EP3089755A1 - Vlp, procédés pour leur obtention et applications de ceux-ci - Google Patents

Vlp, procédés pour leur obtention et applications de ceux-ci

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Publication number
EP3089755A1
EP3089755A1 EP15702968.7A EP15702968A EP3089755A1 EP 3089755 A1 EP3089755 A1 EP 3089755A1 EP 15702968 A EP15702968 A EP 15702968A EP 3089755 A1 EP3089755 A1 EP 3089755A1
Authority
EP
European Patent Office
Prior art keywords
structural protein
virus
polynucleotide
protein
trv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15702968.7A
Other languages
German (de)
English (en)
Inventor
José Francisco Rodríguez Aguirre
Diego Marcelo GUÉRIN AGUILAR
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fundacion Biofisica Bizkaia
Euskal Herriko Unibertsitatea
Original Assignee
Fundacion Biofisica Bizkaia
Euskal Herriko Unibertsitatea
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Filing date
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Application filed by Fundacion Biofisica Bizkaia, Euskal Herriko Unibertsitatea filed Critical Fundacion Biofisica Bizkaia
Priority to EP15702968.7A priority Critical patent/EP3089755A1/fr
Publication of EP3089755A1 publication Critical patent/EP3089755A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/005—Trypanosoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/22011—Dicistroviridae
    • C12N2770/22023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/22011—Dicistroviridae
    • C12N2770/22034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/22011—Dicistroviridae
    • C12N2770/22041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/22042—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • the present invention belongs to the field of virus and virus-like particles (VLPs) as it relates to VLPs derived from Triatoma virus (TrV) comprising a heterologous polypeptide, a process for obtaining a Triatoma virus VLP modified to express a heterologous protein of interest, polynucleotides comprising the necessary elements for generation of recombinant TrV virions and the process for obtention of said polynucleotides.
  • VLPs virus and virus-like particles
  • VLPs are potent inducers of immune response for both themselves and also against any antigen displayed on them.
  • VLPs Virus-like particles
  • VLPs are structures built in an organized and geometrically regular manner from many polypeptide molecules of one or more types. Being comprised of more than one molecule, VLPs can be referred to as being supramolecular. VLPs lack the viral genome and, therefore, are noninfectious. VLPs can be produced in large quantities by heterologous expression and can be easily purified. The geometry of a VLP typically resembles the geometry of the source virus particle. VLPs are being exploited in the area of vaccine production because of their structural properties, their ease in large scale preparation and purification, and their non-infectious nature.
  • VLPs include the self-assemblages of capsid or nucleocapsid polypeptides of hepatitis B virus, Sindbis virus, hepatitis C virus, rotavirus, Norwalk virus, retroviruses, retrotransposons and human papilloma virus. Plant-infecting virus capsid polypeptides also self-assemble into VLPs in vitro and in vivo.
  • VLPs have also proven amenable to the insertion or fusion of foreign antigenic sequences, allowing the production of chimeric VLPs exposing the foreign antigen on their surface.
  • Other VLPs have been used as carriers for foreign antigens, including non-protein antigens, via chemical conjugation.
  • WO 2011057134 Al Methods for generating VLPs linked to antigens through protein-protein interaction have been described in WO 2011057134 Al .
  • antigen presenting systems based on VLP particles have been described in the art.
  • WO 1996030523 A2 describes an antigen presentation system based on retrovirus- like particles
  • WO 2011091279 A2 describes antigen presentation based on calicivirus VLPs
  • WO 2010118424 A2 describes a VLP based on papilloma virus LI major capsid protein comprising a neutralizing epitope of a papillomavirus L2 protein
  • WO 2011102900 Al describes VLPs that display one or more truncated, re-engineered or remodeled hemagglutinin molecules on their surface.
  • TrV-VLP insect viral pathogen Triatoma virus
  • TrV-VLP insect viral pathogen Triatoma virus
  • TRWLP This antigen presenting system
  • TrV-VLP allows epitope exposure on the internal or external surface of the VLPs by insertion or substitution of amino acids, thus facilitating their recognition by the immune system and/or increasing their immunogenicity.
  • production of TrV-VLPs bearing different epitopes may occur through the expression in insect cells by infection of recombinant baculoviruses.
  • the present invention relates to a VLP comprising
  • Dicistroviridae family or the viral structural proteins VPl , VP2, and VPO of a virus of the Dicistroviridae family, or the structural protein precursor (PI) from a virus of the Dicistroviridae family, or a functionally equivalent variant any thereof, and
  • heterologous polypeptide is provided as a fusion protein with at least one of said viral structural proteins.
  • the present invention relates to a polynucleotide encoding a fusion protein selected from the group consisting of
  • a fusion protein comprising a structural protein from a virus of the Dicistroviridae family selected from the group consisting of VPl, VP2, VP3, VP4 and VPO, or a functionally equivalent variant thereof, and a heterologous polypeptide, or
  • a fusion protein comprising a structural protein precursor (PI) from a virus of the Dicistroviridae family, or a functionally equivalent variant thereof, and a heterologous polypeptide.
  • the invention relates to a vector comprising the polynucleotide encoding a fusion protein as above. In a further aspect, the invention relates to a host cell comprising the polynucleotide encoding a fusion protein or the vector as above.
  • the present invention relates to a process for obtaining a Dicistroviridae family virus VLP modified to express a heterologous polypeptide of interest comprising the steps of:
  • step (ii) maintaining the cells under conditions suitable for the expression of the polynucleotide introduced in the cell in step (i).
  • the present invention relates to the VLP obtainable by the process above.
  • the present invention relates to a vaccine or immunogenic composition comprising the VLP as above.
  • the present invention relates to a VLP, to a polynucleotide, or to a vector as mentioned above for use in the treatment of an infectious disease caused by a pathogenic organism and/or an immune reaction caused by an allergen from which the heterologous peptide is derived.
  • the present invention relates to a R A polynucleotide comprising in the 5 ' to 3 ' direction:
  • the invention relates to a DNA polynucleotide having at least one strand which is complementary to the R A polynucleotide as above, further comprising a transcriptional regulatory region which regulates the expression of said DNA polynucleotide
  • the present invention relates to a process for obtaining an infectious Triatoma virus (TrV) comprising the steps of:
  • step (ii) maintaining the cells under conditions suitable for the expression of the polynucleotide introduced in the cell in step (i).
  • the invention relates to an infectious Triatoma virus (TrV) obtained by the process above.
  • the invention relates to a method for killing insects comprising the administration of an infectious Triatoma virus (TrV) as above.
  • TrV infectious Triatoma virus
  • FIG. 1 Negatively stained transmission electron micrograph of TrV VLPs obtained according to the invention (magnification 80,000x).
  • the VLPs are of about 30 nm in diameter and were obtained by means of contacting the host cell with two polynucleotides.
  • the first polynucleotide corresponds to the sequence encoding the TrV structural proteins (PI), and the second polynucleotide comprises the sequence encoding for the non- structural protein precursor of TrV (NS).
  • FIG. 1 Negatively stained transmission electron micrograph of TrV VLPs obtained according to the invention (magnification 80,000x).
  • the VLPs are of about 50 nm in diameter and were obtained by means of contacting the host cell with only one polynucleotide sequence encoding for the TrV structural protein precursor PI .
  • FIG. 1 Immuno-stimulatory properties of TrV VLPs. Mouse spleen cells were immunized in vitro with TrV, empty TrV and recombinant TrV VLPs. The data showed that the recombinant VLP has good immunostimulatory properties.
  • Figure 4. Accessibility of reactive groups on the TrV VLPs external surface. Fluorescein is conjugated to Lys residues exposed on the surface of the VLP.
  • TrV Triatoma virus
  • BAP biotin acceptor peptide
  • 5 '-end designates the end of a nucleotide strand that has the fifth carbon in the sugar-ring of the deoxyribose at its terminus.
  • 3 '-end designates the end of a nucleotide strand that has the hydroxyl group of the third carbon in the sugar-ring of the deoxyribose at its terminus.
  • biotin acceptor peptide or "BAP”, as used herein, relates to a peptide with the capacity to bind to biotin or to an analog thereof (e.g. 2-iminobiotin, etc.).
  • BAP biotin acceptor peptide
  • a biomolecule e.g. a protein molecule
  • biotin are well known in the art (Bayer and Wilchek 1992 Methods in Mol Biol 10, 143).
  • biotin-binding molecule relates to a member of a binding pair that binds to biotin.
  • the biotin-binding molecule is avidin, streptavidin (SA) or SA or avidin fragments which retain substantial binding activity for biotin, such as at least 50 percent or more of the binding affinity of native SA.
  • avidin refers to a glycoprotein found in egg whites and in tissues of birds, reptiles and amphibians protein and which has the capacity to bind to biotin with high affinity as well as any expressed or engineered form of the avidin biotin-binding molecule, such as streptavidin, neutravidin and the like.
  • avidin includes both avidin found naturally in the eggs of Gallus gallus (NCBI accession numbers NM_205320.1 / GL45384353en, release as of 14 May 2013) as well as the orthologues of said protein in other species.
  • Streptavidin corresponding to the protein from Streptomyces avidinii (accession number CAA00084.1 in GenBank, release as of 28 January 1993), as well as the orthologues, homologues and fragments of streptavidin defined in the same manner as avidin.
  • Streptavidin comprises 4 subunits each of which contains a binding site for biotin.
  • Streptavidin (SA) or avidin fragments which retain substantial binding activity for biotin, such as at least 50 percent or more of the binding affinity of native SA or avidin, respectively, may also be used.
  • the affinity of the avidin variant for biotin is of at least 10 15 M “1 , 10 14 M “1 , 10 13 M “1 , 10 12 M “1 , 10 10 M “1 or 10 9 M “1 .
  • Avidin and estreptavidin variants suitable for use in the present invention include, without limitation:
  • CSA Core streptavidin
  • Mutants with reduced immunogenicity such as mutants mutated by site- directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes,. See Meyer et al, Protein Sci. 2001 10: 491-503.
  • Variants resulting from the chemical modification of avidin such as those resulting from the complete or partial modification of glycosylation and fragments thereof as well as the completely deglycosylated avidin variant known as neutravidin.
  • Modified streptavidin dimers such as those described in WO06058226, The protein with biotin binding capacity as described in WO04018509, Streptavidin having a higher affinity for biotin as described in WO9840396, - The modified streptavidin and avidin molecules as described in
  • streptavidin with modified affinity as described in WO9624606.
  • the terms “avidin” and “streptavidin” as used herein are intended to encompass biotin-binding fragments, mutants and core forms of these binding pair members.
  • Different avidin variants are commercially available, such as Extravidin (Sigma-Aldrich), NeutrAvidin (Thermo Scientific), NeutrAvidin (Invitrogen) and NeutraLite (Belovo).
  • Extravidin Sigma-Aldrich
  • NeutrAvidin Thermo Scientific
  • NeutrAvidin Invitrogen
  • NeutraLite Belovo
  • nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences can be found, for example, in GenBank Accession Nos. X65082; X03591; NM --205320; X05343; Z21611; and Z21554.
  • binding means the physical association between a first species and a second species, for example, the binding of a ligand by a receptor, the binding of an antigen by an antibody, the binding of a nucleic acid by a nucleic acid binding protein, etc.
  • cleavage or "cleavage activity”, as used herein, relates to R A cleavage performed by a ribozyme in specific RNA sites.
  • the ribozyme is an autocatalytic ribozyme.
  • Dicistroviridae family relates to a family of Group IV (positive sense single stranded RNA) arthropod-infecting viruses, particularly insect- infecting viruses.
  • Some insects commonly infected by dicistro viruses include, without limitation, aphids, leafhoppers, flies, bees, ants, crickets, triatomines and silkworms. Viruses of this family are characterized by the location of their structural protein genes at the 3' end of the genome, and by having two genomic segments.
  • the Dicistroviridae family includes Aparavirus and Cripavirus genuses.
  • the insect from the Dicistroviridae family is an insect from the Cripavirus genus, more particularly a Triatoma virus (TrV) or a Cricket paralysis virus (CrPV), preferably TrV.
  • epitope refers to that portion of a given immunogenic substance that is the target of, i.e., is bound by, an antibody or cell-surface receptor of a host immune system that has mounted an immune response to the given immunogenic substance as determined by any method known in the art.
  • an epitope may be defined as a portion of an immunogenic substance that elicits a humoral or cellular, response, in particular an antibody response or a T-cell response in an animal, as determined by any method available in the art (see, for example, Geysen et al, Proc Natl Acad Sci (1984) Vol.81 3998-4002).
  • An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • immunogenic substance such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • epitope may also be used interchangeably with “antigenic determinant” or “antigenic determinant site”.
  • expression cassette refers to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell.
  • fusion protein as used herein, relates to proteins generated by gene technology which consist of two or more functional domains derived from different proteins.
  • a fusion protein may be obtained by conventional means (e.g. by means of gene expression of the nucleotide sequence encoding for said fusion protein in a suitable cell).
  • the fusion protein of the invention comprises a heterologous polypeptide of interest and at least one viral structural protein, in particular at least one viral structural protein of the Triatoma virus selected from the group comprising VPO, VP1, VP2, VP3 and VP4.
  • GK1 relates to a synthetic antigen peptide that is comprised by the S3pvac vaccine against Porcine cysticercosis, altogether with peptides KETcl and KETcl2.
  • the term ' 'haematophagous relates to bloodsucking insects, in particular flying insects, which are commonly attracted to a potential living blood source, such as mammals.
  • Haematophagous insects include, without limitation, mosquitoes, phlebotomine sand flies, simuliid black flies, horse flies and midges.
  • the haematophagous insect is a member of the genus Cimex, including Cimex lectularius (commonly known as bed bug), Cimex pipistrelli, Cimex pilosellus, and Cimex adjunctus.
  • the haematophagous insect is a member of the Culicidae family (a mosquitoe).
  • the haematophagous insect is a member of the Reduviidae family, particularly of the Triatominae subfamily, more particularly Triatoma infestans, Triatoma dimidiata, Triatoma protracta and Rhodnius prolixus.
  • heterologous polypeptide relates to any polypeptide which is to be present in a VLP or virion according to the present invention, including, without limitation, an antigen, a toxin or an enzyme.
  • Non-limiting illustrative examples of antigens which can be used as heterologous polypeptides of interest in the present invention include:
  • ⁇ Peptides or proteins capable of (suitable or designed for) inducing an immune response against an infectious disease, such as an infectious disease in animals caused by pathogenic microorganisms of animals, including humans, for example, virus, bacteria, fungi and infectious parasites, relevant in human or animal health.
  • the proteins or peptides capable of inducing an immune response can be recombinant proteins or peptides, identical or similar to the natural antigens of a specific microorganism.
  • infectious virus include virus of the families: Arteriviridae, Retroviridae, Picornaviridae, Calciviridae, Togaviridae, Flaviridae, Coronoviridae, Rhabdoviradae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae (parvovirus), Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, etc.
  • antigens which can be used according to the present invention include but are not limited to HIV antigens, gpl20 antigen, hepatitis B surface antigen, rotavirus antigens such as VP4 and VP7, influenza virus antigens such as hemagglutinin or nucleoprotein, thymidine kinase herpes simplex antigen, etc.
  • Non- limiting illustrative examples of bacteria include both Gram positive bacteria, e.g., Pasteurella sp., Staphylococcus sp., Streptococcus sp., etc., and Gram negative bacteria, e.g., Escherichia coli, Pseudomonas sp., Salmonella sp., etc.
  • Specific examples of infectious bacteria include: Helicobacter pylori, Borelia burgdorferi, Legionella pneumoplailia, Mycobacteria sp. (e.g., M. tuberculosis, M. avium, M. intracellular, M. kansaii, M.
  • Non-limiting illustrative examples of infectious fungi include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis and Candida albicans.
  • protozoa are included among the infectious parasites, such as Plasmodium sp., protozoa causing malaria, e.g. P. falciparum, P. malariae, P. ovale, P. vivax, etc., Leishmania sp., protozoa causing leishmaniasis, e.g., L. major, L. donovani, L. infantum, L. braziliensis, L. panamensis, L.
  • infectious parasites such as Plasmodium sp., protozoa causing malaria, e.g. P. falciparum, P. malariae, P. ovale, P. vivax, etc.
  • Leishmania sp. protozoa causing leishmaniasis, e.g., L. major, L. donovani, L. infantum, L. braziliensis, L. panamensis, L.
  • mexicana etc.
  • Toxoplasma gondii Schistosoma sp., etc.
  • parasitic nematodes such as Dirofilaria immitis, protozoa causing the Chagas disease, e.g. Tripanosoma cruzi, protozoa causing African trypanosomiasis, e.g. Tripanosoma brucei, etc.
  • antigens for these parasites include Plasmodium spp. circumsporozoite antigen; Plasmodium spp. merozoite surface antigen; Leishmania spp. gp63, etc.
  • ⁇ Peptides or proteins associated with tumors or cancers capable of (suitable or designed for) inducing an immune response against a tumor or cancer cell therefore the heterologous polypeptide of interest can be used in the treatment of cancers by means of the stimulation of an antigen-specific immune response against a tumor antigen.
  • Non-limiting illustrative examples of cancers which could be potentially treated according to the teachings of the present invention include bile duct cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, stomach cancer, intraepithelial neoplasias, lymphomas, liver cancer, lung cancer (e.g., small and non-small cell lung cancer), melanoma, neuroblastomas, mouth cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin cancer, testicular cancer, thyroid cancer and renal cancer, as well as other carcinomas and sarcomas.
  • a person skilled in the art can select tumor antigens or antigenic determinants for the treatment of cancers in view of the state of the art [Renkvist et al., Cancer Immunol. Immunother. 50:3-15 (2001)], said antigens and antigenic determinants being included within the scope of the present invention.
  • antigens or antigenic determinants include: Her2 (breast cancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA (medullary thyroid cancer); CD52 (leukemia); human melanoma gplOO protein; human melanoma melan- A/MART- 1 protein; tyrosinase; NA17-A nt protein; MAGE-3 protein; p53 protein; HPV16E7 protein; and antigenic fragments of said peptides or proteins.
  • allergen relates to a peptide or protein to which a subject is sensitive and causes an immune reaction, for example, allergen extracts of pollens, allergen extracts of insects, allergen extracts of food or food products, components present in saliva, insect claws or stings which induce a sensitivity reaction in a subject, components present in plants which induce a sensitivity reaction in a subject, etc.
  • Non-limiting illustrative examples of allergens include protein extracts of pollens, e.g., of Lolium perenne, Poa pratense, Phleum pratense, Cynodon dactylon, Festuca pratensis, Dactylis glomerata, Secale cereale, Hordeum vulgare, Avena sativa, Triticum sativa, Artemisia vulgaris, Chenopodium album, Plantago lanceolata, Taraxacum vulgare, Parietaria judaica, Salsola kali, Urtica dioica, Olea europea, Platanus sp., Cupressus sp., etc.; protein extracts of insects, e.g., of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acari such as Acarus siro, Blomia tropicalis, Euroglyphus maynei, Glyciphagus domestic
  • autoantigen relates to peptides or proteins encoded by the DNA of the subject and products generated by proteins or RNA encoded by the DNA of the subject. Examples of autoantigens are described in WO 02/56905.
  • host cell refers to a cell into which a nucleic acid of the invention, such as a polynucleotide or a vector according to the invention, has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the term “intergenic region” or IGR as used herein, relates to a stretch of non- coding DNA and R A sequences located between genes.
  • immunogenic composition refers to a composition that elicits an immune response in a subject that produces antibodies or cell-mediated immune responses against a specific immunogen.
  • antigenic composition refers to a composition that can be recognized by a host immune system.
  • an antigenic composition contains epitopes that can be recognized by humoral or cellular components of a host immune system.
  • infectious disease relates to diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases may be caused by viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis, and the like.
  • viruses including adenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpe
  • Infectious diseases may also be caused by bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersinia pestis, and the like.
  • bacteria including Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacterium
  • Infectious diseases may also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium marneffei, and the like. Infectious diseases may also be caused by protozoa and parasites such as chlamydia, kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.
  • KETcl relates to a synthetic antigen peptide originally identified in a Taenia crassiceps cDNA library, and that is comprised by the S3pvac vaccine against Porcine cysticercosis, altogether with peptides GK-1 and KETcl2.
  • KETcl2 relates to a synthetic antigen peptide that is comprised by the S3pvac vaccine against Porcine cysticercosis, altogether with peptides GK-1 and KETcl .
  • ligand does not involve any particular size or another structural technical feature different from that of said ligand being capable of interacting and binding to the heterologous polypeptide present in the VLP and/or the infective virions of the invention; therefore, said ligand contains a region of interaction with the specific binding domain which is present in the heterologous polypeptide contained in the VLP or the virion of the invention.
  • the "heterologous polypeptide"-"ligand” pair acts as a specific binding pair, e.g., of the type of biotin-avidin, epitope-antibody or functional fragment thereof (e.g., a Fab or F(ab')2 fragment, an scFv (single-chain antibody), a chimeric antibody, etc.), etc., in which each of said peptide insert and ligand form the members of said specific binding pair, each of them containing the binding domains or the corresponding regions of interaction; thus, depending on the heterologous polypeptide present in the VLP or virion of the invention the ligand which must be present in the product of interest will be chosen, and, vice versa, depending on the ligand which is to be bound to the VLP or virion of the invention, the heterologous polypeptide which must be present in said VLP or virion will be chosen.
  • a specific binding pair e.g., of the type of biotin-avidin, epitope
  • the binding of the ligand to the heterologous polypeptide is performed directly.
  • a product of interest with the capacity to bind to said polypeptide present in the VLP or virion of the invention will be used, such that said heterologous polypeptide and product of interest form a specific binding pair.
  • heterologous polypeptide comprises an amino acid tag (e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.); likewise when said peptide insert comprises a peptide epitope which can be recognized by an antibody (e.g., c-myc-tag, etc.), the product of interest is an antibody or a functional fragment thereof; etc.
  • amino acid tag e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.
  • peptide insert comprises a peptide epitope which can be recognized by an antibody (e.g., c-myc-tag, etc.)
  • the product of interest is an antibody or a functional fragment thereof; etc.
  • the binding of said ligand to the heterologous polypeptide can be performed indirectly, i.e., by means of using a "linker", or molecule comprising, on one hand, a first region of interaction with the binding domain contained in the heterologous polypeptide present in the VLP or virion and, on the other hand, a second region of interaction (binding domain) with a region of interaction present in the ligand.
  • a linker or molecule comprising, on one hand, a first region of interaction with the binding domain contained in the heterologous polypeptide present in the VLP or virion and, on the other hand, a second region of interaction (binding domain) with a region of interaction present in the ligand.
  • the product of interest binds to the VLP or virion through a structure of the type: heterologous polypeptide -linker-ligand-product of interest.
  • Illustrative non-limiting examples of this embodiment include the use of avidin or an analog thereof (linker) when the heterologous polypeptide present in the VLP or virion comprises a BAP; etc.
  • the term "avidin” includes any avidin, of a eukaryotic or prokaryotic origin, and its variants which maintain the capacity to bind to biotin; in a particular embodiment, the ligand present in the conjugate of the invention comprises a monomeric variant of avidin (mAV).
  • non-structural protein as used in the context of the present invention in relation to a virus of the Dicistroviridae family, in particular to a Triatoma virus, relates to proteins encoded by the viral genome which do not form the capsid of said virus.
  • the dicistrovirus genome has two open reading frames, namely ORF1 and ORF2, which encodes the non-structural protein precursor (NS) and the structural protein precursor (PI), respectively.
  • Dicistrovirus, in particular TrV non-structural proteins include the viral polymerase, the viral protease and the viral helicase.
  • non-structural protein precursor or "NS”, as used in the present invention in relation to a virus of the Dicistroviridae family, in particular to a Triatoma virus, relates to the non-structural polyprotein precursor encoded by the 5' ORF1 viral genome and, particularly, corresponding to the region 549-5936 of Triatoma virus complete RNA genome according to the sequence NC 003783.1 (NCBI database as of April 28 th , 2010), to the region 549-5936 of the sequence AF 178440 from the non- structural and capsid protein precursors (NCBI database as of April 14 th , 2010), and according to Czibener C et al. 2000 J Gen Virology 81 : 1149-1154.
  • TrV non-structural polyprotein precursor The amino acid sequence of the TrV non-structural polyprotein precursor is located in NCBI database under accession number NP_620562.1 (1795 aa, NCBI database as of April 28 th , 2010). From this non-structural protein precursor, a RNA helicase, a peptidase and a RNA- dependent RNA polymerase (which catalyzes the synthesis of the RNA strand complementary to a given RNA template) are produced.
  • polynucleotide relates to a polymer formed by a variable number of monomers wherein the monomers are nucleotides, including ribonucleotides as well as deoxyribo nucleotides.
  • the polynucleotides include monomers modified by methylation as well as unmodified forms.
  • polynucleotide and nucleic acid are used indiscriminately in the present invention and include mRNA, cDNA and recombinant polynucleotides.
  • polynucleotides are not limited to polynucleotides as they appear in nature, and also include polynucleotides where unnatural nucleotide analogues and inter- nucleotide bonds appear.
  • Non- limitative examples of this type of unnatural structures include polynucleotides wherein the sugar is different from ribose, polynucleotides wherein the phosphodiester bonds 3 '-5' and 2'-5' appear, polynucleotides wherein inverted bonds (3 '-3' and 5 '-5') appear and branched structures.
  • polynucleotides of the invention include unnatural inter-nucleotide bonds such as peptide nucleic acids (PNA), locked nucleic acids (LNA), C1-C4 alkylphosphonate bonds of the methylphosphonate, phosphoramidate, C1-C6 alkylphosphotriester, phosphorothioate and phosphorodithioate type.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • C1-C4 alkylphosphonate bonds of the methylphosphonate phosphoramidate
  • C1-C6 alkylphosphotriester phosphorothioate
  • phosphorodithioate type unnatural inter-nucleotide bonds
  • polypeptide refers to polymers of amino acids of any length.
  • the polymer may be linear, cyclic, or branched.
  • the polymer may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass amino acid polymers that have been modified, for example, via sulfonation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, ubiquitination, or any other manipulation, such as conjugation with a labeling component.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • paraflagelar rod protein or "PFR” relates to a protein identified in T. cruci flagellum and specific to kinetoplastidis. This protein has been isolated in T. brucei and Leishmania species as well.
  • the paraflagellar rod protein 2 (PFR2) gene was first identified in T. cruzi by pre-embedding immunoelectron microscopy with a gold- tagged secondary antibody (Saborio JL et al. 1989 J Biol Chem 264(7):4071-5), and it is believed to be a member of a multi-gene family of nearly 30 protozoan parasites.
  • the protein has a highly organized three-dimensional structure, been found only in kinetoplastids, euglenoids, and some dino flagellates, and is responsible for parasite cell motility (Abdille MH et al. 2008 Exp Parasitol 118: 614-618).
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a "constitutive" promoter is a promoter that is active under most physiological and developmental conditions.
  • an “inducible” promoter is a promoter that is regulated depending on physiological or developmental conditions.
  • a “tissue specific” promoter is only active in specific types of differentiated cells/tissues.
  • exemplary suitable promoters according to the invention include, without limitation, polyhedrin (polh) promoter or baculo virus immediate early (iel) promoter, plO promoter or the basic protein promoter.
  • ribozyme or "RNA enzyme” or “catalytic RNA” refers to an RNA- based enzyme capable of targeting and cleaving particular base sequences in DNA or, more typically, RNA.
  • preferred ribozymes comprise autocatalytically cleaving ribozymes.
  • Ribozymes include, without being limited to, hammerhead ribozyme (HamRz), Varkud satellite (VS) ribozyme, hairpin ribozyme, hepatitis delta virus (HDV) ribozyme (HDVRz) (Been MD & Wickham GS 1997 Eur J Biochem 247: 741-753), mammalian CPEB3 ribozyme, GlmS glucosamine-6-phosphate activated ribozyme and beta-globin co-transcriptional cleavage (CoTC) ribozyme.
  • the ribozyme is the hammerhead ribozyme (HamRz) or the hepatitis delta virus ribozyme (HDVRz).
  • structural protein as used in the present invention in the context of the Triatoma virus proteins, relates to those proteins forming the viral capsid of TrV.
  • the dicistro virus genome has two open reading frames, namely ORF1 and ORF2, which encodes the non-structural proteins (NS) and the structural protein precursor (PI), respectively.
  • TrV structural proteins include VP1, VP2, VP3 and VP4.
  • VPO is the precursor ofVP3 and VP4.
  • structural protein precursor or PI, as used in the present invention in relation to Triatoma virus, relates to a protein precursor encoded by the region 6109- 8715 of Triatoma virus RNA genome according to the sequence NC_003783.1 (NCBI database as of April 28 , 2010), by the region 6109-8715 of the sequence AF 178440 from the non-structural and capsid protein precursors (NCBI database as of April 14 th , 2010), and according to Czibener C et al. 2000 J Gen Virology 81 : 1149-1154.
  • the amino acid sequence of the structural protein precursor is located at NCBI database under accession number NP_620563.1 (868 aa, NCBI database as of April 28 th , 2010).
  • the structural protein precursor is processed during viral biogenesis to yield the different polypeptides that form the viral capsid, namely, VP1, VP2, VP3, VP4 and VP0 (see Agirre et al, 2011, Virology, 409: 91-101).
  • transcription regulatory region refers to a nucleic acid sequence which is capable of governing the expression of another nucleic acid sequence operatively linked thereto, such as a gene of interest.
  • the transcription control sequence preferably, is a DNA sequence.
  • the transcriptional control sequence comprises a first promoter and, optionally, at least one binding site for a transcriptional repressor wherein said first promoter and said binding site for a transcriptional repressor are arranged so that the binding of the transcriptional repressor to said binding site inhibits the transcriptional activity of the promoter.
  • TrV Triatoma virus
  • Hemiptera:Reduviidae family Hemiptera, genus Reduviidae
  • Chagas disease also called American trypanosomiasis
  • This disease is a major health problem in Latin America, where it is endemic and affects about eight million people, 30-40% of whom develop cardiomyopathy and/or digestive megasyndromes.
  • TrV The aetiological agent of Chagas disease is the protozoan parasite Trypanosoma cruzi, which infects the insect vector, which in turn infects vertebrate hosts during blood meals.
  • TrV is a member of the Cripavirus genus (type: Cricket Paralysis Virus, CrPV) in the Dicistroviridae family, which is a family of small ssRNA(+) viruses of invertebrates.
  • the members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance.
  • the Triatoma virus belongs to the group of RNA viruses requiring a RNA polymerase for its replication, i.e. is a virus the genome of which is formed by an RNA molecule and which, unlike other R A viruses the genome of which first undergoes a reverse transcription to give rise to a DNA which is used as a template for the replication, is replicated as a result of an RNA-dependent RNA polymerase which is encoded by the genome of the virus itself.
  • Triatomine also known as conenose bugs, kissing bugs, assassin bugs, as used herein, relates to the members of Triatominae, a subfamily of Reduviidae family. Most of the 130 or more species of this subfamily are haematophagous, i.e. feed on vertebrate blood; a very few species feed on other invertebrates. They are mainly found and widespread in the Americas, with a few species present in Asia, Africa, and Australia. These bugs usually share shelter with nesting vertebrates, from which they suck blood.
  • Triatoma infestans which is the main vector of the Chagas disease in Cambodia
  • Chaco region located among Paraguay, north of Argentina and south-east of Brasil
  • the terms "under the operative control” and “operatively linked” are used herein interchangeably to refer to two nucleic acids are either physically linked or are functionally linked so that at least one of the nucleic acids can act on the other nucleic acid.
  • the transcription control sequence of the present invention and a nucleic acid sequence to be expressed are operatively linked if the expression of the nucleic acid sequence can be governed by the said transcription control sequence. Accordingly, the transcription control sequence and the nucleic acid sequence to be expressed may be physically linked to each other, e.g., by inserting the transcription control sequence at the 5 'end of the nucleic acid sequence to be expressed.
  • the transcription control sequence and the nucleic acid to be expressed may be merely in physical proximity so that the transcription control sequence is functionally linked to the nucleic acid sequence to be expressed.
  • the transcription control sequence and the nucleic acid to be expressed are, preferably, separated by not more than 1,500 bp, 500 bp, 300 bp, 100 bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 bp or 5 bp.
  • the term "untranslated region”, as used herein, also known as “UTR region”, relates to either of two sections, one on each side of a coding sequence of a strand of mRNA.
  • the "5' UTR region” (or leader sequence) is located at the 5' end of the mRNA, and it may contain elements controlling gene expression by way of regulatory elements (including binding sites for proteins that may affect stability or translation of the mRNA, and sequences that promote or inhibit translation initiation). This region begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region.
  • the "3' UTR region” (or trailer sequence) is located at the 3' end of the mRNA, immediately following the translation termination codon.
  • the 3' UTR comprises both binding sites for regulatory proteins as well as microRNAs (miRNAs).
  • the 3'-UTR also has silencer regions which bind to repressor proteins and will inhibit the expression of the mRNA.
  • the 3' UTR may comprise as well AU-rich elements (AREs). Proteins bind AREs to affect the stability or decay rate of transcripts in a localized manner or affect translation initiation.
  • the 3' UTR comprises the sequence AAUAAA, that directs addition of several hundred adenine residues called the poly(A) tail to the end of the mRNA transcript.
  • vaccine relates to an immunogenic composition for in vivo administration to a host, especially a human host, to confer protection against a disease.
  • vector refers to a nucleic acid sequence comprising the necessary sequences so that after transcribing and translating said sequences in a cell a viral particle is generated. Said sequence is operably linked to additional segments that provide for its autonomous replication in a host cell of interest.
  • the vector is an expression vector, which is defined as a vector, which in addition to the regions of the autonomous replication in a host cell, contains regions operably linked to the genome of the invention and which are capable of enhancing the expression of the products of the genome according to the invention.
  • the vectors of the invention can be obtained by means of techniques widely known in the art.
  • viral particle refers to a whole viral particle and not to a protein subunit or peptide.
  • Viral particles consist of two or three parts: the genetic material of the virus made from either DNA or RNA; a protein coat that protects these genes; and, in some cases, an envelope of lipids that surrounds the protein coat when they are outside a cell.
  • the shape of the viral particle ranges from simple helical and icosahedral forms to more complex structures, depending on the virus.
  • virus-like particle also referred to as "VLP”
  • VLP virus-like particle
  • capsid proteins capsid proteins
  • the authors of the present invention have developed a system for antigen presentation based on VLP derived from the Triatoma virus (TrV).
  • the present invention relates to a virus-like particle (VLP) comprising
  • heterologous polypeptide (ii) a heterologous polypeptide, wherein said heterologous polypeptide is provided as a fusion protein with at least one of said viral structural proteins.
  • the virus of the Dicistroviridae family is
  • a VLP according to the invention is an oligomeric structure resulting from the assembly of a determined number of TrV capsid structural proteins.
  • the VLPs are formed by the assembly of VPl, VP2, VP3 and VP4.
  • the VLP according to the invention comprises the structural proteins VPl, VP2, VP3 and VP4 of a virus of the Dicistroviridae family, preferably of a Triatoma virus (TrV), wherein said structural proteins VPl, VP2 and VP3 are in a stoichiometry of 1 : 1 : 1.
  • the VLP according to the invention comprises 60 units of VPl, 60 units of VP2 and 60 units of VP3.
  • the VLP is formed by an assembly of the VPl, VP2, VP3 and VP4 polypeptides.
  • the VP3 and VP4 polypeptides appear as an unprocessed polyprotein known as VPO.
  • VPO unprocessed polyprotein
  • the VLPs result from the assembly of the VPl, VP2 and VPO polypeptides.
  • the average size of the VLPs according to the invention is about 30 nm.
  • the VLP is is formed by the structural protein precursor PI from a virus of the Dicistroviridae family, preferably of a Triatoma virus (TrV).
  • the average size of the VLPs according to the invention is about 50 nm.
  • TrV The molecular description of the capsid of TrV is located at Protein Data Bank under accession number 3NAP (Protein Data Bank as released on May 30th, 2013; DOI: 10.2210/pdb3nap/pdb).
  • the amino acid sequence of TrV PI capsid polyprotein precursor comprises 868 residues and is located at UniProt database under accession number Q9QEY5 (UniProt database version 40, as of April 3, 2013).
  • the sequence of the polynucleotide encoding the nonstructural protein precursor and capsid protein precursor of Triatoma virus is located in NCBI database under accession No. AF178440 (as of April 14 th , 2000, 9010 bp).
  • the complete genome of the Triatoma virus is located at NCBI database under accession number NC 003783.1 (as of April 28 th , 2010, 9010 bp).
  • the VLP according to the present invention may further comprise one or more TrV non-structural proteins, wherein said TrV nonstructural protein is selected from the group comprising a polymerase, a protease and a helicase or a combination thereof.
  • the capsid proteins of the VLPs are provided as a fusion protein comprising the sequence of the capsid protein and a heterologous polypeptide.
  • the fusion protein comprises the viral structural protein and the heterologous polypeptide connected directly or indirectly, wherein the viral structural protein may be located N-terminal to the heterologous protein or wherein the viral structural protein may be located C-terminal to the heterologous protein.
  • the fusion protein comprises the sequence of the viral polypeptide which in interrupted by one or more copies and at one or more positions by the sequence of the heterologous polypeptide.
  • the fusion protein is interrupted at one position by a single copy of the heterologous polypeptide.
  • the fusion protein is interrupted at one position by several copies located in tandem of the heterologous polypeptide. In one embodiment, the fusion protein is interrupted at more than one position by one copy at each position of the same heterologous polypeptide. In one embodiment, the fusion protein is interrupted at several positions and contains at each position a different heterologous polypeptide.
  • the capsid protein variants forming the VLPs of the present invention are designed so that they are still capable of forming VLPs, i.e. that their assembly properties are not altered by the presence of the one or more copies of the heterologous polypeptide.
  • Methods available in the art for determining whether a capsid protein variant is capable of forming VLPs are well-known in the art. For instance, a polynucleotide encoding a modified PI structural precursor protein comprising one or more modified capsid protein can be transfected into a cell together with the NS precursor protein as described in the examples of the present invention.
  • the cell will produce VLPs that can be characterized using any technique known in the art, including the techniques used for the characterization of TrV and described by Agirre et al. ⁇ supra , native mass spectrometry (NMS) and atomic force microscopy (AFM) as described by Snidjer et al. (Snidjer J et al.
  • the capsid protein variants show a viral capsid assembly activity of at least by 60%, preferably by 70%, advantageously by 80%>, more preferably by 90%>, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99% of the activity obtained with the complete protein.
  • the heterologous polypeptide may have a length which can vary within a wide range, for example, between 1 and 100 amino acid residues; advantageously, said peptide insert has less than 40 amino acid residues, preferably less than 30 amino acid residues, more preferably less than 20 amino acid residues, for example, between 1 and 15 amino acids.
  • the heterologous polypeptide forming part of the capsid protein variant will be capable of interacting with a polypeptide of interest, so that once the variant capsid protein forms the VLPs, the VLPs will form complexes with the polypeptide of interest.
  • the stoichiometry of the resulting complex will depend on the capsid component which has been modified.
  • the capsid protein variant is the VP1 , VP2 or VP3 polypeptide. Since the VLPs contain 60 copies of each of these polypeptides, the resulting VLP will contain up to 60 heterologous polypeptides available for binding to a polypeptide of interest.
  • stoichiometry with regard to the polypeptide of interest per VLP.
  • more than one capsid protein in the VLP is provided as a capsid protein variant.
  • the resulting complexes between VLPs and polypeptides of interest may have stoichiometries of 120: 1 , 180: 1 or more.
  • the VLP of the invention comprises a heterologous polypeptide provided as a fusion protein with at least one of the viral VPO, VP1, VP2, VP3 and VP4 structural proteins, wherein said heterologous polypeptide is a first member of a binding pair.
  • the heterologous polypeptide is a first member of a binding pair, and said polypeptide comprises an amino acid sequence with the capacity to bind to a second member of a binding pair such as a ligand, and does not involve any particular size or any other structural technical feature different from that of said peptide (which comprises a specific binding domain) being capable of binding to a ligand (which comprises the region of interaction corresponding to said specific binding domain present in the peptide); Therefore, virtually any heterologous peptide with the capacity to bind to a ligand can be used in the present invention.
  • said peptide can be a member of a specific binding pair, for example, an amino acid tag (e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.), a peptide epitope which can be recognized by an antibody (e.g., c-myc-tag, etc.), a biotin acceptor peptide (BAP), a linear domain of interaction with another protein or proteins (e.g. SH3 proteins, signal transduction proteins, etc.), a post-translational modification sequence (e.g. myristoylation, methylation, phosphorylation, etc.), a sequence of transport to a cell compartment, etc.
  • an amino acid tag e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.
  • a peptide epitope which can be recognized by an antibody e.g., c-myc-tag, etc.
  • BAP biotin acceptor peptide
  • the heterologous polypeptide in the VLP of the invention is a first member of a binding pair, particularly a first member of a binding pair selected from the group comprising a biotin acceptor peptide (BAP), a peptide comprising the tripeptide Arg-Gly-Asp, a peptide comprising the sequence XBBXBX (SEQ ID NO:22), wherein X represents any amino acid and B represents Arg or Lys, and a peptide comprising the sequence XBBBXXBX (SEQ ID NO:23), wherein X represents any amino acid and B represents Arg or Lys.
  • BAP biotin acceptor peptide
  • the heterologous polypeptide comprises the RGD motif (Ernst W et al. 2006 J Biotechnol 126: 237-240).
  • the heterologous polypeptide comprises the XBBXBX motif (SEQ ID NO:22) (Verrecchio A et al. 2000 J Biol Chem 275(11): 7701-7707), wherein X represents any aminoacid and B represent a basic amino acid selected from the group consisting of Arg (R) and Lys (K).
  • the heterologous polypeptide comprises the XBBBXXBX motif (SEQ ID NO:23) (Verrecchio A et al. 2000 J Biol Chem 275 : 7701- 7707) wherein X represents any aminoacid and B represent a basic amino acid selected from the group consisting of Arg (R) and Lys (K).
  • the first member of the binding pair is the biotin acceptor peptide (BAP).
  • BAP biotin acceptor peptide
  • Illustrative, non-limitative, examples of BAP are mentioned in EP71 1303 and include peptides comprising the following amino acid sequence (SEQ ID NO: 24):
  • Xi is any amino acid
  • X 2 is an amino acid different from Leu, Val, He,
  • Trp, Phe, or Tyr X 3 is Phe or Leu; X 4 is Glu or Asp; X 5 is Ala, Gly, Ser, or Thr; X 6 is Gin or Met; X 7 is He, Met, or Val; X 8 is Glu, Leu, Val, Tyr, or He; X 9 is Trp, Tyr, Val, Phe, Leu, or He; and X 10 is any amino acid different from Asp or Glu.
  • amino acid sequence of BAP is the following 15 amino acid sequence:
  • BAP is recognized by biotin ligase (BL) encoded by Escherichia coli gene BirA.
  • BL biotin ligase
  • BirA biotin ligase
  • the heterologous polypeptide comprises at least one epitope.
  • the heterologous polypeptide comprises at least one epitope from an antigen selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, an environmental antigen or a tumor antigen.
  • the epitope is selected from the group comprising PFR-2, PFR- 3, RAP-2, RAP-3, KETcl , GK-1 , KETcl2, HSP-70.
  • Viral antigens susceptible of being included in the VLP according to the invention include, without limitation, the antigens of HIV- 1 , (such as tat, nef, gpl20 or gpl60, gp40, p24, gag, env, vif, vpr, vpu, rev), human herpes virus, (such as gH, gL, gM, gB, gC, gK, gE or gD or derivatives thereof) or immediate early protein such as ICP27, ICP47, ICP4, ICP36 of VHS 1 or VHS2, cytomegalovirus, especially human cytomegalovirus, (such as gB or derivatives thereof), Epstein Barr virus (such as gp350 or derivatives thereof), varicella zoster virus (such as gpl, II, III and IE63), or of a hepatitis virus such as hepatitis B virus (for example surface antigen of he
  • Bacterial antigens susceptible of being included in the VLP according to the invention include, without limitation, antigens of Neisseria spp, including N. gonorrhea and N. meningitidis (transferrin-binding proteins, lactoferrin-binding proteins, PilC and adhesins); antigens of S. pyogenes (such as M proteins or fragments thereof and C5A protease); antigens of S. agalactiae, S. mutans; H.
  • Moraxella spp including M catarrhalis, also known as Branhamella catarrhalis (such as high and low molecular weight invasins and adhesins); antigens of Bordetella spp, including B. pertussis (for example Parapertussis and B. bronchiseptica (such as pertactin, tetanus toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, fimbriae); antigens of Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M.
  • B. pertussis for example Parapertussis and B. bronchiseptica (such as pertactin, tetanus toxin or derivatives thereof, filamentous hemagglutinin, adenylate cyclase, fimbria
  • ESAT6 antigen 85A, -B or -C, MPT 44, MPT59, MPT45, HSPIO, HSP65, HSP70, HSP 75, HSP90, PPD of 19kDa [Rv3763], PPD of 38kDa [Rv0934]
  • antigens of Escherichia spp including enterotoxic E. coli (for example colonization factors, thermolabile toxin or derivatives thereof, heat stable toxin or derivatives thereof), antigens of enterohemorrhagic E. coli and enteropathogenic E.
  • coli for example shiga toxin-like toxin or derivatives thereof
  • antigens of Vibrio spp including V. cholera (for example cholera toxin or derivatives thereof); antigens of Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica (for example a Yop protein); antigens of Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C. jejuni (for example toxins, adhesins and invasins); antigens of Salmonella spp, including S. typhi, S. paratyphi, S.
  • choleraesuis S. enteritidis
  • Listeria spp. including L. monocytogenes
  • Helicobacter spp including H. pylori (for example urease, catalase, vacuolating toxin)
  • antigens of Pseudomonas spp including P. aeruginosa
  • Staphylococcus spp. including S. aureus, S. epidermidis
  • Enterococcus spp. including E. faecalis, E. faecium
  • Clostridium spp. including C. tetani (for example tetanus toxin and derivative thereof); antigens of C.
  • botulinum for example botulinum toxin and derivative thereof
  • antigens of C. difficile for example Clostridium toxins A or B and derivatives thereof
  • antigens of Bacillus spp. including B. anthracis (for example anthrax toxin and derivatives thereof); Corynebacterium spp., including C. diphtheriae (for example diphtheria toxin and derivatives thereof); antigens of Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB); antigens of B. garinii (for example OspA, OspC, DbpA, DbpB), B.
  • afzelii for example OspA, OspC, DbpA, DbpB
  • antigens of B. andersonfi for example OspA, OspC, DbpA, DbpB
  • antigens of B. hermsii for example OspA, OspC, DbpA, DbpB
  • antigens of B. hermsii for example OspA, OspC, DbpA, DbpB
  • Ehrlichia spp. including E. equi and the human granulocytic ehrlichiosis agent
  • Rickettsia spp including R. rickettsii
  • Chlamydia spp. including C. trachomatis (for example MOMP, heparin-binding proteins); antigens of Chlamydia pneumoniae (for example MOMP, haparin-binding proteins), antigens of C.
  • T. pallidum for example the uncommon outer membrane proteins
  • antigens of T. denticola, T. hyodysenteriae for example SAG2, SAGS, Tg34
  • antigens of M for example SAG2, SAGS, Tg34
  • tuberculosis such as Rv2557, Rv2558, RPFs: Rv0837c, Rvl884c, Rv2389c, Rv2450, Rvl009, aceA (Rv0467), PstSl, (Rv0932), SodA (Rv3846), Rv2031c of 16 kDal, Tb Ral2, Tb ⁇ 9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCCl); antigens of Chlamydia (such as the high molecular weight protein (HWMP), ORF3 (document EP 366 412) and possible membrane proteins (Pmp); antigens of Streptococcus spp, including S.
  • HWMP high molecular weight protein
  • ORF3 document EP 366 412
  • Pmp membrane proteins
  • pneumoniae PsaA, PspA, streptolysin, cho line-binding proteins, the pneumolysin protein antigen, and detoxified mutant derivatives thereof); antigens derived from Haemophilus spp., including H. influenzae type B (for example PRP and conjugates thereof); antigens of non-classifiable H. influenzae (such as OMP26, high molecular weight adhesins, P5, P6, D protein and D lipoprotein, and fimbrin and fimbrin-derived peptides, or multiple copy variants or the fusion proteins thereof).
  • H. influenzae type B for example PRP and conjugates thereof
  • antigens of non-classifiable H. influenzae such as OMP26, high molecular weight adhesins, P5, P6, D protein and D lipoprotein, and fimbrin and fimbrin-derived peptides, or multiple copy variants or the fusion proteins thereof).
  • Fungal antigens susceptible of being included in the VLP according to the invention include, without limitation, fungal antigenic components of Candida; fungal antigens of Histoplasma such as heat shock protein 60 (HSP60) and other fungal antigenic components of Histoplasma; of Pneumocystis spp., including P. carinii; fungal antigens of cryptococci such as capsular polysaccharides and other fungal antigenic components of cryptococci; fungal antigens of coccidia such as spherule antigens and other fungal antigenic components of coccidia; antigens of Candida spp., including C. albicans; of Cryptococcus spp., including C. neoformans; and fungal antigens of Tinea such as tricophitin and other fungal antigenic components of coccidia.
  • Prokaryotic antigens susceptible of being included in the VLP according to the invention include, without limitation, antigens of Plasmodium spp., such as P. falciparum and antigens derived from Plasmodium falciparum (such as RTS.S, TRAP, MSP1, AMA1, MSP3, EBA, GLURP, PFR-2, PFR-3, RAP1, RAP2, sequestrin, PfEMPl, Pf332, LSA1, LSA3, STARP, SALSA, PffiXPl, Pfs25, Pfs28, PFS27/25, Pfsl6, Pfs48/45, Pfs230 and the analogs thereof in Plasmodium spp.); as well as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamet surface antigens, blood type antigen pf, 55/RESA and other plasmoid antigenic components; antigen
  • vaginalis antigens of Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; the antigen of Leishmannia and other antigens of Leishmania such as gp63, lipophosphoglycan and their associated-protein and other antigenic components of Leishmania; antigens of Giardia spp., including G. lamblia; and antigens of Trypanosoma cruzi such as the 75-77-kDa antigen, the 56 kDa-antigen and other antigenic components of Trypanosoma.
  • Allergens or environmental antigens susceptible of being included in the VLP according to the invention include, without limitation, an antigen derived from allergens produced naturally such as pollen allergens (pollen allergens of trees, herb, undergrowth and grass), insect allergens (inhaled allergens, allergens from saliva and from posion), allergens from the dander and hair of animals, and food allergens.
  • an antigen derived from allergens produced naturally such as pollen allergens (pollen allergens of trees, herb, undergrowth and grass), insect allergens (inhaled allergens, allergens from saliva and from posion), allergens from the dander and hair of animals, and food allergens.
  • Important pollen, tree, grass and herb allergens originate from taxonomic ordes of Fagales, Oleales, Finales and Platanaceae including among other birch (Betula), alder (Alnus), hazel (Corylus), hard beam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), banana (Platanus), the order of Poales including, among others, grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum, the orders of Asterales and Urticales including among others herbs of the genera Ambrosia, Artemisia and Parietaria.
  • allergenic antigens which can be used include the allergens from house dust mites of the genera Dermatophagoides and Euroglyphus, storage mites for example Lepidoglyphys, Glycyphagus and Tyrophagus, allergens from cockroaches, midges and fleas for example Blatella, Periplaneta, Chironomus and Ctenocepphalides, allergens from mammals such as cat, dog and horse, birds, allergens from poison including those originating from bites or stings of insects such as those of the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps and ants (superfamily Formicoidae).
  • allergenic antigens which can be used include allergens from the inhalation of fungi such as those of the genera Alternaria and Cladosporium.
  • Tumor antigens susceptible of being included in the VLP according to the invention include, without limitation, MAGE, MART-l/Melan-A, gplOO, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophin b, colorrectal-associated antigen (CRC)-0017-1A/GA733, carcinoembryonic antigen (CEA) and its antigenic epitopes CAP-1 and CAP-2, etv6, amll, prostate-specific antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cells /CD3-C, strand receptor, MAGE family of tumor antigens (for example, MAGE-A1, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A
  • the epitope is selected from the group comprising epitopes from PFR-2, PFR-3, RAP-2, RAP-3, KETcl, GK- l, KETcl2, HSP-70.
  • the heterologous polypeptide is a PFR-2 epitope.
  • the PFR-2 epitope is selected from sequences:
  • the heterologous polypeptide is a PFR-3 epitope.
  • the PFR-3 epitope is selected from sequences: - PFR3 4 ⁇ - 4Jb : FVSCCGELTV (SEQ ID NO:4),
  • the heterologous polypeptide is a RAP-2 epitope.
  • the RAP-2 epitope is selected from sequences:
  • RAP-2 HABP 26229 RSV NVISK KTLGLRKRSS (SEQ ID NO: 10).
  • the heterologous polypeptide is a RAP-3 epitope.
  • the RAP3 epitope comprises a sequence that is selected from sequences:
  • heterologous polypeptide is a KETcl epitope which comprises the sequence APMSTPSATSVR (SEQ ID NO: 17).
  • the heterologous polypeptide is a GK-1 epitope which comprises the sequence GYYYPSDPNTFTAPPYSA (SEQ ID NO: 18).
  • the heterologous polypeptide is a KETcl 2 epitope which comprises the sequence GNLLLSCL (SEQ ID NO: 19).
  • the heterologous polypeptide is a HSP70 epitope which comprises a sequence selected from TLLTIDGGI (SEQ ID NO:20) and TLQPVERV (SEQ ID NO:21).
  • the resulting VLP is bound to a second member of a binding pair via interaction of said second member of a binding pair with the first member of a binding pair comprised by the VLP.
  • the first member of the binding pair is the biotinylated BAP and the second member of the binding pair is a molecule comprising a biotin-binding region.
  • Molecules comprising a biotin-binding region according to the invention include, without limitation, avidin, an avidin analog, streptavidin, and streptavidin analog.
  • the VLP according to the invention comprises a heterologous polypeptide, wherein said polypeptide allows the formation of complexes between the VLP and a product of interest.
  • This product of interest can interact directly with the heterologous polypeptide or can be modified by means of the presence of a ligand showing affinity for the heterologous polypeptide such that the product of interest interacts with the heterologous polypeptide through said ligand.
  • the term "product of interest” includes any molecule with biological activity in its broadest sense, i.e., any substance which, when administered to a subject, interacts with its receptor in the site of action and exerts a determined effect.
  • the product of interest can be of a peptide (protein) nature or a non-peptide nature, for example, a protein, an antibody or a functional fragment thereof, a hormone, an enzyme, a lipid, a sugar, a non-peptide drug, a nucleic acid, etc.
  • Illustrative non-limiting examples of products of interest include any type of bioactive molecules such as synthetic molecules (e.g., acetylsalicylic acid, paracetamol, tranexamic acid, metoprolol, etc.), natural products (e.g., taxol, taxane derivatives, paclitaxel, epibatidine, atropine, etc.), oligonucleotides (e.g., augmerosen), aptamers (e.g., pegaptanib), proteins (e.g., interleukins, interferons, erythropoietins, coagulation factors, insulins, etc.), antibodies (e.g., erbitux, rituxan, herceptin, avastin, remicade, humira, xolair, etc.), peptides (e.g., calcitonins, vasopressin analogs (e.g., arginine
  • said product of interest is of a peptide nature; in this case, its size can vary within a wide range, from a few amino acids to hundreds of amino acids.
  • Said peptide product of interest can be virtually any peptide, regardless of its origin (eukaryotic, prokaryotic, viral, etc.), which can be recombinantly expressed, for example, a peptide antigen, such as a viral, bacterial, microbial, tumor antigen, etc., against which an immune response is to be induced in a subject (such as an animal, including humans); an enzyme, such as an enzyme intended to supplement a function in which an organism is deficient; a peptide drug intended to prevent or treat a pathological condition in a subject; an antibody or a functional fragment thereof; etc.
  • a peptide antigen such as a viral, bacterial, microbial, tumor antigen, etc.
  • said peptide product of interest is a peptide useful in vaccination, therapy or diagnosis, such as an epitope or antigenic determinant capable of inducing an immune response in a subject (e.g., animals, including humans) against diseases caused by viruses, bacteria, parasites or any other type of microorganism, or against tumor diseases, or is a peptide useful in therapy (prevention and/or treatment) of other pathologies, for example, heparin, a cell growth factor, an interleukin, an interferon, a protein of interaction with a membrane receptor, a peptide of interaction with a specific cell marker, etc.
  • a peptide useful in vaccination, therapy or diagnosis such as an epitope or antigenic determinant capable of inducing an immune response in a subject (e.g., animals, including humans) against diseases caused by viruses, bacteria, parasites or any other type of microorganism, or against tumor diseases, or is a peptide useful in therapy (prevention and/or treatment) of other path
  • said product of interest is a product of a non-peptide nature.
  • Said product of interest of a non-peptide nature can be virtually any compound of a non-peptide nature, for example, a polynucleotide, a non-peptide antigen, such as allergens, viral, bacterial, microbial, tumor antigen, etc., against which an immune response is to be induced in a subject (such as an animal, including humans); a non-peptide drug intended to prevent or treat a pathological condition in a subject; a lipid with the capacity to interact with a molecule of interest; a sugar with the capacity to interact with a molecule of interest; etc.
  • said non-peptide product of interest is a compound useful in vaccination, therapy or diagnosis, such as a non-peptide antigenic determinant capable of inducing an immune response in a subject (e.g., animals, including humans) against diseases caused by viruses, bacteria, parasites or any other type of microorganism, or against tumor diseases, or is a compound useful in the therapy (prevention and/or treatment) of pathologies of another type.
  • a subject e.g., animals, including humans
  • diseases caused by viruses, bacteria, parasites or any other type of microorganism, or against tumor diseases or is a compound useful in the therapy (prevention and/or treatment) of pathologies of another type.
  • the product of interest can interact with the heterologous polypeptide of the VLP of the invention through a ligand.
  • ligand relates to a molecule which is capable of interacting specifically and binding to the heterologous polypeptide insert present in the VLPs of the invention.
  • the product of interest can be of a peptide (protein) nature or a non-peptide nature.
  • the expression “capable of interacting specifically” or “specific interaction” describes the interaction between a first species and a second species characterized in that the nature of the binding is such that an antibody, a receptor or a nucleic acid binding protein binds to its corresponding binding partner but does not substantially bind to other species.
  • binding or “binding to” means the physical association between a first species and a second species, for example, the binding of a ligand by a receptor, the binding of an antigen by an antibody, the binding of a nucleic acid by a nucleic acid binding protein, etc.
  • ligand does not involve any particular size or another structural technical feature different from that of said ligand being capable of interacting and binding to the heterologous polypeptide present in the VLP of the invention; therefore, said ligand contains a region of interaction with the specific binding domain which is present in the heterologous polypeptide contained in the VLP of the invention.
  • the "heterologous polypeptide"-"ligand” pair acts as a specific binding pair, e.g., of the type of biotin-avidin, epitope-antibody or functional fragment thereof (e.g., a Fab or F(ab')2 fragment, an scFv (single-chain antibody), a chimeric antibody, etc.), etc., in which each of said peptide insert and ligand form the members of said specific binding pair, each of them containing the binding domains or the corresponding regions of interaction; thus, depending on the heterologous polypeptide present in the VLP of the invention the ligand which must be present in the product of interest will be chosen, and, vice versa, depending on the ligand which is to be bound to the VLP of the invention, the peptide insert which must be present in said VLP will be chosen.
  • a specific binding pair e.g., of the type of biotin-avidin, epitope-antibody or functional fragment thereof (e.g
  • the binding of the ligand to the heterologous polypeptide insert is performed directly.
  • a product of interest with the capacity to bind to said heterologous polypeptide present in the VLP of the invention will be used, such that said polypeptide insert and product of interest form a specific binding pair.
  • polypeptide insert comprises an amino acid tag (e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.); likewise when said polypeptide insert comprises a peptide epitope which can be recognized by an antibody (e.g., c-myc-tag, etc.), the product of interest is an antibody or a functional fragment thereof; etc.
  • amino acid tag e.g., a histidine tag (his-tag), an arginine tag (arg-tag), etc.
  • arg-tag arginine tag
  • the product of interest is an antibody or a functional fragment thereof; etc.
  • the binding of said ligand to the peptide insert can be performed indirectly, i.e., by means of using a "linker", or molecule comprising, on one hand, a first region of interaction with the binding domain contained in the heterologous polypeptide present in the VLP and, on the other hand, a second region of interaction (binding domain) with a region of interaction present in the ligand.
  • a linker or molecule comprising, on one hand, a first region of interaction with the binding domain contained in the heterologous polypeptide present in the VLP and, on the other hand, a second region of interaction (binding domain) with a region of interaction present in the ligand.
  • the product of interest binds to the VLP through a structure of the type: heterologous polypeptide- linker-ligand-product of interest.
  • this embodiment include the use of avidin or an analog thereof (linker) when the peptide insert present in the VLP comprises a BAP; etc.
  • the term "avidin” includes any avidin, of a eukaryotic or prokaryotic origin, and its variants which maintain the capacity to bind to biotin; in a particular embodiment, the ligand present in the conjugate of the invention comprises a monomeric variant of avidin (mAV).
  • the VLP comprises a heterologous polypeptide easily detectable including, without limitation, luciferase, (green/red) fluorescent protein and variants thereof, such as EGFP (enhanced green fluorescent protein), RFP (red fluorescent protein, such as DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue green fluorescent protein), YFP (yellow fluorescent protein), ⁇ -galactosidase or chloramphenicol acetyltransferase, and the like.
  • luciferase green/red fluorescent protein
  • RFP red fluorescent protein, such as DsRed or DsRed2
  • CFP cyan fluorescent protein
  • BFP blue green fluorescent protein
  • YFP yellow fluorescent protein
  • ⁇ -galactosidase or chloramphenicol acetyltransferase and the like.
  • the heterologous polypeptide can be a polypeptide of therapeutic interest such that the VLPs of the present invention can be used for the in vitro expression of said polypeptide or for the treatment of diseases requiring the expression of said polypeptide.
  • sequences of therapeutic interest which can be incorporated in the VLP of the invention include but are not limited to those polypeptides encoded by genes or cDNAs encoding erythropoietin (EPO), leptins, corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), prolactin-releasing hormone (PRH), melatonin-releasing hormone (MPvH), prolactin- inhibiting hormone (PIH), somatostatin, adrenocorticotropic hormone (ACTH), somatotropin or growth hormone (GH), luteinizing hormone (LH), follicle- stimulating hormone (F
  • the invention contemplates also VLPs wherein the TrV VP1, VP2, VP3, and VP4, capsid proteins are not the native proteins as they appear in the TrV in nature but functionally equivalent variants thereof.
  • a structural protein of the Triatoma virus is understood as any sequence having additions, substitutions, deletions or combinations thereof in its amino acid sequence and/or which has been chemically modified with respect to said sequence and which substantially maintains the function of said structural protein, particularly the capacity of assembling into a viral capsid.
  • Suitable assays for determining viral capsid assembly capacity are known by the skilled person and include electron microscopy, criomicroscopy, etc. as disclosed for example in Bottcher B et al. 1998 EMBO J 17(23):6839-45; and Yadav SS et al. 2012 Virology 429(2): 155-162.
  • the functional equivalent variants of the structural proteins of the Triatoma virus show the aforementioned viral capsid assembly activity at least by 60%, preferably by 70%, advantageously by 80%>, more preferably by 90%>, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • “functional equivalent variants" of the nonstructural protein precursor NS of the Triatoma virus show the helicase, peptidase and/or RNA-dependent RNA polymerase activities at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • the VLP according to the present invention may or may not contain a product of interest bound to the heterologous polypeptide present in said VLP through a ligand with the capacity to bind to said heterologous polypeptide, or alternatively, through a dual linker, i.e., with two different binding domains, one with the capacity to bind to said heterologous polypeptide and another one with the capacity to bind to said ligand to which the product of interest is bound.
  • the heterologous polypeptide insert can be found in any position with respect to the structural protein, in particular TrV structural protein, to which it is fused.
  • the invention contemplates a fusion protein comprising the structural protein the C-terminus of which is fused to the N-terminus of the heterologous polypeptide, as well as a fusion protein comprising the structural protein the N-terminus of which is fused to the C- terminus of the heterologous polypeptide.
  • the heterologous polypeptide is inserted inside the amino acid sequence of at least one viral structural protein, wherein said viral structural protein is TrV VP1, VP2, VP3, VP4 or VPO.
  • the introduction of said heterologous polypeptide in said at least one TrV structural protein must be performed such that it does not substantially alter the self-assembly capacity of said structural protein; and, furthermore, said heterologous polypeptide must be located in a region of said capsid structural protein accessible to the solvent in the oligomeric structures formed after the self-assembly of the capsid.
  • the fusion protein maintains the amino and carboxyl termini of said structural capsid protein and the heterologous polypeptide is included inside the amino acid sequence of said structural capsid protein, for example, between two contiguous amino acids of said capsid protein or, alternatively, replacing one or more amino acids of said capsid protein.
  • said heterologous polypeptide is located between 2 contiguous amino acids of the structural capsid protein, i.e., between the "x" residue and the "x+1" residue of the amino acid sequence of said structural capsid protein; whereas, in another particular embodiment, due to the strategy followed for the introduction of the heterologous polypeptide, one or more residues located in the regions adjacent to the point of insertion can be lost. Furthermore, given that the heterologous polypeptide must be located in a region of said structural capsid protein accessible to the solvent in the VLPs formed according to the invention, the heterologous polypeptide must be inserted in a suitable region.
  • Particularly suitable regions of the structural protein for introducing the peptide insert are the loops present in said structural capsid proteins, particularly those loops which, in the oligomeric structures that they form, are exposed to the solvent.
  • the suitable site of insertion of the heterologous polypeptide of interest according to the invention in a TrV structural capsid protein it is convenient to know the atomic structure of the capsid protein to be genetically modified.
  • the atomic structure of a structural protein can be determined by means of using X-ray diffraction.
  • suitable computer programs such as the UCSF Chimera program (http://www.cgl.ucsf.edu/chimera/) for example, allows locating the regions of the protein under study which can potentially be genetically modified.
  • said structural protein for the insertion of the heterologous polypeptide of interest is selected from the group comprising VPl, VP2, VP3, VP4, VPO and variants thereof.
  • Sequences of VPl, VP2 and VP3 are located under Accession No. Q9QEY5 (UniProt database version 40, as of April 3, 2013, residues 1-255 corresponding to VP2, residues 313-597 corresponding to VP3 and residues 598-868 corresponding to VPl) and VP4 sequence is described in Agirre J et al. 2011 Virology 409: 91-101 (residues 256-312 in Q9QEY5).
  • the heterologous polypeptide of interest is inserted inside the amino acid sequence of at least one TrV structural protein, is bound to the amino terminal end of at least one TrV structural protein and/or is bound to the carboxy terminal end of at least one TrV structural protein wherein said at least one TrV structural protein is the same TrV structural protein or is a different TrV protein.
  • regions for insertion/substitution in TrV structural proteins include the following regions: 1) in VPl protein
  • Glu274 in VP3 sequence C-terminal region located after a structural ⁇ -strand Ser583-Glu586 (Ser271-Glu274 in VP 3 sequence), and aa chain with unknown structure after Pro588 (Pro276 in VP3 sequence).
  • the heterologous polypeptide is located in VPl structural protein, replacing the VPl region from Ala681 to Trp688, replacing the VPl region from Asp841 to Thr854 and/or by after Leu860 in
  • VPl protein according to Q9QEY5 Uniprot sequence, in VP2 structural protein, replacing the VP2 region from Thr97 to Lysl03, and/or after Ala251 in VP2 protein, according to Q9QEY5 Uniprot sequence,
  • said heterologous polypeptide is bound to the amino or carboxyl terminus of at least one TrV capsid structural protein. Therefore, in a particular embodiment, said heterologous polypeptide is bound to the amino terminus of said TrV capsid protein, whereas, in another particular embodiment, said heterologous polypeptide is bound to the carboxyl terminus of said TrV capsid protein.
  • the TrV capsid protein comprises two heterologous polypeptide bound to said protein, wherein each of said heterologous polypeptide is bound to each of the amino and carboxyl termini of said capsid protein.
  • the VLP of the invention comprises more than one heterologous polypeptide, wherein said more than one heterologous polypeptide is provided as a fusion protein with one or more TrV structural proteins.
  • the VLP of the invention comprises several heterologous polypeptides, wherein said several heterologous polypeptides are provided as fusion proteins with one or more TrV structural proteins.
  • Polynucleotide encoding a fusion protein (first polynucleotide of the invention).
  • Vectors and host cells thereof are provided.
  • the present invention relates to a polynucleotide (first polynucleotide of the invention) encoding a fusion protein, wherein said fusion protein comprises
  • a structural protein from a virus of the Dicistroviridae family selected from the group consisting of VP1, VP2, VP3, VP4 and VP0, or a functionally equivalent variant thereof, and a heterologous polypeptide or
  • a structural protein precursor (PI) from a virus of the Dicistroviridae family, or a functionally equivalent variant thereof, and a heterologous polypeptide or
  • the virus of the Dicistroviridae family is the TrV.
  • viruses of the Dicistroviridae family have been previously described in the context of the VLP of the invention and include VP1, VP2, VP3, VP4 and VPO.
  • the polynucleotide of the invention encodes a fusion protein comprising the complete Triatoma virus structural protein precursor PI, or a functional equivalent variant thereof.
  • TrV structural proteins precursor PI is encoded by the TrV ORF2 viral genome (nucleotides 6109-8715, according to Czibener C et al. 2000 J Gen Virology 81 : 1149-1154, and GenBank accession number AF 178440).
  • the polynucleotide of the invention further comprises a nucleotide sequence encoding the non-structural protein NS of the Triatoma virus according to GenBank accession number AF 178440 or a functionally equivalent variant thereof.
  • TrV polyprotein precursor of non- structural proteins is encoded by the 5' ORF1 TrV viral genome (nucleotides 549-5933, according to Czibener C et al. 2000 J Gen Virology 81 : 1149-1154, and GenBank accession number AF 178440).
  • TrV non- structural proteins include the viral polymerase, the viral protease and the viral helicase.
  • the heterologous polypeptide comprised by the fusion protein encoded by the first polynucleotide of the invention is inserted inside the amino acid sequence of at least one TrV structural protein, is bound to the amino terminal end of at least one TrV structural protein and/or is bound to the carboxy terminal end of at least one TrV structural protein wherein said at least one TrV structural protein is the same TrV structural protein or is a different TrV protein.
  • heterologous polypeptide comprised by the fusion protein encoded by the polynucleotide of the invention is located at a position selected from the group consisting of:
  • heterologous polypeptides according to the invention have been previously described in the context of the VLP of the invention.
  • the heterologous polypeptide is a first member of the binding pair.
  • the heterologous polypeptide comprises at least one epitope.
  • the first member of the binding pair is selected from the group consisting of a biotin acceptor peptide (BAP), a peptide comprising the tripeptide Arg-Gly-Asp, a peptide comprising the sequence XBBXBX (SEQ ID NO:22), wherein X represents any aminoacid and B represents Arg or Lys, and a peptide comprising the sequence XBBBXXBX (SEQ ID NO:23), wherein X represents any aminoacid and B represents Arg or Lys.
  • BAP biotin acceptor peptide
  • the heterologous polypeptide comprises a sequence comprising the RGD motif (Ernst W et al. 2006 J Biotechnol 126: 237-240), that comprises the tripeptide arginyl-glycyl-aspartic acid (Arg-Gly-Asp).
  • the heterologous polypeptide comprises the XBBXBX motif (SEQ ID NO:22) (Verrecchio A et al. 2000 J. Biol. Chem. 275(11): 7701-7707), wherein X represents any aminoacid and B represent a basic amino acid selected from the group consisting of Arg (R) and Lys (K).
  • the heterologous polypeptide comprises the XBBBXXBX motif (SEQ ID NO:23) (Verrecchio A et al. 2000, J. Biol. Chem. 275(11): 7701-7707) wherein X represents any aminoacid and B represent a basic amino acid selected from the group consisting of Arg (R) and Lys (K).
  • the epitope is selected from an antigen selected from the group comprised by a viral antigen, a bacterial antigen, a fungal antigen, an environmental antigen or a tumor antigen.
  • the epitope is selected from the group comprising epitopes from PFR-2, PFR-3, RAP-2, RAP-3, KETcl, GK-1, KETcl2 and HSP-70.
  • the PFR-2 epitope of the VLP of the invention comprises a sequence that is selected from sequences:
  • the PFR-3 epitope of the VLP of the invention comprises a sequence that is selected from sequences:
  • the RAP-2 epitope of the VLP of the invention comprises a sequence that is selected from sequences:
  • - RAP-2 HABP 26225 IKK PFLRVLNKASTTTHAT (SEQ ID NO:08), - RAP-2 HABP 26235: FLAEDFVELFDVTMDCYSRQ (SEQ ID NO:09), and
  • RAP-2 HABP 26229 RSVNNVISKNKTLGLRKRSS (SEQ ID NO: 10).
  • the RAP-3 epitope of the VLP of the invention comprises a sequence that is selected from sequences:
  • the epitope KETcl of the VLP of the invention comprises the sequence APMSTPSATSVR (SEQ ID NO: 17).
  • the epitope GK-1 of the VLP of the invention comprises the sequence GYYYPSDPNTFTAPPYS A (SEQ ID NO : 18).
  • the epitope KETcl2 of the VLP of the invention comprises the sequence GNLLLSCL (SEQ ID NO: 19).
  • the epitope from HSP70 of the VLP of the invention is selected from sequences TLLTIDGGI (SEQ ID NO:20) and TLQPVERV (SEQ ID NO:21).
  • polynucleotide encoding a fusion protein comprising a structural protein from TrV and a heterologous polypeptide according to the present invention may be conveyed in a vector in order to allow adequate propagation of said polynucleotide. Therefore, the invention also contemplates that the polynucleotide encoding a fusion protein comprising a structural protein from the Triatoma virus and a heterologous polypeptide as described above is comprised by a vector (vector of the invention).
  • the invention relates to a vector comprising a polynucleotide encoding a fusion protein comprising a structural protein from the Triatoma virus and a heterologous polypeptide, wherein said polynucleotide has been described previously.
  • suitable vectors include prokaryotic expression vectors (e.g. pUC18, pUC19, Bluescript and their derivatives), mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phages and shuttle vectors (e.g. pSA3 and pAT28), yeast expression vectors (e.g.
  • vectors of the type of 2 micron plasmids include integration plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors (e.g. the pAC series and pVL series vectors), plant expression vectors, such as vectors of expression in plants (e.g. pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors), and eukaryotic expression vectors based on viral vectors (e.g.
  • adenoviruses viruses associated to adenoviruses as well as retroviruses and lentiviruses
  • non-viral vectors e.g. pSilencer 4.1-CMV (Ambion®, Life Technologies Corp., Carlsbad, CA, US), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKS V- 10, pBPV- 1 , pML2d and pTDTl).
  • pSilencer 4.1-CMV Ambion®, Life Technologies Corp., Carlsbad, CA, US
  • pcDNA3, pcDNA3.1/hyg pHCMV/Zeo
  • Vectors may further contain one or more selectable marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds (e.g. hyg encoding hygromycin resistance (GenBank accession no. AF025746; or AF025747) and Kan R (GenBank accession no. U75323), genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g.
  • the vectors of the present invention may carry a non-antibiotic selection marker, including, for instance, genes encoding a catabolic enzyme which enables the growth in medium containing a substrate of said catabolic enzyme as a carbon source.
  • a catabolic enzyme includes, but is not restricted to, lacYZ encoding lactose uptake and beta- galactosidase (GenBank accession nos. J01636, J01637, K01483, or K01793).
  • selection markers that provide a metabolic advantage in defined media include, but are not restricted to, galTK (GenBank accession no. X02306) for galactose utilization, sacPA (GenBank accession no. J03006) for sucrose utilization, trePAR (GenBank accession no. Z54245) for trehalose utilization and xylAB (GenBank accession nos. CAB 13644 and AAB41094) for xylose utilization.
  • the selection can involve the use of antisense mRNA to inhibit a toxic allele, for instance the sacB allele (GenBank accession no. NP 391325).
  • Vectors according to the invention can also comprise at least one heterologous sequence encoding a cytokine, which is used to elicit augmented host responses to the immunogen vector.
  • the particular cytokine encoded by the vector is not critical to the present invention includes, but not limited to, interleukin-4 (herein referred to as "IL- 4"), IL-5, IL-6, IL-10, IL-12 p40, IL-12 p70, TGFp and TNFa.
  • Vectors according to the invention can also comprise a promoter region, which can be used to regulate the transcription of the polynucleotide of the invention.
  • Said promoter region can comprise a constitutive promoter, i.e., a promoter which directs the transcription in a basal manner, or an inducible promoter, in which the transcriptional activity requires an external signal.
  • Suitable constitutive promoters for regulating the transcription are, among others, the CMV promoter, the SV40 promoter, the DHFR promoter, the mouse mammary tumor virus (MMTV) promoter, the elongation factor la (EFla) promoter, the albumin promoter, the ApoAl promoter, the keratin promoter, the CD3 promoter, the immunoglobulin heavy or light chain promoter, the neurofilament promoter, the neuron- specific enolase promoter, the L7 promoter, the CD2 promoter, the myosin light chain kinase promoter, the HOX gene promoter, the thymidine kinase promoter, the RNA Polymerase II promoter, the MyoD gene promoter, the phosphoglycerokinase (PGK) gene promoter, the low-density lipoprotein (LDL) promoter, the actin gene promoter.
  • the CMV promoter the SV40 promoter, the DHFR promoter
  • the inducible promoters which can be used in the context of the present invention are preferably those which respond to an inducing agent, which show nil or negligible basal expression in the absence of inducing agent and which are capable of promoting the activation of the gene located in the 3' position.
  • the inducible promoters are classified as Tet on/off promoters (Gossen, M. and H. Bujard (1992) Proc.Natl.Acad.Sci.USA, 89:5547- 5551; Gossen, M. et al, 1995, Science 268: 1766-1769; Rossi, F.M.V. and H.M. Blau, 1998, Curr. Opin.
  • the vector can contain additional expression-regulating elements such as start and stop signals, cleavage sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. Generally, said elements are chosen depending on the host cells which are to be used.
  • the vector can additionally contain a transcription terminator sequence, such as the terminator of the bovine or human growth hormone gene, the terminator of the SV40 early genes, the terminators of the beta globin genes.
  • a transcription terminator sequence such as the terminator of the bovine or human growth hormone gene, the terminator of the SV40 early genes, the terminators of the beta globin genes.
  • the polynucleotides and vectors according to the invention may be provided within host cells.
  • the present invention also contemplates a host cell (host cell of the invention) comprising the first polynucleotide of the invention encoding a fusion protein comprising a structural protein from a virus of the Dicistroviridae family, particularly the Triatoma virus, and a heterologous polypeptide as described above as well as a host cell comprising the vector of the invention comprising said polynucleotide as described above.
  • the host cell can be a bacterium, wherein said bacterium can be a gram negative bacterial cell, this term intended to include all facultative anaerobic Gram-negative cells of the family Enterobacteriaceace such as Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, Enterobacter, Erwinia, Kluyvera, Serratia, Cedecea, Morganella, Hafhia, Edwardsiella, Providencia, Proteus and Yersinia.
  • the bacterium is a gram positive bacterial cell of the genus Mycobacteriaceae such as M. bovis- CG, M. leprae, M. marinum, M.
  • the host cell can be an insect cell including, without limitation, Sf9 cells, SF21 cells, SF+ cells, High- Five cells, or insect larval cells.
  • the host cell is an insect cell, more particularly a High Five (H5) (BTI-TN-5B1-4) insect cell, commercially available from, for example, Invitrogen.
  • the polynucleotide DNA of interest can be integrated or incorporated into the host cell genome and is referred to as integrated DNA or integrated DNA of interest.
  • the DNA of interest can be introduced stably into and expressed in a host cell (i.e. production of foreign proteins is carried out from the DNA of interest present in the host cell).
  • the DNA of interest is integrated into the host cell DNA, as a result of homologous recombination.
  • a recombinant plasmid is used for introduction of the DNA of interest into the host cells and for stable integration of the DNA into the host cell genome.
  • the recombinant plasmid used includes: 1) host cell sequences (referred to as plasmid-borne host cell sequences) necessary for homologous recombination to occur (between plasmid-borne mycobacterial sequences and sequences in the mycobacterial genome); 2) DNA sequences necessary for replication and selection; and 3) the DNA of interest (e.g. DNA encoding a selectable marker and DNA encoding a protein or polypeptide of interest).
  • the recombinant plasmid is introduced, using known techniques, into the host cells. Therapeutic methods of the virus-like particle, first polynucleotide and vector of the invention
  • the present invention relates to a vaccine or immunogenic composition
  • a vaccine or immunogenic composition comprising a VLP of the invention as described above, the first polynucleotide of the invention as described above, or a vector of the invention as described above.
  • the pharmaceutical compositions according to the present invention comprise vaccines and immunogenic compositions containing the VLP, first polynucleotide, or vector of the invention.
  • said pharmaceutical composition comprises a VLP according to the invention, wherein said VLP comprises a heterologous polypeptide in such a way that an immune response against said antigenic heterologous polypeptide is induced.
  • said pharmaceutical composition comprises a first polynucleotide according to the invention, wherein said polynucleotide encodes a fusion protein comprising a heterologous polypeptide in such a way that an immune response against said antigenic heterologous polypeptide is induced.
  • said pharmaceutical composition comprises a vector according to the invention, wherein said vector encodes a fusion protein comprising a heterologous polypeptide in such a way that an immune response against said antigenic heterologous polypeptide is induced.
  • the VLP, first polynucleotide or vector of the invention may be tested according to methods well-known in the art. For example, tests for several variables (e.g. toxicity, virulence, safety) are carried out in suitable animal models (e.g. mice, guinea pigs), some of which are usually immunocompromised. The ability of the vaccine preparations to elicit an immune response is tested likewise in suitable animal models (e.g. mice, non-human primates). In addition, protection studies involving vaccination, boosting and subsequent challenges are also carried out using suitable animal models (e.g. mice, guinea pigs, non- human primates), so that the contribution of the unspecific protection and immunity conferred by the vector can be depicted.
  • suitable animal models e.g. mice, guinea pigs, non- human primates
  • the amount of the VLP, first polynucleotide of vector of the present invention to be administered will vary depending on the species of the subject, as well as the disease or condition that is being treated.
  • the subject is a mammal. More preferably, the subject is a human.
  • the subject to which the VLP, first polynucleotide or vector, and pharmaceutical compositions thereof of the present invention are administered is a non-human mammal.
  • the subject is a non- human primate, cow, goat, cat, dog, pig, buffalo, badger, possum, deer, or bison.
  • the invention relates to a VLP as described above, to a first polynucleotide as described above, or to a vector of the invention as described above, for use in the treatment of an infectious disease caused by a pathogenic organism and/or an immune reaction caused by an allergen from which the heterologous peptide of the VLP is derived.
  • the invention relates to a method for the treatment of an infectious disease caused by a pathogenic organism that comprises the administration to a subject in need thereof of the VLP of the invention comprising a heterologous polypeptide derived from said pathogenic organism, a first polynucleotide as described above, or a vector of the invention as described above. More alternatively, the invention relates to the use of the VLP of the invention, to a first polynucleotide as described above, or to a vector of the invention as described above, in the manufacture of a medicament for the treatment of an infectious disease caused by a pathogenic organism from which the heterologous peptide of the VLP is derived.
  • said VLPs comprise a heterologous polypeptide, wherein said polypeptide is an epitope of a viral antigen, a bacterial antigen, a fungal antigen, an allergen or environmental antigen or a tumor antigen. Therefore, diseases that may be treated or prevented by using the VLPs and pharmaceutical compositions of the present invention include without limitation diseases involving viruses, bacteria, fungi or any other agent comprising said antigens and including as well, without limitation, diseases affecting non-human mammals such as bovine tuberculosis, porcine reproductive respiratory syndrome, coccidiosis, leptospirosis, infectious laryngotracheitis, and leishmaniasis.
  • the heterologous polypeptide of the VLP of the invention is a trypanosoma immunogen, in which case the VLP of the invention and/or compositions thereof are used for the treatment of trypanosomiasis.
  • the heterologous polypeptide of the VLP of the invention is a Trypanosoma cruzi immunogen, in which case the VLP of the invention and/or compositions thereof are used for the treatment and/or prevention of Chagas disease.
  • the heterologous polypeptide of the VLP of the invention is a plasmodium immunogen, in which case the VLP of the invention and/or compositions thereof are used for the treatment and/or prevention of malaria.
  • Suitable plasmodium antigens which may be used in the present invention are sporozoite surface protein 2 (TRAP/SSP2), liver-stage antigen (LSA in particular LSA3), Pf exported protein 1 (Pf Expl)/Py hepatocyte erythrocyte protein (PyHEP17), and Pf antigen 2 (where Pf represents Plasmodium falciparum and Py represents Plamsodium yoelii), sporozoite and liver stage antigen (SALSA), sporozoite threonine and asparagines-rich (STARP), circumsporozoite protein (CSP), merozoite surface protein 1 (MSP2), in particular merozoite surface protein 1 (MSP-1), merozoite surface protein 2 (M
  • Antigens of the sexual stage which may be used in the present invention are sexual stage and sporozoite surface antigen, antigen Pfg27/25, antigen QF122, 11-1 polypeptide, gametocyte-specific surface protein (Pfs230) ookinete surface protein (P25), chitinase, multidrug resistance protein (MRP).
  • the antigens may derive from any of the five species of Plasmodium parasites known to infect human: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi and Plasmodium falciparum.
  • the heterologous polypeptide of the VLP of the invention is a taenia immunogen, particularly an epitope from Taenia crassiceps (causing murine cysticercosis and that has been shown to protect pigs against T. solium cysticerosis) in which case the VLP of the invention and/or compositions thereof are used for the treatment of porcine cysticercosis.
  • Suitable immunogens include, without limitation, KETcl, GK-1, and KETcl2, as described previously.
  • the immunogen is a mycobacteria immunogen, in which case the recombinant mycobacteria is used for the treatment of a disease caused by the mycobacteria infection.
  • Suitable mycobacteria immunogen include, without limitation, the 85A, 85B or 85C antigens from Mycobacterium tuberculosis or Mycobacterium bovis or a variant thereof.
  • the vaccines and compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to generate a cellular immune response, and degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of about one microgram to about one milligram, preferably about 1 microgram and more preferably about 5 micrograms, and more preferably 100 micrograms active ingredient per kilogram bodyweight individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration.
  • the vaccines of the invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (a) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, mouthwash or hydrogels, (b) parenteral administration, for instance, by subcutaneous, intramuscular or intravenous injection of, for example, a sterile solution or suspension, (c) intracavity administration (e.g. intraperitoneal instillation), intravesical (i.e. urinary bladder) instillation, intrathecal administration, (d) intraorgan administration (e.g. intraprostatical administration), (e) topical application (i.e.
  • oral administration such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes, mouthwash or hydrogels
  • parenteral administration for instance, by subcutaneous, intramuscular
  • Changes in cell activity or cell proliferation can be used to determine whether the selected amounts are an effective amount for the particular combination of compounds.
  • the regimen of administration also can affect what constitutes an effective amount. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation.
  • the TrV- VLP can be obtained by means of the expression of a nucleotide sequence encoding TrV structural proteins VP1, VP2 VP3, VP4, and/or VPO, or a nucleotide sequence encoding TrV structural protein precursor PI, and the nucleotide sequence encoding the fusion protein comprising the heterologous polypeptide of interest and at least one of said TrV structural proteins in suitable host cells.
  • the invention relates to a process for obtaining a VLP, comprising culturing a host cell containing a polynucleotide comprising the nucleotide sequence encoding the structural proteins and the nucleotide sequence encoding the heterologous protein of interest, under conditions allowing the expression of said polynucleotide.
  • the present invention relates to a process for obtaining a Dicistroviridae family virus VLP modified to express a heterologous protein of interest, comprising the steps of:
  • step (i) contacting a host cell with a first polynucleotide which encodes a fusion protein comprising a heterologous polypeptide of interest and the structural protein precursor (PI) of a virus of the Dicistroviridae family or a functionally equivalent variant thereof, and (ii) maintaining the cells under conditions suitable for the expression of the polynucleotide introduced in the cell in step (i).
  • a first polynucleotide which encodes a fusion protein comprising a heterologous polypeptide of interest and the structural protein precursor (PI) of a virus of the Dicistroviridae family or a functionally equivalent variant thereof
  • the virus of the Dicistroviridae family is
  • a host cell and a polynucleotide are contacted under conditions suitable for the entry of said polynucleotide into the cell, wherein said polynucleotide encodes a fusion protein comprising
  • Suitable polynucleotides for use in the first step have been described in the context of the polynucleotides of the invention and are equally useful for the method for the generation of VLPs according to the present invention.
  • the conditions suitable for the entry of the polynucleotide in the host cell are well known for the person skilled in the art and include the microinjection of a composition comprising the polynucleotide or, alternatively, contacting the cell with a co-precipitate of the polynucleotide and calcium phosphate or calcium chloride or, alternatively, using transfection techniques such as DEAE-dextran, lipofection, electroporation as well as an entire series of transfection kits based on the previous techniques and which are commercially available.
  • the polynucleotide can form part of a viral vector, in which case the conditions suitable for the entry of the polynucleotide in the cell include contacting the cell with said viral vector.
  • Viral vectors suitable for the introduction of the polynucleotide in the cell include but are not limited to adenoviral vectors, adeno-associated vectors, retroviral vectors, lentiviral vectors, vectors based on herpes simplex or vectors based on alphaviruses.
  • the polynucleotide is introduced into the cell using a vector, more preferably using a bacmid vector (baculovirus shuttle vector).
  • the polynucleotide encoding a fusion protein comprising a heterologous protein of interest and the structural protein precursor (PI) of the Triatoma virus or a functionally equivalent variant thereof may further comprise additional elements for expression of the heterologous protein in an adequate host cell. Therefore, in a particular embodiment, the polynucleotide comprises a promoter region to regulate the transcription of said polynucleotide. Said promoter is selected from a constitutive promoter and an inducible promoter, which have been previously described in the context of the first polynucleotide of the present invention. In a particular embodiment, the polynucleotide comprises a polyhedrin promoter.
  • the polynucleotide comprises additional expression-regulating elements such as start and stop signals, cleavage sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), transcription terminator sequences, etc.
  • additional expression-regulating elements such as start and stop signals, cleavage sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), transcription terminator sequences, etc.
  • said elements are chosen depending on the host cells which are to be used
  • the efficiency of the transfection will depend on a series of factors including the type of cell, the number of passages, the state of confluence as well as the transfection conditions (time, form of preparation of the liposomes or of the precipitates, etc.). All these parameters can be determined and adjusted by means of routine experimentation.
  • the step (i) of the method of the invention further comprises contacting the host cell used in said step (i) with one or more additional polynucleotides, under conditions suitable for the entry of said one or more additional polynucleotides into the cell, wherein said one or more additional polynucleotides comprise the nucleotide sequence encoding the non-structural (NS) protein precursor the Triatoma virus or a functionally equivalent variant thereof.
  • NS non-structural
  • Suitable heterologous polypeptides have been described previously in the context of the TrV-VLP of the invention.
  • the heterologous polypeptide comprises a first member of a binding pair.
  • VLPs will thus be obtained in which the capsid will be modified by the presence of multiple copies of said ligand, which will allow the formation of conjugates between the VLPs and a compound of interest which is capable of binding specifically to said encoding sequence, such as a second member of a binding pair.
  • This modification of the VLPs will allow addressing the particles to a target organ or cell of interest having on its surface molecules with the capacity to bind specifically to said heterologous sequence.
  • said first member of a binding pair is selected from the group consisting of a peptide comprising the tripeptide Arg-Gly-Asp, a peptide comprising the sequence XBBXBX (SEQ ID NO:22), wherein X represents any aminoacid and B represents Arg or Lys, and a peptide comprising the sequence XBBBXXBX (SEQ ID NO:23), wherein X represents any aminoacid and B represents Arg or Lys and a biotin acceptor peptide (BAP).
  • the heterologous polypeptide is an epitope.
  • the heterologous polypeptide is an epitope of a viral antigen, a bacterial antigen, a fungal antigen, an allergen or environmental antigen or a tumor antigen.
  • Preferred epitopes according to the invention are selected from the group comprising PFR-2, PFR-3, RAP-2, RAP-3, KETcl, GK-1, KETcl2 and HSP-70. Particular preferred sequences for these epitopes have been described previously.
  • heterologous polypeptide is BAP. In a more particular embodiment, the heterologous polypeptide is biotinylated BAP.
  • the nucleotide sequence encoding the heterologous polypeptide comprised by the fusion protein may be inserted in the nucleotide sequence encoding structural protein precursor PI of the Triatoma virus or functionally equivalent variant thereof at different positions.
  • said nucleotide sequence encoding the heterologous polypeptide is inserted at:
  • said at least one TrV structural protein is the same TrV structural protein or is a different TrV structural protein, and/or wherein said heterologous polypeptide is the same polypeptide or is different polypeptide.
  • the nucleotide sequence encoding the heterologous polypeptide is located between nucleotide positions which result in a fusion protein wherein the heterologous polypeptide is located at a position selected from the group consisting of:
  • the host cell is maintained under conditions suitable for the expression of the polynucleotide introduced in said cell in the previously described step (i) of the process.
  • Conditions suitable for the expression of the polynucleotide in the host cell include those conditions that allow the optimal growth of said host cell and those conditions that allow the protein expression in said host cell. Said culture conditions are typically different for each type of host cell. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the host cells and with the amount of VLP desired to be prepared. Again, those conditions are well known and can readily be determined in specific situations. Additionally, specific culture conditions can be obtained from the examples herein.
  • the polynucleotide encoding a fusion protein comprising a heterologous polypeptide and a the structural protein precursor (PI) of the Triatoma virus or a functionally equivalent variant and the polynucleotide which comprises a sequence encoding the non-structural protein precursor (NS) of the Triatoma virus or a functionally equivalent variant thereof are provided in the same polynucleotide or in different polynucleotides.
  • the method of the invention comprises, in a first step, contacting a host cell with a polynucleotide under conditions suitable for the entry of said polynucleotide into the cell, wherein said polynucleotide encodes a fusion protein comprising
  • polynucleotide further comprises a nucleotide sequence encoding the non-structural protein precursor (NS) of the Triatoma virus or a functionally equivalent variant thereof.
  • the method of the invention comprises, in a first step, contacting a host cell with polynucleotides under conditions suitable for the entry of said polynucleotides into the cell, wherein a first polynucleotide encodes a fusion protein comprising
  • At least a second polynucleotide further comprises a nucleotide sequence encoding the non-structural protein precursor (NS) of the Triatoma virus or a functionally equivalent variant thereof.
  • sequences necessary for the generation of the VLPs by the process of the invention include the sequences encoding the structural capsid proteins (VP1, VP2, VP3 and VP4; or VP1 , VP2 and VPO). Therefore, it is possible to contact the cell used in step (i) with polynucleotides encoding each of said structural proteins.
  • the person skilled in the art will understand that use can be made of the genomic organization itself of the Triatoma virus wherein all the proteins necessary for the formation of the capsids are synthesized in the form of a polyprotein comprising both the aforementioned structural capsid proteins and the non-structural capsid proteins (structural protein precursor PI and the non-structural NS protein precursor) but which are necessary for processing the polyprotein.
  • the method of the invention would comprise contacting the cell with a polynucleotide comprising the sequence encoding said polyprotein.
  • steps (i) and (ii) of the process of the invention for obtaining a Triatoma virus VLP modified to express a heterologous protein of interest is to provide the cell with all the components necessary for the formation of said VLPs, therefore they can be carried out sequentially or simultaneously.
  • the process of the invention for obtaining a modified Triatoma virus VLP may further comprise a step which comprises recovering the VLPs from the cell culture used in the previous steps.
  • the particles can be recovered from the cells or from the supernatant of the culture medium.
  • the step of recovering requires the separation of the cells from the culture medium by any method known by the persons skilled in the art (trypsinization and centrifugation or filtration) and the rupture of said cells in an inert solution (freezing/thawing cycles, homogenization, sonication, cavitation, use of detergents and the like).
  • the heterologous polypeptide of interest comprised by the fusion protein is a first member of a binding pair. Therefore, additionally or alternatively, the process of the invention for obtaining a modified Triatoma virus VLP may further comprise a step which comprises contacting the VLPs with a second member of a binding pair under conditions adequate for the interaction between said first and second members of the binding pair to occur. Conditions adequate for interaction such as the temperature, pH, adequate buffers, humidity, time for contact between the both members, binding pair components concentration, particle size, blocking solutions, washing steps, etc. may be adjusted as necessary by the skilled person to obtain an optimal product.
  • the heterologous polypeptide comprised by the fusion protein is a first member of a binding pair, wherein said first member of a binding pair is biotinylated BAP, and the second member of the binding pair is a molecule comprising a biotin-binding region, wherein said molecule containing a biotin-binding region is selected from the group comprising avidin, an avidin analog, streptavidin, and streptavidin analog.
  • the present invention relates as well to the VLPs obtained by a process for obtaining a VLP of a virus of the Dicistroviridae family, particularly TrV, modified to express a heterologous protein of interest as it has been described above.
  • the present invention relates as well to vaccine or immunogenic compositions comprising the VLPs as above, wherein said VLPs have been obtained by the process of the invention as previously described.
  • the present invention relates as well to the VLPs as above, wherein said VLPs have been obtained by the process of the invention as previously described, for use in the treatment of an infectious disease caused by a pathogenic organism and/or an immune reaction caused by an allergen from which the heterologous peptide of the VLP is derived.
  • the invention relates to a method for the treatment of an infectious disease caused by a pathogenic organism and/or an immune reaction caused by an allergen that comprises the administration to a subject in need thereof of the VLP obtained by the process of the invention as previously described.
  • the invention relates to the use of the VLP obtained by the process of the invention as previously described, in the manufacture of a medicament for the treatment of an infectious disease caused by a pathogenic organism and/or an immune reaction caused by an allergen from which the heterologous peptide of the VLP is derived.
  • Second polynucleotide of the invention is derived from Second polynucleotide of the invention.
  • the present invention relates to a R A polynucleotide comprising in the 5 ' to 3 ' direction:
  • Element (i) of the R A polynucleotide of the invention is an R A sequence encoding a first autocatalytic ribozyme capable of cleaving the sequence containing said ribozyme at the 3' end of the ribozyme.
  • the first autocatalytic ribozyme capable of cleaving the sequence containing said ribozyme at the 3' end of the ribozyme is the hammerhead ribozyme (HamRz).
  • the hammerhead ribozyme comprises the sequence corresponding to GenBank accession number L35892.1 (125 nucleotides, NCBI database as of March 7 th , 2000).
  • Element (ii) in the RNA polynucleotide of the invention is the 5' UTR of the TrV virus genome.
  • the 5' UTR of the TrV virus genome comprises the RNA sequence as in region 1-548 of the sequence NC_003783.1.
  • the 5' -UTR is a functional equivalent variant thereof of the RNA sequence as in region 8716-9010 of the sequence NC 003783.1.
  • a functional equivalent variant of the untranslated region of the Triatoma virus genome in particular of the 5' UTR, is understood as any sequence having additions, substitutions, deletions or combinations thereof in its nucleotide sequence and/or which has been chemically modified with respect to said sequence and which substantially maintains the function of the non-translated regulatory sequences, particularly the capacity to promote the replication of the sequences located in the 3' direction mediated by the RNA-dependent RNA polymerase, the capacity to promote the translation of the sequences located in the 3' direction and/or the capacity to promote the encapsidation of nucleic acids containing said regulatory sequences in the VLPs.
  • Suitable assays for determining the capacity of a determined sequence to promote the transcription or the expression of sequences located in the 3' direction are, for example, the assay described by Nagarajan and Kibenge (J. Virol. Methods., 1998 72:51-58).
  • the variants of the non-translated 5' region of the Triatoma virus show said capacity to regulate the transcriptional expression of the nucleotide sequence operatively coupled in the 3 ' position at least by 60%, preferably by 70%, advantageously by 80%, more preferably by 90%>, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • Element (iii) of the R A polynucleotide according to the invention is the sequence encoding the non-structural protein precursor NS of the Triatoma virus or a functionally equivalent variant thereof.
  • the sequence encoding the non-structural protein precursor NS of the Triatoma virus or a functionally equivalent variant thereof comprises the RNA sequence as in region 549-5936 of the sequence NC 003783.1 , or a functionally equivalent variant thereof.
  • a nonstructural protein precursor NS of the Triatoma virus is understood as any sequence having additions, substitutions, deletions or combinations thereof in its nucleotide sequence and/or which has been chemically modified with respect to said sequence and which substantially maintains the function of said non- structural protein precursor, particularly the capacity to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis if the non- structural protein is an helicase, the proteolytic capacity if the non-structural protein is an peptidase, and/or the capacity to catalyze the synthesis of the RNA strand complementary to a given RNA template if the non-structural protein is an RNA-dependent RNA polymerase.
  • Suitable assays for determining helicase activity include, without limitation, electrophoresis (Venkatesan M et al. 1982 J Biol Chem 257: 12426-34; Matson SW et al. 1983 J Biol Chem 258: 14017-24), measure of the sensitization of labeled duplex DNA to single-strand specific nucleases, such as SI or exonuclease I, resulting in the production of ssDNA during unwinding (Palas KM & Kushner SR 1990 J Biol Chem 265:3447-54) and electron microscophy (Runyon GT et al. 1990 Proc Natl Acad Sci. 87:6383-7).
  • Suitable assays for determining peptidase activity are known by the skilled person and include without limitation, fluorescence resonance energy transfer (FRET) assays, spectrophotometric assays and fluorometric assays.
  • Suitable assays for determining RNA-dependent RNA polymerase activity include, without limitation, radioactive assays and fluorometric assays, and are disclosed in, for example, Okazaki T et al. 1964 J Biol Chem 239:259-68; Johanson KO et al. 1980 J Biol Chem 255(22): 10984- 90; US 5635349; Seville M et al. 1996 Biotechniques 21(4): 664-672; and Ma C et al.
  • the functional equivalent variants of the non-structural protein precursor NS of the Triatoma virus show the aforementioned helicase, peptidase and/or R A-dependent R A polymerase activities at least by 60%, preferably by 70%>, advantageously by 80%>, more preferably by 90%), more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • Element (iv) of the RNA polynucleotide according to the invention is the intergenic region of the genome of TrV virus.
  • the intergenic region of the genome of the Triatoma virus comprises the RNA sequence as in region 5937-6108 of the sequence NC 003783.1.
  • element (iv) is a functionally equivalent variant of the region 5937-6108 of the sequence NC 003783.1.
  • RNA polynucleotide having the above elements is introduced into a cell, determining whether the sequence downstream of the intergenic region is expressed by detecting the protein encoded by said sequence.
  • Triatoma virus show the aforementioned translation promoting activity of at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably by 97% and even more preferably 98%, advantageously 99% of the translation promoting activity of the native intergenic region.
  • Element (v) of the RNA polynucleotide according to the invention is a sequence encoding the structural protein precursor PI of the Triatoma virus or a functionally equivalent variant thereof.
  • element (v) comprises the R A sequence as in region 6109-8715 of the sequence NC 003783.1, or a functionally equivalent variant thereof.
  • “functionally equivalent variant" of structural protein precursor PI of the Triatoma virus is understood as any sequence having additions, substitutions, deletions or combinations thereof in its nucleotide sequence and/or which has been chemically modified with respect to said sequence and which substantially maintains the function of said structural protein precursor, particularly the capacity of giving raise to structural proteins able to assemble into a viral capsid and/or allow encapsidation of said structural proteins.
  • Suitable assays for determining viral capsid assembly capacity are known by the skilled person and include electron microscopy, criomicroscopy, etc. as disclosed for example in Bottcher B et al. 1998 EMBO J 17(23):6839-45; and Yadav SS et al.
  • the functional equivalent variants of the structural protein precursor PI of the Triatoma virus show the aforementioned viral capsid assembly activity at least by 60%, preferably by 70%, advantageously by 80%>, more preferably by 90%>, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • RNA polynucleotide of the invention is the 3' UTR of the
  • the 3' UTR of the TrV virus genome comprises the RNA sequence as in region 8716-9010 of the sequence NC 003783.1.
  • the 5' -UTR is a functional equivalent variant thereof of the RNA sequence as in region 8716-9010 of the sequence NC 003783.1.
  • a functional equivalent variant of the untranslated region of the Triatoma virus genome in particular of the 3' UTR, is understood as any sequence having additions, substitutions, deletions or combinations thereof in its nucleotide sequence and/or which has been chemically modified with respect to said sequence and which substantially maintains the function of the non-translated regulatory sequences, particularly polyadenylation, translation efficiency, localization, and stability of the mRNA.
  • the variants of the non-translated 3' region of the Triatoma virus show said capacity of polyadenylation, translation efficiency, localization, and stability of the mR A of the nucleotide sequence operatively coupled at least by 60%, preferably by 70%, advantageously by 80%>, more preferably by 90%>, more preferably by 95%, even more preferably by 97% and even more preferably by 98%, advantageously by 99%.
  • the R A polynucleotide of the invention comprises as well an R A sequence encoding a second autocatalytic ribozyme capable of cleaving the sequence containing said ribozyme at the 5 ' end of the ribozyme element (vii) of the RNA polynucleotide of the invention).
  • the second autocatalytic ribozyme capable of cleaving the sequence containing said ribozyme at the 5' end of the ribozyme is the hepatitis delta virus ribozyme (HDVRz).
  • the hepatitis ⁇ virus ribozyme (HDVRz) is capable of being self-processed such that, once the polynucleotide of the invention has been transcribed, the hepatitis ⁇ virus ribozyme acts on the RNA strand, causing its elimination from the RNA strand. It is thus possible to exactly control the sequence of the terminal 3' region, length of the RNA molecule, since it is enough to include the sequence encoding the hepatitis ⁇ virus ribozyme immediately in the 3 ' position with respect to the nucleotide which is to form the terminal nucleotide of the RNA molecule.
  • the HDVRz comprises the sequence corresponding to positions 1-88 as in the sequence located in GenBank under accession number AY261457 (NCBI database as of March 1 st , 2004).
  • RNA polynucleotide according to the invention can be single stranded RNA (ssRNA), or double stranded RNA (dsRNA).
  • ssRNA single stranded RNA
  • dsRNA double stranded RNA
  • the invention relates to a DNA polynucleotide comprising at least one strand which is complementary to the RNA polynucleotide of the invention as described above.
  • the DNA polynucleotide of the invention can be single stranded DNA (ssDNA), or double stranded DNA (dsDNA).
  • the DNA polynucleotide of the invention further comprises a transcriptional regulatory region which regulates the expression of said DNA polynucleotide. Therefore, the DNA polynucleotide of the invention comprises at least one strand which is complementary to the RNA sequence encoding the first polynucleotide of the invention.
  • a "sequence complementary to a determined sequence” is understood as the reverse of a sequence resulting from replacing each nucleotide in said determined sequence by the complementary nucleotide according to the rules established by Watson-Crick. Therefore, two sequences are complementary when they can be bound to one another in an antiparallel direction (the 5' end of one of them with the 3' end of the other one and each A, T(U), G and C of a sequence is paired, respectively, with a T(U), A, C and G.
  • the reverse sequence is understood to mean a nucleic acid sequence in which the order of nucleotides is reversed as compared to the original sequence. For example, reverse sequence to AAGAG is GAGAA.
  • coding strand refers to the DNA strand which is complementary to the template strand. Such coding strand has the same base sequence as the mRNA (although with thymine replaced by uracil) and corresponds to the codons that are translated into protein.
  • template strand refers to the DNA strand that is used as a template for the synthesis of mRNA. Such template strand has a complementary sequence to the mRNA sequence.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof.
  • the severe hybridization conditions comprise, for example, a concentration comprised between 0.15 M and 0.9 M of NaCl and a temperature comprised between 20°C and 65°C.
  • the very severe hybridization conditions comprise, for example, a concentration comprised between 0.02 M and 0.15 M of NaCl and a temperature comprised between 50°C and 70°C.
  • the DNA polynucleotide of the present invention can be a single stranded DNA (ssDNA), wherein said strand is complementary to the sequence of the RNA polynucleotide as described above, and therefore would have the previously indicated sequence of the Triatoma virus RNA genome read in the reverse direction (3' to 5') and replacing each U, A, C and G of said genome with A, T, G and C, respectively.
  • ssDNA single stranded DNA
  • the DNA polynucleotide of the present invention is double stranded DNA (dsDNA), wherein said DNA polynucleotide comprises a first strand as above (which is complementary to the sequence of the RNA polynucleotide as above) and a second strand complementary to said first strand.
  • dsDNA double stranded DNA
  • the second DNA polynucleotide of the invention is under the operative control of a transcription regulatory region, wherein said transcription regulatory region regulates the expression of said DNA polynucleotide.
  • said transcription regulatory region comprises a promoter region.
  • the DNA polynucleotide of the invention is operatively coupled to the transcription regulatory region.
  • operatively coupled relates to an ordering of elements wherein each of said elements is arranged such that it performs its usual function.
  • a gene is operatively coupled to a determined promoter when the promoter is capable of activating the transcription of said gene in the presence of the suitable enzymes.
  • the promoter does not have to be contiguous to the sequence of the gene provided that it maintains the transcription- activating function.
  • the gene will be considered to be under operative control if its transcription is activated by said promoter.
  • the promoters which can be used to regulate the transcription of the DNA polynucleotide of the invention can be constitutive promoters, i.e., promoters which direct the transcription in a basal manner or inducible promoters in which the transcriptional activity requires an external signal.
  • Suitable constitutive promoters for regulating the transcription are, among others, the CMV promoter, the SV40 promoter, the DHFR promoter, the mouse mammary tumor virus (MMTV) promoter, the elongation factor la (EFla) promoter, the albumin promoter, the ApoAl promoter, the keratin promoter, the CD3 promoter, the immunoglobulin heavy or light chain promoter, the neurofilament promoter, the neuron- specific enolase promoter, the L7 promoter, the CD2 promoter, the myosin light chain kinase promoter, the HOX gene promoter, the thymidine kinase promoter, the RNA Polymerase II promoter, the MyoD gene promoter, the phosphoglycerokinase (PGK) gene promoter, the low-density lipoprotein (LDL) promoter, the actin gene promoter.
  • the CMV promoter the SV40 promoter, the DHFR promoter
  • the inducible promoters which can be used in the context of the present invention are preferably those which respond to an inducing agent, which show nil or negligible basal expression in the absence of inducing agent and which are capable of promoting the activation of the gene located in the 3' position.
  • the inducible promoters are classified as Tet on/off promoters (Gossen, M. and H. Bujard (1992) Pioc.Natl.Acad.Sci.USA, 89:5547-5551; Gossen, M. et al, 1995, Science 268: 1766-1769; Rossi, F.M.V. and H.M. Blau, 1998, Curr. Opin.
  • the promoter is the polyhedrin promoter.
  • the second DNA polynucleotide of the invention can contain additional expression-regulating elements such as start and stop signals, cleavage sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), transcription terminator sequences, etc. Generally, said elements, are chosen depending on the host cells which are to be used.
  • the sequence encoding the structural protein precursor PI of the Triatoma virus or the functionally equivalent variant thereof further comprises a polynucleotide sequence encoding a heterologous polypeptide which results in a fusion protein comprising at least one of the structural proteins of the Triatoma virus and the heterologous polypeptide. More particularly, the polynucleotide sequence encoding the heterologous polypeptide is located within the sequence of the sequence encoding the structural protein precursor PI of the Triatoma virus so that, after expression, the heterologous polypeptide is located at a position selected from the group consisting of
  • the heterologous polypeptide comprises a first member of a binding pair.
  • the first member of a binding pair is selected from the group consisting of a polypeptide comprising the tripeptide Arg-Gly-Asp, a peptide comprising the sequence XBBXBX (SEQ ID NO:22), wherein X represents any aminoacid and B represents Arg or Lys, and a peptide comprising the sequence XBBBXXBX (SEQ ID NO:23), wherein X represents any aminoacid and B represents Arg or Lys, and a biotin acceptor peptide (BAP).
  • the heterologous polypeptide comprises at least one epitope of an antigenic polypeptide.
  • Epitopes according to the invention have been previously described and include epitopes comprised by a viral antigen, a bacterial antigen, a fungal antigen, an environmental antigen and a tumor antigen.
  • the antigens are selected from the group comprising PFR-2, PFR-3, RAP-2, RAP-3, KETcl, GK-1, KETcl2, and HSP-70.
  • the polynucleotide sequence encoding the structural protein precursor PI of the Triatoma virus or the functionally equivalent variant thereof further comprises a polynucleotide sequence encoding a heterologous polypeptide which results in a fusion protein comprising at least one of the structural proteins of the Triatoma virus and the heterologous polypeptide.
  • the polynucleotide sequence encoding the heterologous polypeptide is located within the sequence of the sequence encoding the structural protein precursor PI of the Triatoma virus so that, after expression, the heterologous polypeptide is located at a position selected from the group consisting of
  • the present invention also contemplates a process for obtaining an infectious Triatoma virus (TrV) based on the second polynucleotide of the invention as described above. Therefore, the present invention relates to a process for obtaining an infectious Triatoma virus (TrV), wherein said process comprises the following steps:
  • step (ii) maintaining the cells under conditions suitable for the expression of the polynucleotide introduced in the cell in step (i).
  • the host cell is an insect cell, preferably a High Five insect cell.
  • step (i) further comprises contacting the host cell with a polynucleotide encoding the NS non-structural protein precursor. This will allow increasing the intracellular concentration of the proteins needed for the replication of the viral genome and for the processing of the PI precursor protein.
  • the host cells are maintained under conditions suitable for the expression of the polynucleotide previously introduced.
  • the infectious TrV particles are recovered.
  • Methods for recovery of viral particles include those methods for the recovery of VLPs explained above, included the method described in Agirre et al. (supra).
  • the invention relates as well to those infectious TrV particles which are obtained by this method.
  • the invention relates as well to a method for killing insects comprising the administration of an infectious Triatoma virus (TrV) obtained by a process as described previously.
  • TrV Triatoma virus
  • the insects susceptible of being killed by the method of the invention are hematophagous insects, preferably hematophagous insects which are vectors of diseases, more preferably which are vectors of human diseases.
  • the insects susceptible of being killed by the method of the invention are insects which are susceptible of being infected by an infectious TrV virion obtained by the process of the invention for obtaining recombinant infectious TrV virions.
  • said insect is an hematophagous insect, more particularly an hematophagous insect member of the Triatominae family, preferably a triatome insect, which is a vector of the Chagas disease.
  • the hematophagous insect susceptible of being killed by the method of the invention is an insect of the Cimicidae family, preferably from Cimex genus, more preferably Cimex lectularius.
  • PFRs Paraflagellar rod proteins located at the Tripanosoma cruzi flagellum and specific to the kinetoplastids, have been reported as vaccine candidates.
  • the CD8+ T cells immune response against specific antigens have shown to efficiently control the spread of T. cruzi in murine experimental infection.
  • HLA-A human leukocyte antigen-A
  • PFR-2 and PFR-3 human leukocyte antigen-A
  • PFR3481-489 six human leukocyte antigen-A (HLA-A)*02:01-restricted epitopes, which are efficiently processed and presented in the context of the natural infection of T. cruzi, have been identified in the PFR-2 and PFR-3 from T. cruzi.
  • These proteins contain at least six cytotoxic CD8+ T cell epitopes restricted to the HLA-A*02:01 molecule, named PFR219-28, PFR2156-163, PFR2449-457, PFR3428-436, PFR3475-482 and PFR3481-489, which are processed and presented during the natural infection of Chagas disease.
  • amino acid sequences of these peptides are (from N-terminal to C-terminal):
  • PFR219-28 AVPEVTDVTL (SEQ ID NO:01)
  • PFR3428-436 FVSCCGELTV (SEQ ID NO:04)
  • RAP-2 rhoptry-associated proteins
  • RAP-2 is a ligand used by merozoites to invade RBCs.
  • This protein has four RBC binding sequences defined by high-activity binding peptides (HABPs) numbered as 26220, 26225, 26229, and 26235 (3).
  • HABPs high-activity binding peptides
  • RAP-3 shows two HABPs, and these peptides are named 33860 and 33873.
  • amino acid sequences of peptides HABPs are (from N-terminal to C- terminal):
  • RAP-2 HABP 26220 NHF S S ADELIKYLEKTNINT (SEQ ID NO:07)
  • RAP-2 HABP 26225 IKK PFLRVLNKASTTTHAT (SEQ ID NO:08)
  • RAP-2 HABP 26235 FLAEDFVELFDVTMDCYSRQ (SEQ ID NO: 09)
  • RAP-2 HABP 26229 RSVNNVISKNKTLGLRKRSS (SEQ ID NO: 10)
  • RAP-3 HABP 33860 FNHF SN VDE AIE YLKGLNIN (SEQ ID NO: l 1)
  • RAP-3 HABP 33873 KNRTYALPKVKGFRFLKQLF (SEQ ID NO: 12)
  • TrV mutants carrying selected T. cruzi and P. falciparum epitopes. These epitopes are exposed to the solvent at the external TrV capsid surface. This is done either by inserting one of more peptides on the VP 1-4 structural proteins or by substitution these amino acid portion in the wt TrV sequence (GenBank AF 178440). Depending of the number of insertions, these VLPs will have a minimum of 60 copies of a single epitope, 120 copies by inserting 2 epitopes, and so on.
  • Regions for the insertion/substitution of the exogenous epitopes are:
  • Trp91 Stretch of 9 amino acid long located between two structural ⁇ - strands (Pro79-Tyr82).
  • TrV-VLPs Triatoma virus-Virus Like Particles
  • ATAAGAATGCGGCCGCC7MAGTAGTGGACTCTGAAGTCG-3' SEQ ID NO: 14
  • primers incorporated EcoRI, and Notl restriction sites (underlined), respectively, ATG initiation codon (bold) and CTA stop codon (italic).
  • Gene fragment encoding NS (nucleotides 549 to 5,933 from TrV full-length RNA; NCBI reference sequence: NC 003783) was also obtained by RT-PCR using the following primers: 5 ' - AT AAGAATGCGGCCGC ATGGAATTTTTGCGAAAATTCTTAATTCC-3 ' (SEQ ID NO: 15) and 5 -CCGCTCGAGrC4CATAGTCAAGTCCGTAGG-3 ' (SEQ ID NO: 16). These primers incorporated Notl and Xhol restriction sites (underlined), respectively, ATG initiation codon (bold) and CTA stop codon (italic).
  • PCR products were treated with the corresponding restriction enzymes and cloned under the polyhedrin promoter into different pFastBacl plasmids (Invitrogen). Two recombinant plasmids were obtained: pFastBacl-Pl and pFastBacl-NS.
  • Recombinant plasmids were transformed into DHlOBac Escherichia coli strain (Invitrogen), in which sequences between Tn7R and Tn7L from the recombinant plasmids were site-specific transposed to the bacmids.
  • These recombinant bacmids (Pl- bacmid and NS-bacmid) were isolated, screened by PCR and used for transfection of High Five (H5) insect cells (Invitrogen). Transfection was carried out by adding the transfection mixture (recombinant bacmid, lipofectamine 2,000 (Invitrogen) and TC- 100 insect medium (Invitrogen)) to a 60% confluent monolayer of H5 cells.
  • TrV-VLPs by co-infection with PI and NS TrV-VLPs were produced by co-infecting 90%> confluent monolayer of H5 cells cultured in TC-100 medium supplemented with 2% (v/v) FCS at 28°C with both PI and NS recombinant baculoviruses at a Multiplicity Of Infection (MOI) of 1 in both cases.
  • MOI Multiplicity Of Infection
  • lysis buffer 50mM Tris-HCl pH 7, 500mM NaCl, ImM MgCl 2 and 0.25% (v/v) Igepal CA-630 (Sigma-Aldrich)
  • lysis buffer 50mM Tris-HCl pH 7, 500mM NaCl, ImM MgCl 2 and 0.25% (v/v) Igepal CA-630 (Sigma-Aldrich)
  • a Protease Inhibitor Cocktail tablet Roche.
  • Cells were lysed by incubation in lysis buffer for 20 min and by applying 3 freeze-thaw cycles. The cell lysate was centrifuged at 15,000g for 20 min to pellet cell debris and supernatant was centrifuged through a 20%> (w/v) sucrose cushion in water at 100,000g for 4h.
  • the white pellet was resuspended in lmL of NMT buffer (50mM Tris- HCl pH 7, lOmM NaCl and ImM MgCl 2 ) and then loaded on the top of a 35mL discontinuous 10-30%) (w/v) sucrose gradient. The centrifugation was carried out at 100,000g for 2h and 30mins.
  • TrV-VLPs are shown in Figure 1.
  • TrV-VLPs Expression and purification of TrV-VLPs by infection only with PI
  • TrV-VLPs were also produced by infecting 90% confluent monolayer of H5 cells cultured in TC-100 medium supplemented with 2% (v/v) FCS at 28°C with PI recombinant baculo virus at a Multiplicity of Infection (MOI) of 1. Purification was carried out as described above. TrV-VLPs are shown in Figure 2.
  • TrV ORFs namely NS and PI
  • rBV recombinant baculovirus
  • NCBI Reference Sequence: NC 003783.1 flanked by the hammerhead ribozyme (HamRz) and the hepatitis delta virus ribozyme (HDVRz) at its 5' and 3' ends, respectively.
  • This construct is synthesized in vitro and cloned into the pFastBac plasmid (Invitrogen) under the control of the BV polyhedrin promoter.
  • the presence of the HamRz and HDVRz elements flanking the TrV genomic sequences is designed to ensure the correct trimming, carried out by the mentioned ribozymes, of the RNAs generated by transcription of the described construct during baculovirus replication to release RNAs with 5 ' and 3 ' identical to those found in the TrV genome.
  • the resulting plasmid (named pFB HmRz-TrV-HDVRz) is used to generate a recombinant baculovirus, BV HmRz-TrV-HDVRz, harboring the described TrV construct by following the Bac-to-Bac protocol (Invitrogen; http://tools.invitrogen.com/content/sfs/manuals/bactobac_man.pdf).
  • the BV HmRz- TrV-HDVRz virus is grown and titrated in HighFive cells using the Bac-to-Bac protocols (Invitrogen).
  • the BV HmRz-TrV-HDVRz virus is used to infect HighFive cells. At different times post-infection, cells are collected and used to generate the or to assess the presence of: i) TrV RNAs of the expected size by Northern blot analysis using TrV- specific 32P-labeled probes; ii) TrV polypeptides by Western blot analysis using specific antibodies against structural TrV polypeptides; and iii) TrV.
  • TrV RNA, structural proteins, virus particles and infective virus in extracts from coinfected are tested as described above.
  • Example 3 Immuno- stimulatory properties of TrV VLPs Spleen cells were obtained from female Mus musculus CD1 mice between 5 and 8 weeks of age, without any previous stimulation. 1 x 10 6 cells were cultured in vitro in RPMI medium, supplemented with 10% of calf fetal serum at 37 °C and with 5 % C0 2 .
  • Group 1 PBS
  • Group 2 NMT buffer
  • Group 3 1 ⁇ g lipopolysaccharide (LPS) from Escherichia coli
  • Group 4 1 ⁇ g of TrV VLPs
  • Group 5 1 ⁇ g of empty TrV
  • Group 6 1 ⁇ g of TrV.
  • the dye used was Fluorescein (FL).
  • the experimental conditions were the following:
  • n-VLPs + FL samples were purified by size exclusion (Sephadex G25) using PBS buffer at pH 7.2. Fluorescence emission spectra of the samples were registered ( Figure 4), showing that emission increases as the dye concentration is rised. No fluorescence was observed in the control sample.
  • TrV VLPs can be decorated with molecules able to react to NH2 + groups.

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Abstract

La présente invention concerne le domaine des virus et des particules pseudo-virales (VLP) en ce qui concerne des VLP dérivées de virus Triatoma (TrV) comprenant un polypeptide hétérologue, un procédé d'obtention d'une VLP de virus Triatoma modifié pour exprimer une protéine hétérologue d'intérêt, des polynucléotides comprenant les éléments nécessaires pour la génération de virions TrV recombinants et le procédé pour l'obtention desdits polynucléotides. L'invention concerne en outre un procédé d'obtention de particules de TrV infectieuses.
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