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

Abstract

To disclose nucleic acid molecules and compositions comprising one or more nucleic acid sequences that encode a consensus Ebolavirus glycoproteinimmunogens.SOLUTION: The coding sequences optionally include operable linked coding sequence that encodes a signal peptide in some cases. Immunomodulatory methods and methods of inducing an immune response against Ebolavirus are disclosed. Method of preventing Ebolavirus and methods of treating individuals infected with Ebolavirus are disclosed. Consensus Ebolavirus proteins are disclosed.SELECTED DRAWING: Figure 1A-1C

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 332,372, filed May 5, 2016, U.S. Provisional Application No. 62 / 402,519, filed September 30, 2016, and U.S. Provisional Application No. 62 / 483,979, filed April 11, 2017, each of which is incorporated by reference in its entirety.

[0002] The present invention relates to vaccines for inducing an immune response, preventing filovirus infection and / or treating individuals infected with filoviruses, in particular Ebola virus.The present invention relates to consensus Ebola virus proteins and nucleic acid molecules encoding same. [Background technology]

[0003] The Filoviridae family is a non-segmented, single-stranded RNA virus that comprises two distinct genera, Marburg virus (MARV) and Ebola virus (EBOV). Each member can cause a severe and highly fatal hemorrhagic fever disease for which no curative or licensed vaccine exists (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;andTowner JS,et al.(2006)Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. J Virol 80:6497-6516).

[0004] With a case fatality rate of up to 90%, these diseases are said by the World Health Organization (WHO) to be "among the most virulent viral diseases known to mankind". The US Centers for Disease Control and Prevention classifies them as "Category A bioterrorism weapons" because they pose a certain potential threat to national security if weaponized (Burki TK (2011) USA focuses on Ebola vaccine but research gaps remain. Lancet 378:389). These "high priority" weapons could theoretically be easily transmitted, cause high mortality, have a significant impact on public health, cause panic, and require special measures for public health preparedness (CDC (2011) Bioterrorism Agents / Diseases. Atlanta: Centers for Disease Control and Prevention).

[0005] Hemorrhagic fevers are acute infectious diseases that can be easily transmitted between humans and non-human primates by direct contact with infected body fluids, blood, and tissues, but no carrier state exists (Feldmann H.,et al.(2003)Ebola virus:from discovery to vaccine. Nat Rev Immunol 3:677-685). In outbreak situations, reuse of medical equipment, limited-resource medical facilities, and untimely application of preventive measures can increase disease transmission and lead to amplification of infections in the medical environment.

[0006] The natural reservoir of these zoonotic pathogens is likely to be 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 likely being a more amplifying host, and thus the manner in which the virus first emerges at the onset of an outbreak is through human contact with infected animals. The unpredictable emergence of endemic disease in the Philippines, potentially in Europe, and primarily in Africa is an additional major public health concern (Outbreak news.(2009)Ebola Reston in pigs and humans,Philippines. Wkly Epidemiol Rec 84:49-50).

[0007] Experiments are being conducted to measure the protective efficacy and the ability of the vaccine to induce broad CTL, including experiments in rodent preclinical trials (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 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 LA, Schmaljohn CS (2011).Progress in recombinant DNA-derived vaccines for Lassa virus and filoviruses. Virus Res 162:148-161)。.

[0008] 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 have shown promise, but these include primarily recombinant adenoviruses and vesicular stomatitis viruses. Non-infectious strategies such as recombinant DNA and Ag-conjugated virus-like particle (VLP) vaccines have also demonstrated levels of preclinical efficacy and are generally considered safer than virus-based platforms. Virus-specific antibodies, when administered passively, can be protective when administered either before or shortly after infection (Gupta M, Mahanty S, Bray M, Ahmed R, Rollin PE (2001).Passive transfer of antibodies protects immunocompetent and immunodeficient 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 the efficacy was largely related to the lytic function of adoptively transferred CD8+ T cells. However, detailed analysis of this response elicited by protective vaccines has rarely been reported.

[0009] Development of countermeasures will ultimately require an improved understanding of protective immune correlates and how they are modulated during infection. This has proven difficult when infected individuals with filovirus diseases are unable to achieve early immune responses. These rapid hemorrhagic diseases result in immune dysregulation, as indicated by the lack of virus-specific Ab responses and a large reduction in total T cell numbers, leading to uncontrolled viral replication and multi-organ infection and failure. Conversely, survivors of Ebola virus (EBOV) disease show an early and transient IgM response, followed rapidly by increasing levels of virus-specific IgG and CTL. These findings suggest that humoral and cellular immune responses play a role in conferring protection against 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, mounting evidence indicates that T cells play a key role in providing protection, with potency largely linked to the functional phenotype of CD8+ T cells. Although these recent studies highlight the importance of T cells in providing protection, their exact contribution remains uncharacterized and controversial, and detailed analysis of this response with protective vaccines has rarely been reported. Summary of the Invention

[0010] Provided is an isolated nucleic acid molecule comprising one or more nucleic acid sequences encoding a first consensus Zaire Ebolavirus envelope glycoprotein immunogen (ZEBOVCON), a nucleic acid encoding a second consensus Zaire Ebolavirus envelope glycoprotein immunogen (ZEBOVCON2), or a nucleic acid encoding a ZEBOV Guinea 2014 outbreak envelope glycoprotein immunogen (ZEBOVGUI).

[0011] In one embodiment, ZEBOVCON includes an amino acid sequence that is at least 95% homologous to SEQ ID NO:1, a fragment of an amino acid sequence that is at least 95% homologous to SEQ ID NO:1, an amino acid sequence that is at least 99% homologous to SEQ ID NO:1, a fragment of an amino acid sequence that is at least 99% homologous to SEQ ID NO:1, the amino acid sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1.

[0012] In one embodiment, ZEBOVCON2 includes an amino acid sequence at least 95% homologous to SEQ ID NO:68, a fragment of an amino acid sequence at least 95% homologous to SEQ ID NO:68, an amino acid sequence at least 99% homologous to SEQ ID NO:68, a fragment of an amino acid sequence at least 99% homologous to SEQ ID NO:68, the amino acid sequence of SEQ ID NO:68, or a fragment of SEQ ID NO:68.

[0013] In one embodiment, ZEBOVGUI includes an amino acid sequence that is at least 95% homologous to SEQ ID NO:67, a fragment of an amino acid sequence that is at least 95% homologous to SEQ ID NO:67, an amino acid sequence that is at least 99% homologous to SEQ ID NO:67, a fragment of an amino acid sequence that is at least 99% homologous to SEQ ID NO:67, the amino acid sequence of SEQ ID NO:67, or a fragment of SEQ ID NO:67.

[0014] In some embodiments, the fragment comprises at least 600 amino acids, at least 630 amino acids, or at least 660 amino acids.

[0015] In one embodiment, ZEBOVCON is linked to an IgE signal peptide. In one embodiment, ZEBOVCON2 is linked to an IgE signal peptide. In one embodiment, ZEBOVGUI is linked to an IgE signal peptide.

[0016] In one embodiment, the nucleic acid encoding ZEBOVCON comprises a nucleic acid sequence at least 95% homologous to SEQ ID NO: 69, or a fragment thereof. In one embodiment, the nucleic acid encoding ZEBOVGUI comprises a nucleic acid sequence at least 95% homologous to SEQ ID NO: 72, or a fragment thereof. In one embodiment, the nucleic acid encoding ZEBOVCON2 comprises a nucleic acid sequence at least 95% homologous to SEQ ID NO: 70, or a fragment thereof.

[0017] In one embodiment, the nucleic acid encoding ZEBOVCON includes a nucleic acid transcribed from a DNA sequence at least 95% homologous to SEQ ID NO: 69, or a fragment thereof. In one embodiment, the nucleic acid encoding ZEBOVGUI includes a nucleic acid transcribed from a DNA sequence at least 95% homologous to SEQ ID NO: 72, or a fragment thereof. In one embodiment, the nucleic acid encoding ZEBOVCON2 includes a nucleic acid transcribed from a DNA sequence at least 95% homologous to SEQ ID NO: 70, or a fragment thereof.

[0018] Also provided are compositions comprising one or more nucleic acid sequences encoding one or more of ZEBOVCON, ZEBOVCON2, and ZEBOVGUI. In one embodiment, the composition comprises two or more nucleic acid sequences encoding two or more of ZEBOVCON, ZEBOVCON2, and ZEBOVGUI. In one embodiment, the composition comprises two nucleic acid molecules. In one embodiment, the composition comprises three or more nucleic acid sequences encoding ZEBOVCON, ZEBOVCON2, and ZEBOVGUI. In one embodiment, the composition comprises three nucleic acid molecules.

[0019] The present invention also provides novel sequences for producing immunogens in mammalian cells or viral vectors.

[0020] 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 includes 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 generally 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 generally 600 or more, 630 or more, or 660 or more amino acid residues in length. The amino acid sequence of the consensus Sudan Ebola virus envelope glycoprotein immunogen comprises 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 generally 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 generally 600 or more, 630 or more, or 660 or more amino acid residues in length. 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 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 an amino acid sequence homologous to SEQ ID NO:3 is typically 600 or more, 637 or more, or 670 or more amino acids in length. The amino acid sequence may optionally include a leader sequence, such as an IgE leader.

[0021] 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 virus 1 consensus envelope glycoprotein immunogen, a nucleic acid sequence encoding a Marburg virus 2 consensus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a Marburg virus 3 consensus envelope glycoprotein immunogen. The amino acid sequence of the consensus Zaire Ebola virus envelope glycoprotein immunogen can 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 generally 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 generally 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 generally 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 generally 600 or more, 630 or more, or 660 or more amino acids long. The amino acid sequence of the Marburg Marburg virus first 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 generally 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 generally 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 generally 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 generally 600 or more, 637 or more, or 670 or more amino acids long. The amino acid sequence of the Marburg virus 3 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 generally 95% or more, 96% or more, 97% or more, 99% or more, or 99% or more homologous to SEQ ID NO:6. A fragment of SEQ ID NO:6 or a fragment of an amino acid sequence homologous to SEQ ID NO:6 is generally 600 or more, 637 or more, or 670 or more amino acids in length. 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 generally 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 generally 600 or more, 637 or more, or 670 or more amino acids in length.The amino acid sequence may optionally include a leader sequence, such as the IgE leader.

[0022] Also provided are compositions comprising a nucleic acid sequence encoding a consensus Ebola virus envelope glycoprotein immunogen and a nucleic acid sequence encoding a consensus Ebola virus envelope glycoprotein immunogen. The amino acid sequence of the consensus Ebola virus envelope glycoprotein immunogen can 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 generally 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 generally 600 or more, 630 or more, or 660 or more amino acid residues in length. 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 generally 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 generally 600 or more, 630 or more, or 660 or more amino acids in length. The amino acid sequence may optionally include a leader sequence, such as an IgE leader.

[0023] 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 nucleic acid sequence homologous to SEQ ID NO:64. A nucleic acid sequence homologous to SEQ ID NO:64 is generally 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 generally encodes a consensus Zaire Ebola virus envelope glycoprotein immunogen of 600 residues or more, 630 residues or more, or 660 residues or more encoded by SEQ ID NO:64. Nucleic acid sequences encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen include 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 nucleic acid sequence homologous to SEQ ID NO:65. A nucleic acid sequence homologous to SEQ ID NO:65 is generally 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 generally encodes amino acids of the consensus Sudan Ebola virus envelope glycoprotein immunogen of 600 residues or more, 630 residues or more, or 660 residues or more 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 nucleic acid sequence homologous to SEQ ID NO:66. A nucleic acid sequence that is homologous to SEQ ID NO:66 is generally greater than 95%, greater than 96%, greater than 97%, greater than 99%, or greater than 99% homologous to SEQ ID NO:66.Fragments of SEQ ID NO: 66 or fragments of amino acid sequences homologous to SEQ ID NO: 66 generally encode amino acids of the Marburg virus Angola 2005 envelope glycoprotein immunogen of 600 or more, 630 or more, or 670 or more residues 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.

[0024] Each of the different nucleic acid sequences may be on a single nucleic acid molecule, each may be on a separate nucleic acid molecule, or in various permutations. The nucleic acid molecule may be a plasmid.

[0025] The compositions may be formulated for delivery to an individual using electroporation.

[0026] 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.

[0027] The composition may be used in a method of inducing an immune response against a filovirus, which may be selected from the group consisting of: Marburg virus, Ebola virus Sudan, and Ebola virus Zaire.

[0028] A method of treating an individual diagnosed with a filovirus is provided comprising administering to the individual a therapeutically effective amount of a composition, the filovirus being selected from the group consisting of Marburg virus, Ebola virus Sudan, and Ebola virus Zaire.

[0029] A method for preventing filovirus infection in an individual is provided, the method 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.

[0030] The present invention provides a composition comprising 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]

[0031] [Figure 1A-1C] Multivalent vaccine construction strategy and expression experiment in Example 1 are shown. Phylogenetic trees of MGP (top), SGP (bottom right), and ZGP (bottom left) are shown. Significant support values ​​are indicated (*) as verified by bootstrap analysis. A consensus strategy was adopted for ZGP and SGP immunogens (CON VACCINE). Scale bars indicate the distance of amino acids per site, and analysis was performed using MEGA version 5 software. GP transgenes were commercially synthesized, gene optimized, and subcloned into modified pVAX1 mammalian expression vectors. After transfection of HEK293T cells, antigen expression was analyzed by Western immunoblotting and FACS. Western immunoblotting results are shown in Figure 1B, and FACS results are shown in Figure 1C. As a comparative control, rVSV expressing MGP, SGP, or ZGP were run simultaneously with each GP sample, and species-specific anti-GP1 mAb was used for detection. Sizes are shown in (kDa). FACS results are shown in Figure 1C. For FACS, transfected cells were indirectly stained with mouse-derived GP-specific serum reagents, followed by extensive washing and staining with goat anti-mouse IgG and MHC class I. Western immunoblotting and FACS experiments were repeated at least three times with similar results. The significance of the unrooted phylogenetic trees was determined by maximum likelihood and verified by bootstrap analysis, with significant support values ​​(≥80%; 1,000 bootstrap replicates) determined by MEGA version 5 software. [Figure 2A-2H] Results of the experiment in Example 1 are shown in which complete protection against MARV and ZEBOV challenge was observed. Animal survival data is shown in Figures 2A and 2E. Figure 2A shows that trivalent vaccinated animals survived MARV challenge while all control animals died by day 10. Figure 2E shows that trivalent vaccinated animals survived ZEBOV challenge while all control animals died by day 7. Percent weight change data for vaccinated and control animals when challenged with MARV versus vaccination is shown in Figure 2B. The y-axis shows weight change as shown in Figure 2F. The light solid line shows trivalent vaccinated animals. The light dashed line shows TriAVE, the average result of trivalent vaccinated animals. The dark solid line shows control animals. The dark dashed line shows ControlAVE, the average result of control animals. The light solid and light dashed lines remain stable on the graph on the days following challenge, showing no significant weight loss among vaccinated animals. The dark solid and black dashed lines on the graph from days 0 to 9 following challenge ending with dagger show animals that died of disease by day 10. Data on percent weight change for vaccinated and control animals upon challenge with ZEBOV versus vaccination is shown in FIG. 2F. The y-axis shows weight change in %. The light solid line shows trivalent vaccinated animals. The light dashed line shows TriAVE, the average result for trivalent vaccinated animals. The dark solid line shows control animals. The dark dashed line shows ControlAVE, the average result for control animals. The light solid and light dashed lines remained stable on the graphs on the days following challenge, indicating no significant weight loss among vaccinated animals. The dark solid and black dashed lines on the graphs for days 0-6 following challenge terminated with daggers indicate animals that died of disease by day 8 (n=3 for gpMARV and n=6 for gpZEBOV). Binding Abs (FIG. 2C and FIG. 2G) and NAbs (FIG. 2D and FIG. 2H) were measured in sera from vaccinated animals before and after the first (1×) and second (2×) vaccination. Analysis was performed on pooled sera. *p<0.1;***p<0.001;****p<0.0001. [Figure 3A-3C]Results from Example 1 showing the induction of neutralizing antibodies are shown. B cell responses were evaluated in mice (n=5 / group) 20 days after each of two vaccinations with 40 μg E-DNA vaccination, 3 weeks apart. Figure 3A shows serum GP-specific IgG responses from vaccinated (solid line) or pre-bled (dotted line) mice measured by ELISA. Data are summarized in Figure 3B. All responses from animals immunized with pEBOS and pEBOZ were measured against sucrose-purified ZGP, as SGP was not available for this study. IgG responses from pMARV-immunized mice were measured against MARV-Ozolin GP or negative control sucrose-purified Nipah G protein. Neutralizing activity of serum samples was measured against ZEBOV-EGFP, SUDV-Boniface and MARV-Angola in a BSL-4 facility, and NAb titers are shown in Figure 3C. NAbs against SUDV-Boniface were assayed based on cytopathic effect (CPE) on CV-1 cells, and NAbs against MARV-Angola were assayed using an immunofluorescence assay. Means are shown in Figures 3B and 3C, error bars represent SEM. Analysis of groups 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-4D]The broad T cell responses generated by vaccination are shown. H-2b (light bars) and H-2d (dark bars) mice (n=5 / group) were immunized twice with either pMARV, pEBOS or pEBOZ DNA and IFNγ responses were measured by IFNγ ELISPOT assay. Splenocytes harvested 8 days after the second immunization were incubated in the presence of individual GP peptides (15-mers with 9 amino acid overlap) and results are shown in stacked bar graphs. Epitope-containing peptides were identified (≥10 AVE spots and ≥80% response rate) that were confirmed by flow cytometry and characterized populations of total activated IFNγ+ and CD44+ CD4+ and / or CD8+ T cells (Tables 1-6) and peptide numbers of positive inducers are shown above the bars. Peptides containing only CD4+ epitopes, only CD8+ epitopes (*) and dual CD4+ and CD8+ epitopes (**) are numbered. Putative shared and / or partial epitopes were examined for consecutive positive peptide responses (Tables 1-6). Figure 4B shows the broad T cell responses generated by vaccination. An amino acid similarity plot comparing the GP sequences from MARV, SUDV, and ZEBOV viruses shown in Figure 1A is shown. Figure 4C shows the broad T cell responses generated by vaccination. Putative domains within ZEBOV GP (GenBank#VGP_EBOZM) are shown. SP, signal peptide; RB, receptor binding; MUC, mucin-like region; FC, furin cleavage site; TM, transmembrane region. In Figure 4D, the broad T cell responses generated by vaccination are shown. Each subdominant (dark shading) and immunodominant (light shading) T cell epitope response is 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-5D]Data from an experiment evaluating protective "single dose" vaccination-elicited neutralizing Abs and CTLs are shown. H-2k mice (n=10 / group) were vaccinated once intramuscularly with pEBOZ E-DNA and then challenged 28 days later with mEDBOV 1,000 LD50 in a BSL-4 facility. Mice were weighed daily to monitor disease progression. Animal survival data in Figure 5A. Vaccinated animals survived the challenge, while control animals died by day 7. Figure 5B shows data on percent weight change in challenged animals. Data from immunized animals are shown in solid lines; mean data from immunized animals are shown in dashed lines. Data from control animals are shown in solid lines; mean data from control animals are shown in dashed lines. The light solid and light dashed lines remain stable within approximately 85%-120% range on the graphs the days after challenge, indicating no significant weight loss among vaccinated animals. Dark solid and dark dashed lines on graphs of days 0-6 post-challenge terminated with daggers indicate animals that died of disease by day 7. Nabs measured before challenge; data shown in Figure 5C. T cell responses after a single pEBOZ immunization measured by FACS are summarized as AVE% of total CD44+ / IFNγ+ CD4+ (dark) or CD8+ (light) cells in Figure 5D. Th1-type effector markers were assessed (TNF and T-bet) and CD44+ / IFNγ+ CD4+ and CD8+ T cell data were compared to total T cell data 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 repeated twice with similar results. Error bars represent SEM. [Figure 6] FIG. 1 shows GP-specific T cell gating as disclosed in Example 1. [Figure 7A-7B]1 shows that the vaccination experiment of Example 1 generated robust T cells. [Figure 8A-8B] FIG. 1 shows T cell induction by "single dose" vaccination as disclosed in Example 1. [Figure 9] Vaccination strategies against Ebola are presented. Ebola viral glycoproteins are the primary target for vaccines. Currently, three vaccines are currently in clinical trials that are protective, immunogenic, and have single-dose protection in non-human primates (NHPs). However, these vaccines have the potential to develop anti-vector immunity, have shown adverse reactions in human clinical trials, have uncertain duration of memory responses, and may not be suitable for all populations. [Figure 10] FIG. 7 shows EBOV glycoprotein vaccine construction and formulation strategies and expression experiments. [Figure 11] 1 shows the experimental results of Example 7 demonstrating that a single immunization of a DNA vaccine is immunogenic in mice. [Figure 12] 1 shows the results of Example 7 demonstrating that a single immunization is fully protective against a lethal mouse-adapted Ebola virus challenge in mice. [Figure 13] 1 shows the results of Example 7, demonstrating that individual GP DNA vaccine constructs induce strong memory responses in mice. [Figure 14] 1 shows the results of Example 7 demonstrating the efficacy of a GP DNA vaccine in non-human primates. [Figure 15] 1 shows the results of Example 7 demonstrating that the GP DNA vaccine formulation is immunogenic in NHPs. [Figure 16] 1 shows the results of Example 7 demonstrating that a GP DNA formulation vaccine protects against a lethal Zaire Ebola virus (Makona) challenge. [Figure 17] The EBOV-001 Phase I clinical trial (NCT02464670) strategy is shown. [Figure 18] 1 shows the results of Example 7 demonstrating EBOV-001 seroconversion in a Phase I clinical trial. [Figure 19]1 shows the results of Example 7 demonstrating the induction of Ebola GP-specific T cell responses (ELISpot) in a representative patient. [Figure 20] 1 shows the results of Example 7, which shows a comparison with other Ebola vaccine platforms currently in clinical trials. [Figure 21] 1 shows a description of the cohorts and subject demographics of the cohorts for the ELISA experiment of Example 8. [Figure 22] 1 shows the results of Example 8 showing ELISA titers by cohort and time points for cohorts 1-3. [Diagram 23] 1 shows the results of Example 8 showing ELISA titers by cohort and time points for cohorts 4 and 5. [Figure 24] 1 shows the results of Example 8 showing the ELISA summary for all cohorts. [Diagram 25] 1 shows a description of the cohorts and subject demographics of the cohorts for the ELISpot experiment of Example 8. [Figure 26] 1 shows the results of Example 8 showing subject responses to peptide pools from Cohort 1. [Figure 27] 1 shows the results of Example 8 showing subject responses to peptide pools from Cohort 2. [Figure 28] 1 shows the results of Example 8 showing subject responses to peptide pools from Cohort 3. [Figure 29] 1 shows the results of Example 8 showing subject responses to peptide pools from Cohort 4. [Diagram 30] 1 shows the results of Example 8 showing subject responses to peptide pools from Cohort 5. [Diagram 31] 1 shows the results of Example 8 showing ELISpot summary by cohort. [Diagram 32] 1 shows the results of Example 8 showing ELISpot summary by cohort. [Diagram 33] 1 shows the results of Example 8 showing an analysis of vaccine responders versus all subjects. [Diagram 34] 1 shows the results of Example 8 showing an analysis of vaccine responders for subjects with baseline outliers removed. [Diagram 35] 1 shows a description of the cohorts and subject demographics of the cohorts for the ICS study of Example 8. [Diagram 36] 1 shows the results of Example 8 showing a cohort-based Wilcoxon pairwise analysis of the ICS study. [Figure 37] FIG. 1 shows the results of Example 8 showing ICS analysis of cohort 3 (INO4201 ID) cytokines in CD4+ T cells. [Figure 38] 1 shows the results of Example 8 showing ICS analysis of Cohort 3 cytokines in CD8+ T cells. [Figure 39] 1 shows the results of Example 8 showing a detailed analysis of vaccine responders for each subject in cohorts 1-3. [Diagram 40] 1 shows the results of Example 8 showing a detailed analysis of vaccine responders for each subject in cohorts 4-5. [Diagram 41] 1 shows the results of Example 8 showing median responses by cohort and pool. [Diagram 42] 1 shows the results of Example 8 showing mean responses by cohort and pool. [Diagram 43] Results are shown showing that bivalent and trivalent DNA vaccines induce long-lasting immune responses in vivo in cynomolgus macaques. Serum and peripheral blood mononuclear cells (PBMCs) were collected 2 weeks after each DNA injection and monthly after the final injection. Total IgG endpoint titers against Guinea GP and Mayinga GP were assayed by ELISA. Cellular immune responses to Ebola GP after DNA immunization. PMBCs were collected from NHPs and assayed by ELISPOT-IFNγ for T cell responses after stimulation with pools of GP peptides. [Fig. 44A-44C]Results showing protection in cynomolgus macaques by bivalent and trivalent DNA vaccines against lethal EBOV challenge are shown. (Figure 44A): Survival. Animals were challenged with 1000 TCID50 of lethal Guinea Macauca C07 EBOV and survival was monitored for 28 days post-challenge. (Figure 44B): Clinical scores. Clinical signs of disease were monitored throughout the course of infection. (Figure 44C): Viral load. Viremia during the course of infection was assayed by TCID50 assay from blood. [Diagram 45] Quantitative ELISA assessing the amount of ZEBOV-specific IgG antibodies in the serum of each vaccinated subject at the start of the study and two weeks after each immunization using the ADI Human Anti-Ebola Zaire Virus Glycoprotein IgG Kit. Statistical analysis was performed using a two-tailed Wilcoxon signed rank test. [Figure 46] Interferon gamma ELISpot responses are shown. At study entry and 2 weeks after each immunization, PBMCs were isolated from vaccinated subjects and stimulated with Ebola peptides spanning the full-length GP in an IFNg ELISpot assay. The graph represents EBOV-GP-specific spot-forming units per million PBMCs. The line within the box represents the median response. Individual dots represent outlier data. [Figure 47] Experimental results are presented demonstrating long-term immunogenicity with an Ebola GP DNA vaccine formulation in non-human primates (NHPs), with robust antibody responses observed for more than 6 months after vaccination. [Figure 48]

[0036] Figure 2 shows a memory study of intramuscular delivery of EBOV GP DNA vaccine. NHPs were immunized with bivalent or trivalent EBOV GP DNA vaccine formulations over a 3-month period. Immune responses were followed for 12 months after the last dose. A 1-year booster was administered at month 13. [Figure 49] Figure 1 shows total IgG endpoint titers in animals administered the trivalent DNA GP formulation. An increase in total IgG antibody response was observed after one year of boosting. [Figure 50]Figure 1 shows total IgG endpoint titers in animals administered bivalent or trivalent DNA GP formulations. Increased total IgG antibody responses were observed after one year of boosting. [Figure 51] ELISPOT of trivalent DNA GP formulation. Increased IFNγ ELISPOT response after 1 year booster. Booster strength is not as high as in the 3 injection group. [Figure 52] ELISPOT of bivalent or trivalent DNA GP formulations. Increased IFNγ ELISPOT response after 1 year boost. Highly potent boost in single immunization group. [Figure 53] A summary of IFNγ ELISPOT results is shown. After the boost, 17 / 19 animals increased their T cell response. 1 / 19 animals maintained the same level of T cell response. 1 / 19 animals had a poor T cell response after the boost, but this animal had a consistently high T cell response over the past year in the 3 injection groups. [Figure 54] An exemplary IFNγ ELISPOT for animals in the single immunization group at 12 months prior to the 1-year boost is shown. [Figure 55] An exemplary IFNγ ELISPOT for animals in the single immunization group after one year of boosting is shown. [Figure 56] Results of an IM memory study are shown. The EBOV GP DNA vaccine induces a long-lasting immune response with robust recall after a one-year boost. An intradermal delivery study is performed to test the immune response of the intradermal EBOV GP DNA vaccine. [Figure 57] An overview of the EBOV DNA vaccine clinical trial (EBOV-001) is shown. Healthy volunteers will receive a three-dose regimen of INO-4201, INO-4202, INO-4212, or INO-4212 and INO-9021. [Figure 58] A comparison of binding antibodies between EBOV-001 and rVSV EBOV is shown. All EBOV-001 cohorts showed a significant increase at both weeks 6 and 14 compared to week 0. [Figure 59]

[0036] Figure 1 shows an overview of the intradermal delivery of the EBOV GP DNA vaccine trial, with additional cohorts to explore dosing, dosing regimens, and the use of IL-12 DNA as an immune adjuvant. [Figure 60] Results are shown for the ID cohort at week 14. Seroreactivity was observed in 125 / 127 (98.4%) subjects in all ID cohorts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one aspect of the invention, the consensus antigen desirably provides improved transcription and translation, including having one or more of the following: a low GC content leader sequence to increase transcription; mRNA stability and codon optimization; removal of possible cis-acting sequence motifs (i.e., internal TATA boxes).

[0033] In some embodiments of the invention, it is desirable to generate consensus antigens that generate broad immune responses across multiple strains, including having one or more of the following: incorporate all available full-length sequences; computer generate sequences that utilize the most commonly occurring amino acids at each position; and increase cross-reactivity between strains.

[0034] Diversity among the Filoviridae is relatively high. Intensive efforts have been made aiming to develop a universal and broadly reactive filovirus vaccine that would ideally protect against multiple species that cause the highest human mortality. However, this has proven difficult due to the relative diversity of the Filoviridae. EBOV is currently classified into five different species: Zaire Ebolavirus (ZEBOV), Sudan Ebolavirus (SUDV), Reston Ebolavirus (RESTV), Bundibugyo Ebolavirus (BDBV) and Tai Forest Ebolavirus (TAFV; formerly Côte d'Ivoire Ebolavirus), with the first two being candidates with the highest lethality and the highest potential to be weaponized. Diversity among Marburg viruses (MARV) is low, and they can also range in lethality up to 90%. Currently, there is only one taxonomic species, Marburg Marburgvirus (formerly Lake Victoria Marburgvirus), but recent revisions have proposed to include two more, including Rabinvirus (RAVV). Adding to the complexity of multivalent vaccine development, MARV and EBOV are highly diverse, with approximately 67% diversity at the nucleotide level. Furthermore, the phylogenetic diversity of filovirus GPs is also very high (overall 82%). These suggest the possibility of evolution of filoviruses, as shown by the recent emergence of BDBV in 2007. Thus, due to the relative diversity that exists among Filoviridae, it seems that the development of an effective multivalent filovirus vaccine will require a mixture of immunogenic components.

[0035] We developed a synthetic polyvalent filovirus DNA vaccine against Marburg virus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV).The novel polyvalent filovirus vaccine employs a multidrug approach and is composed of three DNA plasmids encoding the envelope glycoprotein (GP) genes of Marburg virus (MARV), Sudan ebolavirus (SUDV), or Zaire ebolavirus (ZEBOV). As filovirus vaccine candidates, DNA (DNA)-based enhanced platforms show many advantages due to 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 gene optimized and subcloned into a modified mammalian expression vector and then delivered using in vivo electroporation (EP).

[0036] Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, while murine T cell responses were extensively analyzed using the novel modified assays described herein. T cell responses were extensively analyzed, including the use of novel methods for epitope identification and characterization described herein. This model provides an important preclinical tool for testing protective immune correlates that can be applied to existing platforms.

[0037] Vaccination in preclinical rodent studies elicited potent and robust neutralizing antibodies (NAb) and CTL expressing T h The vaccine induced type 1 markers and was fully protective against MARV and ZEBOV challenge. A comprehensive T cell analysis (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) using the novel modified assay described herein demonstrated a large number of cytotoxic T lymphocytes, epitope breadth, and T h Representing type 1 marker expression. A total of 52 novel T cell epitopes were identified (19 of 20 MARV epitopes, 15 of 16 SUDV epitopes, and 18 of 22 ZEBOV epitopes) from two mice of different genetic backgrounds, which mainly occurred in highly conserved regions of the respective glycoproteins (GPs). These data represent the most comprehensive report of preclinical glycoprotein epitopes to date.

[0038] In formulating a strategy to provide protection against multiple species with the highest human mortality, we focused on MARV, SUDV, and ZEBOV. Due to their relative diversity, we hypothesized that the development of a multivalent filovirus vaccine would require a cocktail of components that can be rapidly and easily adapted depending on future outbreak strains and / or species. Although the overall diversity of EBOV is approximately 33%, the amino acid identity rises substantially when SUDV and ZEBOV are analyzed separately (approximately 94% identity within each species). Therefore, as shown in Figure 1A, we designed a two-component strategy to cover the most lethal EBOV, a plasmid GP vaccine for SUDV and another plasmid GP vaccine for ZEBOV. Because the GP diversity between each species is relatively low (5.6% for SUDV and 7.1% for ZEBOV), we developed a consensus immunogen, a strategy previously shown to enhance protection between diverse lineages of influenza and HIV. These GP sequences were consensus for all reported outbreak sequences (GenBank) as determined by alignment with Vector NTI software (Invitrogen, CA, USA). Four amino acid non-consensus residues 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 had the highest human mortality of any Filoviridae outbreak (n=425), while Mbomo / Mbanza was selected because it was the most recent and deadly outbreak with published sequence data. The consensus GPs for SUDV (SUDV CON VACCINE) and ZEBOV (ZEBOV CON VACCINE) were strains aligned phylogenetically intermediate to their parent.

[0039] Identification of proteins in Figure 1A are 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_EBOZ 5;Yambuku(Ekron)'76:VGP_EBOEC;Yambuku(Mayinga)'76:VGP_EBOZM;Kasai'08:AER59712;Kassai'07:AER59718;Etoumbi'05:ABW34742;Mbomo / Mbandza'03:ABW34743.

[0040] The sequence listing provided herein includes a list of 72 sequences, including:

[0041] SEQ ID NO:1 is the amino acid sequence of the consensus Zaire Ebola virus envelope glycoprotein immunogen, ZEBOV CON (CONGP1).

[0042] SEQ ID NO:2 is the amino acid sequence of SUDV CON, which is the consensus Sudan Ebola virus envelope glycoprotein immunogen.

[0043] SEQ ID NO:3 is the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein and the amino acid sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen, MARV or MARV ANG.

[0044] SEQ ID NO:4 is the amino acid sequence of MARV CON1, which is the first consensus Marburg virus envelope glycoprotein immunogen.

[0045] SEQ ID NO:5 is the amino acid sequence of MARV CON2, which is the second consensus Marburg virus envelope glycoprotein immunogen.

[0046] SEQ ID NO:6 is the amino acid sequence of MARV CON3, which is the third consensus Marburg virus envelope glycoprotein immunogen.

[0047] SEQ ID NOs:7 to 25 are peptides derived from MARV ANG.

[0048] SEQ ID NOs: 26 to 41 are peptides derived from SUDV CON.

[0049] SEQ ID NOs: 42 to 62 are peptides derived from ZEBOV CON.

[0050] SEQ ID NO: 63 is the sequence of the IgE signal peptide: MDWTWILFLVAAATRVHS.

[0051] SEQ ID NO:64 is the nucleotide sequence insert in plasmid pEBOZ encoding the consensus Ebola Zaire virus envelope glycoprotein immunogen.

[0052] SEQ ID NO:65 is the nucleotide sequence insert in plasmid pEBOS encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

[0053] SEQ ID NO:66 is the nucleotide sequence insert in plasmid pMARZ ANG encoding the Marburg virus Angola 2005 envelope glycoprotein.

[0054] SEQ ID NO:67 is the amino acid sequence of ZEBOVGUI (Guinea GP), which is the consensus Zaire Ebolavirus envelope glycoprotein immunogen isolated from the 2014 outbreak in Guinea.

[0055] SEQ ID NO:68 is the amino acid sequence of ZEBOVCON2 (CONGP2), which is the second consensus Zaire Ebola virus envelope glycoprotein.

[0056] SEQ ID NO:69 is the nucleotide sequence insert in plasmid pZEBOVGUI encoding the consensus Zaire Ebolavirus envelope glycoprotein immunogen isolated from the 2014 outbreak in Guinea.

[0057] SEQ ID NO:70 is the nucleotide sequence insert of plasmid pEBOZCON2 encoding the second consensus Zaire Ebola virus envelope glycoprotein.

[0058] SEQ ID NO:71 is the nucleotide sequence of plasmid pEBOZCON2 encoding the second consensus Zaire Ebola virus envelope glycoprotein.

[0059] SEQ ID NO:72 is the nucleotide sequence insert of plasmid pZEBOVGUI encoding the Zaire Ebola virus envelope glycoprotein immunogen isolated from the 2014 outbreak in Guinea.

[0060] In some embodiments, the strategy uses coding sequences of three filovirus immunogens selected from: MARV, SUDV, ZEBOV, ZEBOVGUI, and ZEBOVCON2. The MARV immunogen is the glycoprotein of the Angola 2005 isolate. The SUDV ZEBOV, ZEBOVGUI, and ZEBOVCON2 consensus glycoprotein sequences were designed.

[0061] In some embodiments, the strategy uses the coding sequences of five filovirus immunogens. Three MARV immunogens are provided. The consensus glycoproteins Ozolin, Musoke, or Rabin from the three clusters are designed. These three MARV immunogens are targets of immune response together with the designed SUDV, ZEBOV or ZEBOVCON2, ZEBOVGUI consensus glycoprotein sequences.

[0062] In some embodiments, the strategy uses coding sequences of six filovirus immunogens. Four MARV immunogens were provided and three consensus glycoproteins derived from three clusters were designed. These three MARV immunogens are targets of the immune response along with the designed SUDV, ZEBOV and ZEBOVCON2, ZEBOVGUI consensus glycoprotein sequences, the MARV immunogens being glycoproteins of the Angola 2005 isolate.

[0063] As a filovirus vaccine candidate, DNA vaccines show numerous advantages, including rapid and inexpensive upscale production, stability at room temperature, and ease of transportation, all of which further strengthen this platform from an economic and geographical standpoint. Due to the synthetic nature of the plasmids, Ag sequences can be rapidly and easily modified in response to newly emerging species and / or expanded to include additional vaccine components and / or regimens for rapid response under outbreak situations. For example, the MARV strategy herein can be easily expanded for broader coverage by co-administration of additional plasmids encoding consensus MARV GP (MGP) immunogens against other phylogenetic clusters.

[0064] Although "first generation" DNA vaccines have low immunogenicity, recent technological advances have dramatically improved immunogenicity in clinical trials. Optimization of plasmid DNA vectors and the Ag genes they encode has led to increased in vivo immunogenicity. When highly concentrated plasmid vaccine formulations are administered with in vivo electroporation, a technique in which plasmids are driven into transiently permeabilized cells using brief square-wave electrical pulses within the vaccination site, cellular uptake and subsequent Ag expression are substantially amplified. In theory, cocktails of DNA plasmids can be assembled to direct highly specialized immune responses to any number of variable Ags. Immunity can be further induced by co-delivery of plasmid molecular adjuvants encoding species-specific cytokine genes and "consensus engineering" of Ag amino acid sequences to help direct vaccine-induced immunity against specific strains. This strategy has been shown to enhance protection among diverse lineages of influenza virus and HIV. In part due to these technological advances, immunizations involving these DNA vaccines are highly versatile and highly customizable.

[0065] 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 this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0066] For the purposes of reciting numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision, for example, in the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range of 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.

[0067] Adjuvant As used herein, "adjuvant" can mean any molecule added to a DNA plasmid vaccine described herein to enhance the antigenicity of one or more consensus filovirus immunogens encoded by the DNA plasmid and encoding a nucleic acid sequence as described below.

[0068] b.Antibodies "Antibody" may refer to antibodies of the classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, as well as single chain, bispecific, diabody, bivalent, 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 a desired epitope or a sequence derived therefrom.

[0069] C. coding sequence As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) that comprises nucleotides that code for a protein. A coding sequence may also include a DNA sequence that codes for an RNA sequence. The coding sequence may further include start and stop signals operably linked to regulatory elements including a promoter and a polyadenylation signal that are 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 include an initiation codon that optionally codes for an N-terminal methionine or a signal peptide, such as an IgE or IgG signal peptide.

[0070] D. Genetic constructs As used herein, a "genetic construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that encodes a protein, such as an immunogen. The genetic construct may also refer to a DNA molecule transcribed from an RNA molecule. The coding sequence includes start and stop signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, that are capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that contains the necessary regulatory elements operably linked to a coding sequence that encodes a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0071] e. complement As used herein, "complement" or "complementary" can mean that a nucleic acid exhibits Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0072] f. consensus or consensus sequence As used herein, "consensus" or "consensus sequence" can refer to a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of the alignment of multiple subtypes of a particular filovirus antigen, which can be used to induce broad immunity against a particular filovirus antigen subtype or serotype.

[0073] 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 may 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 contains SEQ ID NO:64.

[0074] 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 may 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.

[0075] The Marburg virus Angola 2005 envelope glycoprotein is not a consensus, but a protein sequence derived from an isolate. This protein has the sequence of SEQ ID NO:3. The Marburg virus Angola 2005 envelope glycoprotein immunogen refers to SEQ ID NO:3, a fragment of SEQ ID NO:3, a variant of SEQ ID NO:3, and a fragment of a variant 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 may be referred to as pMARV or pMARV-ANG. The coding sequence of the Marburg virus Angola 2005 envelope glycoprotein immunogen includes SEQ ID NO:66, a fragment of SEQ ID NO:66, a variant of SEQ ID NO:66, and a fragment of a variant of SEQ ID NO:66. The plasmid pMARV ANG contains SEQ ID NO:66.

[0076] 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 from the Rabin cluster consensus (Rabin, Darva (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 may be referred to as pMARV-RAV.

[0077] 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 from the Ozolin cluster consensus (Ozolin, Uganda (01Uga07), and Dahba (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 be referred to as pMARV-OZO.

[0078] 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 of 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 may be referred to as pMARV-MUS.

[0079] Consensus Zaire Ebolavirus GP envelope glycoprotein immunogen refers to SEQ ID NO:67, fragments of SEQ ID NO:67, variants of SEQ ID NO:67 and fragments of variants of SEQ ID NO:67. (ZEBOVGUI or ZEBOVGUI VACCINE). A plasmid containing the coding sequence of SEQ ID NO:67 may be referred to as pZEBOVGUI or pEBOZGUI. Coding sequences for consensus Zaire Ebolavirus Guinea 2014 envelope glycoprotein immunogen include SEQ ID NO:69, fragments of SEQ ID NO:69, variants of SEQ ID NO:69 and fragments of variants of SEQ ID NO:69. Plasmid pEBOZGUI contains SEQ ID NO:69.

[0080] The second consensus Ebola virus Zaire envelope glycoprotein immunogen is referred to as SEQ ID NO:68, fragments of SEQ ID NO:68, variants of SEQ ID NO:68, and fragments of variants of SEQ ID NO:68. (ZEBOV2 or ZEBOVCON2 or ZEBOVCON2 VACCINE). A plasmid containing the coding sequence of SEQ ID NO:68 may be referred to as pEBOVCON2. The second consensus Ebola virus Zaire envelope glycoprotein immunogen coding sequences include SEQ ID NO:70, fragments of SEQ ID NO:70, variants of SEQ ID NO:70, and fragments of variants of SEQ ID NO:70. Plasmid pEBOVCON2 contains SEQ ID NO:70.

[0081] g. Constant current As used herein, "constant current" defines the electrical current that a tissue, or cells defining the tissue, receive or experience for the duration of an electrical pulse delivered to the tissue. The electrical pulse is delivered from an electroporation device as described herein. This current remains at a constant amount in the tissue for the life of the electrical pulse because the electroporation devices provided herein preferably include a feedback element having instantaneous feedback. The feedback element measures the resistance of the tissue (or cells) for the duration of the pulse and can cause the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current in the tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0082] h. Current feedback or feedback As used herein, "current feedback" or "feedback" are 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 varying 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 initiating the pulse sequence or electrical treatment. Feedback may be accomplished by the electroporation components of the electroporation device, e.g., a controller, such that an electrical circuit within the electroporation device continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) to a preset current, and continuously adjusts 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.

[0083] i.Distributed current As used herein, "distributed current" may refer to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein that minimizes or preferably eliminates the occurrence of electroporation-related thermal stress to any area of ​​tissue being electroporated.

[0084] j. Electroporation "Electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, may refer to the use of transmembrane electric field pulses to generate microscopic pathways (pores) in biological membranes. The presence of these microscopic pathways allows the passage of biological molecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and water, from one side of the cell membrane to the other.

[0085] k. Feedback mechanism As used herein, a "feedback mechanism" 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 energy pulse to achieve a pre-set value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0086] l.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 having 98% or greater homology to a consensus sequence described herein with or without a signal peptide and / or a methionine at position 1, a protein having 99% or greater homology to a consensus sequence described herein, and a protein having 100% homology to a consensus sequence described herein. Fragments may or may not include, for example, a fragment of a filovirus immunogen linked to a signal peptide, e.g., an immunoglobulin signal peptide, e.g., an IgE signal peptide or an IgG signal peptide.

[0087] Any fragment of ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2 or a variant thereof, in each case with or without a signal peptide and / or methionine at position 1, is intended to be complementary to the specific full length ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, MARV-MUS The fragment 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 CON, ZEBOVGUI or ZEBOVCON2 or a variant thereof. A fragment refers to a polypeptide of a fragment that is 100% identical to the sequence ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2, in each case with or without a signal peptide and / or methionine at position 1. Fragments also refer to fragments of variants, i.e. polypeptides that are 95% or more, 98% or more, or 99% or more homologous to the sequences ZEBOV CON, SUDV CON, MARV ANG, MARV-RAV CON, MARV-OZO CON, MARV-MUS CON, ZEBOVGUI or ZEBOVCON2, in each case with or without a signal peptide and / or methionine at position 1. The 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, 67-68, and may further include a signal peptide, such as an immunoglobulin signal peptide, that is not included when calculating the percentage of homology. In some embodiments, the fragments of SEQ ID NOs: 1-6, 67-68 are linked to a signal peptide, for example an immunoglobulin signal peptide, such as an IgE signal peptide or an IgG signal peptide.Fragments can include fragments of SEQ ID NOs: 1-6, 67-68 that contain 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: 1-6, 67-68. If a signal peptide is present, it is not included when calculating the percent homology.

[0088] In some embodiments, a fragment of SEQ ID NOs: 1-6, 67-68 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 NOs: 1-6 or a variant thereof. In some embodiments, a fragment of SEQ ID NOs: 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 less consecutive amino acids of any of SEQ ID NOs: 1-6, 67-68 or a variant thereof.

[0089] "Fragment" also refers to a fragment of a nucleic acid sequence encoding a filovirus immunogen, which encodes a fragment of a filovirus immunogen capable of eliciting an immune response to a filovirus in a mammal by recognizing a specific filovirus antigen. Fragments of a nucleic acid fragment encoding a filovirus immunogen or variant thereof, in each case with or without a signal peptide and / or methionine at position 1, include 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, or 99% or more of the length of the specific full-length nucleic acid sequence encoding a filovirus immunogen or variant thereof. The fragments may include fragments of nucleotide sequences encoding polypeptides that are 98% or more, 99% or more, or 100% identical to the filovirus immunogens set forth in SEQ ID NOs: 1-6, 67-68, and may further include a signal peptide, such as an immunoglobulin signal peptide, that is not included when calculating percent homology. In some embodiments, fragments of nucleotide sequences encoding fragments of SEQ ID NOs: 1-6, 67-68 are linked to a signal peptide, for example an immunoglobulin signal peptide, such as an IgE signal peptide or an IgG signal peptide. The coding sequence for the signal peptide is present but is 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 variants 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, 67-68 or variants thereof.In some embodiments, a fragment of a nucleotide sequence encoding a fragment of SEQ ID NOs:1-6, 67-68 or a variant thereof may include a sequence encoding 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 NOs:1-6, 67-68 or variants thereof.

[0090] In some embodiments, the fragment is a fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72. A fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, in each case with or without a signal peptide and / or methionine at position 1, can 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. A fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 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, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, and further includes a signal peptide, such as an immunoglobulin signal peptide, which is not included in calculating the percentage of homology. A fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 can 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 SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, or a variant thereof.A fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 may comprise a sequence encoding 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 less contiguous amino acids of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, or variants thereof. In some embodiments, the fragment is a fragment of an RNA transcribed or encoded from SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72.

[0091] m. "the same" As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences may mean that the sequences have a specified percentage of identical residues over a specified region. To calculate this percentage, the two sequences may be suitably aligned, the two sequences may be compared over a specified region, the number of positions at which identical residues occur between both sequences may be determined to give the number of matching positions, the number of matching positions may be divided by the total number of positions in the specified region, and the result may be multiplied by 100 to calculate the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more sticky ends such that only a single sequence is included in the specified comparison region, 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) may be considered equivalent. Identity may be calculated by hand calculation or may be calculated using computer sequence algorithms such as BLAST and BLAST 2.0.

[0092] Impedance "Impedance" as used herein may be utilized when discussing feedback mechanisms and may be converted to a current value according to Ohm's law, so that it can be compared to a preset current.

[0093] Immune response As used herein, "immune response" can refer to activation of a host's immune system, e.g., a mammalian immune system, in response to the introduction of a filovirus consensus antigen via one or more of the provided DNA plasmid vaccines. The immune response can be a cellular or humoral response, or both.

[0094] p.Nucleic acid As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to at least two nucleotides covalently linked to each other. By describing a single strand, the sequence of the complementary strand is also defined. Thus, a nucleic acid also encompasses the complementary strand of the described single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes 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, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0095] The nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, and may contain a combination of deoxyribonucleotides and ribonucleotides, including combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis or by recombinant methods.

[0096] q. Operably linked As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter that is spatially connected to the gene. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene that controls the promoter in the gene from which the promoter is derived. As is well known in the art, changes in this distance may be accommodated without loss of promoter function.

[0097] r. Promoter As used herein, a "promoter" may refer to a synthetic or naturally occurring molecule capable of effecting, activating, or enhancing the 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 modify its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the transcription start site. Promoters may be derived from sources including viral, bacterial, fungal, plant, insect, and animal. A promoter may regulate the expression of genetic components in the cells, tissues, or organs in which expression occurs, or in the developmental stages in which expression occurs, constitutively or differentially, or in response to external stimuli, such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0098] s. Stringent hybridization conditions As used herein, "stringent hybridization conditions" can refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) in, for example, a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and therefore will vary from situation to situation. Stringent conditions are those that meet the melting temperature (T) for a particular sequence at a defined ionic strength pH. m ) is selected to be about 5 to 10°C lower than the T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess, hence T m (In this case, 50% of the probes are occupied at equilibrium). Stringent conditions include a salt concentration of less than about 1.0 M sodium ion, for example, about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of at least about 30° C. for short probes (e.g., about 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions may also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following conditions: This involves incubation at 42°C with 50% formamide, 5x SSC and 1% SDS, or alternatively incubation at 65°C with 5x SSC, 1% SDS and washing at 65°C with 0.2x SSC and 0.1% SDS.

[0099] t. substantially complementary As used herein, "substantially complementary" 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.

[0100] u. Substantially identical As used herein, "substantially identical" can mean that a first and 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, that the first sequence is substantially complementary to the complement of the second sequence.

[0101] v. Mutant A "variant," as used herein with respect to a nucleic acid, can mean (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 to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0102] A "variant" as used in reference to a peptide or polypeptide can refer to a protein that has an amino acid sequence that differs by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant can also refer to a protein that has an amino acid sequence that is substantially identical to a reference protein that has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing one amino acid with another amino acid with similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as usually involving minor changes. Such minor changes can be identified in part by considering the hydropathic index of amino acids, 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 a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In some embodiments, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By considering the hydrophilicity of amino acids in the context of a peptide, the maximum local average hydrophilicity of the peptide can be calculated, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, the contents of which are incorporated herein by reference. As is understood in the art, substitutions of amino acids with similar hydrophilicity values ​​result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydropathic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with these findings, it is understood that substitutions of amino acids that are compatible with biological function depend on the relative similarity of the amino acids, particularly the relative similarity of the side chains of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. The "mutants" are preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to SEQ ID NOs: 1 to 6, 67 to 68.

[0103] "Variants" of 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, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 that code for the same amino acid sequence as the amino acid sequence encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 can be 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 of RNA transcribed by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 that code for the same amino acid sequence as the amino acid sequence encoded by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 can be 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. A variant may also be a variant of RNA transcribed by SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72 that encodes a protein that is a variant of the protein encoded by RNA transcribed by SEQ ID NO:64, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, having an amino acid sequence substantially identical to the reference protein and that retains at least one biological activity, typically with an amino acid sequence that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous. A variant may also be a variant of an RNA transcribed by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72, which has an amino acid sequence substantially identical to the reference protein and which retains at least one biological activity, typically having an amino acid sequence that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the protein encoded by an RNA transcribed by SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72.

[0104] W. Vector As used herein, "vector" may refer to a nucleic acid sequence that includes a replication origin.Vector may be a plasmid, bacteriophage, bacteria, viral vector, artificial chromosome or yeast artificial chromosome.Vector may be a DNA or RNA vector.Vector may be a self-replicating extrachromosomal vector or a vector that is integrated into the host genome.

[0105] 2. Protein Provided herein is a filovirus immunogen that can be used to induce broad immunity against multiple subtypes or serotypes of a particular filovirus antigen. The consensus filovirus antigen can include the consensus amino acid sequence of Marburg virus filovirus glycoprotein MARV RAV immunogen, the consensus amino acid sequence of Marburg virus filovirus glycoprotein MARV OZO immunogen, the consensus amino acid sequence of Marburg virus filovirus glycoprotein MARV MUS immunogen, the isolated amino acid sequence of Marburg virus filovirus glycoprotein MARV ANG, the consensus amino acid sequence of Zaire Ebola virus glycoprotein ZEBOV immunogen, the consensus amino acid sequence of Zaire Ebola virus glycoprotein ZEBOV2014 immunogen, the isolated amino acid sequence of Zaire Ebola virus glycoprotein ZEBOVCON2 immunogen, and the consensus amino acid sequence of Sudan Ebola virus glycoprotein SUDV immunogen. In some embodiments, the immunogen may include a signal peptide derived from a different protein, such as an immunoglobulin protein, for example, an IgE signal peptide or an IgG signal peptide.

[0106] The amino acid sequences of the immunogens include SEQ ID NOs: 1 to 6, 67 to 68 and variants thereof, and fragments of SEQ ID NOs: 1 to 6, 67 to 68 and variants thereof, and optionally include a signal peptide, such as an IgE or IgG signal peptide.

[0107] 3. Coding sequence that encodes a protein Coding sequences encoding the proteins described herein may be generated using routine methods. Compositions including a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a second consensus Zaire Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen envelope glycoprotein immunogen, a nucleic acid sequence encoding a consensus Sudan Ebola virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a Marburg MRV virus Angola 2005 envelope glycoprotein immunogen, a nucleic acid sequence encoding a first consensus Marburg MRV virus envelope glycoprotein immunogen, a nucleic acid sequence encoding a second consensus Marburg MRV virus envelope glycoprotein immunogen, and a nucleic acid sequence encoding a third consensus Marburg MRV virus envelope glycoprotein immunogen may be generated based on the disclosed amino acid sequences.

[0108] The nucleic acid sequence may encode a full-length consensus Zaire Ebola virus envelope glycoprotein immunogen, a full-length consensus second Zaire Ebola virus envelope glycoprotein immunogen, a full-length consensus Guinea Ebola virus envelope glycoprotein immunogen, a full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a full-length Marburg MRB virus Angola 2005 envelope glycoprotein immunogen, a full-length first consensus Marburg MRB virus envelope glycoprotein immunogen, a full-length second consensus Marburg MRB virus envelope glycoprotein immunogen, or a full-length third consensus Marburg MRB virus envelope glycoprotein immunogen. The nucleic acid sequence may include a sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:67, or SEQ ID NO:68. The nucleic acid sequence may comprise SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72. In one embodiment, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence described herein. For example, the nucleic acid may comprise an RNA sequence transcribed by the DNA sequence of SEQ ID NO:64, 65, 66, 69, 70, or 72, a fragment thereof, or a variant thereof. The nucleic acid sequence may optionally comprise a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0109] The nucleic acid sequence may encode a fragment of a full-length consensus Ebolavirus Zaire envelope glycoprotein immunogen, a fragment of a second full-length consensus Ebolavirus Zaire envelope glycoprotein immunogen, a fragment of a full-length consensus Ebolavirus Zaire Guinea envelope glycoprotein immunogen envelope glycoprotein immunogen, a fragment of a full-length consensus Ebolavirus Sudan envelope glycoprotein immunogen, a fragment of a full-length Marburg virus Angola 2005 envelope glycoprotein immunogen, a fragment of a first full-length consensus Ebolavirus Zaire envelope glycoprotein immunogen, a fragment of a second full-length consensus Ebolavirus Zaire envelope glycoprotein immunogen, or a fragment of a third full-length consensus Ebolavirus Zaire 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, SEQ ID NO:6, SEQ ID NO:67, or SEQ ID NO:68. The nucleic acid sequence may comprise a fragment of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72. Fragment sizes are disclosed as described herein in the section entitled "Fragments." The nucleic acid sequence may optionally comprise a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0110] The nucleic acid sequence may encode a protein homologous to a full-length consensus Ebola virus envelope glycoprotein immunogen, a full-length consensus Ebola virus envelope glycoprotein immunogen, a full-length consensus Guinea virus envelope glycoprotein immunogen, a full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a protein homologous to a full-length Marburg virus envelope glycoprotein immunogen, a protein homologous to a full-length consensus Angola 2005 Marburg virus envelope glycoprotein immunogen, a protein homologous to a full-length consensus 1 Marburg virus envelope glycoprotein immunogen, a protein homologous to a full-length consensus 2 Marburg virus envelope glycoprotein immunogen, and a protein homologous to a full-length consensus 3 Marburg virus envelope glycoprotein immunogen. The nucleic acid sequence may include 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, SEQ ID NO:6, SEQ ID NO:67, or SEQ ID NO:68. The nucleic acid sequence may be homologous to SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72. The degree of homology is discussed herein, such as in the section referring to variants. The nucleic acid sequence may optionally include a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0111] The nucleic acid sequence may be a protein homologous to a fragment of the full-length consensus Zaire Ebola virus envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length second consensus Zaire Ebola virus envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length consensus Sudan Ebola virus envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length consensus Marc ... The nucleic acid sequence may encode a protein homologous to a fragment of the Burgmarburg virus Angola 2005 envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length first consensus Marburgmarburg virus envelope glycoprotein immunogen, a protein homologous to a fragment of the full-length second consensus Marburgmarburg virus envelope glycoprotein immunogen, or a protein homologous to a fragment of the full-length third consensus Marburgmarburg virus envelope glycoprotein immunogen. The nucleic acid sequence may include 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, SEQ ID NO:6, SEQ ID NO:67, or SEQ ID NO:68. The nucleic acid sequence may include a fragment homologous to SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:72. The degree of homology is discussed herein, such as in the section referring to variants. The nucleic acid sequence can optionally include a coding sequence encoding a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0112] SEQ ID NO:64 is the nucleotide sequence insert in plasmid pEBOZ encoding the consensus Ebola Zaire virus envelope glycoprotein immunogen.

[0113] SEQ ID NO:65 is the nucleotide sequence insert in plasmid pEBOS encoding the consensus Sudan Ebola virus envelope glycoprotein immunogen.

[0114] SEQ ID NO:66 is the nucleotide sequence insert in plasmid pMARZ ANG encoding the Marburg virus Angola 2005 envelope glycoprotein.

[0115] SEQ ID NO:69 is the nucleotide sequence insert in plasmid pEBOZGUI encoding the consensus Zaire Ebola virus GP envelope glycoprotein immunogen.

[0116] SEQ ID NO:70 is the nucleotide sequence insert of plasmid pEBOZCON2 encoding the second consensus Zaire Ebola virus envelope glycoprotein.

[0117] 4. Vector Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" includes a nucleic acid capable of infecting, transfecting, transiently or permanently transducing a cell. It will be appreciated that a vector can be a naked nucleic acid or a nucleic acid complexed with a protein or lipid. The vector optionally includes viral or bacterial nucleic acid, and / or protein, and / or membrane (e.g., cell membrane, viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which a piece of DNA can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see U.S. Pat. No. 5,217,879), and the like, and include both expression and non-expression plasmids. When a recombinant microorganism or cell culture is described as carrying an "expression vector," this includes both extrachromosomal circular and linear DNA, and DNA integrated into the host chromosome(s). When a vector is maintained by a host cell, the vector may be stably replicated as an autonomous structure during mitosis or alternatively, may be integrated into the host's genome.

[0118] A plasmid The plasmid may contain nucleic acid sequences encoding one or more of the various immunogens disclosed above, including coding sequences encoding synthetic consensus antigens capable of eliciting an immune response against a phyloprotein.

[0119] A single plasmid may contain the coding sequence for a single phylloprotein immunogen, the coding sequence for two phylloprotein immunogens, the coding sequence for three phylloprotein immunogens, the coding sequence for four phylloprotein immunogens, the coding sequence for five phylloprotein immunogens, or the coding sequence for six phylloprotein immunogens. A single plasmid may contain the coding sequence for a single phylloprotein immunogen that can be formulated together. In some embodiments, the plasmid may contain coding sequences encoding IL-12, IL-15, and / or IL-28.

[0120] The plasmid may further comprise a start codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The start codon and stop codon may be in frame with the coding sequence.

[0121] The plasmid may also include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as 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 may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the entire contents of which are incorporated herein by reference.

[0122] The plasmid may also include a polyadenylation signal that may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, Calif.).

[0123] The plasmid may contain an enhancer upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer from CMV, FMDV, RSV, or EBV. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the entire contents of each of which are incorporated herein by reference.

[0124] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid within a cell. The plasmid may be pVAX-1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, allowing high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0125] The plasmid may also contain regulatory sequences that may be adequate for gene expression in the 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.

[0126] 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 IgE or IgG.

[0127] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which may be used for protein production in Escherichia coli (E. coli). The plasmid may be pYES2 (Invitrogen, San Diego, CA), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may be a plasmid in the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, CA), which may be used for protein production in insect cells. The plasmid may be pcDNAI or pcDNA3 (Invitrogen, San Diego, CA), which may be used for protein production in mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0128] B. RNA vector In one embodiment, the nucleic acid is an RNA molecule. Thus, in one embodiment, the invention provides an RNA molecule encoding one or more of the envelope glycoprotein (GP) genes of Marburg Marburg virus (MARV), Sudan Ebola virus (SUDV) or Zaire Ebola virus (ZEBOV). The RNA may be positive stranded. Thus, in some embodiments, the RNA molecule may be translated by the cell without the need for an intervening replication step such as reverse transcription. The RNA molecule useful in the invention may have a 5' cap (e.g., 7-methylguanosine). This cap may facilitate translation of the RNA in vivo. The 5' nucleotide of the RNA molecule useful in the invention may have a 5' triphosphate group. In capped RNA, this may be linked to the 7-methylguanosine via a 5' to 5' bridge. The RNA molecule may have a 3' poly-A tail. It may also have a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecule useful in the invention may be single stranded.

[0129] C. Linear vector Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that can be efficiently delivered to a subject by electroporation and express one or more desired antigens. The LEC may be linear DNA lacking any phosphate backbone. The DNA may encode one or more antigens. The LEC may include a promoter, an intron, a stop codon, a polyadenylation signal. Expression of the antigen may be controlled by a promoter. The LEC may be free of antibiotic resistance genes and / or a phosphate backbone. The LEC may be free of other nucleic acid sequences unrelated to the desired antigen gene expression.

[0130] The LEC can be derived from any plasmid that can be linearized. The plasmid can be capable of expressing an antigen. The plasmid can be pVAX, pcDNA3.0, or provax, or any other expression vector that expresses DNA and allows the cell to translate the sequence into an antigen that is recognized by the immune system.

[0131] The LEC can be derived from any plasmid that can be linearized. The plasmid can be capable of expressing an antigen. The plasmid can be pVAX, pcDNA3.0, or provax, or any other expression vector that expresses DNA and allows the cell to translate the sequence into an antigen that is recognized by the immune system.

[0132] D. Viral Vectors In one embodiment, a viral vector capable of delivering the nucleic acid of the present invention to a cell is provided herein. The expression vector can be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001), Ausubel et al. (1997), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers. (See, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193.) Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0133] 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.

[0134] The composition may comprise a nucleic acid molecule, such as a plasmid, that collectively comprises a coding sequence for a single filoprotein antigen selected from the group consisting of one or more of a consensus Zaire Ebola virus envelope glycoprotein immunogen, a second consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Zaire Ebola virus Guinea 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.

[0135] The composition comprises a nucleic acid sequence encoding a combination of: 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 and a second consensus Zaire Ebola virus envelope glycoprotein immunogen; or a consensus Zaire Ebola virus envelope glycoprotein immunogen and a consensus Zaire Ebola virus Guinea 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 and a second consensus Zaire Ebola virus envelope glycoprotein immunogen; or a consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen and consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen; or consensus Zaire Ebola virus envelope glycoprotein immunogen, second consensus Zaire Ebola virus envelope glycoprotein immunogen, first consensus Marburg virus envelope glycoprotein immunogen, second consensus Marburg virus envelope glycoprotein immunogen or consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, first consensus Marburg virus envelope glycoprotein immunogen, second consensus Marburg virus envelope glycoprotein immunogen and third consensus Marburg virus envelope glycoprotein immunogen;or consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen, first consensus Marburg virus envelope glycoprotein immunogen, second consensus Marburg virus envelope glycoprotein immunogen and third consensus Marburg virus envelope glycoprotein immunogen; or consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen Ebola virus envelope glycoprotein immunogen, Marburg virus Angola 2005 envelope glycoprotein, first consensus Marburg virus envelope glycoprotein immunogen, second consensus Marburg virus envelope glycoprotein immunogen and third consensus Marburg virus envelope glycoprotein immunogen; or consensus Zaire Ebola virus envelope glycoprotein immunogen, consensus Sudan Ebola virus envelope glycoprotein immunogen envelope glycoprotein immunogen, consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, Marburg MARburg virus Angola 2005 envelope glycoprotein, first consensus Marburg MARburg virus envelope glycoprotein immunogen, second consensus Marburg MARburg virus envelope glycoprotein immunogen and third consensus Marburg MARburg virus envelope glycoprotein immunogen; or consensus Zaire Ebola virus envelope glycoprotein immunogens, consensus Sudan Ebola virus envelope glycoprotein immunogen, second consensus Zaire Ebola virus envelope glycoprotein immunogen, Marburg MMR virus Angola 2005 envelope glycoprotein, first consensus Marburg MMR virus envelope glycoprotein immunogen, second consensus Marburg MMR virus envelope glycoprotein immunogen and third consensus Marburg MMR virus envelope glycoprotein immunogen;

[0136] Each coding sequence for each phyloprotein immunogen is preferably contained on a separate plasmid.

[0137] Thus, a composition comprising nucleic acid sequences encoding a consensus Zaire Ebolavirus envelope glycoprotein immunogen and a consensus Sudan Ebolavirus envelope glycoprotein immunogen; a consensus Zaire Ebolavirus envelope glycoprotein immunogen and a second consensus Zaire Ebolavirus envelope glycoprotein immunogen; or a consensus Zaire Ebolavirus envelope glycoprotein immunogen and a consensus Zaire Ebolavirus Guinea envelope glycoprotein immunogen may be present on a single plasmid, but are preferably present on two separate plasmids.

[0138] A composition comprising a nucleic acid sequence 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; or a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen and a second consensus Zaire Ebola virus envelope glycoprotein immunogen; or a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen and a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen; may be present on one plasmid or may be present on two plasmids in any permutation, but is preferably present on three separate plasmids.

[0139] The compositions 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 MMR virus envelope glycoprotein immunogen, the second consensus Marburg MMR virus envelope glycoprotein immunogen and the third consensus Marburg MMR virus envelope glycoprotein immunogen may be present 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 are preferably present on five separate plasmids.

[0140] The compositions comprising nucleic acid sequences encoding the consensus Ebola virus envelope glycoprotein immunogen, the second consensus 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 present 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 are preferably present on five separate plasmids.

[0141] The compositions comprising nucleic acid sequences encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, the consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, the first consensus Marburg MMRburg virus envelope glycoprotein immunogen, the second consensus Marburg MMRburg virus envelope glycoprotein immunogen and the third consensus Marburg MMRburg virus envelope glycoprotein immunogen may be present 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 are preferably present on five separate plasmids.

[0142] A composition comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a second consensus Zaire Ebola virus envelope glycoprotein immunogen, a 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 may be present 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, or on four plasmids in any permutation, but preferably on six separate plasmids.

[0143] A composition comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, a 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 may be present 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, or on four plasmids in any permutation, but preferably on six separate plasmids.

[0144] A composition comprising nucleic acid sequences encoding the consensus Zaire Ebola virus envelope glycoprotein immunogen, the consensus Sudan Ebola virus envelope glycoprotein immunogen, the Marburg MARburg virus Angola 2005 envelope glycoprotein, the first consensus Marburg MARburg virus envelope glycoprotein immunogen, the second consensus Marburg MARburg virus envelope glycoprotein immunogen, and the third consensus Marburg MARburg virus envelope glycoprotein immunogen may be present 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, or on five plasmids in any permutation, but preferably on six separate plasmids.

[0145] A composition comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a second 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 Angola virus envelope glycoprotein immunogen, a second consensus Marburg Angola virus envelope glycoprotein immunogen and a third consensus Marburg Angola virus envelope glycoprotein immunogen may be present 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, or on five plasmids in any permutation, or on six plasmids in any permutation, but are preferably present on seven separate plasmids.

[0146] A composition comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a 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 may be present 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, or on five plasmids in any permutation, or on six plasmids in any permutation, but are preferably present on seven separate plasmids.

[0147] Similarly, a composition comprising a nucleic acid sequence encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen, a second consensus Zaire Ebola virus envelope glycoprotein immunogen, a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a Marburg MARburg virus Angola 2005 envelope glycoprotein, a first Marburg MARburg virus envelope glycoprotein immunogen, a second Marburg MARburg virus envelope glycoprotein immunogen, a third consensus Marburg MARburg virus envelope glycoprotein immunogen may be present 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, or on five plasmids in any permutation, or on six plasmids in any permutation, or on seven plasmids in any permutation, but preferably on eight separate plasmids.

[0148] 6. Vaccines Provided herein is a vaccine capable of generating an immune response in a mammal against filoviruses, particularly Marburg virus, Ebola virus Sudan and / or Ebola virus Zaire. The vaccine may comprise each plasmid as described above. The vaccine may comprise multiple plasmids, or a combination thereof. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

[0149] 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 and a consensus Zaire Ebola virus Guinea envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebola virus envelope glycoprotein immunogen and a second consensus Zaire 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 second consensus Zaire 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 consensus Zaire Ebola virus Guinea 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 second consensus Zaire 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 the consensus Ebola virus Zaire envelope glycoprotein immunogen, the consensus Ebola virus Zaire Guinea envelope glycoprotein immunogen and the Marburg virus Angola 2005 envelope glycoprotein.The vaccine may be used to deliver a nucleic acid molecule encoding a consensus 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. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus envelope glycoprotein immunogen, a second consensus 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. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebolavirus Zaire envelope glycoprotein immunogen, a consensus Ebolavirus Zaire Guinea envelope glycoprotein immunogen, a consensus Ebolavirus Sudan 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. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus envelope glycoprotein immunogen, a Zaire Ebola virus 2014 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, a third consensus Marburg virus envelope glycoprotein immunogen.The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus Zaire envelope glycoprotein immunogen, a consensus Ebola virus Zaire Guinea envelope glycoprotein immunogen, a first consensus Marburg virus envelope glycoprotein immunogen, a second consensus Marburg virus envelope glycoprotein immunogen, or a third consensus Marburg virus envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Zaire Ebolavirus envelope glycoprotein immunogen, a consensus Sudan Ebolavirus Guinea envelope glycoprotein immunogen, a 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, a third consensus Marburg MARburg virus envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus envelope glycoprotein immunogen, a second consensus Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a Marburg virus Angola 2005 envelope glycoprotein, a first consensus Marburg virus envelope glycoprotein immunogen, a second consensus Marburg virus envelope glycoprotein immunogen, a third consensus Marburg virus envelope glycoprotein immunogen.The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus envelope glycoprotein immunogen, a consensus Ebola virus Guinea envelope glycoprotein immunogen, a consensus Ebola virus envelope glycoprotein immunogen, a Marburg virus Angola 2005 envelope glycoprotein, a first consensus Marburg virus envelope, a second consensus Marburg virus envelope glycoprotein immunogen and a third consensus Marburg virus envelope glycoprotein immunogen. The vaccine may be used to deliver a nucleic acid molecule encoding a consensus Ebola virus envelope glycoprotein immunogen, a second consensus Ebola virus envelope glycoprotein immunogen, a consensus Ebola virus Guinea envelope glycoprotein immunogen, a consensus Ebola virus envelope glycoprotein immunogen, a consensus Sudan Ebola virus envelope glycoprotein immunogen, a Marburg virus Angola 2005, a first consensus Ebola virus envelope glycoprotein immunogen, a second consensus Ebola virus envelope glycoprotein immunogen and a third consensus Ebola virus envelope glycoprotein immunogen. The vaccine is preferably a composition comprising a plasmid.

[0150] The vaccine may further comprise a pharma- ceutically acceptable excipient. The pharma-ceutically acceptable excipient may be a functional molecule as a vehicle, adjuvant, carrier, or diluent. The pharma-ceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs such as monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0151] The transfection facilitating agent is a polyanion, polycation, such as poly-L-glutamate (LGS), or a lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably the poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs such as monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and may be administered with the genetic construct using hyaluronic acid. In some embodiments, the DNA plasmid vaccine may also include a transfection facilitating agent, such as a lipid, a liposome, such as a lecithin liposome or other liposomes known in the art, such as a DNA liposome mixture (see, e.g., WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, such as poly-L-glutamate (LGS), or a lipid. 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.

[0152] The pharma- ceutically acceptable excipient may be one or more adjuvants, which may be other genes expressed from the same or alternative plasmids or delivered as proteins in combination with the above plasmids in the vaccine.One or more of the adjuvants may be a protein and / or a nucleic acid encoding a protein 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), epithelial thymus expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, e.g., where the signal sequence or the coding sequence encoding the signal sequence has been deleted and optionally a different signal peptide such as from IgE, or IgE, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-lα, 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 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, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP IL-15 with different signal peptides derived from K, SAP-1, JNK, interferon response genes, 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 the 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 described in PCT Application No. PCT / US1997 / 019502 and corresponding U.S. patent application Ser. No. 08 / 956,865, and U.S. Provisional Application No. 61 / 569600, filed Dec. 12, 2011. 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. PCT / 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. PCT / 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. PCT / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding U.S. 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. PCT / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding U.S. 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. PCT / US11 / 024098, which is incorporated herein by reference.Examples of chemokine CTACK, TECK and MEC constructs and sequences are disclosed in PCT Application No. PCT / US2005 / 042231 and corresponding U.S. Patent Application No. 11 / 719,646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulatory agents are disclosed in U.S. Patent Application No. 10 / 560,653, each of which is incorporated herein by reference. Examples of DR5 and other immunomodulatory agents are disclosed in U.S. Patent Application No. 09 / 622452, each of which is incorporated herein by reference.

[0153] The vaccine may further comprise a genetic vaccine facilitator such as those described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, the entire contents of which are incorporated by reference.

[0154] The vaccine comprises consensus antigen and plasmid in an amount of about 1 ng to about 100 mg, about 1 μg to about 10 mg, or preferably about 0.1 μg to about 10 mg, or more preferably about 1 mg to about 2 mg. In some preferred embodiments, the pharmaceutical composition of the present invention comprises about 5 ng to about 1000 μg of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 10 ng to about 800 μg of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 μg of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 μg of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 μg, about 100 to about 200 μg, about 1 ng to 100 mg, about 1 μg to about 10 mg, about 0.1 μg to about 10 mg, about 1 mg to about 2 mg, about 5 ng to about 1000 μg, about 10 ng to about 800 μg, about 0.1 to about 500 μg, about 1 to about 350 μg, about 25 to about 250 μg, or about 100 to about 200 μg of the consensus antigen or its plasmid.

[0155] The vaccine may be formulated according to the mode of administration to be used. The injectable vaccine pharmaceutical composition may be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may include a vasoconstrictor. The isotonic solution may include phosphate buffered saline. The vaccine may further include a stabilizer such as gelatin or albumin. Stabilization of the vaccine formulation with LGS or polycations or polyanions, etc., may make the formulation stable for extended periods of time at room or ambient temperature.

[0156] The vaccine is stable at room temperature (25° C.) for more than one week, in some embodiments for more than two weeks, in some embodiments for more than three weeks, in some embodiments for more than four weeks, in some embodiments for more than five weeks, and in some embodiments for more than six weeks. In some embodiments, the vaccine is stable for more than one month, more than two months, more than three months, more than four months, more than five months, more than six months, more than seven months, more than eight months, more than nine months, more than ten months, more than eleven months, or more than twelve months. In some embodiments, the vaccine is stable for more than one year, more than two years, or more than five years. In one embodiment, the vaccine is stable under refrigeration (2-8° C.). Thus, in one embodiment, the vaccine does not require a refrigeration chain. A vaccine is stable if it retains its biological activity for a sufficient period of time to allow for its intended use (e.g., generation of an immune response in a subject). For example, for vaccines that are stored and shipped, etc., it may be desirable for the vaccine to be stable for months to years.

[0157] 7. Vaccine delivery method Provided herein are methods of vaccine delivery to provide consensus antigen genetic constructs and proteins that contain epitopes that make them particularly effective against filovirus, particularly Marburg virus, Ebola virus Sudan and / or Ebola virus Zaire immunogens that can elicit an immune response. Vaccine or vaccination delivery methods may be provided to induce therapeutic and prophylactic immune responses. The vaccination process may generate an immune response in a mammal against filovirus, particularly Marburg virus, Ebola virus Sudan and / or Ebola virus Zaire. The vaccine may be delivered to an individual to modulate the activity of the mammal's immune system and enhance the immune response. The delivery of the vaccine may be the 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. Delivery of the vaccine may be used to induce or elicit an immune response in a mammal against filoviruses, particularly Marburg virus, Ebola virus Sudan and / or Ebola virus Zaire, by administering to the mammal a vaccine as described above.

[0158] In the delivery of vaccines and plasmids into mammalian cells, the transfected cells express and secrete consensus antigens for each of the injected plasmids from the vaccine. These proteins are recognized as foreign by the immune system and antibodies are generated against them. These antibodies are maintained by the immune system and allow an effective response to subsequent infections with filoviruses, particularly Marburg virus, Ebola virus Sudan and / or Ebola virus Zaire.

[0159] The vaccine may be administered to a mammal to elicit an immune response in the mammal, which may be a human, a primate, a non-human primate, cow, cattle, sheep, goat, antelope, bison, buffalo, bison, cow, deer, hedgehog, elephant, llama, alpaca, mouse, rat, and chicken.

[0160] Combination treatment The vaccine may be a chemokine that is capable of expressing a specific chemokine, such as CCL20, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, e.g., with the signal sequence deleted, and optionally with the IgE signal peptide, MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, Mad CAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, A po-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP The present invention may also be administered in combination with genes encoding IL-15 and / or other proteins containing different signal peptides, such as K, SAP-1, JNK, interferon response genes, 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 vaccine may be administered in combination with one or more of the following nucleic acid molecules and / or proteins: a nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising a coding sequence encoding one or more of CCL20, IL-12, IL-15, IL-28, CTACK, TECK, MEC and RANTES, or a functional fragment thereof, and a protein selected from the group consisting of CCL02, an IL-12 protein, an IL-15 protein, an IL-28 protein, a CTACK protein, a TECK protein, a MEC protein or a RANTES protein, or a functional fragment thereof.

[0161] The vaccine may be administered by different routes, such as orally, parenterally, sublingually, transdermally, rectally, mucosally, topically, by inhalation, buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. For veterinary use, the composition may be administered in an appropriately acceptable formulation, according to normal veterinary practice. A veterinarian can easily determine the most appropriate dosing regime and route of administration for a particular animal. The vaccine may be administered by traditional syringe, needleless injection device, "microparticle bombardment gene gun", or other physical methods, such as electroporation (EP), "hydrodynamic methods", or ultrasound.

[0162] Vaccine plasmids may be delivered to mammals by several well-known techniques including in vivo electroporation, liposome-mediated, nanoparticle-facilitated, DNA injection (also called DNA vaccination) with or without recombinant vectors such as recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccinia. Consensus antigens may be delivered by DNA injection and in vivo electroporation.

[0163] B. Electroporation Administration of the vaccine by 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 effective to form reversible pores in the cell membrane, and preferably the energy pulse is a constant current close to a preset current input 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 elements of the electroporation device, such as a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. The electroporation method can use 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.

[0164] The electroporation component may function as one element of the electroporation device, and other elements may be separate elements (or components) that communicate with the electroporation component. The electroporation component may function as more than one element of the electroporation device, and may communicate with other elements of the electroporation device that are still separate from the electroporation component. Elements of the electroporation device that are part of an electromechanical or mechanical device may not be limited to those that can function as one device or as separate elements that communicate with each other. The electroporation component may deliver an energy pulse that generates a constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having multiple electrodes in a spatial arrangement, which receives an energy pulse from the electroporation component and delivers the same to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral during delivery of the energy pulse and measures the impedance at the desired tissue and communicates the impedance to the electroporation component. The feedback mechanism may receive the measured impedance and can adjust the energy pulse delivered by the electroporation component to maintain the constant current.

[0165] The electrodes may deliver energy pulses in a distributed pattern through control of the electrodes under a programmed sequence, the programmed sequence being input by a user into the electroporation component. The programmed sequence may include a plurality of pulses delivered in sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one neutral electrode that measures impedance, and a subsequent pulse of the plurality of pulses being delivered by a different one of the at least two active electrodes with one neutral electrode that measures impedance.

[0166] The feedback mechanism may be implemented in either hardware or software. The feedback mechanism may be implemented by an analog 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 neutral electrode may measure the impedance at the desired tissue and communicate that impedance to the feedback mechanism, which then adjusts the energy pulse in response to that impedance and maintains a constant current at a value similar to the preset current. The feedback mechanism may continuously and instantaneously maintain a constant current within the delivery period of the pulse of energy.

[0167] Examples of electroporation devices and electroporation methods that may facilitate delivery of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli, et al., and U.S. Patent Publication No. 2005 / 0052630 to Smith, et al., the entire contents of which are incorporated herein by reference. Other electroporation devices and methods that may be used to facilitate delivery of the DNA vaccines include those provided in U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 USC 119(e) to U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, the entire contents of which are incorporated herein by reference.

[0168] Draghia-Akli, et al., U.S. Patent No. 7,245,963, describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in a body or plant. The modular electrode system may include a plurality of needle electrodes, a hypodermic needle, an electrical connector providing a conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. A user can grasp the plurality of needle electrodes mounted on a support structure and firmly insert the electrodes into a selected tissue in a body or plant. The biomolecules are then delivered to the selected tissue through the hypodermic needle. The programmable constant current pulse controller is activated 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.

[0169] U.S. 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 array of electrodes and pulse parameters input under user control, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of U.S. Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0170] The electrode arrays and methods 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. Depending on the configuration of the electrode array, the needle (delivering the selected biomolecule) is inserted completely into the target organ and the electrode is injected perpendicular to the target tissue in a pre-delineated area. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0171] In addition, as anticipated in some embodiments incorporating electroporation devices and their use, there are electroporation devices described in the following patents: 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, 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 including inventions disclosed in U.S. Patent No. 6,697,669, issued February 24, 2004, which relate to delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which relates to methods of DNA injection, are contemplated herein. All of the above-mentioned patents are incorporated herein by reference.

[0172] c. DNA plasmid preparation method Provided herein is a method for preparing the DNA plasmid comprising the DNA vaccine discussed herein.The DNA plasmid after the final subcloning step into mammalian expression plasmid can be used to inoculate cell culture in large-scale fermentation tanks using methods known in the art.

[0173] DNA plasmids for use with the EP device of the present invention can be formulated or manufactured using a combination of known equipment and techniques, but are preferably manufactured using the optimized plasmid manufacturing techniques described in co-pending U.S. Provisional Patent Application No. 60 / 939,792, filed May 23, 2007, which is subject to licensing. In some instances, DNA plasmids for use in these tests can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques include and incorporate a variety of equipment and protocols generally known to those of skill in the art, including those described in U.S. Provisional Patent Application No. 60 / 939,792, as well as those described in U.S. Patent No. 7,238,522, which issued July 3, 2007, which is subject to licensing. 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 by reference. EXAMPLES

[0174] The present invention is further illustrated in the following examples. It should be understood that these examples are illustrative of preferred embodiments of the present invention and are provided for illustrative purposes only. From the above description and these examples, a person skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, it is clear from the above description that a person skilled in the art can make various modifications to the present invention in addition to the invention described herein. Such modifications are also intended to fall within the scope of the claims.

[0175] Example 1 method Plasmid vaccine construction The pMARV, pEBOS, and pEBOZ plasmid DNA constructs encode the full-length GP protein. For pEBOS and pEBOZ, an amino acid consensus strategy was used, while for pMARV, a type-matched sequence (GenBank#VGP_MABVR) from the 2005 Angolan epidemic strain was used (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 predominant 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 and RNA optimization to enhance protein expression (GenScript, Piscataway, NJ)) and commercially synthesized (GenScript, Piscataway, NJ) and then subcloned into a modified pVAX1 mammalian expression vector (Invitrogen, Carlsbad, CA) under the control of the cytomegalovirus immediate 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 adopted because GP diversity among MARVs is much higher in comparison (approximately 70% identity). For MARV coverage, we chose to utilize the sequence of MGP derived from the 2005 outbreak in Angola (GenBank #VGP_MABVR), as this was responsible for the largest and most deadly MARV epidemic to date.This sequence differed by more than 10% from either Musoke, Popp, and Leiden (10.6% diversity), or from the closest cluster of related strains, including Uganda (01Uga07), Darba (05DRC99 and 07DRC99), and Ozolin (10.3% diversity).The three-plasmid strategy formed the basis of a novel trivalent-multivalent filovirus vaccine strategy.

[0176] Transfection and immunoblotting Human Embryonic Kidney (HEK) 293T cells were cultured, transfected, and harvested. Briefly, cells were grown in DMEM containing 10% FBS, 1% Pen-strep, sodium pyruvate, and L-glutamine. Cells were cultured in 150 mm Corning dishes and incubated at 37 °C for 3 h at 5% CO. 2 Cells were grown to 70% confluence overnight in a 37°C incubator containing 10% CO. 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 protocols and then incubated for 24–48 h. Cells were harvested in ice-cold PBS, washed by centrifugation, and then pelleted for Western immunoblot or FACS analysis. Standard Western blotting was used, and a GP-specific MAb for GP1 detection was generated. Data from Western immunoblotting experiments are shown in Figure 1B. Data from FACS analysis are shown in Figure 1C.

[0177] 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 Jackson Laboratory (Bar Harbor, ME), while Hartley guinea pigs were obtained 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 at PHAC and the Canadian Council on Animal Health guidelines for the housing and care of laboratory animals, and after appropriate review and approval by the above institutions, as recommended by the Guide for the Care and Use of Laboratory Animals of NIH. UPenn and NML adhere to NIH policies on animal welfare, the Animal Welfare Act, and all other applicable federal, state, and local laws.

[0178] Mice were immunized intramuscularly with 40 μg of plasmid resuspended in water by needle injection, while guinea pigs were immunized intradermally with 200 μg each at three separate vaccination sites. EP was performed at the same sites immediately after vaccination. 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, delivering two constant current pulses of 0.1 A at 52 msec / pulse separated by a 1-second delay.

[0179] In the lethal challenge study, 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 by intraperitoneal injection with MARV-Angora (681 TCID50 / animal). Briefly, guinea pig-adapted MARV was generated by serial passage of wild-type MARV-Angora in outbred adult female Hartley guinea pigs. Seven days after inoculation, animals were euthanized and livers were harvested and homogenized. The homogenate was then injected intraperitoneally into naive adult guinea pigs, and the process was repeated until the animals lost weight, hair, and became infected similar to EBOV-adapted guinea pigs. For the mouse lethal challenge test (Kobinger GP,et al.(2006). Chimpanzee adenovirus vaccine protects against Zaire Ebola virus. Virology 346:394-401), 1,000LD of mouse-adapted ZEBOV was injected intraperitoneally. 50 Mice were injected intraperitoneally with 200 μl (10 FFU / animal). All animals were weighed daily and disease progression was monitored for at least 18 days for mice and 22 days for guinea pigs using an approved score sheet. All infection work was performed in a "Biosafety Level 4" (BSL4) facility at the NML, PHAC.

[0180] ELISA and neutralization assays Antibody (Ab) titers were measured using 96-well ELISA plates coated with either sucrose-purified MARV Ozolin GP or ZGP, or using negative control sucrose-purified Nipah G protein at a concentration of 1:2,000. Briefly, plates were 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 (at dilutions of 1:100, 1:400, 1:1,600, and 1:6,400 in PBS containing 5% skim milk and 0.5% Tween-20). After incubation for 1 hour 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 (diluted 1:2,000) and further incubated for 1 hour at 37°C in a humidified container. After washing, 100 μl of AbST (2,2'-azino-bis(3-ethylbenzazolin-6-sulfonic acid) and peroxidase substrate (Cedarlane) were added to visualize Ab binding. Plates were incubated for 30 min at 37°C and then read at 405 nm. Positive binding results were characterized as >3 SD subtraction of positive control from negative control serum.

[0181] ZEBOV neutralization assays were performed. Briefly, serum was collected from immunized mice and guinea pigs inactivated at 56 °C for 45 min, and each sample (1:20, 1:40, etc. for mice and 1:50 for guinea pigs in 50 μl of DMEM) was mixed with an equal amount of ZEBOV expressing an EGFP reporter gene (ZEBOV-EGFP) (100 transducing units / well according to EGFP expression) and incubated at 37 °C for 90 min. The mixture was then transferred to subconfluent VeroE6 cells in 96-well flat-bottom plates and incubated at room temperature for 5–10 min. Control wells were infected with an equal 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 plate was incubated at 37 °C, 5% CO 2 The mixture was incubated at RT for 48 hours.

[0182] Alternatively, neutralization of MARV-Angola 368 was assessed using an immunofluorescence assay. Primary rabbit anti-MARV Ab and secondary goat anti-rabbit IgG FITC-conjugated Ab were used for detection. Neutralizing antibodies (NAbs) against SUDV Boniface were assayed based on cytopathic effect (CPE) on CV-1 cells. Cells were incubated with equal amounts of immune 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 sample dilutions showing >50% reduction in the number of green cells compared to the control were scored as positive for NAbs. Alternatively, NAbs against SUDV-Boniface were assayed based on cytopathic effect (CPE) on CV-1 cells. All infectivity tests were performed in the BSL4 laboratory at NML, PHAC.

[0183] Isolation of splenocytes Mice were sacrificed 8–11 days after the final immunization, and spleens were harvested. Briefly, spleens were placed in RPMI1640 medium (Mediatech Inc., Manassas, VA) supplemented with 10% FBS, 1× anti-antibody (Invitrogen), and 1× 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 with PBS, and then resuspended in RPMI medium for use in ELISPOT or FACS assays.

[0184] ELISPOT assay A standard IFNγ ELISPOT assay was performed. Briefly, 96-well plates (Millipore, Billerica, MA) were coated with anti-mouse IFN-γ capture antibody and incubated at 4°C for 24 h (R&D Systems, Minneapolis, MN). The next day, plates were washed with PBS and then blocked with blocking buffer (1% BSA and 5% sucrose in PBS) for 2 h. Splenocytes (1–2 × 10 5 Cells / well) were plated in triplicate and incubated at 37°C, 5% CO 2 The wells were stimulated overnight with PBS and in the presence of either RPMI1640 (negative control), ConA (positive control), or GP peptides, either individually (15-mers spanning the length of each GP, overlapping by 9 amino acids) or pooled altogether (final concentration 2.5 μg / ml). After 18–24 h of stimulation, the plates were washed with PBS and then incubated with biotinylated anti-mouse IFN-γ mAb (R&D Systems, Minneapolis, MN) for 24 h at 4°C. Next, the plates were washed again with PBS and streptavidin-alkaline phosphatase (MabTech, Sweden) was added to each well and incubated for 2 h at room temperature. Finally, the plates were washed again in PBS and then BCIP / NBT Plus substrate (MabTech) was added to each well for 5–30 min for spot development. As soon as the development process was complete by visual inspection, 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).

[0185] For comprehensive analysis of T cell breadth, a standard IFNγ ELISPOT was modified herein as previously described in Shedlock DJ, et al. (2012). supra. Identification and measurement of subdominant and immunodominant T cell epitopes were assessed by stimulating splenocytes with individual peptides as opposed to total peptide pools or matrix peptide pools; traditional practices of pooling peptides for sample preservation, such as the use of matrix array pools, reduce assay sensitivity because total functional responses in pools containing multiple epitope-displaying peptides effectively reduce assay resolution, i.e., "drone out" to lower magnitudes. Therefore, modified ELISPOT was performed with individual peptides (15-mers overlapping by 9 amino acids; final concentration 2.5 μg / ml) across each GP immunogen. Peptides containing T cell epitopes were identified (≥10 AVE IFNγ+ spots and ≥80% animal response rate; summarized in Tables 1-6) and subsequently confirmed functionally and phenotypically by FACS. No shared or partial epitopes were identified, and neither the FACS data nor the web-based epitope prediction software suggested the presence of conserved CD4+ or CD8+ T cell epitopes within the consecutive peptides. Here, possible shared / partial T cell epitopes were addressed for all cases of consecutive peptide responses, as identified by a modified ELISPOT assay. Cells were stimulated with each of the consecutive peptides individually and paired in combination for direct comparison, and were defined as "shared / partial" if the combined response was not greater than either of the two individual responses. It should also be noted that the epitope responses presented here may not have been completely comprehensive, since the "15-mer with 9 amino acid overlap" algorithm for generating peptides is biased towards full coverage of CD8 T cell epitopes and may underestimate CD4 T cells 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.

[0186] Flow cytometry Splenocytes were added to a 96-well plate (1 × 10 6 Cells / well) were stimulated for 5-6 h with either individual peptides or a "minimal peptide pool" (final concentration 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). Splenocytes and transfected 293T were first pre-stained with the LIVE / DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen). For spleens, 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 Bioscience, San Jose, CA), washed three times with PBS+1% FBS, permeabilized with BD Cytofix / Cytoperm™ kit and then stained intracellularly for 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. Three times immunized H-2 bIndirect staining was performed after 30 min incubation at 4°C in PBS+1% FBS containing the indicated mouse-derived GP-specific polyclonal serum reagents (1:200 dilution), which were generated by pooling sera from mice with their respective DNA vaccines or pVAX1 empty vector control. Cells were then stained with FITC-conjugated goat anti-mouse IgG (BioLegend, San Diego, CA), washed extensively, and then stained with MHC class I (HLA-ABC; PE-Cy7; clone G46-2.6; BD). All cells were fixed with 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 markers CD19 and viability dye.

[0187] Figure 6 shows GP-specific T cell gating. Functional and phenotypic analysis of peptides containing T cell epitopes identified by ELISPOT was performed by FACS gating on total lymphocytes, live (LD) CD3+ cells that were negative for CD19 and LIVE-DEAD (dump channel), singlets (excluding cell doublets), CD4+ and CD8+ cells, activated cells (CD44+), and peptide-specific IFNγ-producing T cells.

[0188] statistical analysis The significance of the unrooted phylogenetic trees was determined by maximum likelihood and verified by bootstrap analysis, with significant support values ​​(≥80%; 1,000 bootstrap replicates) determined by MEGA version 5 software. Analysis of groups was completed by matched two-tailed independent t-tests, and survival curves were analyzed by log-rank (Mantel-Cox) tests. All values ​​are means ± SEM, and statistical analysis was performed with GraphPad Prism (La Jolla, CA).

[0189] result Vaccine construction and expression Phylogenetic analysis revealed that the EBOV GP (94.4% in SUDV and 92.9% in ZEBOV) is relatively conserved, whereas the MARV GP (MGP) is more diverse (approximately 70% conserved). Therefore, a consensus strategy determined by alignment of the predominant ZEBOV and SUDV GP amino acid sequences was employed for the EBOV GP, whereas a type-matching strategy using the 2005 Angola outbreak sequence, the largest and most lethal MARV outbreak, was used for MARV. Each GP transgene was gene-optimized, commercially synthesized, and then subcloned into a modified pVAX1 mammalian expression vector. Taken together, a three-plasmid strategy formed the basis of a novel multivalent-filovirus vaccine strategy.

[0190] HEK293T cells were transfected separately with each plasmid, and GP expression was assessed by Western immunoblotting and FACS. A protein of approximately 130 kDa was observed in each case in cell lysates taken 48 h after transfection using species-specific anti-GP1 mAbs for detection. The results are shown in Figure 1B. As a comparative control, recombinant vesicular stomatitis viruses (rVSVs) expressing each GP were loaded in parallel lanes. GP expression on the cell surface was then analyzed 24 h after transfection by indirect staining with GP-specific or control polyclonal sera by FACS. The results are shown in Figure 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, and positive sera did not react with pVAX1-transfected cells (data shown for pEBOZ). As expected for EBOV GP, cell surface expression leads to sterically occlusive recognition of surface MHC class I as well as β1 integrin (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).

[0191] Full protection against MARV and ZEBOV challenges To determine the efficacy of protection, 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. Although primary isolates of MARV and EBOV cause non-lethal disease in guinea pigs, a small number of passages in this host selects mutants capable of causing fatal disease with pathological features similar to those seen in filovirus-infected primates. Similarly, mice are also widely used in filovirus vaccine development, but unlike the guinea pig model, immunodetection reagents are widely available to assess immune and T cell responses. 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 seen in primates infected with EBOV.

[0192] Guinea pigs (n=24) were immunized twice intradermally with 200 μg of each plasmid (pEBOZ, pEBOS, and pMARV) at three separate vaccination sites or the pVAX1 empty vector control (n=9), and then boosted 1 month later with the same vaccine. Animals were immunized in a BSL-4 facility with 1000 LD of guinea pig-adapted MARV-Angora (gpMARV) (n=9) or ZEBOV (gpZEBOV) (n=15). 50The animals were challenged with gpMARV 28 days after the second immunization and observed and weighed daily. The results are shown in Figures 2A-2H. Vaccinated animals were fully protected, whereas control-vaccinated animals died by day 10 post-challenge with gpMARV (n=3; P=0.0052) or by day 7 post-challenge with gpZEBOV (n=6; P=0.0008) (Figures 2A and 2E). Furthermore, vaccinated animals were protected from weight loss (Figures 2B and 2F; P<0.0001). Vaccine-induced Abs likely contributed to protection, as GP-specific Abs 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 similar results were repeated twice, and error bars in Figures A-2H represent SEM. Group analysis was completed by matched two-tailed independent t-test, and survival curves were analyzed by log-rank (Mantel-Cox) test.

[0193] The plasmid vaccine was highly immunogenic To better characterize the immune correlates promoted by the protective DNA vaccines (plasmids pEBOZ, pEBOS, and pMARV, also called trivalent DNA vaccines), a wide range of immune detection reagents are available employing a mouse model that is widely used as a screening and "proof of concept" tool for filovirus vaccine development. First, B cell responses were assessed by H-2 immunization 20 days after each of the two vaccinations, 3 weeks between injections of 40 μg of each monovalent DNA vaccine, and 10 days after the first vaccination. dMice (n=5 / group) were evaluated for GP-specific IgG. Data from these experiments are shown in Figures 3A-3C. As shown in Figures 3A and 3B, little GP-specific IgG was observed in the prebleed control samples, but a significant increase was detected in all animals after vaccination. Purified SGP was not available, so purified ZGP was used as a surrogate. IgG from SUDV-vaccinated mice bound to ZGP, demonstrating vaccine-induced Ab generation and cross-species recognition capabilities. Furthermore, seroconversion occurred in 100% of vaccinated animals after one immunization, and the response was significantly increased thereafter by homologous boosting; AVE reverse end-point dilution titers increased 22.1-fold in pMARV-immunized mice and 3.4- and 8.6-fold in pEBOS- and pEBOZ-vaccinated animals, respectively. Samples were then assayed for neutralization of ZEBOV, SUDV-Boniface, and MARV-Angola in a BSL-4 facility. Results of the neutralization assays are shown in Figure 3C. A significant increase in NAb titers was detected after vaccination in all animals.

[0194] Mice of two different genetic backgrounds (H-2 d and H-2 b Mice (n=5 / group) were immunized with 40 μg of each plasmid pEBOZ, pEBOS and pMARV, boosted homologously 2 weeks later and sacrificed 8 days later for T cell analysis. The results of a novel modified ELISPOT assay to assess global vaccine-induced T cell responses in which splenocytes were stimulated with individual peptides as opposed to matrix pools are shown in Figure 4A. DNA vaccination induced robust IFNγ+ responses recognizing diverse 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 demonstrated low background responses from control wells (H-2 b In the IFNγ-producing SFC / 10 6 Spleen cells and H-2 dThe results shown in Figure 4A indicate that vaccination with pMARV inhibits H-2 b Induces nine measurable epitopes in mice and H-2 d pEBOS induced 11, pEBOS induced 9 and 8, and pEBOZ generated 10 and 12 in these strains, respectively. b Although five of the nine epitopes (55.6%) derived from mice were CD8+, these accounted for approximately 57.3% of the total MGP-specific IFNγ+ response as measured by both ELISPOT and FACS confirmation and phenotypic analysis. Similarly, only 33% and 38% of confirmed epitopes were expressed by pEBOS-immunized H-2 cells, respectively. b and H-2 d In both mouse strains, the CD8+ T cell response was estimated to be approximately 56%, whereas FACS estimates were approximately 50%. b and H-2 d The total CD8+ responses were lower in pEBOZ-vaccinated animals, ranging from 33% to 57% (33% for both strains by ELISPOT and 33% for H-2 by FACS). b and H-2 d In mice, it was measured as 6% and 57%, respectively.

[0195] A single immunodominant epitope was detected in both mouse strains fed pEBOS, roughly defined as the one that generated at least a two-fold increase in IFNγ response to the highest subdominant epitope; pMARV was the only mouse strain that generated a single immunodominant epitope that generated at least a two-fold increase in IFNγ response to the peptide MGP. 25-39 (#5), MGP 67-81 (#12), MGP 181-195 (#31) and MGP 385-399 4 H-2s in (#65) b Restricted immunodominant CD8+ epitopes, as well as MGP 151-171 H-2 in (#27) dAs shown in Figure 4B and Figure 4C, four of these epitopes are located within highly conserved regions of MARV GP1, three are located within the putative receptor binding domain, and only one is located within the variable mucin-like region (MGP1). 385-399 (#65)). pEBOS contains the highly conserved region of GP1, H-2 b Mouse and H-2 d SUDV GP (SGP) in mice 19-33 (#4) and SGP 241-255 (#41). However, pEBOZ immunization stimulated the CD8+ epitope present in H-2 d In mice, three immunodominant epitopes were identified within the receptor-binding domain and the mucin-like region, respectively (ZEBOV GP receptor-binding domain (GP) 139-153 A CD8-restricted epitope located in (#24), as well as two CD4-restricted epitopes ZGP 175-189 (♯30) and ZGP 391-405 (#66)). H-2 b In mice, only one immunodominant epitope was defined, which included both CD4+ and CD8+ epitopes (#89) and resided 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; total AVE subdominant responses, as measured by a modified ELISPOT assay, were significantly higher in H-2 mice immunized with pMARV, pEBOS, and pEBOZ. b In mice, the percentages were approximately 12%, 62%, and 74%, respectively, and H-2 d Responses in mice were 47%, 50% and 34%, respectively.

[0196] Finally, total GP-specific T cell responses were measured by FACS using stimulation with a minimal peptide pool containing only the identified epitope-containing peptides. A robust response was detected in each of the vaccinated animals, which in the majority of cases included both activated CD4+ and CD8+ T cells. The response was GP-specific, as little IFNγ production was observed with the control peptide (h-Clip), correlating well with the ELISPOT data. The only example in which immunization did not significantly induce CTLs as measured by FACS was H-2 mice vaccinated with pMARV, confirming that the epitopes identified by ELISPOT were not CD8-restricted. d In mice, the GP-specific T cell responses were highly immunogenic 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 use of traditional matrix array peptide pools for epitope identification may have overlooked the highly diverse subdominant T cell responses characterized herein.

[0197] T cell responses were measured by FACS for reactivity to a minimal peptide pool containing all positively identified peptides for each GP. Figure 7A shows DNA vaccine-induced T cell responses from a representative animal, with IFNγ-producing CD4+ (right) and CD8+ (left) cells gated. FACS plots are shown. Incubation in the presence of h-CLIP peptide served as a negative control (control). Figure 7B summarizes the results of the gated cells in Figure 7A as the mean % of total CD44+ / IFNγ+CD4+ or CD8+ cells, with error bars representing SEM. Experiments were repeated at least twice with similar results.

[0198] "Single-dose" protection in mice Next, vaccine efficacy against ZEBOV challenge was evaluated in a preclinical mouse model. Because of the strong NAb induction and protection data observed, mice were vaccinated only once. Mice (H-2 k (n=10 / group) were immunized with 40 μg of pEBOZ DNA and 28 days later administered 1,000 LD 50 Protection was assessed by challenging the mice with mouse-adapted ZEBOV (mZEBOV) from the vaccinated mice. By day 7 post-challenge, all control animals had succumbed to infection, whereas Figure 5A shows that DNA-vaccinated mice were fully protected (P=0.0002). Furthermore, as shown in Figure 5B, control mice showed a progressive loss of body weight until death (P<0.0001).

[0199] To better understand the mechanism of DNA-induced protection in the "single dose" model, we next evaluated the generation of NAb and T cells. Assessed 25 days after vaccination and 3 days before challenge, a significant (P<0.0001) increase was detected in all vaccinated animals (n=10 / group), as shown in Figure 5C. Reverse endpoint dilution titers ranged from 19 to 42, 27.3±2.5.

[0200] We next broadened the analysis to evaluate the generation of ZGP-specific T cells and compare responses in mice immunized with pEBOZ alone or with the trivalent formulation. IFN-γ production (n=5) was assessed after 11 days by FACS using the total ZGP peptide pool; data are shown in Figure 5D. IFNγ-producing T cells were detected in all animals and were specific for the ZGP peptide, since stimulation with the control peptide did not induce cytokine production. Immunization with either the monovalent or trivalent formulation induced robust IFNγ T cell responses, which were not significantly different when compared, P=0.0920.

[0201] CTLs may be important in eliminating 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 the additional effector cytokine, TNF, and the growth restriction factor, T hThe T-box transcription factor TBX21 (T-bet), known to correlate with type 1 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 approximately 61% and 33% of activated CD4+ and CD8+ T cells, respectively, produced TNF in addition to IFNγ. Furthermore, most 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.

[0202] 8A and 8B show T cell induction by "single dose" vaccination. H-2 T cell induction after a single pEBOZ immunization or a single trivalent vaccination containing the three vaccine plasmids at separate sites as measured by FACS. k T cell responses in mice are shown (a) and summarized (b) as AVE% of CD44+ / IFNγ+ CD4+ (purple) or CD8+ (orange) cells. 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 SEM; ns, not significant.

[0203] 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 two DNA vaccinations in guinea pigs and a "single dose" DNA vaccination in mice against mZEBOV. To date, genetic vaccination of guinea pigs has involved 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 DNA 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 Venezuelan equine encephalitis virus. Vaccine 21:4071-4080), but at least three vaccinations were required to achieve full protection with both methods.The improved protection herein may be due to the induction of potent Abs, since a single DNA vaccination produced GP-specific IgG binder titers comparable 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 after a single dose, respectively, 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). Virus-like particles show potential as pan-filovirus vaccines for both Ebola and Marburg virus infections (Vaccine 23:3033-3042). Furthermore, recombinant adenovirus (rAd) approaches induced lower ZGP-specific NAb titers (endpoint dilution titers 53 versus 88 herein) than those from 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 current platforms.Overall, these data indicate that DNA vaccination can induce binding and neutralizing Abs comparable to non-replicating viral platforms and may help to partially explain the strong guinea pig survival data herein.

[0204] The generation of NAbs by protective DNA vaccination may have benefited from the mature GP structure that expressed the transgene. In vitro transfection studies confirmed that the vaccine-encoded GP was highly expressed, posttranslationally cleaved (Fig. 1B), transported to the cell surface, and sterically blocked immune detection of cell surface molecules (Fig. 1C). Thus, the vaccine immunogen formed herein is likely to have matured into a heterotrimeric spike that would otherwise function on virion assembly during infection. This may be important for the generation and display of virologically relevant neutralizing determinants that are subsequently important 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). In this regard, expression of native anchor structures may therefore be superior to soluble derivatives in their 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).

[0205] To better characterize the T cell responses driven by the protective vaccine, we performed immunogenicity and efficacy studies in mice to determine complete "single-dose" protection against mZEBOV by DNA vaccination (Figures 5A-5D). To date, the most effective platform that confers complete protection in this model is VLP or rAd vaccination ((Kobinger GP,et al.(2006) supra;Choi et al.(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 ((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;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) 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)). 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 significantly limited in these studies, being restricted to splenocyte stimulation with either two (Warfield KL,(2007), supra) or one (Warfield KL,et al.(2005) supra) peptides previously described to contain the ZGP T cell epitope. (Warfield KL,et al.(2005) supra; Olinger GG,et al.(2005) supra; Kobinger GP,et al.(2006), supra; Sun Y,et al.(2009). Choi,JH,et al.(2012). Herein, we report the induction of robust and broad CTLs by protective vaccination, as extensively analyzed by a novel modified T cell assay (Figure 4A and Tables 1-6). A total of 52 novel T cell epitopes were identified, including a number of immunodominant epitopes that are predominantly present in highly conserved regions of GP. Of the total 22 ZGP epitopes identified, only 4 were previously reported. Moreover, one of the 20 MGP epitopes (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 1 of the 16 SGP epitopes had been previously described. Thus, this is the most comprehensive report of preclinical GP epitopes to date, describing GP epitopes from multiple filoviruses in mice of two different genetic backgrounds.

[0206] Another novel finding from these analyses was the assessment of vaccine-induced parathyroid T cell responses, which accounted for a significant proportion of the broad T cell responses, ranging broadly from 12% to 74% (Figure 4D). This may be particularly important as subdominant responses can contribute significantly to protection. Thus, determining the specific contribution of subdominant and immunodominant epitope T cell responses to protection may prove beneficial in the future. Of note, these responses may have been overlooked using traditional matrix array peptide pools for epitope identification. Thus, limited epitope detection in previous studies may be directly related to low 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.

[0207] Although the debate over immune correlates for filoviruses remains controversial, the data generated by this highly immunogenic approach provide a unique opportunity to study T cell immunity elicited by a protective vaccine. DNA vaccination herein induced potent ZGP-specific T cells, the majority of which express high levels of T-bet, which has been shown to correlate with T cell cytotoxicity in humans. hIt is characterized by type 1 polyfunctional CTLs. Previous standalone 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 shown to induce robust CD8+ T cells in NHPs and in the clinic. 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 rapid response during Filoviridae biothreat situations and outbreaks. Furthermore, this model approach provides an important tool to study protective immune correlates against filovirus disease and can be applied to existing platforms to guide future strategies.

[0208] 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).

[0209] Example 3 A five plasmid vaccine is provided. The first plasmid contains a nucleic acid sequence encoding ZEBOV CON, the Zaire Ebola virus consensus immunogen, SEQ ID NO: 1. The second plasmid contains a nucleic acid sequence encoding SUDV CON, the Sudan Ebola virus consensus immunogen, SEQ ID NO: 2. The third plasmid contains a nucleic acid sequence encoding Marburg Marburg virus-Rabin cluster consensus (MARV-RAV CON), SEQ ID NO: 4, with Marburg Marburg virus Rabin, Derba (09DRC99) and Uganda (02Uga07Y). The fourth plasmid contains a nucleic acid sequence encoding Marburg Marburg virus-Ozolin cluster consensus (MARV-OZO CON), SEQ ID NO: 5, with Ozolin, Uganda (01Uga07) and Derba (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) according to (Musoke, Popp, and Leiden).

[0210] Example 4 Five plasmid vaccines are provided. The first plasmid contains 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 contains 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 contains a nucleic acid sequence encoding a Marburg Marburg virus Rav consensus based on SEQ ID NO: 4, a Marburg Marburg virus-Rabin cluster consensus (MARV-RAV CON) modified to include an IgE signal peptide at the N-terminus of the consensus Marburg Marburg virus-Rav immunogen using Marburg Marburg virus Rabin Dhaka (09DRC99) and Uganda (02Uga07Y). The fourth plasmid contains a nucleic acid sequence encoding a Marburg virus-Ozo consensus based on SEQ ID NO:5, a Marburg virus-Ozorin cluster consensus (MARV-OZO CON) using Ozorin, Uganda (01Uga07), and Darva (05 and 07DRC99), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Ozo immunogen. The fifth plasmid contains a nucleic acid sequence encoding a Marburg virus-Musoke cluster consensus (MARV-MUS CON) using a Marburg virus-Mus consensus based on SEQ ID NO:6, (Musoke, Popp, and Leiden), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Mus immunogen.

[0211] Example 5 Six plasmid vaccines are provided. The first plasmid contains a nucleic acid sequence encoding ZEBOV CON, Zaire Ebola virus consensus immunogen, SEQ ID NO: 1. The second plasmid contains a nucleic acid sequence encoding SUDV CON, Sudan Ebola virus consensus immunogen, SEQ ID NO: 2. The third plasmid contains a nucleic acid sequence encoding Marburg Marburg virus-Rabin cluster consensus (MARV-RAV CON), SEQ ID NO: 4, Marburg Marburg virus-Rabin cluster consensus (MARV-RAV CON), with Marburg Marburg virus-Rabin, Derba (09DRC99) and Uganda (02Uga07Y). The fourth plasmid contains a nucleic acid sequence encoding Marburg Marburg virus-Ozorin cluster consensus (MARV-OZO CON), SEQ ID NO: 5, Marburg Marburg virus-Ozorin cluster consensus (MARV-OZO CON), with Ozorin, Uganda (01Uga07) and Derba (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) using (Musoke, Popp, and Leiden). The sixth plasmid contains SEQ ID NO:3, a nucleic acid sequence encoding the Marburg virus Angola 2005 isolated glycoprotein immunogen.

[0212] Example 6 Five plasmid vaccines are provided. The first plasmid contains a nucleic acid sequence encoding a Zaire Ebola virus consensus immunogen based on ZEBOV CON of 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 contains a nucleic acid sequence encoding a Sudan Ebola virus consensus immunogen based on SUDV CON of 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 contains a nucleic acid sequence encoding a Marburg Marburg virus Rav consensus based on SEQ ID NO: 4, a Marburg Marburg virus-Rabin cluster consensus (MARV-RAV CON) using Marburg Marburg virus Rabin Dhaka (09DRC99) and Uganda (02Uga07Y), modified to include an IgE signal peptide at the N-terminus of the consensus Marburg Marburg virus-Rav immunogen. The fourth plasmid contains a nucleic acid sequence encoding a Marburg virus-Ozorin cluster consensus (MARV-OZO CON) using the Marburg virus Ozo consensus, Ozorin, Uganda (01Uga07), and Darva (05 and 07DRC99) based on SEQ ID NO:5, modified to include an IgE signal peptide at the N-terminus of the consensus Marburg virus-Ozo immunogen. The fifth plasmid contains a nucleic acid sequence encoding a Marburg virus-Musoke cluster consensus (MARV-MUS CON) using the Marburg virus Mus consensus, (Musoke, Popp, and Leiden) based on SEQ ID NO:6, 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 MARV ANG, a Marburg virus Angola 2005 isolated glycoprotein immunogen based on SEQ ID NO:3, modified to include an IgE signal peptide at the N-terminus of the Marburg virus Angola immunogen.

[0213] Example 7 Herein, we describe three synthetic Zaire Ebola virus (EBOV) glycoprotein (GP) expressing DNA vaccine formulations: two designed based on GP sequence alignment (1976-2014) and a third construct adapted to the 2014 outbreak strain. Plasmid IL-12 (pIL-12) was also included as an adjuvant to further enhance the cellular immune response. Multivalent GP DNA vaccine formulations were administered to macaques following a DNA-DNA prime-boost immunization regimen. Macaques (n=3 or 4 / group) received multivalent GP DNA formulations + pIL-12 by intramuscular delivery followed by electroporation. Differences in immunogenicity were assayed and protection monitored at different doses, regimens (2, 3, 4, and 5 injections), and different intervals between subsequent doses. Both antibody and T cell responses were observed in 83% of animals 2 weeks after the first injection and in 100% of animals after the second injection. Macaques were challenged with a lethal dose of the EBOV Guinea Macona outbreak strain (1000 pfu, 7-U virus) and monitored for 28 days post-infection. 100% of animals receiving at least three injections spaced 4 weeks apart survived the lethal challenge. Animals were fully protected from signs of disease and showed no elevated blood chemistries. Interestingly, 50% of animals receiving two injections survived the lethal challenge. Surviving animals showed minimal signs of disease, suggesting that with further optimization, full protection with two injections is potentially achievable. Further optimization studies in mice found that a single injection was 100% protective and induced a long-term immune response 8 months after vaccination.

[0214] method Development of EBOV GP DNA vaccine formulations Vaccines currently in clinical trials include rVSVΔG / ZEBOVGP, ChAd3 prime+MVA boost, and MVA pan-filovirus. Although these vaccines are immunogenic, protective in non-human primates (NHPs), and provide single-dose protection, they develop anti-vector immunity, have an uncertain duration of memory response, and have resulted in adverse reactions in human clinical trials, and may not be suitable for all populations. Therefore, additional platforms with cleaner safety profiles that can induce strong immune responses against heterologous Zaire Ebola virus would be highly beneficial (Figure 9).

[0215] Three EBOV DNA constructs were designed: ZEBOV (1976-1996) (ConEBOVGP#1), ZEBOV (2002-2008) (ConEBOVGP#2), and a consensus sequence of matched ZEBOV sequences from the 2014 Guinea outbreak (Guinea GP). Five vaccines were produced, a monovalent vaccine containing only a single DNA construct, a bivalent vaccine formulation of ConEBOVGP#1 and Guinea GP, and a trivalent formulation containing all three, ConEBOVGP#1, ConEBOVGP#2, and Guinea GP (Figure 10).

[0216] result A single immunization of DNA vaccines is immunogenic in mice Following electroporation (EP) of the vaccine formulations, BALB / c mice were given a single intramuscular (IM) immunization. Total IgG antibody titers and ELISPOT-IFNγ were measured on day 28. Each vaccine formulation generated a robust IgG response. Polyfunctional CD4+ and CD8+ T cells secreting IFNγ, IL2, and TNFα were similar for all vaccines and formulations (Figure 11).

[0217] A single immunization is fully protective against lethal mouse-adapted Ebola virus challenge in mice BALB / c mice immunized with bivalent or trivalent vaccine formulations were given 1000LD 50The mice were lethally challenged with a heterologous Ebola virus of the same name. The challenge strain was mouse-adapted Ebola Mayinga 1976. Mice vaccinated with the control plasmid pVax1 rapidly lost weight and were not alive by day 7. However, no mice vaccinated with the bivalent or trivalent vaccine formulations died up to day 20 (Figure 12).

[0218] Individual GP DNA vaccine constructs induce robust memory responses in mice. BALB / c mice received 3xIM 40μg immunizations of the monovalent vaccine followed by electroporation on days 0, 28 and 84. IgG antibody titers, INFγ, CD4+CD44+ memory T cells, and CD8+CD44+ memory T cells were measured. Robust immune responses were detectable several months after the last injection (Figure 13).

[0219] GP DNA vaccine formulations are immunogenic in NHPs Cynomolgus macaques were used as NHPs because they are a model for Ebola vaccine efficacy and lethal challenge. Cynomolgus macaques were administered either the bivalent formulation with Rhesus pIL12 adjuvant or the trivalent formulation with Rhesus pIL12 adjuvant IM followed by EP. Different injection regimens to understand immunogenicity. Group 1 was given two IM-EP injections of the bivalent formulation, 4 weeks apart. Group 2 was given two IM-EP injections of the trivalent formulation, 4 weeks apart. Group 3 was given three IM-EP injections of the trivalent formulation. Samples for immunogenicity studies were collected monthly and for one month after the last dose. (Figure 6). Each DNA vaccine formulation induced a robust anti-Ebola GP antibody response (GMT>10 3 ) and induced anti-GP T cell responses. The immune response was boosted after each injection (Figure 14).

[0220] GP DNA-formulated vaccine protects against lethal Zaire Ebola virus (Makona) challenge Cynomolgus macaques from groups 1, 2 and 3 were challenged with 1000 TCID50 Guinea-Macona 2014C07 virus (7-U reference strain) 28 days after the final DNA immunization. Animals were monitored for 28 days after challenge. At 10 days after challenge, no control animals survived, whereas at 28 days after challenge, 4 / 4 of the group 3 animals survived, 4 / 8 of the group 2 animals survived, and 3 / 4 of the group 1 animals survived (Figure 15). Surviving animals had no signs of significant disease. They also maintained normal CBC and enzyme levels. Bivalent and trivalent DNA vaccines delivered by IM-EP induce long-term antibody and T cell responses detectable for more than 3 months after the final DNA injection (Figure 43).

[0221] Three doses of trivalent EBOV GP DNA vaccine are 100% protective against lethal EBOV challenge. Two doses of bivalent EBOV GP DNA vaccine provide 75% protection. Overall, the data support further study of DNA vaccines delivered by IM-EP for possible administration against Ebola and other infectious pathogens.

[0222] EBOV-001 Phase I Clinical An open-label study of INO-4212 (with or without INO-9012) was conducted. INO-4212 was administered IM or ID followed by electroporation in healthy volunteers. Safety and immunological assessments were monitored. Intradermal and intramuscular delivery were compared. A total of 69 subjects were included. ELISA analysis was performed prior to immunization and at weeks 2, 6, and 14 (baseline). Serology was defined as a positive IgG antibody response against the Ebola Zaire glycoprotein.

[0223] INO-4201 is a DNA vaccine formulated with the consensus envelope glycoprotein of Zaire Ebola virus (ConEBOVGP#1) generated using the envelope glycoprotein sequences of the 1976, 1994, 1995, 1996, 2003, 2005, 2007, and 2008 outbreak strains and driven by the human CMV promoter (hCMV promoter) with the bovine growth hormone 3'-terminal polyadenylation signal (bGH polyA). pGX4201 was generated by cloning the synthetic consensus envelope glycoprotein gene of Zaire Ebola virus into pGX0001 at the BamHI and XhoI sites.

[0224] The ConEBOVGP#1 (ConGP1) sequence was constructed by generating a consensus envelope glycoprotein sequence of Zaire Ebola virus using the envelope glycoprotein sequences of outbreak strains from 1976, 1994, 1995, 1996, 2003, 2005, 2007, and 2008. Briefly, a consensus GP sequence was first generated based on six envelope sequences from outbreak strains from 1976, 1994, 1995, 1996, 2003, and 2005. Three non-consensus residues at positions 377, 430, and 440 were then weighted in the 2003, 2005, 2007, and 2008 strains, as these were the most recent deadly outbreaks with published sequence data. The GenBank accession numbers of the selected outbreak strain GP sequences are: Q05320, P87671, AAC57989, AEK25495, ABW34743, P87666, AER59718, AER59712, ABW34742, AAL25818. After obtaining the consensus GP1 sequence, an upstream Kozak sequence was added to the N-terminus. Furthermore, the codon usage of this gene was adapted to the codon bias of Homo sapiens genes to achieve higher levels of expression. Furthermore, RNA optimization was also performed, avoiding very high (>80%) or very low (<30%) GC content and cis-acting sequence motifs such as internal TATA boxes, chisites, and ribosome entry sites. The synthesized ConGP1 was digested with BamHI and XhoI and cloned into an expression vector.

[0225] INO-4202 is a DNA vaccine formulated with a DNA plasmid driven by the human CMV promoter (hCMV promoter) and the 3'-terminal adenylation signal of the bovine growth hormone (bGH polyA) poly-HCMV promoter and expressing the envelope glycoprotein of Zaire Ebola virus isolated from the 2014 outbreak in Guinea (Guinea GP).

[0226] INO-4212 is a bivalent vaccine of INO-4201 and INO-4202.

[0227] Figure 17 shows the vaccine formulation schedule, route and dose for each cohort. After the first injection, 15% or less of patients were seropositive. After the second injection, 50-100% of patients were seropositive. After the third injection, 79-100% of patients were seropositive (Figure 18). Two representative patients with moderate or high IFNγ ELISpot showed specific T cell responses (Figure 19).

[0228] While other Ebola vaccine platforms, including NIAID VRC / GSK and rVSV / ZEBOVGP, are currently in clinical trials, the current bivalent and trivalent vaccines described herein have advantages not seen with other vaccine platforms. For example, bivalent or trivalent vaccines can be administered IM or ID, whereas other vaccine platforms are administered IM only. Importantly, NIAID VRC / GSK and rVSV / ZEBOVGP exhibit side effects including fever, fatigue, arthralgia, and lymphopenia, whereas bivalent and trivalent vaccines do not. However, it should be noted that some of the side effects of rVSV / ZEBOVGP and ChAd3 / MVAGP overlap with symptoms of Ebola. Additionally, the bivalent and trivalent vaccines confer antibody titers that are 1-2 orders of magnitude greater than rVSV / ZEBOVGP and ChAd3 / MVAGP (Figure 20).

[0229] Subjects (n=15) were assigned to receive INO-4201 as two separate 1 mg (0.1 mL) ID (Mantoux) injections at 2 mg DNA / dose using a CELLECTRA®-3P device, followed by EP. Subjects received a three-dose sequence immunizing at weeks 0, 4, and 12 (0-4-12 week schedule). Antibodies specific to EBOV glycoprotein (GP) were measured by binding ELISA from sera of vaccinated subjects. Reverse endpoint titers above day 0 are shown 2 weeks after each immunization (Figure 22). 100% of subjects vaccinated with INO-4201 seroconverted after two immunizations (Figure 22, cohort 3, and Figure 24).

[0230] Example 8 Immune response data for three EBOV DNA constructs described in Example 7 are described: ZEBOV (1976-1996) (ConEBOVGP#1 or INO-4201), the consensus sequence for ZEBOV (2002-2008) (ConEBOVGP#2), and the matched ZEBOV sequence from the 2014 Guinea outbreak (Guinea GP). Five vaccines were developed, a monovalent vaccine containing only a single DNA construct, a bivalent vaccine formulation containing either ConEBOVGP#1 and Guinea GP, and a trivalent formulation containing all three ConEBOVGP#1, ConEBOVGP#2, and Guinea GP. Sixty-nine subjects from cohorts 1-5 were included in the analysis of immune responses by ELISA, and 75 subjects from cohorts 1-5 were included in the analysis of immune responses by ELISpot (Figures 21, 25).

[0231] ELISA titers by cohort and time point Anti-EBOVR titers were measured for each cohort at weeks 2, 6, and 14. By week 6, each cohort saw an increase in antibody titers above day 0 (Figures 22-23). ​​There was little to no reactivity to the first dose in each cohort. Dose 2 initiates seroconversion with cohorts 3 and 5 showing the greatest frequency. Dose 3 results in >90% seroconversion in 4 / 5 cohorts. Cohorts 3 and 5 show 100% seroconversion at this time point (Figure 24).

[0232] Subject response to peptide pools Cohort responses were analyzed by peptide pool (Figures 26-33). To analyze ELISpot outliers, the day 0 values ​​of each pool and the total EBOV response were used to create an outlier threshold (mean day 0 value + (3 x STDEV of day 0)). This threshold should encompass 99% of a normally distributed population. All subjects who displayed baseline values ​​greater than the outlier threshold were removed and a response criterion was generated with the remaining subjects.

[0233] ICS analysis 47 subjects from all cohorts were included in the analysis, with cohort 5 being underrepresented (Figure 35). ICS analysis was performed at baseline and week 14. A single EBOV peptide pool consisting of pools 1-4 is used for stimulation. Analysis of T cell activity in the form of IFNg or TNFa production from both CD4 and CD8 compartments suggests a significant increase in TNFα in both CD4 and CD8 compartments as well as an increase in TNFα and / or IFNg in cohort 3 only (Wilcoxon matched pairs analysis, two-tailed).

[0234] Summary of Immunity 100% of patients in cohort 3 (ID) seroconverted after 2 doses. 92% of patients in cohort 5 (IM+IL12) seroconverted after 2 doses and 100% after 3 doses. Other cohorts showed a maximum of 67% after 2 doses and as high as 93% after 3 doses.

[0235] Analyzing all patients: the best response frequencies were in cohorts 2 and 4, 53% and 57%, respectively. Cohort 3 showed a 40% response. The addition of IL-12 in cohort 5 did not seem to affect the response rate (47%). Analyzing patients excluding the 8 outlying outliers, the response frequencies were 84.6% and 76.9% in cohorts 2 and 4, respectively. Cohort 3 showed a 64.3% response. The addition of IL-12 in cohort 5 did not seem to affect the response rate (53.3%).

[0236] Both CD4 and CD8 T cells showed higher expression of TNFα and TNFα and / or IFNg in cohort 3 (statistically significant vs. baseline, Wilcoxon matched-pair test, two-tailed).

[0237] Immunization with INO-4201 was well tolerated in healthy volunteers, with no grade 3 or grade 4 SAEs. INO-4201 induced robust Ebola GP-specific antibodies (GMT 46,968) and resulted in 100% seroconversion by binding ELISA after two doses of INO-4201. INO-4201 administration was consistent with interferon-gamma (IFNγ) ELISpot (10 6 INO-4201 produced EBOV GP-specific T cell responses as assessed by immunoglobulin E (IgE) of 295.3 SFU per PBMC and significant increases in IFNγ or TNFα production in both the CD8+ and CD4+ T cell compartments. Intradermal administration of INO-4201 using the Cellectra device was both well tolerated and immunogenic, as assessed by both humoral and cellular EBOV GP-specific immunoassays. These results indicate that INO-4201 is a strong candidate for further clinical development as a prophylactic Ebola vaccine.

[0238] Example 9 - Sustained humoral and cellular immune responses in cynomolgus monkeys following administration of Zaire Ebola virus (EBOV) GP DNA vaccine delivered by intramuscular electroporation Herein, we present a novel Ebola virus disease (EVD) DNA vaccine with a clean safety profile and that is serum independent, allowing for possible repeated vector administration. Three novel synthetic Zaire Ebola virus (EBOV) GP DNA vaccines were designed and developed in bivalent or trivalent formulations. Both EBOV-GP DNA vaccines were highly protective (75-100%) against lethal EBOV Macona C07 challenge in cynomolgus macaques. Animals (n=4-5 / group) in the different regimens were followed to monitor long-term immunogenicity following DNA immunization. All NHPs rapidly seroconverted. NHPs have resistant total IgG antibody titers and T cell responses to EBOV GP antigens, including responses in polyfunctional CD4 and CD8 T cell and memory subset populations expressing IFNγ, IL2, and TNFα (Figures 47-59). Together, the data strongly support the delivery of EBOV-GP DNA vaccines for the generation of robust memory immune responses and protection.

[0239] The EBOV-GP Dan vaccine induces long-term immune responses, with strong recall responses after a one-year booster immunization (Figures 49-50). The recall response was significantly higher in the group given a single IM injection (Figure 50).

[0240] Example 10 The peptide sequences and nucleic acid sequences of the peptides are presented here. [Table 1] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6 splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ). [Table 2] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6 splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ). [Table 3] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ). [Table 4] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6 splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ). [Table 5] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6 splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ). [Table 6] Epitope-containing peptides were analyzed by IFNγ ELISPOT (SFC of ≥10 / 10 6splenocytes and a response rate of ≥80) and confirmed by FACS (≥3–5 × 10 4 Each response was further characterized by FACS (CD4 and / or CD8 expression by CD3+ / CD44+ / IFNγ+ cells). Predicted CD8+ epitopes are underlined (highest consensus % rank by IEDB) and refer to previous epitopes. Immunodominant epitopes are indicated ( * ).

[0241] [1] 1. An isolated nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of a nucleic acid encoding a first consensus Ebola virus envelope glycoprotein immunogen (ZEBOVCON), a nucleic acid encoding a second consensus Ebola virus envelope glycoprotein immunogen (ZEBOVCON2), and a nucleic acid encoding a ZEBOV Guinea 2014 outbreak envelope glycoprotein immunogen (ZEBOVGUI), said ZEBOVCON comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1, or a fragment of an amino acid sequence that is at least 95% identical to SEQ ID NO:1; said ZEBOVGUI comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:67, or a fragment of an amino acid sequence that is at least 95% identical to SEQ ID NO:67; and The isolated nucleic acid molecule, wherein the ZEBOVCON2 comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:68, or a fragment of an amino acid sequence that is at least 95% identical to SEQ ID NO:68. [2] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVCON comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:1, or a fragment of an amino acid sequence that is at least 99% identical to SEQ ID NO:1. [3] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVCON comprises the amino acid sequence of SEQ ID NO:1, or a fragment of SEQ ID NO:1. [4] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVGUI comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:67, or a fragment of an amino acid sequence that is at least 99% identical to SEQ ID NO:67. [5] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVGUI comprises the amino acid sequence of SEQ ID NO:67 or a fragment of SEQ ID NO:67. [6] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVCON2 comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:68, or a fragment of an amino acid sequence that is at least 99% identical to SEQ ID NO:68. [7] 2. The isolated nucleic acid molecule of claim 1, wherein the ZEBOVCON2 comprises the amino acid sequence of SEQ ID NO:68 or a fragment of SEQ ID NO:68. [8] 2. The isolated nucleic acid molecule of item 1, wherein the fragment of the amino acid sequence that is at least 95% homologous to SEQ ID NO:1 comprises at least 600 amino acids, at least 630 amino acids, or at least 660 amino acids. [9] 2. The isolated nucleic acid molecule of item 1, wherein the fragment of the amino acid sequence at least 95% identical to SEQ ID NO: 67 comprises at least 600 amino acids, at least 630 amino acids, or at least 660 amino acids.

[10] 2. The isolated nucleic acid molecule of item 1, wherein the fragment of the amino acid sequence at least 95% identical to SEQ ID NO: 68 comprises at least 600 amino acids, at least 630 amino acids, or at least 660 amino acids.

[11] 2. The isolated nucleic acid molecule of item 1, wherein the ZEBOVCON is linked to an IgE signal peptide.

[12] 2. The isolated nucleic acid molecule of item 1, wherein the ZEBOVGUI is linked to an IgE signal peptide.

[13] 2. The isolated nucleic acid molecule of item 1, wherein the ZEBOVCON2 is linked to an IgE signal peptide.

[14] 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid encoding ZEBOVCON comprises a nucleic acid sequence of SEQ ID NO: 69 that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 69, or a fragment thereof.

[15] 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid encoding ZEBOVGUI comprises a nucleic acid sequence of SEQ ID NO: 72 or a fragment thereof that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 72.

[16] 2. The isolated nucleic acid molecule of item 1, wherein the nucleic acid encoding ZEBOVCON2 comprises a nucleic acid sequence of SEQ ID NO: 70 or a fragment thereof that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 70.

[17] A composition comprising the nucleic acid molecule of item 1.

[18] 19. The composition of claim 18, wherein the composition comprises two nucleic acid molecules.

[19] 19. The composition of claim 18, wherein the composition comprises three nucleic acid molecules.

[20] 2. The composition of claim 1, wherein the nucleic acid molecule is a plasmid. [twenty one] 21. The composition of any of items 1 to 20, formulated for delivery to an individual using electroporation. [twenty two] 21. The composition according to any of items 1 to 20, further comprising a nucleic acid sequence encoding one or more proteins selected from the group consisting of IL-12, IL-15 and IL-28. [twenty three] 21. A method for inducing an immune response against a filovirus, comprising administering to an individual the composition according to any one of items 1 to 20 in an amount effective to induce an immune response in said individual. [twenty four] 21. A method for inducing an immune response against Ebola virus, comprising administering to an individual the composition according to any one of items 1 to 20 in an amount effective to induce an immune response in the individual. [twenty five] 21. A method for treating an individual diagnosed with Ebola virus, comprising administering to the individual a therapeutically effective amount of the composition according to any one of items 1 to 20.

[26] 21. A method for treating an individual diagnosed with Ebola virus, comprising administering to the individual a therapeutically effective amount of a composition according to any of items 1 to 20.

[27] 21. A method for preventing Ebola virus infection in an individual, comprising administering to the individual a prophylactically effective amount of the composition according to any one of items 1 to 20.

[28] 21. A method for preventing Ebola virus infection in an individual, comprising administering to the individual a prophylactically effective amount of the composition according to any one of items 1 to 20.

Claims

[Claim 1] The invention described in the specification.

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