BR112020008198A2 - adenovirus and its uses - Google Patents

Abstract

  The present invention relates to adenoviral nucleic acid sequences and adenoviral vectors comprising said nucleic acid sequences. The adenoviral vectors conferred can be used to induce a protective immune response in a subject.

Description

Descriptive report of the Invention Patent for "ADENO-VIRUS AND ITS USES".

FIELD OF THE INVENTION

[0001] The present invention relates to biotechnology. More particularly, the field and use of adenoviral vectors, such as defective adenoviral vectors in replication to administer antigens and elicit an immune response in hosts.

BACKGROUND OF THE INVENTION

[0002] Recombinant adenoviral vectors are widely applied for gene therapy and vaccine applications. AdV-5 vector-based vaccines have been shown to elicit potent immune and protective responses in a variety of animal models (see, for example, WOZ2001 / 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 vaccines based on recombinant AdV-5 vectors is likely to be limited by the high seroprevalence of neutralizing antibodies specific for AdV-5 (NAbs) in human populations. The existence of anti-AdV-5 immunity has been shown to substantially suppress the immunogenicity of AdV-5-based vaccines in studies in mice, rhesus monkeys and humans.

[0003] A promising strategy to circumvent the existence of pre-existing immunity in individuals previously infected or treated with the most common human adenovirus, for example, AdV-5, involves the development of recombinant vectors from adenovirus sero types. who do not find such pre-existing immunities. One of these strategies is based on the use of simian non-human adenoviruses, since these typically do not infect human beings and exhibit low seroprevalence in human samples. Simian nonhuman viruses are applicable for human use, since it has been shown that these viruses can infect human cells in vitro (WO2003 / 000283; WO2004 / 037 189).

[0004] Thus, there is a need in the field for alternative adenoviral vectors that can be produced in large quantities, that do not find pre-existing immunities in the host, but that are still immunogenic and capable of inducing a strong immune response. against antigens encoded by the heterologous nucleic acids inserted in the vector.

BRIEF SUMMARY OF THE INVENTION

[0005] Isolated nucleic acid sequences encoding hexon polypeptides are conferred. In certain embodiments, the hexon polypeptide or a functional derivative thereof, comprises a polypeptide encompassing hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 1.

[0006] In certain embodiments, the hexon polypeptide comprises a polypeptide comprising hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1. In certain modalities, the hexon polypeptide or its functional derivative 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 to along its entire length, with the amino acid sequence of SEQ ID NO: 2.

[0007] Isolated nucleic acid sequences encoding a fiber polypeptide or functional derivative thereof are also provided. In certain embodiments, the fiber polypeptide comprises at least one of a fiber button polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10; a fiber axis 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 the entire period. its length, with the amino acid sequence of SEQ ID NO: 3.

[0008] Modalities of the invention also include isolated fiber and hexon polypeptides encoded by the fiber and hexon nucleic acid sequences of the invention.

[0009] In this document, isolated nucleic acids comprising a hexon nucleic acid sequence encoding at least one of the hexon polypeptides described in this document and a fiber nucleic acid sequence encoding at least one are also provided. of the fiber polypeptides described in this document. In certain embodiments, vectors comprising the isolated nucleic acids described in this document are given in this document. In one embodiment, the vector is a viral vector. In another modality, the vector is an expression vector. In a preferred embodiment, the vector is an adenoviral vector. More preferably, the vector further comprises a transgene.

[0010] Recombinant cells comprising the vectors described in this document are also checked. These cells can be used for production of recombinant protein, expression of recombinant protein or production of vectors or viral particles. Vector production methods are also checked. The methods comprise (a) the growth of the recombinant cell described in this document under conditions for the production of the vector; and (b) isolating the vector from the recombinant cell.

[0011] In certain modalities, immuno compositions are conferred

noogenic substances comprising the vectors described in this document. Methods are also provided to induce an immune response in a subject in need of it, comprising administering the immunogenic compositions to the subject described in this document.

[0012] In certain embodiments, adenoviral vectors comprising (a) at least one transgene are conferred; and (b) a nucleic acid sequence encoding a hexon polypeptide according to embodiments of the invention. The hexon polypeptide can, for example, comprise a polypeptide comprising hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical, over its entire length , with the amino acid sequence of SEQ ID NO: 1. The hexon polypeptide can, for example, comprise an amino acid sequence having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the hexon polypeptide comprises an 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, with the amino acid sequence of SEQ ID NO: 2.

[0013] In certain embodiments, adenoviral vectors comprising (a) at least one transgene are conferred; and (b) a nucleic acid sequence encoding a fiber polypeptide according to embodiments of the invention. In certain embodiments, the fiber polypeptide can comprise at least one of a fiber button polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10; a fiber axis 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 modalities,

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 , with the amino acid sequence of SEQ ID NO: 3. Modalities of the invention also include adenoviral vectors comprising (a) at least one transgene; (b) a nucleic acid sequence encoding a hexon polypeptide according to embodiments of the invention; and (c) a nucleic acid sequence encoding a fiber polypeptide according to embodiments of the invention.

[0014] In certain modalities, the adenoviral vectors given in this document are adenovirus vectors with replication defect (rAd). In one embodiment, adenoviral vectors can comprise an E1 deletion. In certain modalities, the adenoviral vectors given in this document may still comprise an E3 deletion. Adenoviral vectors can be simian adenoviral vectors comprising adenoviral nucleic acid sequences from one or more simian adenoviruses (SAdV), such as chimpanzee adenoviruses (for example, ChAd3); gorilla adenovirus; or rhesus adenovirus (for example, rhAd51, rhAd52 or rhAd53). Adenoviral vectors can be human adenoviral vectors comprising adenoviral sequences from one or more human adenoviruses (for example, hAdV-4, hAdV-5, hAdV-26, hAdV-35). Preferably, the adenoviral vector is a chimeric adenoviral vector comprising one or more human adenoviral nucleic acid sequences. Human adenoviral nucleic acid sequences can, for example, be from human adenovirus 4 (hAdV-4), human adenovirus 5 (hAdV-5), human adenovirus 26 (hAdV-26) or human adenovirus 35 (hAdV -35). Adenoviral vectors can, for example, comprise a human adenovirus 5

(hAdV-5) E4 orf6 and orf 6/7.

[0015] In certain modalities, the transgene is located adjacent to an inverted terminal repetition (ITR). In certain modalities, a transgene is located in, or adjacent to, the E1 deletion, in, or adjacent to, the E3 deletion and / or in, or adjacent to, ITR, for example, between the E4 region and the right ITR ( RITR).

[0016] In certain embodiments, the adenoviral vectors given in this document comprise a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0017] Immunogenic compositions or vaccines are also provided comprising the adenoviral vectors described in this document and a pharmaceutically acceptable carrier. Methods are also provided to induce an immune response in a subject who needs it. The methods comprise administration to the subject of the vaccines described in this document. Methods of producing a vaccine are still checked. The methods comprise combining an adenoviral vector described herein with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The preceding summary, as well as the following detailed description of preferred modalities of this application, will be better understood when read in conjunction with the attached drawings. However, it must be understood that the order is not limited to the precise modalities shown in the drawings.

[0019] Figure 1 shows the cellular and humeral immune responses induced by BLY6.FLuc and Ad49.FLuc. Figure 1A shows the experimental configuration. Figure 1B shows the cellular immune response induced by Ad26.FLuc, BLY6.FLuc and Ad49.FLuc against the vector-encoded antigen (ie Fluc, firefly luciferase), as determined by the interferon gamma ELISPOT analysis (IFN- [)). The y-axis shows the number of Point-Forming Units (SFU) per 10th splenocyte and the dotted line indicates the percentage 95% of the mean stimuli.

[0020] Figure 2 shows cellular and humoral immune responses induced by BLY6.RSVF-2A-GLuc. Figure 2A shows the experimental configuration. Figure 2B shows a graph of the results of a virus neutralization test (VNA) carried out eight weeks after immunization with Ad26.RSVF-2A-GLuc or BLY6.RSVF-2A-GLuc in three different concentrations ( 10%, 10th and 10thºvp), or with Ad26.FLuc or BLY6.FLuc at 10 * ºvp. Figure 2C shows the cell immune responses induced by Ad26.RSVF-2A-GLuc and BLY6.RSVF-2A-GLuc after immunization, as determined by IFN-y ELISPOT analysis. Figure 2D shows a graph of IgG-binding antibodies specific for RSVF induced by Ad26.RSVF-2A-GLuc or BLY6.RSVF-2A-GLuc in the serum of immunized mice 8 weeks after immunization.

[0021] Figure 3 shows homologous and heterologous adenovirus neutralization titers induced in mice immunized with Ad35, Ad26, Ad49, Ad5, Ad4, and BLY6 adenoviral vectors.

[0022] Figure 4 shows the seroprevalence of Ad35, Ad26, Ad5 and BLY6 in 200 serum samples from adult human cohort, aged between 18 and 55 years old, residing in the United States (USA) and European Union (EU). The neutralization titles measured on these values in relation to each vector were divided into four categories (<16 (negative), 16 to 300, 300 to 1,000, 1,000 to 4,000 and> 4,000), represented in the graphs, as indicated .

[0023] Figure 5 shows a schematic of plasmid PBLY6.dE1.dE3 (SEQ ID NO: 8).

[0024] Figure 6 shows a schematic of the plasmid PBLY6.dE1.dE3.51Xp (SEQ ID NO: 9).

[0025] Figure 7 shows an alignment of the polygon sequences

hexon BLY6 peptide (SEQ ID NO: 2) and hexon polypeptide SAdV-30-1 (SEQ ID NO: 4).

[0026] Figure 8 shows the productivity of the new BLY6.Fluc vector in the sSPER.C6 production cell line.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is based, at least in part, on the isolation and identification of a new gorilla adenovirus isolate, allocated to E adenovirus species, as well as on the construction and evaluation of vaccine vectors comprising nucleic acids encoding variable regions of the hexon and fiber polypeptides of said gorilla adenovirus. Adenoviral vectors are capable of eliciting an immune response and, in addition, have low seroprevalence in humans. Adenoviral vectors can be formulated for vaccines and used to induce protective immunity against specific antigens of interest.

[0028] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is incorporated in this document as a reference in its entirety. The discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is intended for the purpose of providing context for the invention. Such a discussion is not an admission that any or all of these matters are part of the preceding technique with respect to any of the described or claimed inventions.

[0029] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by the person skilled in the art to which the present invention belongs. Otherwise, certain terms used in this document have the meanings as presented in the specification.

[0030] It should be noted that, as used in this document and the appended claims, the singular forms "one", "one" and "o / a" - include references in the plural unless the context clearly determines another mode.

[0031] Unless stated otherwise, any numerical values, such as a concentration or range of concentrations described in this document, should be understood as being modified in all cases by the term "about" . Thus, a numerical value typically includes + 10% of the displayed value. For example, a concentration of 1 mg / ml includes 0.9 mg / ml to 1.1 mg / ml. Likewise, a concentration range from 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used in this document, the use of a numerical range expressly includes all possible sub-ranges, all individual numerical values within that range, including whole numbers within such ranges and fractions of the values unless the context clearly indicates in another way.

[0032] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood as referring to all elements in the series. Those skilled in the art will recognize, or be able to determine using, no more than routine experimentation, many equivalents of the specific embodiments of the invention described in this document. Such equivalents are intended to be encompassed by the invention.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains" or "containing", or any other variation of themselves, will be understood as meaning the inclusion of a mentioned integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open. For example, a composition, a mixture, a process, a method, an article or a device comprising a list of elements is not necessary.

limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article or device. Also, unless expressly stated otherwise, "or" refers to an "or" of inclusion and not an "or" of exclusion. For example, a condition A or B is met by any of the following: A is true (or is present) and B is false (or is not present), A is false (or is not present) and B is true (or is present), and both A and B are true (or are present).

[0034] As used in this document, the conjunctive term "and / or" between multiple elements recited is understood to encompass both individual and combined options. For example, where two elements are joined by "and / or", a first option refers to the applicability of the first element, without the second. A second option concerns the applicability of the second element without the first. A third option concerns the applicability of the first and second elements together. Any of these options is understood to fall within meaning, and therefore meets the requirements of the term "and / or" as used in this document. Concurrent applicability of more than one of the options is also understood as falling within the meaning, and, thus, satisfies the requirement of the term "and / or".

[0035] As used herein, the term "consists of" or variations such as "consist of" or "consisting of", as used throughout the specification and the claims, indicate the inclusion of any integer or group of mentioned integers, but that no additional integers or groups of integers can be added to the specified method, structure or composition.

[0036] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of", as used throughout the specification and the claims, indicates the inclusion of any integer - ro or group of integers mentioned, and the optional inclusion of any integer or group of integers mentioned that does not materially change the basic or new properties of the specified method, structure or composition. See M.P.E.P. $ 2111.03.

[0037] As used in this document, "subject" means any animal, preferably a mammal, most preferably a human being, that will be or has been vaccinated by a method according to an embodiment of the invention. The term "mammal", as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably a human being .

[0038] The words "right", "left", "lower" and "upper" designate directions in the drawings to which reference is made.

[0039] It should also be understood that the terms "about", "approximately", "generally", "substantially" and similar terms used in this document when referring to a dimension or characteristic of a component of the preferential invention, indicate that the dimension / characteristic described is not a strict limit or parameter and does not exclude small variations of the same that are functionally the same or similar, as would be understood by a person of ordinary knowledge in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (for example, rounding errors, measurement or other systemic errors, manufacturing tolerances, etc.) would not vary the least significant digit.

[0040] The terms "identical" or "identity" percentage, in the context of two or more nucleic acids or polypeptide sequences (for example, hexon and fiber polypeptides and polynucleotides that encode them), if refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum match, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0041] For sequence comparison, typically a sequence acts as a reference sequence, with which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm are designated. The algorithm comparison sequence then calculates the percentage of identity sequence for the test sequence (s) in relation to that of the reference sequence, based on the designated program parameters.

[0042] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by Needleman & Wunsch's homology alignment algorithm, J. Mol. Biol. 48: 443 (1970), for the search for a similarity method by Pearson & Lipman, Proc. Nat '.. Acad. Sci. EUA85: 2444 (1988), for computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or visual inspection (see generally, Current Protocols in Molecular Biology, FM Auelel et a /., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (Supplement in

1995) (Ausubel)).

[0043] Examples of algorithms that are suitable for the percentage determination of the sequence of identity and similarity of sequence 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 software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first involves the identification of high score of sequence pairs (HSPs) by identifying words of short length W in the query sequence, which either match or satisfy some positive T score limits when aligned with a word of the same length in a sequence database. T is referred to as the scoring limit for the neighboring word (Altschul et al, supra). These initial neighbor word accesses act as points to initiate searches in order to find longer HSPs that contain them. These word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0044] Accumulated scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for incompatible residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word accesses in each direction is interrupted when: the cumulative alignment score drops out by the quantity X since its maximum value reached; the cumulative score tends to zero, or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached. The

BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4 and a comparison of both strings. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expectation (E) of 10, and the BLOSUM6? 2 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci USA 89: 10915 (1989)).

[0045] In addition to the calculation of the percentage sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Alschul, Proc. Nat '. Acad. Sci. USA 90: 5873-5787 (1993)). A measure of the similarity conferred by the BLAST algorithm is the lowest probability of sum (P (N)), which gives an indication of the probability by which a match between two nucleotides or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest probability sum in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than than about 0.01, and most preferably less than about 0.001.

[0046] An additional indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactively with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under severe conditions, as described below.

[0047] As used in this document, the term "protective immunity" or "protective immune response" means that the vaccinated subject is able to control an infection with the pathogen against which the vaccination was carried out. The pathogen can, for example, be an antigenic gene product or antigenic protein, or a fragment thereof. Usually, the subject having developed a "protective immune response" only develops mild to moderate clinical symptoms or does not even have symptoms. Usually, a subject having a "protective immune response" or "protective immunity" against a particular agent will not die due to infection with that agent.

[0048] 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 improve an immune response to the adenoviral vectors of the invention.

[0049] As used herein, the term "antigenic gene product or its fragment" or "antigenic protein" can include a bacterial, viral, parasitic or fungal protein or a fragment thereof. Preferably, an antigenic protein or antigenic gene product is capable of generating a protective immune response in a host, for example, inducing an immune response against a disease or infection (for example, a bacterial, viral, parasitic or fungal disease or infection) , and / or production of immunity (that is, vaccination) of a subject against a disease or infection, which protects the subject against the disease or infection. Adenoviral Vectors

[0050] Exposure to certain adenoviruses resulted in immune responses against certain adenoviral serotypes, which can affect the efficacy of adenoviral vectors. Since human adenovirus infections are common in humans, the prevalence of neutralizing antibodies against human adenoviruses in human populations is high. The presence of such neutralizing antibodies in individuals can be expected to reduce the effectiveness of a gene transfer vector based on a human adenoviral structure. One way to circumvent the reduction in efficacy is to replace the epitopes in the adenoviral capsid proteins that are the targets of neutralizing antibodies. The target sequences in the capsid proteins can be replaced by protein sequences from other adenoviruses that are of low prevalence and, therefore, against which neutralizing antibodies are rare in human populations.

[0051] A "capsid protein" refers to a protein in the capsid of an adenovirus (eg, BLY6, HAdV-4) or a fragment or functional derivative thereof, which 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 an adenovirus full or full length capsid protein. In other embodiments, the capsid protein is a fragment or derivative of a full-length adenovirus capsid protein. In certain embodiments, the hexon, penton and fiber encoded by an adenoviral vector of the invention have the same or different adenoviral structure (ie, a BLY6 hexon and BLY6 fiber, a BLY6 hexon and a human adenovirus fiber, a human adenovirus hexon and a BLYB6 fiber, etc.).

[0052] A "hexon polypeptide" refers to adenovirus hexon coat proteins, their functional and derived fragments.

[0053] A "fiber polypeptide" refers to adenovirus fiber proteins, their functional fragments and derivatives.

[0054] A target of neutralizing antibodies against adenovirus is the main coating protein, the hexon protein. Replacing the hexon protein or the variable sequences within the hexon protein, which define the serotype and bind neutralizing antibodies, with the hexon protein or variable sequences within the hexon protein from adenoviruses that are rare in the human population, such like the gorilla sequences described in this document, it can allow the construction of adenovirus vectors that would be less susceptible to neutralization by antibodies commonly found in humans.

[0055] A second target of neutralizing antibodies against adenovirus is fiber protein. Replacing the fiber protein or the variable sequences within the fiber protein with the fiber protein or variable sequences within the fiber protein from adenoviruses that are rare in the human population, such as the gorilla sequences described in this document, it can also allow the construction of adenovirus vectors that would be less susceptible to neutralization by antibodies commonly found in humans. A combination of fiber substitution and hexon substitutions described above can provide additional resistance to neutralization by antibodies commonly present in human populations.

[0056] 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 comprising at least one of the isolated nucleic acid sequences.

[0057] A "functional derivative" of a polypeptide refers appropriately to a modified version of a polypeptide, for example, in which one or more amino acids of the polypeptide can be deleted, inserted, modified and / or replaced . A derivative of an unmodified adenoviral capsid protein is considered to be functional if, for example: (a) an adenovirus comprising the capsid protein derived within its capsid retains substantially the same or lesser seroprevalence compared to an adenovirus comprising the protein of the unmodified capsid; and / or (b) an adenovirus comprising the capsid protein derived within its capsid retains substantially the same or greater infectivity of the host cells compared to an adenovirus comprising the unmodified capsid protein; and / or (c) an adenovirus comprising the capsid protein derived within its capsid retains substantially the same or greater immunogenicity as compared to an adenovirus comprising the unmodified capsid protein; and / or (d) an adenovirus comprising the capsid protein derived within its capsid retains substantially the same or a higher level of transgene productivity compared to an adenovirus comprising the unmodified capsid protein.

[0058] An "adenoviral vector" refers to a recombinant vector derived from, or containing at least a portion of, an adenoviral genome.

[0059] In preferred embodiments, the isolated nucleic acid sequences encode hexon polypeptides or a functional derivative thereof, comprising a polypeptide encompassing hypervariable regions.

hexon compounds having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 1. In certain embodiments, hexon polypeptides comprise a polypeptide encompassing hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1. In preferred modes, the hexon polypeptide or its functional derivative 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 to along its entire length, with the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the hexon polypeptide or its functional derivative comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, over its entire length, with the amino acid sequence of SEQ ID NO: 2.

[0060] In preferred embodiments, the isolated nucleic acid sequences encode fiber polypeptides or a functional derivative thereof. The fiber polypeptide can, for example, comprise at least one of a fiber button polypeptide comprising the amino acid sequence of SEQ ID NO: 10; a fiber axis polypeptide comprising 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 a BLY6 fiber polypeptide (SEQ ID NO: 3) or an amino acid sequence that is at least 95% identical, over its entire length, with the amino acid sequence of SEQ ID NO: 3.

[0061] In preferred embodiments, an isolated nucleic acid comprising a hexon nucleic acid sequence encoding at least one of the hexon polypeptides described herein and a nucleic acid sequence encoding at least one of the fiber polypeptides described herein is provided. document.

[0062] In preferred embodiments, vectors are conferred, preferably adenoviral vectors, comprising at least one of an isolated hexon nucleic acid sequence and / or an isolated fiber nucleic acid sequence according to the modalities of the invention. Adenoviral vectors can, for example, comprise 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 a polypeptide comprising hypervariable regions of hexon described herein and the fiber polypeptide comprises a fiber polypeptide described in this document.

[0063] Typically, an adenoviral vector of the invention comprises the complete recombinant adenoviral genome in, for example, a plasmid, cosmid or baculovirus vector. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. DNA can be double-stranded or single-stranded.

[0064] One skilled in the art will recognize that elements obtained from several serotypes can be combined into a single adenoviral vector, for example, human or simian adenovirus. In this way, a chimeric adenovirus vector that combines desirable properties of different serotypes can be produced. Thus, in some modalities, a chimeric adenovirus vector of the invention can combine the absence of pre-existing immunity from the hexon polypeptide and / or simian fiber sequences with the

city of antigen delivery and high-level presentation of existing adenoviral vectors, such as rAd4, rAd5, rAd26 or rAd35.

[0065] The advantages of adenoviral vectors for use as vaccines include ease of handling, good large-scale manufacturability and an excellent safety record based on many years of experience in research, development, production and clinical trials with numerous vectors adenoviral drugs that were reported. Adenoviral vectors that are used as vaccines generally provide a good immune response to the protein encoded by the transgene, including a cellular immune response. An adenoviral vector according to the invention can be based on any type of adenovirus and, in certain embodiments, is a human adenovirus, which can be of any group or serotype. In preferred embodiments, the recombinant adenovirus is based on a human adenovirus in group A, B, C, D, E, F or G. In other preferred embodiments, the recombinant adenovirus is based on a human adenovirus serotype 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 can be of any serotype. In certain embodiments, the recombinant adenovirus is based on a 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 SA7TP.

[0066] In a more preferred embodiment, the chimpanzee adenovirus vector of the second composition is ChAdV3. The recombinant chimpanzee adenovirus serotype 3 (ChAd3 or cAd3) is a subgroup C adenovirus with similar properties to that of human adenovirus serotype 5 (Ad5). ChAd3 has been shown to be safe and immunogenic in human studies that have evaluated candidate vaccines for hepatitis C virus (HCV) (Barnes E, et al. 2012 Science trans-

lational medicine 4: 115ra1). ChAd3-based vaccines have been reported to be capable of inducing an immune response comparable to a human vectorized Ad5 vaccine. See, for example, Peruzzi D, et al. 2009 Vaccine 27: 1293-300 and Quinn KM, et al. 2013 J Immunol 190: 2720-35; WO 2005/07 1093; WO2011 / 0130627, etc.

[0067] Adenoviral vectors, methods for their construction and methods for their propagation, are well known in the art and are described in, for example, Pat. U.S. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191 and 6,113,913 and Thomas Shenk, "Ade- noviridae and their Replication", MS Horwitz, "Adenoviruses," in Virology, BN Fields et al., eds., 3rd ed., Raven Press, Ltd., New York (1996), and other references mentioned in this document. Typically, the construction of adenoviral vectors involves the use of conventional molecular biology techniques, such as those described in, p. eg, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, New York (1989), Watson et al., Recombinant DNA, 2nd ed., Scientific American Books (1992), and Ausubel et al., Curriculum Protocols in Molecular Biology, Wiley Interscience Publishers, New York (1995), and other references mentioned in the present document.

[0068] In certain embodiments, the adenoviral vector comprises an E1 deletion and / or an E3 deletion. An E1 or E3 deletion can, for example, include a complete deletion of the gene or a partial deletion, which makes the product of the E1 or E3 gene functionally defective. Thus, in certain modalities, adenovirus is poorly replicated, for example, since it contains a deletion in the E1 region of the genome. As is known to the person skilled in the art, in case of deletions of essential regions of the adenovirus genome,

the functions encoded by these regions must be provided in trans, preferably by the producing cell, that is, when parts or all of the E1, E2 and / or E4 regions are deleted from the adenovirus, these must be present in the producing cell , for example integrated into its genome, or in the form of so-called auxiliary adenoviruses or auxiliary plasmids. The adenovirus can also have a deletion in the E3 region, which is not necessary for replication, and so that deletion does not have to be complemented. One or more of the E1, E2, E3 and E4 regions can also be inactivated by other means, such as the insertion of a transgene of interest (usually linked to a promoter) in the regions to be inactivated.

[0069] The producer cell (sometimes also referred to in the art and in this document as a "packaging cell" or "complementary cell") that can be used can be any producer cell in which a desired adenovirus can be propagated. For example, the propagation of recombinant adenovirus vectors is carried out in producer cells that complement adenovirus deficiencies. Such producer cells preferably have at least one adenovirus E1 sequence in their genome and are thus capable of complementing recombinant adenoviruses with a deletion in the E1 region. Any complementary E1 producing cell can be used, such as human retinal cells immortalized by E1, eg, 911 or PER.C6 cells (see U.S. patent 5,994,128), E1-transformed amniocytes (See EP patent 1230354), A549 cells transformed with E1 (see, eg, WO 98/39411, U.S. patent 5,891,690), GH329: HeLa (Gao et a / l., 2000, Hum Gene Ther 11: 213-19), 293 and the like. In certain embodiments, the producing cells are, for example, HEK293 cells or PER.C6 cells or 911 cells or IT293SF cells and the like. The production of adenoviral vectors in producer cells is reviewed in (Kovesdi et al., 2010, Viruses 2:

1681-703).

[0070] In certain embodiments, the adenoviral vector is a chimeric adenoviral vector comprising one or more human adenoviral nucleic acid sequences. Human adenoviral nucleic acids can, 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, an E1-deficient adenoviral vector comprises the E4-orf6 coding sequence of a human Ad5 adenovirus. This allows for the propagation of such adenoviruses in well-known complementary cell lines that express the Ad5 E1 genes, such as, eg, 293 cells or PER.C6 cells (see, eg, Fallaux et a /. , 1998, Hum Gene Ther 9: 1909-17, Havenga et al., 2006, J Gen Virol 87: 2135-43; WO 03/104467, incorporated herein in its entirety by reference).

[0071] In certain embodiments, the adenoviral vector comprises a transgene. A "transgene" refers to a heterologous nucleic acid, which is a nucleic acid that is not naturally present in the vector and, according to the present invention, the transgene can encode an antigenic gene product or antigenic protein that elicits an immune response on the subject. The transgene can, for example, be introduced into the vector by conventional molecular biology techniques. The transgene can, for example, be cloned into an E1 or E3 region deleted from an adenoviral vector, or in the region between the E4 region and the riTR. A transgene is generally operationally linked to expression control sequences. In preferred embodiments, the transgene is inserted into a transgene insertion site.

[0072] If necessary, the nucleic acid sequences encoding hexon and / or fiber polypeptides according to the modalities of the invention and / or the transgene can be optimized by mode codon.

to ensure adequate expression in the treated host (eg human). Codon optimization is a technology widely applied in the art.

[0073] The transgene may be under the control of (ie, operatively linked to) an adenovirus-derived promoter (for example, the Major Late Promoter) or may be under the control of a heterologous promoter. Examples of suitable heterologous promoters include the CMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within an expression cassette.

[0074] In preferred embodiments, the adenoviral vector comprises a nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. Immunogenic compositions

[0075] Immunogenic compositions are compositions comprising an immunologically effective amount of purified or partially purified human or simian adenoviral vectors (e.g. gorilla) for use in the invention. Said compositions can be formulated as a vaccine (also referred to as an "immunogenic composition") according to methods well known in the art. Such compositions can include adjuvants in order to enhance immune responses. The optimal proportions of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present invention.

[0076] Immunogenic compositions according to embodiments of the present invention can be made using methods known to those skilled in the art for the purposes of the present invention. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saline solution or glycols, such as ethylene glycol, propylene glycol or polyethylene-

glycol can be included.

[0077] The immunogenic compositions useful in the invention may comprise adjuvants. Adjuvants suitable for co-administration in accordance with the invention should be those that are potentially safe, well tolerated and effective in people including QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL- 1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, AS01, ASO3, ASOA4, AS15, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Beta-fectin, Alum, and MF59.

[0078] Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines, such as alpha-interferon, gamma interferon, platelet-derived growth factor (PDGF), granulocyte colony stimulating factor (g-CSF) , granulocyte macrophage colony stimulating factor (QMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL- |, I1L-2, I1L-4, IL-6, IL-8, 11 -10, and 11-12 or encoding their nucleic acids.

[0079] The compositions of the invention may comprise an excipient, carrier, buffer, stabilizer or other pharmaceutically acceptable materials 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 precise nature of the carrier or other material may depend on the route of administration, for example, intramuscular, subcutaneous, oral, intravenous, cutaneous, intra-mucous (for example, intestinal), intranasal or intraperitoneal. Method for Inducing Protective Immunity

[0080] Another general aspect of the invention relates to a method of inducing an immune response in a subject in need of it. The methods may, for example, comprise administering to the subject a vaccine comprising an adenoviral vector described herein and a pharmaceutically acceptable carrier. This document also provides methods of producing a vaccine. The methods comprise combining an adenoviral vector described herein with a pharmaceutically acceptable carrier.

[0081] Any of the immunogenic compositions according to the modalities of the invention, including, but not limited to, those described in this document, can be used in the methods of the invention as a vaccine.

[0082] The administration of immunogenic compositions / vaccines comprising the vectors is typically intramuscularly or subcutaneously. However, other modes of administration, such as intravenous, cutaneous, intradermal or nasal injection can also be provided. Intramuscular administration of the immunogenic compositions can be achieved using a needle to inject a suspension of the adenovirus vector. An alternative is to use a needle-free injection device to administer the composition (using, for example, Biojector "Y) or a lyophilized powder containing the vaccine.

[0083] For intravenous, cutaneous or subcutaneous injection, or injection at the affliction site, the vector will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has adequate pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles, such as Sodium Chloride Injection, Ringer Injection, Ringer Lactate Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as needed. A slow release formulation can also be used.

[0084] Typically, administration will have a prophylactic goal of generating an immune response against an antigen of interest (for example, a bacterial, viral, parasitic and / or fungal pathogen) before infection or the development of symptoms. Diseases and disorders that can be treated or prevented according to the invention include those in which an immune response can play a protective or therapeutic role. In other modalities, adenovirus vectors can be administered for post-exposure prophylaxis.

[0085] Immunogenic compositions containing human or ape adenovirus vectors (for example, gorilla) are administered to a subject, giving rise to an immune response to the antigen of interest in the subject. An amount of a composition sufficient to induce a detectable immune response is defined as an "immunologically effective dose" or an "effective amount" of the composition. The immunogenic compositions of the invention can induce a humoral as well as a cell-mediated immune response. In a typical embodiment, the immune response is a protective immune response.

[0086] The actual amount administered, and the rate and time of course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, for example, decisions about dosage etc., is within the responsibility of general practitioners and other doctors, or in a veterinary context of a veterinarian, and typically takes into account the disorder being treated, the condition of the individual patient, the place of administration, the method of administration and other factors known to professionals. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

[0087] After the production of adenovirus vectors and the optional formulation of such particles in compositions, the vectors can be administered to an individual, particularly a human or other primate. Administration can be in humans or another mammal, for example, mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog or cat. Administration to a non-human mammal does not need to be for a therapeutic purpose, but it can be for use in an experimental context, for example, in the investigation of immune response mechanisms for adenovirus vectors.

[0088] In an exemplary regimen, the adenoviral vector is administered (for example, intramuscularly) in a volume ranging from about 100 uL to about 10 mL, containing concentrations of about 10 to 10 "? Virus particles / Preferably, the adenoviral vector is administered in a volume varying between 0.1 and 2.0 ml. For example, the adenoviral vector can be administered with 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 in a concentration of about 107 vp / mL, 108 vp / mL, 10th vp / mL, 10th vp / mL, 5x10 * º vp / mL, 10 * º vp / mL, or 10%? vp / mL Typically, the adenoviral vector is administered in an amount of about 10º to about 10º? viral particles (vp) to a human being during an administration, more typically in an amount of about 10 ° to about 10? vp.

[0089] The initial vaccination can be followed by a booster or a stimulus from a vaccine / composition comprising the same adenoviral vector encoding an antigen of interest or a vaccine / composition comprising a different adenoviral vector encoding the same antigen of interest .

[0090] 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 comprise metal or plastic foil, such as a blister pack. The kit, packaging or dispenser may be accompanied by instructions for administration.

[0091] The compositions of the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated.

MODALITIES

[0092] The invention also provides the following non-limiting modalities.

[0093] Mode 1 is an isolated nucleic acid sequence encoding a hexon polypeptide or functional derivative thereof, comprising a polypeptide comprising hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 1.

[0094] Mode 2 is an isolated nucleic acid sequence encoding a hexon polypeptide comprising a polypeptide encompassing hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1.

[0095] Mode 3 is the nucleic acid sequence isolated from any of modalities 1-2, wherein the hexon polypeptide or its functional derivative 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, with the amino acid sequence of SEQ ID NO: 2.

[0096] Mode 4 is an isolated nucleic acid sequence comprising any of the modalities 1-3 nucleic acid sequences and further comprising an isolated nucleic acid sequence encoding a fiber polypeptide or a functional derivative thereof.

[0097] Mode 5 is the isolated nucleic acid sequence of mode 4, wherein the fiber polypeptide comprises a fiber button polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10.

[0098] Mode 6 is the isolated nucleic acid sequence of mode 4, wherein the fiber polypeptide comprises a fiber axis polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0099] Mode 7 is the isolated nucleic acid sequence of mode 4, wherein the fiber polypeptide comprises a fiber tail polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 12.

[00100] Mode 8 is the nucleic acid sequence isolated from any of the modalities 4-7, wherein 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, along its entire length, with the amino acid sequence of SEQ ID NO: 3.

[00101] Mode 9 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a fiber button polypeptide sequence, wherein the fiber button polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 10.

[00102] Mode 10 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a fiber axis polypeptide sequence, wherein the fiber axis polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11.

[00103] Mode 11 is an isolated nucleic acid sequence encoding a fiber polypeptide comprising a sequence

fiber tail polypeptide, wherein the fiber tail polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12.

[00104] Mode 12 is the nucleic acid sequence isolated from any of the modalities 9-11, wherein the fiber polypeptide comprises the amino acid sequence of a BLY6 fiber polypeptide (SEQ ID NO: 3).

[00105] Mode 13 is a vector comprising nucleic acid of any of modalities 1-12.

[00106] Modality 14 is the vector of modality 13, being an adenoviral vector and still comprising a transgene.

[00107] Mode 15 is a recombinant cell comprising the mode 13 or 14 vector.

[00108] Mode 16 is a method of producing a vector, comprising: (a) the growth of the mode 15 recombinant cell under conditions for the production of the vector; and (b) isolating the vector from the recombinant cell.

[00109] Modality 17 is an immunogenic composition comprising the vector of modality 14.

[00110] Modality 18 is a method to induce an immune response in a subject in need of it, comprising administering to the subject the immunogenic composition of the modality

17.

[00111] Mode 19 is an adenoviral vector comprising (a) at least one transgene; and (b) a nucleic acid sequence co-complicating a hexon polypeptide comprising a polypeptide encompassing hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1.

[00112] Mode 20 is an adenoviral vector comprising (a) at least one transgene; and (b) a co-nucleic acid sequence

impairing a hexon polypeptide comprising the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2).

[00113] Mode 21 is an adenoviral vector comprising (a) at least one transgene; a nucleic acid sequence encoding a hexon polypeptide comprising the amino acid sequence of a BLY6 hexon polypeptide (SEQ ID NO: 2); and (c) a nucleic acid sequence encoding a fiber polypeptide comprising the amino acid sequence of a BLY6 fiber polypeptide (SEQ ID NO: 3).

EXAMPLES Example 1: Generation of vectors deleted in E1 and E3 based on a new adenovirus isolated BLY6

[00114] A new isolated gorilla adenovirus, BLY6 (also called JAd1-WT), has been identified and sequenced. This isolated gorilla adenovirus was found to belong phylogenetically to human adenovirus E (HAdV-E) species. The complete genome nucleotide sequence of BLYG6 was determined to be SEQ ID NO: 5. Description of the single plasmid system used for the generation of BLY6-based Ad vectors

[00115] —pBLY6.dE1.dE3 (SEQ ID NO: 8; Figure 5) and pBLY6.dE1.dE3.51XP (SEQ ID NO: 9; Figure 6) are full-length carrier plasmids, genomes of adenoviral vectors deleted in E1 and E3 based on isolated BLYG6. The Ad vector genome sequences contained within these plasmids are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. Within each of these plasmids, the adenoviral vector genome is flanked by two Swal restriction enzyme sites (that is, a Swal site is located at each end of the vector's genome). These Swal sites are intended to facilitate excision of the Ad vector genome from the plasmid structure before viral rescue by complementary cell transfection

suitable E1 cells (such as HEK293, 911 and PER.C6 cells). The genomes of the Ad vector comprised by these plasmids still contain certain restriction enzyme sites introduced at the E1 deletion site, in the E3 deletion and adjacent to the right inverted terminal repeat (RITR). These restriction enzyme sites were selected to be unique in the context of the plasmids of the complete Ad genome. They represent "transgene insertion sites" that allow easy construction, by standard molecular cloning techniques, of Ad vectors carrying one or more transgene expression cassettes inserted into any of the respective sites or any combination thereof. Ad vector designs and plasmid constructions are described in more detail in the sections below.

BLY6-based Ad vector genome design

[00116] The genomes of the Ad vector based on BLY6 were each designed to comprise an E1 deletion, an E3 deletion, different transgene insertion sites and a replacement for the native E4 open reading frame (orf) 6 and orf6 / 7 by that of human adenoviruses 5 (HAdV-5). The E1 region of each adenovirus was deleted and replaced by a transgene insertion site comprising a sequence of restriction enzyme sites AsiSI. The E3 region of each adenovirus was deleted and replaced with a transgene insertion site comprising a sequence of the Fsel restriction enzyme site. Another transgene insertion site was created by inserting a sequence from the Pacl restriction enzyme site adjacent to the right inverted terminal repeat (ITR) of each adenovirus. BLY6 sequences comprising the coding sequences E4 orf6 and orf6 / 7 have been replaced by SEQ ID NO: 6. This substitution sequence comprises the human adenovirus 5 (HAdV-5) E4 coding sequences orf6 and orf6 / 7 (base pairs 32914-34077 of the AC 000008 sequence from GenBank).

[00117] Two types of deletions in the E1 region were designed and built. The genome of the BLY6-based Ad vector comprised of pBLY6.dE1.dE3 carries a deletion of the E1 region corresponding to the removal of nucleotides 453 to 3,016 of SEQ ID NO: 5. On the other hand, the genome of the BLY6-based Ad vector comprised by PBLY6.dE1.dE3.5I1XP carries a greater sequence deletion comprising the E1 region that removes all E1 coding sequences from BLYG6 (that is, the nucleotides 453 to 3,366 of SEQ ID NO: 5). In addition, this last genome of the Ad vector was also designed to carry a replacement of the stretch of the non-coding sequence between the coding sequences E1B 55K and plX by HAdV-5 (that is, sequences corresponding to nucleotides 3.367 to 3,454 of SEQ ID NO: 5 were replaced by nucleotides 3,510-3,608 of GenBank AC 000008 (i.e., SEQ ID NO: 7)). Construction of unique plasmids comprising BLY6-based Ad vector genomes

[00118] The pBLY6.dE1.dE3 (SEQ ID NO: 8) was built by several steps of gene synthesis (performed by GenScript) and standard molecular cloning procedures. First, a fragment of DNA from

3,586 bp (SEQ ID NO: 15) containing the left end of the desired Ad vector genome (ie encompassing the E1 deletion mentioned above) was synthesized and ligated, as a Mfel-Ndel restriction fragment, into digested pBR322 in EcoRI and Ndel (GenBank accession number - J0O1749.1), leading to intermediate plasmid 1 of BLY6. Second, a 4,138 bp fragment (SEQ ID NO: 16) containing the right end of the desired Ad vector genome (ie, encompassing the aforementioned E3 deletion, partial replacement of the E4 sequence and transgene insertion site adjacent to the rlTR) was synthesized and ligated, as a BamHI-Ndel restriction fragment, into the BLY6 intermediate plasmid 1 digested in BamHlI and Ndel, leading to the BLY6 intermediate plasmid 2. Third, a 4,563 bp fragment (SEQ ID NO: 17) containing an average genome fragment of the Ad vector was synthesized and ligated as a BamHI-Mfel restriction fragment on BLY6 intermediate plasmid 2 digested in BamHlI and Mfel, leading to the plasmid BLY6 intermediate 3 (SEQ ID NO: 18). Fourth, the 18,987 bp BsrGI-BsrGI restriction fragment from the BLY6 viral genome (SEQ ID NO: 5) was ligated into the BLY6 intermediate plasmid 3 digested in BsrGl, leading to the final plasmid pBLY6.dE1.dE3 (SEQ ID NO : 8).

[00119] pBLY6.dE1.dE3.5I1XP (SEQ ID NO: 9) was constructed in the same way as pBLY6.dE1.dE3, except that the BLY6 intermediate plasmid 3 mentioned above (SEQ ID NO: 18) it was first modified to contain the desired E1 deletion and insertion of the Ad5 plX promoter. This was done by synthesizing a 638 bp fragment (SEQ ID NO: 19) which was subsequently ligated as an AsiS | -Agel restriction fragment into the BLY6 intermediate plasmid 3 digested in AsiS | I and Agel.

[00120] pBLY6.FLuc (SEQ ID NO: 20) and pBLY6.RSVF-2A-GLuc (SEQ ID NO: 21) are plasmids derived from pBLY6.dE1.dE3 in which each houses an Ad vector genome based in BLYG6 equipped with a transgene expression cassette inserted at the E1 deletion site. The Ad vector genome sequences carried within these plasmids are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. PBLY6.FLuc carries a transgene expression cassette for firefly luciferase (FLuc). This cassette is triggered by the main cytomegalovirus immediate early promoter (ie, the "CMV promoter") and contains a polyadenylation signal derived from SV40. pBLY6.RSVF-2A-Gluc carries a transgene expression cassette for "RSV-Fa2-2A-GLuc" (RSVF-2A-GLuc), which is a chimeric protein composed of the A2 fusion glycoprotein from the respiratory syncytial virus strain, a 2A peptide from the FMD virus and Gaussia luciferase (GLuc). Like the Fluc cassette, this cassette is activated as a CMV promoter and carries a SVA40 polyadenisation signal. In addition, that cassette contains in its 5 'untranslated region a sequence comprising intron 2 of the human apolipoprotein A1 gene. The expression cassettes Fluc and RSVF-2A-gluc were each constructed by various standard gene synthesis and molecular cloning steps, after which they were ligated into the single enzyme restriction site of pBLY6.dE1.dE3, generating pBLY6.FLuc and pPBLY6.RSVF-2A-Gluc, respectively.

Generation and production of adenoviral vectors based on BLY6

[00121] The adenoviral vectors BLY6.Fluc (also called JAdDINVTOO3) and BLY6.RSVF-2A-Gluc (also called JAd1NVTOO1), which respectively comprises the genomic sequences of the adenoviral vector SEQ ID NO: 22 and SEQ ID NO: 23, were generated by transfection of the corresponding Ad vector genome plasmids (ie, PpBLY6.Fluc and pBLY6.RSVF-2A-Gluc) in PER.C6 cells that complement E1. Before transfection in PER.C6 cells that were grown as adherent cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 mM MgCl2, the plasmids of the Ad vector genomes. were digested with Swal in order to release the respective genomes of the plasmid adenoviral vector. Transfections were performed according to standard procedures using the Lipofecamine transfection reagent (Invitrogen; Carlsbad, CA). After harvesting viral rescue transfections, the viruses were further amplified by several successive rounds of infection in PER.C6 cell cultures. The viruses were purified from crude viral crops using a two-step cesium chloride density gradient ultracentrifugation procedure (CsCIl), 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 (PV) titers were measured by a spectrophotometry-based procedure described earlier (Maizel et al., "The polypeptides of adenovirus: |. 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 the new adenoviral vectors

[00122] Examples 2 and 3 describe experiments carried out to evaluate the immunogenicity of the new adenoviral vectors based on BLY6 generated there. In these experiments, the new vectors were evaluated for their ability to induce human and cellular immune responses against antigens (model) encoded by vectors in mice after intramuscular immunization. The vectors were tested using two different antigens: Firefly Luciferase (FLuc) and RSV-Fa2-2A-GLuc (RSVF-2A-GLuc). RSVF-2A-GLuc is a chimeric protein composed of the A2 fusion glycoprotein of the respiratory syncytial virus strain, a foot-and-mouth disease virus 2A peptide and Gaussia luciferase (GLuc). Each vector was compared side by side with 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 in document as Ad49) carrying the same transgenic cassette encoding the antigen. Immune responses against the respective antigens were measured using well-known immunological assays, such as enzyme-linked "immunospot" assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA) and, in the case of the RSVF- antigen 2A-GLuc, a neutralization test for the syncytial virus

piratory (VNA). Example 2: Cellular immune responses induced by BLY6.FLuc

[00123] In order to assess the cellular immunogenicity of the new adenoviral BLY6 vector, Balb / C mice were immunized by intramuscular injection with Ad26.FLuc, Ad49.Fluc (positive controls), a firefly luciferase expressing the vector BLY6 (BLY6.FLuc), or with an adenovector not encoding a transgene (empty Ad26). Two doses of vector were tested for administration: 10th and 10th viral particles (vp) per mouse. Two weeks after immunization, the mice were sacrificed and the splenocytes were stimulated during the night with a set of peptides with 15mer overlapping Fluc (experimental configuration in Figure 1A). Cellular immune responses were determined by an ex vivo ELISPOT assay measuring the relative number of IFN-γ-secreting cells (Figure 1B). The results show that in the high dose (10º) immunization, the cellular immune responses induced by BLY6 were almost as high as the response observed for Ad26.Fluc. In contrast, in the lowest dose (10º) immunization, BLY6.Fluc gave a stronger response than Ad26.Fluc.

[00124] In general, cellular immune responses induced by the recombinant adenoviral BLY6 expressing FLuc of the invention clearly indicate a potent immunogenicity of this vector in mice. Example 3: Cellular and humoral immune responses induced by BLY6.RSVF-2A-GLuc

[00125] The immunogenicity of the new adenoviral vector BLY6 was further evaluated using RSV-Fa2-2A-GLuc (RSVF-2A-GLuc) as a vector-encoded vaccine antigen (model). The Balb / C mice were immunized intramuscularly with Ad26.RSVF-2A-GLuc (positive control) or BLY6.RSVF-2A-GLuc (both at 108, 10 and 10

viral cells per mouse), or with Ad26.FLuc or BLY6.FLuc (both 10th viral particles per mouse). The mice were sacrificed after eight weeks and blood and splenocyte samples were collected (Figure 2A). Different immune parameters were assessed as described below.

[00126] A virus neutralization assay was performed in order to assess the ability of BLY6.RSVF-2A-GLuc to represent the virus neutralizing antibodies to the syncytial virus. Figure 2B represents the VNA titers of strain A2 of the respiratory syncytial virus (RSV A2) measured for serum samples taken eight weeks after immunization. Each dot represents a mouse; the bars represent the group mean and the dotted line corresponds to the lower limit of quantification (LLOQ = 6.88; title of the average end point of the linearity samples). The results show that the 10th vp dose immunizations with BLY6.RSVF-2A-GLuc gave rise to higher RSV A2 neutralization titers than those found for the reference vector Ad26 encoding the same antigen. The titers against BLY6.RSVF-2A-GLuc were detected mainly at the highest dose used for immunization, 10 * º vp. As expected, no specific RSV A2 responses were detected against adenovectors encoding firefly luciferase.

[00127] The induction of cellular immunity against the vector-encoded antigen was evaluated by an ELISPOT assay specific to RSV-Faz. To that end, eight weeks after immunization, splenocytes from immunized mice were isolated and stimulated during the night with overlapping 15mer peptides spanning the RSV-Fa2 protein and cellular immune responses were determined by ex-vivo ELISPOT assay. measuring the relative number of cells secreting IFN-y. The data show that antigen-specific cellular immune responses induced by the new BLY6 vector encoding RSVF-

2A-GLuc were dose dependent and, per dose, similar in magnitude to that induced by the reference vector, Ad26.RSVF-2A-Gluc (Figure 2C). As expected, no specific RSVF-Fa response was measured from splenocytes of mice immunized with adenovectors encoding firefly Luciferase.

[00128] The ability of vectors expressing RSVF-2A-Gluc to represent RSV-Fa2-specific IgG antibodies was assessed by ELISA. Sera collected 8 weeks after immunization of mice immunized with Ad26 (positive control) and BLY6 vectors expressing RSVF-2A-Gluc transgene or firefly luciferase (control) were tested in an IgG antibody ELISA. anti-RSV Faz. Specifically, this ELISA detects IgG antibodies capable of binding to a recombinant stable pre-fusion protein RSV-Fa2 (pre-RSV-F). The results show that BLY6.RSVF-2A-GLuc caused higher dose-dependent IgG antibody titers, higher than those induced by Ad26.RSVF-2A-GLuc (Figure 2D) . As expected, no specific antibody titers for RSV-Fa2 were detected in sera from mice immunized with vectors encoding only firefly luciferase.

[00129] Together the data show that the BLY6 vector induced a potent humoral and cellular immune response against the encoded antigens, similar or higher than that induced by the reference vector based on HAdV-26. These immune responses clearly indicate a potent immunogenicity of the BLY6 vector in mice. Example 4: Serological cross-neutralization assessment between new adenoviral vectors and existing ones

[00130] For their potential usefulness as new adenoviral vaccine vectors, the new adenoviral BLY6 vectors created in this document would preferably be serologically distinct from existing adenoviral vectors currently under development as vaccine vectors, such as vectors based on serotypes of human adenoviruses HAdV-5 and HAdV-35. Therefore, cross-neutralization tests were performed between the new adenoviral vectors BLY6 and several existing vectors based on HAdV-4, HAdV-5, HAdV-26, HAdV-35 and HAdV49. To that end, mouse antisera, each created against one of these adenoviral vectors, were tested against each of the different vectors in an adenovirus neutralization assay. The mouse antisera used for this assay were collected from Balb / C mice, two or eight weeks after their immunization with 10 vector particles per mouse. The adenovirus neutralization assay was performed as previously described (Spangers et al.

2003. J.Clin. Microbiol. 41: 5.046-5.052). Briefly, starting at a 1:16 dilution, the sera were diluted 2 times in series and then pre-mixed with adenoviral vectors expressing firefly luciferase (FLuc) and subsequently incubated overnight with A549 cells ( in the multiplicity of infection of 500 virus particles per cell). Levels of luciferase activity in infected cell lines, measured 24 hours after infection, represented efficiencies of vector infection. The neutralization titers against a given vector were defined as the highest serum dilution capable of giving a 90% reduction in the vector's infection efficiency. The neutralization titles were arbitrarily divided into the following categories: <16 (without neutralization), 16 to 200, 200 to 2,000 and>

2,000. The results do not show much cross-neutralization between the vectors tested (Figure 3). There was only a slight one-way cross-neutralization seen between vectors BLY6 and Ad26, with the antiserum BLY6 exhibiting a neutralization titer against Ad26 of

16.12 (ie, just above the lower detection limit of 16) and the antiserum Ad26 does not exhibit a neutralization titer against BLY6. Thus, the new adenoviral vector BLY6 did not exhibit any cross-neutralization, or just very light, with the human adenoviral vectors included in the tested panel, that is, Ad26, Ad35, Ad49, Ad5 and Ad4. Therefore, this vector could potentially be used in combination with one or more of these or other distinct adenoviral vectors in sequential immunizations, for example, in the context of a heterologous primary booster vaccination regimen or, alternatively or in the context of a series of two or more consecutive vaccination regimes against different diseases or antigens. Example 5: Seroprevalence of new adenoviral vectors in human populations

[00131] Important for their potential use as effective vaccine vectors is that the new adenoviral vectors described in this document are not affected by the high levels of pre-existing humoral antiviral immunity in target vaccine populations. Therefore, the BLY6 vector was evaluated for its seroprevalence in 200 serum samples from adult human cohort, aged between 18 and 55 years old, residing in the United States (USA) and the European Union (EU). The vector was tested for neutralization by human serum samples, performing a standard adenovirus neutralization assay, as performed in Example 3 and described previously (Spangers et al. 2003. J.Clin. Microbiol. 41: 5046 -5052). Briefly, starting at a 1:16 dilution, the sera were diluted 2 times in series and then premixed with the adenoviral vectors expressing the firefly luciferase (FLuc) and subsequently incubated for overnight with A549 cells (at a multiplicity of infection of 500 virus particles per cell). Levels of luciferase activity in infected cell lysates, measured 24 hours after infection, represented efficiencies of vector infection. The neutralization titers against a given vector were defined as the highest serum dilution capable of giving a 90% reduction in the vector's infection efficiency. The neutralization titles were arbitrarily divided into the following categories: <16 (without neutralization), 16 to 300, 300 to 1,000, 1,000 to 4,000 and> 4,000.

[00132] The results indicate that the adenovirus BLY6 vector has a considerably lower seroprevalence in the studied human subjects than the control vector Ad5 and the reference vectors Ad26 and Ad35 (Figure 4). In addition, the positive neutralization titers that were seen against the new BLY6 vectors were generally quite low, mainly not more than 300. On the other hand, most of the positive neutralization titers found against Ad26 and Ad5 were higher than 300.

[00133] Taken together, the data above indicate that the pre-existing humoral anti-immunity immunity against BLY6 vectors can be considered to be low in the evaluated vaccine target populations, suggesting that these vectors have potential as effective vaccine vectors in these populations . Example 6: Productivity of the adenoviral vector in the PER.C6 cell suspension

[00134] Adenovirus vectors to be used in clinical trials and beyond need to be rapidly produced for elevated titers on a scalable, serum-free adenovirus production platform. PER.C60 cells adapted to the suspension, also referred to in this document as PER.C6 cells of suspension or SPER.C6, represent this platform since they have been shown to support the large-scale manufacture of adenoviral vectors in bioreactors, reaching large amounts of high grade clinical grade vector preparations, for example, vectors deleted in E1 based on HAdV-26 or HAdV-35 (EP 2536829 B1, EP 2350268 B1).

[00135] As an initial assessment of whether the new vectors described in this document would fit into production processes based on sPER.C6 cells, small scale vector productivity experiments were performed on cultured sPER.C6 cells in shaker flasks. These productivity experiments were performed using the Fluc-coded version of the new Ad BLY6 vector described in Example 1. Taken as a reference control was the Ad26.Fluc vector based on HAdV-26. The PER.C6 cell cultures in suspension, sown in shaker flasks at a density of 1x10º cells / mL, in a total volume of 10 mL of PERMEXCISO medium (available from Lonza) supplemented with 4-glutamine mM (Lonza), were infected with different vectors in different proportions of virus particles (PV) to cell and then incubated for 4 days. The different VP to cell ratios used for infection were 70, 150 and 900. Samples of infected cell cultures were collected every day and the VP titers were determined in these samples by a quantitative PCR-based protocol (gPCR) that employs primers and specific probe for the CMV promoter (which is present in all tested vectors). This protocol involves DNAse treatment of test samples preceding qPCR in order to remove any vector-free DNA (that is, vector genomes that are not packaged in viral particles).

[00136] The productivity results obtained for the new vector BLY6.Fluc are shown in Figure 8. BLY6.Fluc exhibited higher PV titers than the reference control vector Ad26.Fluc in all proportions of PV infection for cell and harvest time points tested. These results demonstrate good productivity of the new BLY6 vector in a cell culture model in suspension without serum based on sSPER.C6.

[00137] Collectively, studies of humoral and cellular immune responses induced by the new recombinant BLY6-based adenoviral vectors of the invention, as presented above, clearly indicate potent immunogenicity of these vectors in mice. Furthermore, the vectors have been shown not to induce responses of cross-neutralization antibodies, or only very limited, against certain candidates for existing adenoviral vaccine vectors (for example, Ad26 and Ad35) or vice versa. In addition, the new vectors showed low seroprevalence in humans. Finally, the new vectors can be easily produced at high yields. The combination of low seroprevalence, immunogenicity and potent productivity suggests that the new adenoviral vectors of the invention may be useful as new candidates for vaccine vectors against a variety of pathogens and may also be useful in gene therapy and / or diagnosis .

[00138] It will be appreciated by experts in the art that changes can be made in the modalities described above without departing from its broad inventive concept. Consequently, it is understood that this invention is not limited to the particular modalities described, but is intended to encompass modifications within the spirit and scope of the present invention as defined by the present description.

Claims (17)

1. Isolated nucleic acid sequence encoding a hexon polypeptide or a functional derivative thereof, characterized by the fact that it comprises a polypeptide comprising hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence which is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 1. 2. Isolated nucleic acid sequence according to claim 1, characterized in that the hexon polypeptide comprises a polypeptide comprising hypervariable regions of hexon having the amino acid sequence of SEQ ID NO: 1. 3. Isolated nucleic acid sequence according to claim 2, characterized in that the hexon polypeptide or its functional derivative comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that it is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 2. Isolated nucleic acid sequence according to any one of claims 1 to 3, characterized in that it further comprises a nucleic acid sequence encoding a fiber polypeptide or a functional derivative thereof. An isolated nucleic acid sequence according to claim 4, characterized in that the fiber polypeptide comprises at least one selected from the group consisting of: (1) a fiber button polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10, (2) a fiber axis polypeptide sequence 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, preferably, the fiber polypeptide or a functional derivative thereof comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical, along its entire length, with the amino acid sequence of SEQ ID NO: 3. 6. Isolated nucleic acid sequence encoding a fiber polypeptide, characterized by the fact that it comprises at least one selected from the group consisting of: (1) a fiber button polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 10; (2) a fiber axis polypeptide sequence 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. An isolated nucleic acid sequence according to claim 6, characterized in that the fiber polypeptide comprises the amino acid sequence of SEQ ID NO: 3. 8. Vector, characterized by the fact that it comprises nucleic acid, as defined in any one of claims 1 to 7. 9. Vector, according to claim 8, characterized by the fact that it is an adenoviral vector and also comprises a transgene. 10. Adenoviral vector according to claim 9, characterized by the fact that the adenoviral vector further comprises at least one of an E1 deletion and an E3 deletion. 11. Adenoviral vector according to claim 9 or 10, characterized in that the adenoviral vector is a chimeric adenoviral vector comprising one or more human adenoviral nucleic acid sequences, preferably human adenoviral nucleic acid sequences are the from at least one of human adenovirus 4, human adenovirus 5, human adenovirus 26 or human adenovirus 35. 12. Adenoviral vector according to any of claims 9 to 11, characterized by the fact that the adenoviral vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 . 13. Recombinant cell, characterized by the fact that it comprises the vector, as defined in any one of claims 8 to 12. 14. Method for producing a vector, characterized in that it comprises: (a) growth of the recombinant cell, as defined in claim 13, under conditions of production of the vector; and (b) isolating the vector from the recombinant cell. 15. Immunogenic composition, characterized by the fact that it comprises the adenoviral vector, as defined in any of claims 9 to 12. 16. Method to induce an immune response in a subject in need of it, characterized by the fact that it comprises the administration to the subject of the immunogenic composition, as defined in claim 15. 17. A vaccine production method, characterized in that it comprises the combination of an adenoviral vector, as defined in any one of claims 9 to 12, with a pharmaceutically acceptable carrier.