ES2370674T3 - E2 POLYPEPTIDE OF PAPILOMAVIRUS USED FOR VACCINATION. - Google Patents

Abstract

Vector comprising nucleic acid molecules encoding at least two E2 papillomavirus polypeptides, wherein said nucleic acid molecules encoding E2 do not comprise a portion of 40 or more contiguous nucleotides showing a percentage of identity of 75% or greater at 75% between any two of said nucleic acid molecules encoding E2.

Description

E2 papillomavirus polypeptide used for vaccination.

The present invention relates to a vector comprising nucleic acid molecules encoding at least two papillomavirus E2 polypeptides, wherein said nucleic acid molecules encoding said E2 do not comprise a portion of 40 or more contiguous nucleotides showing a percent identity of 75% or greater than 75% between any two of said nucleic acid molecules encoding E2. The invention is of very special interest in the field of immunotherapy, and more particularly for treating patients suffering from a persistent papillomavirus infection.

Papillomaviruses have been isolated in several higher organisms, in which they infect the epithelial tissues of the skin and mucous membranes. Currently, more than 100 human papillomavirus (HPV) genotypes have been identified in humans (Stoler, Int. J. Gynecol. Path. 19: 16-28, 2000), which can be classified into genotypes of " under risk " usually associated with benign tumors (for example HPV-6 and HPV-11) and genotypes of "high risk" that are associated with lesions with the potential to progress to premalignant lesions (for example cervical intraepithelial neoplasia, CIN) and finally malignant tumors. For example, more than 99% of cervical cancers contain HPV DNA and five HPV (HR-HPV) genotypes of "high risk" have been identified. as the main cause, detecting HPV-16 and HPV-18 in approximately 70% of invasive cervical cancers diagnosed worldwide (Clifford et al., Br. J. Cancer 88: 63-73, 2003), while HPV -31, HPV-33 and HPV-45 explain an additional 10% (Cohen et al., Science 308: 618-621, 2005).

Papillomaviruses are small-sized DNA viruses surrounded by a protein capsid (see, for example, Pfister, 1987, in: The papovaviridae: The Papillomaviruses, edition of Salzman and Howley, Plenum Press, New York, pages 1 to 38 ). The genome is a circular double-stranded DNA of approximately 7,900 base pairs consisting of three functional regions, the early (E), late (L) and long control (LCR) regions. The CSF contains transcription regulatory sequences, such as enhancers and promoters. The late region encodes the structural proteins L1 and L2, respectively the major and minor capsid proteins, while the early region encodes regulatory proteins (E1-E7), predominantly present in the nucleus, which control viral replication, transcription and cellular transformation

The E1 protein is the largest (HPV-16 E1 has a length of 649 amino acids) and the most conserved encoded by the papillomavirus genome. E1 is a DNA-binding phosphoprotein with an ATP-dependent helicase activity that requires dimerization and interaction with E2 to stimulate viral replication (Desaintes and Demeret, Semin. Cancer Biol. 7: 339-347, 1996; Wilson et al. ., Virus Gene 24: 275-290, 2002). The helicase activity has been located in the C-terminal domain of E1 and the DNA binding domain, in the central domain. The E2 protein (HPV-16 E2 has a length of 365 amino acids) is a multifunctional DNA binding phosphoprotein that regulates viral gene transcription and controls DNA replication (Bechtold et al.,

J. Virol. 77: 2021-2028, 2003). The regulation of viral transcription requires the dimerization and binding of E2 dimers to an E2 binding site (ACCN6GGT consensus sequence), the context of which determines whether viral transcription is transactive or repressed (Ham et al., Trends Biochem Sci. 16: 440-444, 1991; McBride et al., J. Biol. Chem. 266: 18411-18444, 1991). E2 also provides repression of the HP97-16 p97 promoter, which controls the expression of the E6 and E7 oncoproteins. Finally, E2 is involved in the distribution of the viral genome in daughter cells. The N-terminal domain of HPV-16 E2 is responsible for transactivation, interaction with E1 and stimulation of replication, while the C-terminal domain is involved in DNA binding and dimerization (McBride et al., Proc. Natl. Acad. Sci. USA 86: 510-514, 1989). The protein encoded by E4 binds and alters the cytoplasmic keratin network and plays a role in viral maturation. It is not yet known exactly what the function of the E5 protein is. The E6 and E7 proteins are implicated in the oncogenic transformation of HR-HPV infected cells (Kanda et al., J. Virol. 62: 610613, 1988; Vousden et al., Oncogene Res. 3: 1-9, 1988; Bedell et al., J. Virol. 61: 3635-3640, 1987) through the binding of these viral proteins to p53 tumor suppressor and retinoblastoma (Rb) cellular gene products, respectively (reviewed in Howley, Papillomaviruses and their replication, pages 2045 to 2076, in: BN Fields,

D.M. Knipe and P.M. Howley (editors), Virology, 3rd edition, Lippincott-Raven Press, New York, N.Y., 1996).

HPV infection is one of the most frequent sexually transmitted infections and approximately 25% of sexually active adults are infected with HPV (Woodman et al., The Lancet 357: 1831-1836, 2001). Approximately 80% of the subjects manage to spontaneously eradicate the virus in 6 to 12 months (Ho et al., N. Engl. J. Med. 338: 423-428, 1998). However, in the remaining 20%, HPV infection progresses to premalignant CIN lesions that, if not diagnosed, can lead to invasive cancers (O'Shaughnessy et al., Clinical Cancer Research 2: 314-346, 2002). The neoplasm-inducing mechanism apparently involves the integration of the HPV genome into cell chromosomes (Cullen et al., J. Virol. 65: 606-612, 1991). In most cases, the above leads to the disruption of HPV genomic DNA in the E1 / E2 region, to the release of the E6 / E7 promoter from the repressive effect of E2 and consequently to the positive regulation of the expression of E6 and E7 Already the cellular transformation.

Currently, prophylactic vaccines intended to prevent HPV infection are close to commercialization. They present as capsid proteins expressed on the viral surface, in order to block the virus before it enters the host cells mainly by inducing neutralizing antibodies. They are generally based on recombinant production L1 proteins that spontaneously reassemble forming VLP (virus-like particles). Two vaccines for HPV manufactured by Merck and GlaxoSmithKline (GSK) have successfully completed phase III clinical trials showing an efficacy of almost 100% in the prevention of cervical infections of a specific type. The GSK vaccine comprises a mixture of HPV-16 and HPV-18 VLPs, while Merck has also included HPV-6 and HPV-11 VLPs, which cause genital warts.

However, subjects already infected with HPV cannot select for prophylactic vaccination and therapeutic vaccines would be of interest to treat infected patients at risk of developing lesions with oncogenic potential. Because the oncogenic activity has been attributed to the expression of the HPV E6 and E7 genes in infected cells, much effort has been devoted to blocking their expression or inducing a cellular immune response against such transforming gene products. Numerous approaches have been described in the literature, for example based on the use of antisense RNA (Steele et al., Cancer Res. 53: 2330-2337, 1993; He et al., Cancer Res. 57: 3993-3999, 1997 ; Choo et al., Gynecol. Oncol. 78: 293-301, 2000), ribozymes (Chen et al., Cancer Gen. Ther. 3: 18-23, 1996; Pan et al., Mol. Ther. 9: 596-606, 2004), siRNA (Butz et al., Oncogene 22: 5938-5945, 2003; Koivusalo et al., Mol. Pharmacol. 68: 372-382, 2005), immunogenic peptides (Feltkamp et al., Eur J. Immunol. 23: 2242-2249, 1993), plasmids encoding E6 and / or E7 (Peng et al., J. Virol. 78: 8468-8476, 2004) and viral vectors (WO 90/10459 and 99/03885; Kaufmann et al., Clinical Cancer Res. 8: 3676-3685, 2002).

Because it is expressed at an early stage of HPV infection, the E2 protein represents a second potential target for therapeutic vaccination. Preclinical studies in rabbits infected with rabbit cottontail papillomavirus (CRPV) have shown protection against implemented lesions associated with papillomavirus after E2 expression. For example, the administration of a recombinant adenovirus vector expressing CRPV E2 protein resulted in CRPV-induced papilloma removal and infection, probably through cell-mediated immunity (Brandsma et al., J. Virol. 78: 116-123, 2004). The administration of DNA encoding the E1 and E2 proteins of CRPV was also effective in preventing or at least delaying the development of carcinoma from skin papillomas induced by CRPV (Han et al., J. Virol. 74 : 9712-9716, 2000), although it was shown that protective immunity depended on the route of administration (Han et al., Vaccine 18: 2937-2944, 2000). The therapeutic potential of E2 to treat papillomavirus-associated lesions was also demonstrated in human subjects exposed to HPV. Immunity of specifically anti-E2 helper T cells was frequently detected in healthy subjects (De Jong et al., Cancer Res. 62: 472-479, 2002), while immunity of altered CD4 + T cells against E2 and E6 was observed in women with cervical cancers associated with HPV-16 (De Jong et al., Cancer Res. 64: 5449-5455, 2004).

Phase II clinical trials are currently being conducted in patients with high-grade CIN 2 and 3 associated with HPV using a recombinant MVA (modified Ankara virus) vector encoding a bovine papillomavirus E2 protein (BPV). Viral particles are injected directly into CIN lesions and the production of E2 in cells expressing E6 and E7 is expected to lead to their apoptosis. Indeed, a regression of CIN lesions was observed in the majority of treated patients. However, in some cases, viral DNA was not removed and lesion recurrence was detected 1 year later (García-Hernández et al., Cancer Gene Ther. 13: 592-597, 2006).

HPV is expected to continue to be a serious global health threat for many years due to the persistent nature of the infection, its high prevalence and the significant morbidity of HPV-induced cancers. Indeed, it has been shown that women with persistent HR-HPV infection have a significantly higher risk, up to 200 times higher, of developing CIN lesions compared to uninfected women or women who manage to eliminate the virus spontaneously (Bory et al ., Int. J. Cancer 102: 519-525, 2002).

HPV infection is usually detected after abnormal screening (for example, a Pap smear). At present, the only medical advantage of the diagnosis of HPV infection is the implementation of more frequent monitoring in order to detect lesions (for example CIN2 / 3 high grade) as soon as they occur, which in this case can removed by ablative procedures such as electrosurgical loop excision (LEEP) and conical biopsy (conization). These procedures are globally efficient at 90%; however, they present a risk of obstetric complications (for example with an impact on the reproductive potential of women of childbearing age). In addition to not being totally satisfactory from a medical point of view, they also imply discomfort for the patient (anxiety).

Therefore, there is a need to develop a vaccine to treat patients with persistent HPV infection, especially in view of the high risk in this population of progression to premalignant lesions and finally to cancer.

In this way, the present invention represents a significant advance in this context. The present description provides a non-invasive and safe procedure that offers earlier protection against infections caused by HR-HPV genotypes. It has the advantage of providing a treatment for infected patients before the lesions associated with papillomavirus occur and, consequently, of reducing the risks associated with conventional ablative procedures (for example obstetric complications), increasing comfort for the patient (for example reducing anxiety associated with the recognition of injuries). It is important that the present invention could also allow the reduction of the risk of future incidents of HPV reinfection through the eradication of infectious papillomavirus and related isolates thereof.

The presence of homologous sequences in a vector comprising nucleic acid molecules encoding at least two E2 papillomavirus polypeptides is expected to negatively influence its stability, especially during the vector production stage. Recombination between homologous sequences can occur, possibly leading to the loss of the part between the two homologous sequences.

In WO 92/16636, said problem is addressed by arranging a pair of homologous nucleotide sequences in the viral vector so that they are inverted relative to each other.

In the vector of the invention, this technical problem is solved by providing the embodiments defined in the claims.

Other aspects and aspects, features and additional advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are provided for the purpose of publication.

In accordance with the foregoing, in a first aspect, the present invention provides a vector comprising nucleic acid molecules encoding at least two papillomavirus E2 polypeptides, wherein said E2 encoding nucleic acid molecules do not comprise a part of 40 or more contiguous nucleotides showing an identity of 75% or greater than 75% between any two of said nucleic acid molecules encoding E2.

As used herein throughout the application, the terms " a " and "one" are used in the sense of "at least one", "at least a first", "one or more" or "a plurality" of the compounds or steps referenced, unless the context indicates otherwise. For example, the expression "an E2 polypeptide of papillomavirus" it comprises a unique type of papillomavirus E2 polypeptide or a plurality of papillomavirus E2 polypeptides that includes a mixture thereof.

The term "and / or" as used herein includes the meaning of " and ", " or " and "all or any other combination of the elements connected by said term".

The term "around" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

The terms " amino acids " and "waste" they are synonyms and comprise natural amino acids, as well as amino acid analogs (for example, unnatural, synthetic and modified amino acids, including optical isomers D or L).

The terms " polypeptide ", " peptide " and "protein" As used herein they interchangeably refer to polymers of amino acid residues comprising nine or more amino acids linked by peptide bonds. The polymer may be linear, branched or cyclic and may comprise natural amino acids and / or amino acid analogs and may be interrupted by non-amino acids. By way of general indication, in the event that the amino acid polymer is long (for example of more than 50 amino acid residues), it is preferably referred to as a polypeptide or protein, while in the case that it has a length of 50 amino acids or higher, is called "peptide".

The expressions " nucleic acid ", " nucleic acid molecule ", " polynucleotide " and "nucleotide sequence" they are used interchangeably herein and define a polymer of any length of polydeoxyribonucleotide (DNA) (eg cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixture thereof) or molecules thereof polyiribonucleotide (RNA) (for example mRNA or antisense RNA) or mixed polyiribo-polydeoxyribonucleotides. They comprise polynucleotides of a chain or double chain, linear or circular, natural or synthetic. In addition, a polynucleotide may comprise unnatural nucleotides, such as methylated nucleotides and nucleotide analogs (see US Patent Nos. 5,525,711, No. 4,711,955 or EP-A-302 175 as examples of modifications) and may be interrupted by non-nucleotide components. If present, modifications of the nucleotides can be made before or after polymerization.

As used herein, in its use to define products, compositions and methods, the expression "comprising" It is intended to refer to the fact that the products, compositions and methods include the referenced components or stages, although not excluding others. The expression "consisting essentially of" It is intended to refer to the exclusion of other components or stages of any essential significance. Thus, a composition consisting essentially of the indicated components would not exclude trace components or pharmaceutically acceptable carriers. The expression "consisting of" it refers to the exclusion of more than trace elements from other components or stages. For example a polypeptide "consists of" an amino acid sequence in the event that the polypeptide does not contain another amino acid than the indicated amino acid sequence. A polypeptide "consists essentially of" an amino acid sequence in the event that said amino acid sequence is present in conjunction with only a few additional amino acid residues, typically between about 1 and about 50 additional residues. A polypeptide "comprises" an amino acid sequence in case the amino acid sequence is at least part of the final amino acid sequence of the polypeptide. Said polypeptide may have between a few and several hundred additional amino acid residues. Such additional amino acid residues can play a role in polypeptide trafficking, facilitate the production or purification of the polypeptides and prolong the half-life, among other aspects. The same applies to nucleotide sequences.

The expression "host cell" It should be broadly understood so that it comprises isolated cells, a group of cells, as well as a particular organization of cells, for example in a tissue or organ. Said cells may be primary, transformed or cultured cells. They can be prokaryotic (for example Escherichia coli), yeast (for example Saccharomyces cerevisiae, Saccharomyces pombe or Pichia pastoris), eukaryotic (for example, insect, plant and mammalian cells, including humans). The expression "host cell" it includes cells that can be or have been receptors of the nucleic acid molecule, of the vector or of the infectious viral particle used in the present invention and the progeny of said cells.

The expression "host organism" it refers to a vertebrate, in particular a member of the mammalian species and especially domestic animals, farm animals, competition animals and primates, including humans. Preferably, the host organism is a patient suffering from a persistent papillomavirus infection caused by at least one papillomavirus.

The term " papillomavirus " It refers to a virus that belongs to the subfamily papilomavirinae. The definition includes animal papillomaviruses of origin, which include, but are not limited to, cows, horses, rabbits, sheep, dogs, nonhuman primates and rodents, as well as human papillomavirus (HPV).

The term " HPV " it refers more specifically to the papillomavirus of origin of the human species and / or to which a human being can infect. More than 100 HPV genotypes have been identified today and numbered according to the chronological order of isolation. Conventionally the classification of HPV is based on the degree of relationship of their genomes. A phylogenetic tree has been constructed from the alignment of the available nucleotide sequences (Van Ranst et al., J. Gen. Virol. 73: 2653, 1992; De Villiers et al., Virology 324: 17-27, 2004 ). HPV can be classified as "high risk" (HR-HPV) and "low risk" (LR-HPV). HR-HPV refers to HPV closely associated with cell transformation that can lead to lesions with the potential to progress to malignant lesions. Types of HR-HPV include, but are not limited to, HPV-16, HPV-18, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, HPV-68, HPV-70 and HPV

85. LR-HPV refers to HPV that has a weak cellular transformation potential, which can lead to benign lesions, such as warts with a low potential for progress to malignant lesions. LR-HPV types include, but are not limited to, HPV-6 and HPV-11.

Papillomaviruses can be isolated, cloned or derived from any source in nature. Such sources include biological samples, cultured cells, as well as recombinant materials. As used herein, a "biological sample" It comprises a variety of samples collected from a host organism that has been exposed to a papillomavirus that can be used as a source of papillomavirus or in a diagnostic or follow-up test. In the context of the invention, a biological sample may have been manipulated in any way after collection, such as by treatment with reagents, solubilization or enrichment for certain components (for example polypeptides or nucleic acid molecules). The definition includes biological liquids (for example blood, plasma or sera), liquid samples (for example vaginal samples, cervical fluids and cytological samples), solid tissue samples (for example tissue sections, biopsy specimens) and tissue cultures. The expression "cultured cells" it comprises the cells in culture (for example the CaSki cells available from the ATCC), cell supernatants and cell lysates. Recombinant materials comprise, in a non-limiting manner, papillomaviruses (for example available at depository institutions), the papillomavirus genome, genomic or cDNA libraries, plasmids containing one or more papillomavirus genome fragments or any vector of the technique. Previous that is known to include these elements.

For general information, the nucleotide sequences of several papillomavirus genomes and the amino acid sequences of the encoded polypeptides have been described in the literature and are available in specialized databases, for example Genbank accession numbers NC_01526 and K02718 in relation to HPV-16; NC_001375 and X05015 in relation to HPV-18; J04353 in relation to HPV-31; M12732 in relation to HPV-33; NC_001529 in relation to HPV-35; NC_001535 in relation to HPV-39; X74479 in relation to HPV-45; NC_001533 in relation to HPV-51; NC_001592 in relation to HPV-52; X74483 in relation to HPV-56; D90400 in relation to HPV-58; NC_001635 in relation to HPV-59; X67160 and M73258 in relation to HPV-68; U21941 in relation to HPV-70 and AF131950 in relation to HPV-85.

As used herein, the expression "papillomavirus E2 polypeptide" it comprises native E2 polypeptides (ie, expressed from an E2 ORF in a natural source of papillomavirus), modified E2 polypeptides and immunogenic peptides thereof.

An immunogenic E2 peptide has at least 9 amino acids and this term includes E2 epitopes (for example specific amino acid motifs that can induce or activate an immune response through a route mediated by MHC class I and / or II, such as indicated in EP 523 395), a multi-epitope construct (for example as described in WO 2005/089164) and truncated E2 polypeptides. Truncation can vary between 1 and 300 amino acid residues that can be contiguous or not and be located at the N-terminal and / or C-terminal end and / or internally.

The E2 polypeptide encoded by the nucleic acid molecule comprised in the vector of the invention can originate from any papillomavirus genotype, with particular preference for an HR-HPV genotype such as one selected from the group consisting of the listings above, and more particularly HPV-16, HPV-18, HPV-33 or HPV-52 or any combination thereof (for example both HPV-16 and HPV-18). A large number of native E2 polypeptides have been described in the literature, for example HPV-18 E2 in Cole et al. (J. Mol. Biol. 193: 599-608, 1987), HPV-16 E2 in Seedorf et al. (Virology 145: 181-185, 1985) and Kennedy et al. (J. Virol. 65: 2093-2097, 1991), HPV-31 E2 in Goldsborough et al. (Virology 171: 306-311, 1989), HPV-33 E2 in Cole et al. (J. Virol. 58: 991-995, 1986) and E2 of BPV-1 of bovine papillomavirus in Chen et al. (Nature 299: 529-534, 1982) and in Danos et al. (J. Virol. 46: 557-566, 1983). By way of illustration, the amino acid sequence of HPV-16 E2 polypeptide is provided in SEQ ID NO: 1.

However, the present invention is not limited to said exemplary sequences. Indeed, the nucleotide and amino acid sequences may vary between different papillomavirus isolates and this natural genetic variation is within the scope of the invention, as well as one or more unnatural modifications such as those indicated below. The term "modification" it includes the deletion, substitution or addition of one or more nucleotide residues or any combination of these possibilities. In the case that several modifications are included, they may refer to consecutive waste and / or non-consecutive waste. One or more modifications can be made to the nucleic acid molecule used in the invention by various ways known to the person skilled in the art, such as site-directed mutagenesis (for example using the SculptorTM in vitro mutagenesis system from Amersham, Les Ullis , France), PCR mutagenesis, DNA reorganization and synthetic chemical techniques (for example resulting in a synthetic nucleic acid molecule).

The modification or modifications included in the present invention comprise silent modifications that do not modify the amino acid sequence of the encoded papillomavirus E2, as well as the modifications that result in the encoded E2 polypeptide, resulting in a modified amino acid sequence with respect to the corresponding native .

Silent modifications at the level of the encoded E2 polypeptide are typically carried out by replacing one or more codons of the E2 coding sequence with one or more coding codons of the same amino acid. While a single codon encodes the Met or Trp residue, it is well known in the art that 6 different codons can be used to encode arginine, leucine or serine, and four different ones to encode alanine, glycine, proline, Threonine and valine. In this way, it is possible to modify the nucleotide sequence encoding E2 without altering the amino acid sequence. Preferably, said modifications are intended to improve the expression of papillomavirus E2 polypeptide in a given host cell or organism, for example in mammalian host cells, including human ones. Representative examples of such modifications include, but are not limited to, the suppression of one or more codons used infrequently by codon optimization, the suppression of negative sequence elements that foreseeably negatively influence expression levels and / or the suppression of homologous sequences that predictably negatively influence the stability of the nucleic acid molecule or vector used in the present invention.

Typically, codon optimization is carried out by replacing one or more "native" codons. (for example HPV) corresponding to a codon infrequently used in the host cell of interest by one or more codons coding for the same amino acid that is most frequently used. It is not necessary to replace all native codons corresponding to codons used infrequently because an increased level of expression can be achieved even with partial substitution. In addition, some deviations may be made regarding strict adherence to the optimized use of codons to allow the introduction of one or more restriction sites in the resulting nucleic acid molecule. Such optimized codon nucleic acid molecules are described in the literature, for example in WO 01/14416, WO 02/08435 and WO 03/018055.

Representative examples of negative sequence elements that it is suitable to suppress in the context of the invention comprise, in a non-limiting manner, regions that have a very high GC content (> 80%) or very low (<30%); regions that have a very high AT content; unstable direct or inverted repeat sequences; secondary RNA structures; and / or internal cryptic regulatory elements such as internal TATA boxes, chi sites, ribosomal entry sites and / or donor / acceptor processing sites.

The presence of homologous sequences in the vector according to the invention will foreseeably influence its stability, especially during the production stage of the vector. Recombination can occur between the homologous sequences, possibly leading to the loss of the part between the two homologous sequences. As used herein, the expression "homologous sequences" refers to nucleotide sequences that retain a high degree of identity with each other over at least 40, advantageously at least 45, preferably at least 50, more preferably at least 55, or even more preferably at least 59 consecutive nucleotides. A high degree of identity is 75% or more than 75%, advantageously 80% or more than 80%, preferably 85% or more than 85%, preferably 90% or more than 90%, more preferably 95% or more than 95% , still more preferably 97% or greater than 97% (for example a sequence identity of 100%). The percentage of identity between two nucleotide sequences is a function of the number of identical positions shared by the sequences, considering the number of holes that need to be entered for optimal alignment and the length of each hole. Various computer programs and mathematical algorithms are available in the art to determine percentages of identity between nucleotide sequences, such as the GCG Wisconsin package.

Homology between two homologous nucleotide sequences is suppressed by degeneration of the use of codons in at least one of the homologous sequences, so that the percentage of identity between previously homologous sequences is reduced to less than 75%. The above can be accomplished by replacing one

or more codons &quot; natives &quot; (for example HPV) present in the homologous part by one or more codons coding for the same amino acid. It is not necessary to degenerate all native codons because the homology can be sufficiently reduced with partial replacement. Such modifications are particularly useful in the case where the nucleic acid molecule or vector used in the invention encodes two papillomavirus polypeptides, the nucleotide and amino acid sequences of which are relatively conserved (for example sequences encoding 2 or more polypeptides E2, such as HPV-16 and HPV-18 E2 polypeptides or containing a common part (eg, the coding sequences of HPV-16 E1 and E2, which share 59 nucleotides). A representative example of such an embodiment is provided in SEQ ID NO: 6, providing an example of degenerate sequences corresponding to the part of 59 nucleotides present in both E1 coding sequences and E2 coding sequences.

Modifications that translate to the level of the encoded E2 polypeptide result in the mutation of one or more amino acid residues of the E2 polypeptide. Advantageously, the modified E2 polypeptide retains a high degree of amino acid sequence identity with the corresponding native polypeptide along the full-length amino acid sequence or a fragment thereof (for example, at least 9 in length, 20, 50, 100, 200 or 300 amino acids), which is 75% or more than 75%, advantageously greater than 80%, preferably greater than 85%, preferably greater than 90%, more preferably greater than 95%, even more preferably greater than 97% (for example a sequence identity of 100%). The percentage of identity between two polypeptides is a function of the number of identical positions shared by the sequences, considering the number of holes that need to be entered for optimal alignment and the length of each hole. Various computer programs and mathematical algorithms are available in the art for determining the percentages of identity between amino acid sequences, such as, for example, the W2H HUSAR program and the Blast program (for example Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997; Altschul et al., FEBS J. 272: 5101-5109, 2005), available from NCBI.

In one embodiment, the nucleic acid molecule comprised in the vector of the invention is modified to encode a defective E2 polypeptide in at least one of the biological activities of a native E2 polypeptide, and more particularly defective for activation. of viral replication and / or transcription. The amino acids that are critical for such biological activities can be identified by routine methods, such as structural and functional analysis and a person skilled in the art can easily determine the type of mutation or mutations that can reduce or cancel a given biological activity. It is well known in the art that residues involved in the transcriptional activation and replication activities of E2 are located within the N-terminal part, while the C-terminal part is responsible for the recognition of E2 binding sites in the Viral and dimerization DNA. As used herein, the N-terminal part of E2 includes the first 220 amino acid residues starting from the Met initiator. For example, site-directed mutagenesis or PCR techniques can be performed to delete or replace one or more of the amino acid residues in the N-terminal part of E2 that regulate viral replication, so as to significantly reduce or nullify the function. or E2 replication functions and a defective E2 polypeptide is generated for papillomavirus genome replication. Alternatively or in combination, one or more of the amino acid residues within the N-terminal part of E2 responsible for transcriptional activation can be removed or substituted so as to significantly reduce or eliminate the ability of E2 to activate transcription from papillomavirus promoters. Representative examples of defective E2 polypeptides suitable in the literature available to those skilled in the art are described, for example in Demeret et al. (Nucleic Acids Res. 23: 4777-4784, 1995), Sakai et al. (J. Virol. 70: 1602-1611, 1996), Brokaw et al. (J. Virology 70: 23-29, 1996) and Ferguson et al. (J. Virology 70: 4193-4199, 1996). The reduction or lack of E2 replication and transcriptional activation activities can be readily determined in appropriate assays using standard methods known to those skilled in the art (Sakai et al., J. Virol. 70: 1602-1611, 1996).

A preferred replication defective E2 polypeptide encoded by the nucleic acid comprised in the vector of the invention originates from HPV-16 and comprises the amino acid sequence shown in SEQ ID No. 1 except that at least the Glu residue in the position 39 (E39) has been modified, for example replaced by any amino acid residue other than Glu. Another preferred E2 polypeptide defective for transcriptional activation originates from HPV-16 and comprises the amino acid sequence shown in SEQ ID No. 1 except that at least the Ile residue at position 73 (I73) has been modified, by example substituted by any amino acid residue other than Ile. An even more preferred defective E2 polypeptide for both replication and transcriptional activation originates from HPV-16 and comprises the amino acid sequence shown in SEQ ID No. 1 except that at least the residue Glu (E39) in the position 39 and the Ile residue at position 73 (I73) have been modified, for example substituted by any amino acid residue other than Glu and Ile at the respective positions 39 and 73. More preferably, the Glu residue at position 39 and / or the Ile residue at position 73 is replaced by an Ala residue (E39A and / or I73A). In the context of the invention, said defective E2 polypeptide can originate from any papillomavirus and is within the skill of the person skilled in the art to adapt the modifications described in relation to HPV-16 E2 to an E2 polypeptide originated from of another papillomavirus genotype (for example the amino acid residue or residues located in a position equivalent to position 39 and / or position 73 of HPV-16 E2 can be identified by comparison between sequences and modified by standard techniques). By way of illustration, said residues correspond respectively to Glu in position 43 and Ile in position 77 in E2 of HPV-18, Glu in position 39 and Ile in position 73 in HPV-33 and HPV-52 .

In another embodiment, the nucleic acid molecule comprised in the vector of the invention is modified so as to encode a fusion between a papillomavirus E2 polypeptide and one or more fusion partners, at the N-terminal end, at the end C-terminal or at both ends of E2. The fusion partner can originate from a papillomavirus or not. The fusion can be carried out by genetic means, that is to say by fusion within the same reading frame of the nucleotide sequences encoding the E2 polypeptide and those coding for the fusion partner or partners, so that the expression of the coding sequences fused results in a single polypeptide. Fusion can be direct (i.e., without any additional amino acid residue between them) or by a linker peptide to link the E2 polypeptide with the fusion partner or partners. The presence of a connecting molecule can facilitate the correct formation, folding and / or functioning of the fusion protein. Suitable connecting molecules according to the invention have a length of between 2 and 30 amino acids and are composed of amino acid residues such as glycine, serine, threonine, asparagine, alanine and / or proline (see, for example, Wiederrecht et al., Cell 54 : 841, 1988; Aumailly et al., FEBS Lett. 262: 82, 1990; and Dekker et al., Nature 362: 852, 1993).

Non-papillomavirus binding partners comprise non-limiting calreticulin (Cheng et al., J. Clin. Invest. 108: 669-678, 2001), Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen et al., Cancer Res. 60: 1035-1042, 2000), ubiquitin (Rodríguez et al., J. Virol. 71: 8497-8503, 1997) and bacterial toxin, such as the translocation domain of exotoxin A of Pseudomonas aeruginosa (ETA (dIII)) (Hung et al., Cancer Res. 61: 3698-3703, 2001).

Suitable papillomavirus fusion partners can be any papillomavirus polypeptide, sooner or later, or any fragment thereof. A preferred fusion partner originates from an early HPV 5 polypeptide selected from the group consisting of E1, E2, E4, E5, E6 and E7 or a mixture thereof. The E2 polypeptide and the papillomavirus fusion partner can originate from the same papillomavirus genotype, such as the fusion of HPV-16 E1 and E2 polypeptides. Alternatively, the E2 polypeptide and the papillomavirus fusion partner can originate from different papillomavirus genotypes, a representative example being the fusion of the HPV-16 and E2 HPV-18 E2 polypeptides.

Independently or in combination with the modification or modifications defined above, the nucleic acid molecule included in the vector of the invention may further comprise additional modifications that are beneficial for the processing, stability and / or solubility of the encoded E2 polypeptide, for example suppression from one or more potential cleavage sites, the suppression of one or more potential glycosylation sites and / or the presentation of the encoded E2 polypeptide on the surface of the expression host cells. For example, the suppression of a potential glycosylation site can be achieved by identifying a potential N-glycosylation site (for example comprising an Asn-Val-Ser-Val motif) and substituting one or more amino acid residues for a different residue (for example replacing the Ser residue with a Gly or Ala residue), providing an E2 polypeptide that cannot be glycosylated when expressed in a eukaryotic host cell or organism.

In a preferred embodiment, the nucleic acid molecule comprised in the vector of the invention is modified so as to encode an E2 polypeptide presented on the membrane, in order to improve the presentation of MCH class I and / or MHC Class II, and thus its potential immunogenicity in the host cell or organism. It has previously been shown that the presentation on the membrane allows to improve the therapeutic efficacy of the E6 and E7 polypeptides of HPV-16 (see, for example, WO 99/03885). The papillomavirus E2 polypeptide is a nuclear protein, although no typical nuclear localization signal has been identified. The membrane presentation can be achieved by fusion of the E2 polypeptide with a secretory sequence (ie, a signal peptide) and a membrane anchor sequence. Such sequences are known in the art. Briefly, the secretory sequences are generally present at the N-terminal end of the membrane-presented or secreted polypeptides and begin their passage into the endoplasmic reticulum (ER). They comprise 15 to 35 essentially hydrophobic amino acids that are then removed by a specific endopeptidase located in the ER, providing the mature polypeptide. The membrane anchor sequences are usually highly hydrophobic in nature and serve to anchor the polypeptides to the cell membrane (see, for example, Branden and Tooze, in: Introduction to Protein Structure, pages 202-215, NY Garland, 1991).

The choice of membrane anchoring and / or secretory sequences that can be used in the context of the present invention is very broad. They can be obtained from any membrane-anchored and / or secreted polypeptide (eg cellular or viral polypeptides), such as rabies virus glycoprotein, HIV virus envelope glycoprotein or measles virus F protein, or They can be synthetic. The preferred insertion site of the secretory sequence is the N-terminal end located downstream of the translation start codon and that of the membrane anchor sequence is the C-terminal end, for example immediately upstream of the stop codon. In addition, a linker peptide can be used to bind the secretory sequence and / or the membrane anchor sequence to the E2 polypeptide.

The E2 polypeptide with a membrane target encoded by the nucleic acid comprised in the vector of the present invention is preferably modified by fusion with the secretory and membrane anchoring sequences of the rabies virus glycoprotein, as illustrated in the Examples section, attached.

According to a particularly preferred embodiment, the nucleic acid molecule encoding an E2 polypeptide comprises, consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID No. 2. For information, the polypeptide of SEQ ID No. 2 comprises the HPV-16 E2 polypeptide (between positions 24 and 387) defective for replication and transactivation activities (modifications of Glu residue at position 61 and Ile residue at position 95 by Ala residues corresponding to Glu e residues Ile, respectively at positions 39 and 73 of the native E2 polypeptide) fused with the glycoprotein secretory (between positions 2 and 23) and membrane anchor (between positions 388 and 453) sequences of the rabies virus.

The present invention relates to an infectious vector or particle comprising nucleic acid molecules encoding at least two E2 polypeptides originating from two different papillomavirus genotypes, in which said nucleic acid molecules encoding E2 do not comprise a portion of 40 or more contiguous nucleotides showing an identity percentage of 75% or greater between any two of said E2-encoding nucleic acid molecules, and the use of said infectious vector or particle for the preparation of a drug for the treatment of the papillomavirus infection, especially of persistent HR-HPV infection. In an advantageous embodiment, "at least two" it is 2, 3 or 4 and each of the encoded E2 polypeptides originates from HR-HPV of different genotypes. Independently or in combination, the E2 polypeptides are preferably modified as indicated above (for example to be defective for replication and / or transcriptional activation and / or membrane presentation). Nucleic acid molecules encoding at least two E2 polypeptides can be placed under independent regulatory sequences or can be fused together to express themselves in a single polypeptide chain. As indicated herein, representative examples include a vector comprising nucleic acid molecules encoding E2 polypeptides of HPV-16 and HPV-18, as well as a vector comprising nucleic acid molecules encoding polypeptides. E2 of HPV-16, HPV-18, HPV-33 and HPV-52, located under independent regulatory sequences. The nucleotide sequences encoding E2 degenerate so as to show a homology percentage below 75% of each other, said nucleic acid molecules encoding E2 not comprising a portion of 40 or more (for example 50, 55, 59, 70 or even more) contiguous nucleotides showing an identity percentage of 75% or greater than 75% between any two of said E2-encoding nucleic acid molecules.

In a specific embodiment of the present invention, the infectious vector or particle indicated herein may also be used in combination with one or more additional polypeptides or with one or more nucleic acids, vectors or infectious particles encoding said polypeptide or additional polypeptides. Preferably, the additional polypeptide or polypeptides is capable of intensifying the therapeutic activity provided by the active agent indicated above. The nucleic acid encoding said polypeptide or additional polypeptides may be inserted into the vector of the invention or into a separate vector such as one of those indicated herein and its expression may be placed under the control of appropriate regulatory sequences, such as those indicated. In the present memory. The additional polypeptide or polypeptides may be of papillomavirus origin or of non-papillomavirus origin.

Additional non-papillomavirus polypeptides include cytokines (for example IL-2, IL-7, IL-15, IL-18, IL21, IFNy) and suicide gene products (for example HSV-1 thymidine kinase indicated in Caruso et al., Proc. Natl. Acad. Sci. USA 90: 7024-7028, 1993; FCU-1 indicated in WO 99/54481).

Additional papillomavirus polypeptides comprise, in a non-limiting manner, any HPV early polypeptide (or fragment) selected from the group consisting of E1, E2, E4, E5, E6 and E7 or any mixture thereof.

In one aspect of the invention, the additional papillomavirus polypeptide or polypeptides can originate from the same papillomavirus genotype as that of the E2 polypeptide encoded by the vector indicated above.

Alternatively, in another aspect of the invention, the additional papillomavirus polypeptide or polypeptides may originate from a papillomavirus genotype different from that of the E2 polypeptide encoded by the vector indicated above.

The additional papillomavirus polypeptide can be native or modified compared to the corresponding native sequence. For example, the polypeptide or additional polypeptides can be modified so as to reduce

or nullify their respective biological activity (for example an enzymatic activity) while preserving antigenic activity. The exemplary modifications illustrated below are provided with respect to HPV-16 papillomavirus polypeptides, although the person skilled in the art may transpose these exemplary mutations to the corresponding polypeptides of other papillomavirus genotypes. In addition, the additional papillomavirus polypeptide can be further modified so that it is present in the cell membrane as indicated above in relation to E2, as well as in WO 99/03885.

A suitable example of an additional papillomavirus polypeptide is a modified E1 polypeptide comprising a

or more mutations compared to the corresponding native E1 polypeptide, so that it is defective for the stimulation of viral replication. Preferably, the modified E1 polypeptide comprises the mutation of any of the residues at positions 412, 439, 482 and / or 496 of the native E1 polypeptide, such as the W439R, Y412F, G482D and G496R variants of HPV-16 E1 described by Yasugi et al. (J. Virol. 71: 5942-5951, 1997), with particular preference for the G482D variant comprising the amino acid sequence of the HPV-16 native E1 polypeptide except in the substitution of the Gly residue at position 482 with an Asp residue (for example a modified E1 polypeptide having the sequence shown in SEQ ID NO: 3). Another exemplary modified E1 polypeptide comprises HPV-18 E1 polypeptide with a substitution of the Gly residue at position 489 with an Asp residue.

Another suitable example of additional papillomavirus polypeptides is a modified E6 polypeptide that is not oncogenic and has been altered for binding to the tumor suppressor cell p53 gene product. Yet another suitable example of additional papillomavirus polypeptides is a modified E7 polypeptide that is not oncogenic and that has been altered by binding to the tumor suppressor Rb cell gene product. Such non-oncogenic variants are described in, for example, Pim et al. (Oncogene 9: 1869-1876, 1994), Munger et al. (EMBO J. 8: 4099-4105, 1989), Crook et al. (Cell 67: 547-556, 1991), Heck et al. (Proc. Natl. Acad. Sci. USA 89: 4442-4446, 1992) and Phelps et al. (J. Virol. 66: 2148-2427, 1992). A preferred non-oncogenic E6 polypeptide originates from HPV-16 and exhibits the deletion of residues 118 to 122 (CPEEK) (+1 representing the first amino acid of the native HPV-16 E6 polypeptide from the first Met residue). A preferred non-oncogenic E7 polypeptide originates from HPV-16 and has a deletion of residues 21 to 26 (DLYCYE) (represented +1 the first amino acid of the HPV-16 native E7 polypeptide).

Preferably, the additional papillomavirus polypeptide for use in the invention, independently or in combination, is selected from the group consisting of the polypeptides comprising any of the amino acid sequences provided in SEQ ID NO: 3 to 5. More specifically, SEQ ID NO. 3 provides the amino acid sequence of an HPV-16 E1 polypeptide presented in a defective membrane for replication activity (G482D). SEQ ID No. 4 provides the amino acid sequence of a non-oncogenic and membrane-presented E6 polypeptide of HPV-16 and SEQ ID No. 5, the amino acid sequence of a non-oncogenic and membrane-presented E7 HPV16 polypeptide.

In the native context (for example the HPV-16 or HPV-18 genome), the 3 'end of the nucleic acid molecule encoding E1 overlaps at the 5' end of the nucleic acid molecule encoding E2 to along 59 nucleotides. According to a preferred embodiment, the part of the nucleic acid molecule encoding E1 that overlaps with the nucleic acid molecule encoding E2 is modified so as to show an identity percentage lower than 75% with the overlapping E2 sequences . Preferably, the modifications are carried out in the nucleic acid molecule encoding E1 at the nucleotide level by degenerating the use of the codons and are silent at the amino acid level, that is, said modifications are not translated into the polypeptide. E1 of encoded papillomavirus. A representative example of the modifications that can be introduced in the 59 bp portion present at the 3 'end of the HPV-16 E1-encoding nucleic acid molecule and overlapping in the native context with the part is provided in SEQ ID 6 5 'of the HPV-16 E2 encoding nucleic acid molecule.

Preferred vectors according to the invention comprise a vector selected from the group consisting of:

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide and further comprising: (ii) a nucleic acid molecule encoding an HPV-18 E2 polypeptide,

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-33 E2 polypeptide,

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-18 E2 polypeptide, and (iii) a nucleic acid molecule encoding an HPV-52 E2 polypeptide,

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-33 E2 polypeptide, and (iii) a nucleic acid molecule encoding an HPV-52 E2 polypeptide,

-

a vector comprising: (i) a nucleic acid molecule encoding an E2 polypeptide of HPV-16, (ii) a nucleic acid molecule encoding an E2 polypeptide of HPV-18, (iii) a nucleic acid molecule encoding of an HPV-33 E2 polypeptide, and (iv) a nucleic acid molecule encoding an HPV-52 E2 polypeptide,

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-16 E1 polypeptide, (iii) a nucleic acid molecule encoding of an HPV-18 E2 polypeptide, and (iv) a nucleic acid molecule encoding an HPV-18 E1 polypeptide,

-

a vector comprising nucleic acid molecules encoding HPV-16 E1, E2, E6 and E7 polypeptides and HPV-18 E1, E2, E6 and E7 polypeptides.

Preferably, the encoded E2 polypeptide or polypeptides are membrane present and are defective for replication and transcriptional activation activities. Preferably, the HPV-16 E2 polypeptide comprises, alternatively consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID NO: 2 and / or the HPV-18 E2 polypeptide comprises, alternatively consists essentially or alternatively consists of the sequence of amino acids shown in SEQ ID No. 29 and / or the HPV-33 E2 polypeptide comprises, alternatively, consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID No. 30 and / or the HPV-52 E2 polypeptide comprises, alternatively consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID No. 31.

Independently or in combination, the encoded E1 polypeptide or polypeptides are membrane present and defective for replication activity. Preferably, the HPV-16 E1 polypeptide comprises, alternatively consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID NO: 3 and / or the HPV-18 E1 polypeptide comprises, alternatively consists essentially or alternatively consists of the sequence of amino acids shown in SEQ ID NO: 32. The encoded E6 and / or E7 polypeptide or polypeptides are membrane present and are non-oncogenic. Preferably, the HPV-16 E6 polypeptide comprises, alternatively consists essentially or alternatively consists of the amino acid sequence shown in SEQ ID NO: 4, and / or the HPV-16 E7 polypeptide comprises, alternatively consists essentially or alternatively consists of the sequence of amino acids shown in SEQ ID No. 5.

More preferably, the HPV-16 E2 polypeptide nucleic acid molecule comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID NO: 8, and / or the HPV-18 E2 polypeptide nucleic acid molecule. comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID No. 33 and / or the nucleic acid molecule encoding the HPV-33 E2 polypeptide comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID No. 34 or in SEQ ID No. 35, and / or the nucleic acid molecule encoding the HPV-52 E2 polypeptide comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID No. 36 or in SEQ ID No. 37, and / or The nucleic acid molecule encoding the HPV-16 E1 polypeptide comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID NO: 7 (degenerate sequences to reduce the homology with the overlapping part of E2 of HPV-16) and / or the nucleic acid molecule encoding the E1 polypeptide of HPV-18 comprises, consists essentially or consists of the nucleotide sequence shown in SEQ ID No. 38 (degenerate sequence to reduce homology with the E1 coding sequences of HPV-16).

As discussed hereinbefore, the nucleotide sequences encoding E2 are degenerated, so that they show a homology percentage of less than 75% to each other, wherein the nucleic acid molecules encoding E2 do not comprise a part of 40 or more (for example 45, 50, 55, 59, 70 or even more) contiguous nucleotides showing an identity percentage of 75% or greater than 75% between any two of said E2-encoding nucleic acid molecules. The nucleotide sequences indicated above complete said embodiment.

The nucleic acid molecule or molecules comprised in the infectious viral vector or particle of the present invention can be generated using accessible sequence data from the art and sequence information provided herein. It can be isolated directly from HPV-containing cells (for example CaSKi cells available from ATCC under the accession number CRL-1550) or from any papillomavirus source as defined above, by conventional molecular biology or PCR techniques and, if necessary, it can be further modified as defined herein by routine mutagenesis techniques (for example to optimize expression in a particular host cell, to generate a defective variant, etc.). Alternatively, the nucleic acid molecule or molecules comprised in the vector of the invention can also be generated by chemical synthesis in an automated process (for example they can be assembled from overlapping synthetic oligonucleotides as described in, for example, Edge, Nature 292 : 756, 1981; Nambair et al., Science 223: 1299, 1984; Jay et al.,

J. Biol. Chem. 259: 6311, 1984).

In another embodiment, the nucleic acid molecule or molecules comprised in the infectious viral vector or particle of the present invention are in a form suitable for the expression of the encoded polypeptide or polypeptides in a host cell or organism, which implies that The nucleic acid molecule or molecules are placed under the control of one or more regulatory sequences necessary for their expression.

As used herein, the expression "regulatory sequences" refers to any sequence that allows, contributes or modulates the expression of the nucleic acid molecule in a given host cell, including replication, duplication, transcription, splicing, translation, stability and / or transport of the nucleic acid or derivative of the same (ie mRNA) in the host cell. Such regulatory sequences are well known in the art (see, for example, Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, 1990). The person skilled in the art will appreciate that the choice of regulatory sequences may depend on factors such as the type of vector, the host cell, the level of expression desired, etc. In the context of the present invention, the regulatory sequences are functionally linked to the nucleic acid molecule to be expressed. The expression "operably linked" It is intended to refer to the fact that the nucleic acid molecule is linked to the regulatory sequences in a manner that allows its expression in a host cell or organism.

The promoter is of particular importance and the present invention comprises the use of constitutive promoters that direct the expression of the nucleic acid molecule in many types of host cell and those that direct the expression only in certain host cells or in response to specific events.

or exogenous factors (for example temperature, the addition of a nutrient, hormone or other ligand). Suitable promoters have been well described in the literature and viral promoters such as RSV promoters (Rous sarcoma virus), SV40 (simian virus 40), CMV (cytomegalovirus) and MLP (major late promoter can be mentioned specifically. ). Preferred promoters for use in a poxvirus vector include, but are not limited to, Vaccinia 7.5K, H5R, TK, p28, p11 and K1L promoters, chimeric promoters between early and late poxviral promoters, as well as synthetic promoters such as indicated in Chakrabarti et al. (Biotechniques 23: 1094-1097, 1997), Hammond et al. (J. Virological Methods 66: 135-138, 1997) and Kumar and Boyle (Virology 179: 151-158, 1990).

The person skilled in the art will appreciate that the regulatory sequences that control the expression of the nucleic acid molecule may further comprise additional elements for the correct initiation, regulation and / or termination of transcription (for example polyA transcription termination sequences), mRNA transport (for example nuclear localization signal sequences), for processing (i.e. splicing signals), for stability (for example introns and 5 'and 3' non-coding sequences) and for translation (for example sequences tripartite leader, ribosomal binding sites, Shine-Dalgarno sequences, etc.) in the host cell or organism.

In the context of the present invention, one or more copies of the nucleic acid molecule may be comprised in said infectious viral vector or particle used according to the present invention.

The term &quot; vector &quot; as used herein it defines viral as well as non-viral vectors (for example plasmid DNA), including extrachromosomal vectors (for example episomes), multicopy and integrating vectors (i.e., which are incorporated into host chromosomes). Particularly important in the context of the invention are vectors for use in gene therapy (ie, which can introduce the nucleic acid molecule into a host organism), as well as expression vectors for use in various expression systems. In reference to a viral vector, the term &quot; vector &quot; as used herein refers to any nucleic acid molecule that comprises at least one element of viral origin, including a complete viral genome, a portion thereof or a modified viral genome as indicated below, as well as viral particles generated therefrom (for example a viral vector packaged in a viral capsid to produce infectious viral particles).

Suitable non-viral vectors include plasmids such as pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 (Seed, Nature 329: 840, 1987), pVAX and pgWiz (Gene Therapy System Inc., Himoudi et al., J. Virol. 76: 12735-12746, 2002).

Viral vectors can originate from a variety of different viruses, and especially from a virus selected from the group consisting of retroviruses, adenoviruses, adenovirus-associated viruses (AAVs), poxviruses, herpesviruses, measles viruses and viruses. sparkling. Viral vectors may be competent for replication or may be genetically disabled so that they are defective in replication or altered replication. The expression "competent for replication" as used herein, it comprises the viral vectors selective for replication and conditionally replicating that are manipulated to replicate better or selectively in specific host cells (for example in tumor cells).

In one aspect, the vector is an adenoviral vector (for a review see "Adenoviral vectors for gene therapy", Editors D. Curiel and J. Douglas, Academic Press, 2002). It can originate from a variety of human or animal sources and any serotype of adenoviral serotypes 1 to 51 can be used. Human adenoviruses 2 (Ad2), 5 (Ad5), 6 (Ad6), 11 (Ad11) are particularly preferred. , 24 (Ad24) and 35 (Ad35). These adenoviruses are available from the American Type Culture Collection (ATCC, Rockville, Md.) And have been the subject of numerous publications describing their sequence, organization and production methods, allowing the person skilled in the art to make applications of them ( See, for example, US Patent Nos. 6,133,028, No. 6,110,735, WO 02/40665, WO 00/50573, EP 1016711; Vogels et al., J. Virol. 77: 8263- 8271, 2003).

The adenoviral vector may be competent for replication. Numerous examples of adenoviral vectors competent for replication are readily available to the person skilled in the art (Hernández-Alcoceba et al., Human Gene Ther. 11: 2009-2024, 2000; Nemunaitis et al., Gene Ther. 8: 746 -759, 2001; Alemany et al., Nature Biotechnology 18: 723-727, 2000). For example, they can be constructed from a wild-type adenoviral genome by deletion of the CR2 domain of E1A (for example, WO 00/24408) and / or by substitution of the native E1 and / or E4 promoters with specific promoters of tissue, tumor or cell state (see, for example, US Patent No. 5,998,205, WO 99/25860, US Patent No. 5,698,443, WO 00/46355, WO 00/15820 and WO 01/36650).

Alternatively, the adenoviral vector is defective for replication (see, for example, WO 94/28152; Lusky et al., J. Virol. 72: 2022-2032, 1998). Preferred adenoviral vectors for replication are defective for E1 (see, for example, US Patent Nos. 6,136,594 and 6,013,638), with a deletion of E1 extending approximately between positions 459 and 3,328 or between approximately positions 459 and 3,510 (referring to the sequence of human adenovirus type 5 disclosed in GeneBank under accession number M 73260 and in Chroboczek et al., Virol. 186: 280-285, 1992). The cloning capacity can be further enhanced by deletion of the additional part or parts of the adenoviral genome (for example in the non-essential region of E3 or in other non-essential regions of E2 or E4). The insertion of the nucleic acid molecule used in the invention can be carried out by homologous recombination at any location of the adenoviral genome as described in Chartier et al. (J. Virol. 70: 48054810, 1996). Preferably, it is inserted in place of the E1 region. It can be in direction orientation

or antisense regarding the natural direction of transcription of the region in question.

In another preferred aspect, the vector of the invention is a poxvirus vector (see, for example, Cox et al., In: "Viruses in Human Gene Therapy", editor J.M. Hos, Carolina Academic Press). It can be obtained from any member of Poxviridae, in particular of the canary poxvirus (for example ALVAC, as described in WO 95/27780), of the avian poxvirus (for example TROVAC as described in Paoletti et al. , Dev. Biol. Stand. 84: 159163, 1995) or Vaccinia virus, the latter being preferred. Suitable Vaccinia viruses include, but are not limited to, the Copenhagen strain (Goebel et al., Virol. 179: 247-266 and 517-563, 1990; Johnson et al., Virol. 196: 381-401, 1993) , the Wyeth strain, NYVAC (see WO 92/15672 and Tartaglia et al., Virology 188: 217232, 1992) and the highly attenuated modified Ankara (MVA) strain (Mayr et al., Infection 3: 6-16, 1975).

The basic technique for inserting the nucleic acid molecule and associated regulatory elements necessary for expression into a poxvirus genome is described in numerous documents accessible to those skilled in the art (Paul et al., Cancer Gene Ther. 9: 470-477, 2002; Piccini et al., Methods of Enzymology 153: 545-563, 1987; US Patent No. 4,769,330, No. 4,772,848, No. 4,603,112, No. 5,100,587 and No. 5,179,993). Typically, homologous recombination is performed between overlapping sequences (i.e., flanking the desired insertion site) present both in the viral genome and in a plasmid carrying the nucleic acid to be inserted. The nucleic acid molecule comprised in the vector of the invention is preferably inserted into a non-essential locus of the poxviral genome, so that the recombinant poxvirus remains viable and infectious. Non-essential regions are non-coding intergenic regions or any gene for which inactivation or deletion does not significantly alter viral growth, replication or infection. The insertion in an essential viral locus can also be contemplated, provided that the defective function is provided in trans during the production of the viral particles, for example by the use of a helper cell line carrying the complementary sequences corresponding to those deleted. in the poxvirus genome.

When using the Vaccinia Copenhagen virus, the nucleic acid molecule is preferably inserted into the thymidine kinase (tk) gene (Hruby et al., Proc. Natl. Acad. Sci. USA 80: 3411-3415, 1983; Weir et al. , J. Virol. 46: 530-537, 1983). However, other insertion sites are also suitable, for example in the hemagglutinin gene (Guo et al., J. Virol. 63: 4189-4198, 1989), in the K1L locus, in the u gene (Zhou et al. , J. Gen. Virol. 71: 2185-2190, 1990) or at the far left of the Vaccinia virus genome, where a variety of spontaneous or artificial deletions have been published in the literature (Altenburger et al., Archives Virol. 105: 15-27, 1989; Moss et al., J. Virol. 40: 387-395, 1981; Panicali et al., J. Virol. 37: 1000-1010, 1981; Perkus et al., J. Virol 63: 3829-3836, 1989; Perkus et al., Virol. 179: 276-286, 1990; Perkus et al., Virol. 180: 406-410, 1991).

When using the MVA, the nucleic acid molecule can be inserted into any of the deletions I to VII identified present in the genome of the MVA (Antoine et al., Virology 244: 365-396, 1998), as well as in the locus D4R, although insertion in deletion II or III is preferred (Meyer et al., J. Gen. Virol. 72: 1031-1038, 1991; Sutter et al., Vaccine 12: 1032-1040, 1994).

When using the avian poxvirus, although insertion into the thymidine kinase gene can be considered, the nucleic acid molecule is preferably introduced into the intergenic region between ORF 7 and 9 (see, for example, EP 314 569 and the patent US 5,180,675).

Preferably, the vector of the invention is a Vaccinia virus vector, especially with an MVA vector. More preferably, the nucleic acid molecule or molecules are inserted in deletion III and, finally, in the opposite direction, especially if said nucleic acid molecules are under the control of the same promoter. It is preferred that the nucleic acid molecule or molecules encoding E2 and / or E7 be placed under the H5R promoter of Vaccinia and the nucleic acid molecule or molecules encoding E1 and / or E6 under the control of the p7.5K promoter.

The present description also discloses the use of a vector complexed with lipids or polymers forming particulate structures, such as liposomes, lipoplejos or nanoparticles. Such technologies are available in the art (see, for example, Arangoa et al., Gene Ther. 10: 5-14, 2003; Eliaz et al., Gene Ther. 9: 1230-1237, 2002, and Betageri et al. ., "Liposome drug delivery systems", Technomic Publishing Company, Inc., 1993).

The present invention also relates to infectious viral particles comprising the aforementioned vectors and their use as defined herein.

Typically, said viral particles are produced in an appropriate cell line grown under suitable conditions and using methods well known in the art. No attempt is made herein to describe in detail the various known methods for the production of infectious viral particles.

In the event that the viral vector is defective, infectious particles are usually produced in a complementation cell line or through the use of a helper virus, which provides non-functional viral genes in trans. For example, 293 cells (Graham et al., J. Gen. Virol. 36: 59-72, 1997), as well as PER cells, are included among the suitable cell lines for complementing adenoviral vectors with E1 deletion. -C6 (Fallaux et al., Human Gene Ther. 9: 1909-1917, 1998) and derivatives of PER-C6. Appropriate cells for propagating the poxvirus vectors are avian cells, and more preferably primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs. Producer cells can be grown in fermentation bioreactors, flasks and conventional Petri dishes, under appropriate conditions of temperature, pH and oxygen content.

Infectious viral particles can be recovered from the culture supernatant or cells after lysis. They can be further purified according to standard techniques (chromatography, ultracentrifugation as described, for example, in WO 96/27677, 98/00524, 98/22588, 98/26048, 00/40702, EP 1016700 and WO 00/50573) .

The present description also discloses the use of viral vectors or particles that have been modified to allow preferential targeting to a particular target cell (see, for example, Wickam et al., J. Virol. 71: 8221-8229, 1997 ; Arnberg et al., Virol. 227: 239-244, 1997; Michael et al., Gene Therapy 2: 660-668, 1995; WO 94/10323, WO 02/96939 and EP 1 146 125). A characteristic feature of targeted vectors and viral particles is the presence on their surface of a ligand capable of recognizing and binding to a cellular component and exposed on the surface, such as a specific marker of a cell type (for example an infected cell by HPV), a tissue specific marker (for example a specific epithelial cell marker), as well as a viral antigen (for example HPV). Examples of suitable ligands include antibodies or fragments thereof directed to an HPV antigenic domain. Cellular targeting can be carried out by genetically inserting the ligand into a polypeptide present on the surface of the virus (eg adenoviral fiber, pentone, pIX or product of the p14 Vaccinia gene).

Also described are host cells comprising the infectious viral vectors or particles indicated above for use as defined herein.

The infectious viral vectors and particles of the invention can be introduced into the host cell by any method known in the art. Such methods include, but are not limited to, microinjection (Capechi et al., Cell 22: 479-488, 1980), CaPO4-mediated transfection (Chen and Okayama, Mol. Cell Biol. 7: 2745-2752, 1987 ), DEAE-dextran-mediated transfection, electroporation (Chu et al., Nucleic Acid Res. 15: 1311-1326, 1987), lipofection / fusion of liposomes (Felgner et al., Proc. Natl. Acad. Sci USA 84: 7413-7417, 1987), particle bombardment (Yang et al., Proc. Natl. Acad. Sci. USA 87: 9568-9572, 1990), gene guns, transduction, viral infection, as well as direct administration in a host organism by various means. In addition, as discussed above, they can be used in association with transfection reagents in order to facilitate their introduction into the host cell or organism, such as polycationic polymers (for example chitosan, polymethacrylate, PEI, etc.) and cationic lipids (for example DC-ChoI / DOPE, lipofectin and transfectam, now available from Promega).

In another aspect of the present invention, the infectious viral vector or particle indicated above (also referred to herein as "active agent") or any combination thereof is comprised in a composition.

Advantageously, the composition is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of the active agent or agents, a pharmaceutically acceptable carrier. As used herein, a "therapeutically effective amount" it is a sufficient dose for the relief of one

or more symptoms normally associated with the disease or condition to be treated. For example, a therapeutically effective amount could be that amount necessary to induce an immune response or to activate the immune system, resulting in the development of an anti-HPV response. As used herein, a "pharmaceutically acceptable vehicle" It is intended to include some or all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, and absorption retarding agents, and the like, compatible with pharmaceutical administration.

Preferably, the composition of the invention is formulated for human or animal use. It is preferably included in an isotonic, hypotonic or weakly hypertonic diluent and has a relatively low ionic strength. Representative examples of suitable diluents include sterile water, physiological saline (for example sodium chloride), Ringer's solution, glucose, trehalose or sucrose solutions, Hank's solution and other physiologically balanced aqueous saline solutions (see, for example, the most current edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). In addition, the composition can be buffered at a physiological or slightly basic pH (for example between about pH 7 and about pH 9). Suitable buffers, but not limited to, include phosphate buffer (for example PBS), bicarbonate buffer and Tris-HCl buffer. By way of illustration, formulations particularly adapted to the invention include:

or 1 M sucrose, 150 mM NaCl, 1 mM MgCl 2, Tween-80 54 mg / l, 10 mM Tris-HCl, pH 8.5 (especially if the active agent is an adenoviral vector),

or 10 mg / ml mannitol, 1 mg / ml HSA, 20 mM Trips, pH 7.2 and 150 mM NaCl, and

or physiological saline solution.

Such formulations are particularly suitable for maintaining the stability of the composition at freezing temperature (for example -70 ° C or -20 ° C), for cooling (for example 4 ° C) or at room temperature. The composition of the invention can also be formulated in solid form. Solid compositions (for example in the form of dry or lyophilized powders) can be obtained by a process that involves vacuum drying and lyophilization. They are usually reconstituted in a suitable vehicle before use.

The composition may also contain one or more pharmaceutically acceptable excipients to provide desirable pharmaceutical or pharmacodynamic properties, including, for example, the modification or maintenance of pH, osmolarity, viscosity, transparency, color, sterility, stability, dissolution rate of the formulation, the modification or maintenance of the release or absorption in an animal or human organism, the induction of transport through a mucosal barrier or penetration into a particular organ. For example, a composition suitable for vaginal administration may finally include one or more absorption enhancers that are useful for increasing the pore size of the mucosal membranes.

In addition, the composition of the invention may comprise one or more adjuvants suitable for systemic or mucosal application in humans. Preferably, the adjuvant can stimulate immunity against the active agent, especially T-cell mediated immunity, for example through Toll-like receptors (TLR), such as TLR-7, TLR-8 and TLR-9. Representative examples of useful adjuvants include, but are not limited to, alum, mineral oil emulsion such as Freund's complete and incomplete solutions (IFA), lipopolysaccharides or a derivative thereof (Ribi et al., Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, pages 407-419, 1986), saponins such as QS21 (Sumino et al., J. Virol. 72: 4931-4939, 1998; WO 98/56415), compounds of imidazoquinoline such as imiquimod (Suader, J. Am. Acad. Dermatol. 43: S6-S11, 2000), derivative 1H-imidazo (4,5-c) quinolon-4-amine (Aldara ™) and the related compound S-27609 (Smorlesi, Gene Ther. 12: 1324-1332, 2005), cytosine phosphate guanosine oligodeoxynucleotides, such as CpG (Chu et al., J. Exp. Med. 186: 1623, 1997; Tritel et al., J. Immunol. 171: 2358-2547, 2003) and cationic peptides such as IC-31 (Kritsch et al., J. Chromatogr. Anal. Technol. Biomed. Life Sci. 822: 263-270, 2005).

The composition of the invention can be administered by a variety of modes of administration, including systemic, topical and mucosal administration. Systemic administration can be carried out by any means, for example by subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intravascular or intraarterial injection. Injections can be carried out with conventional syringes and needles, or by any appropriate device available in the art. Mucosal administration can be carried out orally, nasally, intratracheally, intrapulmonally, intravaginally or intrarectally. Topical administration can be carried out using transdermal means (eg patches and the like). The composition of the invention is preferably formulated in a form suitable for injection, with intramuscular or subcutaneous administration being preferred.

The appropriate dose can be adapted as a function of various parameters, in particular the mode of administration, the active agent used, the age, health and weight of the host organism, the type of concurrent treatment and / or the frequency of the treatment. The person skilled in the art may routinely perform further calculations necessary to determine the appropriate dose for treatment in light of the relevant circumstances. For general guidance, the appropriate dose for adenoviral particles ranges between about 105 and about 1013 iu (infectious units), preferably between about 107 and about 1012 iu, and preferably between about 108 and about 1011 iu. The suitable dose for the MVA particles ranges between about 104 and about 1010 pfu (plaque forming particle), preferably between about 105 and about 109 pfu, and preferably between about 106 and about 108 pfu. Plasmid vectors can be administered in doses between 10 μg and 20 mg, and preferably between 100 μg and 2 mg.

In addition, administration can take place in a single dose or, alternatively, in multiple doses according to standard protocols, doses and regimens for several hours, days and / or weeks. In addition, administration can be performed by bolus injection or continuous infusion. For example, the host organism can be treated with at least two (for example between 2 and 10) administrations of the nucleic acid molecule, vector, infectious particle or composition indicated above. Preferably, a first series of administrations is carried out sequentially within a period of time between a few days and 4 weeks, followed by a second series of administrations (for example one or two administrations) carried out one to 6 months later. of the last administration of the first series. The period of time between each administration of the second series can be between a few days and 4 weeks. In a preferred embodiment, the first series of administrations comprises three sequential administrations at intervals of one week and the second series comprises an administration 4 to 6 months after the first series. As a general guide, in the case where infectious MVA particles are used according to the present invention, administration is preferably carried out subcutaneously with a dose of MVA particles comprised between 106 and 5x108 pfu.

As indicated above, the vector, infectious viral particle or composition indicated herein, or combination thereof, is preferably administered after exposure of the host organism (the patient) to at least one papillomavirus and before detection / occurrence of a papillomavirus associated lesion. In other words, host organisms with HPV infection but which have not yet progressed to neoplasia are suitable candidates for use according to the present invention to prevent or reduce the likelihood of progression to neoplasia and ultimately to cancer.

The term &quot; exhibition &quot; it refers to the contact of a host organism with at least one papillomavirus that allows infection to occur. Several diagnostic methods are now available in the art, allowing diagnosis of papillomavirus infection. For example, a biological sample from a host organism may be collected at risk of papillomavirus infection and analyzed for the presence of papillomavirus, viral nucleic acids (eg DNA or mRNA) and / or viral antigens. Such methods include, but are not limited to, PCR, in situ hybridization, immunofluorescence, ELISA and several diagnostic tests now available. Suitable representative examples of the assay include the LiPA system (WO 99/14377; products of Labo Biomedical, The Netherlands), the Hybrid Capture II® assay (HCII, Digene Corp., USA), which allows the detection of DNA from 13 HR-HPV), the Linear Array® assay (Roche, which allows DNA genotyping of 37 HPV genotypes), the Thin Prep system (Cytyc Corporate, Marlborough, MA), PreTect-HPV Proofer® (NorChip AS, Norway , which allows the detection of E6 / E7 mRNA for HPV-16, HPV-18, HPV-31, HPV-33 and HPV-45), the PCR / RT-PCR systems and real-time PCR described in Pretet et al. (J. Clin. Virol. 31: 140-147, 2004) or in Monnier-Benoit et al. (J. Clin. Virol. 31: 140-147, 2006). Suitable primers are known to those skilled in the art or can be easily synthesized based on the nucleotide sequence of the detected papillomavirus.

As used herein, a "lesion associated with papillomavirus" refers to any disease

or condition caused by papillomavirus infection. This expression includes premalignant as well as malignant lesions. Representative examples of premalignant lesions include, but are not limited to, low, moderate or high grade intraepithelial neoplasia that can be detected in various tissues, such as CIN, vulvar intraepithelial neoplasia (VIN) and intraepithelial neoplasia (AIN) , penile intraepithelial neoplasia (PIN) and vaginal intraepithelial neoplasia (VaIN). Representative examples of malignant lesions include, but are not limited to, cervical carcinoma, anal carcinoma, vaginal cancer, penile cancer and oral cancer. Premalignant and malignant lesions associated with papillomavirus can be visualized by direct examination (for example by colposcopy, finally after the application of acetic acid) or diagnosed with methods commonly used by the clinical physician (for example screening by Pap smear or detection of abnormal cells of cytological samples). For example, a local biopsy of acidophilic regions or lesions that are visualized by colposcopy can be performed and examined for morphological abnormalities (for example epithelial hyperplasia, marked nuclear abnormalities, etc.). In the context of the invention, papillomavirus associated lesions do not comprise mild abnormalities such as ASCUS (atypical squamous cells of undetermined significance).

According to a preferred embodiment, the infectious viral vector or particle described herein, or composition or combination thereof is used to treat a persistent infection caused by at least one papilloma, especially an HR-HPV, with particular preference for HPV-16, HPV-18, HPV-33 or HPV-52 or any combination thereof (for example, both HPV-16 and HPV-18). In the context of the invention, the E2 polypeptides encoded in the vector according to the invention may originate from the infectious papillomavirus or a papillomavirus that reacts cross-linked with the infectious papillomavirus.

Due to the conservation of the amino acid sequences in the E2 polypeptides of various HR-HPV, cross reactivity between HPV16 and HPV31, HPV33, HPV35, HPV52 and HPV58 could be expected. In one aspect, the present invention uses a vector, infectious viral particle or composition according to the invention as described herein encoding an E2 polypeptide originated from HPV-16, for the treatment of patients suffering from an infection caused. at least one of HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58.

Similarly, cross reactivity can be expected between HPV-18 and HPV-39, HPV-45, HPV-51, HPV-56, HPV-59, HPV-68, HPV-70 and HPV-85. In another aspect, the present invention used a nucleic acid molecule, vector, infectious viral particle or coding composition of an E2 polypeptide originating from HPV-18, for the treatment of patients suffering from an infection caused by at least one among HPV-18, HPV-39, HPV-45, HPV-51, HPV-56, HPV-59, HPV-68, HPV-70 and HPV-85.

As used herein, a "persistent papillomavirus infection" corresponds to the asymptomatic stage of papillomavirus infection in a host organism that has not achieved spontaneous viral eradication after exposure to papillomavirus. A persistent papillomavirus infection has been established in the event that papillomavirus is detected, or at least one of its elements (for example nucleic acids, antigens and the like) in the host organism in 2 successive tests separated by several months, for example at least 6 months, advantageously at least 8 months, preferably at least 10 months, and more preferably at least 12 months, while no clinical evidence is observed (for example, premalignant and / or malignant lesions associated with papillomavirus). The asymptomatic stage is characterized by normal cytology (although mild abnormalities such as ASCUS are tolerated). For example, persistent papillomavirus infection has been established in patients who show a positive HCII trial at intervals of approximately 6 months although they have a normal Pap smear.

In one embodiment, the vector, infectious viral particle or composition indicated above is used according to the modalities indicated herein to induce or activate an immune response in the treated host organism compared to the non-use of said active agents.

The induced immune response or activity may be specific and / or non-specific, and may be humoral and / or cell mediated. The humoral responses include the production of antibodies against at least one papillomavirus polypeptide, while the cellular response includes a helper T cell response and / or CTL and / or the stimulation of cytokine production. Preferably, the induced immune response or activity is effective in providing an antiviral response against at least one of the infectious papillomaviruses.

The ability of the vector, infectious viral particle or composition indicated above to induce or activate an immune response in a treated host organism can be evaluated in vitro or in vivo by a variety of assays that are standard in the art (for a general description of the techniques available to evaluate the induction and activation of an immune response, see, for example, Coligan et al., 1992 and 1994, Current Protocols in Immunology, edited by J. Wiley & Sons Inc., National Institute of Health). The measurement of cellular immunity can be carried out by measuring cytokine profiles secreted by activated effector cells, including those derived from CD4 + and CD8 + T cells (for example the quantification of IL-10 or IFNy producing cells by ELIspot) , by determining the activation status of immunological effector cells (for example T cell proliferation assays by a classical [3 H] thymidine incorporation), by testing for antigen specific T lymphocytes in a sensitized subject (for example specific peptide lysis in a cytotoxocity assay). The ability to stimulate a cellular response could also be evaluated, for example, in syngeneic mice (for example H2Db) or in transgenic mice (for example HLA A2 and HLA B7) by ELISPOT, analytical techniques based on tetramers or other standard techniques for analysis of T-cell mediated immunity. The humoral response can be determined by antibody binding and / or competition assays (see, for example, Harlow, Antibodies, Cold Spring Harbor Press, 1989). For example, immunological tools, for example ELISA, can be developed to detect anti-E2 antibodies in the treated host organism.

In another embodiment, the vector, infectious viral particle or composition indicated above is used according to the modalities indicated herein, to provide an antiviral response against at least one of the infectious papillomaviruses compared to the non-use of said agents. assets.

As used herein, an "antiviral response" It refers to a reduction or elimination of symptoms usually associated with a papillomavirus infection in the treated organism. For example, an antiviral response can be determined from the ability of the aforementioned active agent or agents to control viral infection, reduce or eliminate at least infectious papillomavirus and / or reduce or eliminate infected cells or those expressing sequences. papillomavirus genes (especially the potentially oncogenic E6 and E7 genes). The foregoing can be evaluated as a significant reduction or lack of a detectable level of indicated markers of papillomavirus infection, for example papillomavirus, viral nucleic acids and / or viral antigens, in a biological sample collected from the host organism under treatment, compared to the situation prior to use. The reduction or elimination is indicated by comparing the level of said marker or markers measured at the time point after cessation of use according to the invention, with the level of the same marker or markers measured before said use, which represents the untreated infection. In a preferred embodiment, the antiviral response allows no papillomavirus, viral nucleic acids and / or detectable antigens measured in a biological sample collected from the host organism under treatment for several months after cessation of use according to the invention.

An antiviral response can also be determined from the ability of the aforementioned active agent or agents to significantly reduce the incidence, size and / or severity of the lesions that typically develop in papillomavirus infected organisms. As used herein, "reduce" It refers to preventing, delaying, altering, slowing, delaying and / or postponing the development of the incidence, size and / or severity of papillomavirus associated lesions as defined herein. The ability of the previously active active agent to reduce the incidence, size and / or severity of papillomavirus associated lesions can be assessed by periodic monitoring of the treated host organism. Preferably, the reduction is sufficient so that the treated host organism does not develop any papillomavirus-associated lesions (for example histologically confirmed CIN lesions) for at least one year, advantageously for at least 2 years, preferably for at least 3 years, and more preferably for at least 5 years, after completing the use according to the invention. More preferably, the use according to the invention makes it possible to delay or eliminate the need for an ablative procedure (for example conization) in the treated host organism.

As a general indication, the period of time that separates the last administration of the active agent in the host organism and the detection of the antiviral response may vary depending on the history of papillomavirus infection, the modalities of use and / or the organism. host under treatment. Preferably, it is a matter of three months to several years, especially preferably for at least 3 months, advantageously at least 4 months, preferably at least 5 months, preferably at least 6 months, and more preferably at least one year. For example, an antiviral response is determined if it is detected that the host organism that was positive before treatment for HPV DNA detected in a cervical sample, for example, by Digen HC-II, is negative for the same papillomavirus. at least 6 months after the last administration of the active agent indicated above.

In one embodiment, the use according to the present invention can be carried out in conjunction with a

or more conventional therapeutic modalities. A multiplicity of therapeutic approaches provide the host organism with a broader-based intervention. The administration thereof may be prior, concomitant or subsequent to the administration of the vector, infectious viral particle or composition used in the invention.

In another embodiment, the use according to the invention can be carried out according to a first-dose therapeutic reinforcement modality comprising the sequential administration of one or more initial doses and one or more reinforcements. Typically, the first dose and the booster dose using different vehicles that comprise or encode at least one common immunogenic domain. The initial dose is initially administered in the host organism and the booster dose is subsequently administered in the same host organism after a variable period of between one day and twelve months. The use according to the invention may comprise one to ten sequential administrations of the initial dose followed by one to ten sequential administrations of the booster dose. In addition, the initial dose and the booster dose can be administered at the same site or at alternative sites by the same route or by different routes of administration, for example subcutaneous injection of an MVA vector, intramuscular injection for a DNA plasmid. and for an adenoviral vector.

In the context of the invention, the vector, infectious viral particle or composition indicated above can be used to initiate or reinforce or both initiate and reinforce an anti-papillomavirus immune response. For example, the adenovirus vector or particles as defined above can be used as an initial dose and MVA vector or particles as defined above as a booster, or vice versa. It is also possible to use the vector, infectious viral particle or composition indicated above in combination with any prior art material encoding or comprising an antigenic domain in common with the composition of the invention. The source of said material is broad and includes, but is not limited to, peptides, proteins (for example a recombinantly produced E2 polypeptide), viral vectors, plasmid DNA, protein particles such as viral type particles, cellular materials such as irradiated cells, etc. .

The invention has been described by way of illustration, and it should be construed that the terminology that has been used is intended to be used by way of description and not limitation. Obviously, many modifications and variations may be made to the present invention from the teachings provided above. Therefore, it should be interpreted that, within the scope according to the appended claims, the invention can be practiced in a different way from that specifically described herein.

Legends of the figures

Figure 1 illustrates a schematic representation of the defective and membrane-presented plasmid pTG17408 encoding an E2 polypeptide.

Figure 2 illustrates a schematic representation of plasmid pTG17409 encoding a defective HPV16 E1 polypeptide for replication and presenting in membrane, the nucleotide sequence of which has degenerated into the 59 nucleotide part common to the HPV-E2 coding sequence. 16.

Figure 3 illustrates the E1-specific IFN and Th1 response detected by ELISPOT in mice that have received an injection of MVATG17408 (MVA-E2) or negative control MVA-N33.

The following examples are provided by way of illustration of the present invention.

Examples

The constructs indicated below were prepared according to the general genetic engineering and molecular cloning techniques detailed in Maniatis et al. (Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) or according to the manufacturer's recommendations when using a commercial kit. PCR amplification techniques are known to those skilled in the art (see, for example, PCR protocols - A guide to methods and applications, published by Innis, Gelfand, Sninsky and White, Academic Press, 1990). Recombinant plasmids carrying the ampicillin resistance gene were replicated in E. coli C600 (Stratagene), BJ5183 (Hanahan, J. Mol. Biol. 166: 557-580, 1983) and NM522 in agar or liquid medium supplemented with 100 μg / ml of antibiotic. The strain BJ5183 is preferably used in the event that cloning is carried out by homologous recombination (Bubek et al., Nucleic Acids Res. 21: 3601-3602, 1993).

The recombinant Vaccinia virus constructs are prepared according to conventional technology in the field, described in the documents indicated above and in Mackett et al. (Proc. Natl. ACad. Sci. USA 79: 7415-7419, 1982) and in Mackett et al. (J. Virol. 49: 857-864, 1984). The gpt selection gene (xanthine guanine phosphoribosyltransferase) from E. coli (Falkner and Moss, J. Virol. 62: 1849-1854, 1988) is used to facilitate the selection of recombinant Vaccinia viruses.

Example 1: Construction of a recombinant MVA vector expressing the HPV-16 E2 gene

Cloning of the HPV16 E2 gene

The nucleotide sequences encoding E2 of HPV16 were cloned from the genomic DNA isolated from CaSki cells (ATCC No. CRL-1550). The E2 gene was amplified using primers OTG16809 (SEQ ID No. 9) and OTG16810 (SEQ ID No. 10). The resulting fragment was digested with BamHI and EcoRI and inserted into pGEX2T (Amersham Biosciences) cut with the same enzymes, resulting in pTG172329. The sequencing of the cloned E2 gene showed five mutations compared to the sequence of the HPV16 E2 prototype (described in GenBank No. NC-01526). Two mutations were silent and the three non-silent mutations (T210I, S219P and K310T) were corrected using the site-directed mutagenesis kit QuikChange (Stratagene), resulting in pTG17268.

HPV-16 E2 polypeptide modification

The nucleotide sequences of E2 incorporated into pTG17268 were modified by site-directed mutagenesis in order to generate an E2 variant of HPV-16 (E39A and 173A) called E2 *. More specifically, the E2 replication function was canceled by replacing the Glu residue in position 39 with a Wing and the transactivation function by replacing the Ile residue in position 73 with a Wing. The resulting plasmid pTG17318 comprises the modified sequences encoding E2 * of HPV-16.

HPV-16 E2 * was further modified by fusion at its N-terminal end with a signal peptide and at its C-terminal end with membrane anchor sequences derived from the glycoprotein of the rabies virus isolate (GenBank No. ay009097 ), so that the presentation of HPV-16 E2 * in the expressing host cells was directed to the surface of the plasma membrane. Nucleotide sequences (SEQ ID No. 8) encoding the defective E2 membrane-presented variant called SS-E2 * -TMR were reassembled by triple PCR using the following primers: OTG17500 (SEQ ID No. 11), OTG17501 (SEQ ID No. 12), OTG17502 (SEQ ID No. 13), OTG17503 (SEQ ID No. 14), OTG17504 (SEQ ID No. 15) and OTG17505 (SEQ ID No. 16). The reassembled sequence was inserted into a vector derived from pBS (Stratagene), providing pTG17360, and then cloned into a transfer plasmid Vaccinia downstream of the pH5R promoter (Rosel et al., J. Virol. 60: 436-449, 1986), resulting in pTG17408 (Figure 1).

The transfer plasmid was designed to allow insertion of the nucleotide sequence to be transferred by homologous recombination into deletion III of the MVA genome. It originates from plasmid pTG1E (described in Braun et al., Gene Ther. 7: 1447-1467, 2000), in which the flanking sequences (BRG3 and BRD3) surrounding the deletion III of the MVA, the sequences were cloned of which were obtained by PCR from the MVATGN33.1 DNA (Sutter and Moss, Proc. Natl. Acad. Sci. USA 89: 10847-10851, 1992). The transfer plasmid also contained a fusion between the increased green fluorescent protein of Aequorea victoria (eGFP gene, isolated from pEGP-C1, Clontech) and the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt gene) under promoter control Synthetic early-late Vaccinia p11K7.5 virus (courtesy of R. Wittek, University of Lausanne). The synthesis of xanthine-guanine phosphoribosyltransferase allows the recombinant MVA GPT + to form plaques in a selective medium containing mycophenolic acid, xanthine and hypoxanthine (Falkner et al., J. Virol. 62: 1849-1854, 1988) and eGFP allows visualization of the recombinant MVA plates. The eGPP-GPT selection marker is placed between two homologous sequences in the same orientation. After achieving clonal selection, the selection marker is easily removed by several passes without selection, allowing the growth of eGPP-GPT recombinant MVA.

Construction of a recombinant MVA expressing the HPV-16 SS-E2 * -TMR gene

Generation of the MVATG17408 virus was carried out by homologous recombination in primary chicken embryonic fibroblasts (CEF) infected with MVATGN33.1 (at an MOI of 0.1 pfu / cell) and transfected with pTG17408 (according to standard DNA precipitation with calcium phosphate). Viral selection was carried out by purification of plates in three rounds in the presence of a selective medium containing mycophenolic acid, xanthine and hypoxanthine. As indicated above, the selection marker was then removed by passing in a non-selective medium. The absence of contamination by parental MVA was verified by PCR.

E2 expression analysis was carried out by western blotting. CEFs were infected at an MOI of 0.2 with MVATG17408 and after 24 hours, the cells were harvested. Western blot analysis was carried out using commercial anti-E2 monoclonal antibody TVG271 (Abcam). The expression of a protein with an apparent molecular weight of 55 kDa was detected, while the theoretical molecular weight of E2 * -TMR was 48.9 kDa. After treatment of the cell extracts with endoglucosidase F, a reduction in the size of the recombinant protein was observed, suggesting that E2 * -TMR was modified by N-glycosylation.

Example 2: Construction of a recombinant MVA expressing the HPV-16 E1 gene

Nucleotide sequences encoding the HPV16 E1 polypeptide were cloned from CaSki cell DNA (ATCC No. CRL-1550). More specifically, the E1 gene was amplified in two parts Ela (nt1 to 1,102) and EIb (nt 1,001 to 1,950). Primers OTG16811 (SEQ ID No. 17) and OTG16814 (SEQ ID No. 18) were used to amplify the E1a fragment, which was digested with BamHI and EcoRI and inserted into pGEX2T cut with the same enzymes, resulting in pTG17240. The EIb fragment was generated using OTG16813 (SEQ ID No. 19) and OTG16812 (SEQ ID No. 20) and digested with BamHI and EcoRI before inserting it into pGEX2T, resulting in pTG17241. Sequencing showed 4 mutations compared to the sequence of the HPV-16 prototype E1 (described in GenBank No. NC-01526). One mutation was silent and the three non-silent mutations present in Ela (K130Q, N185T and T220S) were corrected by site-directed mutagenesis. Next, the entire E1 gene was reassembled by cloning the corrected EIa fragment into pTG17241, digested with BsrGI and EcoRI. The resulting plasmid was named pTG17289.

In the HPV-16 genome, the last 59 nucleotides of the E1 gene are identical to the first 59 nucleotides of the E2 gene. The presence of these homologous sequences can generate homologous recombination events and thus, instability during the production stages of an MVA vector encoding E1 and E2. Therefore, said part of the coding sequences of E1 was modified by degeneration of the use of codons so as to reduce sequence homology with the coding sequence of E2. The degenerate sequence was obtained by amplification of the 3 'end of the E1 gene using degenerate primers OTG17408 (SEQ ID No. 21) and OTG17409 (SEQ ID No. 22). The amplified fragment was digested with NsiI and BglII and inserted into pTG17289 cut with the same enzymes, resulting in pTG17340.

The degenerate E1 sequences of HPV-16 were also mutated by site-directed mutagenesis in order to nullify the replication function of the encoded E1 polypeptide, by replacing the Gly residue at position 482 of the HPV-16 E1 with an Asp residue. (G482D, also referred to herein as E1 *), resulting in pTG17373.

The Eldeg * sequences of HPV-16 were also modified to direct the expression of the encoded polypeptide to the surface of the plasma cell by fusion with the signal peptide and membrane anchoring sequences derived from the glycoprotein of the virus isolate. rabies (described in GenBank No. M38452). The sequence of SS-EIdeg * -TMR was reconstituted by triple PCR using the following primers: OTG17560 (SEQ ID No. 23), OTG17561 (SEQ ID No. 24), OTG17562 (SEQ ID No. 25), OTG17563 (SEQ ID No. 26) , OTG17564 (SEQ ID No. 27) and OTG17565 (SEQ ID No. 28). The resulting sequence (SEQ ID NO: 7) was inserted into a vector derived from pBS (Stratagene), providing pTG17404. Next, the sequence of SS-EIdeg * -TMR was cloned into the transfer plasmid as described in Example 1, downstream of the p7.5K promoter (Cochran et al., J. Virol. 54: 30-37 , 1985), giving rise to pTG17409 (Figure 2).

The generation of MVATG17409 viruses was carried out in CEF by homologous recombination as described in Example 1.

Example 3: Construction of a recombinant MVA expressing the HPV-16 E1 and E2 genes

The sequenced SS-EIdeg * -TMR controlled by the p7.5K promoter was isolated from pTG17409 and inserted into pTG17408, resulting in pTG17410.

The generation of MVATG17410 viruses was carried out in CEF by homologous recombination as described in example 1.

Example 4: Construction of a recombinant MVA expressing the E1, E2, E6 and E7 genes of HPV-16

The HPV-16 E7 gene was isolated and modified as described in WO 99/03885 to encode a non-oncogenic and membrane-directed E7 polypeptide, which is illustrated in SEQ ID NO: 5. The non-oncogenic mutation was carried carried out by deletion of amino acid residues 21 to 26 (DLYCYE) and membrane targeting by fusion to the signal peptide and membrane anchoring sequences of the rabies virus glycoprotein. The resulting sequence was cloned under the control of the early-late promoter pH5R. Next, the expression cassette was introduced into pTG17410, generating pTG17482.

The HPV-16 E6 gene was isolated and modified as described in WO 99/03885 to encode a non-oncogenic and membrane-directed E6 polypeptide, which is illustrated in SEQ ID NO: 4. A mutation was carried out. non-oncogenic by deletion of amino acid residues 118 to 122 (CPEEK) and membrane targeting by fusion to the signal peptide and membrane anchoring sequences of measles virus F protein. The resulting sequence was cloned under the control of the p7.5K promoter. Next, the expression cassette was introduced into pTG17482, generating pTG17483.

The generation of NWATG174783 was carried out as described in Example 1.

Example 5: Evaluation of the HPV-16 E1 specific Th1 response in mice

The HPV-16 E2 specific Th1 response was evaluated by ELISPOT in mice injected with MVATG17408 (MVA-E2). MHC class I peptides restricted to human HLA-A0201 and genotype-specific H2b-restricted, predicted by SYFPEITHI and BIMAS, were selected to analyze IFNy producing cells in response to E2. These peptides are shown in Table I below.

position

sequence Name SYFPEITHI score BIMAS score

E2-HPV16 / H2-Db

129  MHYTNWTHI M9I 24 67

280

 NCNSNTTPI N9I 2. 3 117

51

 FKHINHQVV F9V 19 <10

71

QAIELQLTL Q9L 19 30

348

SQVKIPKTI S9I 19 39

E2-HPV16 / HLA-A0201

138 YICEEASVTV Y9V 26 <10

69

 ALQAIELQL A9L twenty twenty-one

7

RLNVCQDKIL R10L twenty <10

93

 TLQDVSLEV T9V 19 285

E2-HPV18 / H2-Db

166  KEGYNTFYI K9I 24 <10

40

 IRWENAIFF I9F 22 <10

281

 LCSGNTTPI L9I 22 84

344

 TKFLNTVAI T9I 22 58

196

 NVIDCNDSM N9M fifteen 61

Female C57B1 / 6 mice were immunized subcutaneously 3 times (days 0, 7 and 14) with 5x107 pfu of MVATGN33 (negative control) or MVATG17408 (MVA-E2). Spleens were removed on day 21 after the first immunization and an IFNy ELISPOT was performed using the above-indicated peptides at a concentration

5 of 5 μg / ml. ELISPOT was carried out using the ELISPO PLUS IFN and Mabtech AB mouse kit (Mabtech, France) following the manufacturer's instructions. The spots were counted using the Elispot Bioreader 4000 Pro-X reader (BIOSYS-GmbH; Serlabo, France). Results were obtained after subtraction of the irrelevant background of peptides.

10 As illustrated in Figure 3, the four peptides had a significant number of IFNy-producing splenocytes, compared to the background response measured in animals vaccinated with MVA-N33. All such peptides were specific to HPV16. F9V and N9I were defined in the context of mouse H2-Db, and A9L and T9V were defined in the context of human HLA-A201. It should be noted that the A9L peptide has been induced to induce a specific CTL response (Konya et al., J. Gen. Virol. 78: 2615-20, 1997).

In conclusion, as demonstrated by ELISPOT, subcutaneous injection of MVATG17408 induces a T-cell response against HPV16 in vaccinated mice.

Example 6: Construction of a recombinant MVA vector expressing HPV-18 E1 and E2 genes (MVATG17582)

The HPV-18 E1 and E2 genes were reconstituted as synthetic genes and the oligonucleotides were designed to reduce homology to a level below 75% between the 59 nucleotide portion present at both the 3 'end of the HPV E1 sequence -18 native and at the 5 'end of the HPV-18 E2 sequence and to introduce the

25 mutations that nullify the enzymatic functions of the HPV-18 E1 and E2 gene product (EI: G489D; E2: E43A and 177A). The synthetic HPV-18 E1 sequence was also designed to reduce the percentage of homology between the homologous parts shared by the native HPV-16 and HPV-18 sequences to a level below 75%. To this end, the nucleotide sequences of the E1 and E2 genes of HPV-16 and HPV-18 were aligned and oligonucleotides were designed to reduce homology to less than 6 consecutive nucleotides.

The HPV-18 degE1 * sequence was reconstituted by assembling 50 oligonucleotides and cloned into a pBS vector, resulting in pTG17473. Next, the E1 sequence was fused to the nucleotide sequence encoding the signaling peptides from measles virus F protein (SS-18E1deg * -TMF) by triple PCR using primers OTG15315 (SEQ ID NO: 39). ), OTG17881 (SEQ ID No. 40),

35 OTG17882 (SEQ ID No. 41), OTG17883 (SEQ ID No. 42), OTG17884 (SEQ ID No. 43) and OTG17885 (SEQ ID No. 44). The resulting fragment (SEQ ID No. 38) encoding the SS18degE1 * -TMF polypeptide was cloned into an MVA transfer vector under the control of the p7.5K promoter, generating pTG17521.

The degE2 * sequence of HPV-18 was reconstituted by assembling 26 oligonucleotides and cloned into a

40 pBS vector, resulting in pTG17498. Fusion with the signal and membrane anchor peptides of the rabies virus glycoprotein (strain ERA, GenBank No. M38452) was carried out by triple PCR using primers OTG17875 (SEQ ID No. 45), OTG17876 (SEQ ID No. 46), OTG17877 (SEQ ID No. 47), OTG17878 (SEQ ID No. 48), OTG17879 (SEQ ID No. 49) and OTG17880 (SEQ ID No. 50). The resulting fragment (SEQ ID No. 33) encoding the SS-18E2 * -TMR polypeptides was inserted into the MVA transfer plasmid downstream of the

PH5R promoter, resulting in pTG17552. Finally, cassette p7.5K-SS-EIdeg * -TMF was isolated from pTG17521 and inserted into pTG17552, resulting in pTG17582.

The generation of recombinant MVATG17521, MVATG17552 and MVATG17582 was performed as described above. After infection of the cultured cells, the expression of the 50 HPV-18 E1 polypeptide of the MVA constructs was confirmed by western blotting using sera obtained from rabbits

immunized

Example 7: Construction of a multivalent recombinant MVA vector expressing HPV16 and HPV-18 E1 and E2 genes (MVATG17583)

The p7.5K-SS-18EIdeg * -TMF cassette and the pH5R-SS-18E2 * -TMR cassette were introduced in pTG17410 (which contained the p7.5K-SS-16EIdeg * -TMR cassette and pH5R-SS-16E2 * - TMR) and the resulting transfer plasmid was named pTG17583. The generation of MVATG17583 was carried out as described above. Following infection with MVA TG17583 of cultured cells, expression of HPV-16, HPV-16 and HPV-18 E1 polypeptides was confirmed by western blotting using sera obtained from immunized rabbits.

Example 8: Construction of a recombinant MVA vector expressing the HPV-33 E2 gene

A synthetic gene encoding the HPV-33 E2 polypeptide was synthesized by Geneart (Regensburg, Germany). The synthetic sequence was designed in order to: (i) reduce the percentage of homology to a level below 75% with respect to the E2 genes of HPV-16, HPV-18 and HPV-52 (if possible, reduce the homologous portions to less than 6 consecutive nucleotides), and (ii) introduce mutations that nullify the enzymatic functions of the HPV-33 gene product (E39A and I73A).

Next, the HPV-33 degE2 * sequence was fused with the nucleotide sequence encoding the signal and membrane anchor peptides of the rabies virus glycoprotein (ERA strain, GenBank # M38452). The above was carried out by triple PCR using primers OTG18962 (SEQ ID No. 51), OTG18963 (SEQ ID No. 52), OTG18964 (SEQ ID No. 53), OTG18965 (SEQ ID No. 54), OTG18966 (SEQ ID No. 55 ) and OTG18967 (SEQ ID No. 56). The resulting fragment (SEQ ID No. 35) encoding the SS-33degE2 * -TMR polypeptide was cloned into an MVA transfer vector under the control of the p7.5K promoter, and viral particles were generated as described above.

Example 9: Construction of a recombinant MVA vector expressing the HPV-52 E2 gene

A synthetic gene encoding the HPV-52 E2 polypeptide was synthesized by Geneart (Regensburg, Germany). The synthetic sequence was designed in order to: (i) reduce the homology percentage to a level below 75% with E2 genes of HPV-16, HPV-18 and HPV-33 (homologous portions were preferably reduced to less than 6 consecutive nucleotides), and (ii) introduce mutations that nullify the enzymatic functions of the HPV-52 gene product (E39A and I73A).

Next, the synthetic sequence of E2 * deg of HPV-52 was fused with the nucleotide sequences encoding the signal peptides and membrane anchoring of the measles virus F protein (providing SS-52E2 * deg-TMF) by triple PCR using primers OTG18968 (SEQ ID No. 57), OTG18969 (SEQ ID No. 58), OTG18970 (SEQ ID No. 59), OTG18971 (SEQ ID No. 60), OTG18972 (SEQ ID No. 61) and OTG18973 (SEC ID No. 62).

The resulting fragment (SEQ ID No. 37) encoding the SS-52E2 * deg-TMF polypeptide was inserted into an MVA transfer plasmid downstream of the p7.5K promoter and viral particles were generated as indicated above.

Example 10: Construction of a multivalent recombinant MVA vector expressing the E2 gene of HPV-16, HPV-18, HPV-33 and HPV-52

The pH5R-SS-18E2 * -TMR cassette encoding the HPV-18 E2 polypeptide presented in membrane and enzymatically defective (isolated from pTG17552), the cassette p7.5K-SS-33degE2 * -TMR encoding the HPV E2 polypeptide -33 presented in membrane and enzymatically defective and cassette p7.5K-SS-52degE2 * -TMF encoding HPV E2 polypeptide presented in membrane and enzymatically defective were introduced in pTG17408 (containing cassette pH5R-SS-16E2 * -TMR ) and viral particles were generated as described above.

Example 11: Animal model for the evaluation of the therapeutic efficacy of recombinant MVA constructs

The CRPV model is the only laboratory model in which virally induced papillomas persist despite immunocompetence and evolve under selective host pressure in invasive and metastatic squamous cell carcinomas (Brandsma, Intervirology 37: 189-190, 1994, and Brandsma, Animal models for human papillomavirus vaccine development, pages 69 to 78, 1996, in: C. Lacey (editor), Papillomavirus reviews: current research on papillomaviruses, Leeds University Press, Leeds, United Kingdom). The MVA expressing E2 could be tested in this model in order to evaluate its therapeutic efficacy with respect to persistent papillomavirus. However, due to the lack of cross-protection between HPV and CRPV antigens, CRPV-specific recombinant MVA constructs were generated with the CRPV antigens (disclosed

in GenBank under accession number NC_001541) replacing its HPV-16 counterparts. More specifically, the following recombinant MVAs were constructed: MVATG17535 comprising inserted in deletion III the expression cassette comprising the nucleotide sequence encoding the CRPV polypeptide E2 (reference sequence) fused with the signal and membrane anchor peptides of measles virus 5 F glycoprotein (SR-CRPVE2-TMF) placed under the control of the pH5R promoter; MVATG17534 comprising inserted in deletion III the expression cassette comprising the nucleotide sequence encoding the CRPV polypeptide E1 (reference sequence) fused with the signal and membrane anchoring peptides of the rabies virus glycoprotein (SR -CRPVE1-TMR) located under the control of the p7.5K promoter, and MVATG17562 comprising inserted in deletion III the E2 expression cassettes of CRPV (pH5R-SR

10 CRPVE2-TMF) and E1 of CRPV (p7.5K-SR-CRPVE1-TMR).

White New Zealand female rabbits were inoculated with CRPV DNA on day 1 using particle-mediated DNA transfer with the Helios gene gun system (Biorad). 1.6 μm gold particles were coated with viral DNA as indicated by the manufacturer and administered at a rate of 0.1 μg per site at three sites and 0.5 μg

15 per site at three other sites on the rabbit's back. On days 2, 9 and 16, recombinant CRPV MVA was injected intradermally into the rabbit's back. Wart development was monitored weekly for 8 to 12 weeks.

At the end of the experiment, rabbits and epidermal sites were sacrificed into which DNA was injected from

20 CRPV and no papillomas were removed and frozen. DNAs were extracted and the presence of CRPV DNA was tested by PCR analysis to determine if vaccination with MVA had induced viral elimination.

Sequence listing

<110> TRANSGENE S.A. BAUDIN Martine

5 BALLOUL Jean-Marc WILD Nathalie

<120> Papillomavirus 10 vaccine <130> TG182 EXT

<150> EP07360004.1

<151> 2007-01-30 15 <150> EP07360018.1

<151> 2007-05-15

<160> 62 20 <170> PatentIn version 3.3

<210> 1

<211> 365

<212> PRT 25 <213> Human papillomavirus type 16

<400> 1 <210> 2

<211> 453 5 <212> PRT

<213> artificial sequence

<220>

<223> HPV-16 E2 variant defective and presented in membrane 10

<400> 2

<210> 3

<211> 737

<212> PRT 5 <213> artificial sequence

<220>

<223> E1 variant of HPV-16 replication defective and presented in the membrane

10 <400> 3

<210> 4 5 <211> 243

<212> PRT

<213> artificial sequence

<220> 10 <223> E6 variant of HPV-16 non-oncogenic and presented in the membrane

<400> 4

<210> 5

<211> 185

<212> PRT

<213> artificial sequence

<220>

<223> E7 variant of HPV-16 non-oncogenic and presented in the membrane

<400> 5

<210> 6

<211> 59

<212> DNA

<213> artificial sequence 15

<220>

<223> Portion of 59 nucleotides of the degenerate HPV-16 E1 coding sequence to decrease homology with the overlapping HPV-16 E2 coding sequence

20 <400> 6

atggtgattc attacctaca ttcaagtgcg tatctggtca gaacacaaat actttgtga 59

<210> 7 25 <211> 2214

<212> DNA

<213> artificial sequence

<220>

<223> sequence encoding a defective and membrane-directed HPV-16 E1 polypeptide comprising the degenerate sequence of 59 nucleotides

<400> 7 <210> 8

<211> 1362 5 <212> DNA

<213> artificial sequence

<220>

<223> nucleotide sequence encoding the defective HPV-16 E2 polypeptide directed to the membrane 10

<400> 8

fifteen

<210> 9 <211> 34 <212> DNA <213> artificial sequence

twenty

<220> <223> sense primer

<400> 9

25

aaacccggat ccatggagac tctttgccaa cgtt 3. 4

30

<210> 10 <211> 49 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 10

aaacccgaat tcaagcttag atcttcatat agacataaat ccagtagac

49

<210> 11 <211> 33 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 11

aaacccggat ccatggtacc acaagcgctg tta

33

<210> 12 <211> 53 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 12

tctctttatg ttttggaaaa ttcccaatag agactctttg ccaacgttta aat

53

<210> 13 <211> 53 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 13

atttaaacgt tggcaaagag tctctattgg gaattttcca aaacataaag aga

53

<210> 14 <211> 49 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 14

cagtgtctac tggatttatg tctatatatg ttcttctctc tgctggaac

49

<210> 15 <211> 49 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 15

gttccagcag agagaagaac atatatagac ataaatccag tagacactg

49

<210> 16 <211> 44 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 16

aaacccagat cttcaaagac gtgtttcgcc tccactctta tgag

44

<210> 17 <211> 34 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 17

aaacccggat ccatggctga tcctgcaggt acca

3. 4

<210> 18 <211> 34 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 18

aaacccgaat tccattatcg taggcccatt gtac

3. 4

<210> 19 <211> 32 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 19

aaacccggat ccgagacacg ccagaatgga ta

32

<210> 20 <211> 50 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 20

aaacccgaat tcaagcttag atcttcataa tgtgttagta ttttgtcctg

fifty

<210> 21 <211> 88 <212> DNA <213> artificial sequence

<220>

<223> antisense primer

<400> 21

<210> 22 <211> 21 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 22

gatgctacag tgccctgttg g

twenty-one

<210> 23 <211> 35 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 23

aaacccaagg atccatggta ccgcaagccc tgcta

35

<210> 24 <211> 52 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 24

ttcccctctg tttcggtaag tttcctatag ctgatcctgc aggtaccaat gg

52

<210> 25 <211> 52 <212> DNA <213> artificial sequence

<220> <223> sense antifitter

<400> 25

ccattggtac ctgcaggatc agctatagga aacttaccga aacagagggg aa

52

<210> 26 <211> 50 <212> DNA <213> artificial sequence

<220> <223> sense primer

<400> 26

tatctggtca gaacacaaat actttgtacg tactgctatc ggcaggcacg

fifty

<210> 27 <211> 50 <212> DNA <213> artificial sequence

<220> <223> antisense primer

<400> 27

cgtgcctgcc gatagcagta cgtacaaagt atttgtgttc tgaccagata

fifty

<210> 28 <211> 42 <212> DNA <213> artificial sequence

<220> <223> sense antifitter

<400> 28

aaacccaaag atcttcacag gcgggtctct ccacctgatt tg

42

<210> 29 <211> 453 <212> PRT <213> artificial sequence

<220> <223> E2 polypeptide replication defective and presented on the HPV-18 membrane (SS-18 E2 * -TMR)

<400> 29

<212> PRT

<213> artificial sequence

<220> 10 <223> E2 polypeptide replication defective and presented on the HPV-33 membrane (SS-33 E2 * -TMR)

<400> 30 5 <210> 31

<211> 457

<212> PRT

<213> artificial sequence

10 <220>

<223> E2 polypeptide replication defective and presented on the HPV-52 membrane

<400> 31 15

<210> 32

<211> 746 5 <212> PRT

<213> artificial sequence

<220>

<223> replication defective E1 polypeptide and presented on the HPV-18 membrane 10

<400> 32 5 <210> 33

<211> 1362

<212> DNA

<213> artificial sequence

10 <220>

<223> nucleotide sequence encoding a replication defective HPV-18 E2 polypeptide and presented on the membrane (degenerated to reduce homology with the sequence encoding HPV-16 E2)

<400> 33 15

<212> DNA

<213> artificial sequence

<220>

10 <223> nucleotide sequence encoding a replication defective HPV-33 E2 polypeptide (degenerate sequence)

<400> 34

<210> 35

<211> 1326 20 <212> DNA

<213> artificial sequence

<220>

<223> nucleotide sequence encoding a replication-defective HPV-33 E2 polypeptide and presented in the membrane (SS-33E2 * -TMR) (degenerate sequence)

<400> 35 5 <210> 36

<211> 1107

<212> DNA

<213> artificial sequence

10 <220>

<223> Nucleotide sequence encoding a defective E2 polypeptide in HPV-53 replication ((52degE2 *) (degenerate sequence)

<400> 36 15

<210> 37 20 <211> 1374

<212> DNA

<213> artificial sequence

<220>

<223> Nucleotide sequence encoding a replication defective HPV-52 E2 polypeptide and presented on the SS-52degE2 * membrane (degenerate sequence)

<400> 37 <210> 38

<211> 2241 5 <212> DNA

<213> artificial sequence

<220>

<223> Nucleotide sequence encoding an HPV-18 E1 defective in replication and presented in the

10 membrane (SS-18E1 * -TMF) (degenerated to reduce homology with the HPV16 nucleotide sequence encoding E1)

<400> 38

<210> 39

<211> 33 <212> DNA <213> artificial sequence

<220> <223> primer oTG15315 to reconstitute the sequence encoding HPV-18 SS-6E1 * deg-TMF

<400> 39

ggggagatct atgggtctca aggtgaacgt ctc

33

<210> 40 <211> 39 <212> DNA <213> artificial sequence

<220> <223> first primer oTG17881 to reconstitute the sequence encoding HPV-18 SS-E1 * deg-TMF

<400> 40

gtgccttctg ggtctgcgcc ccaatggatt tgaccggtg

39

<210> 41 <211> 37 <212> DNA <213> artificial sequence

<220> <223> first primer oTG17882 to reconstitute the sequence encoding HPV-18 SS-E1 * deg-TMF

<400> 41

ggtcaaatcc attggggcgc agacccagaa ggcacag

37

<210> 42 <211> 42 <212> DNA <213> artificial sequence

<220> <223> first primer oTG17883 to reconstitute the sequence encoding HPV-18 SS-E1 * deg-TMF

<400> 42

cagaatcaca ggcctcttgg tttatcgagc actagcatag tc

42

<210> 43 <211> 39 <212> DNA <213> artificial sequence

<220> <223> first primer oTG17884 to reconstitute the sequence encoding HPV-18 SS-E1 * deg-TMF

<400> 43

gctagtgctc gataaaccaa gaggcctgtg attctgtcc

39

<210> 44 <211> 32 <212> DNA

<213> artificial sequence

<220> <223> sense primer oTG17885 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 44

gggggcggcc gctcagagcg accttacata gg

32

<210> 45 <211> 31 <212> DNA <213> artificial sequence

<220> <223> sense primer oTG17875 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 45

ggggagatct atggttcctc aggctctcct g

31

<210> 46 <211> 39 <212> DNA <213> artificial sequence

<220> <223> sense primer oTG17876 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 46

gttttgggaa attccctatt cagacaccga aggaaaccc

39

<210> 47 <211> 43 <212> DNA <213> artificial sequence

<220> <223> sense primer oTG17877 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 47

gtttccttcg gtgtctgaat agggaatttc ccaaaacaca atg

43

<210> 48 <211> 39 <212> DNA <213> artificial sequence

<220> <223> sense primer sense oTG17878 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 48

gtgggataca tgacaatgta tgtattactg agtgcaggg

39

<210> 49 <211> 38 <212> DNA <213> artificial sequence

<220>

<223> sense primer oTG17879 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF

<400> 49

ctgcactcag taatacatac attgtcatgt atcccacc

38

<210> 50 <211> 29 <212> DNA <213> artificial sequence

<220> <223> sense primer oTG17880 to reconstitute the sequence encoding HPV-18 SS-E2 * deg-TMF <400> 50

gggggcggcc gctcacagtc tggtctcac

29

<210> 51 <211> 36 <212> DNA <213> artificial sequence

<220> <223> primer oTG18962 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 51

cccaaaggat ccaccatggt accgcaagcc ctgcta

36

<210> 52 <211> 52 <212> DNA <213> artificial sequence

<220> <223> primer oTG18963 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 52

ttcccctctg tttcggtaag tttcctatag aggaaatatc agcacgcttg aa

52

<210> 53 <211> 52 <212> DNA <213> artificial sequence

<220> <223> primer oTG18964 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 53

ttcaagcgtg ctgatatttc ctctatagga aacttaccga aacagagggg aa

52

<210> 54 <211> 49 <212> DNA <213> artificial sequence

<220> <223> primer oTG18965 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 54

gataagtacc ggattcatga ccttatacgt actgctatcg gcaggcacg

49

<210> 55 <211> 49 <212> DNA <213> artificial sequence

<220> <223> primer oTG18966 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 55

cgtgcctgcc gatagcagta cgtataaggt catgaatccg gtacttatc

49

<210> 56 <211> 56 <212> DNA <213> artificial sequence

<220> <223> primer oTG18967 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 56

aaaaccccgc atgcgcggcc gcaagctatc acaggcgggt ctctccacct gatttg

56

<210> 57 <211> 37 <212> DNA <213> artificial sequence

<220> <223> primer oTG18968 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 57

aaacccgaga tctaccatgg gtctcaaggt gaacgtc

37

<210> 58 <211> 46 <212> DNA <213> artificial sequence

<220> <223> primer oTG18969 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 58

cccaccggtc aaatccattg gggcgaatcg ataccggcac ggttaa

46

<210> 59 <211> 46 <212> DNA <213> artificial sequence

<220> <223> primer oTG18970 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 59

ttaaccgtgc cggtatcgat tcgccccaat ggatttgacc ggtggg

46

<210> 60 <211> 43 <212> DNA <213> artificial sequence

<220> <223> primer oTG18971 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 60

gttatacaag gtgtcatgtc attgggttta tcgagcacta gca

43

<210> 61 <211> 43 <212> DNA <213> artificial sequence

<220>

<223> primer oTG18972 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 61

tgctagtgct cgataaaccc aatgacatga caccttgtat aac

43

<210> 62 <211> 53 <212> DNA <213> artificial sequence

<220> <223> primer oTG18973 to reconstitute the sequence encoding SS-33E2 * -TMR

<400> 62

aagcttgcta gccaccggtg gggccgcggc cgctcagagc gaccttacat agg

53

Claims (23)

1. Vector comprising nucleic acid molecules encoding at least two E2 papillomavirus polypeptides, wherein said nucleic acid molecules encoding E2 do not comprise a portion of 40 or more contiguous nucleotides showing an identity percentage of 75% or greater than 75% between any two of said nucleic acid molecules encoding E2.

2.

Vector according to claim 1, wherein said nucleic acid molecule (s) encoding E2 encodes an N2 polypeptide originating from an HR-HPV selected from the group consisting of HPV16, HPV-18, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV- 66, HPV-68, HPV-70 and HPV-85, preferably originated from HPV-16, HPV-18, HPV-33 or HPV-52 or any combination thereof.

3.

Vector according to any one of claims 1 to 2, wherein said nucleic acid molecules are modified so as to encode a defective E2 polypeptide for at least one activity of activation of replication and / or transcription.

Four.

Vector according to claim 3, wherein said E2 polypeptide originates from HPV-16 and comprises the amino acid sequence depicted in SEQ ID No. 1, except that at least the Glu residue at position 39 (E39) and / or the Ile residue at position 73 (I73) is replaced by any amino acid residue other than Glu and Ile at the respective positions 39 and 73, preferably with a Ala residue.

5.

Vector according to any one of claims 1 to 4, wherein said nucleic acid molecules are modified so as to encode an E2 polypeptide presented on the membrane.

6.

Vector according to claim 5, wherein said E2 polypeptide presented on the membrane was modified by fusion of said papillomavirus E2 polypeptide with a secretory and membrane anchoring sequence, wherein said secretory and / or anchoring sequences to the membrane. The membrane originates preferentially from the rabies virus glycoprotein, the HIV virus envelope glycoprotein or the measles virus F protein or are synthetic.

7.

Vector according to claim 6, wherein said nucleic acid molecule encodes an E2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

8.

Vector according to any one of claims 1 to 7, wherein said vector comprises nucleic acid molecules encoding at least two E2 polypeptides originating from different papillomavirus genotypes.

9.

Vector according to any one of claims 1 to 8, wherein said vector is selected from the group consisting of:

-

a vector comprising (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide and further comprising (ii) a nucleic acid molecule encoding an HPV-18 E2 polypeptide;

-

a vector comprising (i) a nucleic acid molecule encoding an E2 polypeptide of HPV-16, (ii) a nucleic acid molecule encoding an E2 polypeptide of HPV-18, and (iii) a nucleic acid molecule encoding of an HPV-33 E2 polypeptide;

-

a vector comprising (i) a nucleic acid molecule encoding an E2 polypeptide of HPV-16, (ii) a nucleic acid molecule encoding an E2 polypeptide of HPV-18, and (iii) a nucleic acid molecule encoding of an HPV-52 E2 polypeptide;

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-33 E2 polypeptide, and (iii) a nucleic acid molecule coding for an HPV-52 E2 polypeptide;

-

a vector comprising: (i) a nucleic acid molecule encoding an E2 polypeptide of HPV-16, (ii) a nucleic acid molecule encoding an E2 polypeptide of HPV-18, (iii) a nucleic acid molecule encoding of an HPV-33 E2 polypeptide, and (iv) a nucleic acid molecule encoding an HPV-52 E2 polypeptide;

-

a vector comprising: (i) a nucleic acid molecule encoding an HPV-16 E2 polypeptide, (ii) a nucleic acid molecule encoding an HPV-16 E1 polypeptide, (iii) a nucleic acid molecule encoding of an HPV-18 E2 polypeptide, and (iv) a nucleic acid molecule encoding an HPV-18 E1 polypeptide;

-

a vector comprising nucleic acid molecules encoding HPV-16 E1, E2, E6 and E7 polypeptides, and HPV-18 E1, E2, E6 and E7 polypeptides.

10.

Vector according to claim 9, wherein said HPV-16 E2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2; and / or said HPV-18 E2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 29; and / or said HPV-33 E2 polypeptide comprises the amino acid sequence shown in SEQ ID No. 30, and / or said HPV-52 E2 polypeptide comprises the amino acid sequence shown in SEQ ID No. 31.

eleven.

Vector according to claim 9 or 10, wherein said HPV-16 E1 polypeptide comprises the amino acid sequence shown in SEQ ID No. 3, and / or said HPV-18 E1 polypeptide comprises the amino acid sequence shown in SEQ ID No. 32; and / or said HPV-16 E6 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; and / or said HPV-16 E7 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5.

12.

Vector according to any of claims 9 to 11, wherein said nucleic acid molecule encoding the HPV-16 E2 polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or said nucleic acid molecule encoding the E2 polypeptide of HPV-18 comprises the nucleotide sequence shown in SEQ ID NO: 33; and / or said HPV-33 E2 polypeptide nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No. 34 or SEQ ID No. 35; and / or said HPV-52 polypeptide E2 nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No. 36 or SEQ ID No. 37; and / or said nucleic acid molecule encoding the HPV-16 E1 polypeptide comprises the nucleotide sequence shown in SEQ ID No. 7 and / or said HPV-18 polypeptide E1 encoding nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No. 38.

13.

Vector according to any one of claims 1 to 12, wherein said vector is a poxvirus vector generated from a canary poxvirus, an avian poxvirus or a Vaccinia virus, preferably a Vaccinia virus selected from the Copenhagen strain, the Wyeth strain , NYVAC or the modified Ankara strain (MVA).

14.

Vector according to claim 13, wherein said viral vector is a) derived from a Vaccinia Copenhagen virus and wherein said nucleic acid molecule is inserted into the thymidine kinase gene, or b) derived from a Vaccinia MVA virus and wherein said nucleic acid molecule is inserted in deletion III.

fifteen.

Vector according to any of claims 13 to 14, wherein said nucleic acid molecule (s) encoding E2 and / or E7 is placed under the H5R promoter of Vaccinia and molecule (s) ) of nucleic acids encoding (E) and / or E6 under the control of the p7.5K promoter.

16.

Infectious viral particle comprising the vector according to any of claims 1 to 15.

17.

Composition comprising a vector according to any one of claims 1 to 15 or infectious viral particle according to claim 16 or any combination thereof, preferably formulated in a form suitable for injection, in particular by intramuscular or subcutaneous administration.

18.

Use of a vector according to any of claims 1 to 15, an infectious viral particle according to claim 16, or a composition according to claim 17, in the preparation of a drug for the treatment of a host organism suffering from persistent papillomavirus infection. caused by at least one papillomavirus, preferably at least one HR-HPV.

19.

Use according to claim 18, for the treatment of a host organism suffering from an infection caused by at least one of HPV16, HPV31, HPV33, HPV35, HPV52 and HPV58, wherein said vector comprises a nucleic acid molecule encoding an E2 polypeptide originated from HPV-16.

twenty.

Use according to claim 18, for the treatment of a host organism suffering from an infection caused by at least one of HPV-18, HPV-39, HPV-45, HPV-51, HPV-56, HPV-59, HPV -68, HPV-70 and HPV-85, wherein said vector comprises a nucleic acid molecule encoding an E2 polypeptide originated from HPV-18.

twenty-one.

Use according to any of claims 18 to 20, wherein the drug must be administered in a first series of administrations performed sequentially within a period of time between a few days and 4 weeks, followed by a second series of administrations performed in one 6 months after the last administration of the first series, the first series of administrations preferably comprising three sequential administrations at weekly intervals and the second series comprising an administration in 5 or 6 months following the first series.

22

Use according to any of claims 18 to 21, wherein said drug is intended for use a) to provide an antiviral response against at least one of the infectious papillomaviruses, or

b) to induce or activate an immune response in an animal or human organism.

2. 3.

Vector according to any one of claims 1 to 15, an infectious viral particle according to claim 16

or a composition according to claim 17, for use in a) the treatment of a persistent papillomavirus infection in an animal or human organism, b) the induction or activation of an immune response in an animal or human organism, or c) the provision of an antiviral response in an animal or human organism.