first and second recombinant adenovirus vectors

专利摘要:
FIRST AND SECOND RECOMBINANT ADENOVIRUS VECTORS. The present invention provides adenovirus vectors (serotype 26 and serotype 35) that encode filovirus antigens. Adenovirus vectors can be used to induce protective immune responses against filovirus infection.
公开号:BR112013014712B1
申请号:R112013014712-1
申请日:2011-12-14
公开日:2021-03-02
发明作者:Nancy J. Sullivan；Gary J. Nabel；Clement Asiedu；Cheng Cheng；Maria Grazia Pau；Jaap Goudsmit
申请人:The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services；Crucell Holland B.V.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] This invention relates to adenoviral vectors to induce protective immunity against filovirus infection. BACKGROUND OF THE INVENTION
[0002] Replicating 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 phyloviruses, as well as other non-viral 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): e177; Radosevic, et al., ( 2007); Santra, et al., (2009) Vaccine 27 (42): 5837-45. Adenovirus-based vaccines have several advantages as vaccines for humans because they can be produced in high titers under GMP conditions and have been proven to be safe and immunogenic in human (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. Although most initial vaccine work was conducted using rAd5 due to its significant potency in producing CD8 + T cell responses and broad antibodies, 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 can restrict the use of rAd5 in clinical applications for many vaccines that are currently in development including Ebola virus (EBOV) and Marburg virus (MARV).
[0003] To circumvent the problem of pre-existing immunity to rAd5, several alternative vectors are currently under investigation. These include adenoviral vectors derived from rare human serotypes and vectors derived from other animals such as chimpanzees (Vogels, et al., (2003) J. Virol. 77 (15): 8263-71; Abbink, et al., (2007 ) J. Virol. 81: 4654-63; Santra, (2009)). Research on the use of animal-derived adenoviral vectors is relatively nascent, whereas human adenoviruses have the advantages of having biology and tropism on well-characterized human cells, and also documented manufacturability (Vogels, et al., (2007) J. Gen. Virol. 88 (Pt 11): 2915-24.). Immunogenicity of these vectors and their potential as vaccines have been demonstrated in animal models, mainly as combinations of initial vaccination - booster vaccination with heterologous vectors (Abbink, et al., 2007; Shott et al., (2008) Vaccine 26: 2818- 23).
[0004] Adenovirus seroprevalence frequencies are cohort dependent (Mast, et al., (2010) Vaccine 28 (4): 950-7) but among a large group of 51 human adenoviruses tested, Ad35 and Ad11 were the most rarely neutralized by sera from 6 geographic locations (Vogels, et al., 2003). It has been shown that rAd35 vaccines are immunogenic in mice, non-human primates, and humans, and are able to circumvent immunity to Ad5 (Barouch, et al., (2004) J. Immunol. 172 (10): 6290-7 ; Nanda, et al., (2005) J. Virol. 79 (22): 14161-8; Ophorst, et al., (2006) Infect. Immun. 74 (1): 313-20; Thorner, et al. , (2006) J. Virol. 80 (24): 1200916 .; Rodriguez, et al., (2009) Vaccine 27 (44): 6226-33). RAd35 vectors grow with high titers in cell lines suitable for the production of clinical-grade vaccines (Havenga, et al., (2006) J. Gen. Virol. 87 (Pt 8): 2135-43), and have been formulated for injection and also stable inhalable powder (Jin, et al., Vaccine 28 (27): 4369-75). These vectors show efficient transduction of human dendritic cells (de Gruijl, et al., (2006) J. Immunol. 177 (4): 2208-15; Lore, et al., (2007) J. Immunol. 179 (3 ): 1721-9), and thus have the ability to mediate antigen presentation and release at a high level. Ad26, from subgroup D, is another adenovirus selected because of its ability to circumvent pre-existing immunity to Ad5. Although Ad26 seroprevalence may be significant in a certain adult population, Ad26 neutralizing antibody titers remain noticeably lower than Ad5 neutralizing antibody titers (Abbink, et al., 2007; Mast, et al., 2010 ). Studies have shown that rAd26 can be grown with high titers in complementary E1 cell lines of Ad5 suitable for the manufacture of these vectors on a large scale and in a clinical degree (Abbink, et al., 2007), and it has been shown that this vector induces cell-mediated and humoral immune responses in initial vaccination strategies - booster vaccination (Abbink, et al., 2007; Liu, et al., (2009) Nature 457 (7225): 87-91). BRIEF SUMMARY OF THE INVENTION
[0005] The present invention is based, at least in part, on the discovery that vectors rAd35 and rAd26 under single inoculation and also combinations of initial vaccination - heterologous booster vaccinations generate protective immune responses against filovirus infection.
[0006] The present invention therefore provides isolated recombinant adenovirus vectors comprising nucleic acid encoding a phylovirus antigen, wherein the adenovirus vector comprises an adenovirus 26 capsid protein (e.g., an rAd26 vector), or a adenovirus 35 capsid protein (e.g., an rAd35 vector). The adenovirus vector is typically defective in replication.
[0007] The filovirus antigenic protein is usually a glycoprotein from an Ebola virus or a Marburg virus. The Ebola virus can be of any species, for example, Zaire or Sudan / Gulu. Exemplary nucleic acids encoding suitable filovirus antigens are shown in SEQ ID NO: 1 and SEQ ID NO: 2.
[0008] The invention also provides isolated nucleic acid molecules that encode recombinant adenovirus vectors of the invention. Nucleic acids typically comprise an expression cassette comprising a CMV promoter operably linked to a polynucleotide sequence that encodes the filovirus antigenic protein. The polynucleotide sequence encoding the filovirus antigenic protein can be SEQ ID NO: 1 or SEQ ID NO: 2.
[0009] The invention additionally provides immunogenic compositions comprising the isolated adenovirus vectors of the invention. The immunogenic composition can additionally comprise an adjuvant.
[00010] Methods of inducing an immune response against a filovirus antigen in a patient are also provided. The methods comprise administering to the patient an immunologically effective amount of the adenovirus vector of the invention. Usually, the adenovirus vector is administered intramuscularly.
[00011] In some embodiments, the vectors are administered as an initial vaccination followed by a booster vaccination. For example, the initial vaccination may be an administration of an adenovirus vector comprising an adenovirus capsid protein 26 and the booster vaccination may be an administration of an adenovirus vector comprising an adenovirus capsid protein 35. DEFINITIONS
[00012] An "adenovirus capsid protein" refers to a protein on an adenovirus capsid (eg, Ad 26 or Ad 35) that is involved in determining the serotype and / or tropism of an adenovirus specific. Adenoviral capsid proteins typically include 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 Ad26 or Ad35 capsid protein. In certain embodiments, the hexon, penton and fiber proteins are either Ad26 or Ad35.
[00013] The terms "adjuvant" and "immunostimulant" are used interchangeably here, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to the adenovirus vectors of the invention.
[00014] The term "corresponding to", when applied to the positions of amino acid residues in sequences, means that it corresponds to the positions in a plurality of sequences when the sequences are optimally aligned.
[00015] The terms "identical" or "percent identity", in the context of two or more nucleic acid or polypeptide sequences, (e.g., adenovirus capsid protein of the invention 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 match, as measured using one or more of the following sequence comparison algorithms or by visual inspection.
[00016] An "isolated" nucleic acid molecule or an "isolated" adenovirus vector is a nucleic acid molecule (eg, DNA or RNA) or a virus, which has been removed from its natural environment . For example, recombinant DNA molecules contained in a vector are considered to be isolated for the purposes of the present invention. Other examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or DNA molecules (partially or substantially) purified in solution. Isolated RNA molecules include RNA transcripts in vivo or in vitro RNA from the DNA molecules of the present invention. Nucleic acid molecules isolated according to the present invention additionally include such synthetically produced molecules.
[00017] "Operatively linked" indicates that two or more segments of DNA are brought together in such a way that they work in harmony for their intended purposes. For example, coding sequences are operably linked at the promoter in the correct reading matrix such that transcription starts at the promoter and proceeds along the coding segment (s) to the terminator.
[00018] A "polynucleotide" is a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases typically read from the 5 'end to the 3' end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. When the term is applied to double-stranded molecules it is used to denote the total length and it will be understood that it is equivalent to the term "base pairs".
[00019] A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, produced either naturally or synthetically. Polypeptides smaller than 50 amino acid residues are commonly called "oligopeptides".
[00020] The term "promoter" is used herein as recognized in the art to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and the initiation of transcription of an operably linked coding sequence. Promoter sequences are typically found in 5 'non-coding regions of genes.
[00021] A "protein" is a macromolecule comprising one or more polypeptide chains. A protein can also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined here in terms of their main amino acid structures; substituents such as carbohydrate groups are generally not specified, but may nevertheless be present.
[00022] The phrase "substantially identical" in the context of two nucleic acids or polypeptides of the invention (e.g., adenovirus capsid proteins or filovirus antigens), refers to two or more sequences or subsequences that have at least 60%, more preferably 65%, even more preferably 70%, even more preferably 75%, even more preferably 80%, and much more preferably 90-95% nucleotide identity or amino acid residues, when compared and aligned for maximum matching , as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists with respect to a region of the sequences that is at least about 50 residues in length, more preferably with respect to a region of at least about 100 residues, and much more preferably the sequences are substantially identical with respect to at least about 150 residues. In a much more preferred embodiment, the sequences are substantially identical with respect to the entire length of the coding regions.
[00023] For sequence comparison, typically a sequence acts as a reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and the sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) in relation to the reference sequence, based on the program's designated parameters.
[00024] Optimal alignment of sequences for comparison can be conducted, eg, 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), for research using the 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 Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see in general, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).
[00025] Examples of algorithms that are suitable for determining the 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. The computer program for performing BLAST analyzes is publicly available at the National Center for Biotechnology Information. This algorithm first involves identifying high scoring sequence pairs, HSPs by identifying short-length words W in the query sequence, which either coincide with or satisfy some threshold score of positive value T when aligned with a word of coincident length in a database string. T is called the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word results are then extended in both directions along each sequence as the cumulative alignment score can be increased. Cumulative scores are calculated using, for the nucleotide sequences, the parameters M (reward score for a matching residues pair; always> 0) and N (penalty score for residues mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extensions of the word results in each direction are interrupted when: the cumulative alignment score decreases by an amount X from its maximum achieved value; the cumulative score reaches zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of any 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 a predefined parameters a word length (wordlength) (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both tapes . For amino acid sequences, the BLASTP program uses predefined parameters as a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
[00026] In addition to calculating the percentage sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90 : 5873-5787 (1993)). A similarity measurement provided by the BLAST algorithm is the lowest sum probability (P (N)), which provides an indication of the probability by which a coincidence between two amino acid or nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lower sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and much more preferably less than about 0.0001.
[00027] Another 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, if the two peptides differ only in 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.
[00028] The term "substantially similar" in the context of the capsid proteins or filovirus antigens of the invention indicates that a polypeptide comprises a sequence with at least 90%, preferably at least 95% sequence identity with the reference sequence in compared to a 10-20 amino acid comparison window. Percentage of sequence identity is determined by comparing two sequences optimally aligned with respect to a comparison window, the portion of the polynucleotide sequence in the comparison window being comprised of additions or deletions (ie, gaps, gaps) compared to reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the identical amino acid residue or nucleic acid base (a) occurs in both sequences to give the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to give the percentage of sequence identity. BRIEF DESCRIPTION OF THE DRAWINGS
[00029] Figure 1. Organization of vectors and genetic grouping of adenovirus. (A) Phylogenetic tree showing the relationship of adenovirus hexon sequences. The different subgroups A through F are indicated and serotypes 26 and 35 of human adenovirus are highlighted. The tree was built using the ClustalX package's neighbor-joining method (Larkin et al., 2007) and designed using the Phylip Phylogeny Inference package version 3.68. Confidence values are displayed in internal branches as a percentage of 1,000 times the resampling (bootstrap). (B) Schematic overview of the genome of recombinant Ad26 and Ad35 recombinant vectors. Both vectors have a complete E1 deletion and contain an expression cassette containing the EBOV glycoprotein gene under the control of the CMV promoter. Other deletions were made in the E3 regions and the respective E4 orf6 sequences were replaced by the Ad5 E4orf6 sequences to facilitate the replication of these vaccine vectors in complementary E5 Ad5 cell lines such as PER.C6® cells.
[00030] Figure 2. EBOV challenge and immune responses induced by rAd35-GP vaccine (using Ad35BSU.Ebo.GP (Z) FL.wt). (A) The amount of anti-EBOV GP IgG in plasma samples obtained 3 weeks after vaccination of unmodified cynomolgus monkeys with Ad5 (gray bars) and Ad5 immune cynomolgus monkeys (black bars), of rAd35 vaccinated cynomolgus monkeys -GP was determined by ELISA. EC90 antibody titers were determined as described in Methods. (B, C) Frequencies of antigen-specific CD4 + and CD8 + T-lymphocyte responses listed in the subsets of memory cells by ICS for IL-2 (CD4) or TNF-D (CD8), and analysis by flow cytometry after stimulation of PBMC 3 weeks after vaccination. (D) Plasma levels of liver enzyme AST in monkeys vaccinated with rAd35-GP (blue, unmodified with Ad5; red, immune to Ad5) and control (black) after infectious challenge with 1,000 PFU of ZEBOV.
[00031] Figure 3. Dose response for rAd35-GP induction of immune responses in monkeys (using Ad35BSU.Ebo.GP (Z) FL.wt and Ad35BSU.Ebo.GP (S / G) FL). (A) GP-specific IgG antibody titers (EC90) determined by ELISA in plasma obtained from monkeys 3 weeks after vaccination with 1010 or 1011 viral particles from each of the rAd35-GP vectors (Ad35BSU.Ebo.GP (Z) FL .wt and Ad35BSU.Ebo.GP (S / G) FL). (B, C) Antigen-specific CD4 + and CD8 + T-cell frequencies assessed by ICS as in Figure 2. Gray bars, 1010 rAd35-GP viral particles; black bars, 1011 rAd35-GP viral particles. (D, E) Plasma levels of liver enzyme AST after challenge with 1,000 PFU of ZEBOV. Blue, 1010 rAd35-GP viral particles; red, 1011 rAd35- GP viral particles and black, control.
[00032] Figure 4. Ability of rAd26-GP vectors to induce antigen-specific T-lymphocyte and antibody responses in cynomolgus monkeys (using Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S /G)FL.wt). (A) IgG antibody titers (EC90) determined by ELISA, and (B, C) GP-specific CD4 + and CD8 + T cell frequencies, (D) AST liver enzymes in plasma are shown for individual cynomolgus monkeys in two separate studies. Study 1 subjects received a vaccine dose of either 1010 or 1011 rAd26 viral particles from each of the rAd26-GP (Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL vectors. wt) and Study 2 the subjects received a dose of 1012 viral particles from each of the vectors rAd26-GP (Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt).
[00033] Figure 5. Kaplan-Meier survival curves for monkeys vaccinated with rAd26-GP (using Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt). Unvaccinated control animals and animals vaccinated with rAd26-GP were infected four weeks after vaccination with 1,000 PFU of ZEBOV in two separate challenge experiments as shown in Figure 4. Panel A: Black lines show unvaccinated individuals and dark blue lines show individuals vaccinated with Ad26 at the indicated doses. Panel B: a group of individuals vaccinated with rAd35 (light blue) is shown for potency comparison with Ad26 at a dose of 1011 viral particles from each of the rAd26-GP vectors (Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt). Panel C: historical survival of monkeys vaccinated with Ad5 (red) compared to individuals vaccinated with 1012 viral particles from each of the vectors rAd26-GP (Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt).
[00034] Figure 6. Comparison of immune responses from initial vaccination and booster vaccination after vaccinations with rAd26-GP / rAd35-GP (using Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S /G)FL.wt followed by Ad35BSU.Ebo.GP (Z) FL.wt and Ad35BSU.Ebo.GP (S / G) FL). (A) The amount of anti-EBOV GP IgG in plasma samples obtained 3 weeks after vaccination of unmodified cynomolgus monkeys with Ad5 (gray bars) and ad5-immune cynomolgus monkeys (black bars), rin35-GP vaccinated cynomolgus monkeys determined by ELISA. EC90 antibody titers were determined as described in Methods. (B, C) Frequencies of antigen-specific CD4 + and CD8 + T-lymphocyte responses listed in the subsets of memory cells by ICS for IL-2 (CD4) or TNF-π (CD8), and analysis by flow cytometry after stimulation of PBMC 3 weeks after vaccination. (D, E) EBOV challenge results. Plasma levels of liver enzyme AST (D), and Kaplan-Meier survival curve (E) for monkeys vaccinated with rAd26-GP / rAd35-GP after infectious challenge with 1,000 PFU of ZEBOV (blue, rAd26-GP / rAd35-GP ; red, Ad5- GP and black, unvaccinated control). DETAILED DESCRIPTION
[00035] The present invention is based, at least in part, on the discovery that vectors rAd26 and rAd35 under single inoculation and also combinations of initial vaccination - heterologous booster vaccines generate protective immune responses against filovirus infection. In particular, the present invention provides evidence that combinations of initial vaccination - heterologous booster vaccinations and (in particular, initial vaccination with Ad26 followed by booster vaccination with Ad35) are surprisingly effective in generating protective immune responses. The surprising effectiveness of these combinations of initial vaccination - booster vaccination could not have been predicted at the time of the invention. Thus, the present invention provides recombinant adenoviral vectors (rAd35 or rAd26) that express filovirus antigens. Adenoviral vectors can be formulated as vaccines and used to induce protective immunity against filovirus infections either alone or in combinations of initial vaccination - booster vaccination. FILOVIRUS ANTIGENS
[00036] The Ebola viruses, and the genetically related Marburg virus, are phyloviruses associated with highly lethal hemorrhagic fever outbreaks in humans and primates in North America, Europe, and Africa (Peters, CJ et al. In: “Fields Virology ", eds. Fields, BN et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996; Peters, CJ 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 ordered genes. Among viral proteins, envelope glycoprotein exists in two alternative forms, a secreted 50-70 kiloDaltons (kDa) protein (sGP) and a 130 kDa transmembrane glycoprotein (GP) generated by editing RNA that mediates viral entry (Peters, CJ et al. In: Fields Virology, eds. Fields, BN et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996; Sanchez, A. et al. 1996 PNAS USA 93: 36023607). Other structural gene products include nucleoprotein (NP), matrix proteins VP24 and VP40, presumed non-structural proteins VP30 and VP35, and viral polymerase (reviewed in Peters, CJ et al. In: Fields Virology, eds. Fields , BN et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996).
[00037] Nucleic acid molecules can encode the structural gene products of any species of phylovirus. There are five species of Ebola virus: Zaire (typical species, here also called ZEBOV), Sudan (also called here SEBOV), Reston, Bundibugyo, and Ivory Coast. There is a unique species of Marburg virus (also called MARV here).
[00038] The specific antigen expressed in the vectors of the invention is not a critical aspect of the present invention. The adenoviral vectors of the invention can be used to express proteins comprising an antigenic determinant of a wide variety of filovirus antigens. In a typical and preferred embodiment, the vectors of the invention include nucleic acid encoding the transmembrane form of the viral glycoprotein (GP). In other embodiments, the vectors of the invention can encode the secreted form of the viral glycoprotein (SGP), or the viral nucleoprotein (NP).
[00039] A person skilled in the art will recognize that the nucleic acid molecules encoding the filovirus antigenic protein can be modified, e.g., the nucleic acid molecules presented here can be mutated, as long as the modified expressed protein produces a immune response against a pathogen or disease. Thus, as used herein, the term "filovirus antigenic protein" refers to a protein that comprises at least one antigenic determinant of a filovirus protein described herein. The term includes filovirus antigens (i.e., gene products from a filovirus), and also recombinant proteins that comprise one or more filovirus antigenic determinants.
[00040] In some embodiments, the protein can be mutated so that it is less toxic to cells (see, eg, WO / 2006/037038). The present invention also includes vaccines comprising a combination of nucleic acid molecules. For example, and without limitation, nucleic acid molecules encoding GP, SGP and NP from Ebola strains from Zaire, Sudan and Côte d'Ivoire can be combined in any combination, in a vaccine composition. ADENOVIRAL VECTORS
[00041] As noted above, exposure to certain adenoviruses has resulted in immune responses against certain adenoviral serotypes, which can affect the efficacy of adenoviral vaccines. The present invention provides adenoviral vectors comprising capsid proteins from two rare serotypes: Ad26 and Ad35. In the typical modality, the vector is an rAd26 or rAd35 virus.
[00042] Thus, the vectors of the invention comprise an Ad26 or Ad35 capsid protein (e.g., a fiber, penton or hexon protein). A skilled person will recognize that it is not necessary for an entire Ad26 or Ad35 capsid protein to be used in the vectors of the invention. Thus, chimeric capsid proteins that include at least 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 each of the fiber, penton, and hexon proteins are derived from a different serotype, provided that 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 derived from Ad35.
[00043] A knowledgeable person will recognize that elements derived from multiple serotypes can be combined into a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable 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 temperature stability, assembly, anchoring, production yield, redirected or improved infection, DNA stability in the cell. target, and the like.
[00044] In certain embodiments, the recombinant adenovirus vector of the invention is derived mainly or entirely from Ad35 or Ad26 (i.e., the vector is rAd35 or rAd26). In some embodiments, i adenovirus is deficient in replication, eg because it contains a deletion in the E1 region of the genome. For the adenovirus of the invention, being derived from Ad26 or Ad35, it is typical to exchange the E4-orf6 encoding sequence of the adenovirus for the E4-orf6 of a human subgroup C adenovirus such as Ad5. This allows for the propagation of such adenovirus in well-known complementary cell lines that express the Ad5 E1 genes, for example 293 cells, PER.C6 cells, and the like (see, eg, Havenga et al, 2006, J Gen. Virol 87: 213543; WO 03/104467). In certain embodiments, the adenovirus is a serotype 35 human adenovirus, with a deletion in the E1 region in which the nucleic acid encoding the antigen has been cloned, and with an Ad4 orf6 E4 region. In certain embodiments, the adenovirus is a serotype 26 human adenovirus, with a deletion in the E1 region in which the nucleic acid encoding the antigen has been cloned, and with an Ad4 orf6 E4 region. For the Ad35 adenovirus, it is typical to retain the 3 'end of the open reading frame E1B 55K in the adenovirus, for example 166 bp directly upstream of the open reading frame pIX or a fragment comprising this such as a 243 bp fragment directly downstream of the pIX initiation codon, marked at the 5 'end by a Bsu36I restriction site, because this increases the stability of the adenovirus because the promoter of the pIX gene is partially residing in this area (see, eg Havenga et al, 2006, supra WO 2004/001032).
[00045] The preparation of recombinant adenoviral vectors is well known in the art. The preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81 (9): 4654-63. Exemplary Ad26 genome sequences are found in Gene Bank Access EF 153474 and SEQ ID NO: 1 of WO 2007/104792. Preparation of rAd35 vectors is described, for example, in U.S. Patent Number 7,270,811 and in Vogels et al., (2003) J. Virol. 77 (15): 8263-71. An exemplary Ad35 genome sequence is found in Gene Bank Access AC_000019.
[00046] Typically, a vector of the invention is produced using a nucleic acid comprising the entire 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. DNA can be double-stranded or single-stranded.
[00047] The adenovirus vectors of the invention are typically defective in replication. In these modalities, the virus is made defective in replication by the deletion or inactivation of critical regions, such as the E1 region. The regions can be substantially deleted or inactivated by, for example, insertion of the gene of interest (usually linked in a promoter). In some embodiments, the vectors of the invention may contain deletions in other regions, such as the E2, E3 or E4 regions or insertions of heterologous genes linked in a promoter. For E2 and / or E4 mutated adenoviruses, generally complementary cell lines-E2 and / or - E4 are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus do not need to be complemented by the cell line, because E3 is not required for replication.
[00048] A packaging cell line is typically used to produce sufficient amounts of adenovirus vectors of the invention. A packaging cell is a cell that comprises those gels that have been deleted or inactivated in a defective replicating vector, thus allowing the virus to replicate within the cell. Suitable cell lines include, for example, PER.C6, 911, 293, and E1 A549.
[00049] As noted above, a wide variety of filovirus antigenic proteins can be expressed in the vectors of the invention. If required, the heterologous gene encoding the filovirus antigenic protein can be codon-optimized to ensure appropriate expression in the treated host (eg, human). Codon-optimization is a technology widely applied in the technique. Typically, the heterologous gene is cloned into the E1 and / or E3 region of the adenoviral genome.
[00050] The heterologous phylovirus gene may be under the control of (i.e., operably linked to) an adenovirus-derived promoter (e.g., the Major Late Promoter) or may 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.
[00051] As noted above, the adenovirus vectors of the invention can comprise a wide variety of phylovirus antigens known to those skilled in the art. Table 1 provides a summary of exemplary vectors of the invention. Table 1
IMMUNOGENIC COMPOSITIONS
[00052] Purified or partially purified adenovirus vectors of the invention can be formulated as a vaccine (also called an "immunogenic composition") according to methods well known in the art. Such compositions can include adjuvants to enhance the immune response. The optimal proportions of each component in the formulation can be determined by techniques well known to those skilled in the art.
[00053] The preparation and use of immunogenic compositions are well known in the art. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, vegetable or animal oils, mineral oil or synthetic oil. Physiological saline solution, dextrose solution or solution of another saccharide or glycols such as ethylene glycol, propylene glycol or poly (ethylene glycol) may be included.
[00054] The compositions are suitable for single administrations or a series of administrations. When given as a series, inoculations subsequent to the initial administration (initial administration) are given to reinforce the immune response and are typically called booster inoculations. The compositions of the invention can be used as an antigen-initiated booster composition using any of a variety of different starting compositions, or as the starting composition. Thus, an aspect of the present invention provides an initial and / or boosting immune response to an antigen in an individual. For example, in some preferred embodiments, an initial administration of an adenoviral vector of the invention (e.g., rAd26) is followed by a booster inoculation of the second adenoviral vector (e.g., rAd35).
[00055] The timing of administering reinforcement compositions is well within the skill of the art. Booster compositions are administered weeks or months after administration of the initial composition, for example, for example, about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks or one to two years.
[00056] The compositions of the invention may comprise other filovirus antigens or initial or booster inoculations may comprise other antigens. The other antigens used in combination with the adenovirus vectors of the invention are not critical to the invention and can be, for example, filovirus antigens, nucleic acids expressing them, virus-like particles (VLPs), or vectors of the prior art. Such viral vectors include, for example, other adenoviral vectors, vaccinia virus vectors, avipox vectors such as poultry contagious epithelioma vectors, canary contagious epithelioma vectors, herpes virus vectors, vesicle stomatitis virus vectors, or vectors of vesicle stomatitis, or vectors alphavirus. A skilled person will recognize that the immune compositions of the invention can comprise multiple antigens and vectors.
[00057] The antigens in the respective initial and reinforcement compositions (however many reinforcement compositions are used) need not be identical, but must share antigenic determinants. As noted above, the immunogenic compositions of the invention can comprise adjuvants. Adjuvants suitable for coadministration according to the present invention should be those 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.
[00058] Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines such as interferon-alpha, interferon-gamma, platelet derived growth factor, PDGF, granulocyte colony stimulating factor (granulocyte-colony stimulating factor, gCSF), granulocyte-macrophage colony stimulating factor (granulocyte macrophage colony stimulating factor, gMCSF), tumor necrosis factor (tumor necrosis factor, TNF), epidermal growth factor (epidermal growth factor, EGF ), IL-I, IL-2, IL-4, IL-6, IL-8, IL-IO, and IL-12 or nucleic acids encoding them.
[00059] As noted above, the compositions of the invention may comprise a stabilizer, buffer, vehicle, pharmaceutically acceptable excipient or other pharmaceutically acceptable materials well known to those skilled in the art. Such Materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the vehicle or other material may depend on the route of administration, eg, oral, intravenous, cutaneous or subcutaneous, intramucosal (eg, intestinal), intranasal, intramuscular, or intraperitoneal routes. Administration is typically intramuscular.
[00060] Intramuscular administration of immunogenic compositions can be performed using a needle to inject a suspension of the adenovirus vector. An alternative is to use a needle-free injection device to administer the composition (using, for example, Biojector (TM)) or a freeze-dried powder containing the vaccine.
[00061] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affection, the adenovirus vector will be in the form of a parenterally acceptable aqueous solution that is pyrogen free and has adequate pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride for Injection, Ringer's Solution for Injection, Lactated Ringer's Solution for Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, if required. A slow release formulation can also be used.
[00062] Typically, administration will have a prophylactic goal to generate an immune response against a filovirus antigen before infection or the development of symptoms. Diseases and disorders that can be treated or prevented in accordance with the present invention include those in which an immune response can play a protective or therapeutic role. In other modalities, adenovirus vectors can be administered for post-exposure prophylaxis.
[00063] Immunogenic compositions containing adenovirus vectors are administered to an individual, producing an antifilovirus immune response in the individual. An amount of a composition sufficient to induce a detectable immune response is defined as an "immunologically effective dose". As shown below, the immunogenic compositions of the invention induce both humoral and cell-mediated immune responses. In a typical embodiment, the immune response is a protective immune response.
[00064] The actual amount administered, and the rate and course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, eg, dosage decisions etc., is within the responsibility of general practitioners and other physicians, or within the veterinary context, and typically considers the disorder to be treated, the condition of the individual patient, the release site, the method of administration and other factors known to doctors. Examples of the techniques and protocols mentioned above can be found in "Remington's Pharmaceutical Sciences, 16th edition", Osol, A. ed., 1980.
[00065] After the production of adenovirus vectors and the optional formulation of such particles in compositions, the adenovirus vectors can be administered to an individual, particularly a human or another primate. Administration can be to humans, or another mammal, eg, mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. Release to a non-human mammal need not be for a therapeutic purpose, but it can be for use in an experimental context, for example in investigating mechanisms of immune responses to the adenovirus vector.
[00066] In an exemplary regimen, the adenovirus vector is administered (eg, intramuscularly) within the range of about 100 μl to about 10 ml of saline containing concentrations of about 104 to 1012 viral particles / ml . Typically, the adenovirus vector is administered in an amount of about 109 to about 1012 viral particles (pv) to a human subject during an administration, more typically from about 1010 to about 1012 pv. An initial vaccination can be followed by a booster vaccination as described above. The composition may, if desired, be presented in a kit, package or dispenser, which may contain one or more unit dosage forms containing the active ingredient. The kit, for example, can comprise metallic or plastic foil, such as a blister pack. The kit, package, or dispenser may be accompanied with administration instructions.
[00067] The compositions of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated. EXAMPLES
[00068] The following examples are offered to illustrate, but not to limit the claimed invention.
[00069] There are distinct advantages associated with either single injection immunization or initial immunization - booster immunization depending on the need for immediate immunity versus long-term immunity considering when optimizing immunization regimes. Outbreaks of EBOV and outbreaks of other phyloviruses tend to occur suddenly and spread rapidly among populations in which medical facilities are scarce. Thus, under these circumstances, short vaccine regimens may be desirable. For this reason, vaccinations with a single injection with rAd5 vectors containing EBOV nucleoprotein (NP) and glycoprotein (GP) genes have been developed in non-human primates (Sullivan, et al., 2006). Such vaccines have been shown to produce strong immune responses within a month (Sullivan, et al., 2003), probably due to the high levels of expression of the inserts and the tropism of Ad5 for dendritic cells. On the other hand, long-term protective immunity is likely to require an initial vaccination regimen - booster vaccination comprising two or more administrations that can induce durable T-cell memory. Therefore, we planned a series of experiments to test the immunogenicity and potency for both single inoculation and combinations of initial vaccination - booster vaccination using rAd35 and rAd26 vectors, and the results of these studies are presented here. MATERIALS AND METHODS
[00070] Generation of rAd Ebola vaccines. Vaccine vectors of rAd26 and rAd35 with low seroprevalence deleted E1 / E2 expressing EBOV GPs were constructed, grown and purified as previously described (Abbink, et al., 2007). An Ad5 vector, not expressing EBOV GP, was constructed, grown and purified by the same method and used to induce immunity against Ad5 in selected animals as indicated in each experiment. EBOV GP inserts spanning the open reading matrices of Zaire species (SEQ ID NO: 1) and Sudan / Gulu (SEQ ID NO: 2) were cloned under transcriptional control of the human CMV promoter and the SV- polyadenylation sequence 40 on a plasmid containing the left position of the Ad genome, including the left IRT and the packaging signal. Cotransfection of this plasmid with a cosmid containing the remaining Ad sequence (E3-deleted) in PER.C6® cells gave a replication-deficient recombinant Ad26 or Ad35 vaccine vector with E1 / E3-deleted. To facilitate the replication of rAd26 and rAd35 vectors in PER.C6® cells, the native E4 orf6 regions were replaced by the Ad4 E4orf6 sequence (Havenga, et al., 2006). The rAd residues were plaque purified and one plate each was expanded to a production scale of approximately 2.4 L. A two-stage cesium chloride ultracentrifugation procedure was used to purify the rAd EBOV vectors. Purified rAd EBOV vaccines were stored as single-use aliquots below -65 ° C. Viral particle titers were determined by measuring the optical density at 260 nm (Maizel, et al., 1968 Virology 36 (1): 115-25). Infectivity was assessed by TCID50 using 911 cells (Fallaux, et al., (1996) Hum. Gene Ther. 7 (2): 215-22). Adenovirus-mediated EBOV GP expression was assessed by infection of A549 cells followed by analysis of culture lysates in Western blot. The identity of the purified vectors was confirmed by PCR and the complete transgenic regions, using flanking sequences, were checked using DNA sequencing.
[00071] Phylogenetic analysis. The phylogenetic tree was constructed using the full-length adenovirus amino acid sequences. The amino acid sequences were aligned using the Clustal X program (Larkin, et al., (2007) Bioinformatics 23 (21): 29478) and the tree was constructed using the Clustal X neighbor joining method and the tree was resampled 1,000 times . The tree was visualized and plotted using the Drawtree program from Phylip Phylogeny Inference package version 3.68.
[00072] Safety and animal challenge study. Animal experiments were conducted in full compliance with all relevant federal procedural rules and NIH guidelines. Cynomolgus monkeys (Macaca fascicularis) aged 3-5 years and weighing between 2 and 3 kg were obtained from Covance for all studies. The monkeys were housed individually and regularly enriched as recommended by the “Guide for the Care and Use of Laboratory Animals” (“DHEW number NIH 86-23”). The animals were anesthetized with ketamine before blood sampling or vaccination. Each vaccine group in this study contained three cynomolgus monkeys, and each control group contained a single cynomolgus monkey. Four weeks after vaccination against EBOV, the animals were transferred to the maximum confinement laboratory “Maximum Containment Laboratory (BSL-4)” for infection with a target dose of 1,000 PFU of Zaire EBOV released intramuscularly into the caudal thigh. The ZEBOV challenge stock was prepared from a human fatality in the 1995 outbreak in former Zaire. The animals remained there until the study was completed. While in the BSL-4 installation the monkeys were fed and checked at least once a day
[00073] Animal studies performed on BSL-4 bioconfining at USAMRIID have been approved by the “USAMRIID Institutional Animal Care and Use Committee”. Animal research was conducted in accordance with the Animal Welfare Act and other federal statutes and regulations related to animals and animal experiments and adheres to the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facilities used were fully accredited by the “Association for Assessment and Accreditation of Laboratory Animal Care International”.
[00074] Animal immunization. Individuals received intramuscular vaccinations in the bilateral deltoid muscles by needle and syringe with doses and vectors indicated in each experiment. The animals selected, as indicated in each experiment, were pre-immunized with 1011 PFU of an empty Ad5 vector to induce immunity against Ad5. Antibody titers against Ad5 determined by ELISA were established in these animals prior to vaccination against EBOV.
[00075] anti-EBOV GP IgG ELISA. Poly (vinyl chloride) plates for ELISA (Dynatech, Vienna, VA, or Nunc, Rochester, NY) were coated with 100 μl of antigen per well and incubated at 4 ° C overnight. Subsequent incubations were performed at room temperature. EBOV GP with deleted transmembrane (EBOV GPΔTM) generated by transient transfection mediated by calcium phosphate from 293T cells served as the antigen. The plates were washed six times with PBS containing Tween 20 after coating with antigen. Test sera were serially diluted to 7 concentrations ranging from 1:50 to 1: 50,000 and added to antigen-coated wells for 60 minutes. The plates were washed six times followed by incubation with detection antibody, goat anti-human IgG (H + L; Chemicon / Millipore, Billerica, MA) conjugated to horseradish peroxidase. Substrate Sigma Fast o-Phenylene-diamine dihydrochloride (Sigma, St. Louis, MO) was added to the wells and the optical density was determined (450 nm). A pre-vaccination serum sample for each animal was taken each time the assay was performed. A sample of positive control serum from a single animal with a known anti-GP Zaire EBOV IgG response was taken each time the assay was performed. Titles determined by ELISA minus background interfering values are expressed as EC90, values of reciprocal optical density, which represents the dilution in which there is a 90% decrease in antigen binding.
[00076] Intracellular cytokine staining. Samples of whole blood from cynomolgus monkeys were subjected to density gradient centrifugation on Ficoll to isolate peripheral blood mononuclear cells (PBMC). Approximately 1 x 106 cells were stimulated in 100 Dl of RPMI medium containing 10% thermally inactivated fetal bovine serum for 6 hours at 37OC with anti-CD28 (clone CD28.2) and -CD49d (clone L25) antibodies (BD Biosciences), Brefeldin-A (Sigma-Aldrich, St. Louis, MO), and either DMSO or a collection of peptides spanning the entire open reading matrix of Zaire EBOV GP. The peptides were 15 mere superimposed by 11 amino acids reconstituted in new sterile DMSO at a final concentration of 2.5 üg / ml for each peptide. For each sample equivalent, an aliquot was stimulated with SEB as a positive control. After six-hour stimulation, PBMC were stained with a mixture of antibodies against lineage markers (CD3-Cy7-APC, SP34-2 clone (BD Biosciences), CD4-QD605 clone M-T477 (BD Biosciences), CD8-TRPE clone RPA-T8, CD95 Cy5-PE, clone DX2 (BD Biosciences), CD45RA QD655, clone 5H3, at room temperature for 20 min The antibodies CD45RA QD655 and CD8-TRPE were conjugated according to standardized protocols as previously described (Koup et al. "2010 Priming Immunization with DNA Augments Immunogenicity of Recombinant Adenoviral Vectors for Both HIV-1 Specific Antibody and T-Cell Responses." PLoS One 5 (2): e9015. doi: 10.1371 / journal.pone.0009015). washes the cells were fixed and permeabilized with Cytofix / Cytoperm (BD Biosciences) followed by staining with antibodies against TNFα-APC cytokines, clone MAb11 (BD Biosciences), and IL-2 PE, clone MQ17H12 (BD Biosciences). ViViD (Invitrogen) was included to allow discrimination between cells alive and dead (Perfetto, et al., (2006) J. Immunol. Methods 313 (1-2): 199-208). Samples were acquired on an LSR II cytometer (BD Biosciences), collecting up to 1,000,000 events and analyzed using computer programs FlowJo 9.1 and SPICE 5.0 (Tree Star). Cytokine positive cells were defined as a percentage within memory subsets of CD4 + and CD8 + T cells. Memory subsets were defined as CD45RA ± / CD95 + or CD28 ± / CD95 +. In the latter case, CD28 Alexa488 (clone 28.2, BioLegend) was used for stimulation instead of unconjugated CD28 mAb.
[00077] Serum biochemistry. For challenge studies, blood was collected from NHP on days 0, 3, 6, 10, 14 and 28 after infection by Zaire EBOV. Total white blood cell counts, white blood cell differences, and red blood cell counts, platelet counts, hematocrit values, total hemoglobin, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentration were determined from blood samples collected in tubes containing EDTA, using a laser-based hematology analyzer (Coulter Electronics, Hialeah, FL, USA). Serum samples were tested for concentrations of aspartate aminotransferase (AST), using a “Piccolo Point-Of-Care Blood Analyzer” (Abaxis, Sunnyvale, CA, USA).
[00078] EBOV detection. Virus titration was performed by plaque assay on Vero cells. Briefly, 10-fold increasing dilutions of plasma samples were adsorbed onto Vero monolayers in double wells (0.2 ml per well); thus, the limit for detection was 25 pfu / ml.
[00079] Statistics. Comparison of anti-GP IgG titers determined by ELISA, and the production of intracellular cytokine by subsets of T cell memory was performed using a bilateral T test in a GraphPad computer program. RESULTS
[00080] Adenovirus vector and phylogeny construction. Genetic rAd5 vaccines for EBOV provide potent protective immunity in monkeys, and have been shown to be safe and immunogenic in clinical trials in humans (Asmuth, et al .; Kibuuka, et al .; Harro, et al., 2009;). Studies in monkeys and humans have shown that pre-existing vector-directed immunity can limit the potency of viral vector-based vaccines (McCoy, et al., 2007; Buchbinder, et al., 2008). Since seroprevalence data suggests that a large proportion of humans worldwide have experienced natural infection with Ad5, we have evaluated other adenovirus serotypes for use as vaccine vectors (Figure 1A). Ad35 Adenovirus, a group B and Ad26, a group D genetically secrete rAd5, Group C, so we hypothesize that vaccine vectors derived from these serotypes will be less sensitive to immunity against Ad5 in primates. Although the Ad35 and Ad26 vectors use receptors distinct from the use of Ad5, they nevertheless demonstrate efficient transduction of monocyte-derived dendritic cells, and circumvent immunity against Ad5 in mice. Therefore, GP inserts of the Zaire or Sudan / Gulu species of EBOV were cloned under transcriptional control of the human CMV promoter in the E1 region of rAd35 and rAd26 vectors (Figure 1B). Both vector genomes have been deleted in the E1 genes with the purpose of rendering them deficient in replication and reducing the potential for recombination in vaccinated individuals.
[00081] Vaccination with rAd35 and induction of immune response in monkeys. Initial studies were conducted with a vaccine against a single species of EBOV encoding GP from Zaire Ebola virus (ZEBOV), GP (Z), to test the ability of rAd35 vectors to induce immune responses in monkeys not modified with Ad5, and also to assess the vector's potency within the context of pre-existing immunity against Ad5. Six cynomolgus monkeys, three unmodified with Ad5 and three immune to Ad5, were each vaccinated intramuscularly with 1010 particles of rAd35-GP (Z) (Ad35BSU.Ebo.GP (Z) FL.wt) by needle injection. Three weeks after vaccination, T-cell responses and antigen-specific antibodies were evaluated in peripheral blood samples obtained from individual subjects. Antibodies against EBOV-GP (Z) assessed by ELISA were induced in all subjects, demonstrating that the rAd35-GP (Z) vectors mediated successful in vivo transduction and efficient antigen presentation (Fig. 2A). Individuals unmodified with Ad5 and immune to Ad35 generated serum antibody titers ranging from approximately 1: 700 to 1: 3,000. These antibody levels are within the range that has been observed for Ad5-based vaccines containing GP (Z) inserts and have exceeded the minimum value (1: 500) that has been associated with immune protection against EBOV infection in an Ad vaccine model. in a monkey (Sullivan, 2009). Although significant antibody titers were induced in all vaccinees, none of the subjects exceeded the threshold titer (ca. 1: 3,500) that predicts 100% protection after administration of Ad5-GP vaccine vectors in monkeys. However, it is notable that the comparison of antibody titers in individuals unmodified with Ad5 versus immune to Ad5 showed that there was no significant difference in mean titers produced between these groups (1: 1,600 versus 1: 1,800 respectively), suggesting that the vectors rAd35 are effective vaccines in individuals who have been exposed to Ad5.
[00082] Cellular immune responses were assessed by intracellular cytokine staining (intracellular cytokine staining, ICS) for either TNF- (CD8 +) or IL-2 (CD4 +) after stimulation of an individual's PBMC with overlapping peptides spanning the open reading matrix of GP (Z) of EBOV. Lymphocyte surface staining using CD45RA and CD95 was performed to assess antigen-specific immune responses in the subpopulations of CD4 + and CD8 + T-cell memory (Fig. 2 B-C). As observed for antibody responses, monkeys vaccinated with the rAd35 vector generated cellular immunity against EBOV-GP, and the frequency of antigen-specific T-cells was not affected by the immune status to Ad5. The magnitude of the classification of cellular responses in both CD4 + and CD8 + lymphocytes between individuals was similar to antibody responses, although the frequency of antigen-specific T-cells for a V3 individual was below detectable levels. Previous studies in monkeys have shown that vaccine vectors based on rAd5 induce CD8 + T-cell frequencies that are dominant over CD4 + responses. In the present study, individuals vaccinated with rAd35 generated CD4 + and CD8 + lymphocytes specific for GP at similar frequencies. However, given the relatively low number of individuals tested, it is possible that differences, if present, could not be revealed. All of these tests together demonstrate that rAd35-GP is immunogenic in cynomolgus monkeys and that the vector's potency for inducing antigen-specific cell-mediated and humoral immune responses is not reduced in individuals with pre-existing immunity to Ad5.
[00083] ZEBOV challenge for monkeys vaccinated with rAd35. Next, we test whether vaccination with rAd35-GP provides protection against infectious challenge with a high dose of ZEBOV. One week after the assessment of the immune responses shown above, the six cynomolgus monkeys vaccinated with rAd35-GP and an unmodified cynomolgus monkey were exposed to 1,000 PFU of the 1995 ZEBOV Kikwit strain by intramuscular injection. Liver enzymes were measured regularly after the infectious challenge because the elevations in these markers are characteristic of productive EBOV infection in monkeys. Circulatory levels of aspartate transaminase (AST) were assessed every 3-4 days during the period of acute infection, during days 10-14 (Fig. 2D), and then on the last day of the 28-day follow-up period (not shown ). Plasma AST remained at baseline levels until day 3 after infection in all subjects, indicating normal liver function immediately after the EBOV infectious challenge. On day 6 after exposure to EBOV, the unvaccinated control individual exhibited a 10-fold increase in enzyme levels indicating active infection in this individual. Blood samples from the two individuals in the group not modified with Ad5 / vaccinated for rAd35 (V1, V3) also exhibited dramatic increases in AST, although the third individual in this V2 group showed only a marginal increase in a single time before resolution of back to baseline levels. Similarly, two of the three individuals in the Ad35 / vaccinated rAd35 (V4, V5) immune group exhibited elevations in AST, although much lower than the unvaccinated control, although one V6 individual remained normal for this infection parameter. Altogether, AST levels were higher in vaccinated Ad5 unmodified individuals than in vaccinated Ad5 immune individuals. It is noteworthy to mention that each individual who remained normal during this clinical observation exhibited the highest antigen-specific antibody and CD8 + responses within the respective vaccine group. Plasma levels of viremia (Fig. 2E) confirmed infection by EBOV in all animals that exhibited elevated AST.
[00084] The results of this experiment showed that rAd35 is immunogenic when administered at a dose of 1010 particles per individual. The vaccine generated protective immune responses, but this dose and regimen were suboptimal for the uniform protection of all individuals. Within vaccine groups, protective immunity was associated with CD8 + T-cell and antibody responses specific to higher magnitude antigens.
[00085] Effects of dose response of rAd35-GP on induction of protective immunity. RAd-based vectors are commonly administered to monkeys at doses ranging from 1010 to 1012 particles. Previous results with rAd5-GP have shown that 1010 viral particles as the minimum dose to achieve 100% protection of cynomolgus monkeys against EBOV infection (Sullivan, 2006). Since the studies described above were performed at the lower end of this dose range and did not result in uniform protection, it is possible that even marginally lower antigen expression in vivo achieved with the rAd35 vector when compared to that of rAd5 vectors could result in responses suboptimal immune systems. Therefore, we asked whether administering a higher dose of vaccine could produce a higher degree of immune protection. The vaccine in this experiment also included rAd35 expressing GP of the Sudan species of EBOV (S / G) for the purpose of comparing efficacy with historical data with rAd5 vaccines that comprised GPs of both Sudan and Zaire species. In the present study, cynomolgus monkeys (n = 3 per group) were vaccinated with 1010 or 1011 viral particles each of Ad35BSU.Ebo.GP (Z) FL.wt and Ad35BSU.Ebo.GP (S / G) FL and immune responses were measured three weeks after vaccination in the previous experiment.
[00086] GP-specific antibodies were generated in all subjects (Figure 3A), an expected result since the vaccine dose was equal to or greater than that in the previous experiment. However, the titers in two individuals vaccinated with 1010 particles in this experiment (V8, V9) were 1: 340 and 1: 500, respectively, levels close to or below the minimum cut previously observed to predict protection in individuals vaccinated against Ad5-GP ( Sullivan, et al., 2009). The maximum antibody titre observed was 1: 2,900 (individual V10) which is equal to the maximum titers observed in the rAd35 individuals shown in Figure 2. Average antibody titers were higher in the 1011 dose group (1: 1,500 versus 1: 700) but the difference did not reach statistical significance (p = 0.02). As seen in the lowest dose group, there was an individual whose titer was close to the cut of the rAd5-GP vaccine for immune protection (individual V11, 1: 540).
[00087] CD4 + and CD8 + T-cell responses were present in both dose groups within three weeks after vaccination. There was no evident dose response in either CD4 + or CD8 + T-cells. Since the kinetics of cellular immune responses vary between individuals, especially in exogamous animals, the measurement of response is not cumulative over time because it is with antibody levels, group trends are sometimes difficult to capture in a single instant. time. Within each individual, antigen-specific T-cell frequencies were higher for CD4 + cells than for CD8 + cells, but when combined with the results of the first experiment, there was no tendency for either CD4 + cell or vector-induced CD8 + cell dominance rAd35-GP in these experiments.
[00088] One week after the assessment of immune responses, all six vaccinated monkeys and an unvaccinated individual were exposed to 1,000 PFU of ZEBOV by intramuscular injection and observed for signs of productive infection. Hemorrhagic manifestations of EBOV infection routinely result in the appearance of a maculopapular rash on the face and extremities of infected monkeys; individuals also typically reduce food intake and become dehydrated. The earlier onset of symptoms for the two unvaccinated individuals occurred on day 6 after exposure to EBOV; each exhibited a total constellation of symptoms by day 7 (data not shown). Table 2 shows the results of infectious challenge and the day of death in non-survivors for both studies with rAd35. Unvaccinated individuals succumbed to the lethal effects of the infection on days 9 and 8 (Experiments 1 and 2, respectively). Vaccinated individuals who died were similar to control, unvaccinated individuals, except that they survived an average of two days longer than controls, suggesting a potential partial immune benefit from vaccination although mortality was finally observed. The number of survivors was higher in individuals who received only rAd35-GP (Z), compared to those who received GP (Z) plus GP (S / G) regardless of the vaccine dose, but differences in survival rates were not significant through any vaccinated groups in the two challenge experiments. Table 2

[00089] Altogether, studies using rAd35 as a vaccine vector with GP (Z) alone or in combination with GP (S / G) showed that antigen presentation and release were sufficient to generate antigen-specific immune responses, but at levels below what is required for absolute immune protection. Lower protective immunity has been associated with antibody levels detected in some animals which is consistent with what has been shown using rAd5 vectors to be of very little protection.
[00090] Potency and immunogenicity of rAd26-GP vaccine against EBOV infection. Next, we evaluated a vaccine based on recombinant Ad26, one of group D adenovirus, because of its ability to generate protective immunity against EBOV infection. This serotype uses the same cell receptor (CD46) as Ad35 but has been shown to generate very little immune responses when used as an initial vaccine vector (Liu, et al., 2009). For these studies, a gradual dose increase was conducted over a range of three orders of magnitude in two separate infectious challenge experiments. In the first study we tested the vaccine in doses of 1010 or 1011 particles for each vector, Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt, and in the second study we used a dose of 1012 particles each. The first study tested the vaccine in cynomolgus monkeys immune to Ad5 in order to assess whether rAd26, like rAd35, could produce antigen-specific immune response in the presence of pre-existing immunity to Ad5. Four Ad5-immune cynomolgus monkeys per group were vaccinated by intramuscular injection, and blood samples were obtained three weeks later to assess cellular and humoral immune responses in circulation against EBOV GP (Fig. 4A). Average circulating anti-GP antibody titers showed a dose response between dose groups; 1: 700 for vaccinees with 1010 particles and 1: 4,500 for individuals receiving 1011 particles (p = 0.06). The mean titer for three of four subjects in the 1010 particle dose group was just above the minimum threshold for immune protection in subjects vaccinated with rAd5 (1: 500), but subject V16 generated only a very low antibody response, 1: 100, which is well below the protection cut predicted for an Ad5-GP vaccine. In contrast, the V19 individual who was vaccinated with 1011 rAd26 vaccine particles generated a very high antibody titer, 1: 10,500, exceeding almost three times the level that has been associated with complete immune protection (1: 3,500), while the others in this vaccine group exhibited intermediate titers that do not definitively predict survival outcome. In study two, four subjects received 1012 particles from each rAd26 vector and generated antibody responses very similar to those in the 1011 particle dose group, with most individual titers being between 1: 1,000-1: 4,000. The average anti-GP antibody titer for this group was 1: 3,000.
[00091] T-cell immune responses (Figure 4B, C) were measured by ICS as in the studies with rAd35 and also tended towards a dose response in study one, but the difference between the 1010 particle and dose groups 1011 particles were not significant (p = 0.12 and 0.26 for CD4 + and CD8 +, respectively). The mean frequencies of antigen-specific CD4 + T-cells were 0.14% for vaccinees receiving 1010 rAd26-GP particles versus 0.24% at the highest vaccine dose, and one vaccinated in the lowest dose group, individual V14 , had an undetectable CD4 + response (Figure 4B). The rAd26 vaccine did not distort cellular immune responses to either CD4 + or CD8 + dominance; the frequencies of CD8 +, 0.13% and 0.25% (vaccine doses of 1010 particles and 1011 particles, respectively) essentially reflected the magnitude of CD4 + responses. In the case of CD8 + T-cells, there were two individuals in the low dose group, V13 and V14, with specific responses to undetectable antigens (Figure 4B, C). In study two with rAd26, mean CD8 + frequencies specific for antigen (0.34%) were higher than CD4 + responses (0.08%) but this evident distortion for CD8 + responses was mainly driven by a single individual , V24, which had very high CD8 + frequencies and low CD4 + responses. In other respects, the total cellular immune responses were similar to those observed in study one with rAd26.
[00092] Infectious challenges with EBOV were performed by IM injection of 1,000 PFU of ZEBOV at 4 weeks after vaccination for each of the rAd26 vaccine studies, and liver enzyme levels were measured to monitor the disease (Figure 4D). Unvaccinated individuals exhibited manifestations of liver damage between days 3 and 6 of infection. All subjects receiving the lowest dose of rAd26 vaccine, 1010 particles, showed similar signs of illness, although AST levels increased at a slower rate. As predicted for this clinical indicator, all individuals in this group succumbed to the lethal effects of infection by day 8 after the ZEBOV challenge (Figure 5A). T-cell and antibody responses were low or undetectable in this group. Differences in the magnitude of immune responses between study dose groups one with Ad26 (1010 and 1011) were generally reflected in survival rates, with a higher survival outcome, 2 out of 4 protected, in individuals vaccinated with 1011 rAd26 particles. -GP (p = 0.01). Ad26 to 1011 particles was superior not only for the lowest dose, but also provided greater protection than rAd35-GP when equalized for dosage (Figure 5B). Finally, rAd26 given in a dose of 1012 particles gave the highest number of survivors (3 out of 4) for any vaccine regimen tested in these studies, and the level of immune protection did not differ significantly from that previously observed with vectors (p = 0.32, Figure 5C); however, this survival rate was obtained using a higher dose for rAd26 than for rAd5, 1012 versus 1010 particles, respectively, suggesting a potential difference in potency between these vectors in this animal model.
[00093] Initial vaccination - heterologous booster vaccination with rAd26 and rAd35 vectors. The dose response characteristics for rAd26-GP-mediated immune protection and the high survival result using 1012 vector particles suggested that this vector efficiently induces GP antigen expression. Since it has been shown for EBOV and other pathogens, in both human and non-human individuals, that initial vaccination - heterologous booster vaccination can produce more potent immunity than single injection immunization (Sullivan Nature 2000; Sampra et al Vaccine 27 (2009 ) 5837-5845; Koup, et al, PLoS One 2010; Geisbert et al., (2010) Virol 84 (19): 10386-94), we asked if rAd26-GP immune responses could be reinforced with a heterologous vector to improve protection against EBOV infection. Four cynomolgus monkeys were inoculated with 1011 particles of each Ad26.Ebo.GP (Z) FL.wt and Ad26.Ebo.GP (S / G) FL.wt. One month later, all subjects received a booster vaccination with the same dose of Ad35BSU.Ebo.GP (Z) FL.wt and Ad35BSU.Ebo.GP (S / G) FL. Immune responses were assessed immediately before boost, and three weeks after boost. Figure 6A shows that antibody responses against EBOV-GP (Z) were efficiently induced by the initial vaccination. Individual subjects generated EC90 antibody titers against GP from 1: 2,700 to 1: 7,100, and the mean titer for the group was 1: 4,000, consistent with the responses observed in the previous study testing 1011 rAd26 as a single inoculation vaccine (1: 4,500). This study included for comparison a single individual inoculated with 1010 particles of each of rAd5-GP (Z) and rAd5-GP (S / G), whose post-vaccination antibody titer was 1: 6,800. Subsequent inoculation of individuals initiated with rAd26-GP with 1011 particles of rAd35-GP vectors boosted antigen-specific antibody levels by approximately an order of magnitude for most vaccinees to an average titer of 1: 32,000, except for individual V27 whose antibody titers from initial post-vaccination were exceptionally high. Interestingly, booster vaccination generated more uniform titers among individuals, with a standard deviation among individuals of just below 10%, compared with initial vaccination titers that exhibited a 54% standard deviation.
[00094] Cellular immune responses were also notably enhanced by the administration of rAd35-GP to monkeys vaccinated with rAd26 (Fig. 6B, C). CD4 + T-cells were reinforced in all but one individual, V25; the mean increase after administration of rAd35-GP was two-fold among all subjects and the boost revealed a measurable response in individual V27 whose response was undetectable prior to boost. Final CD4 + T-cell frequencies after boosting were comparable to those generated by vaccination with rAd5-GP. The reinforcement effect was greater for CD8 + T-cells and all individuals exhibited the reinforcement effect in this cell compartment giving, in two individuals (V27 and V28), responses surpassing those generated by vaccination with rAd5-GP. Mean frequencies of GP8-specific CD8 + T-cells measured by ICS were 0.09% after initial immunization, and increased 4.7 times to 0.41%, 3 weeks after the second vaccination with rAd35-GP.
[00095] Altogether, the immunogenicity results above showed that the rAd35-GP vectors are potent for reinforcing monkeys initiated with rAd26-GP. Antibodies produced against GP after boosting were induced to a medium level that is almost a log higher than the predictive level for 100% immune protection in primates vaccinated with rAd5-GP (Sullivan, et al., 2009). Importantly, reinforcement of rAd35-GP provided a substantial intensification of CD8 + T-cell frequencies, also shown to be associated with immune protection against EBOV infection. Therefore, one week after the assessment of immune responses (4-weeks after booster) all vaccinated individuals and an unvaccinated control monkey were exposed to 1,000 PFU of ZEBOV by intramuscular injection. The control subject exhibited clinical symptoms characteristic of EBOV infection and succumbed to lethal effects on day 6 after the challenge (Figure 6C, D). In contrast, all vaccinated individuals remained normal for circulating AST levels (Figure 6C), and exhibited no evidence of hemorrhagic disease on macropathological assessment at the end of the study (not shown). All four vaccinated individuals survived the infectious challenge and remained symptom-free for the 28 days after the follow-up period until the end of the study. These results showed that the rAd26 / rAd35 vectors administered as an initial vaccination regimen - booster vaccination provides uniform immune protection against ZEBOV infection, and demonstrated the potential usefulness of this approach to achieve additive or synergistic results with combination vaccines. DISCUSSION
[00096] Adenoviruses play well as vaccine vectors for the release of a variety of viral parasitic and bacterial antigens (Lasaro et al., (2009) Mol Ther 17 (8): 1333-9). RAd5 vectors in particular generate potent antigen-specific immune responses in mice, non-human primates, and humans, and as we have seen when EBOV GP is the target antigen. However, preclinical and clinical studies in humans have suggested that the potency of rAd5-based vectors may be compromised in individuals who have previously been exposed to Ad5 if they have a high level of immunity against the vector. The aim of the studies here was to identify rAd vectors that can release the EBOV GP antigen in both individuals not modified with Ad5 and immune to Ad5. Since pre-existing immunity against any viral vector has the potential to limit its effectiveness, we focus our attention on viruses that infect humans relatively rarely, as indicated by the prevalence of HIV-positive individuals and / or low levels of neutralizing antibodies. The rare human adenovirus serotypes, Ad35 and Ad26 were selected for vaccine development in the present experiment.
[00097] Vaccination with monkeys rAd35-GP generated antigen-specific T-cell and antibody responses in individual subjects within the range previously observed when rAd5 was used as the release vector. The mean titer of anti-GP antibody for all vaccinated with rAd35 (dose independent), 1: 1,400 was lower than the mean for all historical individuals vaccinated with 1010 rAd5-GP (1: 11,000, n = 17) , providing an additional indication that the potency of the vector may differ between two serotypes if the antibody titer is the same correlate for rAd35 as it is for rAd5 EBOV vaccines. CD4 + and CD8 + T-cell responses were detectable in most individuals prior to the infectious challenge, although the absolute magnitude could not be compared with the rAd5 vaccines not included in these studies in the absence of PBMC samples for assay connection controls.
[00098] Vaccination with rAd35 vectors effectively induced immune responses of T-lymphocytes and antigen-specific antibodies in individuals unmodified with rAd5 or Immune to rAd5, suggesting that rare serotype vector genomes are sufficiently distant from common serotypes to resist targeted immunity by heterologous vector. This vector performance characteristic will be important to circumvent the pre-existing immunity originating not only from natural viral infection, but also from the use of heterologous vectors in initial immunizations or vaccination against other pathogens. In fact, inoculation of rAd35-GP provided a potent boost of both antibody and cell-mediated responses in monkeys initiated with rAd26-GP. This result was intriguing because it demonstrates an evident difference in vector potency for the induction of primary versus secondary immune responses; the ability of rAd35-GP to reinforce the immune response not predicted by the magnitude of the responses observed after the initial immunization. These data indicate that the rAd35 vector and other rAd vectors have a higher transduction efficiency in certain populations or activation states of higher target dendritic cells, as recently suggested by Lindsay et al., J. Immunol. 185 (3): 1513-21, which may, in this case, be more abundant or accessible during secondary immune responses.
[00099] rAd26 proved to be more potent than rAd35 as a single injection vaccine against EBOV infection, mediating survival in up to 75% of monkeys vaccinated at the highest tested dose. RAd26-GP vaccines have demonstrated an evident dose response for the induction of protective immunity, suggesting that very small improvements in antigen expression could increase the potency of rAd26-based vaccines for uniform protection generated against high challenge doses of EBOV such as those used here. Interestingly, the highest degree of protection offered by the rAd26-GP vectors compared to rAd35-GP at an equal dose (1011 particles) associated with the highest anti-GP titers determined by ELISA, 1: 4,500 versus 1: 1400, respectively. These data highlight the possibility that antibody titers before the challenge may serve as an immune correlate of protection against ZEBOV infection among rAd serotypes in addition to and within vector groups as has been observed for rAd5-GP vaccines. The order of potency for inducing antibody responses predicted the classification for protection between the vector groups.
[000100] The studies here have tested vaccine vectors that have been compared individually and in combination and demonstrate the usefulness of alternative serotype rAds for use as primate vaccine vectors. The results suggest that these vaccines may be much more useful in a combination of initial vaccination - booster vaccination.
[000101] Because of the high magnitude of antigen-specific responses achieved by the initial vaccination - heterologous booster vaccination, it has been proposed that long-lasting immunity can be optimally achieved by the initial vaccination of rAd with DNA (Santra, et al., ( 2005). J. Virol. 79 (10): 6516-22). Since DNA requires multiple primers and does not induce rapid protection similar to that performed by rAd vectors, initial vaccination - heterologous booster vaccination of rAd can provide an optimal opportunity to generate a balance between induction of rapid protective immunity and long-term protective immunity duration.
[000102] It is understood that the examples and modalities described here are for illustrative purposes only and that various modifications or changes in the light of them will be suggested by those skilled in the art and are to be included within the spirit and scope of this application and the scope of the attached claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

权利要求:
Claims (3)
[0001]
1. First and second recombinant adenovirus vectors, characterized by the fact that each comprises a nucleic acid that encodes an antigenic filovirus protein, in which the first adenovirus vector comprises an adenovirus 26 capsid protein, and in which the second adenovirus vector comprises an adenovirus 35 capsid protein, for use in inducing an immune response in an individual against said filovirus antigen, by administering to the individual, as an initial vaccination, an immunologically effective amount of said first vector of recombinant adenovirus; followed by administration to said individual, as a booster vaccination, an immunologically effective amount of said second adenovirus vector, wherein the filovirus antigenic proteins encoded by the first and second recombinant adenovirus vectors share at least one antigenic determinant, in which the antigenic filovirus proteins are encoded by the polynucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
[0002]
2. Recombinant adenovirus vectors according to claim 1, characterized in that the adenovirus vectors are administered intramuscularly.
[0003]
Recombinant adenovirus vectors according to claim 1 or 2, characterized by the fact that the filovirus antigenic protein is a glycoprotein.

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AU2011343798B2|2016-07-14|

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-14| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-01-21| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-07-21| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US42295410P| true| 2010-12-14|2010-12-14|

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US61/422,954|2010-12-14|

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PCT/US2011/064944|WO2012082918A1|2010-12-14|2011-12-14|Adenovirus serotype 26 and serotype 35 filovirus vaccines|

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