| Modified hpv e6 and e7 genes and proteins useful for vaccination -> Monitor Keywords |
|
|
 
Modified hpv e6 and e7 genes and proteins useful for vaccinationRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Antigen, Epitope, Or Other Immunospecific Immunoeffector (e.g., Immunospecific Vaccine, Immunospecific Stimulator Of Cell-mediated Immunity, Immunospecific Tolerogen, Immunospecific Immunosuppressor, Etc.), Amino Acid Sequence Disclosed In Whole Or In Part; Or Conjugate, Complex, Or Fusion Protein Or Fusion Polypeptide Including The Same, Disclosed Amino Acid Sequence Derived From VirusModified hpv e6 and e7 genes and proteins useful for vaccination description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070190074, Modified hpv e6 and e7 genes and proteins useful for vaccination. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. application Ser. No. 10/472,724, filed Jan. 29, 2004, and issued Apr. 10, 2007 as U.S. Pat. No. 7,201,908, which was filed under 35 U.S.C. .sctn.371 and claims priority of International Patent Application No. PCT/EP02/03271, filed Mar. 22, 2002, which in turn claims priority of European Patent Application No. 01107271, filed Mar. 23, 2001, all of which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the invention [0003] The present invention relates to DNA sequences encoding an E6 or E7 fusion protein of HPV, wherein said DNA sequences are characterized by a combination of the following features: original codons are exchanged by codons which lead to an enhanced translation in a mammalian cell, they contain a deletion resulting in the production of a truncated non-functional protein, and they encode a fusion partner which is a highly immunogenic polypeptide capable of enhancing the immunogenicity of the E6- or E7-protein in the mammalian host. Furthermore, this invention relates to the modified E6- or E7-protein encoded by said DNA sequences as well as expression vectors containing said DNA sequences. Finally, the present invention relates to several uses of the above compounds, particularly as effective vaccines useful in treatment or prevention of an HPV infection or a neoplasm associated with HPV infection. [0004] 2. Description of Related Art [0005] Carcinoma of the uterine cervix (cervical cancer, CC) is the second most common cancer in women worldwide and the first in developing countries. CC develops through premalignant intermediate stages of increasing severity known as cervical intraepithelial neoplasia (CIN) grades 1-3, the latter leading to the development of invasive cancer in about 50% of cases over a period of 1-2 decades. More than 11% of the global cancer incidence in women is due to human papillomavirus (HPV) infections. Infection with HPV types 16 and 18 has been associated with the development of CIN and CC, with HPV genotype 16 being the most prevalent viral type to infect the cervix. The E6 and E7 proteins encoded by these HPV types are thought to be involved in the pathogenesis of CC by inducing abnormal cell proliferation. Expression of E6 and E7 is consistently detected in tissue and tumor cells from HPV-associated CCs. Furthermore, the E6 and E7 genes from HPV types 16 and 18 are sufficient for transformation of epithelial cells in culture (zur Hausen, Biochim. Biophys. Acta 1288(2) (1996): F55-78). [0006] There is increasing evidence that the E6 and E7 proteins encoded by HPV types 16 and 18 may be effective immunological targets for tumor rejection by the host. Efforts are being made to develop effective preventive and therapeutic vaccines which may be useful in treatment and prevention of a neoplasm associated to HPV. The different strategies employed so far for inducing an immune responses to proteins of the HPV types 16 and 18 are: (a) Use of synthetic antigenic peptides, (b) Use of recombinant microorganisms (recombinant bacille Calmette-Guerin; BCG), (c) use of DNA vaccines using wild-type viral genes and (d) use of Virus-like particles (VLPs). [0007] However, unfortunately, the above strategies exhibit a variety of disadvantages which so far have hampered the development of a safe and efficient vaccine. As regards the use of synthetic antigenic peptides it has to be stressed that the identification of HPV specific, immunoreactive peptides is very complex. It requires large numbers and quantities of peptides for vaccines to be effective and of a broad spectrum. Moreover, synthetic peptides do not contain posttranslational modifications (e.g., glycosylation, sulfation, phosphorylation) normally found in native proteins and therefore are not efficient enough as vaccines. The BCG based vaccine delivery systems expressing the L1 late protein of HPV 6b or the E7 early protein of HPV 16 have been used as immunogens. However, the immune responses obtained with these systems was even less than those elicited by protein/adjuvant vaccines and, thus, this system is considered unlikely to be useful as a single component vaccine strategy. As regards DNA vaccines it has been observed that the expression of wild-type HPV genes is quite low, even if they are expressed from strong promoters, such as that of the cytomegalovirus (CMV). As regards the use of Virus-like particles (VLPs) it has to be mentioned that true VLPs are made of the L1 (capsid) protein of a specific HPV type. Therefore, they may be only useful as prophylactic rather than as therapeutic vaccines, if ever. Pseudotyped VLPs containing, for instance, epitopes of HPV-16 E7 have also been described and may be useful as prophylactic and therapeutic vaccines. However, an important limitation is that VLPs are produced in insect cells or in yeast. So far, no suitable production systems in mammalian cells have been established. Therefore critical epitopes depending on posttranslational modifications which take place in human cells are lost in these systems. SUMMARY OF THE INVENTION [0008] Therefore, it is the object of the present invention to provide a safe and effective vaccine, preferably a genetic vaccine, for the treatment or prevention of an HPV infection or a neoplasm associated to HPV. [0009] According to the invention this is achieved by the subject matters defined in the claims. The present invention provides DNA sequences for inducing immune response to the E6 and/or E7 proteins of oncogenic HPV in a host animal, preferably by administering vectors containing said DNA sequences, e.g. plasmid vectors, herpes simplex virus type 1 amplicon or recombinant Semliki forest virus vectors. Said DNA sequences encode the HPV proteins as fusion proteins that are immunogenic but are not capable of inducing cell transformation. The DNA sequences of the invention are characterized by the following features: [0010] (a) The DNA sequences of the HPV E6/E7 genes have been modified to make their codon usage closer to that of human genes, (b) the genes have been modified by deletion to make them non-functional, thereby disabling their oncogenic capability (deletions are, preferably, point mutations, because these lead to loss of potentially essential epitopes), (c) the HPV genes have been fused to highly immunogenic proteins to enhance their immunogenicity in the host (these fusions are not expected to result in masking of HPV protein epitopes, since the fragments fused are of sufficient length as to avoid this problem), and, preferably, expression of the HPV genes is provided by recombinant, replication-deficient HSV, SFV or high copy plasmid vectors or combinations of these. [0011] This approach offers a variety of advantages, namely: [0012] (a) Higher expression levels of the HPV protein as a result of the silent mutations introduced in the HPV genes to make their codon usage closer to the human are obtained. This results in a more efficient host response in immunization trials compared to the use of wild-type HPV genes. [0013] (b) The HPV genes and proteins generated by the present invention are expressed in human cells and, unlike proteins expressed in other systems such as bacteria, yeast or insect cells, they contain posttranslational modifications normally found in proteins expressed in human cells. This is crucial for an adequate recognition of the HPV proteins by the host immune system. [0014] (c) Since the HPV proteins are expressed fused to proteins known to be highly immunogenic, they elicit stronger immune responses in the host animal. [0015] (d) The HPV proteins are not cell-transforming neither in vitro nor in the host animal because in no case are they expressed as full-length polypeptides. The HPV fusion genes express incomplete proteins, whose functions are impaired. In addition, the HPV proteins are expressed as fusions to cytoplasmic proteins and therefore they can not reach the nucleus where they exert their functions. [0016] (e) The HPV proteins are, preferably, expressed tagged with a specific sequence, which can be easily detected in Western blots and by immunofluorescence with the help of commercially available antibodies. [0017] (f) The combination of various viral vectors and of these with plasmid vectors ensures a more efficient immunization, since it prevents neutralization of the vector by immune reaction elicited in a previous boost. DETAILED DESCRIPTION OF THE INVENTION [0018] Accordingly, the present invention relates to a DNA sequence encoding an E6 or E7 fusion protein of HPV, wherein said DNA sequence is characterized by a combination of the following features: [0019] (a) at least 20% of the original codons are exchanged by codons which lead to an enhanced translation in a mammalian cell; [0020] (b) it contains a mutation resulting in the production of a truncated non-functional protein; and [0021] (c) it encodes a fusion partner which is a highly immunogenic polypeptide capable of enhancing the immunogenicity of the E6 or E7 protein in the mammalian host. [0022] The expression "orignial codons" refers to the codons found in the corresponding wildtype version of the HPV. [0023] The expression "enhanced translation in a mammalian cell" refers to the genes resulting from introduction of silent mutations in the HPV sequences, as described in the present invention, which create open reading frames consisting entirely of preferred human codons, and thus lead to enhanced expression of the proteins they encode in mammalian cells. [0024] The term "mutation resulting in the production of a truncated non-functional protein" refers to any mutation which leads to the production of a non-functional version of the protein. Preferably, such a mutation leads to a truncated version of the protein. Examples of appropriate mutations include a mutation, wherein at least one codon has been deleted or a mutation leading to premature termination of translation. Such mutation is, e.g., the replacement of a codon encoding a particular amino acid by a stop codon, an insertion or deletion of one or two nucleotides resulting in a frame shift mutation etc. The term "non-functional protein or gene" means that the mutant HPV genes and proteins of the present invention are "nontransforming neither in vitro nor in vivo" meaning that the capability of the E6 or E7 genes and proteins to transform cells to a tumorigenic phenotype has been eliminated as demonstrated by standard tests. The person skilled in the art can easily determine whether a particular mutation leads to an E6 or E7 gene or protein with the desired characteristics, i.e. which is "nontransforming" according to standard procedures. These include: [0025] 1) In vitro: Transformation assays of NIH 3T3 cells and primary human keratinocytes. Transforming genes (oncogenes) have been routinely identified by use of assays in which transformed foci result from transfection of tumor or recombinant DNA into NIH 3T3 cells (Todaro et al., PNAS USA 51: 66-73, 1964; Jainchill et al., J. Virol. 4: 549-553, 1969; Andersson et al., Cell 16: 63-75, 1979). These cells are murine fibroblasts maintained as contact-inhibited, non-tumorigenic cell lines. Transfer of DNA containing an acitivated oncogene will occasionally give rise to foci of morphologically altered cells that have tumorigenic properties. [0026] 2) In vivo: Tumorigenicity tests are routinely performed in immunodeficient mice by inoculation with mouse or human transformed cells. Cells transfected to express HPV E6 and E7 genes and cell lines derived from cervical carcinomas infected by HPV, such as HeLa cells, have been shown to be tumorigenic (Lichy et al., Cell Growth Differ. 3: 541-548, 1992; Stanbridge, Nature 260: 17-20, 1976). [0027] In a preferred embodiment, the DNA sequence of the present invention encodes the HPV E7 protein with the above described characteristics. [0028] In a further preferred embodiment, at least 50% of the original codons of the DNA sequence of the present invention are replaced by codons which lead to an enhanced translation in a mammalian cell; examples of suitable replacements are e.g., shown in FIGS. 1 and 2 SEQ ID NOs: 3 and 1, respectively). [0029] In a further preferred embodiment, the DNA sequence of the present invention contains a frame-shift point mutation leading to premature stop of translation. [0030] The person skilled in the art knows polypeptides or parts thereof which are suitable as fusion partner for the E6 or E7 protein and which are highly immunogenic in mammals, particularly in humans. Examples of suitable polypeptides include: [0031] 1) Hepatitis B virus small envolope protein (HBsAg-S). This protein has the capacity to self-assemble with host-derived membranes to form empty subviral particles, which are released into the lumen of a pre-Golgi compartment and subsequently secreted (Ganem, "Hepadnaviridae and their replication" p2703-37, in Fields, Knipe and Howley (eds.), Fields Virology 3.sup.rd ed., 1996, Lippincott-Raven Publishers, Philadelphia). E6 or E7 can be fused to the C-terminus of the protein which remains exposed on the surface of the subviral particles. [0032] 2) E2 glycoprotein of Semliki forest virus (SFV) . E2 is a spike component of the SFV virion and a major antigen for neutralizing antibodies (Schlesinger and Schlesinger, "Togaviridae: the viruses and their replication" in Fields, Knipe and Howley (eds.), Fields Virology 3.sup.rd ed., 1996, Lippincott-Raven Publishers, Philadelphia) . E6 or E7 can be fused to the N-terminus of the E2 protein that is exposed on the surface of the viral envelope or the plasma membrane of E2-expressing cells. [0033] 3) Human amyloid .beta.-protein precursor (APP). APP is a transmembrane protein with a large extracellular region and a small cytoplasmic tail. It is normally cleaved by protease to yield a 40 amino acid .beta.-peptide (amyloid), which is found in the plaques of patients with Alzheimer's disease, or a smaller fragment called p3, which may associate with extracellular matrix ("Principles of neural Science", Kandel, Schwartz, and Jessell, (eds.) 3.sup.rd ed., 1991, Elsevier, New York) . E6 and E7 can be inserted into the extracellular part of APP and are thought to be released together with the .beta.-peptide or the p3 fragment. [0034] 4) Human chromogranin B (hCgB) . Although hCgB is a protein involved in the regulated secretory pathway, it has been shown to be constitutively secreted in cells without a regulated pathway, such as HeLa cells, upon transfection (Kaether, and Gerdes, FEBS Letters 369: 267-271, 1995). E6 or E7 can be fused to the C-terminus of hCgB. [0035] 5) The bacterial B-galactosidase, known to be highly immunogenic (Fijikawa et. al., Virology 204:789-793, 1994). E6 or E7 can be fused to the N-or the C-terminus of the protein. As the fusion product is a soluble non-membrane protein that may diffuse to the nucleus, E6 or E7 is a deletion (inactive) mutant. Alternatively, a signal peptide is added to the fusion which targets the product to the cell surface. [0036] 6) Fusion of the N-or C-terminal halves of E6 or E7 together and the resulting chimeric polypeptide fused to any of the above proteins. [0037] The present invention particularly, but not exclusively, relates to the E6 and E7 genes and proteins of the HPV-16 and HPV-18 genotypes. lt will be, however, appreciated that the invention extends to variants of such HPV genotypes and other HPV genotypes which may have oncogenic or other pathologic potential. [0038] In a preferred embodiment, the present invention relates to chimeric genes encoding a polyprotein containing E6 and E7 of HPV-16 and E6 and E7 of HPV-18, either complete or as deletion fragments comprising N- or C-terminal halves of such proteins, fused together and to the polypeptides or parts thereof mentioned above. This allows immunization against HPV16 and HPV18 using a single product as immunogen. Continue reading about Modified hpv e6 and e7 genes and proteins useful for vaccination... Full patent description for Modified hpv e6 and e7 genes and proteins useful for vaccination Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Modified hpv e6 and e7 genes and proteins useful for vaccination patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Modified hpv e6 and e7 genes and proteins useful for vaccination or other areas of interest. ### Previous Patent Application: In vivo efficacy of ny-eso-1 plus adjuvant Next Patent Application: Hair growth stimulant composition Industry Class: Drug, bio-affecting and body treating compositions ### FreshPatents.com Support Thank you for viewing the Modified hpv e6 and e7 genes and proteins useful for vaccination patent info. IP-related news and info Results in 0.11076 seconds Other interesting Feshpatents.com categories: Accenture , Agouron Pharmaceuticals , Amgen , AT&T , Bausch & Lomb , Callaway Golf 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
