AU2014101350A4 - Control of virus-based replicon gene expression using aptamers - Google Patents

Abstract

- 16 The invention provides, inter alia, isolated nucleic acid molecules encoding a positive strand self-replicating RNA and further comprising at least two heterologous small molecule responsive aptamers. In related aspects, the invention also provides methods of controllably regulating gene expression (e.g., of an immunogen) by modulating the levels of the small molecule to which the small molecule-responsive aptamer binds.

Description

- 1 CONTROL OF VIRUS-BASED REPLICON GENE EXPRESSION USING APTAMERS FIELD OF THE INVENTION [1] This invention relates to control of virus-based replicon gene expression using aptamers and aptamer-containing nucleic acids, such as riboswitches. BACKGROUND OF THE INVENTION [2] Viral vectors have been considered useful tools for immunotherapy and therapeutic protein expression. Typically the vectors are replication incompetent, yet subjects can still exhibit significant adverse reactions. Accordingly, a need exists for virus-derived vectors that with additional control elements, such as elements that can be modulated in vivo, e.g., by a small-molecule ligand. SUMMARY OF THE INVENTION [3] The invention provides, inter alia, isolated nucleic acid molecules encoding a positive strand self-replicating RNA and further comprising at least two heterologous small molecule responsive aptamers, i.e., that can change conformation upon binding their ligand and lead to a corresponding change in gene expression from the nucleic acid. In some embodiments, the nucleic acid is DNA and in others, it is RNA. In related aspects, the invention also provides methods of controllably regulating gene expression (e.g., of an immunogen, e.g., where the nucleic acid further encodes a heterologous transgene) by modulating the levels of the small molecule to which the small molecule-responsive aptamer binds. BRIEF DESCRIPTION OF THE FIGURES [4] FIG. 1 illustrates constructs used in experiments in alphavirus, and the results of the experiments (bottom bar graph). Up to 4 tetracycline aptamers were inserted into the 5' UTR of the subgenomic RNA of an alphavirus replicon. RNA replicons were electroporated into BHK cells +/- tetracycline and EGFP expression was examined about 18 hr. later. [5] FIGs. 2A-2B provide a schematic of replicon and riboswitches. FIG. 2A is a schematic showing the DNA-launched TC-83 replicon with a riboswitch in the 3' UTR. The replicon is launched from the cytomegalovirus (CMV) promoter. NSP1-NSP4 indicate the non-structural genes. SEAP, secreted alkaline phosphatase. The subgenomic promoter is indicated by the arrow. FIG. 2B is a schematic showing the types of switches inserted into the 3' UTR of the replicon transcript. Color scheme: Ribozyme, blue; theophylline aptamer, orange; switching strand, red; competing strand, green; theophylline, red circle; arrow, cleavage site [6] FIGs. 3A-E show that DNA-launched TC-83 replicon expression can be regulated by riboswitches. DNA-launched replicons containing (A) the wild-type sTRSV ribozyme, (B) an inactive form of the sTRSV ribozyme, (C) the ON riboswitches L2b8, L2b8-t241 or L2b8-a1, (D) - 2 1 to 3 copies of the OFF riboswitch L2bOFF1, or (E) 2 copies of the ON riboswitch L2b8-a1 were transfected into BHK cells and 24 hours later treated with media +/- 4mM theophylline. Supernatants were collected at 0, 8, and 24 hours post theophylline addition and assayed for SEAP expression. [7] FIGs. 4A-4B show that VRP expression can also be regulated by riboswitches. Replicons were introduced into cells following their encapsidation into viral replicon particles (VRPs) using standard transfection methods. FIG. 4A) Replicons containing riboswitches in their 3' UTR were introduced into BHK cells by infection with VRPs at an MOI of 0.1 and treated +/- 4mM theophylline. Supernatants were collected ~20 hours later and assayed for SEAP expression. FIG. 4B) BHK cells were infected with VRP-packaged replicons containing 1 or 3 copies of the OFF switch L2bOFF1 or VRPs containing the inactive ribozyme control. 12 hours later, cells were treated +/- 4mM theophylline and supernatants were collected at 0 and 8 hours post theophylline treatment and assayed for SEAP expression [8] FIGs. 5A-5C show that the IFN response to DNA-launched replicons and VRPs containing riboswitches can be modulated by theophylline addition. Mouse embryonic fibroblasts (MEFs) were transduced with a lentiviral vector expressing the firefly luciferase gene under the control of a minimal CMV promoter and tandem repeats of the interferon stimulated response element (ISRE). These cells were (FIG 5A) transfected with DNA-launched replicons containing riboswitches or (FIG. 5B) infected with VRPs containing riboswitch-bearing replicons at an MOI of 1 and treated +/- 4mM theophylline. The IFN response was determined ~20 hours later by measuring luciferase expression. Fig. 5C: To examine the modulation of the IFN response over time, the MEF cells were transfected with the DNA-launched replicon containing 2 copies of the L2b8-a1 ON switch or the DNA-launched replicon containing the inactive ribozyme control. 24 hours later, cells were treated +/- 4mM theophylline, followed by removal of theophylline after 8 hours, and then treatment again +/- theophylline after another 12 hours. The IFN response was determined at 0, 8, 20, and 28 hours post treatment by measuring luciferase expression [9] FIGs. 6A-6G show that TC-83 virus replication can be controlled by theophylline dependent riboswitches. (FIG. 6A) Schematic showing insertion of riboswitches into both the 3' and 5' UTR of the viral subgenomic RNA. (FIG. 6B) BHK cells were infected at an MOI of 0.01 with a TC-83 virus containing either an inactive sTRSV ribozyme or the L2bOFF1 riboswitch in the 3' and 5' UTR of the subgenomic RNA, and treated with or without 4mM theophylline. Samples of the supernatant were collected over time and assayed for viral titer. (FIG. 6C, FIG. 6D) Cells were collected at 12 hours post-infection and assayed for expression of dsRNA and TC-83 antigen by flow cytometry to measure # of positive cells (FIG. 6C), or relative levels of expression (MFI; FIG. 6D). (FIG. 6E) BHK cells were infected with the indicated viruses at an MOI of 0.01 and incubated for 12 hours. Cells were then treated with or without 4mM - 3 theophylline and samples of the supernatant were collected over time and assayed for viral titer. (FIG. 6F, FIG. 6G) Cells were collected at both 0 and 8 hours post-theophylline addition and assayed for expression of dsRNA and TC-83 antigen by flow cytometry to measure # of positive cells (FIG. 6F), or relative levels of expression (MFI; FIG. 6G). DETAILED DESCRIPTION OF THE INVENTION [10] In a first aspect, the invention provides an isolated nucleic acid molecule i) encoding a positive strand self-replicating RNA and ii) comprising at least two heterologous small molecule responsive aptamers. A "positive strand self-replicating RNA" can be transcribed directly in, for example, a mammalian cell without the need for an intervening reverse transcription reaction and also encodes a RNA-dependent RNA polymerase that can transcribe RNA from the self replicating RNA molecule (i.e., a replicase). Self-replicating RNAs, in some embodiments, are derived from a virus, such as a togavirus (such as alphavirus, such as VEE (Venezuelan equine encephalitis virus, such as TC83) or sindbis virus), including chimeric viruses. In certain embodiments, the self-replicating RNA is derived from a positive strand RNA virus and comprises a deletion in one or more structural genes, e.g., the virus is not able to make complete viral replicon particles without complementation. A "small molecule-responsive aptamer" is a RNA aptamer that specifically binds a specific small molecule, such as theophylline, tetracycline, or neomycin, which leads to a reversible conformational change in the aptamer. The at least two aptamers may be identical or non-identical (e.g., they may be non identical, but respond to the same small molecule, or different small molecules). In some embodiments, the at least two aptamers have sufficient divergence that they recombine with very low frequency. [11] In some embodiments, the nucleic acids provided by the invention comprise at least 3, 4, or more small molecule-responsive aptamers. [12] In certain embodiments, the at least two heterologous small-molecule-responsive aptamers are in the form of aptazymes. An "aptazyme" is an RNA molecule comprising an aptamer appended to a self-cleaving ribozyme so as to operatively control the activity of the ribozyme. In some embodiments, the aptazyme is an on switch (i.e., the ribozyme portion of the molecule is active in the absence of the small molecule bound by the aptamer portion of the molecule while activity of the ribozyme is significantly reduced in the presence of the small molecule bound by the aptamer portion of the molecule), while in other embodiments, the aptazyme is an off switch (i.e., the ribozyme portion of the molecule is substantially inactive in the absence of the small molecule bound by the aptamer portion of the molecule while activity of the ribozyme is significantly increased in the presence of the small molecule bound by the aptamer portion of the molecule). Exemplary ribozymes include sTRSV-derived and HDV derived ribozymes, as well as other ribozymes, including hammerhead ribozymes. Methods of - 4 engineering aptamers are known in the art and are described in, for example, U.S. Patent Nos. 8,158,595 and 8,603,996. [13] In some embodiments the nucleic acids provided by the invention further comprising a heterologous protein-coding sequence. In more particular embodiments, the heterologous protein-coding sequence is an immunogenic protein that, when administered in an effective amount to a vertebrate, produces a protective immune response in the vertebrate to a pathogen selected from a bacterium, a virus, a fungus, or protist or other pathogen. In other particular embodiments, the heterologous protein-coding sequence is a therapeutic protein, such as an antibody, cytokine, or growth factor. In any of the foregoing embodiments, the at least two heterologous small molecule-responsive aptamers may be adjacent to the heterologous protein coding sequence. In more particular embodiments, at least one of the heterologous small molecule-responsive aptamers is disposed at the 5' end of the heterologous protein-coding sequence and at least one of the heterologous small molecule-responsive aptamers is disposed at the 3' end of the heterologous protein-coding sequence. [14] In some embodiments, the small-molecule-responsive aptamers are located in a subgenomic region of the RNA, optionally in a region corresponding to one or more at least partially deleted viral structural proteins. [15] In some embodiments the nucleic acid is a DNA molecule encoding the positive strand self-replicating RNA. In more particular embodiments, the DNA molecule is a plasmid, comprising one or more of an origin of replication, a selectable marker, and a multiple cloning site. In some embodiments, the DNA molecule further comprises a promoter, optionally wherein the promoter is CMV (cytomegalovirus promoter), or a derivative thereof, e.g., at least about: 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, identical to CMV. In certain particular embodiments, the DNA nucleic acid is linear. [16] In other embodiments, the nucleic acid molecule is a positive strand RNA molecule. [17] In some embodiments, the self-replicating RNA is derived from a positive strand RNA virus and comprises at least a partial deletion in a structural gene rendering the virus replication incompetent. [18] In related aspects, the invention provides vertebrate host cells comprising any of the nucleic acids provided by the invention. In some embodiments the vertebrate is a mammal, such as a human. [19] In other related aspects, the invention provides viral replicon particles (VRPs) comprising a nucleic acid that is or encodes a positive strand RNA virus provided by the invention, as well as methods of making the same; e.g., by transfecting a host cell with a DNA-based replicon, optionally wherein the host cell expresses genes needed for VRP formation, such as deleted structural genes.

- 5 [20] In another aspect, the invention provides methods of controllably expressing a protein comprising contacting a vertebrate host cell (either in vitro or in vivo, in a subject) with a nucleic acid or viral replicon particle provided by the invention that contains a heterologous protein coding sequence and modulating a level of the small molecule to which the small molecule responsive aptamers bind. [21] In a related aspect, the invention also provides methods of controllably raising an immune response to an immunogen in a vertebrate subject, comprising administering a nucleic acid or viral replicon particle provided by the invention that includes a heterologous protein-coding sequence encoding an immunogen to the subject, wherein the nucleic acid or viral replicon particle encodes the immunogen. In some embodiments, these methods optionally include one or more steps of modulating a level of the small molecule to which the small molecule responsive aptamers bind. In some embodiments, the raising of the immune response is a prime-boost strategy comprising a single administration of the nucleic acid or viral replicon, followed by modulating the level of the small molecule to which the small molecule-responsive aptamers bind-e.g., by first expressing the protein (prime), reducing expression, then re expressing the protein (boost). Numerous primer boost cycles are envisioned, e.g., 1, 2, 3, 4, 5, or more cycles of prime and boost. [22] For administration of nucleic acids provided by the invention, numerous delivery platforms are known in the art and encompassed by the invention. Exemplary platforms include lipid nanoparticles, cationic nanomulsions, and oil in water emulsions, such as MF59. See, for example, U.S. Patent No. US 6,451,325; W02012/006380; W02013/006837; and W02012/030901. [23] The nucleic acid molecule or VRP provided by the invention can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If immunogens are expressed as separate polypeptides from a replicon then one or more of these may be provided with an upstream IRES or an additional viral promoter element. Alternatively, multiple immunogens may be expressed from a polyprotein that encodes individual immunogens fused to a short autocatalytic protease (e.g. foot-and-mouth disease virus 2A protein), or as inteins. [24] In some embodiments the immunogen elicits an immune response against one of these bacteria: [25] Neisseria meningitidis: useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding protein. A combination of three useful polypeptides is disclosed in Giuliani et al. (2006) Proc Nat/ Acad Sci U S A 103(29):10834-9. [26] Streptococcus pneumoniae: useful polypeptide immunogens are disclosed in W02009/016515. These include, but are not limited to, the RrgB pilus subunit, the beta-N- - 6 acetyl-hexosaminidase precursor (spr0057), spr0096, General stress protein GSP-781 (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal surface adhesin PsaA. [27] Streptococcus pyogenes: useful immunogens include, but are not limited to, the polypeptides disclosed in W002/34771 and W02005/032582. [28] Moraxella catarrhalis. [29] Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3. [30] Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in W02010/119343, such as a hemolysin, esxA, esxB, ferrichrome binding protein (sta006) and/or the sta0l 1 lipoprotein. [31] Clostridium tetani: the typical immunogen is tetanus toxoid. [32] Cornynebacterium diphtheriae: the typical immunogen is diphtheria toxoid. [33] Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in W02006/110413 and W02005/111066. [34] Pseudomonas aeruginosa [35] Streptococcus agalactiae: useful immunogens include, but are not limited to, the polypeptides disclosed in W002/34771. [36] Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in W02005/002619). LcrE (W02006/138004) and HtrA (W02009/109860) are two preferred immunogens. [37] Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in W002/02606. [38] Helicobacterpylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and/or urease (WO03/018054). [39] Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxigenic E.coli (ETEC), enteroaggregative E.coli (EAggEC), diffusely adhering E.coli (DAEC), enteropathogenic E coli (EPEC), extraintestinal pathogenic E.coli (ExPEC) and/or enterohemorrhagic E.coli (EHEC). ExPEC strains include uropathogenic E.coli (UPEC) and meningitis/sepsis-associated E.coli (MNEC). Useful UPEC immunogens are disclosed in W02006/091517 and W02008/020330. Useful MNEC immunogens are disclosed in W02006/089264. A useful immunogen for several E.co/i types is AcfD (W02009/104092). [40] Bacillus anthracis [41] Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in W02007/049155 and W02009/031043.

- 7 [42] Staphylococcus epidermis [43] Clostridium perfringens or Clostridium botulinums [44] Legionella pneumophila [45] Coxiella burnetii [46] Brucella, such as B.abortus, B.canis, B.melitensis, B.neotomae, B.ovis, B.suis, B.pinnipediae. [47] Francisella, such as Fnovicida, F.philomiragia, F.tularensis. [48] Neisseria gonorrhoeae [49] Treponema pallidum [50] Haemophilus ducreyi [51] Enterococcus faecalis or Enterococcus faecium [52] Staphylococcus saprophyticus [53] Yersinia enterocolitica [54] Mycobacterium tuberculosis [55] Rickettsia [56] Listeria monocytogenes [57] Vibrio cholerae [58] Salmonella typhi [59] Borrelia burgdorferi [60] Porphyromonas gingivalis [61] Klebsiella [62] In some embodiments the immunogen elicits an immune response against one of these viruses: [63] Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus, such as the hemagglutinin, neuraminidase or matrix M2 proteins. Where the immunogen is an influenza A virus hemagglutinin it may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15or H16. [64] Paramyxoviridae viruses: immunogens include, but are not limited to, those derived from Pneumoviruses (e.g. respiratory syncytial virus, RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g. measles virus). [65] Poxviridae: immunogens include, but are not limited to, those derived from Orthopoxvirus such as Variola vera, including but not limited to, Variola major and Variola minor. [66] Picornavirus: immunogens include, but are not limited to, those derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2 and/or - 8 type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie A or B virus. [67] Bunyavirus: immunogens include, but are not limited to, those derived from an Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus. [68] Heparnavirus: immunogens include, but are not limited to, those derived from a Heparnavirus, such as hepatitis A virus (HAV). [69] Filovirus: immunogens include, but are not limited to, those derived from a filovirus, such as an Ebola virus (including a Zaire, Ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus. [70] Togavirus: immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. This includes rubella virus. [71] Flavivirus: immunogens include, but are not limited to, those derived from a Flavivirus, such as Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus. [72] Pestivirus: immunogens include, but are not limited to, those derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV). [73] Hepadnavirus: immunogens include, but are not limited to, those derived from a Hepadnavirus, such as Hepatitis B virus. A composition can include hepatitis B virus surface antigen (HBsAg). [74] Other hepatitis viruses: A composition can include an immunogen from a hepatitis C virus, delta hepatitis virus, hepatitis E virus, or hepatitis G virus. [75] Rhabdovirus: immunogens include, but are not limited to, those derived from a Rhabdovirus, such as a Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV). [76] Caliciviridae: immunogens include, but are not limited to, those derived from Calciviridae, such as Norwalk virus (Norovirus), and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus. [77] Coronavirus: immunogens include, but are not limited to, those derived from a SARS coronavirus, avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV). The coronavirus immunogen may be a spike polypeptide. [78] Retrovirus: immunogens include, but are not limited to, those derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus. [79] Reovirus: immunogens include, but are not limited to, those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus.

- 9 [80] Parvovirus: immunogens include, but are not limited to, those derived from Parvovirus B19. [81] Herpesvirus: immunogens include, but are not limited to, those derived from a human herpesvirus, such as, by way of example only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). [82] Papovaviruses: immunogens include, but are not limited to, those derived from Papillomaviruses and Polyomaviruses. The (human) papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one or more of serotypes 6, 11, 16 and/or 18. [83] Adenovirus: immunogens include those derived from serotype 36 (Ad-36). [84] In some embodiments, the immunogen elicits an immune response against a virus which infects fish, such as: infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), koi herpesvirus, salmon picorna-like virus (also known as picorna-like virus of atlantic salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV). [85] Fungal immunogens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; the less common are Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carini, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigeli, Toxoplasma gondii, Penicillium - 10 marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp. [86] In some embodiments the immunogen elicits an immune response against a parasite from the Plasmodium genus, such as P.falciparum, P.vivax, P.malariae or P.ovale. Thus the invention may be used for immunising against malaria. In some embodiments the immunogen elicits an immune response against a parasite from the Caligidae family, particularly those from the Lepeophtheirus and Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi. [87] In some embodiments the immunogen elicits an immune response against: pollen allergens (tree-, herb, weed-, and grass pollen allergens); insect or arachnid allergens (inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens); animal hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), plane tree (Platanus), the order of Poales including grasses of the genera Lolium, Ph/eum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including herbs of the genera Ambrosia, Artemisia, and Parietaria. Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such as cat, dog and horse, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants (Formicoidae). [88] In some embodiments the immunogen is a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-1 2 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), - 11 HLA-A2-R1 701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T cell non-Hodgkins lymphoma), BCR-abi (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma melanocyte differentiation antigens such as MART-1/Melan A, gp100, MC1R, melanocyte stimulating hormone receptor, tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom/Mel-40, H Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T cell lymphotropic virus antigens, TSP-1 80, p1 85erbB2, p1 80erbB-3, c-met, mn-23H1, TAG-72 4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV1 8, NB/70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90 (Mac-2 binding protein/cyclophilin C associated protein), TAAL6, TAG72, TLP, TPS, and the like. [89] This invention is further illustrated by the following examples which should not be construed as limiting. EXEMPLIFICATION [90] Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust expression of their encoded antigens and strong immune responses. To further explore their use in vaccination, VEE virus vaccine strain TC-83 replicons were engineered for controllable expression of their encoded antigen. This ability to turn gene expression on or off supports novel prime-boost strategies in which only one injection of the vaccine would permit - 12 controlled priming and boosting of the immune response. To do this, various riboswitches were inserted into the replicon 3' UTR. These riboswitch molecules consist of a sensor domain comprised of an RNA aptamer and an actuator domain comprised of a hammerhead ribozyme. The switches have been engineered to respond to theophylline, leading to modulation of ribozyme activity and, in turn, gene expression. The switch-controlled replicons were tested in three different ways. DNA-launched TC-83 replicons expressing SEAP and containing different riboswitches were transfected into BHK cells with or without theophylline. SEAP expression was effectively regulated by switches engineered to turn expression on (ON switch) or off (OFF switch) with addition of theophylline. Inserting two ON switches (L2b8-al) in tandem led to a 48 fold increase in gene expression upon addition of theophylline. Moreover, riboswitch-regulated gene expression by using viral replicon particles (VRPs) was also tested. After infection of BHK cells and treatment with theophylline, gene expression from VRPs was similarly regulated. To model the effect of the modulation of expression on the immune response, DNA-launched replicons and VRPs containing riboswitches were used to infect p53 knockout mouse embryonic fibroblast cells transduced with a lentiviral vector expressing firefly luciferase under the control of the interferon stimulated response element. Addition of theophylline led to a significant increase or decrease in the type I interferon response to replicons containing ON switches or OFF switches, respectively, as measured by luciferase expression. Finally, these riboswitches were tested as a fail-safe switch to terminate replication of viruses, such as oncolytic and live vaccine viruses, to enhance their safety. To demonstrate this, the TC-83 viral genome was engineered to contain the OFF switch L2bOFF1 in both the 3' and 5' UTR of the subgenomic RNA. When virus was grown in the presence of theophylline, viral replication was reduced by 1 000-fold at 12 hours post infection. These studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for novel and safer vaccination strategies. [91] The results of these studies are shown in the figures and their brief descriptions. In conclusion, various ribozyme-aptamer switch systems were into the genome of a positive-strand RNA virus-based replicon. Both ON and OFF switching of replicon expression was observed, achieving up to a 50-fold change in expression. This system was shown to work for DNA launched replicons, as well as VRP-packaged replicons. The type I IFN immune response was successfully controlled by switching ON or OFF replicon expression in vitro, supporting the conclusion that this may also be the case in vivo. TC-83 virus replication could be be shut down using this riboswitch system, which allows for the development of safer vaccination strategies. [92] It should be understood that for all numerical bounds describing some parameter in this application, such as "about," "at least," "less than," and "more than," the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, - 13 the description "at least 1, 2, 3, 4, or 5" also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera. [93] For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety. [94] Headings used in this application are for convenience only and do not affect the interpretation of this application. [95] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate. Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention, including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for materials that are disclosed, while specific reference of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements A-D is disclosed, then, even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically - 14 contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions. [96] The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art-thus, to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features. [97] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (5)

1. An isolated nucleic acid molecule: (i) encoding a positive strand self-replicating RNA; and (ii) comprising at least two heterologous small molecule-responsive aptazymes.

2. The nucleic acid of claim 1, further comprising a heterologous protein-coding sequence, optionally wherein the heterologous protein-coding sequence encodes an immunogenic protein that, when administered in an effective amount to a vertebrate, produces a protective immune response in the vertebrate to a pathogen selected from a bacterium, a virus, a fungus, a protist or other pathogen.

3. The nucleic acid molecule of claim 1 or claim 2, which is a DNA molecule encoding the positive strand self-replicating RNA.

4. A viral replicon particle comprising the nucleic acid of any one of claims 1 to 3.

5. A method of controllably expressing a protein, said method comprising contacting a vertebrate host cell with the nucleic acid of claim 2 or claim 3 or the viral replicon particle of claim 4, and modulating a level of a small molecule to which a small molecule responsive aptamer of at least one aptazyme binds, optionally wherein the heterologous protein coding sequence encodes an immunogenic protein that, when administered in an effective amount to a vertebrate, produces a protective immune response in the vertebrate to a pathogen selected from a bacterium, a virus, a fungus, a protist or other pathogen, and optionally comprising further modulating the level of the small molecule to which the small molecule-responsive aptamer of the at least one aptazyme binds.