OA19482A - MVA-BN and AD26.ZEBOV or AD26.FILO prime-boost regimen - Google Patents

Abstract

Compositions and methods are described for generating an improved effective immune response against an immunogen in humans. The enhanced immune response, is obtained by using an MVA vector as a prime, an adenovirus vector as a first boost, and an adenovirus vector as a second boost. The compositions and methods can be used to provide a protective immunity against a disease, such as an infection of one or more subtypes of Ebola and Marburg filoviruses, in humans.

Description

Various 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 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 terms 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 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”. 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. In each instance herein any of the terms “comprising”, “consisting essentially of ’ and “consisting of ’ can be replaced with either of the other two terms.

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 vaccinated subject is able to control an infection or a disease related to an antigenic protein or immunogenic polypeptide thereof 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 antigenic protein will not die as a resuit of an infection or disease related to the antigenic protein.

The antigenic protein can be a native protein from a pathogen or a tumor, or a modified protein based on a native protein from a pathogen or a tumor.

As used herein, the term “pathogen” refers to an infectious agent such as a virus, a bacterium, a fungus, a parasite, or a prion that causes disease in its host.

As used herein, the term “enhanced” when used with respect to an immune response, such as a CD4+ T cell response, an antibody response, or a CD8+ T cell response, refers to an increase in the immune response in a human subject administered with a prime-boost combination of réplication incompetent MVA and adenovirus vectors according to the invention, relative to the corresponding immune response observed from the human subject administered with a reverse prime-boost combination, wherein the adenovirus vector is provided as a prime and the MVA vector is provided to boost the immune response, using the same primeboost interval.

As used herein, the term “dominant CD4+ or CD8+T cell response” refers to a T cell immune response that is characterized by observing high proportion of immunogen-specific CD4+ T cells within the population of total responding T cells following vaccination. The total immunogen-specific T-cell response can be determined by an IFN-gamma ELISPOT assay. The immunogen-specific CD4+ or CD8+ T cell immune response can be determined by an ICS assay. For example, a dominant CD4+ T cell response can comprise an antigen spécifie CD4+ T cell response that is more than 50%, such as 51%, 60%, 70%, 80%, 90% or 100% of the total antigen spécifie T-cell responses in the human subject. Preferably, the dominant CD4+ T cell response also represents 0.1% or more, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or more ofthe total cytokine responses in the human subject.

As used herein, the term “enhanced antibody response” refers to an antibody response in a human subject administered with a prime-boost combination of réplication incompetent MVA and adenovirus vectors according to the invention, that is increased by a factor of at least 1.5, 2, 2.5, or more relative to the corresponding immune response observed from the human subject administered with a reverse prime-boost combination, wherein the adenovirus vector is provided as a prime and the MVA vector is provided to boost the immune response, using the same primeboost interval.

As used herein, the term “polyfunctional” when used with respect to CD4+ or CD8+ T cells means T cells that express more than one cytokine, such as at least two of: IL-2, IFNgamma, and TNF-alpha.

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 terms “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 Associates, 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: 3389- 3402, 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 falls 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 perforais 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 ofthe 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 Iess than about 0.1, more preferably Iess than about 0.01, and most preferably Iess 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 fîlovirus antigenic proteins ofthe 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 may 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 invention that heterologous prime-boost combinations, in particular, MVA priming followed by Ad26 boosting, are surprisingly effective in generating protective immune responses in human subjects.

Antigenic proteins

Any DNA of interest can be inserted into the viral vectors described herein to be expressed heterologously from the vectors. Foreign genes for insertion into the genome of a virus in expressible form can be obtained using conventional techniques for isolating a desired gene. For organisms which contain a DNA genome, the genes encoding an antigen of interest can be isolated from the genomic DNA; for organisms with RNA genomes, the desired gene can be isolated from cDNA copies of the genome. The antigenic protein can also be encoded by a recombinant DNA that is modified based on a naturally occurring sequence, e.g., to optimize the antigenic response, gene expression, etc.

In certain embodiments of the invention, MVA-prime and adenovirus-boost combinations of réplication incompetent vectors generate an enhanced immune response to an antigenic protein or an immunogenic polypeptide thereof in a human subject. The antigenic protein can be any antigenic protein related to an infection or disease.

According to embodiments of the invention, the antigenic protein or immunogenic polypeptide thereof can be isolated from, or derived from, a pathogen, such as a virus (e.g., filovirus, adenovirus, arbovirus, astrovirus, coronavirus, coxsackie virus, cytomégalovirus, Dengue virus, Epstein-Barr virus, hepatitis virus, herpesvirus, human immunodeficiency virus, human papilloma virus, human T-lymphotropic virus, influenza virus, JC virus, lymphocytic choriomeningitis virus, measles virus, molluscum contagiosum virus, mumps virus, norovirus, parovirus, poliovirus, rabies virus, respiratory syncytial virus, rhinovirus, rotavirus, rotavirus, rubella virus, smallpox virus, varicella zoster virus, West Nile virus, etc.), a bacteria (e.g., Campylobacter jejuni, Escherichia coli, Hélicobacter pylori, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, etc.), a fungus (e.g., Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, Aspergillus species, etc.), a protozoan (e.g., Plasmodium, Leishmania, Trypanosome, cryptosporidiums, isospora, Naegleria fowleri,

Acanthamoeba, Balamuthia mandrillaris, Toxoplasma gondii, Pneumocystis carinii, etc.), or a cancer (e.g., bladder cancer, breast cancer, colon and rectal cancer, endométrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, etc.).

In some embodiments, nucleic acids express antigenic domains rather than the entire antigenic protein. These fragments can be of any length sufficient to be immunogenic or antigenic. Fragments can be at least four amino acids long, preferably 8-20 amino acids, but can be longer, such as, e.g., 100, 200, 660, 800, 1000,1200, 1600, 2000 amino acids long or more, or any length in between.

In some embodiments, at least one nucleic acid fragment encoding an antigenic protein or immunogenic polypeptide thereof is inserted into a viral vector. In another embodiment, about 2-8 different nucleic acids encoding different antigenic proteins are inserted into one or more of the viral vectors. In some embodiments, multiple immunogenic fragments or subunits of various proteins can be used. For example, several different epitopes from different sites of a single protein or from different proteins of the same species, or from a protein ortholog from different species can be expressed from the vectors.

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 fransmembrane 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 may encode structural gene products of any filovirus species, such as subtypes of Zaïre (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 Marburg virus (also referred to herein as MARV).

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 fïlovirus antigens. In a typical and preferred embodiment, the vectors of the invention include nucleic acid encoding the transmembrane form of the viral glycoprotein (GP). In other embodiments, the vectors of the invention may encode the secreted form of the viral glycoprotein (ssGP), or the viral nucleoprotein (NP).

One of skill will recognize 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.

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 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 Zaïre, 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 farnily 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 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 significant 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: 84962; 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 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 may 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 adenovinis 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 ofthe genome. For the adenoviruses ofthe 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 fines 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 of the pIX open reading frame or a fragment comprising this such as a 243 bp fragment directly upstream of the pIX start codon, marked at the 5’ end by a Bsu36I restriction site, since this increases the stability ofthe adenovirus because the promoter of the 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 invention, the vectors useful for the 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 doublestranded or single-stranded.

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 may 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 adenoviruses. 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 ofthe 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.

In some embodiments, the Adenovirus virus may express genes or portions of genes that encode antigenic peptides. These foreign, heterologous or exogenous peptides or polypeptides can include sequences that are immunogenic such as, for example, tumor-specific antigens (TSAs), bacterial, viral, fungal, and protozoal antigens.

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 codonoptimized 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 invention, the adenovirus vectors useful for the invention can comprise a wide variety of filovirus glycoproteins known to those of skill in the art. In a further preferred embodiment of the invention, the rAd vector© comprises one or more GPs selected from the group consisting of GPs of Zaïre ebolavirus (EBOV), GPs of Sudan ebolavirus (SUDV), GPs of Marburg virus (MARV), and GPs substantially similar thereto. MVA vectors

MVA vectors useful for the 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.

In some embodiments, the MVA virus may express genes or portions of genes that encode antigenic peptides. These foreign, heterologous or exogenous peptides or polypeptides can include sequences that are immunogenic such as, for example, tumor-specific antigens (TSAs), bacterial, viral, fungal, and protozoal antigens.

In other embodiments, 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 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 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)]. It was shown in a variety of animal models that the resulting MVA was avirulent [Mayr, A. & Danner, K. (1978), Dev. Biol. Stand. 41: 225-234], As part ofthe early development of MVA as a low dose 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: 1149-1153] in subjects at risk for adverse reactions from vaccinia. In 1976, MVA derived from MVA-571 seed stock (corresponding to the 571 st 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 low dose 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 238:198-211; 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 various known strains of MVA, since the viruses used essentially differ in their properties, particularly in their growth behavior in various cell lines. Such residual réplication is undesirable for various reasons, including safety concems in connection with use in humans.

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 developed 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 effîciently. MVA-BN is strongly adapted to primary chicken embryo fibroblast (CEF) cells and does not replicate in human cells. fn human cells, viral genes are expressed, and no infectious virus is produced. MVA-BN is classifîed as Biosafety Level 1 organism according to the Centers for Disease Control and Prévention in the United States. Préparations of MVABN and dérivatives hâve been administered to many types of animais, and to more than 2000 human subjects, including immune-defîcient 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. 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].

“Dérivatives” or “variants” of MVA refer to viruses 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 DNAprime/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 well-established 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 invention can be prepared using methods known in the art, such as those described in WO/2002/042480 and WO/2002/24224, the relevant disclosures of which are incorporated herein by references.

In another aspect, réplication déficient MVA viral strains may 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 II, del III, 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 MRC-5 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 ofthe invention, the MVA vector(s) comprise a nucleic acid that encode one or more antigenic proteins selected from the group consisting of GPs of Zaïre 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 fîlovirus protein can be inserted into one or more intergenic régions (IGR) ofthe 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, Iess 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 nudeotide sequences may, additionally or alternatively, be inserted into one or more of the naturally occurring délétion sites, in particular into the main délétion sites I, II, III, IV, V, or VI of the MVA genome. In certain embodiments, less than 5, 4, 3, or 2 of the naturally occurring délétion sites of the recombinant MVA comprise heterologous nudeotide sequences encoding antigenic déterminants of a filovirus envelope glycoprotein and/or a further filovirus protein.

The number of insertion sites of MVA comprising heterologous nudeotide sequences encoding antigenic déterminants of a filovirus protein can be 1, 2, 3, 4, 5, 6, 7, or more. In certain embodiments, the heterologous nudeotide 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 fibroblasts (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 of the 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.

Alternatively, 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 PrS5E promoter, a synthetic or natural 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. 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 may 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 skill 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 polyethylene glycol can be included.

The compositions of the invention can comprise MVA or adenovirus vectors expressing one or more antigenic proteins or immunogenic polypeptides thereof. These antigenic peptides or polypeptides can include any sequences that are immunogenic, including but not limited to, tumor-specific antigens (TSAs), bacterial, viral, fungal, and protozoal antigens. For example, the antigenic protein or immunogenic polypeptide thereof can be derived from a pathogen, e.g., a virus, a bacterium, a fungus, a protozoan, or it can also be derived from a tumor. In one or more preferred aspects, the compositions of the invention comprise MVA or adenovirus vectors expressing one or more antigenic proteins from a filovirus, such as the Ebola and/or Marburg filoviruses.

The filovirus subtypes according to the invention can be any filovirus subtype. In a preferred embodiment, the filovirus subtypes are selected from the group of Zaïre, Sudan, Reston, Bundibugyo, Taï Forest and Marburg. The antigenic proteins can be any protein from any filovirus comprising an antigenic déterminant. In a preferred embodiment the antigenic proteins are glycoproteins or nucleoproteins. The antigenic proteins encoded by the MVA vectors or adenovirus vectors comprised in the first and second composition according to the invention can be any antigenic protein from any filovirus.

In a preferred embodiment, the antigenic proteins encoded by the MVA vectors or adenovirus vectors hâve the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and/or SEQ ID NO: 5.

The immunogenic compositions useful in the invention can comprise adjuvants.

Adjuvants suitable for co-administration in accordance with the 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, PG-026,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), IL-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 efficacy of the active ingrédient. The précisé nature of the carrier or other material may dépend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes.

Method for Enhancing an Immune Response

The invention provides an improved method of priming and boosting an immune response to any antigenic protein or immunogenic polypeptide thereof in a human subject using an MVA vector in combination with an adénoviral vector.

According to one general aspect of the invention, a method of enhancing an immune response in a human subject comprises:

a. administering to the human subject a first composition comprising an immunologically effective amount of a MVA vector comprising a first polynucleotide encoding an antigenic protein or an immunogenic polypeptide thereof for priming the immune response;

b. administering to the subject a second composition comprising an immunologically effective amount of a first adenovirus vector comprising a second polynucleotide encoding the antigenic protein or an immunogenic polypeptide thereof for boosting the immune response; and

c. administering to the subject a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a third polynucleotide encoding the antigenic protein or an immunogenic polypeptide thereof for further boosting the immune response, to thereby obtain an enhanced immune response against the antigenic protein in the human subject.

According to embodiments of the invention, the enhanced immune response comprises an enhanced antibody response against the antigenic protein in the human subject.

Preferably, the enhanced immune response further comprises an enhanced CD4+ response or an enhanced CD8+ T cell response against the antigenic protein in the human subject. The enhanced CD4+ T cell response generated by a method according to an embodiment of the invention can be, for example, an increase or induction of a dominant CD4+ T cell response against the antigenic protein, and/or an increase or induction of polyfunctional CD4+ T cells spécifie to the antigenic protein in the human subject. The polyfunctional CD4+ T cells express more than one cytokine, such as two or more of IFN-gamma, IL-2 and TNFalpha.The enhanced CD 8+ T cell response generated by a method according to an embodiment of the invention can be, for example, an increase or induction of polyfunctional CD8+ T cells spécifie to the antigenic protein in the human subject.

More preferably, the enhanced immune response resulting from a method according to an embodiment of the invention comprises an enhanced CD4+ T cell response, an enhanced antibody response and an enhanced CD8+ T cell response, against the antigenic protein in the human subject.

In one or more embodiments of the invention, one or more MVA vectors are used to prime the immune response, and one or more rAd26 or rAd35 vectors are used to boost the immune response in step b and c of a method according to an embodiment of the invention. The same adenovirus vectors, such as one or more rAd26 or rAd35 vectors, can be used in both the second and third compositions. The second and third compositions can also hâve different adenovirus vectors.

The antigens in the respective priming and first and second boosting compositions 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 subeutaneous. However other modes of administration such as intravenous, cutaneous, intradermal or nasal can be envisaged as well. Intramuscular administration of the 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 injection device to administer the composition (using, e.g., Biojector(TM)) or a freeze-dried powder containing the vaccine.

For intravenous, cutaneous or subeutaneous 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 may also be employed.

Typically, administration will hâve a prophylactic aim to generate an immune response against an antigen before infection or development of symptoms. Diseases and disorders that can be treated or prevented in accordance with the invention include those in which an immune response can play a protective or therapeutic rôle. In other embodiments, the MVA and adenovirus vectors can be administered for post-exposure prophylactics.

The immunogenic compositions containing the MVA vectors are administered to a subject, giving rise to an immune response in the subject. An amount of a composition suffïcient to in 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, 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, 16th édition, Osol, A. ed., 1980.

Following production of MVA and adenovirus vectors and optional formulation of such particles into compositions, the vectors can be administered to an individual, particularly a human.

In one exemplary regimen, the adenovirus vector is administered (e.g., intramuscularly) in a volume ranging between about 100 pl 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 10 to about 10 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 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 2x10¹⁰ vp. In another preferred embodiment, the adenovirus vector is administered in an amount of about 4x10¹⁰ vp. In certain embodiments, adenoviruses are formulated as a trivalent composition, wherein three adenoviruses with each a different insert, are mixed together. In a trivalent composition, each distinct adenovirus is preferably présent in an amount of about 4x10¹⁰ vp. In said trivalent composition, the total number of adenoviras particles per dose amounts to about 1.2x10¹¹ vp. In another preferred embodiment, each distinct adenovirus in the trivalent composition is présent in an amount of about IxlO¹¹ vp. In said trivalent composition the total number of adenovirus particles per dose then amounts to about 3xl0ⁿ vp. The initial vaccination is followed by a boost as described above.

In one exemplary regimen, the MVA vector is administered (e.g., intramuscularly) in a volume ranging between about 100 μΐ to about 10 ml of saline solution containing a dose of about 1x1O⁷TCID5o to lxlO⁹ TCID50 (50% Tissue Culture Infective Dose) or Inf.U. (Infectious

Unit). 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 1x10 TCID50 to 1x10 TCID50 (or Inf.U.) to a human subject during one administration. In a preferred embodiment, the MVA vector is administered in an amount of about 5x10 TCID50 to 5x10 TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about 5xl0⁷ TCID50 (or Inf.U.). In a more preferred embodiment, the MVA vector is administered in an amount of about lxlO⁸ TCID50 (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 4.4x10⁸ TCID50 (or Inf.U.). g In a more preferred embodiment, the MVA vector is administered in an amount of about 5x10 TCID50 (or Inf.U.)

The compositions 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, may comprise métal or plastic foil, such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

The compositions of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially dépendent upon the condition to be treated.

Boosting compositions are administered two or more times, weeks or months after administration of the 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.

Preferably, the initial boosting inoculation is administered 1-12 weeks or 2-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming. In a preferred embodiment, the initial boosting inoculation is administered 4 or 8 weeks after priming. In additional preferred embodiments, the initial boosting is conducted at least 1 week, or at least 2 weeks, or at least 4 weeks after priming. In still another preferred embodiment, the initial boosting is conducted 412 weeks or 4-8 weeks after priming.

In a preferred embodiment of the invention, at least one further boosting inoculation is administered at least 4 weeks after the initial boosting inoculation. In still a preferred embodiment of the invention, the further boosting inoculation is administered at least 5 weeks after the initial boosting inoculation. In yet another preferred embodiment, the further boosting inoculation is administered at least 6 weeks after the initial boosting inoculation. For example, the further boosting inoculation can be administered 6 weeks to 5 years after the boosting step (b), such as 6, 7, 8, 9, 10,11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 weeks, or 7, 8, 9, 10, 11 or 12 months, or 2, 3, 4 or 5 years, after the initial boosting inoculation. Optionally, the further boosting step (c) can be repeated one or more times as needed.

In a more preferred embodiment according to this method, an MVA vector, such as a MVA-BN vector, is used for the priming followed by an initial boosting with an adenovirus vector, such as a rAd26 vector, and a further boosting with an adenovirus vector, such as an rAd26 vector. Preferably, the initial boosting composition is administered 1-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks after priming, and the further boosting composition is administered 4-96 weeks after initial boosting, more preferably 8-60 weeks, or preferably 1060 weeks after initial boosting, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 weeks, or 7, 8, 9, 10, 11 or 12 months, or 2, 3, 4 or 5 years, after the initial boosting inoculation. In a preferred embodiment, the initial boosting composition is administered 8 weeks after priming, and the further boosting composition is administered 360 days after priming. In another preferred embodiment, the initial boosting composition is administered 2 week after priming, and the further boosting composition is administered 360 days after priming. In another preferred embodiment, the boosting composition is administered 4 weeks after priming, and the further boosting composition is administered 360 days after priming. In yet another preferred embodiment, the boosting composition is administered 2 weeks after priming, and the further boosting composition is administered 13 weeks after priming. In yet another preferred embodiment, the initial boosting composition is administered 1 week after priming, and the further boosting composition is administered 13 weeks after priming.

In a preferred embodiment, each of the first and second boosting compositions comprises an Ad26 vector, more preferably encoding the same antigenic protein(s) or immunogenic polypeptide(s) thereof.

In other embodiments, each of the first and second boosting compositions comprises different adenovirus vectors, such as an Ad26 vector and an Ad35 vector, respectively, and the different adenovirus vectors can encode the same or different antigenic proteins or immunogenic polypeptides thereof.

In one embodiment, the invention relates to a method of enhancing an immune response against a tumor in a human subject. The method comprises:

a. administering to the human subject a first composition comprising an immunologically effective amount of a MVA vector comprising a first polynucleotide encoding an antigenic protein produced by a cell of the tumor, a substantially similar antigenic protein, or an immunogenic polypeptide thereof for priming the immune response;

b. administering to the subject a second composition comprising an immunologically effective amount of a first adenovirus vector comprising a second polynucleotide encoding the antigenic protein, the substantially similar antigenic protein, or an immunogenic polypeptide thereof for boosting the immune response; and

c. administering to the subject a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a third polynucleotide encoding the antigenic protein or an immunogenic polypeptide thereof for further boosting the immune response, to thereby obtain an enhanced immune response against the tumor in the human subject. Preferably, the enhanced immune response provides the human subject with a protective immunity against the tumor.

In a preferred embodiment the boosting step b is conducted 1-12 weeks or 2-12 weeks after the priming step a. The boosting step b can also be conducted later than 12 weeks after the priming step a. In additional preferred embodiments, the boosting step b is conducted at least 2 weeks or at least 4 weeks after the priming step a. In still other preferred embodiments, the boosting step b is conducted 4-12 weeks or 4-8 weeks after the priming step a.

In another preferred embodiment of the invention, the further boosting step (c) is conducted at least 4 weeks after the boosting step (b). In a preferred embodiment of the invention, the further boosting step (c) is conducted at least 5 weeks after the boosting step (b). In still another preferred embodiment, the further boosting step (c) is conducted at least 6 weeks after the boosting step (b). For example, the further boosting step (c) can be conducted 6 weeks to 5 years after the boosting step (b), such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24 or 25 weeks, or 7, 8, 9, 10, 11 or 12 months, or 2, 3, 4 or 5 years after the boosting step (b).

In another preferred embodiment, the adenovirus vector in each of the second and third compositions is an Ad26 vector.

The antigenic protein produced by a cell of the tumor can be any tumor antigen. In a preferred embodiment, the tumor antigen is a tumor-specific antigen that is présent only on tumor cells. The tumor antigen can also be a tumor-associated antigen that is présent on some tumor cells and also some normal cells.

According to another embodiment, the invention relates to a method of enhancing an immune response against at least one subtype of filovirus in a human subject. The method comprises:

a. administering to the human subject a first composition comprising an immunologically effective amount of a MVA vector comprising a first polynucleotide encoding an antigenic protein of the at least one filovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof, for priming the immune response;

b. administering to the subject a second composition comprising an immunologically effective amount of a first adenovirus vector comprising a second polynucleotide encoding an antigenic protein of the at least one filovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof, for boosting the immune response; and

c. administering to the subject a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a third polynucleotide encoding an antigenic protein of the at least one filovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof, for further boosting the immune response;

to thereby obtain an enhanced immune response against the at least one subtype of filovirus in the human subject.

Preferably, the enhanced immune response provides the human subject a protective immunity against the at least one subtype of filovirus.

In a preferred embodiment the boosting step b is conducted 1-12 weeks or 2-12 weeks after the first step, more preferably 1, 2, 4, or 8 weeks after priming. In additional preferred embodiments, the boosting step b is conducted at least 1 week or at least 2 weeks after the priming. In still other preferred embodiments, the boosting step b is conducted 4-12 weeks or

4-8 weeks after the priming.

The boosting step can also be conducted later than 12 weeks after priming.

In another preferred embodiment of the invention, the further boosting step (c) is conducted at least 4 weeks after the boosting step (b). In a preferred embodiment of the invention, the further boosting step (c) is conducted at least 5 weeks after the boosting step (b). In still another preferred embodiment, the further boosting step (c) is conducted at least 6 weeks after the boosting step (b). For example, the further boosting step (c) can be conducted 6 weeks to 5 years after the boosting step (b), such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 weeks, or 7, 8, 9, 10, 11 or 12 months, or 2, 3, 4 or 5 years after the boosting step (b).

In another preferred embodiment, the adenovirus vector in each of the second and third compositions is an Ad26 vector.

In yet another preferred embodiment, the antigenic protein is a glycoprotein or a nucleoprotein of a filovirus subtype.

In one embodiment of the invention, the MVA vector in the first composition comprises a polynucleotide encoding antigenic proteins derived from more than one filovirus subtypes. More preferably, the MVA vector in the first composition comprises a polynucleotide encoding four antigenic proteins from four filovirus subtypes having the amino acid sequences of SEQ ID NOs: 1, 2, 4 and 5, or immunogenic polypeptides thereof.

In another embodiment of the invention, each of the second and third compositions comprises at least one adenovirus vector comprising a polynucleotide encoding an antigenic protein derived from a filovirus subtype that is same or different from the filovirus subtype encoded by the MVA vector. For example, the adenovirus vector can comprise a polynucleotide encoding an antigenic protein having the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5. Preferably, each of the second and third compositions can comprise more than one adenovirus vectors encoding more than one antigenic proteins or immunogenic polypeptides thereof from more than one filovirus subtypes. For example, each of the second and third compositions can comprise one to three adenovirus vectors encoding one to three of the antigenic proteins hâve the amino acid sequences of SEQ ID NOs: 1,2 and 3.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention. The following examples are offered to illustrate, but not to limit the claimed invention. Example 2

A randomized, placebo-controlled, observer-blind study is performed to evaluate the safety, tolerability and immunogenicity of a heterologous regimen, which contains: (1) a single dose of MVA-BN-Filo (IxlO⁸ TCID₅₀) or placebo (0.9% saline) as prime on Day 1; (2) a single dose of Ad26.Filo (9x10¹⁰ vp) or placebo as the initial boost on Day 15 (i.e., 2 weeks after Day 1); and (3) a single dose of Ad26.Filo (9xlO¹⁰ vp) or placebo as the second boost on Day 92 (13 weeks after Day 1). The Ad26.Filo is a composition that comprises three monovalent replication-incompetent adénoviral vector serotype 26 (Ad26) vectors each encoding either an EBOV Mayinga GP (SEQ ID NO: 1); a SUDV Gulu GP (SEQ ID NO:2); or a MARV Angola GP (SEQ ID NO:3).

This regimen is tested in 7 healthy subjects, aged between 18 and 50 years (inclusive) who hâve never received an experimental Ebola candidate vaccine before and hâve no known exposure to EBOV or diagnosis of Ebola disease. Out of the 7 subjects in this group 5 received active vaccination and 2 the placebo.

The study consists of a vaccination period in which subjects are vaccinated at their baseline visit (Day 1) followed by a first boost on Day 15, a second boost on Day 92, and a post-boost follow-up period until 1 year post prime. The study vaccination schedules of the relevant group is summarized in Table 4.

Table 4: Study Vaccination Schedule

N n Day 1_________________Day 15___________Day 92___________

- MVA-BN-Filo, 5xl0⁸Inf U Ad26.Filo, 9xlO¹⁰ vp Ad26.Filo, 9xlO¹⁰ vp

- Placebo Placebo Placebo

Immunogenicity is assessed using the immunologie assays summarized in Tables 5 and 6. The exploratory assay package may include, but is not limited to, the listed assays.

Table 5: Summary of Immunologie Assays (Serology)

Assay	Purpose
Secondary endpoints Pseudovirus neutralization assay (psVNA) ELISA	Analysis of neutralizing antibodies to EBOV GP, MARV GP and SUDV GP Analysis of antibodies binding to EBOV GP, MARV GP and SUDV GP
Exploratory endpoints Adenovirus/M V A neutralization assay	Neutralizing antibodies to adenovirus/MVA
EBOV: Ebola virus; ELISA: enzyme-linked immunosorbent assay; GP: glycoprotein; IgG: immunoglobulin G; MARV: Marburg virus; MVA: Modified Vaccinia Ankara; NP: nucleoprotein; SUDV: Sudan virus; TAFV: Tai Forest virus
Table 6: Summary of Immunologie Assays (Cellular)
Assay	Purpose
Exploratory endpoints ELISpot ICS of frozen PBMC	T cell IFN-γ responses to EBOV GP, MARV GP and SUDV GP Analysis of T cell responses to EBOV GP, SUDV GP, MARV GP and/or TAFV NP (including CD4/8, IL-2, IFNγ, TNF-α and/or activation markers)

EBOV: Ebola virus; ELISpot: enzyme-linked immunospot; GP: glycoprotein; ICS: intracellular cytokine staining; IFN: interferon; IL: interleukin; MARV: Marburg virus; NP: nucleoprotein; PBMC: peripheral blood mononuclear cells; SUDV: Sudan virus; TAFV: Tai Forest virus; TNF: tumor necrosis factor

The clinical study is ongoing. Some initial results are described below.

Assessment of immune responses

Immunogenicity has been assessed at baseline (Day 1) and Days 15, 36, 92, 99 and 113 post-prime immunization (i.e. 21 days post-second boost vaccination) using an ELIS A assay to analyze antibodies binding to either EBOV GP, MARV GP or SUDV GP, a psVNA to analyze the neutralizing antibody response against either EBOV GP, MARV GP or SUDV GP 15 and a VNA to analyze the neutralizing antibody response against the Ad26 backbone.

Humoral immune responses against EBOV GP

A binding antibody response against EBOV GP was observed in 20% of the subjects 14 days post-prime (Day 15), with the geometric-mean concentration (GMC) <LLOQ (Figure 1). Twenty-one days post-boost vaccination with Ad26.Filo (post-Dose 2, Day 36), a binding antibody response against EBOV GP was observed in 100% of the subjects with a GMC of 3843 ELISA units/mL (95% CI: 1761; 8388) (Figure 1). GMC stayed in the same range until administration of the third vaccination on Day 92 (2425 ELISA units/mL [95% CI: 1503; 3911]) (Figure 1). Seven days post-Dose 3 (Day 99), a binding antibody response against EBOV GP was observed in 100% of the subjects with a GMC of 13445 ELISA units/mL (95% CI: 4125; 43829) (Figure 1). GMC tended to be slightly increased at Day 113 (15715 ELISA units/mL [95%C1: 7146; 34558]) (Figure 1).

No neutralizing antibody responses against EBOV GP were observed in the subjects 14 days post-prime (Day 15) (neutralizing GMTs <LLOQ) (Figure 1). On Day 92, a neutralizing antibody response against EBOV GP was observed in 100% of the subjects, with a neutralizing GMT of 520 IC₅o (95% CI: 168; 1612) (Figure 1). Seven days post-Dose 3 (Day 99), a neutralizing antibody response against EBOV GP was observed in 100% of the subjects, with a neutralizing GMT of 5589 IC50 (95% CI: 1155; 27051) that was forther increased by Day 113 (7779 IC50 [95%CI: 2604; 23239]) (Figure 1). On Day 180, both the binding and neutralizing antibody responses had declined compared to the peak responses post-third vaccination (Figure 1)·

Humoral immune responses against MARV GP

A binding antibody response against MARV GP was observed in 20% of the subjects 14 days post-prime (Day 15), with GMC<LLOQ (Figure 1). Twenty-one days post-boost vaccination with Ad26.Filo (post-Dose 2, Day 36), a binding antibody response against MARV GP was observed in 100% of the subjects with a GMC of 215 ELISA units/mL (95% CI: 52; 887) (Figure 1). GMC stayed in the same range until administration of the third vaccination on Day 92 (155 ELISA units/mL [95% CI: 36; 665]) (Figure 1). Seven days post-Dose 3 (Day 99), a binding antibody response against MARV GP was observed in 100% of the subjects with a GMC of 510 ELISA units/mL (95% CI: 92; 2827) (Figure 1). GMC tended to stay in the same range at Day 113 (825 ELISA units/mL [95% CI: 230; 2959]) (Figure 1).

No neutralizing antibody responses against MARV GP were observed in the subjects 14 days post-prime (Day 15) (neutralizing GMTs <LLOQ) (Figure 1). On Day 92, a neutralizing antibody response against MARV GP was observed in 20% of the subjects, with a neutralizing GMT below LLOQ (Figure 1). Seven days post-Dose 3 (Day 99), a neutralizing antibody response against MARV GP was observed in 40% of the subjects, with a neutralizing GMT of 51 IC₅o (95% CI: <LLOQ; 275) (Figure 1). Neutralizing GMTs remained similar at Day 113 (55 IC50 [95% CI: <LLOQ; 313]) (Figure 1). On Day 180, both the binding and neutralizing antibody responses had declined compared to the peak responses post-third vaccination (Figure 1).

Humoral immune responses against SUDV GP

A binding antibody response against SUDV GP was observed in 80% of the subjects 14 days post-prime (Day 15), with a GMC of 77 ELISA units/mL (95% CI: <LLOQ; 423) (Figure 1). Twenty-one days post-boost vaccination with Ad26.Filo (post-Dose 2, Day 36), a binding antibody response against SUDV GP was observed in 100% of the subjects with a GMC of 1879 ELISA units/mL (95% CI: 803; 4396) (Figure 1). GMC decreased until administration of the third vaccination on Day 92 (726 ELISA units/mL [95% CI: 401; 1313]) (Figure 1). Seven days post-Dose 3 (Day 99), a binding antibody response against SUDV GP was observed in 100% of the subjects with a GMC of 3186 ELISA units/mL (95% CI: 921; 11020) (Figure 1). GMC was similar at Day 113 (3036 ELISA units/mL [95%CI: 1672; 5513]) (Figure 1).

No neutralizing antibody responses against SUDV GP were observed in the subjects 14 days post-prime (Day 15) (neutralizing GMTs <LLOQ) (Figure 1). On Day 92, a neutralizing antibody response against SUDV GP was observed in 60% of the subjects, with a neutralizing GMT of 63 IC₅₀ (95% CI: <LLOQ; 236) (Figure 1). Seven days post-Dose 3 (Day 99), a neutralizing antibody response against SUDV GP was observed in 80% of the subjects, with a neutralizing GMT of 601 IC50 (95% CI: 52; 6995) (Figure 1). Neutralizing GMTs stayed in the same range at Day 113 (100% responders, neutralizing GMT: 884 IC50 [95%CI: 280; 2788]) (Figure 1).

Neutralizing antibody responses against Ad26 backbone

Neutralizing antibodies directed against Ad26 were measured by VNA at baseline and at Day 92, prior to the third vaccination. At baseline, only 1 (20%) subject had a neutralizing antibody response directed against Ad26 (neutralizing GMT: 23 IC50; 95%CI: <LLOQ; 377). Seventy52 seven days after boost vaccination with Ad26.Filo (Day 92), ail 5 subjects had a neutralizing antibody response directed against Ad26 with a neutralizing GMT of 203 IC50 (95% CI:

<LLOQ; 15674).

Example 3

A randomized, placebo-controlled, observer-blind study is performed in Groups 1-3 to evaluate the safety, tolerability and immunogenicity of a heterologous regimen, which contains: (1) a single dose of MVA-BN-Filo (IxlO⁸ TCID50) or placebo (0.9% saline) as prime on Day 1; (2) a single dose of Ad26.ZEBOV (5x10¹⁰ vp) or placebo as the first boost at different time point of Day 15, 29 or 57 (i.e., 2, 4, or 8 weeks after prime); and (3) a single dose of Ad26.ZEBOV (5xlO¹⁰ vp) as the second boost on Day 360 (i.e., 1 year post-prime). The Ad26. ZEBOV encodes the EBOV Mayinga GP (SEQ ID NO: 1).

Subjects are enrolled in 3 different groups, comprising 18 (Groups 1 to 3) healthy subjects each. Subjects are randomized in a 5:1 ratio to receive active vaccine or placebo throughout the study. The study vaccination schedules in the different groups are summarized in Table 7.

Table 7: Study Vaccination Schedules

Group	N	n	Day 1	Day 15	Day 29	Day 57	Day 360
		15	MVA-BN-Filo	Ad26. ZEBOV			Ad26.ZEBOV
1	18	3	Placebo	Placebo			Placebo
2	18	15 3	MVA-BN-Filo Placebo		Ad26.ZEBOV Placebo		Ad26.ZEBOV Placebo
3	18	15 3	MVA-BN-Filo Placebo			Ad26.ZEBOV Placebo	Ad26.ZEBOV Placebo

N: number of subjects to receive study vaccine

MVA-BN-Filo dose level is IxlO⁸ TCID50 (50% Tissue Culture Infective

Dose) in ail groups;

Ad26.ZEBOV dose level is 5xl0¹⁰ vp (viral particles) in ail groups;

Placebo is 0.9% saline in ail groups

Safety is assessed by collection of solicited local and systemic adverse events, unsolicited adverse events and serions adverse events, and by physical examination. In addition, standard chemistry, hématologie (including coagulation parameters) and urinalysis parameters are assessed at multiple time points.

Immunogenicity is assessed using the immunologie assays summarized in Tables 8 and 9. The exploratory assay package may include, but is not limited to, the listed assays.

Table 8: Summary of Immunologie Assays (Serology)

Assay	Purpose
Secondary endpoints Pseudovirus neutralization assay (psVNA) ELISA	Analysis of neutralizing antibodies to EBOV GP Analysis of antibodies binding to EBOV GP
Exploratory endpoints Adenovirus/MV A neutralization assay Molecular antibody characterization	Neutralizing antibodies to adenovirus/MVA Analysis of anti-EBOV GP, SUDV GP, MARV GP and/or TAFV NP antibody characteristics, including IgG subtyping

EBOV: Ebola virus; ELISA: enzyme-linked immunosorbent assay; GP: glycoprotein; IgG: immunoglobulin G; MARV: Marburg virus; MVA: Modified Vaccinia Ankara; NP: nucleoprotein; SUDV: Sudan virus; TAFV: Tai Forest virus

Table 9: Summary of Immunologie Assays (Cellular)

Assay	Purpose
Secondary endpoints ELISpot	T cell IFN-γ responses to EBOV GP
Exploratory endpoints ICS of frozen PBMC	Analysis of T cell responses to EBOV GP (including CD4/8, IL-2, IFN-γ, TNF-α and/or activation markers)

EBOV: Ebola virus; ELISpot: enzyme-linked immunospot; GP: glycoprotein; ICS: intracellular cytokine staining; IFN: interferon; IL: interleukin; PBMC: peripheral blood mononuclear cells; TNF: tumor necrosis factor

Assessment of immune responses

Immunogenicity has been assessed at multiple time points, the most important being baseline (Day 1), pre-boost 1 (Day 15, 29 or 57), 21 days post-boost 1 (Day 36, 50 or 78), preboost 2 (Day 360), 7 days post-boost 2 (Day 367), 21 days post-boost 2 (Day 381) and approximately one year post-boost 2 (Day 720). An ELISA was used to analyze antibodies binding to EBOV GP, a psVNA to analyze the neutralizing antibody response against EBOV GP, an ELISpot and ICS to analyze the cellular immune responses, a VNA to analyze the neutralizing antibody response against the Ad26 backbone and a plaque réduction neutralization test (PRNT) to analyze the neutralizing antibody response against the MVA backbone.

Assessment of humoral immune responses

Figure 2 shows the Ebola Zaïre glycoprotein spécifie humoral immune response observed in individuals that were administered an MVA-BN-Filo vector as a prime on day 1, followed by a boost with an Ad26.ZEBOV vector either on day 15, day 29 or 57 and a second boost with Ad26.ZEBOV on day 360. A 20 to 23 fold increase in antibody levels was assessed by ELIS A after the second boost at day 360 with Ad26.ZEBOV. The onset of this anamnestic response was very fast and was observed already 7 days after administration of the second boost. At this Day 367, binding antibody responses were much higher compared to the peak responses observed post-dose 2, and continued to increase until 21 days post-boost 2 (Day 381). After Day 381, the response levels gradually decreased towards the end of the study (Day 720) but remained 1.6- to 5.8-fold higher compared to 1 year post-prime. Across ail groups’ the responder rates on Day 720 were between 91% and 100%.

In line with the binding antibody responses, re-exposure to the EBOV GP antigen mimicked by the second boost vaccination on Day 360 induced a marked increase of neutralizing antibody levels 7 days later (Figure 3). The neutralizing antibody responses continued to increase until 21 days post-boost 2 (Day 381), except in the MVA/Ad26 28-day interval group, by when they reached géométrie mean neutralizing antibody concentrations that were 27- to 77-fold higher compared to Day 360 (Figure 3). After Day 381, the response levels gradually decreased but the responder rates remained high (between 92% and 100%) until the end of the study (Day 720) (Figure 3).

Assessment of cellular immune responses

Re-exposure to the EBOV GP antigen mimicked by the third vaccination on Day 360 induced an increase in IFN-y+ responses as measured by ELISpot, however, the magnitude of the responses varied across regimens (Figure 4). The highest médian response post-boost 2 was observed on Day 367 in subjects who received the MVA/Ad26 14-day interval regimen and a third dose with Ad26 on Day 360 (Figure 4). In this group, the médian IFN-y+ response on Day 367 was almost 5 times higher compared to Day 360 (Figure 6). After Day 367, the response level in this group gradually decreased and the responder rate at the end of the study (Day 720) was 80% (Figure 4).

Administration of the second boost vaccination on Day 360 induced a modest increase in the CD4+ T cell responses in ali groups by Day 367, while a modest increase in CD8+ T cell responses was measured in only some but not ail groups by ICS (Figures 5 and 5).

The CD4+ T cell responses mostly remained below or around the peak value of the acute phase. After Day 367, the response levels as well as the responder rates gradually decreased (Figure 5). The CD8+ T cell responses continüed to increase until Day 381 but mostly remained either below or around the peak value ofthe acute phase. After Day 381, the response.

levels and responder rates gradually decreased (Figure 6).

Neutralizing antibody responses against the Ad26 backbone

Neutralizing antibody responses against the Ad26 vector as measured by Ad26 neutralization assay were detected in 13.2% of subjects prior to the first dose of Ad26. After the first Ad26 dose but pre-boost 2, neutralizing antibody responses against the Ad26 vector were observed in 98.8% of the subjects. At 21 days after a second dose of Ad26, 100% of subjects had neutralizing antibodies directed against the Ad26 vector. The géométrie mean titers measured by Ad26 neutralization assay increased after both Ad26 doses (titer range after the first dose: 869 to 2001; titer range after the second dose: 1460 to 8004) (data not shown).

In summary, these examples show that immunization sériés consisting of priming the immune response with an MVA vector, followed by an initial boosting of the immune response with an adenovirus vector, and a further boosting of the immune response with an adenovirus vector resulted in a robust increase in immune response. A robust increase in binding and neutralizing antibody titers was observed after the last immunization with an adenovirus vector, ranging from 20 to 23 and 19 to 43 fold increase, respectively. Importantly, this was associated with an increase in the percentage of responders and was observed across the large range of vaccination intervals studied. A similar effect was observed for the cellular immune response albeit to a more modest magnitude.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

SEQUENCE LISTING

SEQ ID NO:1

Glycoprotein Ebola virus Zaïre, strain Mayinga (Amino Acid sequence):

mgvtgilqlprdrfkrtsfflwviilfqrtfsiplgvihnstlqvsdvdklvcrdklsstnqlr SVGLNLEGNGVATDVPSATKRWGFRSGVPPKWNYEAGEWAENCYNLEIKKPDGSECLPAAPDG IRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKD FFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQL NETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISG QSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGP DNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPAT TTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCN PNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYlEGLMHNQDGLICGLRQLANETTQALQLFLRAT TELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGD NDNWWTGWRQWIPAGIGVTGVIIAVIALFCIC KFVF

SEQ ID NO:2

Glycoprotein Ebola virus Sudan, strain Gulu (Amino Acid sequence):

MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQLVCKDHLASTDQLK SVGLNLEGSGVSTDIPSATKRWGFRSGVPPKWSYEAGEWAENCYNLEIKKPDGSECLPPPPDG VRGFPRCRYVHKAQGTGPCPGDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKET FLQSPPIREAVNYTENTSSYYATSYLEYEIENFGAQHSTTLFKIDNNTFVRLDRPHTPQFLFQL NDTIHLHQQLSNTTGRLIWTLDANINADIGEWAFWENKKNLSEQLRGEELSFEALSLNETEDDD AASSRITKGRISDRATRKYSDLVPKNSPGMVPLHIPEGETTLPSQNSTEGRRVGWTQETI.TET AATIIGTNGNHMQISTIGIRPSSSQIPSSSPTTAPSPEAQTPTTHTSGPSVMATEEPTTPPGSS PGPTTEAPTLTTPENITTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRRQTNTKATGK.cn PNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQLANETTQALQLFLRAT TELRTYTILNRKAIDFLLRRWGGTCRILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDN DDNWWTGWRQWIPAGIGITGIIIAIIALLCVCKLLC

SEQ ID NO:3

Glycoprotein Marburg virus Angola (Amino Acid sequence):

MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPL EASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQ NPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLT STNKYWTSSNGTQTNDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNT TDPNSDDEDLTTSGSGSGEQEPYTTSDAATKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQGWT EPGKTNTTAQPSMPPHNTTTISTNNTSKHNLSTPSVPIQNATNYNTQSTAPENEQTSAPSKTTL LPTENPTTAKSTNSTKSPTTTVPNTTNKYSTSPSPTPNSTAQHLVYFRRKRNILWREGDMFPFL DGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDC DAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRV TTEERTFSLINRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTGWGL GGKWWTSDWGVLTNLGILLLLSIA.VLIALSCICRIFTKYIG

SEQ ID NO:4

Glycoprotein Marburg virus Musoke (Amino Acid sequence):

mkttcflisliliqgtknlpileiasnnqpqnvdsvcsgtlqktedvhlmgftlsgqkvadspl EASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQ NPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLT STNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNT TDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQDAVT ELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTL PPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFL DGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDC DAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRV TTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGL GGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG

SEP ID NO:5

Nucleoprotein Ebola virus Taï Forest / Ivory coast (Amino Acid sequence):

MESRAHKAWMTHTASGFETDYHKILTAGLSVQQGIVRQRVIQVHQVTNLEEICQLIIQAFEAGV DFQESADSFLLMLCLHHAYQGDYKQFLESNAVKYLEGHGFRFEVRKKEGVKRLEELLPAASSGK SIRRTLAAMPEEETTEANAGQFLSFASLFLPKLWGEKACLEKVQRQIQVHSEQGLIQYPTAWQ SVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVIANSVAQARFSGLLIVKTVLDHILQ KTEHGVRLHPLARTAKVKNEVNSFKAALSSLAQHGEYAPFARLLNLSGVNNLEHGLFPQLSAIA LGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQKYAESRELDHLGLDDQEKKILKDFHQKKN EISFQQTTAMVTLRKERLAKLTEAITSTSLLKTGKQYDDDNDIPFPGPINDNENSEQQDDDPTD SQDTTIPDIIVDPDDGRYNNYGDYPSET.ANAPEDLVLFDLEDGDEDDHRPSSSSENNNKHSLTG TDSNKTSNWNRNPTNMPKKDSTQNNDNPAQRAQEYARDNIQDTPTPHRALTPISEETGSNGHNE DDIDSIPPLESDEENNTETTITTTKNTTAPPAPVYRSNSEKEPLPQEKSQKQPNQVSGSENTDN KPHSEQSVEEMYRHILQTQGPFDAILYYYMMTEEPIVFSTSDGKEYVYPDSLEGEHPPWLSEKE ALNEDNRFITMDDQQFYWPVMNHRNKFMAILQHHK

Claims (13)

1. Use of a combination for the préparation of a médicament for inducing an enhanced immune response in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of a MVA vector comprising a first polynucleotide encoding a first antigenic protein or an immunogenic polypeptide thereof;

b. a second composition comprising an immunologically effective amount of a first adenovirus vector comprising a second polynucleotide encoding a second antigenic protein or an immunogenic polypeptide thereof; and

c. a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a third polynucleotide encoding a third antigenic protein or an immunogenic polypeptide thereof, wherein the first composition is for priming the immune response, the second composition is for boosting the immune response, and the third composition is for further boosting the immune response, and wherein the first, second and third antigenic proteins share at least one antigenic déterminant.

2. The use according to claim 1, wherein the enhanced immune response comprises an enhanced antibody response, an enhanced CD8+ T cell response, and/or an enhanced CD4+ T cell response against the at least one antigenic déterminant shared by the first, second and third antigenic proteins in the human subject.

3. The use according to claim 2, wherein the enhanced CD8+ or CD4+ T cell response comprises an increase or induction of a dominant CD8+ or CD4+ T cell response and/or an increase or induction of polyfunctional CD8+ or CD4+ T cells against the at least one antigenic déterminant shared by the first, second and third antigenic proteins in the human subject.

4. The use according to claim 1, wherein the enhanced immune response provides a protective immunity to the human subject against a disease related to at least one of the first, second and third antigenic proteins.

5. The use according to any one of claims 1, 2, 3 and 4 , wherein the second composition is for administration 1-12 weeks, 2-12 weeks, at least 1 week, or at least 2 weeks- after the first composition, and the third composition is for administration 4-96 weeks or at least 4 weeks after the second composition.

6. The use according to any one of claims 1, 2, 3, 4, and 5, wherein the first, second or third antigenic protein is derived from a pathogen or a tumor.

7. The use according to claim 6, wherein the first, second or third antigenic protein is derived from a filovirus, preferably, the first, second and third antigenic proteins are identical or substantially identical.

8. The use according to claim 7, wherein each of the first, second and third antigenic protein independently comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, immunogenic polypeptides thereof, and combinations thereof.

9. The use according to claim 8, wherein the MVA vector comprises a polynucleotide encoding at least one antigenic protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, or an immunogenic polypeptide thereof; preferably, the MVA vector comprises a polynucleotide encoding four 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.

10. The use according to claim 8 or 9, wherein each of the first and the second adenovirus vectors comprises a polynucleotide encoding the antigenic protein having the amino acid sequence of SEQ ID NO: 1.

11. The use according to claim 10, wherein the second and/or third composition further comprises an immunologically effective amount of an adenovirus vector comprising a polynucleotide encoding an antigenic protein having the amino acid sequence of SEQ ID NO:2; and further comprises an immunologically effective amount of an adenovirus vector comprising a polynucleotide encoding an antigenic protein having the amino acid sequence ofSEQ ID NO:3.

12. The use according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, wherein each of the first and second adenovirus vectors is an rAd26 vector, and the MVA vector is MVA-BN vector.

13. Use of a combination for the préparation of a médicament for inducing an enhanced immune response against at least one fîlovirus subtype in a human subject, comprising:

a. a first composition comprising an immunologically effective amount of a MVA vector comprising a first polynucleotide encoding a first antigenic protein of the at least one fîlovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof;

b. a second composition comprising an immunologically effective amount of a first adenovirus vector comprising a second polynucleotide encoding a second antigenic protein of the at least one fîlovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof; and

c. a third composition comprising an immunologically effective amount of a second adenovirus vector comprising a third polynucleotide encoding a third antigenic protein of the at least one fîlovirus subtype, a substantially similar antigenic protein, or an immunogenic polypeptide thereof;

wherein the first composition is for priming the immune response, the second composition is for boosting the immune response, and the third composition is for further boosting the immune response, and wherein the first, second and third antigenic proteins share at least one antigenic déterminant.