EA044526B1 - ADENOVIRUS AND WAYS OF ITS APPLICATION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of biotechnology. More particularly, to the field and applications related to 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, WO2001/02607; WO2002/22080; 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 recombinant AdV-5 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 non-human ape adenoviruses, since they generally do not infect humans and have low seroprevalence in human samples. Non-human ape adenoviruses are suitable for use in humans because these viruses have been shown to infect human cells in vitro (WO 2003/000283; WO 2004/037189).

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 isolated nucleic acid sequences encoding hexon polypeptides. In certain embodiments, a hexon polypeptide or a functional derivative thereof provides a polypeptide spanning the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO:1 or an amino acid sequence that is at least 95% identical throughout its entire length to the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the hexon polypeptide comprises a polypeptide spanning the hexon hypervariable regions and characterized by the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the hexon polypeptide or a functional derivative thereof comprises the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2) or the amino acid sequence which is at least 95% identical over its entire length to the amino acid sequence of SEQ ID NO: 2.

The present invention also provides isolated nucleic acid sequences encoding a fiber polypeptide or a functional derivative thereof. In certain embodiments, the fiber polypeptide comprises at least one of a fiber head polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10; a fiber stalk polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 11; and a fiber tail polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the fiber polypeptide or a functional derivative thereof comprises the amino acid sequence of a BLY6 fiber polypeptide (SEQ ID NO: 3) or an amino acid sequence that is at least 95 % identical over its entire length to the amino acid sequence under SEQ ID NO: 3.

Embodiments of the present invention also provide isolated fiber and hexon polypeptides encoded by the fiber and hexon nucleic acid sequences of the present invention.

Additionally, the present invention provides isolated nucleic acids comprising a hexon nucleic acid sequence encoding at least one of the hexon polypeptides disclosed herein and a fiber nucleic acid sequence encoding at least one of the fiber polypeptides disclosed herein. In certain embodiments, the present invention provides vectors containing isolated nucleic acids described herein. In one embodiment, the vector is a viral vector. In another embodiment, the vector is an expression vector. In one preferred embodiment, the vector is an adenoviral vector. More preferably, the vector further contains a transgene.

The present invention also provides recombinant cells containing the vectors described herein. Such cells can be used to produce recombinant protein, express recombinant protein, or produce vectors or viral particles. The present invention also provides methods for producing a vector. The methods include (a) growing a recombinant cell disclosed herein under conditions that permit production of a vector; and (b) isolating the vector from the recombinant cell.

In certain embodiments, immunogenic compositions are provided containing the vectors disclosed herein. Also provided are methods of inducing an immune response in a subject in need thereof, comprising administering to the subject immunogenic compositions disclosed herein.

In certain embodiments, adenoviral vectors are provided comprising (a) at least one transgene and (b) a nucleic acid sequence encoding a hexon polypeptide in accordance with embodiments of the present invention. A hexon polypeptide may, for example, comprise a polypeptide spanning the hypervariable regions of a hexon characterized by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical throughout its length to the amino acid sequence of SEQ ID NO: 1. Hexon polypeptide may, for example, comprise an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the hexon polypeptide comprises the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2) or an amino acid sequence that is at least 95% identical in its entire length amino acid sequence under SEQ ID NO: 2.

In certain embodiments, adenoviral vectors are provided comprising (a) at least one transgene and (b) a nucleic acid sequence encoding a fiber polypeptide in accordance with embodiments of the present invention. In certain embodiments, the fiber polypeptide may comprise at least one of a fiber head polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10; a fiber stalk polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 11; and a fiber tail polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the fiber polypeptide or a functional derivative thereof comprises the amino acid sequence of a BLY6 fiber polypeptide (SEQ ID NO:3) or an amino acid sequence that is at least 95 % identical over its entire length to the amino acid sequence of SEQ ID NO: 3. Embodiments of the present invention also provide adenoviral vectors containing (a) at least one transgene; (b) a nucleic acid sequence encoding a hexon polypeptide in accordance with embodiments of the present invention; and (c) a nucleic acid sequence encoding a fiber polypeptide in accordance with embodiments of the present invention.

In certain embodiments, the adenoviral vectors provided herein are replication-defective adenoviral (rAd) vectors. In one embodiment, adenoviral vectors may include an E1 deletion. In certain embodiments, adenoviral vectors provided herein may further include an E3 deletion. Adenoviral vectors may be simian adenovirus vectors containing adenoviral nucleic acid sequences from one or more simian adenoviruses (SAdVs), such as chimpanzee adenoviruses (eg, ChAd3); gorilla adenoviruses or rhesus macaque adenoviruses (eg rhAd51, rhAd52 or rhAd53). Adenoviral vectors can be human adenovirus-based vectors containing adenoviral sequences from one or more human adenoviruses (eg, hAdV-4, hAdV-5, hAdV26, hAdV-35). Preferably, the adenoviral vector is a chimeric adenoviral vector containing one or more human adenovirus nucleic acid sequences. Human adenovirus nucleic acid sequences may, for example, be derived from human adenovirus-4 (hAdV-4), human adenovirus-5 (hAdV-5), human adenovirus-26 (hAdV-26) or human adenovirus-35 (hAdV-35) . Adenoviral vectors may, for example, contain orf6 and orf 6/7 E4 of human adenovirus-5 (hAdV-5).

In certain embodiments, the transgene is adjacent to an inverted terminal repeat (ITR). In certain embodiments, the transgene is located in or adjacent to the E1 deletion region, or to the E3 deletion region, and/or in or adjacent to the ITR region, for example, between the E4 region and the right ITR (RITR).

In certain embodiments, the adenoviral vectors provided herein contain the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

- 2 044526

Also provided are immunogenic compositions or vaccines comprising the adenoviral vectors described herein and a pharmaceutically acceptable carrier. Additionally, the present invention provides methods for inducing an immune response in a subject in need thereof. The methods involve administering the vaccines disclosed herein to a subject. Additionally, the present invention provides methods for producing a vaccine. The methods involve combining an adenoviral vector disclosed 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 taken in conjunction with the accompanying drawings. It should be understood, however, that the present application is not limited to the specific embodiments shown in the drawings.

In fig. Figure 1 shows the cellular and humoral immune responses induced by BLY6.FLuc and Ad49.FLuc. In fig. Figure 1A shows the experimental design. In fig. 1B shows the cellular immune response induced by Ad26.FLuc, BLY6.FLuc, and Ad49.FLuc against a vector-encoded antigen (i.e., Fluc, firefly luciferase) as determined by an interferon gamma (IFN-γ) ELISPOT assay. . The y-axis shows the number of spot-forming units (SFU) per 106 splenocytes, and the dotted line indicates the 95% percentile value for environmental stimulants.

In fig. Figure 2 shows the cellular and humoral immune responses induced by BLY6.RSVF-2AGLuc. In fig. Figure 2A shows the experimental design. In fig. 2B shows a graph of the results of a virus neutralization assay (VNA) performed eight weeks after immunization with Ad26.RSVF-2A-GLuc or BLY6.RSVF-2A-GLuc at three different concentrations (10 ⁸ , 109 and 10 ¹⁰ v.p.) or Ad26.FLuc or BLY6.FLuc in the amount of 10 ¹⁰ c. h. In Fig. 2C shows the cellular immune responses induced by Ad26.RSVF-2A-GLuc and BLY6.RSVF-2A-GLuc following immunization as determined by IFN-γ ELISPOT assay. In fig. 2D shows a plot of RSVF-specific IgG binding antibodies induced by Ad26.RSVF-2A-GLuc or BLY6.RSVF-2A-GLuc in the serum of immunized mice 8 weeks after immunization.

In fig. Figure 3 shows the neutralization titers of homologous and heterologous adenoviruses induced in mice immunized with adenoviral vectors Ad35, Ad26, Ad49, Ad5, Ad4 and BLY6.

In fig. Figure 4 shows the seroprevalence for Ad35, Ad26, Ad5, and BLY6 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 (negative), 16 to 300, 300 to 1000, 1000 to 4000, and >4000) presented in the indicated charts.

In fig. 5 shows a diagram of plasmid pBLY6.dE1.dE3 (SEQ ID NO: 8).

In fig. 6 shows a diagram of plasmid pBLY6.dE1.dE3.5IXp (SEQ ID NO: 9).

In fig. 7 shows the results of a sequence alignment between the BLY6 hexon polypeptide (SEQ ID NO: 2) and the SAdV-30-1 hexon polypeptide (SEQ ID NO: 4).

In fig. 8 shows productivity in relation to the new vector

BLY6.Fluc in the sPER.C6 producer cell line.

Detailed Description of the Invention

The present invention is based, at least in part, on the isolation and identification of a new isolate of gorilla adenovirus, classified as adenovirus species E, as well as on the creation and evaluation of vaccine vectors containing nucleic acids encoding the variable regions of the hexon and fiber polypeptides of the specified gorilla adenovirus. Adenoviral vectors are capable of inducing an immune response and, in addition, have a low seroprevalence in humans. 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

- 3 044526 singular forms include 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. Therefore, the numerical value typically includes ±10% of the stated 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% (w/v) 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, having, 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, when using mathematical and industrial principles accepted in the art (for example, rounding, measurement errors or other systematic errors, manufacturing tolerances, etc.), will not change the latter significant figure.

- 4 044526

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, test and reference sequences are entered into the computer, the coordinates of 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 Altschul et al. (1997) 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 similarity between neighboring words (Altschul et al above). 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)).

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 measure of similarity 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.

An additional indication that two nucleic acid sequences or polypeptide sequences are substantially identical is that the polypeptide encoded by

- 5 044526 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.

Adenoviral vectors

Exposure to certain adenoviruses results in the development of immune responses to specific 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 with protein sequences from other adenoviruses that are of low abundance and therefore against which neutralizing antibodies are rare in human populations.

Capsid protein refers to a protein on the capsid of an adenovirus (eg, BLY6, HAdV-4) or a functional fragment or derivative thereof that is involved 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 a fragment or derivative of the full-length capsid protein of an adenovirus. In certain embodiments, the hexon, penton, and fiber encoded by the adenovirus vector of the present invention are derived from the same or different adenoviruses (i.e., BLY6 hexon and BLY6 fiber, BLY6 hexon and human adenovirus fiber, human adenovirus hexon and BLY6 fiber etc.).

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 define the serotype and bind to neutralizing antibodies, the hexon protein or variable sequences in the hexon protein from adenoviruses that are rare in the human population, such as the gorilla adenovirus sequences described herein, may allow the construction adenoviral vectors that would be less susceptible to neutralization by antibodies commonly found in humans.

The second target of neutralizing antibodies to adenoviruses is fiber protein. Replacing fiber protein or variable sequences in fiber protein with fiber protein or variable sequences in fiber protein from adenoviruses that are rare in the human population, such as the gorilla adenovirus sequences described herein, may also allow the construction of adenoviral vectors that would be less susceptible to neutralization of anti- 6 044526 bodies commonly 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 isolated nucleic acid sequences encoding hexon polypeptides and/or fiber polypeptides derived from an isolated simian adenovirus serotype, and adenoviral vectors containing at least one of the isolated nucleic acid sequences.

A functional derivative of a polypeptide preferably refers to a modified version of the polypeptide, for example, where one or more amino acids of the polypeptide may be deleted, inserted, modified and/or replaced. A derivative of an unmodified adenoviral capsid protein is considered functional if, for example:

(a) an adenovirus containing a derived capsid protein in its capsid maintains substantially the same or lower seroprevalence compared to an adenovirus containing an unmodified capsid protein; and/or (b) an adenovirus containing a derived capsid protein in its capsid retains substantially the same or greater infectivity against a host cell compared to an adenovirus containing an unmodified capsid protein; and/or (c) an adenovirus containing a derived capsid protein in its capsid retains substantially the same or greater immunogenicity compared to an adenovirus containing an unmodified capsid protein; and/or (d) an adenovirus containing a derived capsid protein in its capsid retains substantially the same or higher level of enabling transgene production compared to an adenovirus containing an unmodified capsid protein.

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

In preferred embodiments, the isolated nucleic acid sequences encode hexon polypeptides or functional derivatives thereof comprising a polypeptide spanning the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical throughout its entire length to the amino acid sequences of SEQ ID NO: 1. In certain embodiments, the hexon polypeptides comprise a polypeptide spanning the hypervariable regions of a hexon characterized by the amino acid sequence of SEQ ID NO: 1. In preferred embodiments, the hexon polypeptide or a functional derivative thereof comprises the amino acid sequence of a hexon BLY6 polypeptide (SEQ ID NO: 2) or an amino acid sequence that is at least 95% identical throughout its entire length to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, a hexon polypeptide or a functional derivative thereof comprises an amino acid sequence that is at least 95% identical %, at least 96%, at least 97%, at least 98%, or at least 99% identical over its entire length to the amino acid sequence of SEQ ID NO: 2.

In preferred embodiments, the isolated nucleic acid sequences encode fiber polypeptides or functional derivatives thereof. The fiber polypeptide may, for example, comprise at least one of a fiber head polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a fiber stalk polypeptide containing the amino acid sequence of SEQ ID NO: 11; and a fiber tail polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In preferred embodiments, the fiber polypeptide or a functional derivative thereof comprises the amino acid sequence of the BLY6 fiber polypeptide (SEQ ID NO: 3) or an amino acid sequence that is at least 95% identical throughout its entire length to the amino acid sequence under SEQ ID NO: 3.

In preferred embodiments, an isolated nucleic acid is provided comprising a hexon nucleic acid sequence encoding at least one of the hexon polypeptides disclosed herein and a nucleic acid sequence encoding at least one of the fiber polypeptides disclosed herein.

In preferred embodiments, vectors are provided, preferably adenoviral vectors, comprising at least one of an isolated hexon nucleic acid sequence and/or an isolated fiber nucleic acid sequence in accordance with embodiments of the present invention. Adenoviral vectors may, for example, contain at least one transgene and a nucleic acid sequence encoding a hexon polypeptide and/or a fiber polypeptide, wherein the hexon polypeptide comprises a polypeptide comprising the hexon hypervariable regions disclosed herein, and the fiber polypeptide comprises the one described herein herein polypeptide

- 7 044526 fiber.

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. In this way, 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 simian adenovirus 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 used as vaccines generally provide a good immune response to the protein encoded by the transgene, 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 type 1, 3, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50, 67 or SA7P.

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 KM, 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, 60201 91 and 6113913 and Thomas Shenk, Adenoviridae and their Replication, M. S. Horwitz, Adenoviruses, chapters 67 and 68 respectively in Virology, B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996), and other sources mentioned in this document. 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, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d 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 in this 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 position, preferably by the producing cell, i.e. if parts or entire regions of E1, E2 and/or E4 are deleted from an adenovirus, then they must be present in the producing cell, for example, be integrated into its genome or be in the form of a so-called auxiliary adenovirus or auxiliary 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

- 8 044526 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 defects of recombinant adenoviruses with a deletion in the E1 region. Any E1 complementary producing cell may be used, such as human retinal cells immortalized using E1, such as 911 or PER.C6 cells (see US Pat. No. 5,994,128), E1 transformed amniocytes (see EP Pat. No. 1230354), E1-transformed A549 cells (see, for example, WO 98/39411, US patent No. 5891690), GH329:HeLa (Gao et al., 2000, Hum Gene Ther 11: 21319), 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 adenoviral vector comprising one or more human adenovirus nucleic acid sequences. 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 Ad5 adenovirus. 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, nucleic acid sequences encoding hexon and/or fiber polypeptides in accordance with embodiments of the present invention, and/or a transgene, can be codon optimized to ensure proper expression in the treated host (eg, 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 contains the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

Immunogenic compositions

Immunogenic compositions are compositions containing an immunologically effective amount of purified or partially purified human or simian (eg, gorilla) 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.

- 9 044526

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-I, 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

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 human or simian (eg, gorilla) adenovirus vectors are administered to a subject, causing an immune response to the 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, such as 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 of 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.

- 10 044526

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 can be to humans or other mammal, for example, mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. 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 v. h./ml, 10 h.h./ml, 10 ⁹ h.h./ml, 10 ¹⁰ h.h./ml, 5x10 ¹⁰ h. h./ml, 10 ¹¹ h.h./ml or 10 ¹² h.h./ml. Typically, the adenoviral vector is administered in an amount of about 10 to about 10 viral particles (vp) to a human subject per administration, more typically in an amount of about 10 to about 10 vp.

The initial vaccination may be followed by a booster or secondary injection of a vaccine/composition containing the same adenoviral vector encoding the antigen of interest, or a vaccine/composition containing a different 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

The present invention also provides the following non-limiting embodiments.

Embodiment 1 is an isolated nucleic acid sequence encoding a hexon polypeptide or a functional derivative thereof, comprising a polypeptide spanning the hypervariable regions of hexon, characterized by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical throughout the length of the amino acid sequence under SEQ ID NO: 1.

Embodiment 2 is an isolated nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide spanning the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO: 1.

Embodiment 3 is the isolated nucleic acid sequence of any one of Embodiments 1-2, wherein the hexon polypeptide or a functional derivative thereof comprises the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2) or an amino acid sequence that is at least 95% identical along its entire length amino acid sequence under SEQ ID NO: 2.

Embodiment 4 is an isolated nucleic acid sequence comprising any of the nucleic acid sequences of Embodiments 1-3 and further comprising an isolated nucleic acid sequence encoding a fiber polypeptide or a functional derivative thereof.

Embodiment 5 is the isolated nucleic acid sequence of Embodiment 4, wherein the fiber polypeptide comprises a fiber head polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10.

Embodiment 6 is the isolated nucleic acid sequence of Embodiment 4, wherein the fiber polypeptide comprises a fiber stalk polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 11.

Embodiment 7 is the isolated nucleic acid sequence of Embodiment 4, wherein the fiber polypeptide comprises a fiber tail polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 12.

Embodiment 8 is the isolated sequence of any one of embodiments 4-7, wherein the fiber polypeptide or a functional derivative thereof comprises the amino acid sequence of the BLY6 fiber polypeptide (SEQ ID NO: 3) or an amino acid sequence that is at least 95% identical in its entire amino acid length

- 11 044526 sequences under SEQ ID NO: 3.

Embodiment 9 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a fiber head polypeptide sequence, wherein the fiber head polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 10.

Embodiment 10 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a fiber stalk polypeptide sequence, wherein the fiber stalk polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11.

Embodiment 11 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a fiber tail polypeptide sequence, wherein the fiber tail polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12.

Embodiment 12 is the isolated nucleic acid sequence of any one of embodiments 9-11, wherein the fiber polypeptide comprises the amino acid sequence of the BLY6 fiber polypeptide (SEQ ID NO: 3).

Embodiment 13 is a vector containing the nucleic acid of any one of embodiments 1-12.

Embodiment 14 is the vector of Embodiment 13, which is an adenoviral vector and further contains a transgene.

Embodiment 15 is a recombinant cell containing the vector of embodiment 13 or 14.

Embodiment 16 is a method of producing a vector, comprising: (a) growing the recombinant cell of Embodiment 15 under conditions to produce the vector; and (b) isolating the vector from the recombinant cell.

Embodiment 17 is an immunogenic composition comprising the vector of Embodiment 14.

Embodiment 18 is a method of inducing an immune response in a subject in need thereof, comprising administering to the subject the immunogenic composition of Embodiment 17.

Embodiment 19 is an adenoviral vector comprising: (a) at least one transgene and (b) a nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide spanning the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO: 1.

Embodiment 20 is an adenoviral vector comprising: (a) at least one transgene and (b) a nucleic acid sequence encoding a hexon polypeptide comprising the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2).

Embodiment 21 is an adenoviral vector containing: (a) at least one transgene; (b) a nucleic acid sequence encoding a hexon polypeptide containing the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2); and (c) a nucleic acid sequence encoding a fiber polypeptide containing the amino acid sequence of a BLY6 fiber polypeptide (SEQ ID NO: 3).

Examples

Example 1. Creation of E1 and E3 deletion vectors based on a new adenovirus isolate BLY6

A new gorilla adenovirus isolate BLY6 (also designated JAd1-WT) was identified and sequenced. This gorilla adenovirus isolate was found to belong phylogenetically to human adenovirus species E (HAdV-E). The nucleotide sequence of the complete BLY6 genome is determined by SEQ ID NO: 5.

Description of the single plasmid system used to generate BLY6-based Ad vectors pBLY6.dE1.dE3 (SEQ ID NO: 8; FIG. 5) and pBLY6.dE1.dE3.5IXP (SEQ ID NO: 9; FIG. 6 ) are plasmids carrying full-length genomes of adenoviral vectors containing E1 and E3 deletions based on the BLY6 isolate. The genomic sequences of the Ad vector contained in these plasmids are given under SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Within each of these plasmids, the genome of the adenoviral vector is flanked by two SwaI restriction enzyme sites (i.e., one SwaI site is located at each end of the vector genome). Such SwaI sites are designed to facilitate excision of the Ad vector genome from the plasmid backbone prior to virus rescue by transfection of suitable E1-complementing cells (such as HEK293, 911 and PER.C6 cells). The Ad vector genomes contained in these plasmids additionally carry specific restriction enzyme sites introduced at the E1 deletion site, the E3 deletion region, and a region adjacent to the right inverted terminal repeat (RITR). These restriction sites

- 12 044526 enzymes were selected to ensure their uniqueness in the context of plasmids with the complete Ad genome. They are transgene insertion sites that allow the easy construction, using standard molecular cloning techniques, of Ad vectors carrying one or more transgene-containing expression cassettes inserted at any of these appropriate locations or any combination thereof. Ad vector designs and plasmid constructions are described in more detail in the sections below.

BLY6-based Ad vector genome diagram

Each of the BLY6-based Ad vector genomes was designed to include an E1 deletion, an E3 deletion, different transgene insertion sites, and replacement of the native E4 open reading frame (orf) 6 and orf6/7 with that of human adenovirus-5 (HAdV- 5). The E1 region of each adenovirus was removed and replaced with a transgene insertion site containing the AsiSI restriction enzyme site sequence. The E3 region of each adenovirus was removed and replaced with a transgene insertion site containing the FseI restriction enzyme site sequence. Another transgene insertion site was created by inserting a PacI restriction enzyme site sequence into the region adjacent to the right inverted terminal repeat (ITR) of each adenovirus. The BLY6 sequences providing the orf6 and orf6/7 E4 coding sequences were replaced with SEQ ID NO: 6. This replacement sequence provided the human adenovirus-5 (HAdV-5) orf6 and orf6/7 E4 coding sequences (base pairs 32914-34077 of the GenBank sequence No. AS_000008).

Two types of E1 region deletions were designed and constructed. The genome of the BLY6-based Ad vector contained in pBLY6.dE1.dE3 carries a deletion of the E1 region corresponding to the removal of nucleotides 453-3016 from SEQ ID NO: 5. At the same time, the genome of the BLY6-based Ad vector contained in pBLY6. dE1.dE3.5IXP, carries a large sequence deletion spanning the E1 region, which removes all E1 BLY6 coding sequences (ie, nucleotides 453-3366 of SEQ ID NO: 5). In addition, this latter Ad vector genome was further designed to carry a replacement of the non-coding sequence region between the 55K and pIX E1B coding sequences with the HAdV-5 sequence (i.e., the sequences corresponding to nucleotides 3367-3454 of SEQ ID NO: 5, replaced by nucleotides 3510-3608 from GenBank AC_000008 (ie SEQ ID NO: 7)).

Construction of single plasmids containing BLY6-based Ad vector genomes pBLY6.dE1.dE3 (SEQ ID NO: 8) were constructed through several steps of gene synthesis (which was performed by GenScript) and standard molecular cloning procedures. First, a DNA fragment of 3586 bp in size was synthesized. (SEQ ID NO: 15), containing the left end of the desired Ad vector genome (i.e., carrying the above-mentioned E1 deletion), and ligated it as an MfeI-NdeI restriction fragment into EcoRI and NdeI digested pBR322 (GenBank accession number - J01749 .1), which led to the production of intermediate plasmid 1 BLY6. Secondly, a 4138 bp fragment was synthesized. (SEQ ID NO:6) containing the right end of the desired Ad vector genome (i.e., carrying the above-mentioned E3 deletion, a partial replacement of the E4 sequence, and a transgene insertion site located in the region adjacent to the rITR), and ligated it as a restriction fragment BamHI-NdeI into intermediate BLY6 plasmid 1 digested with BamHI and NdeI, resulting in intermediate BLY6 plasmid 2. Thirdly, a 4563 bp fragment was synthesized. (SEQ ID NO: 17), containing the middle fragment of the Ad vector genome, and ligated it as a BamHI-MfeI restriction fragment into BLY6 intermediate plasmid 2 digested with BamHI and MfeI, which resulted in intermediate plasmid 3 BLY6 (SEQ ID NO: 18). Fourth, the BsrGI-BsrGI restriction fragment is 18987 bp long. from the BLY6 viral genome (SEQ ID NO: 5) was ligated into the BsrGI-digested BLY6 intermediate plasmid 3, resulting in the final plasmid pBLY6.dE1.dE3 (SEQ ID NO: 8).

pBLY6.dE1.dE3.5IXP (SEQ ID NO:9) was constructed in the same manner as pBLY6.dE1.dE3, except that the above-mentioned BLY6 intermediate plasmid 3 (SEQ ID NO: 18) was first modified so that it involved the required E1 deletion and pIX Ad5 promoter insertion. This was accomplished by synthesizing a 638 bp fragment. (SEQ ID NO: 19), which was subsequently ligated as an AsiSI-AgeI restriction fragment into the AsiSI and AgeI digested BLY6 intermediate plasmid 3.

pBLY6.FLuc (SEQ GO NO:20) and pBLY6.RSVF-2A-GLuc (SEQ ID NO:21) were plasmids derived from pBLY6.dE1.dE3, each carrying a BLY6-based Ad vector genome equipped with transgenic expression cassette inserted at the location of the E1 deletion. The Ad vector genome sequences carried in these plasmids are provided under SEQ ID NO: 22 and SEQ ID NO: 23, respectively. pBLY6.FLuc carries a transgenic firefly luciferase (FLuc) expression cassette. This cassette is driven by the major immediate-early promoter of cytomegalovirus (ie, the CMV promoter) and contains an SV40-derived polyadenylation signal. pBLY6.RSVF-2A-Gluc carries a transgenic expression cassette for RSV-Fa2-2A-GLuc (RSVF-2A-GLuc), which was a chimeric protein consisting of a fusogenic glycoprotein of respiratory syncytial virus strain A2, a viral peptide foot and mouth disease 2A and Gaussia luciferase (GLuc). Like the Fluc cassette, this cassette is driven by the CMV promoter and carries the SV40 polyadenylation signal. In addition, this cassette contains in its 5' untranslated region a sequence providing intron 2 of the human apolipoprotein A1 gene. Each of the Fluc and RSVF-2A-GLuc expression cassettes were constructed through several standard steps of gene synthesis and molecular cloning, after which they were ligated into a unique AsiSI restriction enzyme site in pBLY6.dE1.dE3 to obtain pBLY6.FLuc and pBLY6.RSVF-2A- Gluc respectively.

Creation and production of adenoviral vectors based on BLY6

The adenoviral vectors BLY6.Fluc (also designated JAd1NVT003) and BLY6.RSVF-2A-Gluc (also designated JAd1NVT001), which contained the adenoviral vector genome sequences SEQ ID NO:22 and SEQ ID NO:23, respectively, were prepared by transfection with the corresponding genome plasmids Ad vectors (i.e., pBLY6.Fluc and pBLY6.RSVF-2A-Gluc) of E1-complemented PER.C6 cells. Before transfection of PER.C6 cells, which were grown as adherent 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 SwaI to release the corresponding genomes of adenoviral vectors from the plasmid. Transfections were performed according to standard procedures using Lipofectamine transfection reagent (Invitrogen; Carlsbad, CA). After collection of viruses rescued by transfection, the viruses were further propagated through several sequential rounds of infection in 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)).

Cellular and humoral immune responses induced by novel adenoviral vectors

Examples 2 and 3 describe experiments performed to evaluate the immunogenicity of the novel BLY6-based adenoviral vectors prepared herein. 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-Fa2-2A-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 reference vector based on human adenovirus type 26 (HAdV-26, also referred to herein as Ad26) or human adenovirus type 49 (HAdV-49, also referred to herein as Ad49), 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 2 Cellular Immune Responses Induced by BLY6.FLuc

To evaluate the cellular immunogenicity of the novel BLY6 adenoviral vector, Balb/C mice were immunized with intramuscular injection of Ad26.FLuc, Ad49.Fluc (positive controls), BLY6 vector expressing firefly luciferase (BLY6.FLuc), or an adenovector not encoding the transgene (empty Ad26). During administration, two doses of the vector were tested: 109 and ¹⁰¹⁰ viral particles (vp) per mouse. Two weeks after immunization, mice were sacrificed and splenocytes were stimulated overnight with a pool of 15-mer overlapping FLuc peptides (experimental design in Fig. 1A ). Cellular immune responses were determined by ex-vivo ELISPOT assay, measuring the relative abundance of IFN-γ-secreting cells (Fig. 1B). The results showed that when immunized at higher doses (10 ¹⁰ ), the cellular immune responses induced by BLY6 were approximately as high as the response observed with Ad26.Fluc. In contrast, when immunized with a lower dose (109), BLY6.Fluc produced a stronger response than Ad26.Fluc.

Overall, the cellular immune responses induced by the FLuc-expressing BLY6 recombinant adenoviral vector of the present invention clearly indicated the strong immunogenicity of this vector in mice.

Example 3 Cellular and Humoral Immune Responses Induced by BLY6.RSVF-2A-GLuc

The immunogenicity of the novel adenoviral vector BLY6 was further assessed using RSVFa2-2A-GLuc (RSVF-2A-GLuc) as a vector-encoded (model) vaccine antigen. Balb/C mice were immunized intramuscularly with Ad26.RSVF-2A-GLuc (positive control) or BLY6.RSVF-2A-GLuc (both at 10 ⁸ , 10 9 and 10 ¹⁰ viral particles per mouse) or

- 14 044526 via Ad26.FLuc or BLY6.FLuc (both at 10 viral particles per mouse). Mice were sacrificed eight weeks later, and blood samples and splenocytes were collected (Fig. 2A). Various immune parameters were assessed as described below.

A virus neutralization assay was performed to evaluate the ability of BLY6.RSVF-2A-GLuc to induce the production of respiratory syncytial virus neutralizing antibodies. In fig. 2B 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. BLY6.RSVF-2A-GLuc resulted in higher RSV A2 neutralization titers than those determined for the Ad26 reference vector encoding the same antigen. Titers against BLY6.RSVF-2A-GLuc were detected mainly at the highest dose used for immunization, which was 10 ¹⁰ w.p. As expected, no RSV A2-specific responses were detected to adenovectors encoding firefly luciferase.

Induction of cellular immunity to vector-encoded antigen was assessed using an ELISPOT assay specific for RSV-F _A2 . 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 IFNs. -γ cells. The data show that antigen-specific cellular immune responses induced by the novel BLY6 vector encoding RSVF-2A-GLuc were dose-dependent and, at each dose, were similar in magnitude to those induced by the reference vector Ad26.RSVF-2A-GLuc (Figure 2C ). 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 after immunization from mice immunized with Ad26 (positive control) and BLY6 vectors expressing the RSVF-2A-GLuc transgene or firefly luciferase (control) was tested for IgG to RSV F _A2 by ELISA. 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 BLY6.RSVF-2A-GLuc dose-dependently induced the production of higher titers of pre-RSV-F-specific IgG antibodies than titers induced by Ad26.RSVF-2A-GLuc (Fig. 2D ). As expected, titers of antibodies specific to RSV-FAA2 were not detected in the serum of mice immunized with vectors encoding only firefly luciferase.

Overall, the data show that the BLY6 vector induced strong cellular and humoral immune responses to the encoded antigens that were similar or higher than those induced by the reference HAdV-26 vector. These immune responses clearly indicated the strong immunogenicity of the BLY6 vector in mice.

Example 4: Evaluation of serological cross-neutralization among new and existing adenoviral vectors

To ensure their potential utility as novel adenoviral vaccine vectors, the novel BLY6 adenoviral vectors generated herein will preferably be serologically different from existing adenoviral vectors that are currently in development as vaccine vectors, such as those based on human adenovirus serotypes HAdV-5 and HAdV-35. Therefore, cross-neutralization tests were performed among the new adenoviral vectors BLY6 and several existing vectors based on HAdV-4, HAdV-5, HAdV-26, HAdV-35 and HAdV49. 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 vector particles per mouse. The adenovirus neutralization assay was performed as 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), 16-200, 200-2000 and > 2000. The results show that there was no significant cross-neutralization among the vectors tested (Fig. 3). Only slight one-way cross-neutralization was observed between the BLY6 and Ad26 vectors, with the BLY6 antiserum showing a neutralization titer against Ad26 of 16.12 (i.e., just above the lower limit of detection of 16), and for the Ad26 antiserum a neutralization titer of 16.12. BLY6 was not observed. Thus, the novel adenovirus vector BLY6 showed no or only very weak cross-neutralization with the human adenovirus vectors included in the tested panel, i.e., Ad26, Ad35, Ad49, Ad5, and Ad4. Therefore, this vector could 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 series of two or more sequential vaccination regimens against various diseases or antigens.

Example 5. 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, the BLY6 vector 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 vector was tested for neutralization by human serum samples using a standard adenovirus neutralization assay as performed in Example 3 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 vector BLY6 had significantly lower seroprevalence in the human subjects studied than the control vector Ad5 and the reference vectors Ad26 and Ad35 (Fig. 4). Moreover, the positive neutralization titers observed against the new BLY6 vectors were generally quite low, generally not exceeding 300. In contrast, the majority of positive neutralization titers observed against Ad26 and Ad5 were greater than 300.

Overall, the above data indicated that pre-existing humoral antivector immunity to BLY6 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 6. Productivity of adenoviral vector in 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 the Fluc-encoding version of the new Ad BLY6 vector described in Example 1. The HAdV-26-based vector Ad26.Fluc was used as a reference control. Suspensions of PER.C6 cell cultures seeded in shake flasks at a density of 1x10 cells/ml in a total volume of 10 ml of PERMEXCIS® medium (available from Lonza) supplemented with 4 mM L-glutamine (Lonza) were infected with different vectors with different ratios of viral particles ( h.p.) per cell and then incubated for 4 days. Various relationships c. h. per cell used for infection were 70, 150 and 900. Every day, samples of infected cell cultures were collected and titers of h.p. 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

-

Claims (11)

free vector DNA (i.e. vector genomes that have not been packaged into viral particles). The productivity results obtained for the new BLY6.Fluc vector are shown in FIG. 8. Higher hs titers were observed for BLY6.Fluc than for the reference control vector Ad26.Fluc at all hs infection ratios tested. per cell and at all time points tested when material was collected. These results demonstrated the good productivity of the new BLY6 vector in the SPER.C6-based serum-free suspension cell culture model. Taken together, the results of the studies on the humoral and cellular immune responses induced by the novel BLY6-based recombinant adenoviral vectors of the present invention, as presented above, clearly indicated the potent immunogenicity of these vectors in mice. In addition, the vectors were demonstrated to induce no or only very small cross-neutralizing antibody responses against certain existing adenoviral vaccine vector candidates (eg, Ad26 and Ad35) or vice versa. 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. CLAIM 1. An isolated nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide spanning the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical throughout its entire length to the amino acid sequence of SEQ ID NO: 1 . 2. The isolated nucleic acid sequence according to claim 1, wherein the hexon polypeptide includes a polypeptide covering the hexon hypervariable regions characterized by the amino acid sequence of SEQ ID NO: 1. 3. The isolated nucleic acid sequence of claim 2, wherein the hexon polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical over its entire length to the amino acid sequence of SEQ ID NO: 2. 4. The isolated nucleic acid sequence according to any one of claims 1 to 3, further comprising a nucleic acid sequence encoding a fiber polypeptide. 5. The isolated nucleic acid sequence of claim 4, wherein the fiber polypeptide comprises at least one sequence selected from the group consisting of: (1) a fiber head polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10, (2) a polypeptide sequence a fiber stem comprising the amino acid sequence of SEQ ID NO: 11, and (3) a fiber tail polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 12, wherein preferably the fiber polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical over its entire length to the amino acid sequence of SEQ ID NO: 3. 6. A vector containing a nucleic acid according to any one of claims 1 to 5. 7. The vector according to claim 6, which is an adenoviral vector and additionally contains a transgene. 8. The adenoviral vector of claim 7, wherein the adenoviral vector further comprises at least one of an E1 deletion and an E3 deletion. 9. The adenoviral vector according to claim 7 or 8, wherein the adenoviral vector is a chimeric adenoviral vector containing one or more human adenovirus nucleic acid sequences, wherein preferably the human adenovirus nucleic acid sequences are derived from at least one of human adenovirus-4, human adenovirus-5, human adenovirus-26 or human adenovirus-35. 10. An adenoviral vector according to any one of claims 7 to 9, wherein the adenoviral vector contains a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14. 11. Recombinant cell containing the vector according to any one of claims 6-10. -