WO2016026919A1 - Vaccins et procédés de fabrication pour le traitement et la prévention de maladies - Google Patents
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- WO2016026919A1 WO2016026919A1 PCT/EP2015/069106 EP2015069106W WO2016026919A1 WO 2016026919 A1 WO2016026919 A1 WO 2016026919A1 EP 2015069106 W EP2015069106 W EP 2015069106W WO 2016026919 A1 WO2016026919 A1 WO 2016026919A1
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Definitions
- the present invention relates to a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen. It further relates to a nucleic acid encoding said vaccine, an expression vector comprising said nucleic acid and a recombinant host cell comprising said expression vector. Further, the present invention relates to a method of manufacturing a vaccine of the present invention, a pharmaceutical composition comprising said vaccine and the use of said vaccine for treatment and/or prevention of a disease associated with said antigen.
- Cancer is a multifaced disease with numerous possibilities of origin.
- infectious agents like bacteria, e.g. the bacterium Helicobacter pylori which causes 5.5% of all cancers.
- Viruses are even more prominent as some play an important role as oncoviruses. Examples are the human papilloma viruses (HPV; cancers of anogenital tract and head and neck region), the hepatitis B and C viruses (HBV, HCV; hepatocellular carcinoma), Epstein-Barr virus (EBV; Burkitt's lymphoma, nasopharynx carcinoma), or the human herpes virus 8 (HHV8; Kaposi's sarcoma).
- HPV human papilloma viruses
- HCV hepatitis B and C viruses
- EBV Epstein-Barr virus
- HHV8 Kaposi's sarcoma
- HR-HPVs human papillomaviruses
- CIN cervical intraepithelial neoplasia
- VLP HPV LI virus like particles
- pl6 INK4a is strongly expressed in virtually all HPV-induced carcinomas and high grade pre-neoplasias, including cervical, vulvar, vaginal, penile, anal and head and neck tumors.
- pi 6TM* ⁇ is only expressed in senescent cells and, thus, is barely found in normal tissues.
- pi 6TM* ⁇ is also found overexpressed in various tumors not associated with an HPV infection or in tumors where HPV has been found but a viral carcinogenesis is not proven, including a fraction of melanoma and non-melanoma skin cancers, lung cancers, esophageal, gastric and colorectal cancers and kidney, bladder, ovarian, endometrial and breast cancer. It is known that mutations of the retinoblastoma tumor suppressor gene result in upregulation of pi()iNK4a expression. However, the underlying mechanisms for the strong pi 6TM* ⁇ expression in these instances are most likely more heterogenic and not finally understood.
- prophylactic vaccines only induce type-specific immunity and have therefore a negligible prophylactic effect on other virus (HPV) types. Furthermore they are costly due to the production in mammalians, respectively insect cells. A cold chain for transportation is necessary as these vaccines are not stable at room temperature which further increases the cost factor.
- a vaccine in which a viral coat protein and a tumor or bacterial antigen are covalently or non-covalently connected elicit a strong humoral and/or cellular immune response against both the viral coat protein and the tumor or bacterial antigen.
- This vaccine can be used inter alia for prophylactic and for combined prophylactic and therapeutic vaccination.
- the antigen is a cellular tumor antigen, it provides the advantage that the therapeutic effect of the vaccine is independent from the type of the virus that induced the respective tumor.
- the present invention provides a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen.
- the present invention provides a nucleic acid encoding the vaccine of the first aspect of the present invention.
- the present invention relates to an expression vector comprising the nucleic acid of the second aspect of the present invention.
- the present invention provides a recombinant host cell comprising the expression vector of the third aspect of the present invention.
- the present invention provides a method for manufacturing a vaccine of the first aspect of the present invention, comprising the steps of a) providing a recombinant host cell and
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the vaccine, the nucleic acid or the expression vector of the present invention, a pharmaceutically acceptable carrier and/or suitable excipient(s).
- the present invention relates to a vaccine of the first aspect, a nucleic acid of the second aspect or an expression vector of the third aspect for use in eliciting a T cell response against said antigen for treating and/or preventing a disease associated with said antigen.
- vaccine as referred to in this specification is a biological preparation that improves immunity to an antigen associated with a particular disease.
- a vaccine stimulates the body's immune system to recognize the antigen as foreign, destroy the antigen or cells displaying this antigen, and keep a record of it, so that the immune system can more easily recognize and destroy any of these antigens, cells displaying these antigens or microorganisms bearing these antigens on its surface that it later encounters.
- Vaccines can be prophylactic (e.g. to prevent or ameliorate (i) the effects of a future infection by a pathogen or (ii) the occurrence of cells displaying said antigen, e.g. tumor cells) or therapeutic (e.g. vaccines against an established cancer).
- a cancer vaccine is a vaccine that treats existing cancer and/or prevents the development of cancer.
- Vaccines that treat existing cancer are known as therapeutic cancer vaccines.
- Some types of cancer, such as cervical cancer and some liver cancers, are caused by viruses (known as oncoviruses), and traditional vaccines against those viruses will prevent those types of cancer.
- human papillomavirus is a DNA virus from the papillomavirus family that is capable of infecting humans. Like all papillomaviruses, HPVs establish productive infections only in keratinocytes of the skin or mucous membranes. In some people subclinical infections will become clinical and may cause benign papillomas (such as warts or squamous cell papilloma) or cancers of the cervix, vulva, vagina, penis, oropharynx and anus. Persistent infection with-high-risk-HPV types which are different from the ones that cause skin warts may progress to precancerous lesions and invasive cancer.
- benign papillomas such as warts or squamous cell papilloma
- Persistent infection with-high-risk-HPV types which are different from the ones that cause skin warts may progress to precancerous lesions and invasive cancer.
- HPV infection is a cause of nearly all cases of cervical cancer. HPVs are commonly transmitted by sexual contact and infect exclusively epithelial cells. Most of the cervical HPV infections are cleared as a result of humoral and cell-mediated immune response. However, some infections persist over long periods of time and can progress to cervical intraepithelial neoplasia (CIN) or cancer. The largest is the alpha group that contains most of the genital / mucosal HPV types. Benign and neoplastic diseases of the anogenital tract include cervical, vaginal, penile and anal cancers. Besides their oncogenic properties in the genital tract, HPVs also infect squamous epithelial tissues in the head and neck region.
- CIN cervical intraepithelial neoplasia
- NMSC non-melanoma skin cancer
- EV epidermodysplasia verruciformis
- Papoviridae as referred to in this specification is mainly associated with various neoplasms in mammals.
- the family of Papovaviridae is split into the families of Papillomaviridae and Polyomaviridae.
- Polyomaviridae as referred to in this specification is a taxonomic family of viruses and comprises polyomavirus species which lead to a variety of tumors.
- papillomaviridae is a taxonomic family of non-enveloped DNA viruses, collectively known as papillomaviruses. Several hundred species of papillomaviruses have been identified infecting all carefully inspected mammals but also other amniotes such as birds, snakes and turtles.
- the virus family is divided into subfamilies selected from the group consisting of Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omikronpapillomavirus, Pipapillomavirus, Thetapapillomavirus, Xipapillomavirus and Zumblepillomavirus.
- subfamilies selected from the group consisting of Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapap
- HPV high risk
- low-risk HPV types e.g. HPV 6 and 11
- HPV16 is the most prevalent type of the high-risk category in the general population and is responsible for approximately half of all cervical cancers, followed by HPV 18 as the second most common HPV type.
- Strains of HPV types responsible for skin and mucous infections are selected from the group consisting of type 1, 2, 3, 4, 6, 7, 8, 10, 11, 16, 18, 22, 31, 32, 33, 35, 39, 45, 51, 52, 56, 58, 59, 60, 63, 66, 68, 73, and 82.
- the "viral coat protein” as referred to in this specification is a structural virus capsid protein of the family of Papoviridea.
- the virus capsid is composed of two late proteins, LI and L2 which play important roles in mediating infectivity.
- the major capsid protein LI naturally occurring as a monomer, is the primary structural element and has the ability to form pentamers also referred to as “capsomeres” which then self-assemble into virus like particles (VLP).
- a VLP is the empty capsid of a virus without genetic material inside.
- VLPs can be produced by genetic engineering and are highly immunogenic: they resemble the native, infectious virions in size and shape and are thereby able to induce high titers of virus-neutralizing antibodies, which provide protection from further virus challenge.
- LI consists of 503 amino acids and has a molecular weight of 56 kDa (HPV 16).
- the minor capsid protein L2 was found to be located in the central internal cavity of the LI pentamer. It has a size of 473 amino acids (HPV16) and a molecular weight of 51 kDa.
- the natural virus capsid consists of 72 LI pentamers (360 LI molecules) and approximately 30 L2 molecules. LI side chains lining the axial cavity are highly conserved between different HPV types whereby the rim of the LI pocket is more variable.
- LI capsids assembled in vitro are the basis of prophylactic vaccines against several HPV types. Compared to other papillomavirus genes, the amino acid sequences of most portions of LI are well-conserved between types. However, the surface loops of LI can differ substantially, even for different members of a particular papillomavirus species
- a “variant” as used herein, can alternatively or additionally be characterized by a certain degree of sequence identity to the parent polypeptide from which it is derived. More precisely, a variant in the context of the present invention exhibits at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity to the reference polypeptide. Preferably, the variants of the present invention exhibit the indicated sequence identity, and preferably the sequence identity is over a continuous stretch of 100, 150, 200, 250, 300 or more amino acids. Most preferably the indicated identity is determined over the entire length of the alignment between the two amino acids, i.e. the reference amino acid and the amino acid that is assessed for its identity.
- Such amino acid sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g.
- sequence identity may be calculated using e.g.
- sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.
- Shuffle-LAGAN Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162
- Markov random fields When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.
- an "antigen" as referred to in the context of the present specification is any substance which provokes an adaptive immune response.
- An antigen may be foreign or toxic to the body or may be a cellular protein that is associated with a particular disease.
- Antigens are recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system and may elicit a humoral or cellular immune response.
- B-cell receptor or T-cell receptor highly variable antigen receptors
- a fraction of the proteins inside cells irrespective of whether they are foreign or cellular, are processed into smaller peptides and presented to by the major histocompatibility complex (MHC).
- MHC major histocompatibility complex
- To a cellular immune response is elicited, if the small peptide fragment is bound by a T-cell receptor.
- Foreign antigens can enter the body from the outside, e.g. by inhalation, ingestion, or injection.
- an antigen comprises one or more epitopes.
- epitope refers to an antigenic determinant, which is part of an antigen and is that part of the antigen that is recognized by molecules and/or cells of the adaptive immune system.
- Epitopes that elicit a humoral immune response are referred to as "B cell epitopes”.
- Epitopes that elicit a cellular immune response are referred to as "T cell epitopes”.
- B cell epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
- the B-cell epitopes of an antigen may be a conformational epitope or a non-conformational, i.e.
- a conformational B-cell epitope is composed of discontinuous sections of the antigens' amino acid sequence. These B-cell epitopes interact with the ligand based on the 3- D surface features and shape or tertiary structure of the antigen. Most B cell epitopes are conformational.
- linear epitopes interact with the ligand based on their primary structure.
- a linear B-cell epitope is formed by a continuous sequence of amino acids from the antigen. Conformational epitopes preferably comprise between 8 and 20 discontinuous amino acids, preferably between 8 and 15 amino acids.
- Linear epitopes have a length of between 6 to 20 amino acids, more preferably between 8 and 15 amino acids.
- T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules.
- T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length.
- a "cellular tumor antigen” is an antigenic substance produced in tumor cells, i.e. it triggers an immune response in the host and which is encoded by the genome of the cell, i.e. is not of viral origin like, e.g. HPV E6 or E7.
- TSA Tumor- specific antigens
- TAA tumor-associated antigens
- many antigens thought to be tumor- specific turned out to be expressed on some normal cells as well.
- the modern classification of tumor antigens is based on their molecular structure and source, e.g.
- Overexpressed antigens comprise cancer-rescue antigens, which are expressed in tumor cells in order to protect the non-tumor cell, but due to an inactivated downstream mechanism circumventing cell growth, fail to protect the non-tumor cells.
- Cancer rescue antigens comprise but are not limited to pl5TM K4b , pl6TM K4a , pl8TM K4c , pl9TM K4d , p21 CIP , p27 KIP , and p57 KIP2 .
- Mutated antigens comprise but are not limited to pRb, Bcr-Abl, mt IDH1.
- pl6 INK4a is a tumor suppressor protein which shows increased expression with worsening grades of cervical intraepithelial neoplasia (CIN) and is strongly overexpressed in HPV induced cervical cancer. Therefore, pl6 INK4a is considered as a marker for early diagnosis and is routinely used as a highly specific marker for CIN and transforming HPV infections. pl6 INK4a overexpression increases with the severity of cytological/histological abnormality. pl6 INK4a is a tumor suppressor protein with a molecular weight of 16 kDa (156 amino acids).
- the protein structure shows four contiguous ankyrin repeats, a protein motif consisting of two alpha helices separated by loops.
- pl6 INK4a is encoded by chromosome 9p21 within the INK4a/ARF locus.
- the INK4 family of CDK inhibitors includes four members with similar biochemical properties: pi 5 M4b , pi 6TM ⁇ , pi 8TM ⁇ ° and piS)iNK4d. Mutations and deletions of ⁇ 15TM ⁇ 4 ⁇ and pl6 INK4a are frequently observed in different malignancies and pl6 mK4:i is one of the most direct links between cell cycle control and cancer. Cyclin-dependent kinases (CDKs) form complexes with cyclins and are thereby activated.
- CDKs Cyclin-dependent kinases
- pl6TM K4 also known as cyclin-dependent kinase inhibitor 2A
- pl6TM K4 acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6. This inhibits their ability to interact with cyclins D and to phosphorylate the retinoblastoma protein (RB).
- HPV oncogene expression and evidence of its deregulation can be monitored through detection of the cellular protein pl6 INK4a as E6 and E7 disrupt cell cycle checkpoints and affect almost all CDK inhibitors which are linked to the Gl and G2 checkpoints.
- pl6 INK4a is released from its negative feedback control by pRB inactivation and KDM6B overexpression, elevated levels of this protein do not lead to cell cycle arrest in tumor cells.
- cyclin dependent kinase inhibitor protein is a protein which inhibits cyclin-dependent kinase. Several function as tumor suppressor genes.
- CDIs cyclin-dependent kinases inhibitors
- CKIs cyclin-dependent kinases inhibitors
- CDKIs cyclin-dependent kinases inhibitors
- nucleic acid comprises polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life.
- Nucleic acids which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group.
- Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C).
- the nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone.
- the sugar is desoxyribose
- the polymer is DNA.
- the sugar is ribose
- the polymer is RNA.
- a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers.
- nucleic acid includes but is not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA.
- a nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a miRNA, siRNA, or a piRNA.
- MiRNAs are short ribonucleic acid (RNA) molecules, which are on average 22 nucleotides long but may be longer and which are found in all eukaryotic cells, i.e.
- MiRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.
- mRNAs target messenger RNA transcripts
- siRNAs sometimes known as short interfering RNA or silencing RNA, are short ribonucleic acid (RNA molecules), between 20-25 nucleotides in length. They are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes.
- RNAi RNA interference
- PiRNAs are also short RNAs which usually comprise 26-31 nucleotides and derive their name from so-called piwi proteins they are binding to.
- the nucleic acid can also be an artificial nucleic acid.
- Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally- occurring DNA or RNA by changes to the backbone of the molecule.
- expression vector also referred to as an expression construct, is usually a plasmid or virus designed for protein expression in cells.
- the vector is used to introduce a specific gene into a target cell, and can use the cell's mechanism for protein synthesis to produce the protein encoded by the gene.
- the expression vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
- the goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins.
- a commonly used expression vector is pGEX-4T2.
- recombinant host-cell as referred to in the context of the present invention is selected from the group consisting of a bacterial, yeast, baculovirus, plant, mammalian host cell.
- Bacterial host cells comprise but are not limited to E. coli, preferably BL21 -competent E. coli, or Bacillus subtilis,
- composition refers to a composition formulated to be suitable for administration to a patient in order to prevent and/ or treat precancerous neoplasia and/or persisting tumors.
- the pharmaceutical composition can be used as a tumor vaccination.
- pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties.
- Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems.
- liquid compositions selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention.
- Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.
- the term broughtdisease associated with the antigen is a proliferative disorder.
- a proliferative disorder is one in which too many of some type of cell are produced.
- leukemia is a proliferative disorder characterized by an abnormal proliferation (production) i.e. overproduction of white blood cells.
- the proliferative disorders can be "pre-cancerous" or "pre -neoplastic” or malign. Examples of such conditions include proliferative disorders or cellular proliferative disorders with a high degree of dysplasia.
- Neoplastic diseases of the anogenital tract include cervical, vaginal, penile and anal cancers.
- HPVs Besides their oncogenic properties in the genital tract, HPVs also infect squamous epithelial tissues in the head and neck region. Furthermore, it was found that viruses from the B super group are involved in the development of non-melanoma skin cancer (NMSC) whereby epidermodysplasia verruciformis (EV) HPV types are most plausibly linked to the development of NMSC.
- NMSC non-melanoma skin cancer
- EV epidermodysplasia verruciformis
- HPV is an inherited disease and patients show an increased susceptible to HPVs of the skin.
- the tumor disease comprises malignant tumors. Malignant tumors are also defined as cancer. Cancer comprises a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancers are selected from the group consisting of carcinomas, sarcomas, neuroendocrine tumors, leukemia and lymphomas, and teratomas.
- the present invention provides in a first aspect a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen.
- the antigen is covalently linked and the antigen is a polypeptide, it is preferred that the covalent link is a peptide bond.
- the antigen may be linked to the C-terminus or N-terminus of the viral coat protein. It is also envisioned that the antigen is inserted into the viral coat protein. In the latter case the antigen is preferably inserted in such that the viral coat protein does not lose its ability to assemble into capsomers. This ability can be determined as outlined below. It is particularly preferred that the antigen is inserted into the viral coat protein. This is particular preferred if the viral coat protein is a LI protein of a human Papilloma virus (HPV).
- Preferred regions for inserting antigens are selected from the group consisting of either at the N-terminus of the protein, the C-terminus of the protein or a region within the C-terminal sequence of the viral coat protein.
- an antigen is inserted at the N-terminus of the viral coat protein.
- the viral coat protein may also be a fragment of a viral coat protein, preferably a LI protein, more preferably a LI protein of a HPV, in which N-terminal amino acids are deleted.
- Preferred N-terminal deletions are outlined below in more detail.
- the deletion comprises 10 to 100 amino acids, i.e. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100.
- the deletion comprises 10 N-terminal amino acids (SEQ ID NO: 4) using LI protein of HPV 16.
- the antigen is covalently or non-covalently coupled to the N-terminus of the fragment of the viral coat protein.
- the viral coat protein may also be a fragment of a viral coat protein, preferably a LI protein, more preferably a LI protein of a HPV, in which C-terminal amino acids are deleted. Preferred C-terminal deletions are outlined in more detail below. More preferably, an antigen is inserted at the C-terminal end of the LI protein wherein the C-terminus is modified by deletion, even more preferably the deletion comprises 10 to 50 deletions, i.e.
- the deletion comprises 29 C-terminal amino acids (SEQ ID NO: 6) using LI protein of HPV 16.
- the antigen is covalently or non-covalently coupled to the C-terminus of the fragment of the viral coat protein.
- the antigen is inserted within a C-terminal region of the protein sequence of the viral coat protein, preferably a LI, more preferably a LI protein of a HPV.
- a particular preferred C-terminal region is a region C-terminal to helix 4 of LI proteins.
- Helix 4 of LI of HPV 16 has the amino acid sequence according to SEQ ID NO: 7.
- SEQ ID NO: 7 To identify the region C- terminal of helix 4 the skilled person can align the sequences of various LI proteins and determine the sequence of a given LI protein that is similar to the sequence of helix 4.
- Such an exemplary alignment between the LI amino acid sequences of four different HPV types is shown in Fig. 12.
- each vaccine can be tested using the methods outlined below for one or both of these functional criteria.
- the antigen may also be non-covalently linked to the viral coat protein.
- Suitable non- covalent links can be formed between various non-covalent binding pairs known in the art, which include, e.g. streptavidin and biotin.
- biotin or streptavidin is covalently linked to the antigen the respective other binding partner is covalently linked to the viral coat protein. This allows the separate production of the viral coat proteins as well as capsomeres or VLPs comprising the VLPs and the subsequent loading of antigens onto the viral coat proteins, capsomeres or VLPs.
- the viral coat protein is in a specific embodiment a protein of the virus family of Papoviridae, preferably of the subfamily of the Papillomaviridae.
- the coat proteins of these viruses typically comprise late protein 1 (LI) and late protein 2 (L2).
- LI late protein 1
- L2 late protein 2
- the viral coat protein used in the context of the vaccine of the present invention is the LI protein.
- the LI protein is from a Papillomaviridae selected from the group consisting of Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omikronpapillomavirus, Pipapilloma virus, Thetapapillomavirus, Xipapillomavirus and Zumblepillomavirus, with Alphapapillomavirus being preferred.
- the viral coat protein is the LI from HPV
- Particular preferred LI proteins are derived from HPV types responsible for skin and mucous infections.
- HPV types comprise 1, 2, 3, 4, 6, 7, 8, 10, 11, 16, 18, 22, 31, 32, 33, 35, 39, 45, 51, 52, 56, 58, 59, 60, 63, 66, 68, 73, and 82.
- Particular preferred types are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 that are known to cause cancer.
- the most preferred types to derive LI protein from are either HPV type 6, 10, 11, 16 or 18 which account for 70% of all cervical cancers.
- LI proteins that can be used as viral coat proteins are selected from the group consisting of SEQ ID NO: 2 (HPV Type 16), SEQ ID NO: 16 (HPV Type 6), SEQ ID NO: 17 (HPV Type 10), SEQ ID NO: 18 (HPV Type 18, and SEQ ID NO: 19 (HPV Type 11) or a variant within at least 80% sequence identity over aligned sequence between SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or , SEQ ID NO: 19 and which enhances the T-cell response to a test antigen fused to the LI protein at least to the same extent as the LI protein SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or , SEQ ID NO: 19, respectively.
- a variant in the context of the present invention exhibits preferably at least at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, and most preferably at least 98% sequence identity to its parent protein over the aligned sequence.
- capsomere a structural subunit of a viral capsid.
- one or more capsomers can form capsids, which are usually stabilized among each other by disulfide bonds between neighboring viral coat proteins, e.g. LI molecules. After expression in eukaryotic and prokaryotic systems, some viral coat proteins like HPV LI, are able to self-assemble into empty VLPs with similar immunogenicity compared to infectious virions.
- VLPs are endocytosed, processed, and presented by APCs to naive T cells.
- the vaccines of the present invention are preferably in the form of capsomeres or VLPs. Capsomeres represent a potentially lower cost alternative to VLP-based vaccines as they can be produced in large amounts from prokaryotic expression systems and appear to be more stable at room temperature then VLPS.
- the present inventors were able to show that the capsid-neutralizing antigenic domains are fully conserved in capsomeres and that antisera generated with capsomeres elicit neutralization titers comparable to that of entire VLPs.
- Certain LI constructs form highly immunogenic capsomeres.
- the LI protein can be modified in different ways and immunogenicity of the constructs was compared.
- a HPV construct with an N-terminal deletion of 10 amino acids, named LIANIO appeared to be the most immunogenic.
- LIANIO of HVP 16 induced antibody titers equivalent to those generated in response to VLPs.
- the term "variant" in relation to the viral coat proteins of the present invention also comprises fragments of the viral coat proteins.
- Fragments can be N-terminally deleted, C- terminally deleted, internally deleted, N- and C-terminally deleted, N-terminally and internally deleted, C-terminally and internally deleted, and N- and C-terminally and internally deleted.
- the deletion(s) are selected in such that they do not alter the ability of the fragment to form capsomeres. This functional property can easily be tested by expressing the respective fragment in a suitable host, e.g. E. coli, and analyzing the cellular extract or (partially) purified protein on a non-denaturing polyacrylamide gel.
- Multimers of the viral coat protein can then be identified and quantified by Western blot with viral coat protein specific antibodies or by protein staining techniques like, e.g. Coomassie or silver staining. The latter is used in particular if purified or partially purified viral coat proteins are used.
- the fragment of the viral coat protein more preferably the fragment of the LI protein of a HPV, most preferably the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 20, e.g. 5, 8, 9, 10, 11, 12, 15 or 20, especially 10 N-terminal amino acids (SEQ ID NO: 4).
- the viral coat protein more preferably the fragment of the LI protein of a HPV, most preferably the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 40, e.g. 5, 40, 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40, especially 29 C-terminal amino acids (SEQ ID NO: 6).
- Preferred combinations are 9 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40 especially 29 C-terminal amino acids.
- Preferred combinations are 10 N-terminal deleted amino acids with a deletion of 5 to 40, e.g.
- the fragments may also comprise internal deletions.
- a preferred deletion is an internal deletion of helix 4.
- the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 40, e.g.
- Preferred combinations are 9 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40 especially 29 C-terminal amino acids and an internal deletion of helix 4.
- Preferred combinations are 10 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C- terminal amino acids and an internal deletion of helix 4.
- Preferred combinations are 11 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C-terminal amino acids and an internal deletion of helix 4.
- the fragments of the present invention are preferably based on either LI protein of HPV 16 or HPV 18.
- the preferred deletions are indicated above with reference to the sequence of the LI protein of HPV Type 16.
- the deletion of 10 amino acids from the N-terminus of LI of HPV Type 16 maintains a protein motive conserved among HPVs, namely T/L V Y V P P.
- the N-terminal deletion of the LI proteins of other HPV Types deletes a region(s) that align with the N-terminal region(s) deleted in LI of HPV Type 16.
- the skilled person can easily determine that the deletion of 70 amino acids of the N-terminus of LI of HPV Type 18 according to SEQ ID NO: 18, leads to the deletion of an N-terminal region that aligns with the 10 N-terminal amino acids of LI of HPV Type 16 according to SEQ ID NO: 2.
- preferred N-terminal deletion fragments of LI of HPV Type 18 lack 55 to 70 amino acids from the N-terminus of the amino acid sequence according to SEQ ID NO: 18.
- the deletion of 29 amino acids from the C-terminus of LI of HPV Type 16 maintains a protein motive conserved among HPVs, namely K F L L/V Q X G X R/K X.
- the C-terminal deletion of the LI proteins of other HPV Types deletes a region that aligns with the C-terminal region(s) deleted in LI of HPV Type 16.
- LI pentamers assemble to a VLP with similar immunogenicity compared to infectious virions. Accordingly, the fragments in a specific embodiment can form capsomeres that can form VLPs.
- the cellular tumor antigen is selected from the group consisting of tumor-specific antigens, preferably lineage-restricted, mutated, posttranslational altered, idiothypic antigens; or tumor-associated antigens, preferably oncofetal, overexpressed, cancer-testis or frame-shift antigens.
- the lineage-restricted antigen is HD39
- the mutated antigen is selected from the group consisting of pRb, Bcr-Abl, IDH1
- the oncofetal antigen is selected from the group consisting of onco-throphoblast glycoprotein, alpha-fetoprotein
- the overexpressed antigen is selected from the group consisting of Her-2, Muc-1, p53 (mutated), Ras, WT-1, preferably the overexpressed antigen is a cancer-rescue antigen.
- antigenic fragments thereof comprising at least one T and/or B cell epitope are comprised.
- the cancer-rescue antigen is selected from the group consisting of a cyclin dependent kinase inhibitor protein, preferably pl5 mK4h , pl6 mK4a , plS mK4c , pl9TM K4d , p21 CIP , CIP2, p27 KIP , p57 KIP2 , more preferably pl6 INK4a .
- the antigen is selected from the group consisting of SEQ ID NO: 10 or fragments thereof comprising at least one T and/or B cell epitope, preferably at least on T cell epitope.
- the present invention provides a nucleic acid encoding the vaccine of the first aspect of the present invention
- the present invention provides an expression vector comprising the nucleic acid of the second aspect.
- the expression vector is selected from the group consisting of a bacterial, yeast, baculovirus, plant and mammalian expression vector, more preferably the expression vector is a bacterial expression vector or a cell-free expression vector. Even more preferably the expression vector is pGEX-4T2. More preferably, the expression vector construct is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, most preferably the vector construct is encoded by SEQ ID NO: 15.
- the present invention provides, a recombinant host cell comprising the expression vector and the nucleic acid.
- the host-cell is selected from the group consisting of a bacterial, yeast, baculovirus, plant, mammalian host cell. More preferably, the host cell is a bacterial host-cell, even more preferably a host-cell from E. coli.
- the present invention provides a method for manufacturing a vaccine of the first aspect of the invention comprising the steps of providing a host cell and expressing the vaccine.
- the vaccine is expressed in a bacterial host cell, preferably in E. coli. It is preferred that the vaccine is expressed in inclusion bodies, preferably in E. coli and the purification of the vaccine includes lysis of the cell and separation of the protein containing inclusion bodies. It was surprisingly shown by the inventors that that particularly stable capsomeres could be isolated from inclusion bodies and thus, the vaccine, if purified from inclusion bodies, shows increased stability against heat which decreases the urgent need of a cooling chain.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the vaccine of the first aspect of the present invention, the nucleic acid of the second aspect, the expression vector of the third aspect and pharmaceutical acceptable carriers and/or suitable excipients.
- the pharmaceutical composition is selected from the group consisting of solid, liquid, semi- solid or transdermal therapeutic systems. It is envisioned that the pharmaceutical compositions of the invention comprise two or more different vaccines, wherein the viral coat protein of one vaccine is of one virus type and the viral coat protein of another vaccine is of another virus types, preferably viral coat proteins of two or more HPV types. Preferred combinations are viral coat proteins are viral coat proteins of HPV 16 and 18, HPV 6, 10, 11, 16 and 18.
- the invention provides a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other for use in eliciting a T cell response against said antigen, a nucleic acid encoding said vaccine, or an expression vector comprising said nucleic acid for treating and/or preventing a disease associated with said antigen.
- a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other for use in eliciting a T cell response against said antigen, a nucleic acid encoding said vaccine, or an expression vector comprising said nucleic acid for treating and/or preventing a disease associated with said antigen.
- the disease is a hyperproliferative disorder.
- Preferred hyperproliferative disorders are a benign, pre-cancerous or malign disorder, preferably of the anal region, bladder, blood, bone, brain, breast, cervix, colon, endometrium, esophagus, kidney, larynx, liver, lung, lymphoma, mouth, ovary, pancreas, penis prostate, rectum, skin, stomach, testis, thyroid, vagina, or vulva, preferably tonsil or pharynx, more preferably of the cervix.
- Benign hyperproliferative disorders can be selected from the group consisting of cervical hyperplasia including warts e.g.
- Pre-cancerous or malign hyperproliferative disorders can be anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, mouth (oral) cancer, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, vaginal cancer, and vulvar cancer, preferably cervical cancer.
- the vaccine the nucleic acid encoding said vaccine, the expression system comprising said nucleic acid or the pharmaceutical composition comprising any of those both treats and prevents the disease.
- the disease is caused by HPV and the viral coat protein is a LI protein of a HPV and the antigen is a cellular tumor antigen.
- HPV16 LI - pl6INK4a capsomere constructs depicted in cartoon format.
- the wild-type LI protein is displayed at the top (SEQ ID NO: 1).
- Modified LI and pl6INK4a alone were used as controls during immunization studies and were therefore also cloned and expressed in E. coli.
- Three constructs containing LI and pl6INK4a were generated. Therefore, the complete pl6INK4a encoding cDNA sequence was cloned a) upstream and b) downstream of a modified HPV16 LI sequence into a pGex-4T-2 expression vector.
- the third construct c) the helix 4 region of LI was replaced by pl6INK4a
- pGex-LlAN10Ah4AC29 modified HPV16 LI, forms capsomeres (SEQ ID NO: 11),
- pGex-pl6INK4a full length human pl6INK4a gene (SEQ ID NO: 12),
- construct 1 contains the full-length human p 16INK4a gene fused to the C terminus of the modified LI (SEQ ID NO: 13),
- construct 2 contains the full-length human pi 6INK4a gene fused to the N terminus of the modified LI (SEQ ID NO: 14),
- pGex-LlAN10Ah4-pl6INK4a-LlAC29 contains the full-length human pl6INK4a gene inserted at the helix 4 position of the modified LI (SEQ ID NO: 15).
- Insoluble LI -containing proteins were purified with a special protocol that included several washing and re-suspension steps.
- the purified inclusion bodies were denatured with 5 % N- Lauroylsarcosine and refolded by dialysis. Thereby, E. coli host cell proteins could be removed and high recovery of the target proteins was ensured.
- the GST-tagged proteins present in the bacterial lysate after French press is shown in Coomassie stained protein gels before and after inclusion body purification. The bacterial lysates show strong overexpression of the target proteins; however the supernatants did not contain suitable amounts as shown before. After several washing and re-suspension steps, the purified inclusion body pellets still contain the majority of the GST-fusion protein. Several smaller bands were observed to co-purify and were identified as truncated target proteins.
- Figure 4 Analysis of protein preparations
- the aggregated and probably misfolded proteins had to be transformed into a soluble, at its best, native form. Therefore the proteins were first denatured and thereby solubilized with the anionic detergent N-Lauroylsarcosine. Subsequent refolding was achieved utilizing dialysis with decreasing concentrations of the detergent and salts. Analysis of protein preparations obtained from inclusion body purification after Lauroylsarcosine- denaturation and refolding of the target proteins. The whole protein content analyzed by coomassie staining, the anti-Ll (MD2H11) and the anti-pl6 (D7D7) western blots are shown.
- the major proportion - respectively the dominant band in the Coomassie gel - is made up of the target protein as analyzed by western blotting. Smaller fragments could also be identified to be predominantly of LI or pl6 origin speaking for truncated, co-purified proteins.
- Figure 4 shows the results of the protein preparations obtained from inclusion bodies. For each of the LI containing constructs, Coomassie staining, anti-Ll and anti-pl6 western blot are shown in comparison. The intense bands at -92 kDa, respectively 75 kDa for GST-LI, indicate the GST-tagged fusion proteins. Below this upper dominant band, several smaller fragments could be identified in all the preparations. Western blot analysis revealed that these fragments must be of LI or pl6 origin, and thereby presenting truncated or degraded target proteins.
- GST-pl6 INK4 a As GST-pl6 INK4 a was expressed in soluble form, it had to be purified in a different way than the insoluble LI capsomeres. All protein lysates were treated equally as far as possible, e.g. they were lysed in the same buffer. Coomassie and western blot analysis of the bacterial lysate after French press, the supernatant containing the soluble protein and the purified GST-pl6 INK4a after GSTrap affinity chromatography are shown. The intense band at 42 kDa in the Coomassie gel was confirmed to be ⁇ 16 ⁇ with western blot analysis. After disruption of the bacterial cells, the supernatant was collected and loaded onto a 5 ml GSTrap FF column (GE Healthcare).
- the GST- pl6 INK4a protein was eluted with 20 mM glutathione to sustain the GST-tag. It was also possible to cleave pl6 INK4a from the GST tag with thrombin. However, to ensure best possible comparability for subsequent immunological in vivo experiments, pl6 INK4a was purified along with its GST-tag, at least for the application as control group protein in the mouse trials.
- the purified capsomeres were analyzed for their structural integrity, sedimentation characteristics and the presence of conformation-specific epitopes by means of transmission electron microscopy, sedimentation analysis and conformation specific ELISA.
- purified protein extracts were subjected to sucrose gradient centrifugation.
- the collected fractions (20 in total, 600 ⁇ each, fraction 1 from the top of the tube) were analyzed by western blotting.
- All expressed and purified fusion proteins contain the modified LI sequence with a deletion of the helix 4 region that prevents assembly into larger particles. Sedimentation analysis of the refolded proteins revealed the presence of capsomeres as demonstrated by western blot analysis with the Ll-specific antibody MD2H11.
- VLPs were found at the bottom of the centrifuge tube whereas the capsomeres accumulated at the top of the sucrose gradient (fractions 1 to 4) due to their low sedimentation coefficient. These sedimentation characteristics indicate the presence of small particles and are in line with previous studies.
- the purified proteins were analyzed by transmission electron microscopy (TEM) and two representative replicas of each sample are shown in Figure 44. All preparations contained large amounts of 10 - 12 nm capsomeric structures which is in line with previous reports. The characteristic donut-like appearance was also observed in some of the capsomeres but this was not a consistent finding. The black dot in the center of the capsomeres originates from the staining of their cylindrical cavity. As we used the C-terminally truncated LI construct this dot was only sporadically visible as described before. The capsomeres were found to be of variable size with a certain tendency to the formation of aggregates.
- TEM transmission electron microscopy
- pl6Ll and Llpl6Ll showed higher grades of agglomeration compared to LI and Llpl6.
- many non-assembled LI monomers were observed as "background noise". This often resulted in EM images with lower resolution as the uranyl acetate staining was challenging.
- the LI protein was administered in lower dose for the control group, as it has to be considered that the 50 ⁇ g fusion protein dose does not contain 50 ⁇ g pure LI but GST, ⁇ TM ⁇ and LI and thereby presents a comparable LI proportion altogether.
- the presence of pl6 mK4:i seems to boost the LI specific T cell response what could be due to the presentation of T helper epitopes in this formulation.
- the number of counted spots is very low and was therefore not normalized for RPMI control. As a number of up to 13 spots was found to be regular assay background, these spot numbers must be considered to be background response that would also have appeared without peptide stimulation. This shows that with using the pool of overlapping pl6 peptides for the ELISpot test, only low numbers of pi 6- specific T cells can be detected.
- Figure 10 shows the HPV16 Ll-ELISpot results split for each group, whereby LI 25 ⁇ g and pi()iNK4 a 50 ⁇ g represent the control groups.
- Grey bars indicate assay negative control spot numbers that were counted from unstimulated (RPMI) cells, black bars indicate Ll -specific T lymphocytes.
- Splenocytes from mice vaccinated with LI or one of the LI containing chimeric constructs produced significantly more IFNy spots when stimulated with the AGVDNRECI peptide compared to unstimulated cells. This is true for all test groups.
- N-terminal region of the HPV LI proteins that is deleted in preferred embodiments of the invention is highlighted in grey.
- C-terminal of the highlighted region is the conserved sequence motive that is preferably kept intact in the N-terminal deletion fragments of HPV LI proteins. This motive is indicated by underline.
- helix 4 of the aligned HPV LI proteins Helix 4 is deleted in preferred embodiments of the invention.
- the C-terminal region of the HPV LI proteins that is deleted in preferred embodiments of the invention is highlighted in grey.
- N-terminal of the highlighted region is the conserved sequence motive that is preferably kept intact in the N-terminal deletion fragments of HPV LI proteins. This motive is indicated by underline.
- Corresponding N-terminal, C-terminal and/or internal regions of LI proteins of other HPV can be determined by the skilled person using standard alignment parameters and programs as set out above.
- Example 1 Purification of HPV16 LI capsomeres and chimeric fusion proteins combining
- HPV16 LI and pl6 INK4a from E. coli Protein expression
- HPV16 LI capsomeres and chimeric capsomeres were purified using an adapted protocol described in the iFOLD® Protein Refolding System 1 User Protocol by Novagene.
- Fresh or frozen cell pellets were re-suspended in IB Resuspension Buffer (5.4.2; 10 ml / 1 g cell paste) supplemented with protease inhibitor cocktail (1 tablet per 50 ml).
- 20 ⁇ Lysonase were added per gram cell pellet and stirred for 15 minutes.
- Bacteria were lysed using a high-pressure homogenizer (French press). Subsequently ATP and MgC12 were added to the lysate to final concentrations of 2 and 5 mM, respectively.
- Triton X-100 was added to a final concentration of 1 % and incubated for 15 minutes on a roller mixer. Cell lysates were centrifuged at 8.000 x g (-10.000 rpm - SS-34 rotor, Sorvall) for 15 minutes and the supernatant was discarded. The resulting pellets were washed with IB Wash Buffer (10 ml buffer / 1 g cell pellet) whereby complete resuspension was achieved with the help of a Potter Elvehjem Homogenizer. The suspension was centrifuged again and the Triton X-100 washing was repeated one more time. After the second washing and centrifugation, remaining Triton X-100 was removed by washing the pellet twice in Resuspension Buffer, as described above. The purified inclusion body pellets were stored at -80°C until further use.
- Example 3 Denaturing of inclusion bodies and refolding of the protein
- the buffer was exchanged by freshly prepared buffer and dialyzed over night at 4°C.
- the refolded protein was then removed from the dialysis tube and further analyzed by electron microscopy. Protein concentration was determined with the 2-D Quant Kit (GE Healthcare).
- Fresh or frozen cell pellets were re-suspended in IB Resuspension Buffer (5.4.2; 10 ml / 1 g cell paste) supplemented with protease inhibitor cocktail (1 tablet per 50 ml). 20 ⁇ Lysonase were added per gram cell pellet and stirred for 15 minutes.
- Bacteria were lysed using a high-pressure homogeniser (French press).
- ATP and MgC12 were added to the lysate to final concentrations of 2 and 5 mM, respectively.
- cell lysates were centrifuged at 8.000 x g (-10.000 rpm - SS-34 rotor, Sorvall) for 15 minutes and the pl6 INK4a containing supernatant was used for further purification solution. Afterwards columns were washed with 20-30 CVs of PBS and stored in 20% ethanol.
- Inclusion bodies were denatured with 5 % N-Lauroylsarcosine, urea (4, 6 and 8 M) and 6 M guanidine hydrochloride.
- the protein lysate was incubated with the respective denaturing agent at RT on a roller mixer until the solution became clear.
- Dialysis conditions for denatured proteins were tested in small scale to determine optimal protein concentration and buffer receipts for refolding. Thereto, a 3,500 Da dialysis membrane was cut into 2 x 2 cm squares and equilibrated in aqua bidest. 1.5 ml Protein LoBind Eppendorf reaction tubes were cut at the 1.5 mark and the lid was filled with 100 ⁇ of the denatured protein; diluted in denaturing buffer if required. Then, the dialysis membrane was clamped between the lid and the remaining reaction tube and put into the dialysis buffer of choice over night at 4°C. The next day, absorption at 340 nm was measured to quantify the amount of precipitated protein. The lower the value, the more soluble protein was present in the solution. This method allows testing of different buffer conditions and protein dilutions in an easy and economy way.
- mice were immunized subcutaneously at the neck using a blue G23 needle. 5 to 50 ⁇ g of recombinant antigen was diluted in PBS and mixed; if necessary, with adjuvant. Mice were usually immunized 3 times at biweekly intervals with an injection volume of 100 ⁇ unless mentioned otherwise. Negative control mice were treated with PBS with purified irrelevant antigens.
- mice were immunized by topical application with 50 ⁇ g recombinant protein formulated in 50% DMSO. Thereto, it was necessary to shave the back of the mice with an electric clipper. Mice were held at their tail and carefully shaved across the grain. Between the first shaving and the first antigen application, three days were paused to heal potential wounding of the skin. Mice were creamed daily for about 1 minute till the mixture was absorbed through the skin. This procedure was carried out 2 x 5 days with 2 days rest in-between. Blood and spleens were sampled 4 days after the last topical application and analyzed for humoral and cellular immune response.
- Example 7 Analysis of humoral and cellular immune responses: VLP-capture ELISA to determine Ll-specific antibodies
- 96-well microtiter plates (Nunc) were coated with 50 ⁇ /well of the purified polyclonal HPV16 Ll-specific serum in a 1:500 dilution in PBS at 4°C overnight. All incubation steps were performed 1 h at 37°C unless mentioned otherwise and plates were washed after each incubation step 4 times with PBS-T. After washing off unbound coating antibody, plates were blocked 1 h with 5 % milk in PBS-T. HPV16 VLPs were diluted 1:500 (0.3 ⁇ & ⁇ in blocking solution) and 50 ⁇ per well were added and incubated. Plates were washed and sera of immunized mice were applied in duplicates in a 1: 10 dilution (100 ⁇ /well).
- the positive control (HPV16 LI monoclonal antibody) was also diluted 1: 10. Incubation was followed by another washing step and a horseradish peroxidase conjugated anti-mouse IgG antibody (50 ⁇ / well; 1:5000) was applied. After 1 h, plates were washed and 50 ⁇ TMB substrate were added to the wells and incubated at room temperature. The reaction was stopped after 5 to 10 minutes with 50 ⁇ per well of 1 M H2S04 and absorbance at 450 nm (reference wavelength: 620 nm) was measured in an ELISA reader. Sera were considered to be LI antibody positive if the absorption value was above cut-off.
- the cut-off value is based on the distribution of absorbance values of control sera and was calculated as the mean value of negative controls plus three times standard deviation.
- Example 8 Analysis of humoral and cellular immune responses: pl6 INK4a -peptide pool ELISA to determine pl6 INK4a -specific antibodies
- pi 6TM K4a specific antibodies were detected with overlapping peptides covering the complete sequence.
- 5 peptide pools consisting of 2 or 3 peptides were used by default.
- 96well microtiter plates (Nunc) were coated with a dilution of 40 ⁇ g per petide per ml in PBS. 50 ⁇ per well were incubated at 4°C overnight. Plates were washed 4 times with PBS-T after each step and incubation was performed for 1 h at 37°C. After peptide coating, remaining binding sites were blocked with 0.5 % casein in PBS-T (coating for 1 h at room temperature.
- mice After washing, sera of immunized mice were applied in duplicates in a 1: 10 dilution (50 ⁇ /well) and incubated. Plates were washed again and a horseradish peroxidase conjugated anti-mouse IgG antibody (50 ⁇ / well; 1:5000) was applied and incubated. TMB substrate was used to detect HRP activity and the reaction was stopped after 30 minutes with 50 ⁇ 1 M H2S04. The yellow reaction product was read at 450 nm in an ELISA reader (reference wavelength: 620nm). Individual sera were scored as antibody-positive or negative using a cut-off value that was calculated as the mean value of negative controls plus three times standard deviation. Sera were considered to be positive if at least one of the tested peptide pools showed a value above cut-off.
- Example 9 Detection of antigen-specific cytotoxic T-lymphocytes by IFNy-ELISpot
- IFNy-ELISpot assay was performed 7 days after the last immunization and all steps on day one and two were carried out under sterile conditions in a flow hood.
- 96well MultiScreen-HA plates (Millipore) were incubated for 5 minutes with 70 ⁇ of 70% Ethanol, washed 4 times with 200 ⁇ PBS /well and coated with 100 ⁇ /well anti-mouse IFNy capture antibody (0.6 ⁇ g per well) diluted in PBS.
- the plates were wrapped in aluminium foil and incubated at 4°C over night or at 37°C for 1 - 2 hours.
- test peptides or the pl6 peptide pool were each tested in triplicates with 10 ng/well antigenic peptide in 100 ⁇ medium.
- a typical loading scheme is depicted in Figure 11, yet it was adapted corresponding to the number of mice and peptides tested.
- After incubation for 18 to 20 hours at 37°C cells were removed and plates were washed 4 times with PBS-0.01% Tween20 and once with PBS.
- 0.1 ⁇ g of a biotinylated rat anti-mouse IFNy antibody diluted in PBS (100 ⁇ ) were added per well and incubated 1 - 2 h at room temperature. After removing the secondary antibody, plates were washed 6 times with PBS-T and once with PBS.
Abstract
La présente invention concerne un vaccin comprenant une protéine d'enveloppe virale et un antigène, la protéine d'enveloppe virale et l'antigène étant liés l'un à l'autre par liaison covalente ou non-covalente, et l'antigène étant un antigène tumoral cellulaire. L'invention concerne en outre un acide nucléique codant pour ledit vaccin, un vecteur d'expression comprenant ledit acide nucléique et une cellule hôte recombinante comprenant ledit vecteur d'expression. La présente invention concerne en outre un procédé de fabrication d'un vaccin de la présente invention, une composition pharmaceutique comprenant ledit vaccin et l'utilisation dudit vaccin pour le traitement et/ou la prévention d'une maladie associée audit antigène.
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