EA045436B1 - ADENOVIRAL VECTORS AND WAYS OF THEIR APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of biotechnology, and more particularly to the field and use associated with adenoviral vectors, such as replication-defective adenoviral vectors for delivering antigens and inducing an immune response in hosts.

BACKGROUND OF THE INVENTION

Recombinant adenovirus vectors are widely used in gene therapy applications and for vaccines. AdV-5 vector vaccines have been shown to induce effective and protective immune responses in a number of different animal models (see, for example, WO 2001/02607; WO 2002/22080; Shiver et al., Nature, 415:331 (2002 ); Letvin et al., Ann. Rev. Immunol. 20:73 (2002); Shiver and Emini, Ann. Rev. Med. 55:355 (2004)). However, the utility of AdV-5 recombinant vector vaccines is likely to be limited by the high seroprevalence of AdV-5-specific neutralizing antibodies (NAbs) in human populations. Studies in mice, rhesus monkeys and humans have shown that the existence of immunity against AdV-5 significantly suppresses the immunogenicity of AdV-5-based vaccines.

One promising strategy to circumvent preexisting immunity in individuals previously infected or treated with the most common human adenovirus, such as AdV-5, involves the development of recombinant vectors based on adenovirus serotypes that are not exposed to such preexisting immune defense mechanisms. One such strategy is based on the use of chimeric adenoviruses, which involve replacing native capsid protein sequences (for example, hexon and/or fiber protein sequences) with capsid protein sequences (for example, hexon and/or fiber protein sequences) from adenoviruses with low (or absent) seroprevalence.

Thus, there is a need in the art for alternative adenoviral vectors that can be produced in large quantities, that do not interfere with pre-existing immune defense mechanisms in the host, but that are nonetheless immunogenic and capable of inducing potent immune responses to antigens encoded by heterologous nucleic acids inserted into the vector.

Brief description of the invention

The present invention provides adenoviral vectors. The adenoviral vector may contain a nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide spanning hexon hypervariable regions comprising an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the adenoviral vector may contain a hexon polypeptide sequence, providing SEQ ID NO: 3 or SEQ ID NO: 4.

In certain embodiments, the adenoviral vector further comprises an E1 deletion. In certain embodiments, the adenoviral vector further comprises an E3 deletion. The adenoviral vector may further comprise human adenovirus-5 (HAdV-5) orf6 E4. The adenoviral vector may, for example, contain a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6.

In certain embodiments, the adenoviral vector further comprises at least one transgene. In certain embodiments, the at least one transgene is located in the E1 deletion region, in the E3 deletion region, and/or in the region adjacent to the right inverted terminal repeat (rITR).

In certain embodiments, the adenoviral vector contains one or more nucleic acid sequences from human adenovirus-26 (Ad26).

The present invention also provides recombinant cells containing the adenoviral vectors described herein. The present invention also provides methods for producing adenoviral vectors. The methods include (a) growing the recombinant cells disclosed herein under conditions to produce an adenoviral vector; and (b) isolating the adenoviral vector from the recombinant cell.

The present invention also provides immunogenic compositions comprising the adenoviral vectors described herein and a pharmaceutically acceptable carrier. The present invention also provides methods for inducing an immune response in a subject in need thereof. The methods involve administering to a subject the immunogenic compositions described herein. The present invention also provides methods for preparing immunogenic compositions, the methods comprising combining the adenoviral vectors described herein with a pharmaceutically acceptable carrier.

Brief description of graphic materials

The foregoing brief description, as well as the following detailed description of preferred embodiments of the present invention, will be better understood when considered in conjunction with the accompanying drawings. It should be understood, however, that the present invention is not limited to the specific embodiments shown in the drawings.

In fig. 1 shows a diagram of substitutions in the hexon sequence for the chimeric hexon containing vectors described herein.

In fig. 1A is a diagram showing the locations in the gene encoding the full-length HAdV-26 hexon (open bar), five hexon gene segments (gray bars), and seven short hypervariable regions (HVRs) (black bars) that were previously replaced from HAdV-5 hexons onto HAdV-48 hexons to produce the chimeric HAdV-5 hexon-containing vector Ad5HVR48(1-7) (Roberts et al., Nature, 441:239-43 (2006)).

In fig. Figure 1B shows the results of partial alignment of the hexon polypeptide sequences of HAdV-26, PtroAdV-1, PtroAdV-12 and PtroAdV-13. The gray bars correspond to the five hexon gene segments that were changed here from HAdV-26 to PtroAdV-1, PtroAdV-12, or PtroAdV-13. Black bars indicate sequences corresponding to the aforementioned previously designated HVRs, which were changed from HAdV-5 to HAdV-48.

In fig. Figure 2 shows a diagram of pAd26 chimeric vectors.

In fig. 2A shows a diagram of the common elements of pAd26.HVRPtr12.luc (SEQ ID NO: 21).

In fig. 2B shows a diagram of the common elements of pAd26.HVRPtr13.luc (SEQ ID NO: 22).

In fig. 3 shows a diagram of the common elements of pAd26.ApoA1.RSVF-2A-GLuc (SEQ ID NO: 29).

In fig. Figure 4 shows a diagram of the homologous recombination strategy that was used to create the adenoviral vectors Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc (in E1 complement cells).

In fig. Figure 5 shows the cellular and humoral immune responses induced by Ad26HVRPtr12.FLuc and Ad26HVRPtr13.FLuc.

In fig. Figure 5A shows the experimental design.

In fig. 5B shows a graph of the immune response induced by Ad26.FLuc, Ad26HVRPtr12.FLuc and Ad26HVRPtr13.FLuc against a vector-encoded antigen (ie, Fluc, firefly luciferase) as determined by interferon gamma (IFN-γ) ELISPOT assay. The y-axis shows the number of spot-forming units (SFU) per 10 splenocytes, and the dotted line indicates the 95% percentile value for environmental stimulants.

In fig. Figure 6 shows the cellular and humoral immune responses induced by Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc.

In fig. Figure 6A shows the experimental design.

In fig. 6B shows the results of an RSV A2 virus neutralization assay (VNA) performed eight weeks after immunization with Ad26.RSVF-2A-GLuc, Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc.

In fig. 6C shows the cellular immune response induced by Ad26.RSVF-2A-GLuc, Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc against vector-encoded RSV F antigen as determined by ELISPOT assay using IFN-γ . The y-axis shows the number of spot-forming units (SFU) per 10 splenocytes, and the dotted line indicates the 95% percentile value for environmental stimulants.

In fig. 6D shows a plot of RSVF-specific IgG binding antibodies induced by Ad26.RSVF-2A-GLuc, Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc in the serum of immunized mice 8 weeks after immunization. The graph shows IgG ELISA titers calculated as final titers (log ₁₀ ).

In fig. Figure 7 shows the neutralization titers of homologous and heterologous adenoviruses induced in mice immunized with adenoviral vectors Ad35, Ad26, Ad5, Ad4, Ad26HVRPtr12 and Ad26HVRPtr13.

In fig. Figure 8 shows the seroprevalence for Ad26, Ad5, Ad26HVRPtr12 and Ad26HVRPtr13 in serum samples obtained from a cohort of 200 adults aged 18 to 55 years living in the United States (US) and the European Union (EU). Neutralization titers measured in these serum samples for each vector were divided into four categories (<16 (no neutralization), 16 to 300, 300 to 1000, 1000 to 4000, and >4000) presented in the charts indicated.

In fig. Figure 9 shows the productivity against the new chimeric capsid-containing vectors AdHVRPtr12.FLuc and Ad26HVRPtr13.FLuc in the sPER.C6 producer cell line.

Detailed Description of the Invention

The present invention is based, at least in part, on the creation of chimeric adenovirus vectors containing a human adenovirus backbone and at least one of the chimeric hexon or fiber polypeptide sequences. Adenoviral vectors are capable of inducing an immune response while maintaining low seroprevalence. Adenoviral vectors can be formulated to produce vaccines and used to induce protective immunity against specific antigens of interest.

Various publications, articles and patents are cited or described in the Background section and throughout the specification; each of these references being incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, products, and the like that have been included herein is intended to provide context for the present invention. Such discussion is not an admission that any or all of this material is part of the prior art with respect to any disclosed or claimed inventions.

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

It should be noted that as used herein and in the accompanying claims, the singular number includes a reference to the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values, such as concentration or concentration range, described herein are in all cases to be understood as modified by the term approximately. Thus, the numerical value typically includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes all values in the range from 0.9 to 1.1 mg/ml. Likewise, the concentration range from 1 to 10% (w/v) includes the range from 0.9 to 11% (w/v). As used herein, the use of a numeric range expressly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractional values, unless the context explicitly states otherwise.

Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will be aware of, or can ascertain by routine experimentation, many equivalents to specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the present invention.

As used herein, the terms provides, provides, includes, comprises, has, has, contains, contains, or any other variations thereof are intended to mean that they include the specified integer or group of integers, but do not exclude any other integer or group of integers numbers and are intended to be non-exclusive or non-limiting. For example, a composition, mixture, process, method, article or device containing a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or integral to such composition, mixture, process, method, article or device. Also, unless explicitly stated otherwise, or refers to an inclusive or rather than an exclusive or. For example, the condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present) ).

As used herein, a connecting term and/or between multiple listed elements is understood to include both individual and combined options. For example, when two elements are connected and/or, the first option refers to the applicability of the first element without the second. The second option refers to the applicability of the second element without the first. The third option relates to the applicability of the first and second elements together. Any of these options is understood to fall within this meaning and, therefore, satisfy the requirement of the term and/or used herein. It is clear that the simultaneous applicability of more than one of the options is understood as falling within the given meaning and, therefore, satisfying the requirement of the term and/or.

As used herein, the term consists of, or variations such as consist of or consisting of, as used throughout the specification or claims, indicate the inclusion of any of the given integers or group of integers, but wherein an additional integer or group integers cannot be added to the specified method, structure, or composition.

As used herein, the term essentially consists of, or variations such as essentially consist of or essentially consisting of, as used throughout the specification or claims, indicate the inclusion of any of the given integers or group of integers and the optional inclusion any given integer or group of integers that does not significantly change the basic or new features of the specified method, structure or composition. See M.R.E.R. § 2111.03.

In this context, subject means any animal, preferably a mammal, most preferably a human, that will be vaccinated or has been vaccinated according to the method in accordance with an embodiment of the present invention. As used herein, the term mammal includes 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 humans.

The words right, left, bottom and top indicate the directions in the graphic materials being referenced.

The terms approximately, approximately, generally, essentially and the like when used in this context in relation to the size or property of a component of a preferred invention should be taken to indicate that the size/property described does not constitute a strictly stated limitation or parameter and does not exclude slight deviations therefrom that are functionally identical or similar, as will be understood by one skilled in the art. At a minimum, references to such numerical parameters will include variations that, using mathematical and industrial principles generally accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), will not alter the same significant figure.

The terms identical or percentage identity in the context of two or more nucleic acid or polypeptide sequences (e.g., hexon and fiber polypeptides and the polynucleotides that encode them) refer to two or more sequences or subsequences that are the same or have a certain percentage of amino acid residues or nucleotides, which are the same, when compared and aligned for maximum agreement, as measured using one of the following sequence comparison algorithms or by visual inspection.

When comparing sequences, usually one 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 the computer, the coordinates of the subsequences are designated, if necessary, and the software parameters of the sequence comparison algorithm are specified. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the specified program parameters.

Optimal sequence alignment for comparison can be carried out, for example, using the local homology search algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), using the Needleman & Wunsch homologous alignment algorithm, J. Mol. Biol. 48:443 (1970), using the Pearson-Lipman similarity search method, 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 Drive, Madison, Wis.) 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) ).

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. The BLAST analysis software is publicly available through the National Center for Biotechnology Information. This algorithm involves initially identifying high similarity score sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T is called the threshold for the similarity index of neighboring words (Altschul et al. supra). These initial matches of neighboring words serve as seeds to begin searches to identify longer HSPs that contain them. Word matches are then extended in both directions along each sequence as much as the cumulative alignment score can be increased.

For nucleotide sequences, aggregate scores are calculated using the parameters M (reward per pair of matching residues; always >0) and N (penalty for mismatched residues; always <0). For amino acid sequences, a substitution matrix is used to calculate the cumulative score. Extension of word matches in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum value achieved; the cumulative score drops to zero or below due to the accumulation of one or more balance alignments with a negative score; or the end of any of the sequences has been reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, BLASTP uses a default word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 comparison matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA , 89:10915 (1989)).

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In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One of the similarity measures produced by the BLAST algorithm is the lowest cumulative probability (P(N)), which indicates the probability with which a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest overall probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptide sequences 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, the polypeptide is usually substantially identical to the second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

As used herein, the term protective immunity or protective immune response means that the vaccinated subject is able to control the infection caused by the pathogen against which the vaccination was administered. The pathogenic agent may, for example, be an antigenic gene product or an antigenic protein or a fragment thereof. Typically, a subject who has developed a protective immune response will develop only mild to moderate clinical symptoms or no symptoms at all. Typically, a subject who has a protective immune response to a particular pathogen or protective immunity to it does not die as a result of infection with the specified pathogen.

The term adjuvant is 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 adenoviral vectors of the present invention.

As used herein, the term antigenic gene product or fragment thereof or antigenic protein may include a bacterial, viral, parasitic or fungal protein or fragment thereof. The antigenic protein or antigenic gene product is preferably capable of eliciting a protective immune response in a host, such as inducing an immune response to a disease or infection (e.g., a bacterial, viral, parasitic or fungal disease or infection) and/or causing the subject to develop immunity to a disease or infection (i.e. vaccination), which protects the subject from disease or infection.

As used herein, the term chimeric means a gene, nucleic acid, protein, peptide or polypeptide that contains two or more genes, nucleic acids, proteins, peptides or polypeptides that are not normally associated with each other. A chimeric gene, nucleic acid, or protein may be a fusion molecule of two or more unrelated sequences (eg, two or more different nucleic acids that encode two or more different proteins). A chimeric gene, nucleic acid, or protein may be a fusion of two or more related sequences (e.g., nucleic acids that encode the same protein but the nucleic acids are derived from different starting material, i.e., one nucleic acid originates from humans and the other nucleic acid originates from apes).

Adenoviral vectors.

Exposure to certain adenoviruses results in the development of immune responses to certain adenoviral serotypes, which may influence the effectiveness of adenoviral vectors. Because human adenovirus infections are common in humans, the prevalence of neutralizing antibodies to human adenoviruses in human populations is high. The presence of such neutralizing antibodies in individuals can be expected to reduce the efficiency of a gene transfer vector based on the human adenovirus backbone. One way to overcome reduced efficacy is to replace epitopes on adenoviral capsid proteins that are targets for neutralizing antibodies. Target sequences on capsid proteins can be replaced by protein sequences from other adenoviruses (eg, simian adenoviruses) that are of low abundance and therefore against which neutralizing antibodies are rarely found in human populations.

Capsid protein refers to a protein on the capsid of an adenovirus or a functional fragment or derivative thereof that is used in determining the serotype and/or tropism of a particular adenovirus. Capsid proteins typically include fiber, penton, and/or hexon proteins. In certain embodiments, the capsid protein is the entire or full-length capsid protein of an adenovirus. In other embodiments, the capsid protein is

- 5 045436 fragment or derivative of the full-length capsid protein of an adenovirus. In certain embodiments, the hexon, penton, and fiber encoded by the adenoviral vector of the present invention are derived from the same or different adenoviruses.

Hexon polypeptide refers to the hexon envelope proteins of the adenovirus, their functional fragments and derivatives.

Fiber polypeptide refers to adenovirus fiber proteins, their functional fragments and derivatives.

One of the targets of neutralizing antibodies to adenoviruses is the major envelope protein, hexon protein. Replacement of the hexon protein or variable sequences in the hexon protein that determine serotype and bind to neutralizing antibodies, hexon protein or variable sequences in the hexon protein from adenoviruses that are rare in the human population may allow the construction of adenoviral vectors that would be less susceptible to neutralization antibodies commonly found in humans.

Hexon hypervariable regions (HVRs) are regions of the hexon polypeptide that exhibit the greatest variability among different adenoviral serotypes. These HVRs are generally thought to correspond to the solvent-exposed surfaces of the hexon protein trimer (in the context of an intact virus particle) and are accordingly expected to act as important determinants of antibody-mediated adenovirus neutralization (Roberts et al., Nature , 441:239-43 (2006)). Therefore, replacing the hexon HVR of a given adenoviral vector with the hexon HVR of an adenovirus with low (or no) seroprevalence in humans represents a possible means of overcoming pre-existing antivector humoral immunity in target human populations. Accordingly, there have been many studies exploring the idea of chimeric hexons, mainly involving the replacement of hexon sequences in HAdV-5-based vectors (Roy et al., J. Virol. 72:6875-9 (1998); Gall et al., J. Virol. 72:10260-4 (1998); Youil et al., Hum. Gene Ther. 13:311-20 (2002); Wu et al. J. Virol. 76:12775-82 (2002); Roy et al., Virology. 333:207-14 (2005); Roberts et al., Nature, 441:239-43 (2006); Bradley et al., J. Virol. 86:1267-72 (2012); Yu et al., Biochem Biophys Res Commun. 421:170-6 (2012); Bruder et al., PLoS One, 7(4):e33920 (2012)).

The second target of neutralizing antibodies to adenoviruses is fiber protein. Replacing the fiber protein with fiber sequences from rare adenoviruses that are of non-human animal origin, more preferably replacing variable sequences in the fiber protein, may also allow the construction of adenoviral vectors that are less susceptible to neutralization by antibodies typically found in humans. Combining the fiber replacements described above with hexon replacements may confer additional resistance to neutralization by antibodies commonly present in human populations.

The present invention provides chimeric adenoviral vectors containing transgenes and chimeric hexon nucleic acid sequences. Adenoviral vectors may, for example, contain a nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide spanning hexon hypervariable regions comprising an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the hexon polypeptide sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4. The adenoviral vector may, for example, contain one or more nucleic acid sequences from human adenovirus-4, human adenovirus-5, human adenovirus-26, or human adenovirus-35. In certain embodiments, the adenoviral vector comprises a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6.

Adenoviral vector refers to a recombinant vector derived from or containing at least a portion of an adenoviral genome.

Typically, the adenoviral vector of the present invention contains the entire genome of a recombinant adenovirus, for example, in a plasmid, cosmid or baculovirus vector. The nucleic acid molecules of the present 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.

One skilled in the art will appreciate that elements derived from multiple serotypes can be combined in a single adenoviral vector, for example based on human or simian adenovirus. Thus, it is possible to obtain a chimeric adenoviral vector that combines the required properties from different serotypes. Thus, in some embodiments, the chimeric adenoviral vector of the present invention may combine the lack of pre-existing immunity to chimeric hexon and/or fiber polypeptide sequences with the high level of antigen delivery and presentation ability of existing adenoviral vectors such as rAd4, rAd5, rAd26 or rAd35.

The advantages of adenoviral vectors for use as vaccines include ease of use, good manufacturability on a large scale, and an excellent safety record based on many years of experience in research, development, manufacturing, and clinical trials with numerous adenoviral vectors that have been reported. Adenoviral vectors that are used as vaccines generally provide a good immune response to the transgene-encoded protein or transgene-encoded antigenic gene product, including a cellular immune response. The adenoviral vector in accordance with the present invention can be based on any type of adenovirus and in some embodiments is based on a human adenovirus, which can belong to any group or serotype. In preferred embodiments, the recombinant adenovirus is based on a human adenovirus from group A, B, C, D, E, F, or G. In other preferred embodiments, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49 or 50. In other embodiments, it is a simian adenovirus, such as a chimpanzee or gorilla adenovirus, which may be of any serotype. In certain embodiments, the recombinant adenovirus is based on chimpanzee adenovirus types 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.

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 human adenovirus serotype 5 (Ad5). In human studies evaluating hepatitis C virus (HCV) vaccine candidates, ChAd3 was shown to be safe and immunogenic (Barnes E, et al., 2012, Science translational medicine, 4:115ra1). It was reported that ChAd3-based vaccines were able to induce an immune response comparable to a vaccine containing a human Ad5-based vector. See, for example, Peruzzi D., et al., 2009, Vaccine, 27:1293-300 and Quinn K.M., et al., 2013, J. Immunol. 190: 2720-35; WO 2005/071093; WO 2011/0130627, etc.

Adenoviral vectors, methods for their construction and methods for their propagation are well known in the art and are described, for example, in US patents No. 5559099, 5837511, 5846782, 5851806, 5994106, 5994128, 5965541, 5981225, 6040174, 6020191 and 6113913 and Thomas Shenk, Adenoviridae and their Replication, M. S. Horwitz, Adenoviruses, chapters 67 and 68 respectively in Virology, W.K. Fields et al., eds., ^3rd ed., Raven Press, Ltd., New York (1996), and other sources cited herein. Typically, the construction of adenoviral vectors involves the use of standard molecular biology techniques, such as those described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, ^2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989 ), Watson et al., Recombinant DNA, ^2nd ed., Scientific American Books (1992) and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995), as well as other sources cited herein document.

In certain embodiments, the adenoviral vector includes an E1 deletion and/or an E3 deletion. A deletion of E1 or E3 may, for example, involve a complete deletion of the gene or a partial deletion, which renders the E1 or E3 gene product functionally defective. Thus, in certain embodiments, the adenovirus is replication defective, for example, because it contains a deletion in the E1 region of the genome. As is known to those skilled in the art, in the case of deletions of essential regions in the adenovirus genome, the functional elements encoded by these regions must be provided in trans, preferably by the producing cell, i.e. if parts or entire sections of E1, E2 and/or E4 are removed from the adenovirus, then they must be present in the producer cell, for example, be integrated into its genome or be in the form of a so-called helper adenovirus or helper plasmids. The adenovirus may also include a deletion in a region of E3 that is not essential for replication and, therefore, such a deletion should not be compensated for. One or more of the E1, E2, E3 and E4 regions can also be inactivated by other means, such as insertion of a transgene of interest (usually linked to a promoter) into the regions to be inactivated.

The producer cell (also sometimes referred to in the art and herein as a 'packaging cell' or 'complementary cell') that can be used can be any producer cell in which the desired adenovirus can be propagated. For example, propagation of vectors based on a recombinant adenovirus is carried out in producer cells that compensate for defects in the adenovirus. Preferably, such producer cells contain in their genome at least an adenovirus E1 sequence, and are thus capable of compensating for the defects of recombinant adenoviruses with a deletion in the E1 region. Any E1-complementary producer cell may be used, such as human retinal cells immortalized with E1, e.g. 911 or PER.C6 cells (see US Pat. No. 5,994,128), E1-transformed amniocytes (see EP Pat. No. 1,230,354) , E1-transformed A549 cells (see, for example, WO 98/39411, US patent No. 5891690), GH329:HeLa (Gao et al., 2000, Hum Gene Ther. 11:213-19), 293, etc. . In certain embodiments, the producer cells are, for example, HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells, or the like. The production of adenoviral vectors in producer cells is described in Kovesdi et al., 2010, Viruses, 2:1681-703.

In certain embodiments, the adenoviral vector is a chimeric

- 7 045436 adenoviral vector containing one or more nucleic acid sequences of a human adenovirus. The human adenovirus nucleic acids may, for example, be selected from human adenovirus-4 (Ad-4), human adenovirus-5 (Ad-5), human adenovirus-26 (Ad-26) or human adenovirus-35 (Ad-35) . In certain embodiments, the E1-deficient adenoviral vector comprises the E4-orf6 coding sequence from human adenovirus Ad5. This allows such adenoviruses to propagate in well-known complementary cell lines that express the E1 genes from Ad5, such as, for example, 293 cells or PER.C6 cells (see, for example, Fallaux et al., 1998, Hum Gene Ther. 9 :1909-17, Havenga et al., 2006, J. Gen. Virol. 87:213543; WO 03/104467, incorporated herein by reference in its entirety).

In certain embodiments, the adenoviral vector contains a transgene. A transgene refers to a heterologous nucleic acid, which is a nucleic acid that is not normally present in the vector, and according to the present invention, the transgene may encode an antigenic gene product or an antigenic protein that elicits an immune response in a subject. For example, a transgene can be introduced into a vector using standard molecular biology techniques. The transgene can, for example, be cloned into the deleted E1 or E3 regions of the adenoviral vector or into the region between the E4 region and the rITR. The transgene is usually operably linked to expression control sequences. In preferred embodiments, the transgene is inserted into a transgene insertion site.

If necessary, the nucleic acid sequence encoding a hexon or fiber in accordance with embodiments of the present invention, and/or a transgene, can be codon optimized to ensure proper expression in the host being treated (eg, a human). Codon optimization is a technology widely used in the art.

The transgene may be under the control of (ie, operably linked to) an adenovirus-derived promoter (eg, a 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. The promoter is preferably located upstream of the gene of interest in the expression cassette.

In preferred embodiments, the adenoviral vector comprises a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6.

Immunogenic compositions.

Immunogenic compositions are compositions containing an immunologically effective amount of purified or partially purified human adenovirus vectors for use in the present invention. These compositions can be formulated as a vaccine (also referred to herein as an immunogenic composition) according to methods well known in the art. Such compositions may include adjuvants to enhance immune responses. The optimal proportions of each component in the composition can be determined using techniques well known to those skilled in the art in light of the present disclosure.

Immunogenic compositions in accordance with embodiments of the present invention can be prepared using methods known to those skilled in the art, taking into account the present disclosure. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petrolatum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included.

The immunogenic compositions used in the present invention may contain adjuvants. Adjuvants suitable for co-administration in accordance with the present invention must be potentially safe, well tolerated and effective in humans, including QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU , TERamide, PSC97B, Adjumer, PG-026, GSK-I, AS01, AS03, AS04, AS15, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum and MF59.

Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines such as interferon alpha, interferon gamma, 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-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12 or nucleic acids encoding them.

The compositions of the present invention may contain a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other 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 specific nature of the carrier or other material may depend on the route of administration, for example, intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (eg, intestinal), intranasal, or intraperitoneal routes.

Method for inducing protective immunity.

- 8 045436

Another general aspect of the present invention relates to a method of inducing an immune response in a subject in need thereof. The methods may, for example, involve administering to the subject a vaccine comprising an adenoviral vector described herein and a pharmaceutically acceptable carrier. The present invention also provides methods for producing a vaccine. The methods involve combining an adenoviral vector described herein with a pharmaceutically acceptable carrier.

In the methods of the present invention, any of the immunogenic compositions in accordance with embodiments of the present invention, including, without limitation, those described herein, can be used as a vaccine.

Administration of immunogenic compositions/vaccines containing vectors is usually carried out intramuscularly or subcutaneously. However, other routes of administration such as intravenous, dermal, intradermal or nasal, etc. may also be contemplated. Intramuscular administration of immunogenic compositions can be carried out using a needle to inject a suspension of the adenoviral vector. An alternative is to use a needle-free injection device to administer the composition (using, for example, Biojector™) or lyophilized powder containing the vaccine.

For intravenous, dermal, subcutaneous or intralesional injection, the vector will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art are quite capable of preparing suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's solution for injection, lactated Ringer's solution for injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included. A time-release formulation can also be used.

Typically, administration will be for prophylaxis to generate an immune response to the antigen of interest (eg, bacterial, viral, parasitic and/or fungal pathogen) prior to infection or development of symptoms. Diseases and disorders that can be treated or prevented in accordance with the present invention include those in which the immune response can play a protective or therapeutic role. In other embodiments, adenoviral vectors can be administered for post-exposure prophylaxis.

Immunogenic compositions containing chimeric vectors based on human adenovirus are administered to a subject, causing an immune response to an antigen of interest in the subject. An amount of the composition sufficient to induce a detectable immune response is defined as an immunologically effective dose or effective amount of the composition. The immunogenic compositions of the present invention can induce both humoral and cellular immune responses. In a typical embodiment, the immune response is a protective immune response.

The actual amount administered, as well as the frequency and duration of administration, will depend on the nature and severity of the phenomenon being treated. Prescribing treatment, e.g. decisions regarding dosage, etc., is the responsibility of general practitioners and other medical practitioners, or the veterinarian in the case of veterinary practice, and will usually take into account the disorder being treated, the condition of the individual patient, the site delivery, route of administration and other factors known to medical practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, ^16th edition, Osol, A. ed., 1980.

Once adenoviral vectors are prepared and such particles are optionally formulated into compositions, the vectors can be administered to an individual, particularly a human or other primate. Administration may be to humans or other mammals, such as mice, rats, hamsters, guinea pigs, rabbits, sheep, goats, pigs, horses, cows, donkeys, monkeys, dogs or cats. Delivery to a non-human mammal may not necessarily be for a therapeutic purpose, but may be for experimental use, for example, in studying the mechanisms of immune responses to adenoviral vectors.

In one exemplary regimen, the adenoviral vector is administered (eg, intramuscularly) in a volume of about 100 μl to about 10 ml, containing concentrations of about 10 ⁴ to 10 ¹² viral particles/ml. Preferably, the adenoviral vector is administered in a volume of 0.1 to 2.0 ml. For example, the adenoviral vector can be administered in a volume of 100 μl, 500 μl, 1 ml, 2 ml. More preferably, the adenoviral vector is administered in a volume of 0.5 ml. Optionally, the adenoviral vector can be administered at a concentration of approximately 10 ⁷ , 10 ⁸ , 109, 10 ¹⁰ , 5 x 10 ¹⁰ , 10 ¹¹ or 10 ¹² ppm/ml. Typically, the adenoviral vector is administered in an amount of from about 109 to about ¹⁰¹² viral particles (vp) to a human subject per administration, more typically in an amount from about ¹⁰¹⁰ to about ¹⁰¹² vp. After the initial vaccination there is a booster injection as described above.

After the initial vaccination, there may be a booster or secondary injection of the vaccine/composition,

- 9 045436 containing the same adenoviral vector encoding the antigen of interest, or a vaccine/composition containing another adenoviral vector encoding the same antigen of interest.

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, may include metal foil or polymer film, such as a blister pack. The kit, package or dispenser may be accompanied by instructions for administration.

The compositions of the present invention can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition being treated.

Embodiments

Embodiment 1 is an adenoviral vector containing a nucleic acid sequence encoding a hexon polypeptide, comprising a polypeptide spanning hexon hypervariable regions, comprising an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2.

Embodiment 2 is the adenoviral vector of Embodiment 1, wherein the hexon polypeptide sequence is SEQ ID NO: 3 or SEQ ID NO: 4.

Embodiment 3 is an adenoviral vector according to embodiment 1 or 2, wherein the adenoviral vector further comprises an E1 deletion.

Embodiment 4 is the adenoviral vector of any one of embodiments 1-3, wherein the adenoviral vector further comprises an E3 deletion.

Embodiment 5 is the adenoviral vector of any one of embodiments 1-4, wherein the adenoviral vector further comprises human adenovirus-5 (HAdV-5) orf6 E4.

Embodiment 6 is an adenoviral vector according to any one of embodiments 1-5, wherein the adenoviral vector comprises a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6.

Embodiment 7 is an adenoviral vector according to any one of embodiments 1-6, wherein the adenoviral vector further comprises at least one transgene.

Embodiment 8 is the adenoviral vector of any one of embodiments 1-7, wherein the transgene is located in the E1 deletion region, in the E3 deletion region, and/or in the region adjacent to the right inverted terminal repeat (rITR).

Embodiment 9 is an adenoviral vector according to any one of embodiments 1-8, wherein the adenoviral vector contains one or more nucleic acid sequences from human adenovirus-26 (Ad26).

Embodiment 10 is a recombinant cell containing the vector of any one of embodiments 1-9.

Embodiment 11 is a method for producing an adenoviral vector, comprising: (a) growing the recombinant cell of Embodiment 10 under conditions to produce the adenoviral vector; and (b) isolating the adenoviral vector from the recombinant cell.

Embodiment 12 is an immunogenic composition comprising the adenoviral vector of any one of embodiments 1-9 and a pharmaceutically acceptable carrier.

Embodiment 13 is a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject the immunogenic composition of Embodiment 12.

Embodiment 14 is a method of producing an immunogenic composition, the method comprising combining the adenoviral vector of any one of embodiments 1-9 with a pharmaceutically acceptable carrier.

Examples

Example 1. Development of adenoviral vectors Ad26HVRPtr1, Ad26HVRPtr12 and Ad26HVRPtr13 containing chimeric hexon.

This example describes the Ad26HVRPtr1, Ad26HVRPtr12 and Ad26HVRPtr13 designs, which are novel HAdV-26-based vectors carrying specific substitutions in the hexon sequence with sequences derived from chimpanzee adenoviruses. These chimeric hexon adenovirus vectors were developed with the goal of creating possible new adenovirus-based (vaccine) vectors that are technologically advanced, serologically distinct from HAdV-26, and against which there is low (or no) pre-existing immunity in human populations.

Ad26HVRPtr1, Ad26HVRPtr12 and Ad26HVRPtr13, containing the genome sequences of the adenoviral vector under SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, were developed as

- 10 045436 containing chimeric hexon versions of the previously described recombinant vector HAdV-26 (WO 2007/104792 A2; Abbink et al., 2007). Thus, these vectors were designed to carry the same E1 deletion, E3 deletion, and E4 orf6 substitution (that of HAdV-5) as previously stated (WO 2007/104792 A2; Abbink et al., 2007 ).

Specific variants of the chimeric hexon containing vectors created and studied in this example, which are described in subsequent examples, are Ad26HVRPtr1.Fluc, Ad26HVRPtr12.Fluc, Ad26HVRPtr13.Fluc, Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2AGLuc, which contain viral genomic the sequences are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. As indicated by their vector names, these vectors were generated to carry, at the location of their E1 deletion, a CMV promoter-driven expression cassette encoding either firefly luciferase (FLuc) or the RSV-Fa2-2A-GLuc chimeric protein (RSVF-2A -GLuc), which is a fusion molecule based on the fusogenic glycoprotein of respiratory syncytial virus strain A2 (RSV-F _2A ), foot-and-mouth disease virus peptide 2A and Gaussia luciferase (GLuc). Both FLuc and RSVF-2A-GLuc expression cassettes are driven by the CMV promoter and carry the SV40 polyadenylation signal. The RSVF-2A-GLuc cassette additionally contains in its 5' untranslated region a sequence providing intron 2 of the human apolipoprotein A1 (ApoA1) gene.

The adenoviral vectors Ad26HVRPtr1, Ad26HVRPtr12 and Ad26HVRPtr13 (containing SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively) were designed to contain chimeric hexon in the sense that certain hexon gene segments in their HAdV-26-based genomes were replaced by corresponding segments of the hexon gene of other adenoviruses. The viruses that served as hexon sequence donors for Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13 were PtroAdV-1, PtroAdV-12, and PtroAdV-13, respectively. These viruses were identified in fecal samples of wild chimpanzees and were classified as human adenovirus species E (HAdV-E) (Wevers et al., J. Virol. 85(20):10774-84 (2011)). Partial hexon-coding gene sequences from these viruses have been made publicly available under GenBank accession numbers JN163971, JN163982, and JN163983, respectively. Considering that the acceptor vector for hexon sequences is based on HAdV-26, i.e. representative of human adenovirus species D (HAdV-D), while the three donor hexon virus sequences belong to HAdV-E, the vectors obtained according to this document are interspecies adenoviral containing chimeric hexon vectors.

The HAdV-26 hexon gene segments that were replaced herein to produce the chimeric hexon vectors Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13 (and their derivatives constructed herein containing transgene expression cassettes) corresponded to nucleotides 18178-18357, 18379-18438, 18 556 -18633, 18685-18723 and 19027-19158 of the complete wild-type HAdV-26 genome deposited under Genbank accession number EF153474 (version 1). These five HAdV-26 hexon gene segments and their corresponding replacement segments derived from PtroAdV-1, PtroAdV-12, or PtroAdV-13 corresponded largely, but not completely, to the hypervariable region (HVR) coding sequences. This is shown in Fig. 1A, where the locations of the five segments, as well as the locations of the previously designated HVRs, are indicated in a schematic representation of the HAdV-26 hexon gene. Moreover, this is illustrated in more detail by the results of amino acid alignments produced using the (partial) hexon polypeptide sequences of HAdV-26, PtroAdV-1, PtroAdV-12 and PtroAdV-13, where specific segments that have been replaced according to this document , are specifically highlighted in parallel with the previously designated HVRs (Fig. 1B).

It should be noted that the five segments of the HAdV-26 hexon gene that were replaced herein to create Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13 did not fully correspond to the sequences providing the hexon HVR that were replaced in previous publications that described containing chimeric hexon HAdV-5-based vectors (Roberts et al., Nature, 441:239-43 (2006); Bradley et al., J. Virol. 86:1267-72 (2012); Yu et al., Biochem. Biophys. Res. Commun. 421:170-6 (2012); Bruder et al., PLoS One. 7(4):e33920 (2012)).

For example, as illustrated in FIG. 1A and 1B, the five hexon gene segments did not exactly match the seven amino acid regions that were previously replaced to create Ad5HVR48(1-7), a chimeric hexon vector containing HAdV-5 and a hexon HVR containing HAdV-48 (Roberts et al., Nature, 441:239-43 (2006)).

The complete nucleotide sequences of the adenoviral vectors Ad26HVRPtr1 Ad26HVRPtr12 and Ad26HVRPtr13 encoding the chimeric hexon genes are given under SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The complete polypeptide sequences of the chimeric hexons for these vectors are given under SEQ ID NO: 12, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Example 2. Molecular construction of plasmids carrying complete genomes of adenoviral vectors Ad26HVRPtr1.Fluc, Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc.

Plasmids containing the vector genome Ad26HVRPtr1, Ad26HVRPtr12 and Ad26HVRPtr13 carrying a promoter-driven FLuc expression cassette in the adenoviral E1 region were constructed from to

- 11 045436 using the same methods and the same strategy as previously described for the creation of the chimeric hexon-containing Fluc encoding vector Ad26.HVR5C (Ma et al., J. Cane. Res. Clin. Oncol. 141(3):419-29 (2015), supplementary Fig. 4). Briefly, the desired changes in the hexon-encoding gene were first introduced in the context of the intermediate hexon gene-transfer shuttle plasmid pHex26-Shuttle.BamHI. This was accomplished using standard gene synthesis and subcloning procedures (conducted through GeneArt (LifeTechnologies, Carlsbad, Calif.)) resulting in modified hexon gene-carrying shuttle plasmids carrying the aforementioned chimeric hexon gene encoding sequences listed under SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 15. The genes encoding the chimeric hexons were then transferred from the hexon gene transfer shuttle plasmids into pAd26.luc.dH by homologous recombination in E. coli BJ5183 (Stratagene/Agilent Technologies, Santa Clara, CA). , a plasmid that carries between two PacI restriction sites the gene of a recombinant HAdV-26-based hexon gene deletion vector, equipped at the location of the E1 deletion therein with a CMV promoter-driven expression cassette encoding Fluc. The above molecular cloning procedures resulted in the creation of plasmids pAd26.HVRPtr1.luc (SEQ ID NO: 20), pAd26.HVRPtr12.luc (SEQ ID NO: 21; Fig. 2A), and pAd26.HVRPtr13.luc (SEQ ID NO: 22; Fig. 2B).

Also, three plasmids encoding matching adenoviral vectors containing the chimeric hexon were constructed exactly as described above. In these three plasmids, named pAd26.HVR5.luc (SEQ ID NO: 17), pAd26.HVR35.luc (SEQ ID NO: 18) and pAd26.HVR52.luc (SEQ ID NO: 19), the above set of five hexon gene segments of HAdV-26 were replaced with the corresponding sets of segments from HAdV-5, HAdV-35 and HAdV-52, respectively. These plasmids contained the nucleotide sequences of the chimeric hexon genes set forth in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively. These hexon genes encode the chimeric hexon polypeptide sequences set forth in SEQ ID NO: 26, SEQID NO: 27 and SEQ ID NO: 28, respectively.

Example 3. Initial assessment of viability, growth efficiency and productivity of adenoviral vectors Ad26HVRPtr1.Fluc, Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc.

Previous studies have shown that chimeric adenoviral vectors incorporating cross-species adenoviral substitutions in the hexon sequence are often nonviable or may exhibit slower growth kinetics and lower yields (Youil et al., Hum. Gene Ther. 13:311-20 ( 2002);Wu et al., J. Virol. 76:12775-82 (2002); Bradley et al., J. Virol. 86:1267-72 (2012); Bruder et al., PLoS One. 7(4 ):e33920 (2012)). Therefore, new hexon-containing chimeric adenoviral (vaccine) vectors must be tested for basic growth properties, production yield, and particle quality.

Chimeric hexon adenoviral vectors designed and constructed herein were evaluated for viability, growth efficiency, productivity, and particle infectivity compared to their parent HAdV-26 vector. For this purpose, adenoviral vectors Ad26HVRPtr1.Fluc, Ad26HVRPtr12.Fluc, and Ad26HVRPtr13.Fluc, as well as reference vectors Ad26HVR5.Fluc, Ad26HVR35.Fluc, and Ad26HVR52.Fluc, were created by transfection in accordance with standard procedures using the transfection-facilitating reagent Lipofectamine (Invitrogen; Carlsbad , California), corresponding to the Ad vector genome-carrying plasmids that are described in Example 2 (i.e. pAd26.HVRPtr1.luc, pAd26.HVRPtr12.luc, pAd26.HVRPtr13.luc, pAd26.HVR5, pAd26.HVR35 and pAd26.HVR52 respectively), E1-complementing PER.55K cells (Vogels et al., J. Virol. 77:8263-71 (2003)), which were cultured in T25 flasks. Before transfections, plasmids carrying the Ad vector genome were digested with PacI, releasing the corresponding adenoviral vector genomes from the plasmid. Cultures of transfected cells were monitored daily to record the day the first viral plaque began to form, as well as the day on which the overall cytopathic effect (CPE) was achieved (see table). At complete CPE, infected cells and media were collected and virus was released through three freeze-thaw cycles. After collection of transfection-rescued viruses, they were further propagated by several successive rounds of infection in E1-complementing cell cultures. Viruses were then isolated from the crude viral biomass (using a two-step cesium chloride (CsCl) density gradient ultracentrifugation procedure) and titers of viral particles (vp) and infectious units (IU/ml) were determined, all using standard methods , which were described earlier (Alba R., Baker A.N., Nicklin S.A. Vector systems for prenatal gene therapy: principles of adenovirus design and production. Methods Mol. Biol. 2012; 891:55-84).

- 12 045436

Rescue efficiencies, final production yields, and h.p./IE ratios observed for chimeric hexon-containing adenoviral vectors

Vector HAdV species that are HVR donors Rescue efficiency virus Characteristics of the purified batch All viral plaques are formed 1st viral plaque (days and. T.) Total quantities about CPE (days etc.) T.) Total output for 25 flasks T150 (v.h.) Attitude c. h. to NOT Ad26.FLuc n.a. Yes 3-5 7-8 Ι,ΙΟχΙΟ ¹⁰ 337 Ad26HVR5.FLuc C Yes 5 eleven 1.05x10 4000 Ad26HVR35.FLuc IN Yes 5 12 1.75χ10 ^1ζ 829 Ad26HVR52.FLuc G No - - - - Ad26HVRPtrl.FLuc E Yes 3 9 3.78x10 1029 Ad26HVRPtrl2.FLuc E Yes 4 8 2.16χ10 ^1ζ 272 Ad26HVRPtrl3.FLuc E Yes 3 7 1.07χ10 ¹⁰ 150

n.a. - not applicable;

p.t. - after transfection.

Of the six chimeric vectors tested, only Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc produced results indicating that modifications to their capsid did not negatively affect vector productivity and infectivity (see table). Viral rescue and growth efficiencies for these two vectors, expressed in terms of the time to onset of plaque formation and the time required to achieve complete CPE (after transfection of viral DNA into E1 complement cells), were in the range of values observed for the parental vector Ad26.Fluc. This was not observed for the remaining chimeric vectors tested, with the exception of Ad26HVRPtr1.Fluc. Moreover, of all the vectors tested, Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc provided the highest yields of viral particles (vp) for large-scale production and purification. Finally, although all other chimeric vectors exhibited higher hs:IE ratios than the parental Ad26.Fluc, Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc were found to have no change in hs:IE ratios.

The four remaining chimeric vectors, i.e. Ad26HVR5.Fluc, Ad26HVR35.Fluc, Ad26HVR52.Fluc, and Ad26HVRPtr1.Fluc, showed varying degrees of reduced productivity and/or infectivity. Ad26HVR52.Fluc was not viable at all (i.e., viral plaques could not be detected after transfection of viral DNA), whereas the other three vectors were successfully rescued. Of these three, Ad26HVR5.Fluc and Ad26HVR35.Fluc clearly exhibited delayed rescue and growth kinetics, whereas Ad26HVRPtr1.Fluc appeared to have approximately the same rescue and growth efficiency as the parent vector. Characterization of purified vector batches revealed that the physical yield of viral particles was particularly affected in the case of Ad26HVR5.Fluc and Ad26HVRPtr1.Fluc, while particle infectivity was greatly reduced in all three of them (as indicated by a higher h.p.:IE ratio observed for these vectors).

In conclusion, it should be noted that Ad26HVRPtr12 and Ad26HVRPtr13, which are chimeric hexon vectors involving interspecies adenoviral substitutions in the hexon sequence, showed good growth and production properties and are therefore considered as promising candidates for the role of new vaccine vectors (from the point of view of manufacturability ). The four remaining chimeric hexon vectors, which were created using the same chimeric hexon design but using a different adenovirus as the donor hexon sequence, exhibited less favorable properties.

Example 4. Creation of adenoviral vectors Ad26HVRPtr12.Fluc and Ad26HVRPtr13.Fluc.

This example describes the construction of chimeric hexon-containing Fluc-encoding adenoviral vectors used in the immunogenicity, seroprevalence, cross-neutralization, and manufacturability experiments described in Examples 6, 8, and 9.

The adenoviral vectors Ad26HVRPtr12.Fluc (also designated Ad26C4NVT005) and Ad26HVRPtr13.Fluc (also designated Ad26C3NVT005), which contained the adenoviral vector genome sequences SEQ ID NO: 8 and SEQ ID NO: 9, respectively, were prepared by transfecting the corresponding plasmids with the Ad vector genome ( i.e. pAd26.HVRPtr12.luc and pAd26.HVRPtr13.luc, respectively) E1-complementing PER.C6 cells. Before transfection of PER.C6 cells, which were grown as adherent cell cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 mM MgCl ₂ , Ad vector genome plasmids were digested with PacI to releasing the corresponding genomes of adenoviral vectors from the plasmid. Transfections were performed according to standard procedures using Lipofectamine transfection reagent (Invitrogen; Carlsbad, CA). After collecting the viruses rescued by transfection, the viruses were further propagated using

- 13 045436 several successive cycles of infection on PER.C6 cell cultures. Viruses were purified from collected crude viral biomass using a two-step cesium chloride (CsCl) density gradient ultracentrifugation procedure as previously described (Havenga et al., Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells , J Gen Virol 87(8):2135-43 (2006). Viral particle titers (vp) were measured using a previously described spectrophotometric procedure (Maizel et al., The polypeptides of adenovirus: I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12, Virology , 36(1): 115-25 (1968)).

Example 5. Creation of adenoviral vectors Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc.

This example describes the construction of chimeric hexon-encoding RSVF-2A-GLuc adenoviral vectors used in the immunogenicity experiments described in Example 7.

The creation of the adenoviral vectors Ad26HVRPtr12.RSVF-2A-GLuc (also designated Ad26C4NVT001) and Ad26HVRPtr13.RSVF-2A-Gluc (also designated Ad26C3NVT001), which contained the adenoviral vector genome sequences SEQ ID NO: 10 and SEQ ID NO: 11, respectively, involved the use the aforementioned plasmids pAd26.HVRPtr12.luc (SEQ ID NO: 21; Fig. 2A) and pAd26.HVRPtr13.luc (SEQ ID NO: 22; Fig. 2B), respectively, as well as the plasmid pAdApt26.ApoAI.RSVF-2A-GLuc ( SEQ ID NO: 29; Fig. 3).

pAdApt26.ApoAI.RSVF-2A-GLuc is a plasmid carrying a fragment of the left edge of the genome from the E1 deletion vector based on HAdV-26, which was described previously (WO 2007/104792 A2; Abbink et al., 2007), which in at the location of the adenoviral deletion, E1 further contains the above-mentioned transgene expression cassette encoding RSV-Fa2-2A-GLuc (RSVF-2A-GLuc). pAdApt26.ApoAI.RSVF-2A-GLuc was constructed through several steps of standard gene synthesis and molecular cloning, which together led to the creation of the indicated RSVF-2A-GLuc cassette and its insertion into pAdApt26, a previously described plasmid carrying the indicated fragment of the left edge of the Ad-genome vector (WO 2007/104792 A2; Abbink et al., 2007).

Adenoviral vectors Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc were created as described below. Plasmids pAd26.HVRPtr12.luc and pAd26.HVRPtr13.luc were digested with restriction enzymes PacI and PsiI to release from these plasmids a specific 28-kb left-deleted fragment of the adenoviral vector genome containing the chimeric hexon sequence. Each of the resulting corresponding cleavage products was separately transfected with PacI-digested pAdApt26.ApoAI.RSVF-2A-GLuc into E1-complementing PER.C6 cells to allow rescue of the corresponding chimeric hexon-containing viruses encoding RSVF-2A-Gluc by homologous recombination between overlapping restriction fragments of the vector genome, as shown in FIG. 4. According to this strategy, homologous recombination occurs in a 2.7 kb region, which represents the overlap between the 6.7 kb Pad-Pad restriction fragment. in pAdApt26.ApoAI.RSVF-2A-GLuc (Fig. 4, top) and a 28-kb PsiI-PacI restriction fragment. in pAd26.HVRPtr12.luc or pAd26.HVRPtr13.luc (Fig. 4, bottom). Transfections were performed according to standard procedures using Lipofectamine transfection reagent (Invitrogen; Carlsbad, CA). Individual isolated plaques due to the two rescued viruses Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-Gluc were further propagated on PER.C6 cells and then purified and titrated as described herein for the Ad26HVRPtr12.FLuc and vectors. Ad26HVRPtr13.Fluc in example 4.

Cellular and humoral immune responses induced by a new adenoviral vector.

Examples 6 and 7 describe experiments performed to evaluate the immunogenicity of the new adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 prepared in accordance with this document. In these experiments, new vectors were evaluated for their ability to induce humoral and cellular immune responses to vector-encoded (model) antigens in mice after intramuscular immunization. The vectors were tested with two different antigens: firefly luciferase (FLuc) and RSV-F _A2 -2A-GLuc (RSVF-2A-GLuc). RSVF-2A-GLuc is a chimeric protein consisting of the fusogenic glycoprotein of respiratory syncytial virus strain A2, foot-and-mouth disease virus peptide 2A, and Gaussia luciferase (GLuc). Each vector was compared in parallel to a human adenovirus type 26 (HAdV-26, also referred to herein as Ad26) reference vector carrying the same antigen-encoding transgene cassette. Immune responses to the respective antigens were measured using well-known immunoassays such as enzyme-linked immunosorbent spot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA) and, in the case of the RSVF-2A-GLuc antigen, respiratory syncytial virus neutralization assay (VNA).

Example 6 Cellular immune responses induced by Ad26HVRPtr12.FLuc and Ad26HVRPtr13.FLuc.

To evaluate the cellular immunogenicity of the new adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13, Balb/C mice were immunized intramuscularly with the Ad26.FLuc (positive control), Ad26HVRPtr12 and Ad26HVRPtr13 vectors expressing FLuc

- 14 045436 (i.e. Ad26HVRPtr12.FLuc and Ad26HVRPtr13.FLuc) or via an adenovector that did not encode FLuc, i.e. empty Ad26. FLuc expression vectors were tested at 10 ⁹ and 10 ¹⁰ viral particles (vp) per mouse, and the empty Ad26 vector was administered at 10 ¹⁰ vp. Two weeks after immunization, mice were sacrificed and splenocytes were isolated (Fig. 5A). Cellular immune responses were determined by an ex-vivo ELISPOT assay, measuring the relative abundance of IFN-γ-secreting cells after stimulation of splenocytes overnight with a pool of 15-mer overlapping FLuc peptides (Fig. 5B). The results showed that when immunized at higher doses (10 ¹⁰ ), the cellular immune responses induced by the Ad26HVRPtr12 and Ad26HVRPtr13 vectors were in the same range of values or exceeded the level of response observed in the case of Ad26.Fluc.

Overall, the cellular immune responses induced by the FLuc-expressing recombinant adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 of the present invention clearly indicated the strong immunogenicity of these vectors in mice.

Example 7 Cellular and humoral immune responses induced by Ad26HVRPtr12.RSVF-2AGLuc and Ad26HVRPtr13.RSVF-2A-GLuc.

The immunogenicity of the novel adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 was further assessed using RSV-Fa2-2A-GLuc (RSVF-2A-GLuc) as the vector-encoded (model) vaccine antigen. Balb/C mice were immunized intramuscularly with Ad26.RSVF-2A-GLuc (positive control), Ad26HVRPtr12.RSVF-2A-GLuc, or Ad26HVRPtr13.RSVF-2A-GLuc at three different concentrations (10 ⁸ each). ⁹ or 10 ¹⁰ w.p. per mouse) or through Ad26.FLuc, Ad26HVRPtr12.FLuc or Ad26HVRPtr13.FLuc in the amount of 10 ¹⁰ w.p. on the mouse). Eight weeks after immunization, mice were sacrificed and blood samples and splenocytes were collected (Fig. 6A). Various immune parameters were assessed as described below.

A virus neutralization assay (VNA) was performed to evaluate the ability of Ad26HVRPtr12.RSVF-2AGLuc and Ad26HVRPtr13.RSVF-2A-GLuc to induce the production of respiratory syncytial virus neutralizing antibodies. In fig. 6B shows VNA titers for respiratory syncytial virus strain A2 (RSV A2) measured from serum samples collected eight weeks after immunization. Each dot represents one mouse; bars represent the group mean and the dotted line represents the lower limit of quantitation (LLOQ=6.88; mean final titer of linear samples). The results show that immunization with a dose of 10 ¹⁰ w.p. Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc resulted in RSV A2 neutralization titers similar to those determined for the reference vector Ad26 encoding the same antigen. Titers induced by all three vectors, Ad26, Ad26HVRPtr12 and Ad26HVRPtr13, encoding RSVF-2A-GLuc, were detected predominantly at the highest dose used for immunization, which was 10 ¹⁰ v.p. As expected, no RSV A2-specific responses were detected to the corresponding adenovectors encoding firefly luciferase.

Induction of cellular immunity to vector-encoded antigen was assessed using an RSV-FA2-specific ELISPOT assay. To this end, eight weeks after immunization, splenocytes were isolated from immunized mice and stimulated overnight with 15-mer overlapping peptides spanning the RSV-F _A2 protein, and cellular immune responses were determined using an ex vivo ELISPOT assay, measuring the relative amount of secreting IFN-γ γ cells. The data shows that antigen-specific cellular immune responses induced by the new vectors Ad26HVRPtr12 and Ad26HVRPtr13, encoding RSVF-2A-GLuc, were dose-dependent and for each dose, respectively, were higher and similar in magnitude to the responses induced by the reference vector Ad26.RSVF-2A- GLuc (Fig. 6C). As expected, the measurements did not detect RSVF-FA2-specific responses among splenocytes from mice immunized with adenovectors encoding firefly luciferase.

The ability of RSVF-2A-GLuc expression vectors to induce the production of RSV-FA2-specific IgG antibodies was assessed by ELISA. Serum collected 8 weeks postimmunization from mice immunized with Ad26 (positive control), Ad26HVRPtr12, Ad26HVRPtr13 expressing the RSVF-2A-GLuc transgene or firefly luciferase (control) was tested by ELISA for anti-RSV-FA2 IgG. Specifically, this ELISA detected IgG antibodies capable of binding to the recombinant stable RSV-F protein RSV-FA2 in the pre-fusion conformation (pre-RSV-F). The results show that Ad26HVRPtr12.RSVF-2A-GLuc and Ad26HVRPtr13.RSVF-2A-GLuc dose-dependently induced titers of pre-RSV-F specific IgG antibodies similar to those induced by Ad26.RSVF-2A- GLuc (Fig. 6D). As expected, titers of antibodies specific to RSV-FA2 were not detected in the serum of mice immunized with vectors encoding only firefly luciferase. The graph shows IgG ELISA titers calculated as final titers (log ₁₀ ). Each dot represents one mouse; bars indicate group mean and dotted line indicates lower limit of quantitation

- 15 045436 (LLOQ), calculated as log ₁₀ 1.36.

Overall, the data show that the novel adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 induced strong cellular and humoral immune responses to the encoded antigens that were similar in magnitude or higher than those induced by the reference HAdV-26 vector. These immune responses clearly indicated the strong immunogenicity of the Ad26HVRPtr12 and Ad26HVRPtr13 adenoviral vectors in mice.

Example 8. Evaluation of serological cross-neutralization among new and existing adenoviral vectors.

To ensure their potential usefulness as new adenoviral vaccine vectors, the new adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 created in accordance with this document will preferably be serologically different from existing adenoviral vectors that are currently in development as vaccine vectors, such as based on human adenovirus serotypes HAdV-5 and HAdV-35. Therefore, cross-neutralization tests were performed among the new adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 and several existing vectors based on HAdV-4, HAdV-5, HAdV-26 and HAdV-35. To this end, mouse antisera, each raised in response to one of these adenoviral vectors, were tested against each of these different vectors in an adenovirus neutralization assay. The mouse antiserum used for this assay was collected from Balb/C mice two to eight weeks after their immunization with 10 ¹⁰ vector particles per mouse. The adenovirus neutralization assay was performed as previously described (Spangers et al., 2003. J. Clin. Microbiol. 41:5046-5052). Briefly, starting at a 1:16 dilution, serum was serially diluted 2-fold, then premixed with adenoviral vectors expressing firefly luciferase (FLuc) and then incubated overnight with A549 cells (at a multiplicity of infection of 500 viral particles per cell). Levels of luciferase activity in infected cell lysates measured 24 h postinfection represented the efficiency of vector infection. Neutralization titers for a given vector were determined as the highest serum dilution capable of reducing the efficiency of infection by the vector by 90%. Neutralization titers were roughly divided into the following categories: <16 (no neutralization), 16–200, 200–2000, and >2000.

The results demonstrated the absence or presence of very low levels of cross-neutralization among the vectors tested (Fig. 7). Minor cross-neutralization was observed for Ad26HVRPtr12 with vectors Ad26 and Ad26HVRPtr13 and for Ad26HVRPtr13 with vectors Ad26 and Ad26HVRPtr12. The reciprocal cross-neutralization titers observed for these vectors were significantly lower than the corresponding homologous neutralization titers obtained for the same vectors. It is important to note that the new vectors Ad26HVRPtr12 and Ad26HVRPtr13 did not demonstrate cross-neutralization with the human adenovirus vectors included in the tested panel, i.e. Ad35, Ad5 and Ad4, with the exception of Ad26, for which cross-neutralization was observed at a very low level. Therefore, each of the new adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 can potentially be used in combination with one or more of these or other individual adenoviral vectors in sequential immunizations, for example, in the context of a heterologous prime-boost vaccination regimen or, alternatively or additionally, in the context of a two-part series or more sequential vaccination regimens against different diseases or antigens.

Example 9. Seroprevalence of new adenoviral vectors in human populations.

Important for their potential use as effective vaccine vectors is that the novel adenoviral vectors described herein are not inhibited by high levels of pre-existing anti-vector humoral immunity in vaccine target populations. For this reason, each of the Ad26HVRPtr12 and Ad26HVRPtr13 vectors was assessed for its seroprevalence in serum samples obtained from a cohort of 200 adults aged 18 to 55 years living in the United States (US) and the European Union (EU). The vectors were tested for neutralization by human serum samples using the standard adenovirus neutralization assay performed in Example 7 and which has been described previously (Spangers et al., 2003. J. Clin. Microbiol. 41:5046-5052). Briefly, starting at a 1:16 dilution, serum was serially diluted 2-fold, then premixed with adenoviral vectors expressing firefly luciferase (FLuc) and then incubated overnight with A549 cells (at a multiplicity of infection of 500 viral particles per cell). Levels of luciferase activity in infected cell lysates measured 24 h postinfection represented the efficiency of vector infection. Neutralization titers for a given vector were determined as the highest serum dilution capable of reducing the efficiency of infection by the vector by 90%. Neutralization titers were roughly divided into the following categories: <16 (no neutralization), 16–300, 300–1000, 1000–4000, and >4000.

The results show that the adenoviral vectors Ad26HVRPtr12 and Ad26HVRPtr13 are characterized by

- 16 045436 had a significantly lower seroprevalence in the human subjects studied than the reference vector Ad5, and a similar seroprevalence to the reference vector Ad26 in these subjects (Fig. 8). Moreover, the positive neutralization titers observed for the new vectors Ad26HVRPtr12 and Ad26HVRPtr13 were generally quite low, generally not exceeding 300. In contrast, the majority of positive neutralization titers observed for both Ad26 and Ad5 were greater than 300 .

Overall, the above data indicated that pre-existing humoral anti-vector immunity to the Ad26HVRPtr12 and Ad26HVRPtr13 vectors could be considered low in the vaccine target populations assessed, suggesting that these vectors have the potential to be effective vaccine vectors in these populations.

Example 10. Productivity of an adenoviral vector in a PER.C6 cell suspension.

Adenoviral vectors for use in clinical trials and beyond must be capable of easy production of high titers in a scalable, serum-free adenovirus production platform. Such a platform is suspension-adapted PER.C6® cells, also referred to herein as suspended PER.C6 or SPER.C6 cells, as they have been shown to support large-scale production of adenoviral vectors in bioreactors, providing large quantities of clinical-grade vector-based drugs. high titer, for example, E1 deletion vectors based on HAdV-26 or HAdV-35 (EP 2536829 B1, EP 2350268 B1).

As an initial assessment of whether the novel vectors described herein would be suitable for SPER.C6 cell-based production methods, small-scale vector productivity experiments were performed on SPER.C6 cells cultured in shake flasks. These productivity experiments were performed using Fluc-encoding versions of the new chimeric vectors Ad26HVRPtr12 and Ad26HVRPtr13 (described in Example 4). The HAdV-26-based vector Ad26.Fluc was used as a reference control. A suspension of PER.C6 cell cultures seeded in shake flasks at a density of 1x106 cells/ml in a total volume of 10 ml of PERMEXCIS® medium (available from Lonza) supplemented with 4 mM L-glutamine (Lonza) was infected with different vectors with different ratios of viral particles ( h.p.) per cell and then incubated for 4 days. Various relationships v.ch. per cell used for infection were 70, 150, and 900. Each day, samples of infected cell cultures were collected and i.p. titers were determined. in these samples using a quantitative PCR (qPCR)-based protocol that involved the use of primers and probe specific to the CMV promoter (which was present in all vectors tested). This protocol involved DNase treatment of test samples prior to qPCR to remove any free vector DNA (i.e., vector genomes that were not packaged into viral particles).

The productivity results obtained for the Ad26HVRPtr12.FLuc and Ad26HVRPtr13.Fluc chimeric vectors are shown in FIG. 9. The two chimeric vectors provided h.p. titers that were equivalent to those obtained for the parental reference vector Ad26.Fluc. These results demonstrated good productivity for each of the new chimeric vectors in the SPER.C6-based serum-free suspension culture model.

Taken together, the results of the studies of humoral and cellular immune responses to the novel recombinant adenoviral vectors of the present invention, which are presented above, clearly indicated the strong immunogenicity of these vectors in mice. In addition, the vectors were demonstrated to induce no or only very little cross-neutralizing antibody responses against certain existing adenoviral vaccine candidate vectors (e.g., Ad26 and Ad35) or vice versa, and also induced only very low cross-neutralizing antibody responses. to each other. Moreover, low seroprevalence in humans was observed for the new vectors. Finally, new vectors can be easily produced with high yields. The combination of low seroprevalence, high immunogenicity and productivity suggests that the novel adenoviral vectors of the present invention may be useful as new vaccine vector candidates for various pathogens and may additionally be useful in gene therapy and/or diagnostics.

Those skilled in the art will appreciate that changes may be made to the embodiments described above without departing from their general inventive spirit. Thus, it is to be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the present disclosure.

Claims (9)

1. Adenoviral vector HAdV-26, including: a) deletion of E1 and E3; b) replacement of human adenovirus-5 (HAdV-5) with E4 orf6; c) a sequence encoding a hexon hypervariable region containing SEQ ID NO: 1 or SEQ ID NO: 2, or a hexon consisting of SEQ ID NO: 3 or SEQ ID NO: 4. 2. The adenoviral vector according to claim 1, where the adenoviral vector contains a nucleic acid sequence selected from SEQ ID NO: 5 or SEQ ID NO: 6. 3. Adenoviral vector according to any one of claims 1, 2, where the adenoviral vector additionally contains at least one transgene. 4. Adenoviral vector according to any one of claims 1-3, where the transgene is located in the E1 deletion region, in the E3 deletion region and/or in the region adjacent to the right inverted terminal repeat (rITR). 5. Recombinant cell containing an adenoviral vector according to any one of claims 1-4. 6. A method for producing an adenoviral vector, including: (a) growing a recombinant cell according to claim 5 under conditions ensuring the production of an adenoviral vector; and (b) isolating the adenoviral vector from the recombinant cell. 7. An immunogenic composition containing an adenoviral vector according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 8. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject an immunogenic composition according to claim 7. 9. A method for producing an immunogenic composition, comprising combining an adenoviral vector according to any one of claims 1-4 with a pharmaceutically acceptable carrier.