OA19176A

OA19176A - Methods and compositions for inducing protective immunity against a Marburg virus infection.

Info

Publication number: OA19176A
Application number: OA1201900012
Authority: OA
Inventors: Benoit Christophe Stephan Callendret; Ramon Roozendaal; Jutta Sytske Hartkoorn-Pasma; Ariane Volkmann; Robin Steigerwald; Ulrike Dirmeier
Original assignee: Bavarian Nordic A/S; Janssen Vaccines & Prevention B.V.
Priority date: 2016-07-15
Filing date: 2017-07-14
Publication date: 2020-03-09

Abstract

Compositions, vaccines and methods using a combination of adenovirus vectors and MVA vectors for inducing protective immunity against a Marburg virus infection are described.

Description

METHODS AND COMPOSITIONS FOR INDUCING PROTECTIVE IMMUNITY AGAINST A MARBURG VIRUS INFECTION

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with govemment support under contract HHSN272200800056C, awardçd by the National Institute of Health. The govemment has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is entitled to priority purs,uant to 35 U.S.C. § 119(e) to U.S. Provisional

Patent Application No. 62/362,831 filed July 15, 2016, the disclosures of which are herein incorporated by reference in their entirety,

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS15 Web as an ASCII formatted sequence listing with a file name “315wo_ST25”, création date of July 11, 2017, and having a size of about 30,152 bytes. The sequence listing submitted concurrently via EFS-Web is part ofthe spécification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions, vaccines and methods for inducing protective immunity against a Marburg virus infection.

BACKGROUND OF THE INVENTION

Ebolaviruses, such as Zaïre ebolavirus (EBOV) and Sudan ebolavirus (SUDV), and the closely related Marburg virus (MARV), are associated with outbreaks of highly léthal Hémorrhagie Fever (HF) in humans and primates, mainly located in Africa with occasional spillover in North America and Europe. These viruses are filoviruses that are known to infect humans and nonhuman primates with severe health conséquences, including death. Filovirus infections hâve resulted in case fatality rates of up to 90% in humans. EBOV, SUDV, and MARV infections cause HF with death often occurring within 7 to 10 days post-infection. HF présents as an acute febrile syndrome manifested by an abrupt fever, nausea, vomiting, diarrhea, maculopapular rash, malaise, prostration, generalized signs of increased vascular permeability, coagulation abnormalities, and dysrégulation of the innate immune response. Much of the disease appears to be caused by dysrégulation of innate immune responses to the infection and by réplication of virus in vascular endothélial cells, which induces death of host cells and destruction of the endothélial barrier.

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Filoviruses can be spread by small particle aérosol or by direct contact with infected blood, organs, and body fluids of human or NHP origin. Infection with a single virion is reported to be suffïcient to cause HF in humans. Presently, there is no therapeutic or vaccine approved for treatment or prévention of HF. Supportive care remains the only approved medical intervention for individuals who become infected with filoviruses.

As the cause of severe human disease, filoviruses continue to be of concem as both a source of naturel infections, and also as possible agents of bioterrorism. The réservoir for filoviruses in the wild has not yet been defînitively identified. Four subtypes of Ebolaviruses hâve been described to cause HF, i.e., those in the Zaire, Sudan, Bundibugyo and Ivory Coast épisodes (Sanchez, A. et al. 1996 PNAS USA 93:3602-3607). These subtypes of Ebolaviruses hâve similar genetic organizations, e.g., negative-stranded RNA viruses containing seven linearly arrayed genes. The structural gene products of Ebolaviruses include, for example, the envelope glycoprotein that exists in two alternative forms, a secreted soluble glycoprotein (ssGP) and a transmembrane glycoprotein (GP) generated by RNA editing that médiates viral entry (Sanchez, et al. 1996 PNAS USA 93:3602-3607).

Marburg virus (MARV) causes Marburg virus disease (MVD), also referred to as Marburg hémorrhagie fever (MHF), in humans and nonhuman primates. The disease names are derived from the German city Marburg, where MARV was first discovered in an outbreak in 1967. Comparative analyses of the GP and viral protein (VP) 35 genes of MARV strains showed that there are two distinct lineages within the Marburg marburgvirus species of MARV, the Marburg lineage and Ravn lineage. There are a number of isolated strains of the Marburg lineage, such as the original MARV isolâtes from the 1967 épisodes in Marburg (Popp and Ratayczak strains), the Ozolin strain from a case in 1975 in South Africa, the Musoke strain (MARV-Musoke) from a 1980 case in Kenya, and a seemingly more pathogenic Angola strain (MARV-Angola). The Ravn lineage includes an isolate from Kenya in 1987 (Ravn strain). See Daddario-DiCaprio et al., J. of Virology, Oct. 2006, p. 9659-9666. The structural proteins of MARV include four proteins (NP, VP35, VP30, and L) that make up the helical nucleocapsid, which is surrounded by a matrix that is composed of the viral proteins VP40 and VP24. The surface of MARV virions is coated with spikes that consist of the structural glycoprotein (GP). The MARV GP plays a rôle in virus entry and pathogenesis and serves as a major and logical target for vaccine strategies. Id.

It has been suggested that immunization can be useful in protecting against filovirus infection because there appears to be less nucléotide polymorphism within filovirus subtypes than among other RNA viruses (Sanchez et al. 1996 PNAS USA 93:3602-3607). Until recently, developments of préventive vaccines against filoviruses hâve had variable results, partly because the requirements for protective immune responses against filovirus infections are poorly understood. Additionally, the large number of fïloviruses circulating within naturel réservoirs complicates efforts to design a vaccine that protects against ail species of fïloviruses.

Currently, there are several vaccine antigen delivery platforms that demonstrated varions levels of protection in non-human primates (NHPs) exposed with high infections doses of fïloviruses. Vaccine candidates are in development based on a variety of platform technologies including réplication competent vectors (e.g. Vesicular Stomatitis Virus; Rabies virus; Parainfluenza Virus); réplication incompetent vectors (Adenovirus, Modified Vaccinia Ankara Virus); protein subunits inclusive of Virus Like Particles expressed in bacterial cells, insect cells, mammalian cells, plant cells; DNA vaccines; and /or live and killed attenuated filovirus (Friedrich et al., 2012). The EBOV glycoprotein GP is an essential component of a vaccine that protects against exposures with the same species of EBOV. Furthermore, inclusion of the GP from F.BOV and SUDV, the two most virulent species of ebolaviruses, can protect monkeys against EBOV and SUDV intramuscular exposures, as well as exposures with the distantly related Bundibugyo (BDBV), Taï Forest ebolavirus (TAFV; formerly known as Ivory Coast or Cote d’ivoire ) species. Likewise, inclusion of the GP from MARV can protect monkeys against MARV intramuscular and aérosol exposures. The development of medical countermeasures for these viruses is a high priority, in particular the development of a PAN-filovirus vaccine - that is one vaccine that protects against ail pathogenic fïloviruses.

Replication-defective adenovirus vectors (rAd) are powerful inducers of cellular immune responses and hâve therefore corne to serve as useful vectors for gene-based vaccines particularly for lentiviruses and fïloviruses, as well as other nonviral pathogens (Shiver, et al., (2002) Nature 415(6869): 331-5; (Hill, et al., Hum Vaccin 6(1): 78-83. ; Sullivan, et al., (2000) Nature 408(6812): 605-9; Sullivan et al., (2003) Nature 424(6949): 681-4; Sullivan, et al., (2006) PLoS Med 3(6): el77; Radosevic, et al., (2007); Santra, et al., (2009) Vaccine 27(42): 5837-45.

Adenovirus-based vaccines hâve several advantages as human vaccines since they can be produced to high titers under GMP conditions and hâve proven to be safe and immunogenic in humans (Asmuth, et al., J Infect Dis 201(1): 132-41; Kibuuka, et al., J Infect Dis 201(4): 600-7; Koup, et al., PLoS One 5(2): e9015. ; Catanzaro, et al., (2006) J Infect Dis 194(12): 1638-49; Harro, et al., (2009) Clin Vaccine Immunol 16(9): 1285-92). While most of the initial vaccine work was conducted using rAd5 due to its significant potency in eliciting broad antibody and CD8+ T cell responses, pre-existing immunity to rAd5 in humans may limit efficacy (Catanzaro, (2006); Cheng, et al., (2007) PLoS Pathog 3(2): e25.; McCoy, et al., (2007) J Virol 81(12): 6594-604.; Buchbinder, et al., (2008) Lancet 372(9653): 1881 -93). This property might restrict the use of rAd5 in clinical applications for many vaccines that are currently in development including Ebolavirus (EBOV) and Marburg virus (MARV).

Replication-defective adenovirus vectors, rAd26 and rAd35, derived from adenovirus serotype 26 and serotype 35, respectively, hâve the ability to circumvent Ad5 pre-existing immunity. rAd26 can be grown to high titers in Ad5 El-complementing cell lines suitable for manufacturing these vectors at a large scale and at clinical grade (Abbink, et al., 2007, Virol 81(9): 4654-63), and this vector has been shown to induce humoral and cell-mediated immune responses in prime-boost vaccine strategies (Abbink, et al., 2007, supra; Liu, et al., (2009) Nature 457(7225): 87-91). rAd35 vectors grow to high titers on cell lines suitable for production of clinical-grade vaccines (Havenga, et al., (2006) J Gen Virol 87(Pt 8): 2135-43), and hâve been formulated for injection as well as stable inhalable powder (Jin, et al., Vaccine 28(27): 4369-75). These vectors show efficient transduction of human dendritic cells (de Gruijl, et al., (2006) J Immunol 177(4): 2208- 15; Lore, et al., (2007) J Immunol 179(3): 1721-9), and thus hâve the capability to médiate high level antigen delivery and présentation.

Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, a member of the généra Orthopoxvirus in the family of Poxviridae. Poxviruses are known to be good inducers of CD8 T cell responses because of their intracytoplasmic expression. However, they are generally believed to be poor at generating CD4 MHC class II restricted T cells (see for example Haslett et al. Journal of Infections Diseases 181 : 1264-72 (2000), page 1268). MVA has been engineered for use as a viral vector for recombinant gene expression or as recombinant vaccine.

Strains of MVA having enhanced safety profiles for the development of safer products, such as vaccines or pharmaceuticals, hâve been developed by Bavarian Nordic. MVA was further passaged by Bavarian Nordic and is designated MVA-BN, a représentative sample of which was deposited on August 30, 2000 at the European Collection of Cell Cultures (ECACC) under Accession No. V00083008. MVA-BN is further described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699), both of which are incorporated by reference herein in their entirety.

MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. MVA-BN is réplication incompetent, meaning that the virus does not replicate in human cells. In human cells, viral genes are expressed, and no infections virus is produced. MVABN is classified as Biosafety Level 1 organism according to the Centers for Disease Control and Prévention in the United States. Préparations of MVA-BN and dérivatives hâve been administered to many types of animais, and to more than 2000 human subjects, including immune-deficient individuals. Ail vaccinations hâve proven to be generally safe and well tolerated. Despite its high atténuation and reduced virulence, in preclinical studies MVA-BN has been shown to elicit both humoral and cellular immune responses to vaccinia and to heterologous gene products encoded by genes cloned into the MVA genome (E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300; A.

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Cosma et al. (2003), Vaccine 22(l):21-9; M. Di Nicola et al. (2003), Hum. Gene Ther. 14(14): 1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res., 10(16):5381-5390).

There is an unmet need for improved vaccines that elicit immune responses against Marburg viruses.

BRIEF SUMMARY OF THE INVENTION

It is discovered in the présent invention that varions prime-boost combinations of réplication incompetent vectors generate effective immune protection against Marburg virus infections.

Accordingly, one general aspect of the présent invention relates to a combination vaccine comprising:

(i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus, together with a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of at least one additional filovirus subtype, together with a pharmaceutically acceptable carrier;

wherein the first antigenic protein and the second antigenic protein share at least one antigenic déterminant, and one of the compositions is a priming composition and the other composition is a boosting composition. Preferably, the first composition is the priming composition and the second composition is the boosting composition, and more preferably, the boosting composition is to be administered 1-12 weeks after the priming composition.

Another general aspect of the présent invention relates to the use of:

a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus; and a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of at least one additional filovirus subtype, for generating a protective immune response against a Marburg virus; wherein the first and second compositions are used for priming and for boosting said immune response, respectively, or vice versa, and the first antigenic protein and the second antigenic protein share at least one antigenic déterminant. Preferably, the first composition is used for priming and the second composition is used for boosting, and more preferably, the boosting composition is administered 1-12 weeks after the priming composition.

The Marburg virus according to the présent invention can be any isolate or strain of the

Marburg marburgvirus species, including but not limited to, the Marburg Popp strain, Ratayczak strain, Ozolin strain, Musoke strain (MARV-Musoke), Angola strain (MARV-Angola), and the

Ravn strain.

The antigenic proteins can be any protein comprising an antigenic déterminant. In a preferred embodiment the antigenic proteins include Marburg virus glycoproteins (GPs) or nucleoproteins (NPs), and the antigenic proteins can further be one or more antigenic protein from at least one additional filovirus, such as Ebola. Other antigenic proteins, such as an NP, VP35, VP30, L, VP40, VP24 or GP of a Marburg virus, can also be used in the invention. The antigenic proteins encoded by the adenovirus vectors or MVA vectors comprised in the first and second composition according to embodiments of the présent invention can include any antigenic protein from any isolate of Marburg virus, preferably, GPs of at least one of MARV-Musoke and MARVAngola.

In a preferred embodiment, the adenovirus vector in the first composition (i) comprises a nucleic acid encoding a first antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3, and the MVA vector in the second composition (ii) comprises a nucleic acid encoding a second antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

In yet another preferred embodiment, the MVA vector in the second composition (ii) comprises a nucleic acid encoding antigenic proteins of at least four filovirus subtypes. Preferably said MVA vector comprises a nucleic acid encoding antigenic proteins from four different filovirus subtypes, and the antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

In a preferred embodiment, the adenovirus vectors used in the first composition is a rAd26 vector. It is contemplated that the methods, vaccines, and compositions described herein can be embodied in a kit. For example, in one embodiment, the présent invention can include a kit comprising:

(i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus, together with a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of one or more additional filovirus subtypes, together with a pharmaceutically acceptable carrier;

Therefore in a preferred embodiment, the présent invention relates to a combination vaccine, a kit or a use wherein the adenovirus vector in composition (i) comprises a nucleic acid encoding a first antigenic protein having the amino acid sequence of SEQ ID NO: 3; and wherein the MVA vector in composition (ii) comprises a nucleic acid encoding antigenic proteins from four different filovirus subtypes and the antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

In a preferred embodiment, the adenovirus vectors comprised in the combination vaccine, kit of the présent invention, or the adenovirus vectors used for generating a protective immune response against at least one Marburg virus are rAd26 or rAd35 vectors. In a preferred embodiment said adenovirus vectors are rAd26 vectors. In a preferred embodiment of this use, the boosting composition is administered 1-12 weeks after the administration of the priming composition.

One additional general aspect of the présent invention relates to a method of inducing an immune response against a Marburg virus in a subject, the method comprising:

a. administering to the subject a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic glycoprotein of a Marburg virus; and

b. administering to the subject a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of one or more additional filovirus subtypes ;

wherein steps (a) and (b) are conducted in either order, and the first antigenic protein and the second antigenic protein share at least one antigenic déterminant.

In another embodiment, the adenovirus vector in the first composition comprises a nucleic acid encoding a first antigenic protein having the amino acid sequence of SEQ ID NO:3, and the MVA vector in the second composition comprises a nucleic acid encoding a second antigenic protein having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

In an even more preferred embodiment, the MVA vector in the second composition comprises a nucleic acid encoding antigenic proteins of at least four filovirus subtypes. Preferably said MVA vector comprises a nucleic acid encoding antigenic proteins from four different filovirus subtypes, and the antigenic proteins hâve the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:

2, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

Therefore, in a preferred embodiment, the présent invention relates to method of inducing an immune response against a Marburg virus in a subject, wherein the adenovirus vector in the first composition comprises a nucleic acid encoding an antigenic protein having the amino acid sequence of SEQ ID NO: 3; and wherein the MVA vector in the second composition comprises a nucleic acid encoding antigenic proteins from four different filovirus subtypes and the antigenic proteins hâve the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

In a preferred embodiment, the adenovirus vectors used in the method of the présent invention are rAd26 or rAd35 vectors.

In another preferred embodiment of the présent invention, step (a) and step (b) of the method are conducted 1-12 weeks apart. One of the steps (a) and (b) is conducted for priming vaccination and the other step for boosting vaccination.

In another preferred embodiment, the priming vaccination step (a) is conducted at week 0, followed by the boosting vaccination step (b) at week 1-10, more preferably at week 6-10 and even more preferably at week 8. In another preferred embodiment, the priming vaccination step (a) is conducted at week 0, followed by the boosting vaccination step (b) at week 1 -4, preferably at week 1,2 or 4.

In another preferred embodiment, the priming vaccination step (b) is conducted at week 0, followed by the boosting vaccination step (a) at week 1-10, more preferably at week 6-10 and even more preferably at week 8. In another preferred embodiment, the priming vaccination step (b) is conducted at week 0, followed by the boosting vaccination step (a) at week 1 -4, preferably at week 1,2 or 4.

In a preferred embodiment of the présent invention, the method comprises a priming vaccination with an immunologically effective amount of an rAd26 vector expressing a first Marburg virus antigenic protein, followed by a boosting vaccination with an immunologically effective amount of one or more vectors different from rAd26, preferably MVA vectors, expressing a second Marburg virus antigenic protein that shares at least one antigenic déterminant with the first Marburg virus antigenic protein, preferably further expressing one or more antigenic proteins from one or more additional filovirus subtypes or substantially similar glycoproteins. The first and second Marburg virus antigenic proteins can be identical or different.

In preferred embodiments of the présent invention, the one or more filoviruses are Ebolaviruses or Marburgviruses. The Ebolavirus can be of any species or isolâtes, for example,

Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV), Reston, Bundibugyo, Taï Forest. The Marburg virus (MARV) may be of any species. The Marburgviruses can be any isolate or strain of the Marburg marburgvirus species, including but not limited to, the Marburg Popp strain Ratayczak strain, Ozolin strain, Musoke strain (MARV-Musoke), and Angola strain (MARVAngola), and the Ravn strain. Exemplary amino acid sequences of suitable filovirus antigenic proteins include, but are not limited to, SEQ ID NO: 1 to SEQ ID NO: 5.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the précisé embodiments shown in the drawings.

In the drawings:

Figure 1 shows the amount of MARV GP-binding antibodies in sérum samples taken from Cynomolgus macaques (Macaca fascicularis) (NHPs) vaccinated with different vaccine combinations as analyzed by a MARV GP-specifïc Enzyme-Linked Immunosorbent Assay (ELIS A): loglO-transformed ELIS A concentrations (ELIS A Units per ml, EU/ml) are shown, and the symbols represent the titers from the individual animais tested, bars represent the mean per group, and dotted line represents the Limit of Détection (LOD) of the assay;

Figure 2 shows the protective efficacy of different vaccine combinations and négative controls (Neg ctrl) in vaccinated NHPs after challenge with Marburg Angola virus; and

Figure 3 is a plot of the kinetic of MARV GP-binding antibodies in sérum samples taken from NHPs vaccinated with different vaccine combinations as analyzed by a MARV GP-specific ELISA, the mean loglO-transformed ELISA concentrations (ELISA Units per ml, EU/ml) with standard déviations are shown.

DETAILED DESCRIPTION OF THE INVENTION

Varions publications, articles and patents are cited or described in the background and throughout the spécification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the présent spécification is for the purpose of providing context for the présent invention. Such discussion is not an admission that any or ail of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, ail technical and scientific tenus used herein hâve the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.

Otherwise, certain terms used herein hâve the meanings as set forth in the spécification. Ail patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictâtes otherwise.

Unless otherwise indicated, the term “at least” preceding a sériés of éléments is to be understood to refer to every element in the sériés. Those skilled in the art will recognize, or be able to ascertain using no more than routine expérimentation, many équivalents to the spécifie embodiments of the invention described herein. Such équivalents are intended to be encompassed by the présent invention.

Throughout this spécification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect or embodiment of the présent invention can be substituted with the term “consisting ofthough less preferred.

When used herein “consisting of ’ excludes any element, step, or ingrédient not specified in the claim element. When used herein, “consisting essentially of ’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

As used herein, the conjunctive term “and/or” between multiple recited éléments is understood as encompassing both individual and combined options. For instance, where two éléments are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second éléments together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the invention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.

As used herein, the term “protective immunity” or “protective immune response” means that the vaccinaied subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a “protective immune response” develops only mild to moderate clinical symptoms or no symptoms at ail. Usually, a subject having a “protective immune response” or “protective immunity” against a certain agent will not die as a resuit of the infection with said agent.

An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad 26 or Ad 35) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adénoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a “Ad26 capsid protein” or a “Ad35 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 or Ad35 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26 or of Ad35. In certain embodiments, the hexon, penton and fiber are of Ad26 or of Ad35.

The ternis “adjuvant” and immune stimulant are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune System. In this context, an adjuvant is used to enhance an immune response to the adenovirus and/or MVA vectors of the invention.

The term “corresponding to”, when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, (e.g., glycoproteins of filovirus and polynucleotides that encode them) refer to two or more sequences or subsequences that are the same or hâve a specified percentage of amino acid residues or nucléotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculâtes the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implémentations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the

Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associâtes, Inc. and John Wiley & Sons, Inc., (1995 Supplément) (Ausubel)).

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 33893402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word ofthe same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

Cumulative scores are calculated using, for nucléotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score fails off by the quantity X from its maximum achieved value; the cumulative score goes to zéro or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X détermine the sensitivity and speed of the alignment. The BLASTN program (for nucléotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucléotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molécules hybridize to each other under stringent conditions, as described below.

The term “substantially similar” in the context of the filovirus antigenic proteins of the invention indicates that a polypeptide comprises a sequence with at least 90%, preferably at least 95% sequence identity to the reference sequence over a comparison window of 10-20 amino acids. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or délétions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or délétions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the resuit by 100 to yield the percentage of sequence identity.

It is discovered in the présent invention that heterologous prime-boost combinations, in particular, Ad26 priming followed by MVA boosting and vice versa, are surprisingly effective in generating protective immune responses against one or more subtypes of Marburg viruses.

Filovirus antigenic proteins

The Ebola viruses, and the genetically-related Marburg virus, are filoviruses associated with outbreaks of highly léthal hémorrhagie fever in humans and primates in North America Europe, and Africa (Peters, C.J. et al. in: Fields Virology, eds. Fields, B.N. et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996; Peters, C.J. et al. 1994 Semin Virol 5:147-154). Although several subtypes hâve been defined, the genetic organization of these viruses is similar, each containing seven linearly arrayed genes. Among the viral proteins, the envelope glycoprotein exists in two alternative forms, a 50-70 kilodalton (kDa) secreted protein (sGP) and a 130 kDa transmembrane glycoprotein (GP) generated by RNA editing that médiates viral entry (Peters, C.J. et al. in: Fields Virology, eds. Fields, B.N. et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996; Sanchez, A. et al. 1996 PNAS USA 93:3602-3607). Other structural gene products include the nucleoprotein (NP), matrix proteins VP24 and VP40, presumed nonstructural proteins VP30 and VP35, and the viral polymerase (reviewed in Peters, C.J. et al. in: Fields Virology, eds. Fields, B.N.

et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996).

The nucleic acid molécules comprised in the adenovirus and MVA vectors can encode structural gene products of any filovirus species, such as subtypes of Zaire (type species, also referred to herein as ZEBOV), Sudan (also referred to herein as SEBOV), Reston, Bundibugyo, and Ivory Coast. There is a single species of Marburgvirus (also referred to herein as MARV) comprising two genetic lineages, the Marburg lineage and Ravn lineage. The Marburg virus according to the présent invention can be any isolate or strain of the Marburg marburgvirus species, including but not limited to, the Marburg Popp strain, Ratayczak strain, Ozolin strain^ Musoke strain (MARV-Musoke), and Angola strain (MARV-Angola), and the Ravn strain. Preferably, the Marburg virus is at least one of MARV-Musoke and MARV-Angola.

The adénoviral vectors and MVA vectors of the invention can be used to express antigenic proteins which are proteins comprising an antigenic déterminant of a wide variety of filovirus antigens. In a typical and preferred embodiment, the vectors of the invention include nucleic acid encoding the transmembrane form ofthe viral glycoprotein (GP). In other embodiments, the vectors of the invention can encode the secreted form of the viral glycoprotein (ssGP), or the viral nucleoprotein (NP).

One of skill will recogmze that the nucleic acid molécules encoding the filovirus antigenic protein can be modified, e.g., the nucleic acid molécules set forth herein can be mutated, as long as the modified expressed protein elicits an immune response against a pathogen or disease. Thus, as used herein, the term “antigenic protein” or “filovirus protein” refers to a protein that comprises at least one antigenic déterminant of a filovirus protein described above. The term encompasses filovirus glycoproteins (i.e., gene products of a filovirus) or filovirus nucleoprotein as well as recombinant proteins that comprise one or more filovirus glycoprotein déterminants. The term antigenic proteins also encompasses antigenic proteins that are substantially similar to the naturally occurring antigenic proteins. For example, as used herein, a GP of a filovirus, such as a GP of a Marburg virus, can be the naturally occurring GP. A GP of a filovirus can also be a protein that is substantially similar to a naturally occurring GP of a filovirus and contains at least one antigenic déterminant of the naturally occurring GP.

In some embodiments, the protein can be mutated so that it is less toxic to cells (see e.g., WO/2006/037038) or can be expressed with increased or decreased level in the cells. The présent invention also includes vaccines comprising a combination of nucleic acid molécules. For example, and without limitation, nucleic acid molécules encoding GP, ssGP and NP of the Zaire, Sudan, Marburg and Ivory Coast/Taï forest Ebola strains can be combined in any combination, in one vaccine composition.

Adenoviruses

An adenovirus according to the invention belongs to the family of the Adenoviridae and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu; in the présent invention a human adenovirus is meant if referred to Ad without indication of species, e.g. the brief notation “Ad5” means the same as HAdV5, which is human adenovirus serotype 5), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV).

Most advanced studies hâve been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, the recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49 or 50. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of one of the serotypes 26 or 35.

An advantage of these serotypes is a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Préparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63, both of which are incorporated by reference herein in their entirety. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792. Préparation of rAd35 vectors is described, for example, in US Patent No. 7,270,811, in WO 00/70071, and in Vogels et al., (2003) J Virol 77(15): 8263-71, ail of which are incorporated by reference herein in their entirety. Exemplary genome sequences of Ad35 are found in GenBank Accession AC_000019 and in Fig. 6 of WO 00/70071.

Simian adenoviruses generally also hâve a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a signifîcant amount of work has been reported using chimpanzee adenovirus vectors (e.g. US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849- 62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other preferred embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39,

40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P.

Adénoviral Vectors rAd26 and rAd35

In a preferred embodiment according to the présent invention the adénoviral vectors comprise capsid proteins from two rare serotypes: Ad26 and Ad35. In the typical embodiment, the vector is an rAd26 or rAd35 virus.

Thus, the vectors that can be used in the invention comprise an Ad26 or Ad35 capsid protein (e.g., a fiber, penton or hexon protein). One of skill will recognize that it is not necessary that an entire Ad26 or Ad35 capsid protein be used in the vectors of the invention. Thus, chimeric capsid proteins that include at least a part of an Ad26 or Ad35 capsid protein can be used in the vectors of the invention. The vectors of the invention can also comprise capsid proteins in which the fiber, penton, and hexon proteins are each derived from a different serotype, so long as at least one capsid protein is derived from Ad26 or Ad35. In preferred embodiments, the fiber, penton and hexon proteins are each derived from Ad26 or each from Ad35.

One of skill will recognize that éléments derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines désirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of the Ad26 and Ad35 serotypes with characteristics such as température stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like.

In certain embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad35 or from Ad26 (i.e., the vector is rAd35 or rAd26). In some embodiments, the adenovirus is réplication déficient, e.g. because it contains a délétion in the El région of the genome. For the adenoviruses of the invention, being derived from Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g. Havenga et al, 2006, J Gen Virol 87: 2135-43; WO 03/104467). In certain embodiments, the adenovirus is a human adenovirus of serotype 35, with a délétion in the El région into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 région of Ad5. In certain embodiments, the adenovirus is a human adenovirus of serotype 26, with a délétion in the El région into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 région of Ad5. For the Ad35 adenovirus, it is typical to retain the 3’ end of the E1B 55K open reading frame in the adenovirus, for instance the 166 bp directly upstream ofthe pIX open reading frame or a fragment comprising this such as a 243 bp fragment directly upstream ofthe pIX start codon, marked at the 5’ end by a Bsu36I restriction site, since this increases the stability ofthe adenovirus because the promoter ofthe pIX gene is partly residing in this area (see, e.g. Havenga et al, 2006, supra; WO 2004/001032).

The préparation of recombinant adénoviral vectors is well known in the art. Préparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al, (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792. Préparation of rAd35 vectors is described, for example, in US Patent No. 7,270,811 and in Vogels et al, (2003) J Virol 77(15): 8263-71. An exemplary genome sequence of Ad35 is found in GenBank Accession AC 000019.

In an embodiment of the présent invention, the vectors useful for the présent invention include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.

Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adénoviral genome (e.g, a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molécules that encode the adénoviral vectors ofthe invention. The nucleic acid molécules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or singlestranded.

The adenovirus vectors useful the invention are typically réplication defective. In these embodiments, the virus is rendered replication-defective by délétion or inactivation of régions critical to réplication of the virus, such as the El région. The régions can be substantially deleted or inactivated by, for example, inserting the gene of interest (usually linked to a promoter). In some embodiments, the vectors of the invention can contain délétions in other régions, such as the E2, E3 or E4 régions or insertions of heterologous genes linked to a promoter. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4-complementing cell lines are used to generate recombinant adenovrruses. Mutations in the E3 région of the adenovirus need not be complemented by the cell line, since E3 is not required for réplication.

A packaging cell line is typically used to produce suffïcient amount of adenovirus vectors of the invention. A packaging cell is a cell that comprises those genes that hâve been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6, 911, 293, and El A549.

As noted above, a wide variety of filovirus glycoproteins can be expressed in the vectors. If required, the heterologous gene encoding the filovirus glycoproteins can be codon- optimized to ensure proper expression in the treated host (e.g, human). Codon-optimization is a technology widely applied in the art. Typically, the heterologous gene is cloned into the El and/or the E3 région of the adénoviral genome.

The heterologous filovirus gene can be under the control of (i.e., operably linked to) an adenovirus-derived promoter (e.g., the Major Late Promoter) or can be under the control of a heterologous promoter. Examples of suitable heterologous promoters include the CMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within an expression cassette.

In a preferred embodiment of the présent invention, the rAd vector comprises the GP of a Marburg virus (MARV) or GPs substantially similar thereto.

MVA vectors

MVA vectors useful for the présent invention utilize attenuated virus derived from Modified Vaccinia Ankara virus which is characterized by the loss of their capabilities to reproductively replicate in human cell lines. The MVA vectors express a wide variety of filovirus glycoproteins as well as other structural filovirus proteins, such as VP40 and nucleoprotein (NP).

In one aspect, the présent invention provides a recombinant modified vaccinia virus Ankara (MVA) comprising a nucléotide sequence encoding an antigenic déterminant of a filovirus glycoprotein (GP), in particular an envelope glycoprotein. In another aspect, the présent invention provides a recombinant MVA vector comprising a heterologous nucléotide sequence encoding an antigenic déterminant of a filovirus glycoprotein, in particular an envelope glycoprotein, and a heterologous nucléotide sequence encoding an antigenic déterminant of a further filovirus protein.

MVA has been generated by more than 570 serial passages on chicken embryo fibroblasts of the dermal vaccinia strain Ankara (Chorioallantois vaccinia virus Ankara virus, CVA; for review see Mayr et al. (1975), Infection 3, 6-14) that was maintained in the Vaccination Institute, Ankara, Turkey for many years and used as the basis for vaccination of humans. However, due to the often severe post-vaccination complications associated with vaccinia viruses, there were several attempts to generate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded in attenuating CVA by over 570 continuous passages in CEF cells (Mayr et al. (1975), Infection 3, 6-14). It was shown in a variety of animal models that the resuiting MVA was avirulent (Mayr, A. & Danner, K. (1978), Dev. Biol. Stand. 41: 225-234). As part of the early development of MVA as a pre-smallpox vaccine, there were clinical trials using MVA-517 in combination with Lister Elstree (Stickl (1974), Prev. Med. 3: 97-101; Stickl and Hochstein-Mintzel (1971), Munch. Med. Wochenschr. 113: 11491153) in subjects at risk for adverse reactions from vaccinia. In 1976, MVA derived from MVA571 seed stock (corresponding to the 571st passage) was registered in Germany as the primer vaccine in a two-stage parentéral smallpox vaccination program. Subsequently, MVA-572 was used in approximately 120,000 Caucasian individuals, the majority children between 1 and 3 years of âge, with no reported severe side effects, even though many of the subjects were among the population with high risk of complications associated with vaccinia (Mayr et al. (1978), Zentralbl. Bacteriol. (B) 167:375-390). MVA-572 was deposited at the European Collection of Animal Cell Cultures as ECACC V94012707.

As a resuit ofthe passaging used to attenuate MVA, there are a number of different strains or isolâtes, depending on the number of passages conducted in CEF cells. For example, MVA-572 was used in a small dose as a pre-vaccine in Germany during the smallpox éradication program, and MVA-575 was extensively used as a veterinary vaccine. MVA as well as MVA- BN lacks approximately 15% (31 kb from six régions) of the genome compared with ancestral CVA virus. The délétions affect a number of virulence and host range genes, as well as the gene for Type A inclusion bodies. MVA-575 was deposited on December 7, 2000, at the European Collection of Animal Cell Cultures (ECACC) under Accession No. V00120707. The attenuated CVA-virus MVA (Modified Vaccinia Virus Ankara) was obtained by serial propagation (more than 570 passages) of the CVA on primary chicken embryo fibroblasts.

Even though Mayr et al. demonstrated during the 1970s that MVA is highly attenuated and avirulent in humans and mammals, certain investigators hâve reported that MVA is not fully attenuated in mammalian and human cell lines since residual réplication might occur in these cells (Blanchard et al. (1998), J. Gen. Virol. 79:1159-1167; Carroll & Moss (1997), Virology 238:198211; U.S. Patent No. 5,185,146; Ambrosini et al. (1999), J. Neurosci. Res. 55: 569). It is assumed that the results reported in these publications hâve been obtained with varions known strains of MVA, since the viruses used essentially differ in their properties, particularly in their growth behavior in varions cell lines. Such residual réplication is undesirable for varions reasons, including safety concems in connection with use in humans.

MVA-BN can attach to and enter human cells where virally-encoded genes are expressed very efficiently. MVA-BN is strongly adapted to primary chicken embryo fibroblast (CEF) cells and does not replicate in human cells. In human cells, viral genes are expressed, and no infections virus is produced. MVA-BN is classified as Biosafety Level 1 organism according to the Centers for Disease Control and Prévention in the United States. Préparations of MVA-BN and dérivatives hâve been administered to many types of animais, and to more than 2000 human subjects, including immune-deficient individuals. Ail vaccinations hâve proven to be generally safe and well tolerated. Despite its high atténuation and reduced virulence, in preclinical studies MVA-BN has been shown to elicit both humoral and cellular immune responses to vaccinia and to heterologous gene products encoded by genes cloned into the MVA genome (E. Harrer et al. (2005), Antivir. Ther. 10(2):285300; A. Cosma et al. (2003), Vaccine 22(1):21 -9; M. Di Nicola et al. (2003), Hum. Gene Ther. 14(14):1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res., 10(16):5381-5390).

Dérivatives” or “variants” of MVA refer to vrruses exhibiting essentially the same réplication characteristics as MVA as described herein, but exhibiting différences in one or more parts of their genomes. MVA-BN as well as a dérivative or variant of MVA-BN fails to reproductively replicate in vivo in humans and mice, even in severely immune suppressed mice. More specifically, MVA-BN or a dérivative or variant of MVA-BN has preferably also the capability of reproductive réplication in chicken embryo fibroblasts (CEF), but no capability of reproductive réplication in the human kératinocyte cell line HaCat (Boukamp et al (1988), J. Cell Biol. 106: 761-771), the human bone osteosarcoma cell line 143B (ECACC Deposit No. 91112502), the human embryo kidney cell line 293 (ECACC Deposit No. 85120602), and the human cervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL-2). Additionally, a dérivative or variant of MVA-BN has a virus amplification ratio at least two fold less, more preferably three-fold less than MVA-575 in Hela cells and HaCaT cell lines. Tests and assay for these properties of MVA variants are described in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US 2006/0159699).

The term “not capable of reproductive réplication” or “no capability of reproductive réplication” is, for example, described in WO 02/42480, which also teaches how to obtain MVA having the desired properties as mentioned above. The term applies to a virus that has a virus amplification ratio at 4 days after infection of less than 1 using the assays described in WO 02/42480 or in U.S. Patent No. 6,761,893, both of which are incorporated by reference herein in their entirety.

The term “fails to reproductively replicate” refers to a virus that has a virus amplification ratio at 4 days after infection of less than 1. Assays described in WO 02/42480 or in U.S. Patent No. 6,761,893 are applicable for the détermination of the virus amplification ratio.

The amplification or réplication of a virus is normally expressed as the ratio of virus produced from an infected cell (output) to the amount originally used to infect the cell in the first place (input) referred to as the “amplification ratio”. An amplification ratio of “1” defines an amplification status where the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells, meaning that the infected cells are permissive for virus infection and reproduction. In contrast, an amplification ratio of less than 1, i.e., a decrease in output compared to the input level, indicates a lack of reproductive réplication and therefore atténuation of the virus.

The advantages of MVA-based vaccine include their safety profile as well as availability for large scale vaccine production. Preclinical tests hâve revealed that MVA-BN demonstrates superior atténuation and efficacy compared to other MVA strains (WO 02/42480). An additional property of MVA-BN strains is the ability to induce substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost régimes when compared to DNA- prime/vaccinia virus boost régimes.

The recombinant MVA-BN viruses, the most preferred embodiment herein, are considered to be safe because of their distinct réplication deficiency in mammalian cells and their wellestablished avirulence. Furthermore, in addition to its efficacy, the feasibility of industrial scale manufacturing can be bénéficiai. Additionally, MVA-based vaccines can deliver multiple heterologous antigens and allow for simultaneous induction of humoral and cellular immunity

MVA vectors useful for the présent invention can be prepared using methods known in the art, such as those described in WQ/2002/042480 and WO/2002/24224, the relevant disclosures of which are incorporated herein by references.

In another aspect, réplication déficient MVA viral strains can also be suitable such as strain MVA-572, MVA-575 or any similarly attenuated MVA strain. Also suitable can be a mutant MVA, such as the deleted chorioallantois vaccinia virus Ankara (dCVA). A dCVA comprises del I, del Π, del ΠΙ, del IV, del V, and del VI délétion sites of the MVA genome. The sites are particularly useful for the insertion of multiple heterologous sequences. The dCVA can reproductively replicate (with an amplification ratio of greater than 10) in a human cell line (such as human 293, 143B, and MRC5 cell lines), which then enable the optimization by further mutation useful for a virus-based vaccination strategy (see WO 2011/092029).

In a preferred embodiment of the présent invention, the MVA vector(s) comprise a nucleic acid that encode one or more antigenic proteins selected from the group consisting of GPs of Zaire ebolavirus (EBOV), GPs of Sudan ebolavirus (SUDV), GPs of Marburg virus (MARV), the NP of Taï Forest virus and GPs or NPs substantially similar thereto.

The filovirus protein can be inserted into one or more intergenic régions (IGR) of the MVA. In certain embodiments, the IGR is selected from IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR 148/149. In certain embodiments, less than 5, 4, 3, or 2 IGRs of the recombinant MVA comprise heterologous nucléotide sequences encoding antigenic déterminants of a filovirus envelope glycoprotein and/or a further filovirus protein. The heterologous nucléotide sequences can, additionally or altematively, be inserted into one or more of the naturally occurring délétion sites, in particular into the main délétion sites I, Π, III, IV, V, or VI ofthe MVA genome. In certain embodiments, less than 5, 4, 3, or 2 of the naturally occurring délétion sites of the recombinant MVA comprise heterologous nucléotide sequences encoding antigenic déterminants of a filovirus envelope glycoprotein and/or a further filovirus protein.

The number of insertion sites of MVA comprising heterologous nucléotide sequences encoding antigenic déterminants of a filovirus protein can be l, 2, 3, 4, 5, 6, 7, or more. In certain embodiments, the heterologous nucléotide sequences are inserted into 4, 3, 2, or fewer insertion sites. Preferably, two insertion sites are used. In certain embodiments, three insertion sites are used. Preferably, the recombinant MVA comprises at least 2, 3, 4, 5, 6, or 7 genes inserted into 2 or 3 insertion sites.

The recombinant MVA viruses provided herein can be generated by routine methods known in the art. Methods to obtain recombinant poxviruses or to insert exogenous coding sequences into a poxviral genome are well known to the person skilled in the art. For example methods for standard molecular biology techniques such as cloning of DNA, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques are described in Molecular Cloning, A laboratory Manual (2nd Ed.) (J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)), and techniques for the handling and manipulation of viruses are described in Virology Methods Manual (B.W.J. Mahy et al. (eds.), Academie Press (1996)). Similarly, techniques and know-how for the handling, manipulation and genetic engineering of MVA are described in Molecular Virology: A Practical Approach (A.J. Davison & R.M. Elliott (Eds.), The Practical Approach Sériés, IRL Press at Oxford University Press, Oxford, UK (1993) (see, e.g., Chapter 9: Expression of genes by Vaccinia virus vectors) and Current Protocols in Molecular Biology (John Wiley & Son, Inc. (1998) (see, e.g., Chapter 16, Section IV: Expression of proteins in mammalian cells using vaccinia viral vector).

For the génération of the various recombinant MVAs disclosed herein, different methods can be applicable. The DNA sequence to be inserted into the virus can be placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the MVA has been inserted. Separately, the DNA sequence to be inserted can be ligated to a promoter. The promoter-gene linkage can be positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a région of MVA DNA containing a non-essential locus. The resulting plasmid construct can be amplified by propagation within E. coli bacteria and isolated. The isolated plasmid containing the DNA gene sequence to be inserted can be transfected into a cell culture, e.g., of chicken embryo fîbroblasts (CEFs), at the same time the culture is infected with MVA. Recombination between homologous MVA DNA in the plasmid and the viral genome, respectively, can generate an MVA modified by the presence of foreign DNA sequences.

According to a preferred embodiment, a cell of a suitable cell culture as, e.g., CEF cells, can be infected with a poxvirus. The infected cell can be, subsequently, transfected with a first plasmid vector comprising a foreign or heterologous gene or genes, preferably under the transcriptional control of a poxvirus expression control element. As explained above, the plasmid vector also comprises sequences capable of directing the insertion of the exogenous sequence into a selected part ofthe poxviral genome. Optionally, the plasmid vector also contains a cassette comprising a marker and/or sélection gene operably linked to a poxviral promoter.

Suitable marker or sélection genes are, e.g., the genes encoding the green fluorescent protein, β- galactosidase, neomycin-phosphoribosyltransferase or other markers, The use of sélection or marker cassettes simplifies the identification and isolation of the generated recombinant poxvirus. However, a recombinant poxvirus can also be identified by PCR technology. Subsequently, a further cell can be infected with the recombinant poxvirus obtained as described above and transfected with a second vector comprising a second foreign or heterologous gene or genes. In case, this gene shall be introduced into a different insertion site of the poxviral genome, the second vector also differs in the poxvirus-homologous sequences directing the intégration of the second foreign gene or genes into the genome of the poxvirus. After homologous recombination has occurred, the recombinant virus comprising two or more foreign or heterologous genes can be isolated. For introducing additional foreign genes into the recombinant virus, the steps of infection and transfection can be repeated by using the recombinant virus isolated in previous steps for infection and by using a further vector comprising a further foreign gene or genes for transfection.

Altematively, the steps of infection and transfection as described above are interchangeable, i.e., a suitable cell can at first be transfected by the plasmid vector comprising the foreign gene and, then, infected with the poxvirus. As a further alternative, it is also possible to introduce each foreign gene into different viruses, co-infect a cell with ail the obtained recombinant viruses and screen for a recombinant including ail foreign genes. A third alternative is ligation of DNA genome and foreign sequences in vitro and reconstitution of the recombined vaccinia virus DNA genome using a helper virus. A fourth alternative is homologous recombination in E.coli or another bacterial species between a vaccinia virus genome, such as MVA, cloned as a bacterial artificial chromosome (BAC) and a linear foreign sequence flanked with DNA sequences homologous to sequences flanking the desired site of intégration in the vaccinia virus genome.

The heterologous filovirus gene can be under the control of (i.e., operably linked to) one or more poxvirus promoters. In certain embodiments, the poxvirus promoter is a Pr7.5 promoter, a hybrid early/late promoter, or a PrS promoter, a PrSSE promoter, a synthetic or naturel early or late promoter, or a cowpox virus ATI promoter.

Immunogenic Compositions

Immunogenic compositions are compositions comprising an immunologically effective amount of purified or partially purified adenovirus or MVA vectors for use in the invention. The vector comprises a nucleic acid encoding an antigenic protein of a Marburg virus. The antigenic protein of a Marburg virus encoded by the adenovirus vector can be the same antigenic protein of a Marburg that is encoded by the MVA vector, or the Marburg virus antigenic proteins encoded by the adenovirus and the MVA vectors can be different but share at least one antigenic déterminant.

In one embodiment ofthe invention, the adenovirus vector comprises a nucleic acid encoding a GP from a Marburg virus, such as the GP of MARV-Angola (e.g., that having the amino acid sequence of SEQ ID NO:3), or MARV- Musoke (e.g., that having the amino acid sequence of SEQ ID NO:4), and the MVA vector comprises a nucleic acid encoding the same GP 15 from the Marburg virus.

In another embodiment of the invention, the adenovirus vector comprises a nucleic acid encoding a GP from one isolate of Marburg virus, such as the GP of MARV-Angola (e.g., that having the amino acid sequence of SEQ ID NO:3), and the MVA vector comprises a nucleic acid encoding a GP from a different isolate of Marburg virus, such as the GP of MARV- Musoke (e.g., 20 that having the amino acid sequence of SEQ ID NO:4), or vice versa.

Said compositions can be formulated as a vaccine (also referred to as an “immunogenic composition ) according to methods well known in the art. Such compositions can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the présent disclosure.

The préparation and use of immunogenic compositions are well known to those of ski 11 in the art. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, minerai oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or 30 polyethylene glycol can be included.

In addition to adenovirus or MVA vectors comprising a nucleic acid encoding an antigenic protein of a Marburg virus, the compositions of the invention can comprise other filovirus antigen(s) or nucleic acid encoding other filovirus antigen(s), or the priming or boosting inoculations can comprise other filovirus antigens or nucleic acid encoding other filovirus antigen(s). The other antigens and nucleic acids expressing the other antigens can be, for example, from one or more additional filovirus subtypes, such as Ebola. The nucleic acid encoding the other filovirus antigen(s) can be included in the same adenovirus or MVA vectors that encode the antigenic protein of a Marburg virus. They can also be présent on other vectors in the compositions or the priming or boosting inoculations.

The immunogenic compositions useful in the invention can comprise adjuvants.

Adjuvants suitable for co-administration in accordance with the présent invention should be ones that are potentially safe, well tolerated and effective in people including QS-21, Detox-PC, MPL- SE, MoGM-CSF, TiterMax-G, CRL- 1005, GERBU, TERamide, PSC97B, Adjumer, PG026,GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines such as alpha-interferon, gamma interferon, platelet derived growth factor (PDGF), granulocyte-colony stimulating factor (gCSF), granulocyte macrophage colony stimulating factor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), LL-I, IL-2, IL-4, IL-6, IL-8, IL-IO, and IL-12 or encoding nucleic acids therefore.

The compositions of the invention can comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfère with the effîcacy of the active ingrédient. The précisé nature of the carrier or other material can dépend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes.

Method for Inducing Protective Immunity Against a Marburg Virus Infection

The présent invention provides a method of priming and boosting an immune response to a Marburg virus in an individual using an adénoviral vector in combination with an MVA vector.

According to one general aspect of the présent invention, a method of inducing an immune response against a Marburg virus in a subject comprises:

a. administering to the subject a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus; and

b. administering to the subject a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of one or more additional filovirus subtypes wherein steps (a) and (b) are conducted in either order, and the first and second antigenic proteins share at least one antigenic déterminant. In a preferred embodiment the later step is conducted I -12 weeks after the first step. In a more preferred embodiment the later step is conducted 4 or 8 weeks after the first step.

In one embodiment of the disclosed methods, an adenovirus vector is used to prime the immune response, and an MVA vector is used to boost the immune response about 1-12 weeks after the priming vaccination. Boosting compositions are generally administered weeks or months after administration ofthe priming composition, for example, about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks or one to two years after administration of the priming composition.

In a preferred embodiment of the présent invention, the adenovirus vectors disclosed herein include a rAd26 or rAd35 vector. In one exemplary embodiment, an rAd26 or rAd35 vector is used to prime the immune response, and an MVA vector is used to boost the immune response, or vice versa.

In a more preferred embodiment according to this method, an rAd26 vector is used for the priming followed by a boosting with an MVA vector. Preferably, the boosting composition is administered 1-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming In a preferred embodiment, the boosting composition is administered 8 weeks after priming Tn another preferred embodiment, the boosting composition is administered 1 week after priming Tn another preferred embodiment, the boosting composition is administered 2 weeks after priming. Tn another preferred embodiment, the boosting composition is administered 4 weeks after priming

In a more preferred embodiment according to this method, an MVA vector is used for the priming followed by a boosting with a rAd26 vector. Preferably, the boosting composition is administered 1-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming Tn a preferred embodiment, the boosting composition is administered 8 weeks after priming Tn another preferred embodiment, the boosting composition is administered 1 week after priming Tn another preferred embodiment, the boosting composition is administered 2 weeks after priming Tn another preferred embodiment, the boosting composition is administered 4 weeks after priming

The présent invention is also related to methods of inducing immune responses against a Marburg virus by priming and boosting the immune responses, preferably with a combination of heterologous vectors.

According to another aspect of the présent invention, a method of inducing an immune response against a Marburg virus in a subject comprises:

a. administering to the subject an immunologically effective amount of a rAd26 or rAd35 vector comprising nucleic acids encoding a first glycoprotein of a Marburg virus; and

b. administering to the subject an immunologically effective amount of one or more MVA vectors comprising one or more nucleic acids encoding a second glycoprotein of a Marburg virus and one or more glycoproteins of additional subtypes of fîloviruses, wherein steps (a) and (b) are conducted in either order, and the first and second glycoprotein share at least one antigenic déterminant.

In one or more embodiments of the described method, one or more of rAd26 or rAd35 vectors are used to prime the immune response, and one or more of MVA vectors are used to boost the immune response, or vice versa.

In a preferred embodiment according to the method herein, one or more of rAd26 vectors are used for the priming followed by a boosting with one or more of MVA vectors. Preferably, the boosting inoculation is administered l-l2 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming.

The antigens of a Marburg virus in the respective priming and boosting compositions (however many boosting compositions are employed) need not be identical, but should share antigenic déterminants or be substantially similar to each other.

Administration of the immunogenic compositions comprising the vectors is typically intramuscular or subcutaneous. However other modes of administration such as intravenous, cutaneous, intradermal or nasal can be envisaged as well. Intramuscular administration ofthe immunogenic compositions can be achieved by using a needle to inject a suspension of the adenovirus vector. An alternative is the use of a needleless inj ection device to administer the composition (using, e.g., Biojector(TM)) or a freeze-dried powder containing the vaccine.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the vector will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of skill in the art are well able to préparé suitable solutions using, for example, isotonie vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required. A slow-release formulation can also be employed. Typically, administration will hâve a prophylactic aim to generate an immune response against a filovirus antigen before infection or development of symptoms. Diseases and disorders that can be treated or prevented in accordance with the présent invention include those in which an immune response can play a protective or therapeutic rôle. In other embodiments, the adenovirus and MVA vectors can be administered for post-exposure prophylactics.

The immunogenic compositions containing the adenovirus vectors are administered to a subject, giving rise to an anti-Marburg virus immune response in the subject. An amount of a composition sufficient to induce a détectable immune response is defined to be an “immunologically effective dose.” As shown below, the immunogenic compositions of the invention induce a humoral as well as a cell-mediated immune response. In a typical embodiment the immune response is a protective immune response.

The actual amount administered, and rate and time-course of administration, will dépend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, I6th édition, Osol, A. ed., 1980.

Following production of adenovirus and MVA vectors and optional formulation of such particles into compositions, the vectors can be administered to an individual, particularly human or other primate. Administration can be to humans, or another mammal, e.g., mouse, rat, hamster^ guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. Delivery to a nonhuman mammal need not be for a therapeutic purpose, but can be for use in an experimental context, for instance in investigation of mechanisms of immune responses to the adenovirus or MVA vectors.

In one exemplary regimen, the adenovirus vector is administered (e.g., intramuscularly) in a volume ranging between about 100 μΐ to about 10 ml containing concentrations of about 10⁴ to 10¹² virus particles/ml. Preferably, the adenovirus vector is administered in a volume ranging between 0.25 and 1.0 ml. More preferably the adenovirus vector is administered in a volume of 0.5 ml.

Typically, the adenovirus is administered in an amount of about 10⁹ to about 10¹² viral particles (vp) to a human subject during one administration, more typically in an amount of about 10¹⁰ to about 10¹² vp. In a preferred embodiment, the adenovirus vector is administered in an amount of about 1x10¹¹ vp. In a preferred embodiment, the adenovirus vector is administered in an amount of about 5x10¹⁰ vp. In another preferred embodiment, the adenovirus vector is administered in an amount of about 0.8x10¹⁰ vp. In another preferred embodiment, the adenovirus vector is administered in an amount of about 2xlO¹⁰ vp. In another preferred embodiment, the adenovirus vector is administered in an amount of about 4xlO¹⁰ vp. The human adenovirus vector can be administered in a concentration of about 10⁷ vp/ml, 10⁸vp/ml, 10⁹vp/ml, 10¹⁰vp/ml, 5xlO¹⁰vp/ml, 10¹¹ vp/ml, or 10¹² vp/ml.

In certain embodiments, adenoviruses are formulated as a trivalent composition, wherein three adenoviruses with each a different insert, are mixed together.

In another exemplary regimen, the MVA vector is administered (e.g. intramuscularly) in a volume ranging between about 100 pl to about 10 ml of saline solution containing a dose of about

1x1O⁷TCID5₀ to IxlO⁹ TCID50 (50% Tissue Culture Infective Dose) or Inf.U. (Infections Unit or

InfU). Preferably, the MVA vector is administered in a volume ranging between 0.25 and 1.0 ml.

More preferably the MVA vector is administered in a volume of 0.5 ml.

Typically, the MVA vector is administered in a dose of about IxlO⁷ TŒD50 to IxlO⁹TCID50 (or Inf.U.) to a human subject during one administration. In a preferred embodiment, the MVA vector is administered in an amount of about 5xl0⁷ TCID50 to 5xl0⁸ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about 5x10⁷ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about IxlO⁸ TCfD50 (or Inf.U.). In another preferred embodiment, the MVA vector is administered in an amount of about 1.9x10⁸ TCID50 (or Inf.U). In yet another preferred embodiment, the MVA vector is administered in an amount of about 4.4xl0⁸ TCID50 (or Inf.U.). In another preferred embodiment, the MVA vector is administered in an amount of about 1,3xl0⁸ TCID50 (or Inf.U). In another preferred embodiment, the MVA vector is administered in an amount of about 1.0 xlO⁸TCID50 (or Inf.U). In another preferred embodiment, the MVA vector is administered in an amount of about 2.5x10⁸ TCID50 (or Inf.U). In a more preferred embodiment, the MVA vector is administered in an amount of about 5x10⁸ TCID₅o (or Inf.U )

The composition can, if desired, be presented in a kit, pack or dispenser, which can contain one or more unit dosage forms containing the active ingrédient. The kit, for example, can comprise métal or plastic foil, such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

Claims

The invention provides also the following non-limiting embodiments:

1. A vaccine combination comprising (i) a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus, together with a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of at least one additional filovirus subtype, together with a pharmaceutically acceptable carrier;

wherein one of the compositions is a priming composition and the other composition is a boosting composition, and the first and second antigenic proteins share at least one antigenic déterminant.
2. The vaccine combination according to embodiment 1, wherein the first and second antigenic proteins are from the same Marburg virus.
3. The vaccine combination according to embodiment 1, wherein the first and second antigenic proteins are from different Marburg viruses.
4. The vaccine combination according to any of embodiments 1-3, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
5. The vaccine combination according to any of embodiments 1 -4, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
6. The vaccine combination according to any of embodiments 1 and 3-5, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3, and the second antigenic protein comprises the amino acid sequence of SEQ ID NO: 4.
7. The vaccine combination according to any of embodiments 1 -6, wherein the first composition comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
8. The vaccine combination according to any one of embodiments 6 and 7, wherein the second composition comprises one or more MVA vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
9. The vaccine combination according to any one of embodiments 1-8, wherein the adenovirus vector is rAd26 or rAd35 vector, and the MVA vector is MVA-BN vector.
10. A vaccine combination comprising:

(i) a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3, together with a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, together with a pharmaceutically acceptable carrier;

wherein the first compositions is a priming composition and the second composition is a boosting composition.
11. The vaccine combination according to embodiment 10, wherein the MVA vector comprises a nucleic acid encoding the second antigenic protein having the amino acid sequence of SEQ ID NO:4.
12. The vaccine combination according to embodiment 10 or 11, wherein the MVA vector comprises a nucleic acid encoding antigenic proteins having the amino acid sequences of SEQ ID NO. 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5; optionally, the first composition further comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
13. A vaccine combination according to any one of embodiments 1-12 for use in generating a protective immune response against at least one Marburg virus in a subject, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.
14. A vaccine combination according to any one of embodiments 1-12 for use in generating a protective immune response against at least one Marburg virus in a subject, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.
15. A method of inducing an immune response against a Marburg virus in a subject, the method comprising:

a. admimstering to the subject a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus; and

b. administering to the subject a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of at least one additional filovirus subtype, wherein steps (a) and (b) are conducted in either order, and the fïrst and second antigenic proteins share at least one antigenic déterminant.
16. The method according to embodiment 15, wherein the first and second antigenic proteins are from the same Marburg virus.
17. The method according to embodiment 15, wherein the first and second antigenic proteins are from different Marburg viruses.
18. The method according to any of embodiments 15-17, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
19. The method according to any of embodiments 15-18, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
20. The method according to any of embodiments 15 and 17-19, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3, and the second antigenic protein comprises the amino acid sequence of SEQ ID NO: 4.
21. The method according to any of embodiments 15-20, wherein the first composition comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
22. The method according to any one of embodiments 20 and 21, wherein the second composition comprises one or more MVA vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
23. The method according to any one of embodiments 15-22, wherein the adenovirus vector is rAd26 or rAd35 vector, and the MVA vector is MVA-BN vector.
24. The method according to any one of embodiments 15-23, wherein step (b) is conducted 112 weeks, preferably 4-8 weeks, after step (a).
25. A method of inducing an immune response against a Marburg virus in a subject, the method comprising:

a. administering to the subject a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3; and

b. administering to the subject a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

wherein step (b) is conducted 1-12 weeks, preferably 4 or 8 weeks, after step (a).
26. The method according to embodiment 25, wherein the MVA vector comprises a nucleic acid encoding the second antigenic protein having the amino acid sequence of SEQ ID NO:4.
27. The method according to embodiment 25 or 26, wherein the MVA vector comprises a nucleic acid encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5; optionally, the first composition further comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO:

1 and SEQ ID NO: 2.
28. A kit comprising:

a. a first composition comprising an immunologically effective amount of an adenovirus vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus, together with a pharmaceutically acceptable carrier; and

b. a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus, preferably the nucleic acid further encoding one or more antigenic proteins of at least one additional filovirus subtype, together with a pharmaceutically acceptable carrier;

wherein one of the compositions is a priming composition and the other composition is a boosting composition, and the first and second antigenic proteins share at least one antigenic déterminant.
29. The kit according to embodiment 28, wherein the first and second antigenic proteins are from the same Marburg virus.
30. The kit according to embodiment 28, wherein the first and second antigenic proteins are from different Marburg viruses.
31. The kit according to any of embodiments 28-30, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
32. The kit according to any of embodiments 28-31, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.
33. The kit according to any of embodiments 28 and 30-32, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3, and the second antigenic protein comprises the amino acid sequence of SEQ ID NO: 4.
34. The kit according to any of embodiments 28-33, wherein the first composition comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
35. The kit according to any one of embodiments 33 and 34, wherein the second composition comprises one or more MVA vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.
36. The kit according to any one of embodiments 28-35, wherein the adenovirus vector is rAd26 or rAd35 vector, and the MVA vector is MVA-BN vector
37. A kit comprising:

(i) a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding a first antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3, together with a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of an MVA vector comprising a nucleic acid encoding a second antigenic protein of a Marburg virus having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, together with a pharmaceutically acceptable carrier;

wherein the first compositions is a priming composition and the second composition is a boosting composition.
38. The kit according to embodiment 37, wherein the MVA vector comprises a nucleic acid encoding the second antigenic protein having the amino acid sequence of SEQ ID NO:4.
39. The kit according to embodiment 37 or 38, wherein the MVA vector comprises a nucleic acid encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5; optionally, the first composition further comprises one or more adenovirus vectors comprising one or more nucleic acids encoding antigenic proteins having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
40. The kit according to any one of embodiments 28-39 for use in generating a protective immune response against at least one Marburg virus in a subject, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.
41. The kit according to any one of embodiments 28-39 for use in generating a protective immune response against at least one Marburg virus in a subject, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.
42. A use of a vaccine combination according to any one of embodiments 1-14 for manufacturing a pharmaceutical composition or médicament for inducing an immune response against a Marburg virus in a subject, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.
43. A use of a vaccine combination according to any one of embodiments 1-14 for manufacturing a pharmaceutical composition or médicament for inducing an immune response against a Marburg virus in a subject, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.
44. A use of a kit according to any one of embodiments 28-39 for manufacturing a pharmaceutical composition or médicament for inducing an immune response against a Marburg virus in a subject, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.
45. A use of a kit according to any one of embodiments 28-39 for manufacturing a pharmaceutical composition or médicament for inducing an immune response against a Marburg virus in a subject, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.