METHODS AND COMPOSITIONS FOR INDUCING PROTECTIVE IMMUNITY AGAINST FILOVIRUS INFECTION AND/OR DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

62/164,909, filed May 21, 2015, the disclosure of which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name "688097-70WO Sequence Listing", creation date of May 20, 2016 and having a size of about 15 kB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This invention relates to compositions, vaccines and methods for inducing protective immunity against filovirus infection and/or disease, particularly protective immunity against infection of Ebolavirus.

BACKGROUND OF THE INVENTION

[0004] Marburg viruses (MARV) and Ebolaviruses (EBOV), such as the Zaire ebolavirus, are associated with outbreaks of highly lethal Marburg Hemorrhagic Fever (MHF) and Ebola Virus Disease (EVD) in humans and primates in Africa, North America and Europe. These viruses are filoviruses that are known to infect humans and non-human primates with severe health consequences, including death. Filovirus infections have resulted in case fatality rates of up to 90% in humans. EBOV infections cause EVD with death often occurring within 7 to 10 days post-infection. EVD presents as an acute febrile syndrome manifested by an abrupt fever, nausea, vomiting, diarrhea, maculopapular rash, malaise, prostration, generalized signs of increased vascular permeability, coagulation abnormalities, and dysregulation of the innate immune response. Much of the disease appears to be caused by dysregulation of innate immune responses to the infection and by replication of virus in vascular endothelial cells, which induces death of host cells and destruction of the endothelial barrier. Filoviruses can be spread by small particle aerosol or by direct contact with infected blood, organs, and body fluids of human or NHP origin. Infection with a single virion is reported to be sufficient to cause EVD in humans. Presently, there is no therapeutic or vaccine approved for treatment or prevention of EVD, although two candidate vaccines have progressed to a phase 2/3 clinical trial ongoing in Liberia. Supportive care remains the primary medical intervention for individuals who become infected with filoviruses and/or who suffer from EVD.

[0005] People remain infectious for Ebola virus disease (EVD) as long as their blood and secretions contain the virus. The high infectivity of blood and secretions puts healthcare workers at particularly high risk during outbreaks, and direct contact with the bodies of deceased victims can also have a role in the transmission of the virus. The incubation period of EVD is 2 to 21 days (7 days on average, depending on the strain) followed by a severe acute viral illness often characterized by the rapid onset of non-specific symptoms such as fever, extreme fatigue, pharyngitis, gastrointestinal complaints, abdominal pain, anorexia, headache, myalgia and/or arthralgia.

[0006] A primary antibody response (IgM) can be detected in the blood of infected persons 2 to 9 days after infection whereas IgG antibodies appear approximately 17 to 25 days after infection. This IgG response coincides with the recovery phase. In survivors of EVD, both humoral and cellular immunity are detected, however, their relative contribution to protection is unknown (Sullivan et al, Ebola virus pathogenesis: implications for vaccines and therapies. J Virol 2003;77:9733-7).

[0007] As the cause of severe human disease, filoviruses continue to be of concern as both a source of natural infections, and also as possible agents of bioterrorism. The reservoir for filoviruses in the wild has not yet been definitively identified. Four subtypes of Ebolaviruses have been described to cause EVD, i.e., those in the Zaire, Sudan, Bundibugyo and Ivory Coast episodes (Sanchez, A. et al. 1996 PNAS USA 93:3602-3607). These subtypes of Ebolaviruses have similar genetic organizations, e.g., negative-stranded RNA viruses containing seven linearly arrayed genes. The structural gene products include, for example, the envelope glycoprotein that exists in two alternative forms, a secreted soluble glycoprotein (ssGP) and a transmembrane glycoprotein (GP) generated by RNA editing that mediates viral entry

(Sanchez, et al. 1996 PNAS USA 93:3602-3607).

[0008] It has been suggested that immunization can be useful in protecting against Ebola infection and/or related disease because there appears to be less nucleotide polymorphism within Ebola subtypes than among other RNA viruses (Sanchez et al. 1996 PNAS USA 93:3602-3607). Until recently, developments of preventive vaccines against filoviruses have had variable results, partly because the requirements for protective immune responses against filovirus infections are poorly understood.

[0009] Currently, there are several vaccine antigen delivery platforms that demonstrated various levels of protection in non-human primates (NHPs) exposed with high infectious doses of filoviruses. Vaccine candidates in development are based on a variety of platform technologies including replication competent vectors (e.g. Vesicular Stomatitis Virus; Rabies virus; Parainfluenza Virus); replication incompetent vectors (Adenovirus, Modified Vaccinia Ankara Virus); protein subunits inclusive of Virus Like Particles expressed in bacterial cells, insect cells, mammalian cells, plant cells; DNA vaccines; and /or live and killed attenuated filovirus (Friedrich et al, 2012). The EBOV glycoprotein GP is an important component of a vaccine that protects against exposures with the same species of EBOV. The development of medical countermeasures for these viruses is a high priority.

[0010] Replication-defective adenovirus vectors (rAd) are powerful inducers of cellular immune responses and have therefore come to serve as useful vectors for gene-based vaccines particularly for lentiviruses and filoviruses, as well as other nonviral pathogens (Shiver, et al , (2002) Nature 415(6869): 331-5; (Hill, et al , Hum Vaccin 6(1): 78-83.; Sullivan, et al, (2000) Nature 408(6812): 605-9; Sullivan et al, (2003) Nature 424(6949): 681-4; Sullivan, et al , (2006) PLoS Med 3(6): el77; Radosevic, et al, (2007); Santra, et al , (2009) Vaccine 27(42): 5837-45). Adenovirus-based vaccines have several advantages as human vaccines since they can be produced to high titers under GMP conditions and have proven to be safe and immunogenic in humans (Asmuth, et al , J Infect Dis 201(1): 132-41 ; Kibuuka, et al, J Infect Dis 201(4): 600-7; Koup, et al , PLoS One 5(2): e9015. ; Catanzaro, et al , (2006) J Infect Dis 194(12): 1638-49; Harro, et al, (2009) Clin Vaccine Immunol 16(9): 1285-92). While most of the initial vaccine work was conducted using rAd5 due to its significant potency in eliciting broad antibody and CD8+ T cell responses, pre-existing immunity to rAd5 in humans can limit efficacy (Catanzaro, (2006); Cheng, et al , (2007) PLoS Pathog 3(2): e25.; McCoy, et al, (2007) J Virol 81(12):6594-604.; Buchbinder, et al, (2008) Lancet 372(9653): 1881-93). This property might restrict the use of rAd5 in clinical applications for many vaccines that are currently in development including Ebolavirus (EBOV).

[0011] Replication-defective adenovirus vectors, rAd26 and rAd35, derived from adenovirus serotype 26 and serotype 35, respectively, have the ability to circumvent Ad5 preexisting immunity. rAd26 can be grown to high titers in Ad5 El -complementing cell lines suitable for manufacturing these vectors at a large scale and at clinical grade (Abbink, et al, 2007), and this vector has been shown to induce humoral and cell-mediated immune responses in prime-boost vaccine strategies (Abbink, et al, 2007; Liu, et al , (2009) Nature 457(7225): 87-91). rAd35 vectors grow to high titers on cell lines suitable for production of clinical- grade vaccines (Havenga, et al , (2006) J Gen Virol 87(Pt 8): 2135-43), and have been formulated for injection as well as stable inhalable powder (Jin, et al , Yaccine 28(27): 4369- 75). These vectors show efficient transduction of human dendritic cells (de Gruijl, et al , (2006) J Immunol 177(4): 2208- 15; Lore, et al , (2007) J Immunol 179(3): 1721-9), and thus have the capability to mediate high level antigen delivery and presentation.

[0012] The prevalence of immunity to human adenovirus also prompted the consideration of simian adenoviruses as vectors. Simian adenoviruses are not known to cause pathology or illness in humans and the prevalence of antibodies to chimpanzee origin adenoviruses is less than 5% in humans residing in the US (Tatsis N. et al. 2007 Mol th: the Journal of the American Society of Gene Therapy 15: 608-17). They exhibit hexon structures homologous to that of human adenoviruses (Bruna-Romero O. et al. 2001 Nat Academy of Sciences of the USA 98: 11491-6).

[0013] Recombinant chimpanzee adenovirus serotype 3 (ChAd3 or cAd3) is a subgroup C adenovirus with properties similar to those of human adenovirus serotype 5 (Ad5). Chimpanzee adenovirus ChAd63 (cAd63) is another simian vector used in order to circumvent issues surrounding pre-existing immunity to AdHu5 in humans. Both the ChAd3 and cAd63 were shown to be safe and immunogenic in human studies evaluating candidate vaccines for hepatitis C virus (HCV) (Barnes, et al. Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man. Science translational medicine 2012;4: 115ral) and malaria (O'Hara et al. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. The Journal of infectious diseases 2012;205:772-81), respectively.

[0014] There is an unmet need for improved vaccines that elicit immune responses against filoviruses, particularly, protective immunity against the more deadly Ebolaviruses, such as EBOV. BRIEF SUMMARY OF THE INVENTION

[0015] It is discovered in the invention that various prime-boost combinations of replication incompetent vectors generate an effective immune response against filovirus infection and/or disease. Specifically, a prime-boost combination of a first composition comprising at least one human adenovirus vector and a second composition comprising a simian adenovirus vector generates improved immune protection.

[0016] Accordingly, one general aspect of the invention relates to a vaccine combination comprising:

(i) a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of a simian (e.g. chimpanzee) adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0017] In a vaccine combination according to an embodiment of the invention, the first antigenic protein and the second antigenic protein can be the same or different proteins.

[0018] Another general aspect of the invention relates to a method of inducing an immune response against a filovirus in a subject comprising:

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

b. administering to the subject a second composition comprising an

immunologically effective amount of a simian (e.g. chimpanzee) adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus,

wherein steps (a) and (b) are conducted in either order.

[0019] In a method according to an embodiment of the invention, the first antigenic protein and the second antigenic protein can be the same or different proteins. [0020] In certain embodiments, the first composition is used for priming said immune response and the second composition is used for boosting said immune response. In other embodiments the second composition is used for priming said immune response and the first composition is used for boosting said immune response.

[0021] In an embodiment of the method, the boosting composition is administered 1-15 weeks after the priming composition. In a preferred embodiment, the priming vaccination is conducted at week 0, followed by a boosting vaccination at week 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. In another preferred embodiment, the priming vaccination is conducted at week 0, followed by a boosting vaccination at month 6, 9, 12 or 24. More preferably, the priming vaccination is conducted at week 0, followed by a boosting vaccination at week 2, 4, 6, 8, 10 or 12.

[0022] The filovirus subtypes according to the invention can be any filovirus. In preferred embodiments of the invention the filovirus is an Ebola virus. In a preferred embodiment, the Ebola virus is of the subtype Zaire.

[0023] The antigenic protein encoded by the adenovirus vectors comprised in the first and second compositions can be any protein from any filovirus (e.g. Marburg or Ebola). In one embodiment, the antigenic protein is a glycoprotein (GP) or a nucleoprotein (NP) of a filovirus. In a preferred embodiment, the first antigenic protein comprises a GP comprising the amino acid sequence of SEQ ID NO: 1, or a protein substantially similar to the GP, such as a GP comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the second antigenic protein comprises a GP comprising the amino acid sequence of SEQ ID NO: l or a protein substantially similar to the GP, such as a GP comprising the amino acid sequence of SEQ ID NO: 3. In a further embodiment, each of the first and second antigenic protein comprises a GP comprising the amino acid sequence of SEQ ID NO: l or a protein

substantially similar to the GP, such as a GP comprising the amino acid sequence of SEQ ID NO: 3.

[0024] It is contemplated that the methods, vaccines, and compositions described herein can be embodied in a kit. For example, in one embodiment, the invention can include a kit comprising:

(i) a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus and a pharmaceutically acceptable carrier;

and (ii) a second composition comprising an immunologically effective amount of an simian (e.g. chimpanzee) adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0025] In said kit, the first antigenic protein and the second antigenic protein can be the same or different proteins.

[0026] In a preferred embodiment, the human adenovirus vector comprised in the first composition of the vaccine combination, method, or kit is a rAd26 or rAd35 vector, more preferably a rAd26 vector. In another preferred embodiment, the simian adenovirus vector comprised in the second composition of the vaccine combination, method, or kit is a chimpanzee adenovirus vector, such as a ChAd3 vector.

[0027] Therefore in one embodiment, the invention relates to a combination vaccine, a kit or a method wherein a first composition comprises an immunologically effective amount of at least one human adenovirus vector, and wherein a second composition comprises an immunologically effective amount of a simian adenovirus vector, such as a chimpanzee adenovirus vector. The first composition comprises an immunologically effective amount of at least one human adenovirus vector comprising a nucleic acid encoding an antigenic protein of a filovirus, and the second composition comprises an immunologically effective amount of a simian adenovirus vector comprising a nucleic acid encoding an antigenic protein of a filovirus that can be the same or different from the antigenic protein encoded by the at least one human adenovirus vector in the first composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0029] In the drawings:

[0030] FIG 1 is a graph which shows the EBOV-GP specific humoral immune response assessed by enzyme-linked immunosorbent assay (ELISA), displayed in ELISA Units per mL, on test samples obtained from subjects from groups Gl and G3, at weeks 1 through 21 of the trial; and

[0031] FIG 2 is a graph which shows the EBOV-GP specific humoral immune response assessed by enzyme-linked immunosorbent assay (ELISA), displayed in ELISA Units per mL, on test samples obtained from subjects from groups G2 and G4, at weeks 1 through 21 of the trial.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Various publications, articles and patents are cited or described in the background and throughout the specification; 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 specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All 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 dictates otherwise.

[0034] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be

encompassed by the invention.

[0035] Throughout this specification 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 ingredient 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 may be replaced with either of the other two terms.

[0036] As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements 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 elements 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."

[0037] As used herein, "subject" means any animal, preferably a mammal, most preferably a human, to whom will be or has been vaccinated 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.

[0038] As used herein, the term "protective immunity" or "protective immune response" means that the vaccinated subject is able to control an infection with the pathogenic agent against which the vaccination was done. Usually, the subject having developed a "protective immune response" develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a "protective immune response" or "protective immunity" against a certain agent will not die as a result of the infection with said agent.

[0039] A "capsid protein" refers to a protein on the capsid of an adenovirus (e.g., Ad26 or Ad35) that is involved in determining the serotype and/or tropism of a particular adenovirus. Capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein an "Ad26 capsid protein" or an "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. [0040] The terms "adjuvant" is 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 vectors of the invention.

[0041] 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 have a specified percentage of amino acid residues or nucleotides 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.

[0042] 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 calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0043] 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 implementations 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 Supplement) (Ausubel)).

[0044] 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 of the 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.

[0045] Cumulative scores are calculated using, for nucleotide 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 zero 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 determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, 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)).

[0046] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin & Altschul, Proc. Nat l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01 , and most preferably less than about 0.001.

[0047] 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 molecules hybridize to each other under stringent conditions, as described below.

[0048] The term "substantially similar" in the context of the filovirus antigenic proteins of the invention indicates that a polypeptide comprises a sequence with at least 90%, preferably at least 95% sequence identity to the reference sequence. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) 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 result by 100 to yield the percentage of sequence identity.

[0049] It is discovered in the invention that various prime-boost combinations of replication incompetent vectors generate an effective immune response against filovirus infection and/or disease. Specifically, a prime-boost combination of a first composition comprising at least one human adenovirus vector and a second composition comprising a simian adenovirus vector, such as a chimpanzee adenovirus vector, generates an improved immune response .

Filovirus antigenic proteins

[0050] The Ebola viruses are filoviruses associated with outbreaks of highly lethal hemorrhagic 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 have been defined, the genetic organization of these viruses is similar, each containing seven linearly arrayed genes. Among the viral proteins, the envelope glycoprotein exists in two alternative forms, a 50-70 kilodalton (kDa) secreted protein (sGP) and a 130 kDa transmembrane glycoprotein (GP), generated by RNA editing that mediates 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).

[0051] The nucleic acid molecules comprised in the adenovirus vectors can encode structural gene products of any filovirus species (Marburg and/or Ebola), such as subtypes of

Zaire (type species, also referred to herein as EBOV), Sudan, Reston, Bundibugyo, and Ivory

Coast, including but not limited to those disclosed in WO2003/028632, the content of which is incorporated herein by reference in its entirety. Nucleic acid molecules comprised in the adenovirus vectors can encode structural gene products of filovirus species involved in a particular outbreak, such as those species reported to be associated with the outbreak of 2014/2015 or any future outbreak.

[0052] The adenoviral vectors of the invention can be used to express antigenic proteins comprising an antigenic determinant of a wide variety of filovirus antigens. In a typical and preferred embodiment, the vectors of the invention comprise nucleic acid encoding the transmembrane form of the viral glycoprotein (GP). In other embodiments, the vectors of the invention can encode the secreted form of the viral glycoprotein (ssGP), or the viral nucleoprotein (NP).

[0053] One of skill will recognize that the nucleic acid molecules encoding the filovirus antigenic protein can be modified, e.g., the nucleic acid molecules 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 determinant of a filovirus protein, such as those described above. The term encompasses filovirus glycoproteins (i.e. , gene products of gene 4 or GP gene of a filovirus) or filovirus nucleoprotein as well as recombinant proteins that comprise one or more filovirus glycoprotein or nucleoprotein determinants. The term antigenic proteins also encompasses antigenic proteins that are substantially similar to the naturally occurring filovirus proteins.

[0054] In some embodiments, the protein can be mutated so that it is less toxic to cells (see e.g. , WO/2006/037038), the content of which is incorporated herein by reference in its entirety, or can be expressed with increased or decreased level in the cells. In a preferred embodiment, nucleic acid molecules encoding GP, ssGP and NP of the Zaire ebolavirus strain are comprised in the first or second composition of the vaccine combination. Preferably, the vector in the first composition of the vaccine combination encodes a GP having the amino acid sequence of SEQ ID NO: 1, or a protein substantially similar to SEQ ID NO: 1, such as a protein comprising the amino acid sequence of SEQ ID NO: 3, preferably the protein is capable of inducing an immune response against a GP having the amino acid of SEQ ID NO: 1. In another preferred embodiment, the vector in the second composition of the vaccine combination encodes a GP having the amino acid sequence of SEQ ID NO: l or an amino acid sequence substantially similar to SEQ ID NO: 1, such as a protein comprising the amino acid sequence of SEQ ID NO: 3, preferably the protein is capable of inducing an immune response against a GP having the amino acid of SEQ ID NO: 1. In a more preferred embodiment, the vector in the first composition of the vaccine combination encodes a GP having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and the vector in the second composition of the vaccine

combination encodes a GP having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Human Adenovirus Vectors

[0055] An adenovirus vector according to the invention is derived from an adenovirus that belongs to the family of the Adenoviridae and preferably is one that belongs to the genus Mastadenovirus . In a preferred embodiment, the adenovirus vector of the first composition is derived from a human adenovirus (HAdV, or AdHu; in the present disclosure 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).

[0056] Most advanced studies have been performed using human adenoviruses. In certain 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 vector is derived from a human adenovirus of one of the serotypes 26 and 35. An advantage of these serotypes is a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Preparation 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: l of WO 2007/104792, the contents of which are incorporated herein by reference in their entirety. Preparation 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, all 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, which are incorporated by reference herein in their entirety.

[0057] In a preferred embodiment, the human adenoviral vectors comprise capsid proteins from two rare serotypes: Ad26 and Ad35. In one embodiment, the vector is a rAd26 or rAd35 virus.

[0058] Thus, the vectors that can be used in the invention comprise an Ad26 or Ad35 capsid protein (e.g. , a fiber, penton or hexon protein). One of skill will recognize that it is not necessary that an entire Ad26 or Ad35 capsid protein be used in the vectors of the invention.

Thus, chimeric capsid proteins that include at least a part of an Ad26 or Ad35 capsid protein can be used in the vectors of the invention. The vectors of the invention can also comprise capsid proteins in which the fiber, penton, and hexon proteins are each derived from a different serotype, so long as at least one capsid protein is derived from Ad26 or Ad35. In preferred embodiments, the fiber, penton and hexon proteins are each derived from Ad26 or each from Ad35.

[0059] One of ordinary skill will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector, for example human or chimpanzee adenovirus. Thus, a chimeric adenovirus vector that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric human adenovirus vector of the invention could combine the absence of pre-existing immunity of the Ad26 and Ad35 serotypes with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like.

[0060] In certain embodiments the recombinant human adenovirus vector useful in the invention is derived mainly or entirely from Ad35 or from Ad26 (i. e. , the vector is rAd35 or rAd26). In some embodiments, the human adenovirus is replication deficient, e.g. because it contains a deletion in the El region of the genome. For the exemplary human adenoviruses of the invention, being derived from Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g. Havenga et al, 2006, J Gen Virol 87: 2135-43; WO 03/104467), which are incorporated by reference herein in their entirety. In certain embodiments, the adenovirus is a human adenovirus of serotype 35, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. In certain embodiments, the adenovirus is a human adenovirus of serotype 26, with a deletion in the El region into which the nucleic acid encoding the antigen has been cloned, and with an E4 orf6 region 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 &¾361 restriction site, since this increases the stability of the 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). [0061] A human adenoviral vector can be prepared using method known in the art in view of the present disclosure. Preparation 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: l of WO

2007/104792. Preparation 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.

[0062] 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.

Simian Adenovirus Vectors

[0063] Simian adenovirus vectors according to the present invention are derived from adenoviruses originated from simians, which, as used herein, include monkeys and apes, but exclude humans. Examples of simians include, but are not limited to, chimpanzee, gorilla, orangutan, etc. Simian adenoviruses generally also have a low seroprevalence and/or low preexisting neutralizing antibody titers in the human population, and a significant amount of work has been reported using simian adenovirus vectors, such as chimpanzee adenovirus vectors (e.g. US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984; Farina et al, 2001, J Virol l5: 11603-13; Cohen a/, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al. , 2007 ^' , Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849- 62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39, all of which are incorporated by reference herein in their entirety). Hence, in other preferred embodiments, the recombinant adenovirus vector according to the invention is based upon a simian adenovirus, e.g. a chimpanzee or bonobo adenovirus. In certain embodiments, the recombinant adenovirus is based upon chimpanzee adenovirus type 1, 3, 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 67 or SA7P.

[0064] In simian adenovirus vectors, such as chimpanzee adenovirus vectors, the El locus can be deleted to render viruses replication deficient and allow transcomplementation on an El AdHu5 complementing cell line (Farina SF, et al. 2001 J Virol 75: 11603-13). An additional attractive observation is that the lack of sequence homology between AdHu5 and simian adenoviruses at the El flanking sequence prevents homologous recombination and production of replication competent virus (Tatsis N, et al. 2006 Gene Ther 13: 421-9). In certain embodiments, the recombinant adenovirus vector is based upon chimpanzee adenovirus type 3 or 63.

[0065] In a preferred embodiment, the simian adenovirus vector of the second composition is a chimpanzee adenovirus vector (ChAdV or AdCh).

[0066] In a more preferred embodiment, the chimpanzee adenovirus vector of the second composition is ChAdV3. Recombinant chimpanzee adenovirus serotype 3 (ChAd3 or cAd3) is a subgroup C adenovirus with properties similar to those of human adenovirus serotype 5 (Ad5). ChAd3 has been shown to be safe and immunogenic in human studies evaluating candidate vaccines for hepatitis C virus (HCV) (Barnes E, et al. 2012 Science translational medicine 4: 115ral). It was reported that ChAd3-based vaccines were capable of inducing an immune response comparable to a human Ad5 vectored vaccine. See, e.g., Peruzzi D, et al. 2009 Vaccine 27: 1293-300 and Quinn KM, et al. 2013 J Immunol 190: 2720-35; WO 2005/071093;

WO2011/0130627, etc.

[0067] A vector useful in the invention can be produced using a nucleic acid comprising the recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.

[0068] The human and simian adenovirus vectors useful to the invention are typically replication defective. In these embodiments, the virus is rendered replication-defective by deletion or inactivation of regions critical to replication of the virus, such as the El region. The regions can be substantially deleted or inactivated by, for example, inserting the gene of interest (usually linked to a promoter). In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions 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 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.

[0069] A packaging cell line is typically used to produce sufficient amount of adenovirus vectors of the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, Procell-92, PER.C6, 911, 293, and El A549. [0070] As noted above, a wide variety of filovirus glycoproteins can be expressed in vectors. If required, the heterologous gene encoding the filovirus glycoproteins can be codon- optimized to ensure proper expression in the treated host (e.g., human). Codon-optimization is a technology widely applied in the art. Typically, the heterologous gene is cloned into the El and/or the E3 region of the human adenoviral genome.

[0071] 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 but are not limited to the CMV promoter, e.g. the hCMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within an expression cassette.

[0072] As noted above, the human or simian adenovirus vectors useful for the invention can comprise a wide variety of filovirus glycoproteins known to those of skill in the art.

[0073] In a preferred embodiment of the invention, the rAd and ChAdV vector(s) comprise one or more GPs of Zaire ebolavirus (EBOV), or GPs substantially similar thereto.

Immunogenic combination

[0074] A vaccination strategy to achieve protective immunity in most recipients with a single vaccination would be desirable in an outbreak setting. Vaccination strategies that achieve durable protective immunity would be desirable for populations in areas of the world where outbreaks occur sporadically. Optimally, one approach would serve both needs, but a different approach may be needed for rapid immunity than is needed for durable immunity.

[0075] One general aspect of the invention relates to a vaccine or immunogenic

combination comprising:

(i) a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of a simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0076] Another aspect of the invention relates to a vaccine or immunogenic combination for inducing protective immunity from filovirus infection and/or disease comprising: (i) a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of a simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus, wherein the second nucleic acid is the same or different than the first nucleic acid, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0077] In a preferred embodiment of the invention, a vaccine combination comprises:

(i) a first composition comprising an immunologically effective amount of rAd26 or rAd35 vector comprising a first nucleic acid encoding a first antigenic protein having the amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of ChAd3 vector comprising a second nucleic acid encoding a second antigenic protein having the amino acid sequence that is at least 90% identical to SEQ ID NO: l, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0078] In a more preferred embodiment of the invention, a vaccine combination comprises:

(i) a first composition comprising an immunologically effective amount of rAd26 vector comprising a first nucleic acid encoding an antigenic protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of ChAd3 vector comprising a second nucleic acid encoding the antigenic protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0079] In another more preferred embodiment of the invention, a vaccine combination comprises:

(i) a first composition comprising an immunologically effective amount of rAd26 vector comprising a first nucleic acid encoding an antigenic protein having the amino acid sequence of SEQ ID NO: 1, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of ChAd3 vector comprising a second nucleic acid encoding the antigenic protein having the amino acid sequence of SEQ ID NO: 3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0080] In another preferred embodiment of the invention, a vaccine combination comprises:

(i) a first composition comprising an immunologically effective amount of rAd26 vector comprising the nucleotide sequence of SEQ ID NO:2, and a pharmaceutically acceptable carrier; and

(ii) a second composition comprising an immunologically effective amount of ChAd3 vector comprising the nucleotide sequence of SEQ ID NO:2, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0081] Another aspect of the invention relates to a kit comprising:

(i) a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and (ii) a second composition comprising an immunologically effective amount of a simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein, wherein the second nucleic acid is the same or different than the first nucleic acid, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

Immunogenic composition

[0082] Immunogenic compositions are compositions comprising an immunologically effective amount of purified or partially purified human or simian (e.g., chimpanzee) adenovirus 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 can include adjuvants to enhance immune responses. The optimal ratios of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.

[0083] The immunogenic compositions according to embodiments of the present invention can be made using methods known to those of skill in the art in view of the present disclosure. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral 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.

[0084] The compositions of the invention can comprise other filovirus antigens or the priming or boosting inoculations can comprise other antigens. The other antigens used in combination with the adenovirus vectors of the invention can be, for example, filovirus antigens and nucleic acids expressing them.

[0085] 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-LAS01, AS03, AS04, AS 15, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59. [0086] 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-10, and IL-12 or encoding nucleic acids therefore.

[0087] 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 interfere with the efficacy of the active ingredient. The first and second compositions can have the same or different

pharmaceutically acceptable carriers. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes.

Method for Inducing Protective Immunity Against Filovirus Infection and/or Disease

[0088] Another general aspect of the invention relates to a method of inducing an immune response against a filovirus in a subject. The method comprises:

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

b. administering to the subject a second composition comprising an

immunologically effective amount of a simian (e.g. chimpanzee) adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of a filovirus, wherein the second antigenic protein is the same or different as the first antigenic protein,

wherein steps (a) and (b) are conducted in either order.

[0089] In certain embodiments, the first composition is used for priming said immune response and the second composition is used for boosting said immune response. In other embodiments, the second composition is used for priming said immune response and the first composition is used for boosting said immune response.

[0090] Any of the immunogenic compositions according to embodiments of the invention, including but not limited to those described herein, can be used in methods of the invention.

[0091] In one embodiment of the disclosed methods, an adenovirus vector is used to prime the immune response and another adenovirus vector is used to boost the immune response about 1-15 weeks after the priming vaccination, for example, the boosting vaccination can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 weeks after the priming vaccination. Additional boosting compositions can be administered weeks or months after the initial boosting administration, for example, about 1 , 2, or 3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks, or 36 weeks, or one to two years after the initial boosting.

[0092] In a preferred embodiment, the human adenovirus vector used according to the present invention includes an rAd26 or rAd35 vector and the simian adenovirus vector includes a chimpanzee adenovirus vector, such as a ChAd3 vector. In one exemplary embodiment, an rAd26 or rAd35 vector is used to prime the immune response, and a ChAd3 vector is used to boost the immune response, or vice versa.

[0093] The antigens in the respective priming and boosting compositions do not necessarily need to be identical, but should preferably share similar antigenic determinants or be substantially similar to each other.

[0094] Administration of the immunogenic compositions comprising the vectors is typically intramuscular or subcutaneous. However other modes of administration such as intravenous, cutaneous, intradermal or nasal can be envisaged as well. Intramuscular administration 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™) or a freeze-dried powder containing the vaccine.

[0095] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the vector will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required. A slow-release formulation can also be employed

[0096] Typically, administration will have a prophylactic aim to generate an immune response against a filovirus antigen before infection or development of symptoms. Diseases and disorders that can be treated or prevented in accordance with the invention include those in which an immune response can play a protective or therapeutic role. In other embodiments, the adenovirus vectors can be administered for post-exposure prophylactics. [0097] The immunogenic compositions containing the human or simian (e.g., chimpanzee) adenovirus vectors are administered to a subject, giving rise to an anti-filovirus immune response in the subject. An amount of a composition sufficient to induce a detectable 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.

[0098] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

[0099] Following production of adenovirus vectors and optional formulation of such particles into compositions, the vectors can be administered to an individual, particularly human or other primate. Administration can be to humans, or another mammal, e.g., mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. Delivery to a non-human mammal need not be for a therapeutic purpose, but can be for use in an experimental context, for instance in investigation of mechanisms of immune responses to the adenovirus vectors.

[00100] In one exemplary regimen, the human adenovirus vector is administered (e.g., intramuscularly) in a volume ranging between about 100 μΐ to about 10 ml containing concentrations of about 10 ⁴ to 10 ¹² virus particles/ml. Preferably, the human adenovirus vector is administered in a volume ranging between 0.1 and 2.0 ml. For example, the human adenovirus vector can be administered with 100 μΐ, 500 μΐ, 1 ml, 2 ml. More preferably the human adenovirus vector is administered in a volume of 0.5 ml. Optionally the human adenovirus vector can be administered in a concentration of about 10 ⁷ vp/ml, 10 ^s vp/ml, 10 ⁹ vp/ml, 10 ¹⁰ vp/ml, 5xl 0 ¹⁰ vp/ml, 10 ¹¹ vp/ml, or 10 ¹² vp/ml.

[0100] Typically, the human adenovirus vector is administered in an amount of about 10 ⁹ to about 10 ¹² viral particles (vp) to a human subject during one administration, more typically in an amount of about 10 ¹⁰ to about 10 ¹² vp. The initial vaccination is followed by a boost as described above. [0101] In another exemplary regimen, the simian (e.g., chimpanzee) adenovirus vector is administered (e.g. , intramuscularly) together with a pharmaceutically acceptable carrier. The simian (e.g., chimpanzee) adenovirus vector can be administered, for example, with saline solution in the range of from about 100 μΐ to about 10 ml of saline solution containing

4 12

concentrations of from about 10 to 10 virus particles/ml. For example, the simian (e.g., Chimpanzee) adenovirus vector can be administered with 100 μΐ, 500 μΐ, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml of saline solution. Optionally the simian (e.g.,

Chimpanzee) adenovirus vector can be administered in a concentration of about 10 ⁴ vp/ml, 10 ⁵ vp/ml, 10 ⁶ vp/ml, 10 ⁷ vp/ml, 10 ⁸ vp/ml, 10 ⁹ vp/ml, 10 ¹⁰ vp/ml, 10 ¹¹ vp/ml, or 10 ¹² vp/ml. Typically, the simian (e.g., Chimpanzee) adenovirus vector is administered in an amount of about 10 to about 10 viral particles (vp) to a human subject during one administration, more typically from about 10 ¹⁰ to about 10 ¹² vp.

[0102] The initial vaccination is followed by a boost as described above. The composition can, if desired, be presented in a kit, pack or dispenser, which can contain one or more unit dosage forms containing the active ingredient. The kit, for example, can comprise metal or plastic foil, such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

[0103] The compositions of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0104] The invention provides also the following non-limiting embodiments:

[0105] 1. A vaccine combination comprising:

a. a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and b. a second composition comprising an immunologically effective amount of a

simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0106] 2. The vaccine combination according to embodiment 1 , wherein the first antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l.

[0107] 3. The vaccine combination according to embodiment 1 or 2 wherein the first antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0108] 4. The vaccine combination according to any one of embodiments 1 to 3, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0109] 5. The vaccine combination according to any one of embodiments 1 to 3, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0110] 6. The vaccine combination according to any one of embodiments 1 to 5, wherein the second antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: 1.

[0111] 7. The vaccine combination according to any one of embodiments 1 to 6, wherein the second antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: 1.

[0112] 8. The vaccine combination according to any one of embodiments 1 to 7, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0113] 9. The vaccine combination according to any one of embodiments 1 to 7, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0114] 10. The vaccine combination according to any one of embodiments 1 to 9, wherein the human adenovirus vector is an rAd26 vector.

[0115] 11. The vaccine combination according to any one of embodiments 1 to 10, wherein the simian adenovirus vector is a chimpanzee adenovirus vector.

[0116] 12. The vaccine combination according to embodiment 11, wherein the chimpanzee adenovirus vector is a ChAd3 vector.

[0117] 13. A vaccine combination comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l, and a pharmaceutically acceptable carrier; and b. a second composition comprising an immunologically effective amount of a

ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a

pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0118] 14. A vaccine combination comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO:3, and a pharmaceutically acceptable carrier; and b. a second composition comprising an immunologically effective amount of a

ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a

pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0119] 15. A vaccine combination comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l, and a pharmaceutically acceptable carrier; and b. a second composition comprising an immunologically effective amount of a

ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO:3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0120] 16. The vaccine combination according to any one of embodiments 1 to 15 for use in generating a protective immune response in a subject in need thereof against at least one filovirus subtype, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.

[0121] 17. The vaccine combination according to any one of embodiments 1 to 15 for use in generating a protective immune response in a subj ect in need thereof against at least one filovirus subtype, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.

[0122] 18. A method of inducing an immune response against a filovirus in a subject in need thereof, the method comprising:

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

b. administering to the subject a second composition comprising an

immunologically effective amount of a simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein of the filovirus, wherein steps (a) and (b) are conducted in either order.

[0123] 19. The method according to embodiment 18, wherein the first antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l.

[0124] 20. The method according to embodiment 18 or 19, wherein the first antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l.

[0125] 21. The method according to any one of embodiments 18 to 20, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0126] 22. The method according to any one of embodiments 18 to 20, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0127] 23. The method according to any one of embodiments 18 to 22, wherein the second antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0128] 24. The method according to any one of embodiments 18 to 23, wherein the second antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0129] 25. The method according to any one of embodiments 18 to 24, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0130] 26. The method according to any one of embodiments 18 to 24, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0131] 27. The method according to any one of embodiments 18 to 26, wherein the human adenovirus vector is a rAd26 vector.

[0132] 28. The method according to any one of embodiments 18 to 27, wherein the simian adenovirus vector is a ChAd3 vector.

[0133] 29. A method of inducing an immune response against a filovirus in a subject in need thereof, the method comprising:

a. administering to the subject a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l, and a pharmaceutically acceptable carrier; and

b. administering to the subject a second composition comprising an

immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a pharmaceutically acceptable carrier, wherein steps (a) and (b) are conducted in either order.

[0134] 30. A method of inducing an immune response against a filovirus in a subject in need thereof, the method comprising:

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

b. administering to the subject a second composition comprising an

immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a pharmaceutically acceptable carrier, wherein steps (a) and (b) are conducted in either order. [0135] 31. A method of inducing an immune response against a filovirus in a subject in need thereof, the method comprising:

a. administering to the subject a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l , and a pharmaceutically acceptable carrier; and

b. administering to the subject a second composition comprising an

immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO:3, and a pharmaceutically acceptable carrier,

wherein steps (a) and (b) are conducted in either order.

[0136] 32. The method according to any one of embodiments 18 to 31 , wherein step (b) is conducted 1 -15 weeks, preferably 1-12 weeks, after step (a).

[0137] 33. The method according to any one of embodiments 18 to 31 , wherein step (b) is conducted 1 -15 weeks, preferably 1-12 weeks, before step (a).

[0138] 34. A kit comprising:

a. a first composition comprising an immunologically effective amount of at least one human adenovirus vector comprising a first nucleic acid encoding a first antigenic protein of a filovirus, and a pharmaceutically acceptable carrier; and

b. a second composition comprising an immunologically effective amount of a simian adenovirus vector comprising a second nucleic acid encoding a second antigenic protein, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0139] 35. The kit according to embodiment 34, wherein the first antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l , preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0140] 36. The kit according to embodiment 34 or 35, wherein the first antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l.

[0141] 37. The kit according to any one of embodiments 34 to 36, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0142] 38. The kit according to any one of embodiments 34 to 36, wherein the first antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0143] 39. The kit according to any one of embodiments 34 to 38, wherein the second antigenic protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0144] 40. The kit according to any one of embodiments 34 to 39, wherein the second antigenic protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: l, preferably is also capable of inducing an immune response to the amino acid sequence of SEQ ID NO: l .

[0145] 41. The kit according to any one of embodiments 34 to 40, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO: l.

[0146] 42. The kit according to any one of embodiments 34 to 40, wherein the second antigenic protein comprises the amino acid sequence of SEQ ID NO:3.

[0147] 43. The kit according to any one of embodiments 34 to 42, wherein the human adenovirus vector is a rAd26 vector.

[0148] 44. The kit according to any one of embodiments 34 to 43, wherein the simian adenovirus vector is a ChAd3 vector.

[0149] 45. A kit comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l, and a pharmaceutically acceptable carrier; and

b. a second composition comprising an immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0150] 46. A kit comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO:3, and a pharmaceutically acceptable carrier; and

b. a second composition comprising an immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l or SEQ ID NO:3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0151] 47. A kit comprising:

a. a first composition comprising an immunologically effective amount of an rAd26 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO: l, and a pharmaceutically acceptable carrier; and

b. a second composition comprising an immunologically effective amount of a ChAd3 vector comprising a nucleic acid encoding an antigenic protein comprising the amino acid sequence of SEQ ID NO:3, and a pharmaceutically acceptable carrier; wherein one of the compositions is a priming composition and the other composition is a boosting composition.

[0152] 48. The kit according to any one of embodiments 34 to 47, for use in generating a protective immune response against at least one filovirus subtype, wherein the first composition is used for priming said immune response and the second composition is used for boosting said immune response.

[0153] 49. The kit according to any one of embodiments 34 to 47, for use in generating a protective immune response against at least one filovirus subtype, wherein the second composition is used for priming said immune response and the first composition is used for boosting said immune response.

[0154] 50. The kit according to any one of embodiments 34 to 49, wherein the priming composition is administered to a subject in need thereof 1 - 15 weeks, preferably 1-12 weeks, before the boosting composition is administered to the subj ect.

EXAMPLES

[0155] The following examples are offered to illustrate, but not to limit the claimed invention.

Human Phase 1 Trial of Prime-Boost Regimen Combining ChAd3-EBO-Z and Ad26.ZEBOV

[0156] A Phase I human clinical study was designed to assess the safety of the heterologous prime-boost regimen combining the monovalent Ebola Zaire candidate vaccines ChAd3-EBO-Z and Ad26.ZEBOV in 4 or 8 week time intervals. The experimental sample was 32 healthy adults aged 18-50.

[0157] ChAd3-EBO-Z is a viral vectored vaccine using a chimpanzee adenovirus as a vector encoding a Zaire strain Ebola glycoprotein. It was administered in a dose of lxlO ¹¹ vp.

Ad26.ZEBOV is a viral vectored vaccine using a human adenovirus as a vector encoding a Zaire strain Ebola glycoprotein. It was administered in a dose of 5xl0 ¹⁰ vp. Each ChAd3-EBO-Z and Ad26.ZEBOV was given intramuscularly, according to the setup shown in Table 1.

Table 1: Experimental Setup for Human Phase 1 Trial of Prime-Boost Reg

Combining ChAd3-EBO-Z and Ad26.ZEBOV

Vaccine

Week 8

Boosting - - Ad26.ZEBOV ChAd3-EBO-Z

Vaccine

[0158] Group 1 (Gl) candidates received a primer vaccine of ChAd3-EBO-Z at week 0 and a boosting vaccine of Ad26.ZEBOV at week 4. Group 2 (G2) candidates received an

Ad26.ZEBOV priming vaccine at week 0 and a boosting vaccine of ChAd3-EBO-Z at week 4. Group 3 (G3) candidates received a primer vaccine of ChAd3-EBO-Z at week 0 and a boosting vaccine of Ad26.ZEBOV at week 8. Group 4 (G4) candidates received an Ad26.ZEBOV priming vaccine at week 0 and a boosting vaccine of ChAd3-EBO-Z at week 8. The follow-up duration of the study is 5 months.

Vaccine materials

[0159] Use of the recombinant, non-replicating ChAd3 vector for construction of the ChAd3-EBO-Z vaccine was based on properties to induce immune responses as readily as rAd5, but with little to no pre-existing immunity to ChAd3 in human populations. The recombinant chimpanzee adenovirus Type-3 vectored Ebola Zaire vaccine (ChAd3-EBO-Z) is a recombinant replication-deficient adenovirus chimpanzee serotype 3 (ChAd3) vector expressing a glycoprotein having the amino acid sequence of SEQ ID NO:3, which is identical to wild-type (WT) Ebola glycoprotein (SEQ ID NO: 1) from the Zaire strain, except having a valine at position 662, instead of an isoleucine. The investigational ChAd3-EBO-Z vaccine was developed using the adenovirus vaccine platform technology. The drug substance was manufactured and labelled under Good Manufacturing Practice (GMP) conditions. ChAd3-EBO- Z was supplied as a liquid in sterile aliquots in 1 ml clear glass vials at a concentration of lxlO ¹¹ vp per ml and in an aliquot of 2 ml at a concentration of lxlO ¹¹ vp per ml.

[0160] The human adenovirus Ad26.ZEBOV, which encodes the WT Ebola GP (SEQ ID NO: 1), was supplied as a liquid in sterile aliquots at a concentration of lxl 0 ¹⁰ vp per ml. The vaccines were stored in a -80 °C freezer.

Vaccination and Experimental Design

[0161] The vaccines were administered intramuscularly (IM) for all groups in the deltoid muscle of either arm. On vaccination day, vaccines were allowed to thaw to room temperature and administered within 1 hour. Depending on dose and concentration, in general, one or more vials of vaccine can be used. Also, in general, one vial can be used for more than one vaccine if administration is performed within the allotted time period post thawing, 1 hour.

[0162] On the vaccination days, before the injection, study subjects underwent clinical evaluation and samples were collected for laboratory tests.

[0163] Ebolavirus specific immunogenicity can be assessed by a variety of immunological assays. In general, the primary immunogenicity outcome measures are ELISA and neutralization antigen-specific assays for antibody responses and intracellular cytokine staining (ICS) assay for T cell responses. For example, immunogenicity can be assessed using the immunologic assays summarized in tables 3 and 4. The exploratory assay package can include, but is not limited to, the listed assays. Exploratory outcome measures include, but are not limited to, ex-vivo ELISPOT and flow cytometry performed with research samples collected at study time points throughout the study as well as other immunogenicity assays and evaluation of genetic factors associated with immune responses. Vaccine-induced mRNA expression profiles before and after vaccination may also be performed as an exploratory evaluation.

[0164] Ebola glycoprotein-specific humoral immune response was be assessed by ELISA.

Table 3: Summary of Immunologic Assays (Serology)

Assay Purpose

Secondary endpoints

Virus neutralization assay Analysis of neutralizing antibodies to EBOV GP

ELISA Analysis of antibodies binding to EBOV GP

Exploratory endpoints

Adenovirus neutralization Neutralizing antibodies to adenovirus

assay

Molecular antibody Analysis of anti-EBOV GP antibody characteristics, characterization including IgG subtyping

Exploratory ELISA Analysis of binding antibodies to a different source of EBOV

GP

EBOV: Ebola virus; ELISA: enzyme-linked immunosorbent assay; GP: glycoprotein; IgG: immunoglobulin G Table 4: Summary of Immunologic Assays (Cellular)

Assay Purpose

Secondary endpoints

Analysis of T cell responses to EBOV GP including CD4-

ICS on fresh PBMC positive and low-magnitude T cell responses (including

CD4/8, IL-2, IFN-γ, TNF-q and/or activation markers)

Exploratory endpoints

ICS of frozen PBMC Analysis of T-cell responses to EBOV GP (including CD4/8,

IL-2, IFN-γ, TNF-a and/or activation markers)

ELISpot of frozen PBMC T-cell IFN-γ responses to EBOV GP

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

ELISA Ebola Glycoprotein Specific IgG Response Assay

[0165] In brief, plates were coated with a recombinant, soluble, affinity purified, trimeric form of the Ebola Makona variant glycoprotein. Antibody responses were measured against trimerised Zaire strain Ebola Glycoprotein. Samples were assayed in triplicate and a reference pool of ZEBOV GP-positive serum was used to form a standard curve on each plate. Arbitrary ELISA units were calculated for each sample using the OD values of the sample and the parameters of the standard curve. A seropositive threshold of 166 ELISA Units was determined from the Mean ELISA Units + 3SD of 59 naive serum samples run on the assay.

[0166] Humoral immune responses were measured for all volunteers until 28 days after administration of the boosting vaccine (B+28), and for 17 of the 32 volunteers until 90 days after administration of the boosting vaccine (B+90) (Gl : n=7, G2: n=5, G3: n=3, G4: n=2). The remaining B+90 samples are run once all visits are completed.

[0167] FIGs 1 and 2 are graphs of the kinetic of the EBOV GP-specific humoral immune response assessed by ELISA. FIG1 summarizes the data obtained from test groups Gl and G3. FIG 2 summarizes the data obtained from test groups G2 and G4. Geometric mean ELISA concentration are displayed in ELISA Units per mL together with their 95% confidence interval. Group 1 candidates (Gl) received a prime immunization with ChAd3 expressing the EBOV GP followed by a boost with Ad26 expressing the EBOV GP 4 weeks after. Group 2 candidates (G2) received a prime immunization with Ad26 followed by a boost with ChAd3 4 weeks after. Group 3 candidates (G3) received a prime immunization with ChAd3 followed by a boost immunization with Ad26 8 weeks later. Group 4 candidates (G4) received a prime

immunization with Ad26 followed by a boost with ChAd3 8 weeks after.

[0168] As shown in the graphs of FIGs 1 and 2, Ebola GP specific immune responses were induced by both Chimpanzee derived and human derived adenoviral vectors expressing the Ebola Zaire GP. Importantly, responses were increased significantly after boosting with the heterologous vector. At the peak of the immune response observed 2 weeks post boost, EBOV GP specific antibody titers increased compared to pre boost level from 3.7 to 7.6 fold when boost immunization was administered 4 weeks after prime immunization (Gl and G2), and from 8.6 to 12.4 fold when administered 8 weeks after (G3 and G4). Overall, a slightly higher immune response was observed when a longer prime boost interval was used. Interestingly, EBOV GP specific humoral immune responses were maintained at 90 days post boost immunization (the latest time point analyzed) at a level exceeding the response observed post prime immunization.

[0169] In conclusion, heterologous prime boost regimens using a chimpanzee derived adenoviral vaccine vector and a human derived adenoviral vaccine vector are able to induce a strong and long lasting immune response, independently of the order of the vaccines as prime or boost immunization.