the above-mentioned plasmids.The one or more adjuvants may be selected from the group consisting of CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymic epithelium expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, including IL-15 having a signal sequence or coding sequence that encodes a deleted signal sequence and optionally including a different signal peptide, such as from IgE, or a coding sequence that encodes a different signal peptide, such as from IgE, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP -1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, and neural growth factor child, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAI L-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP The nucleic acid molecule may be a protein and / or a nucleic acid molecule encoding a protein selected from the group consisting of K, SAP-1, JNK, an interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof, or combinations thereof.In some embodiments, the adjuvant may be one or more proteins and / or nucleic acid molecules encoding proteins selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, or RANTES. Exemplary IL-12 constructs and sequences are disclosed in PCT Application No. PTC / US1997 / 019502, and corresponding U.S. patent application Ser. No. 08 / 956,865 and U.S. Provisional Patent Application No. 61 / 569600, filed Dec. 12, 2011, each of which is incorporated herein by reference. Examples of IL-15 constructs and sequences are disclosed in PCT Application No. PCT / US04 / 18962 and corresponding US Patent Application No. 10 / 560,650, PCT Application No. PCT / US07 / 00886 and corresponding US Patent Application No. 12 / 160,766, and PCT Application No. PCT / US10 / 048827, each of which is incorporated herein by reference. Examples of IL-28 constructs and sequences are disclosed in PCT Application No. PTC / US09 / 039648 and corresponding US Patent Application No. 12 / 936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PTC / US1999 / 004332 and corresponding US Patent Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PTC / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PTC / US1999 / 004332 and corresponding US Patent Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PTC / US11 / 024098, which is incorporated herein by reference.Examples of the constructs and sequences of chemokines CTACK, TECK, and MEC are disclosed in PCT Application No. PTC / US2005 / 042231 and corresponding US Patent Application No. 11 / 719 / 646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulatory agents are disclosed in US Patent Application No. 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulatory agents are disclosed in US Patent Application No. 09 / 622452, which is incorporated herein by reference.

[0113] The vaccine may further comprise a genetic vaccine facilitator as described in US patent application Ser. No. 021 / 579, filed Apr. 1, 1994.

[0114] The vaccine may comprise consensus antigen and plasmid in an amount of about 1 nanogram to about 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the pharmaceutical composition according to the present invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 micrograms to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, or about 100 to about 200 micrograms of the consensus antigen and its plasmid.

[0115] Vaccines can be formulated according to the mode of administration used. The pharmaceutical composition of the injectable vaccine can be sterile, pyrogen-free, and particle-free. Isotonic preparations or solutions can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Vaccines can include vasoconstrictors. Isotonic solutions can include phosphate buffered saline. Vaccines can further include stabilizers, including gelatin and albumin. Stabilization can allow the formulation to be stable for long periods at room temperature or ambient temperature, and can be, for example, LGS, or polycations or polyanions to the vaccine formulation.

[0116] 7. Vaccine delivery method Provided herein are methods for delivering vaccines to provide genetic constructs and proteins of consensus antigens that contain epitopes that are particularly effective immunogenic for filoviruses, specifically Marburg virus, Ebola virus Sudan, and / or Ebola virus Zaire, against which an immune response can be induced. Methods for delivering vaccines or vaccination can be provided to induce therapeutic and prophylactic immune responses. The vaccination process can generate an immune response in a mammal against filoviruses, specifically Marburg virus, Ebola virus Sudan, and / or Ebola virus Zaire. The vaccine can be delivered to an individual to modulate the activity of the mammal's immune system and enhance the immune response. Delivery of the vaccine can be by transfection of the consensus antigen as a nucleic acid molecule that is expressed in a cell and delivered to the surface of the cell, where it is recognized by the immune system and induces a cellular, humoral, or cellular and humoral response. Vaccine delivery can be used to induce or elicit an immune response in a mammal against a filovirus, specifically, Marburg virus, Ebola virus Sudan, and / or Ebola virus Zaire, by administering to the mammal a vaccine as discussed above.

[0117] Delivery of the vaccine and plasmids into mammalian cells causes the transfected cells to express and secrete consensus antigens from the vaccine to the respective plasmids injected. These proteins are recognized as foreign by the immune system and antibodies are made against them. These antibodies are maintained by the immune system, allowing an effective response to subsequent infection with filoviruses, specifically Marburg virus, Ebola virus Sudan, and / or Ebola virus Zaire.

[0118] The vaccine can be administered to a mammal to elicit an immune response in the mammal, which can be a human, a primate, a non-human primate, a cow, a pig, a sheep, a goat, an antelope, a bison, a water buffalo, a bison, a bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0119] Combination treatment The vaccines include CCL20, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymic epithelial expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, and other chemokines with deleted signal sequences and different signal peptides such as IgE signal peptide. Optionally contains IL-15, MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, RANTES, MCP- 1, MIP-1α, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL- 1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR5, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 In some embodiments, the vaccine is administered in combination with one or more of the following nucleic acid molecules and / or proteins, which may be administered in combination with other proteins and / or genes encoding LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof, or combinations thereof, wherein the nucleic acid molecule is selected from the group consisting of nucleic acid molecules comprising a coding sequence encoding one or more of the functional fragments thereof, CCL20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, and RANTES, and wherein the protein is selected from the group consisting of CCL02, IL-12 protein, IL-15 protein, IL-28 protein, CTACK protein, TECK protein, MEK protein, or RANTES protein, or functional fragments thereof.

[0120] The vaccine can be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. For veterinary use, the composition can be administered in a formulation that is appropriately acceptable for normal veterinary practice. Veterinarians can easily determine the administration regimen and route of administration that is most suitable for a particular animal. The vaccine can be administered by traditional syringes, needleless injection devices, "microprojectile guns", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

[0121] Vaccine plasmids can be delivered to mammals by several well-known techniques, including DNA injection with and without in vivo electroporation (also called DNA vaccination), liposome-mediated, nanoparticle-facilitated recombinant vectors such as recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccinia. Consensus antigens can be delivered via DNA injection, accompanied by in vivo electroporation.

[0122] B. Electroporation Administration of the vaccine via electroporation of the vaccine plasmid can be accomplished using an electroporation device that can be configured to deliver an energy pulse to the desired mammalian tissue that is effective to form reversible pores in the cell membrane, preferably the energy pulse is a constant current similar to the current input preset by the user. The electroporation device can include an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include and incorporate one or more of the various components of the electroporation device, including a control device, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory device element, a power source, and a power switch. Electroporation can be accomplished by using an in vivo electroporation device, such as the CELLECTRA EP system (VGX Pharmaceuticals, Blue Bell, PA) or the Elgen electroporator (Genetronics, San Diego, CA), to facilitate transfection of cells with the plasmid.

[0123] The electroporation component can function as one element of the electroporation device, and other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component can function as more than one element of the electroporation device, and may also communicate with other elements of the electroporation device that are separate from the electroporation component. The elements of the electroporation device that are present as part of an electromechanical or mechanical device are not limited as elements that can function as one device or as separate elements that communicate with each other. The electroporation component can deliver an energy pulse that produces a constant current to the desired tissue and includes a feedback mechanism. The electrode assembly can include an electrode array having a plurality of spaced apart electrodes, where the electrode assembly receives an energy pulse from the electroporation component and delivers it to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral during delivery of the energy pulse, measures impedance at the desired tissue, and communicates the impedance to the electroporation component. The feedback mechanism can receive the measured impedance and adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0124] The electrodes can deliver energy pulses in a distributed pattern. The electrodes can deliver energy pulses in a distributed pattern through control of the electrodes by a programmed sequence, the programmed sequence being input by a user into the electroporation component. The programmed sequence can include a plurality of pulses delivered in a sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one indifferent electrode measuring impedance, and subsequent pulses of the plurality of pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode measuring impedance.

[0125] The feedback mechanism can be implemented by either hardware or software. The feedback mechanism can be implemented by an analog closed loop circuit. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous, as determined by available techniques for determining response time). The indifferent electrode can measure impedance at the desired tissue and communicate the impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulse to maintain a constant current at a value similar to the preset current. The feedback mechanism can maintain a constant current continuously or instantaneously during delivery of the energy pulse.

[0126] Examples of electroporation devices and electroporation methods that can enhance delivery of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al., and U.S. Patent Publication No. 2005 / 0052630 by Smith, et al., the contents of which are incorporated herein by reference in their entireties. Other electroporation devices and electroporation methods that can be used to enhance delivery of DNA vaccines include those provided in co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein by reference in their entireties.

[0127] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use for facilitating the introduction of biomolecules into cells of selected tissues in a body or plant. The modular electrode system can include a plurality of needle electrodes, a hypodermic needle, an electrical connector that provides conductive ink from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. An operator can grasp the plurality of needle electrodes loaded on a support structure and firmly insert them into a selected tissue of a body or plant. The biomolecule is then delivered through the hypodermic needle into the selected tissue. The programmable constant current pulse controller is actuated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse promotes the introduction of the biomolecule into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0128] US Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues within a body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between an electrode array based on user control and input of pulse parameters, allowing for storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of US Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0129] The electrode array and method described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 can be adapted to penetrate deep into tissues such as muscle, as well as other tissues or organs. Due to the configuration of the electrode array, the injection needle (delivering the selected biomolecule) is also inserted completely into the target organ, and the injection is applied perpendicular to the target tissue in the area pre-delineated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 are preferably 20 mm long and 21 gauge.

[0130] In addition, there are electroporation devices described in the following patents as contemplated in some embodiments incorporating electroporation devices and their use: U.S. Patent No. 5,273,525 issued December 28, 1993, U.S. Patent No. 6,110,161 issued August 29, 2000, U.S. Patent No. 6,261,281 issued July 17, 2001, and U.S. Patent No. 6,958,060 issued October 25, 2005, and U.S. Patent No. 6,939,862 issued September 6, 2005. Additionally, patents covering subject matter provided in U.S. Patent No. 6,697,669 issued February 24, 2004, which relates to delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064 issued February 5, 2008, which is directed to a method of injecting DNA, are contemplated herein. The above patents are incorporated by reference in their entirety.

[0131] c. DNA plasmid preparation method Provided herein is a method for preparing the DNA plasmids that comprise the DNA vaccines discussed herein, which, after a final subcloning step into a mammalian expression plasmid, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0132] The DNA plasmids used in the EP devices of the present invention can be formulated and manufactured using a combination of known equipment and techniques, but preferably they are manufactured using optimized plasmid manufacturing techniques described in co-pending U.S. Provisional Patent Application No. 60 / 939,792, filed May 23, 2007. In some instances, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques also include or incorporate a variety of equipment and protocols generally known to those of skill in the art in addition to those described in U.S. Patent Application No. 60 / 939,792, including those described in co-pending U.S. Patent No. 7,238,522, filed July 3, 2007. The above referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety. EXAMPLES

[0133] The present invention will be further described in the following examples. It should be understood that these examples, while showing preferred embodiments of the present invention, are presented for illustrative purposes only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications of the present invention to suit various applications and conditions without departing from the spirit and scope of the present invention. Thus, in addition to those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0134] Example 1 method Plasmid vaccine construction The pMARV, pEBOS, and pEBOZ plasmid DNA constructs encode full-length GP proteins. An amino acid consensus strategy was used for pEBOS and pEBOZ, while a type-matched sequence (GenBank #VGP_MABVR) from the 2005 Angola outbreak was used for pMARV (Towner JS, et al. (2006). Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. J Virol 80:6497-6516). The consensus sequence was determined by aligning the mainstream ZEBOV and SUDV GP amino acid sequences and generating a consensus for each. Each vaccine GP gene was genetically optimized for expression in humans, including codon optimization and RNA optimization to enhance protein expression (GenScript, Piscataway, NJ), commercially synthesized, and then subcloned into a modified pVAX1 mammalian expression vector (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus early (CMV) promoter (GenScript, Piscataway, NJ). Modifications include 2A>C, 3C>T, 4T>G, 241C>G, 1,942C>T, 2,876A>-, 3,277C>T, and 3,753G>C. Phylogenetic analysis was performed by multiple alignment with ClustalW using MEGA version 5 software. Alternatively, a consensus strategy was not applied because GP diversity between MARVs was much higher in the comparison (approximately 70% identity). Due to the scope of MARV coverage, we chose to use the MGP sequence (GenBank #VGP_MABVR) from the 2005 outbreak in Angola, which was the single source of the largest and most deadly MARV outbreak to date. This sequence also diverged by more than 10% from any of the closest clusters of related strains, including Musoke, Popp, and Leiden (10.6% divergence), or Uganda (01Uga07), Durba (05DRC99 and 07DRC99), and Ozolin (10.3% divergence).Overall, the three plasmid strategy formed the basis of our novel trivalent multivalent filovirus vaccine strategy.

[0135] Transfection and immunoblotting Human Embryonic Kidney (HEK) 293T cells were cultured, transfected, and harvested. Briefly, cells were grown in DMEM with 10% FBS, 1% Pen-strep, sodium pyruvate, and L-glutamine. Cells were cultured in 150 mm Corning dishes and grown to 70% confluence overnight in a 37° incubator with 5% CO2. Dishes were transfected with 10-25 μg of Filoviridae pDNA using either the Calphos™ Mammalian Transfection Kit protocol (Clonetech) or Lipofectamine™ 2000 reagent (Invitrogen) according to the manufacturer's protocol, then incubated for 24-48 hours. Cells were harvested in cold PBS, centrifuged and washed, and then pelleted for Western immunoblot or FACS analysis. Standard Western blots were used, and GP-specific MAbs were generated for GP1 detection. Data from Western immunoblotting experiments are shown in Figure 1B. Data from FACS analysis are shown in Figure 1C.

[0136] Animals, vaccination, and challenge Adult female C57BL / 6(H-2 b ), BALB / cJ(H-2 d ), and Bl0.Br(H-2 k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), while Hartley guinea pigs were purchased from Charles River (Wilmington, MA). All animal experiments were performed in accordance with the UPenn IACUC and School of Medicine Animal Facility, or the NML Institutional Animal Care Committee of the PHAC and the Canadian Council on Animal Care guidelines for housing and care of laboratory animals, and after relevant checks and approvals by the aforementioned institutions, were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of NIH. UPenn and NML comply with the NIH policy on animal welfare, the Animal Welfare Act, and all other applicable federal, state, and local laws.

[0137] Mice were immunized by intramuscular needle injection with 40 μg of plasmid resuspended in water, while guinea pigs were immunized intradermally with 200 μg each at three separate vaccination sites. Vaccination was followed immediately by EP at the same sites. Briefly, a three-pronged CELLECTRA® adaptive constant current Minimally Invasive Device was inserted approximately 2 mm intradermally (Inovio Pharmaceuticals, Inc., Blue Bell, PA). Square wave pulses were delivered through a triangular three-electrode array consisting of 26-gauge solid stainless steel electrodes, with two constant current pulses of 0.1 Amp separated by a 1 second delay and delivered 52 msec / pulse.

[0138] For lethal challenge studies, challenge was limited to rodent-adapted ZEBOV and MARV. Guinea pigs were challenged with 1,000 LD 50guinea pig-adapted ZEBOV (21.3 FFU / animal) (Richardson JS, Abou MC, Tran KN, Kumar A, Sahai BM, Kobinger GP (2011). Impact of systemic or mucosal immunity to adenovirus on ad-based Ebola virus vaccine efficacy in guinea pigs. J Infect Dis 204 Suppl 3:S1032-1042), or 1,000LD 50 Animals were challenged 28 days after the final vaccination by intraperitoneal injection with MARV-Angola (681 TCID50 / animal). Briefly, guinea pig-adapted MARV was generated by serial passage of wild-type MARV-Angola in outbred adult female Hartley guinea pigs. Seven days after inoculation, animals were euthanized and livers were harvested and homogenized. This homogenate was then injected intraperitoneally into naive adult guinea pigs, and the process was repeated until the animals lost weight, lost their coat, and became susceptible to infection similar to EBOV adaptation in guinea pigs. For mouse lethality studies (Kobinger GP, et al. (2006). Chimpanzee adenovirus vaccine protects against Zaire Ebola virus. Virology 346:394-401), mice were injected with 200 μl of 1,000LD 50 Mouse-adapted ZEBOV was injected intraperitoneally (10 FFU / animal). All animals were weighed daily and monitored for disease progression using approved scoring sheets for at least 18 days for mice and 22 days for guinea pigs. All infection procedures were performed in a "Biosafety Level 4" (BSL4) facility at the NML, PHAC.

[0139] ELISA and neutralization assays Antibody (Ab) titers were determined using 96-well ELISA plates coated with sucrose-purified MARV Ozolin GP or ZGP or negative control sucrose-purified Nipah G protein at a concentration of 1:2,000. Briefly, plates were then incubated for 18 hours at 4°C, washed with PBS and 0.1% Tween-20, and 100 μl / sample of serum was tested in triplicate (1:100, 1:400, 1:1,600, and 1:6,400 dilutions in PBS with 5% skim milk and 0.5% Tween-20). After 1 hour of incubation at 37°C in a humidified container, plates were washed and then 100 μl of goat anti-mouse IgG-conjugated HRP antibody (Cedarlane) was added (1:2,000 dilution) and further incubated for 1 hour at 37°C in a humidified container. After washing, 100 μl of ABST (2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) and peroxidase substrate (Cedarlane) were added to visualize Ab binding. Plates were incubated for 30 min at 37° C., again in a humidified container, and then read at 405 nm thereafter. Positive binding results were characterized by subtracting the positive control from the negative control serum and being greater than 3 standard deviations.

[0140] ZEBOV neutralization assays were performed. Briefly, sera from immunized mice and guinea pigs were inactivated at 56°C for 45 min, and serial dilutions of each specimen (1:20, 1:40, etc. for mice and 1:50 for guinea pigs in 50 μl of DMEM) were mixed with an equal volume of ZEBOV expressing the EGFP reporter gene (ZEBOV-EGFP) (100 transducing units / well, according to EGFP expression) and incubated at 37°C for 90 min. The mixtures were then transferred onto sub-confluent VeroE6 in 96-well flat-bottom plates and incubated at room temperature for 5-10 min. Control wells were infected with the same amount of ZEBOV-EGFP virus without the addition of serum or non-immune serum. Then, 100 μl of DMEM supplemented with 20% FBS was added to each well, and the plates were incubated at 37°C in 5% CO2 for 48 h.

[0141] Alternatively, neutralization of MARV-Angola368 was assessed using immunofluorescence analysis. Normal rabbit anti-MARV Ab and secondary goat anti-rabbit IgG FITC-conjugated Ab were used for detection. Neutralizing Ab (NAb) against SUDV Boniface was analyzed based on cytopathic effect (CPE) on CV-1 cells. Cells were incubated with equal portions of immunized serum and SUDV Boniface for 10 days, then fixed with 10% buffered formalin for 24 hours and examined under a light microscope. EGFP and FITC positive cells were counted in each well, and specimen dilutions showing more than 50% reduction in the number of green cells compared to the control were scored positive for NAb. Alternatively, NAb against SUDV-Boniface was analyzed based on cytopathic effect (CPE) on CV-1 cells. All infection procedures were performed in the BSL4 laboratory at NML, PHAC.

[0142] Splenocyte isolation Mice were sacrificed 8-11 days after the final immunization, and spleens were harvested. Briefly, spleens were placed in RPMI 1640 medium (Mediatech Inc., Manassas, VA) supplemented with 10% FBS, 1X Anti-anti (Invitrogen), and 1X β-ME (Invitrogen). Splenocytes were isolated by mechanical disruption of the spleen using a Stomacher machine (Seward Laboratory Systems Inc., Bohemia, NY), and the resulting product was filtered using a 40 μm cell strainer (BD Falcon). Cells were then treated with ACK lysis buffer (Lonza, Switzerland) for lysis of RBCs for 5 min, washed in PBS, and then resuspended in RPMI medium for use in ELISPOT or FACS analysis.

[0143] ELISPOT analysis Standard IFNγ ELISPOT assays were performed. Briefly, 96-well plates (Millipore, Billerica, MA) were coated with anti-mouse IFN-γ capture antibody and incubated for 24 h at 4 °C (R&D Systems, Minneapolis, MN). The next day, plates were washed with PBS and then blocked for 2 h with blocking buffer (1% BSA and 5% sucrose in PBS). Splenocytes (1–2 × 10 5 Cells / well) were plated in triplicate and stimulated in the presence of either RPMI 1640 (negative control), Con A (positive control), or GP peptides, either individually (spanning a 15-mer overlap of 9 amino acids and their respective GP lengths) or all pooled together (final 2.5 μg / ml), overnight at 37°C in 5% CO2. After 18-24 hours of stimulation, plates were washed in PBS and then incubated with biotinylated anti-mouse IFN-γ mAb (R&D Systems, Minneapolis, MN) for 24 hours at 4°C. Next, plates were washed again in PBS and streptavidin alkaline phosphatase (MabTech, Sweden) was added to each well and incubated for 2 hours at room temperature. Finally, plates were washed again with PBS and then BCIP / NBT Plus substrate (MabTech) was added to each well for 5-30 minutes for spot development. Once the visual development process was complete, the plates were rinsed with distilled water and then dried overnight at room temperature. Spots were counted using an automated ELISPOT reader (Cellular Technology Ltd., Shaker Heights, Ohio).

[0144] For comprehensive analysis of T cell breadth, standard IFNγ ELISPOT was modified herein as previously described in Shedlock DJ et al. (2012). Identification and measurement of subdominant and immunodominant T cell epitopes were assessed by stimulating splenocytes with individual peptides against total or matrix peptide pools. Traditional procedures of peptide pooling for specimen conservation, e.g., using matrix array pools, result in reduced analytical sensitivity, since total functional responses in pools containing peptides representing multiple epitopes effectively reduce the resolution of the analysis, i.e., "drown out" lower scale ones. Therefore, modified ELISPOT was performed with individual peptides (15mer overlaps with 9 amino acids, final 2.5 μg / ml) across each GP immunogen. Peptides containing T cell epitopes were confirmed (average ≧10 IFNγ+ spots and ≧80% animal response rate, summarized in Tables 1-6) and then functionally and phenotypically confirmed by FACS. No common or partial epitopes were identified, and neither the FACS data nor web-based epitope prediction software suggested the presence of conserved CD4+ or CD8+ T cell epitopes within consecutive peptides. Here, potential common / partial T cell epitopes were addressed for all cases of consecutive peptide responses identified by modified ELISPOT analysis. Cells were stimulated with each of the consecutive peptides individually and in pairs in combination for direct comparison, and were defined as "common / partial" if the combined response was not greater than either of the two individual responses. It should also be noted that the epitope responses herein may not be fully comprehensive, since the "15mer overlap by 9 amino acids" algorithm for generating peptides is biased towards the full coverage of CD8 T cell epitopes, which may underestimate CD4 T cell responses due to the nature of class II-restricted epitopes longer than 15 amino acids. Finally, amino acid similarity plots were generated using Vector NTI software, and the results are shown in Figure 4B.

[0145] Flow cytometry Splenocytes were cultured in a 96-well plate (1x10 6 Cells / well) and stimulated for 5-6 h with either individual peptides or the "minimal peptide pool" (final 2.5 μg / ml). Individual peptide stimulation was used for functional validation and phenotypic characterization of all peptides identified by modified ELISPOT (Tables 1-6). First, splenocytes and transfected 293T were pre-stained with LIVE / DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen). For splenocytes, cells were surface stained for CD19 (V450; clone 1D3), CD4 (PE-Cy7; clone RM4-5), CD8α (APC-Cy7; clone 53-6.7), and CD44 (PE-Cy5; clone IM7) (BD Biosciences, San Jose, CA), washed three times in PBS+1% FBS, permeabilized with BD Cytofix / Cytoperm™ kit, and then stained intracellularly with IFNγ (APC; clone XMG1.2), TNF (FITC; clone MP6-XT22), CD3 (PE-cy5.5; clone 145-2C11), and T-bet (PE; clone 4B10) (eBioscience, San Diego, CA). GP expression in transfected 293T cells was assessed 24 hours after transfection. H-2, each immunized three times with its corresponding DNA vaccine or pVAX1 empty vector control bIndirect staining was performed after 30 min incubation at 4°C in PBS + 1% FBS containing the indicated mouse-derived GP-specific polyclonal serum reagent (1:200 dilution) produced by pooling serum from mice. Cells were then stained with FITC-conjugated goat anti-mouse IgG (BioLegend, San Diego, CA), extensively washed, and then stained for MHC class I (HLA-ABC; PE-Cy7; clone G46-2.6; BD). All cells were fixed in 1% paraformaldehyde. All data were collected using an LSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star, Ashland, OR). Splenocytes were gated for activated, IFNγ-producing T cells that were CD3+CD44+, CD4+, or CD8+ and negative for B cell marker CD19 and viability dyes.

[0146] Figure 6 shows GP-specific T cell gating. Functional and phenotypic analysis of peptides containing T cell epitopes confirmed by ELISPOT was performed by FACS gating of total lymphocytes, live (LD) CD3+ cells and LIVE-DEAD (dump channel), singlet (excluding cell doublets), CD4+ and CD8+ cells, activated cells (CD44+), and peptide-specific IFNγ-producing T cells that were negative for CD19.

[0147] statistical analysis Significance for unrooted phylogenetic trees was determined by maximum likelihood estimation and verified by bootstrap analysis, and significant support values ​​(≥80%, 1,000 bootstrap replicates) were determined by MEGA version 5 software. Group analysis was completed by matched two-tailed independent t-tests, and survival curves were analyzed by log-rank (Mantel-Cox) tests. All values ​​are means ± standard error, and statistical analysis was performed by GraphPad Prism (La Jolla, CA).

[0148] result Vaccine construction and expression Phylogenetic analysis showed relative conservation in EBOV GP (94.4% for SUDV and 92.9% for ZEBOV), while MARV GP (MGP) was more divergent (approximately 70% conserved). Therefore, a consensus strategy determined by alignment of the mainstream ZEBOV and SUDV GP amino acid sequences was applied for EBOV GP, while for MARV a type match strategy was used employing the 2005 Angola outbreak sequence, which was the single cause of the largest and most lethal MARV outbreak. Each GP transgene was genetically optimized, commercially synthesized, and then subcloned into a modified pVAX1 mammalian expression vector. Overall, the three-plasmid strategy formed the basis of our novel multivalent filovirus vaccine strategy.

[0149] HEK293T cells were transfected separately with each plasmid, and GP expression was assessed by Western immunoblotting and FACS. A 130 kDa protein was observed for each in cell lysates taken 48 hours after transfection using species-specific anti-GP1 mAbs for detection. The results are shown in FIG. 1B. As a comparative control, recombinant vesicular stomatitis virus (rVSV) expressing each GP was loaded in parallel lanes. GP expression on the cell surface was then analyzed 24 hours after transfection by indirect staining with GP-specific or control polyclonal sera by FACS. The results are shown in FIG. 1C. Cell surface expression was detected for all vaccine plasmids, while little nonspecific binding was observed. Control sera did not react with GP-transfected cells, nor did positive sera react with pVAX1-transfected cells (data for pEBOZ is shown). As predicted for EBOV GP, cell surface expression sterically occluded surface MHC class I and β1-integrin recognition (Francica JR, Varela-Rohena A, Medvec A, Plesa G, Riley JL, Bates P (2010). Steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein. PLoS Pathog 6:e1001098).

[0150] Complete defense against MARV and ZEBOV attacks To determine protective efficacy, a guinea pig preclinical challenge model was employed. Preclinical immunogenicity and efficacy studies were performed herein using guinea pig and mouse models. The guinea pig preclinical model is widely used as a screening and "proof of concept" tool for filovirus vaccine development. Primary isolates of MARV and EBOV cause non-lethal disease in guinea pigs, but minority passage in this host leads to the selection of variants that can cause lethal disease with pathological features similar to those seen in filovirus-infected primates. Similarly, mice are widely used for filovirus vaccine development; however, unlike the guinea pig model, immunodetection reagents for evaluating immune and T cell responses are widely available. Infection with mouse-adapted ZEBOV (mZEBOV) results in disease characterized by high levels of virus in target organs and pathological changes in the liver and spleen similar to those found in EBOV-infected primates.

[0151] Guinea pigs (n=24) were immunized twice intradermally at three separate vaccination sites with 200 μg of each plasmid (pEBOZ, pEBOS, and pMARV) or with the pVAX1 empty vector control (n=9), then boosted 1 month later with the same vaccine. Animals were immunized 28 days after the second immunization with 1,000 LD 50Guinea pigs were challenged with guinea pig-adapted MARV-Angora (gpMARV) (n=9) or ZEBOV (gpZEBOV) (n=15) and then observed and weighed daily. Results are shown in Figures 2A-2H. Vaccinated animals were fully protected, whereas control-vaccinated animals died of gpMARV by 10 days post-challenge (n=3, P=0.0052) or gpZEBOV by 7 days post-challenge (n=6, P=0.0008) (Figures 2A and 2E). In addition, vaccinated animals were protected from weight loss (Figures 2B and 2F, P<0.0001). It is likely that vaccine-induced Ab contributed to protection, as GP-specific Ab in pooled sera showed a significant increase in binding (Figures 2C and 2G) and neutralizing (Figures 2D and 2H) titers. Experiments were performed in a BSL-4 facility and were repeated twice with similar results; error bars in Figures A-H represent standard errors. Group analyses were completed by matched two-tailed independent t-tests, and survival curves were analyzed by log-rank (Mantel-Cox) tests.

[0152] The plasmid vaccine was highly immunogenic To better characterize the immune correlates elicited by the protective DNA vaccines (plasmids pEBOZ, pEBOS, and pMARV, also referred to as trivalent DNA vaccines), we next employed a mouse model that has been widely used as a screening and "proof of concept" tool for filovirus vaccine development and for which a wide range of immune detection reagents are available. First, B cell responses were assessed by H-2 immunization with 20 days after each of the two vaccinations, with a 3-week interval between injections of 40 μg of each monovalent DNA vaccine. dGP-specific IgG was evaluated in mice (n=5 / group). Data from these experiments are shown in Figures 3A-3C. As shown in Figures 3A and 3B, little GP-specific IgG was observed in prebleed control specimens, but a significant increase was detected in all animals after vaccination. Purified SGP was not available, so purified ZGP was used instead. IgG in SUDV-vaccinated mice bound to ZGP, demonstrating vaccine-induced Ab generation capacity and cross-species recognition capacity. In addition, seroconversion occurred in 100% of vaccinated animals after only one immunization, and responses were significantly increased thereafter with similar enhancement. AVE reciprocal endpoint dilution titers were enhanced 22.1-fold in pMARV-immunized mice and 3.4- and 8.6-fold in pEBOS- and pEBOZ-vaccinated animals, respectively. Specimens were then analyzed for neutralization of ZEBOV, SUDV-Boniface, and MARV-Angola in a BSL-4 facility. Results of the neutralization analysis are shown in Figure 3C. A significant increase in NAb titers was detected after vaccination in all animals.

[0153] Mice from two different genetic backgrounds (H-2 d and H-2 b , n=5 / group) were immunized with 40 μg of each plasmid pEBOZ, pEBOS, and pMARV, boosted similarly 2 weeks later, and then sacrificed 8 days later for T cell analysis. Results from a novel modified ELISPOT assay to assess the overall vaccine-induced T cell response in which splenocytes were stimulated with individual peptides against a matrix pool are shown in Figure 4A. DNA vaccination induced robust IFNγ+ responses recognizing a diversity of T cell epitopes (Tables 1-6). All positive epitope-containing peptides were subsequently gated (see Figure 6), confirmed, and further characterized by FACS. This modified ELISPOT approach proved to be extremely sensitive, as there was a low background response from control wells (H-2 b7.2±0.2 IFNγ-producing SFC / 10 6 Splenocytes, and H-2 d The results, shown in Figure 4A, indicate that vaccination with pMARV inhibited the H-2 b 9 in mice and H-2 d pMARV-immunized H-2 induced 11 measurable epitopes, pEBOS induced 9 and 8, and pEBOZ generated 10 and 12. b Five of the nine epitopes (55.6%) derived from mice were CD8+ and accounted for approximately 57.3% of the total MGP-specific IFNγ+ response as measured by both ELISPOT and FACS validation and phenotypic analysis. b and H-2 d In mice, only 33% and 38% of validated epitopes, respectively, were CD8-restricted. However, these epitopes comprised approximately 50-90% of the total response, with CD8+ T cell responses estimated to be approximately 56% in both mouse strains, while FACS revealed that H-2 b and H-2 d The total CD8+ responses were estimated to be 51% and 90% in pEBOZ-vaccinated mice, respectively. Total CD8+ responses were lower in pEBOZ-vaccinated animals, measuring between 33% and 57% (33% for both strains by ELISPOT and 33% for H-2 by FACS). b and H-2 d 6% and 57%) for mice, respectively.

[0154] A single immunodominant epitope was detected in both mouse strains receiving pEBOS, and the immunodominant epitope was generally defined as generating an IFNγ response at least twice as high as the most subdominant epitope. pMARV contains four H-2 b Restricted immunodominant CD8+ epitopes are expressed as peptides 25-39 (#5), MGP 67-81 (#12), MGP 181-195 (#31), and MGP385-399 (#65) and one H-2 d Restricted CD4+ epitope MGP 151-171 Four of these epitopes occurred within highly conserved regions of MARV GP1, and as shown in Figures 4B and 4C, three of these were located within the putative receptor binding domain, but only one was located within the variable mucin-like region (MGP 385-399 (#65)). pEBOS was generated in the H-2 b and H-2 d In mice, the SUDV GP (SGP) and the SUDV GP1 are both located within the highly conserved region of GP1. 19-33 (#4) and SGP 241-255 However, pEBOZ immunization stimulated CD8+ epitopes occurring within the receptor binding domain and mucin-like region, respectively, of H-2 d Three immunodominant epitopes in mice (ZEBOV GP receptor binding domain (GP) 139-153( #24), as well as two CD4-restricted epitopes ZGP 175-189 (#30) and ZGP 391-405 (#66)). Only one immunodominant epitope was identified in the H-2 b A subdominant response was defined in mice that contained both CD4+ and CD8+ epitopes (#89) and occurred within a highly conserved region of GP2. Overall, the diverse epitope hierarchy was consistent and reproducible in each vaccine group. Furthermore, as shown in Figure 4D, subdominant responses accounted for a significant proportion of the total response, and the overall mean subdominant response, as measured by a modified ELISPOT assay, was significantly higher in pMARV-, pEBOS-, and pEBOZ-immunized H-2 mice. b In mice, the percentages were approximately 12%, 62%, and 74%, respectively, whereas in H-2 d Responses in mice were 47%, 50%, and 34%, respectively.

[0155] Finally, total GP-specific T cell responses were measured by FACS using stimulation with a minimal peptide pool containing only validated peptides with validated epitopes. A robust response was detected in each of the vaccinated animals, which in most cases included both activated CD4+ and CD8+ T cells. The response was GP-specific, since almost no IFNγ production was observed with the control peptide (h-Clip) and correlated highly with the ELISPOT data. The only case in which immunization did not induce significant CTL as measured by FACS was in H-2 mice vaccinated with pMARV. d None of the epitopes identified by ELISPOT were confirmed to be CD8-restricted in mice. Collectively, these data indicate that each of the vaccine plasmids was highly immunogenic in mice and generated robust GP-specific T cell responses that recognized a diverse array of T cell epitopes, including immunodominant epitopes within highly conserved regions of GP. Furthermore, the highly diverse subdominant T cell responses characterized herein may have been missed when using conventional matrix array peptide pools for epitope identification.

[0156] T cell responses were measured for reactivity to minimal peptide pools containing all peptides confirmed as positive for their respective GPs by FACS. Figure 7A shows DNA vaccine-induced T cell responses shown from a representative animal, with IFNγ-producing CD4+ (right) and CD8+ (left) cells gated. FACS plots are shown. Incubation with h-CLIP peptide served as a negative control (control). Figure 7B shows the results of the gated cells in Figure 7A summarized as the mean % of total CD44+ / IFNγ+CD4+ or CD8+ cells, with error bars representing standard error. Experiments were repeated at least twice with similar results.

[0157] "Single-dose" protection in mice Next, vaccine efficacy against ZEBOV challenge was evaluated in preclinical mice. Mice were vaccinated only once due to strong NAb induction, and protective data were observed. Mice (H-2 k , n=10 / group) were immunized with 40 μg of pEBOZ DNA, and protection was achieved with 1,000LD 50 The mice were assessed in a BSL4 facility 28 days later by challenge with mouse-adapted ZEBOV (mZEBOV). All control animals succumbed to infection by day 7 post-challenge, whereas Figure 5A shows that DNA-vaccinated mice were completely protected (P=0.0002). In addition, as shown in Figure 5B, control mice exhibited progressive weight loss before death (P<0.0001).

[0158] To better understand the mechanism of DNA-induced protection in the "single dose" model, NAb and T cell generation were next assessed. NAb were assessed 25 days after vaccination and 3 days before challenge, and as shown in Figure 5C, a significant increase (P<0.0001) was detected in all vaccinated animals (n=10 / group). Reciprocal endpoint dilution titers ranged from 19 to 42, 27.3±2.5.

[0159] We next expanded the scope of this analysis to evaluate the generation of ZGP-specific T cells and to compare responses in mice immunized with either pEBOZ alone or the trivalent formulation. IFN-γ production (n=5) was assessed 11 days later by FACS using the total ZGP peptide pool. Data are shown in FIG. 5D. IFNγ-producing T cells were detected in all animals and were specific for the ZGP peptide, since stimulation with a control peptide did not induce cytokine production. Immunization with either the monovalent or trivalent formulation induced robust IFNγ T cell responses, with no significant difference between them (P=0.0920).

[0160] CTLs may be important in the elimination of virus-infected cells (Warfield KL,et al.(2005).Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection.J Immunol 175:1184-1191, Kalina WV,Warfield KL,Olinger GG,Bavari S(2009).Discovery of common marburgvirus protective epitopes in a BALB / c mouse model.Virol J 6:132, Olinger GG,et al.(2005).Protective cytotoxic T-cell responses induced by venezuelan equine encephalitis virus replicons expressing Ebola virus proteins.J Virol 79:14189-14196, Sullivan NJ,et al.(2011).CD8(+)cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates. Nat Med 17:1128-1131, and Geisbert TW,et al.(2010).Vector choice determines immunogenicity and potency of genetic vaccines against Angola Marburg virus in nonhuman primates.J Virol 84:10386-10394), as well as additional effector cytokines, TNF, as well as T hProduction of the T-box transcription factor TBX21 (T-bet), a developmental inhibitor known to correlate with type I CTL immunity and cytotoxicity, was measured, with the following results: for total cells: TNF 2.9 ± 0.8, Tbet 13.0 ± 1.1; for CD4+ / CD44+ / IFNγ+ cells: TNF 61.4 ± 3.1, Tbet 72.6 ± 2.0; for CD8+ / CD44+ / IFNγ+ cells: TNF 33.0 ± 3.3, Tbet 992.1 ± 1.4 ( * p<0.1, *** p<0.001, **** p<0.0001). We found that 61% and 33% of activated CD4+ and CD8+ T cells, respectively, produced TNF in addition to IFNγ. Furthermore, IFNγ-producing T cells expressed high levels of T-bet. Approximately 73% and 92% of CD8+ and CD4+ T cells, respectively, were CD44+ and produced IFNγ after stimulation with ZGP peptide.

[0161] 8A and 8B show T cell induction by "single dose" vaccination. H-2 as measured by FACS after a single pEBOZ immunization or a single trivalent vaccination with the three vaccine plasmids at separate sites. k T cell responses in mice are shown (a) and summarized as the mean % of total CD44+ / IFNγ+CD4+ (purple) or CD8+ (orange) cells (b). Pseudocolor FACS plots are from representative animals and gated on IFNγ-producing CD4+ (right) and CD8+ (left) cells. Incubation with h-CLIP peptide served as a negative control (control). Experiments were performed twice with similar results, error bars represent standard error and ns represents not significant difference.

[0162] Consideration We report the development and evaluation of a multivalent filovirus vaccine in preclinical rodent immunogenicity and efficacy studies. Complete protection against challenge with gpMARV and gpZEBOV was observed after single doses of the two DNA vaccines in guinea pigs and after a "single dose" DNA vaccine in mice against mZEBOV. To date, genetic vaccination of guinea pigs has involved the injection of naked DNA (Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ(2000). Development of a preventive vaccine for Ebola virus infection in primates. Nature 408:605-609) or delivered by gene gun (Dowling W,et al.(2006). The influences of glycosylation on the antigenicity, immunogenicity, and protective efficacy of Ebola virus GP DNA vaccines. J Virol 81:1821-1837, Vanderzanden L,et al.(1998). DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge. Virology 246:134-144, and Riemenschneider J,et al(2003). Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine 21:4071-4080), however, both methods required at least three vaccinations to achieve full protection.The improved protection herein may be due to robust Ab induction, as a single DNA vaccination produced GP-specific IgG binder titers of comparable magnitude to those in protected animals after gene gun vaccination. DNA vaccination induced ZGP- and MGP-specific Ab titers of 3.85 and 2.18 log10, respectively, after a single dose versus 2.7 and 3.0 after three gene gun vaccinations. For comparison with an alternative "single dose" protection strategy in guinea pigs, the Ag-conjugated virus-like particle (VLP) platform produced slightly higher Ab titers than those observed after DNA vaccination (Swenson DL, Warfield KL, Negley DL, Schmaljohn A, Aman MJ, Bavari S (2005). Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections. Vaccine 23:3033-3042). Furthermore, the recombinant adenovirus (rAd) approach induced lower ZGP-specific NAb titers (53 reciprocal end point dilution titer, versus 88 herein) than those from a single DNA vaccination (Kobinger GP, et al. (2006). Chimpanzee adenovirus vaccine protects against Zaire Ebola virus. Virology 346:394-401). Vaccination with rVSV (Jones SM, et al. (2007). Assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis 196 Suppl 2:S404-412) generated ZGP-specific Ab titers similar to the current platform.Collectively, these data demonstrate that DNA vaccination was capable of inducing binding and neutralizing Abs comparable to nonreplicating viral platforms and may help in part to explain the strong guinea pig survival data herein.

[0163] The generation of NAbs by protective DNA vaccination may have benefited from transgene-expressed mature GP structures. In vivo transfection studies confirmed that vaccine-encoded GP was highly expressed, post-translationally cleaved (Fig. 1B), and translocated to the cell surface, sterically blocking immune detection of cell surface molecules (Fig. 1C). Therefore, it was highly likely that the vaccine immunogens formed herein matured into heterotrimeric spikes that were otherwise functional for virion assembly during infection. This may be important for the generation and display of virologically relevant neutralizing determinants that are subsequently crucial for the induction of conformation-dependent Nabs (Dowling W, et al. (2007). Influences of glycosylation on antigenicity, immunogenicity, and protective efficacy of ebola virus GP DNA vaccines. J Virol 81:1821-1837; Shedlock DJ, Bailey MA, Popernack PM, Cunningham JM, Burton DR, Sullivan NJ (2010). Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms. Virology 401:228-235). Thus, from this perspective, expression of native anchor structures may be superior to soluble derivatives in the ability to generate NAbs (Sullivan NJ, et al. (2006). Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs. PLoS Med 3:el77; Xu L, et al. (1998). Immunization for Ebola virus infection. Nat Med 4:37-42).

[0164] To better characterize the T cell responses when driven by a protective vaccine, immunogenicity and efficacy studies in mice were performed to determine "single-dose" complete protection against mZEBOV by DNA vaccination (Figures 5A-5D). Currently, the most effective platform for conferring complete protection in this model is VLP or rAd vaccination (Kobinger GP,et al.(2005).Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection.J Immunol 175:1184-1191, Warfield KL,Swenson DL,Olinger GG,Kalina WV,Aman MJ,Bavari S(2007).Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge.J Infect Dis 196 Suppl 2:S430-437) with or without adjuvant (Sun Y,et al.(2009).Protection against lethal challenge by Ebola virus-like particles produced in insect cells.Virology 383:12-21). al. (2006) SUPRA, Choi JH, et al. (2012). A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs. Mol Pharm 9:156-167, Richardson JS, et al. (2009). Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.PLoS One 4:e5308), or rRABV vaccination (Blaney JE,et al.(2011).Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses.J Virol 85:10605-10616). However, characterization of T cell responses was very limited in these studies and was limited to splenocyte stimulation with either two (Warfield KL,(2007)same as above) or one (Warfield KL,et al.(2005)same as above) previously described to contain ZGP T cell epitopes (Warfield KL,et al.(2005)same as above; Olinger GG,et al.(2005)same as above; Kobinger GP,et al.(2006)same as above; Sun Y,et al.(2009).Choi,JH,et al. (2012). Herein, we report robust and broad CTL induction by protective vaccination, extensively analyzed by a novel modified T cell assay (Figure 4A and Tables 1-6). In total, 52 novel T cell epitopes were identified, including a number of immunodominant epitopes occurring primarily within highly conserved regions of GP. Of the total 22 identified ZGP epitopes, only 4 have been reported previously. Furthermore, only 1 of 20 MGP (Kalina WV, Warfield KL, Olinger GG, Bavari S (2009). Discovery of common marburgvirus protective epitopes in a BALB / c mouse model. Virol J 6:132) and only 1 of 16 SGP epitopes have been described previously. Thus, this is the most comprehensive report to date of preclinical GP epitopes describing GP epitopes from multiple filoviruses in two different mouse genetic backgrounds.

[0165] Another novel finding from these analyses is the assessment of vaccine-induced subdominant T cell responses, which have been shown to comprise a significant proportion of total T cell responses ranging from 12% to 74% (Figure 4D). This may be particularly important as subdominant responses can contribute significantly to protection. Thus, this may prove informative in the future to determine the specific contribution of subdominant and immunodominant epitope T cell responses to protection. Of note, these responses may have been overlooked if traditional matrix array peptide pools for epitope identification had been used. Thus, the limited epitope detection in prior studies may have been directly related to lower levels of vaccine-induced immunity, the use of less sensitive standard assays, and / or the use of peptide sequences and / or algorithms that favor the detection of immunodominant CD8+ epitopes.

[0166] Although immune correlates of protection against filoviruses remain controversial, the data generated by this highly immunogenic approach provide a unique opportunity to study protective vaccine-driven T cell immunity. DNA vaccination herein induced robust ZGP-specific T cells, the majority of which expressed high levels of T-bet. hIt has been characterized by type 1 polyfunctional CTLs and has also been shown to correlate with T cell cytotoxicity in humans. It is clear that previously independent DNA vaccine platforms capable of generating primarily humoral immune responses and cellular immunity biased towards CD4+ T cells may benefit from in vivo EP delivery, which has recently been demonstrated to induce potent CD8+ T cells in NHPs and clinics. It is clear that previously independent DNA vaccine platforms capable of generating primarily humoral immune responses and cellular immunity biased towards CD4+ T cells may benefit from in vivo EP delivery, which has recently been demonstrated to induce potent CD8+ T cells in NHPs and clinics. Thus, the data herein are consistent with this approach as a standalone or prime-boost modality in NHP immunogenicity and efficacy studies. This approach provides an attractive vaccination strategy that can be rapidly and inexpensively modified and / or produced for the Filoviridae biological threat situation and rapid response during development. Additionally, this model approach provides an important tool for studying protective immune correlates against filoviral disease and can be applied to existing platforms to guide future strategies.

[0167] Example 2 A trivalent vaccine is provided that includes three plasmids. The first plasmid includes a nucleic acid sequence encoding a Zaire Ebola virus consensus immunogen based on ZEBOV CON, SEQ ID NO: 1, modified to include an IgE signal peptide at the N-terminus of the Zaire Ebola virus consensus immunogen. The second plasmid includes a nucleic acid sequence encoding a Sudan Ebola virus consensus immunogen based on SUDV CON, SEQ ID NO: 2, modified to include an IgE signal peptide at the N-terminus of the Sudan Ebola virus consensus immunogen. The third plasmid includes a nucleic acid sequence encoding a Marburg Marburg virus Angola (MARV) immunogen based on MARV ANG, SEQ ID NO: 3, modified to include an IgE signal peptide at the N-terminus of the Marburg Marburg virus Angola immunogen.

[0168] Example 3 A five-plasmid vaccine is provided. The first plasmid contains a nucleic acid sequence encoding the Zaire Ebola virus consensus immunogen, ZEBOV CON, SEQ ID NO: 1. The second plasmid contains a nucleic acid sequence encoding the Sudan Ebola virus consensus immunogen, SUDV CON, SEQ ID NO: 2. The third plasmid contains a nucleic acid sequence encoding the Marburg virus Rabin cluster consensus (MARV-RAV CON), SEQ ID NO: 4, using Marburg virus Ravn, Durba (09DRC99), and Uganda (02Uga07Y). The fourth plasmid contains a nucleic acid sequence encoding the Marburg virus Ozolin cluster consensus (MARV-OZO CON), SEQ ID NO: 5, using Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99). The fifth plasmid, (Musoke, Popp, and Leiden), contains SEQ ID NO:6, a nucleic acid sequence encoding the Marburg virus-Musoke cluster consensus (MARV-MUS CON).

[0169] Example 4 A five-plasmid vaccine is provided. The first plasmid comprises a nucleic acid sequence encoding a Zaire Ebola virus consensus immunogen based on ZEBOV CON, SEQ ID NO: 1, modified to include an IgE signal peptide at the N-terminus of the Zaire Ebola virus consensus immunogen. The second plasmid comprises a nucleic acid sequence encoding a Sudan Ebola virus consensus immunogen based on SUDV CON, SEQ ID NO: 2, modified to include an IgE signal peptide at the N-terminus of the Sudan Ebola virus consensus immunogen. The third plasmid comprises a nucleic acid sequence encoding a Marburg virus Rav consensus based on SEQ ID NO: 4, Marburg virus Rabin cluster consensus (MARV-RAV CON), using Marburg virus Ravn, Durba (09DRC99) and Uganda (02Uga07Y), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Rav immunogen. The fourth plasmid, using Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99), contains a nucleic acid sequence encoding a Marburg virus Ozo consensus based on SEQ ID NO:5, the Marburg virus-Ozo cluster consensus (MARV-OZO CON), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Ozo immunogen. The fifth plasmid, using Musoke, Popp, and Leiden, contains a nucleic acid sequence encoding a Marburg virus Mus consensus based on SEQ ID NO:6, the Marburg virus-Mus cluster consensus (MARV-MUS CON), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Mus immunogen.

[0170] Example 5 A six-plasmid vaccine is provided. The first plasmid contains a nucleic acid sequence encoding the Zaire Ebola virus consensus immunogen, ZEBOV CON, SEQ ID NO: 1. The second plasmid contains a nucleic acid sequence encoding the Sudan Ebola virus consensus immunogen, SUDV CON, SEQ ID NO: 2. The third plasmid contains a nucleic acid sequence encoding the Marburg virus Rabin cluster consensus (MARV-RAV CON), SEQ ID NO: 4, using Marburg virus Ravn, Durba (09DRC99), and Uganda (02Uga07Y). The fourth plasmid contains a nucleic acid sequence encoding the Marburg virus Ozolin cluster consensus (MARV-OZO CON), SEQ ID NO: 5, using Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99). The fifth plasmid contains SEQ ID NO:6, a nucleic acid sequence encoding the Marburg virus-Musoke cluster consensus (MARV-MUS CON), (Musoke, Popp, and Leiden). The sixth plasmid contains SEQ ID NO:3, a nucleic acid sequence encoding the Marburg virus Angola 2005 isolate glycoprotein immunogen.

[0171] Example 6 A five-plasmid vaccine is provided. The first plasmid comprises a nucleic acid sequence encoding a Zaire Ebola virus consensus immunogen based on ZEBOV CON, SEQ ID NO: 1, modified to include an IgE signal peptide at the N-terminus of the Zaire Ebola virus consensus immunogen. The second plasmid comprises a nucleic acid sequence encoding a Sudan Ebola virus consensus immunogen based on SUDV CON, SEQ ID NO: 2, modified to include an IgE signal peptide at the N-terminus of the Sudan Ebola virus consensus immunogen. The third plasmid comprises a nucleic acid sequence encoding a Marburg virus Rav consensus based on SEQ ID NO: 4, Marburg virus Rabin cluster consensus (MARV-RAV CON), using Marburg virus Ravn, Durba (09DRC99) and Uganda (02Uga07Y), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Rav immunogen. The fourth plasmid, using Ozolin, Uganda (01Uga07), and Durba (05 and 07DRC99), contains a nucleic acid sequence encoding a Marburg virus Ozo consensus based on SEQ ID NO:5, the Marburg virus-Ozo cluster consensus (MARV-OZO CON), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Ozo immunogen. The fifth plasmid, using Musoke, Popp, and Leiden, contains a nucleic acid sequence encoding a Marburg virus Mus consensus based on SEQ ID NO:6, the Marburg virus-Mus cluster consensus (MARV-MUS CON), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Mus immunogen. The sixth plasmid contains a nucleic acid sequence encoding the Marburg virus Angola 2005 isolate glycoprotein immunogen, based on MARV ANG, SEQ ID NO:3, modified to include an IgE signal peptide at the N-terminus of the Marburg virus Angola immunogen.

[0172] [Table 1]

[0173] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4 CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

[0174] [Table 2]

[0175] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4 CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

[0176] [Table 3]

[0177] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4 CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

[0178] [Table 4]

[0179] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4 CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

[0180] [Table 5]

[0181] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

[0182] [Table 6]

[0183] "Epitope-containing peptides" were identified by IFNγ ELISPOT (≥10 SFC / 10 6 splenocytes and ≥80% response rate), then confirmed by FACS (≥3–5x10 4 CD3+ cells were obtained). Responses for each were further characterized by FACS (CD4 and / CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (best consensus % rank by IEDB) and references to previously described epitopes are indicated. Immunodominant epitopes are indicated ( * ).

Claims

1. 1. A composition comprising: a) a nucleic acid sequence encoding a consensus Ebola virus Zaire envelope glycoprotein immunogen, the amino acid sequence of said consensus Ebola virus Zaire envelope glycoprotein immunogen being selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide; b) a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein being selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide; and c) a nucleic acid sequence encoding a Marburg MARburg virus Angola 2005 envelope glycoprotein immunogen, wherein the amino acid sequence of said Marburg MARburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide.

2. 2. The composition of claim 1, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, and a third plasmid comprising a nucleic acid sequence encoding the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen.

3. a) the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide; b) the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide; 2. The composition of claim 1, wherein the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 635 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:3 comprising 635 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 635 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:3 comprising 635 or more amino acids linked to an IgE signal peptide.

4. 4. The composition of claim 3, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, and a third plasmid comprising a nucleic acid sequence encoding the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen.

5. a) the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide; b) the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide; 4. The composition of claim 3, wherein the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 670 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:3 comprising 670 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 670 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:3 comprising 670 or more amino acids linked to an IgE signal peptide.

6. 6. The composition of claim 5, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, and a third plasmid comprising a nucleic acid sequence encoding the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen.

7. 6. The composition of claim 5, wherein the consensus Zaire Ebola virus envelope glycoprotein immunogen has an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:1 linked to an IgE signal peptide, the consensus Sudan Ebola virus envelope glycoprotein immunogen has an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:2 linked to an IgE signal peptide, and the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen has an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:3 linked to an IgE signal peptide.

8. 8. The composition of claim 7, comprising a first plasmid comprising SEQ ID NO:1 or a nucleic acid sequence encoding SEQ ID NO:1 linked to an IgE signal peptide, a second plasmid comprising SEQ ID NO:2 or a nucleic acid sequence encoding SEQ ID NO:2 linked to an IgE signal peptide, and a third plasmid comprising SEQ ID NO:3 or a nucleic acid sequence encoding SEQ ID NO:3 linked to an IgE signal peptide.

9. 1. A composition comprising: i) a nucleic acid sequence encoding a consensus Ebola virus envelope glycoprotein immunogen, the amino acid sequence of said consensus Ebola virus envelope glycoprotein immunogen being selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide; ii) a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein being selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:2 linked to an IgE signal peptide, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide, iii) a nucleic acid sequence encoding a first consensus Marburg virus envelope glycoprotein immunogen, wherein the amino acid sequence of said first consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:4 (MARV CON1), a fragment of SEQ ID NO:4 comprising 600 or more amino acids, an amino acid sequence 95% identical to SEQ ID NO:4, a fragment of an amino acid sequence 95% identical to SEQ ID NO:4 comprising 600 or more amino acids, SEQ ID NO:4 (MARV CON1) linked to an IgE signal peptide, a fragment of SEQ ID NO:4 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% identical to SEQ ID NO:4 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% identical to SEQ ID NO:4 comprising 600 or more amino acids linked to an IgE signal peptide; iv) a nucleic acid sequence encoding a second consensus Marburg virus envelope glycoprotein immunogen, the amino acid sequence of said second consensus Marburg virus envelope glycoprotein immunogen being selected from the group consisting of SEQ ID NO:5 (MARV CON2), a fragment of SEQ ID NO:5 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:5, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:5 comprising 600 or more amino acids, SEQ ID NO:5 (MARV CON2) linked to an IgE signal peptide, a fragment of SEQ ID NO:5 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:5 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:5 comprising 600 or more amino acids linked to an IgE signal peptide; and v) a nucleic acid sequence encoding a third consensus Marburg virus envelope glycoprotein immunogen, wherein the amino acid sequence of said third consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:6 (MARV CON3), a fragment of SEQ ID NO:6 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:6, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:6 comprising 600 or more amino acids, SEQ ID NO:6 (MARV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:6 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:6 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:6 comprising 600 or more amino acids linked to an IgE signal peptide.

10. 10. The composition of claim 9, comprising: a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen; a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen; a third plasmid comprising a nucleic acid sequence encoding the first consensus Marburg virus envelope glycoprotein immunogen; a fourth plasmid comprising a nucleic acid sequence encoding the second consensus Marburg virus envelope glycoprotein immunogen; and a fifth plasmid comprising a nucleic acid sequence encoding the third consensus Marburg virus envelope glycoprotein immunogen.

11. 10. The composition of claim 9, further comprising a nucleic acid sequence encoding a Marburg virus Angola 2005 envelope glycoprotein immunogen, wherein the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen is selected from SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide.

12. 12. The composition of claim 11, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, a third plasmid comprising a nucleic acid sequence encoding the first consensus MARburg virus envelope glycoprotein immunogen, a fourth plasmid comprising a nucleic acid sequence encoding the second consensus MARburg virus envelope glycoprotein immunogen, a fifth plasmid comprising a nucleic acid sequence encoding the third consensus MARburg virus envelope glycoprotein immunogen, and a sixth plasmid comprising a nucleic acid sequence encoding a MARburg virus Angola 2005 envelope glycoprotein immunogen.

13. i) the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide; ii) the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide; iii) the amino acid sequence of the first consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:4 (MARV CON1), a fragment of SEQ ID NO:4 comprising 630 or more amino acids, an amino acid sequence 98% identical to SEQ ID NO:4, a fragment of an amino acid sequence 98% identical to SEQ ID NO:4 comprising 630 or more amino acids, SEQ ID NO:4 (MARV CON1) linked to an IgE signal peptide, a fragment of SEQ ID NO:4 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% identical to SEQ ID NO:4 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% identical to SEQ ID NO:4 comprising 630 or more amino acids linked to an IgE signal peptide; iv) the amino acid sequence of the second consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:5 (MARV CON2), a fragment of SEQ ID NO:5 comprising 630 or more amino acids, an amino acid sequence 98% identical to SEQ ID NO:5, a fragment of an amino acid sequence 98% identical to SEQ ID NO:5 comprising 630 or more amino acids, SEQ ID NO:5 (MARV CON2) linked to an IgE signal peptide, a fragment of SEQ ID NO:5 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% identical to SEQ ID NO:5 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% identical to SEQ ID NO:5 comprising 630 or more amino acids linked to an IgE signal peptide; 10. The composition of claim 9, wherein the amino acid sequence of the third consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:6 (MARV CON3), a fragment of SEQ ID NO:6 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:6, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:6 comprising 630 or more amino acids, SEQ ID NO:6 (MARV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:6 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:6 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98%* homologous to SEQ ID NO:6 comprising 630 or more amino acids linked to an IgE signal peptide.

14. 14. The composition of claim 13, comprising: a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen; a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen; a third plasmid comprising a nucleic acid sequence encoding the first consensus Marburg virus envelope glycoprotein immunogen; a fourth plasmid comprising a nucleic acid sequence encoding the second consensus Marburg virus envelope glycoprotein immunogen; and a fifth plasmid comprising a nucleic acid sequence encoding the third consensus Marburg virus envelope glycoprotein immunogen.

15. 14. The composition of claim 13, further comprising a nucleic acid sequence encoding a Marburg virus Angola 2005 envelope glycoprotein immunogen, wherein the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:3 comprising 630 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:3 comprising 630 or more amino acids linked to an IgE signal peptide.

16. 16. The composition of claim 15, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, a third plasmid comprising a nucleic acid sequence encoding the first consensus MARburg virus envelope glycoprotein immunogen, a fourth plasmid comprising a nucleic acid sequence encoding the second consensus MARburg virus envelope glycoprotein immunogen, a fifth plasmid comprising a nucleic acid sequence encoding the third consensus MARburg virus envelope glycoprotein immunogen, and a sixth plasmid comprising a nucleic acid sequence encoding a MARburg virus Angola 2005 envelope glycoprotein immunogen.

17. i) the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide; ii) the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide; iii) the amino acid sequence of the first consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:4 (MARV CON1), a fragment of SEQ ID NO:4 comprising 660 or more amino acids, an amino acid sequence 99% identical to SEQ ID NO:4, a fragment of an amino acid sequence 99% identical to SEQ ID NO:4 comprising 660 or more amino acids, SEQ ID NO:4 (MARV CON1) linked to an IgE signal peptide, a fragment of SEQ ID NO:4 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% identical to SEQ ID NO:4 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% identical to SEQ ID NO:4 comprising 660 or more amino acids linked to an IgE signal peptide; iv) the amino acid sequence of the second consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:5 (MARV CON2), a fragment of SEQ ID NO:5 comprising 660 or more amino acids, an amino acid sequence 99% identical to SEQ ID NO:5, an amino acid sequence fragment 99% identical to SEQ ID NO:5 comprising 660 or more amino acids, SEQ ID NO:5 (MARV CON2) linked to an IgE signal peptide, a fragment of SEQ ID NO:5 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% identical to SEQ ID NO:5 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% identical to SEQ ID NO:5 comprising 660 or more amino acids linked to an IgE signal peptide; 10. The composition of claim 9, wherein the amino acid sequence of the third consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:6 (MARV CON3), a fragment of SEQ ID NO:6 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:6, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:6 comprising 660 or more amino acids, SEQ ID NO:6 (MARV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:6 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:6 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:6 comprising 660 or more amino acids linked to an IgE signal peptide.

18. 18. The composition of claim 17, comprising: a first plasmid comprising a nucleic acid sequence encoding the consensus Ebola virus Zaire envelope glycoprotein immunogen; a second plasmid comprising a nucleic acid sequence encoding the consensus Ebola virus Sudan envelope glycoprotein immunogen; a third plasmid comprising a nucleic acid sequence encoding the first consensus Marburg virus envelope glycoprotein immunogen; a fourth plasmid comprising a nucleic acid sequence encoding the second consensus Marburg virus envelope glycoprotein immunogen; and a fifth plasmid comprising a nucleic acid sequence encoding the third consensus Marburg virus envelope glycoprotein immunogen.

19. 18. The composition of claim 17, further comprising a nucleic acid sequence encoding a Marburg MARburg virus Angola 2005 envelope glycoprotein immunogen, wherein the amino acid sequence of the Marburg MARburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:3 comprising 660 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:3 comprising 660 or more amino acids linked to an IgE signal peptide.

20. 20. The composition of claim 19, comprising: a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen; a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen; a third plasmid comprising a nucleic acid sequence encoding the first consensus MARburg virus envelope glycoprotein immunogen; a fourth plasmid comprising a nucleic acid sequence encoding the second consensus MARburg virus envelope glycoprotein immunogen; a fifth plasmid comprising a nucleic acid sequence encoding the third consensus MARburg virus envelope glycoprotein immunogen; and a sixth plasmid comprising a nucleic acid sequence encoding a MARburg virus Angola 2005 envelope glycoprotein immunogen.

21. i) the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON) and SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide; ii) the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON) and SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide; iii) the amino acid sequence of the first consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:4 (MARV CON1) and SEQ ID NO:4 (MARV CON1) linked to an IgE signal peptide; iv) the amino acid sequence of the second consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:5 (MARV CON2) and SEQ ID NO:5 (MARV CON2) linked to an IgE signal peptide; 10. The composition of claim 9, wherein the amino acid sequence of the third consensus Marburg virus envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:6 (MARV CON3) and SEQ ID NO:6 (MARV CON) linked to an IgE signal peptide.

22. 22. The composition of claim 21 , comprising: a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen; a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen; a third plasmid comprising a nucleic acid sequence encoding the first consensus Marburg virus envelope glycoprotein immunogen; a fourth plasmid comprising a nucleic acid sequence encoding the second consensus Marburg virus envelope glycoprotein immunogen; and a fifth plasmid comprising a nucleic acid sequence encoding the third consensus Marburg virus envelope glycoprotein immunogen.

23. 22. The composition of claim 21, further comprising a nucleic acid sequence encoding a Marburg virus Angola 2005 envelope glycoprotein immunogen, wherein the amino acid sequence of said Marburg virus Angola 2005 envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO: 3 (MARV) and SEQ ID NO: 3 (MARV) linked to an IgE signal peptide.

24. 22. The composition of claim 21 , comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, a third plasmid comprising a nucleic acid sequence encoding the first consensus MARburg virus envelope glycoprotein immunogen, a fourth plasmid comprising a nucleic acid sequence encoding the second consensus MARburg virus envelope glycoprotein immunogen, a fifth plasmid comprising a nucleic acid sequence encoding the third consensus MARburg virus envelope glycoprotein immunogen, and a sixth plasmid comprising a nucleic acid sequence encoding a MARburg virus Angola 2005 envelope glycoprotein immunogen.

25. a nucleic acid sequence encoding a consensus Ebola virus envelope glycoprotein immunogen, the amino acid sequence of said consensus Ebola virus envelope glycoprotein immunogen being selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide; and a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, wherein the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide.

26. 26. The composition of claim 25, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, and a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

27. the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:1 comprising 630 or more amino acids linked to an IgE signal peptide; 26. The composition of claim 25, wherein the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 630 or more amino acids, an amino acid sequence 98% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide, an amino acid sequence 98% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 98% homologous to SEQ ID NO:2 comprising 630 or more amino acids linked to an IgE signal peptide.

28. 28. The composition of claim 27, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen, and a third plasmid comprising a nucleic acid sequence encoding the Marburg Marburg virus Angola 2005 envelope glycoprotein immunogen.

29. the amino acid sequence of the consensus Ebola virus Zaire envelope glycoprotein immunogen is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:1 comprising 660 or more amino acids linked to an IgE signal peptide; 26. The composition of claim 25, wherein the amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein is selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 660 or more amino acids, an amino acid sequence 99% homologous to SEQ ID NO:2, a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide, an amino acid sequence 99% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence 99% homologous to SEQ ID NO:2 comprising 660 or more amino acids linked to an IgE signal peptide.

30. 30. The composition of claim 29, comprising a first plasmid comprising a nucleic acid sequence encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, and a second plasmid comprising a nucleic acid sequence encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

31. 26. The composition of claim 25, wherein the consensus Zaire Ebola virus envelope glycoprotein immunogen has an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:1 linked to an IgE signal peptide, and the consensus Sudan Ebola virus envelope glycoprotein immunogen has an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:2 linked to an IgE signal peptide.

32. 32. The composition of claim 31 , comprising a first plasmid comprising SEQ ID NO:1 or a nucleic acid sequence encoding SEQ ID NO:1 linked to an IgE signal peptide, and a second plasmid comprising SEQ ID NO:2 or a nucleic acid sequence encoding SEQ ID NO:2 linked to an IgE signal peptide.

33. 33. The composition of any one of claims 1 to 32, formulated for delivery to an individual using electroporation.

34. The composition of any one of claims 1 to 32, further comprising a nucleic acid sequence encoding one or more proteins selected from the group consisting of IL-12, IL-15, and IL-28.

35. 33. A method of inducing an immune response against a filovirus, comprising administering to an individual a composition according to any one of claims 1 to 32 in an amount effective to induce an immune response in said individual.

36. 33. A method of inducing an immune response against a filovirus selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire, comprising administering to an individual a composition according to any one of claims 1 to 32 in an amount effective to induce an immune response in said individual.

37. 33. A method of treating an individual diagnosed with a filovirus, comprising administering to the individual a therapeutically effective amount of a composition according to any one of claims 1 to 32.

38. 33. A method of treating an individual diagnosed with a filovirus selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire, comprising administering to the individual a therapeutically effective amount of a composition of any one of claims 1-32.

39. 33. A method for preventing a filovirus infection in an individual, comprising administering to the individual a prophylactically effective amount of a composition according to any one of claims 1 to 32.

40. 33. A method for preventing a filovirus infection in an individual, wherein the filovirus is selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire, comprising administering to the individual a prophylactically effective amount of a composition described in any one of claims 1 to 32.

41. A consensus Ebola virus Zaire envelope glycoprotein immunogen, the amino acid sequence of which is selected from the group consisting of SEQ ID NO:1 (ZEBOV CON), a fragment of SEQ ID NO:1 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:1, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids, SEQ ID NO:1 (ZEBOV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:1 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:1 comprising 600 or more amino acids linked to an IgE signal peptide. A consensus Sudan Ebola virus envelope glycoprotein immunogen, the amino acid sequence of said consensus Sudan Ebola virus envelope glycoprotein being selected from the group consisting of SEQ ID NO:2 (SUDV CON), a fragment of SEQ ID NO:2 comprising 600 or more amino acids that is 95% homologous to SEQ ID NO:2, a fragment of an amino acid sequence comprising 600 or more amino acids that is 95% homologous to SEQ ID NO:2, SEQ ID NO:2 (SUDV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:2 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence comprising 95% homologous to SEQ ID NO:2 linked to an IgE signal peptide, and a fragment of an amino acid sequence comprising 600 or more amino acids that is 95% homologous to SEQ ID NO:2 linked to an IgE signal peptide. A Marburg virus Angola 2005 envelope glycoprotein immunogen, the amino acid sequence of which is selected from the group consisting of SEQ ID NO:3 (MARV), a fragment of SEQ ID NO:3 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:3, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids, SEQ ID NO:3 (MARV) linked to an IgE signal peptide, a fragment of SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:3 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:3 comprising 600 or more amino acids linked to an IgE signal peptide. a first consensus Marburg virus envelope glycoprotein immunogen, the amino acid sequence of said first consensus Marburg virus envelope glycoprotein immunogen being selected from the group consisting of SEQ ID NO:4 (MARV CON1), a fragment of SEQ ID NO:4 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:4, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:4 comprising 600 or more amino acids, SEQ ID NO:4 (MARV CON1) linked to an IgE signal peptide, a fragment of SEQ ID NO:4 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:4 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:4 comprising 600 or more amino acids linked to an IgE signal peptide; a second consensus Marburg virus envelope glycoprotein immunogen, the amino acid sequence of which is selected from the group consisting of SEQ ID NO:5 (MARV CON2), a fragment of SEQ ID NO:5 comprising 600 or more amino acids, an amino acid sequence 95% homologous to SEQ ID NO:5, a fragment of an amino acid sequence 95% homologous to SEQ ID NO:5 comprising 600 or more amino acids, SEQ ID NO:5 (MARV CON2) linked to an IgE signal peptide, a fragment of SEQ ID NO:5 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% homologous to SEQ ID NO:5 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% homologous to SEQ ID NO:5 comprising 600 or more amino acids linked to an IgE signal peptide; and 1. A third consensus Marburg virus envelope glycoprotein immunogen, the amino acid sequence of which is selected from the group consisting of SEQ ID NO:6 (MARV CON3), a fragment of SEQ ID NO:6 comprising 600 or more amino acids, an amino acid sequence 95% identical to SEQ ID NO:6, a fragment of an amino acid sequence 95% identical to SEQ ID NO:6 comprising 600 or more amino acids, SEQ ID NO:6 (MARV CON) linked to an IgE signal peptide, a fragment of SEQ ID NO:6 comprising 600 or more amino acids linked to an IgE signal peptide, an amino acid sequence 95% identical to SEQ ID NO:6 linked to an IgE signal peptide, and a fragment of an amino acid sequence 95% identical to SEQ ID NO:6 comprising 600 or more amino acids linked to an IgE signal peptide. A composition comprising two or more proteins selected from the group consisting of:

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