Vaccine and drug delivery by topical application of vectors and vector extracts

Abstract

Disclosed and claimed are methods of non-invasive immunization and drug delivery in an animal and/or methods of inducing a systemic immune or therapeutic response in an animal following topical application of non-replicative vectors, products therefrom and uses for the methods and products therefrom. Also disclosed and claimed are methods of non-invasive immunization and drug delivery in an animal and/or a method of inducing a systemic immune response or systemic therapeutic response to a gene product comprising contacting skin of the animal with cell-free extracts in an amount effective to induce the response, wherein the extracts are prepared by filtration of disrupted cells, wherein the cell comprises and expresses a nucleic acid molecule. Preferably, the cell is temporarily disrupted by sonication, remaining intact and viable after the sonication. Also, methods are disclosed and claimed for enhancing the immunogenicity and efficacy of an epicutaneous vaccine for inducing a systemic immune response to an antigen, in an animal comprising contacting skin of the animal with vaccines admixed with heat-shock protein 27, in an amount effective to induce the response. The methods include contacting skin of the animal with a vector in an amount effective to induce the systemic immune or therapeutic response. The vector can include and express an exogenous nucleic acid molecule encoding an epitope or gene product of interest. The systemic immune response can be to or from the epitope or gene product. The nucleic acid molecule can encode an epitope or antigen of interest and/or a nucleic acid molecule that stimulates and/or modulates an immunological response and/or stimulates and/or modulates expression, e.g., transcription and/or translation, such as transcription and/or translation of an endogenous and/or exogenous nucleic acid molecule; e.g., one or more of influenza hemagglutinin, influenza nuclear protein, influenza M2, tetanus toxin C-fragment, anthrax protective antigen, anthrax lethal factor, anthrax germination factors, rabies glycoprotein, HBV surface antigen, HIV gp120, HIV gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg, botulinum toxin A, and mycobacterium tuberculosis HSP; and/or a therapeutic, an immunomodulatory gene, such as co- stimulatory gene and/or a cytokine gene. The immune response can be induced by the vector expressing the nucleic acid molecule in the animal's cells including epidermal cells. The immune response can also be induced by antigens expressed from the nucleic acid molecule within the vector. The immune response can be against a pathogen or a neoplasm. A prophylactic vaccine or a therapeutic vaccine or an immunological composition can include the vector. The animal can be a vertebrate, e.g., a mammal, such as human, a cow, a horse, a dog, a cat, a goat, a sheep or a pig; or fowl such as turkey, chicken or duck. The vector can be one or more of a viral vector, including viral coat, e.g., with some or all viral genes deleted therefrom, bacterial, protozoan, transposon, retrotransposon, and DNA vector, e.g., a recombinant vector; for instance, an adenovirus, such as an adenovirus defective in its E1 and/or E3 and/or E4 region(s) and/or all adenoviral genes.

We claim:  
     
         1 . A method of non-invasive immunization in an animal and/or a method of inducing a systemic immune response or systemic therapeutic response to a gene product, in an animal, comprising contacting skin of the animal with a non-replicative vector chosen from the group of bacterium, virus, and fungus, wherein the vector comprises and expresses a nucleic acid molecule encoding the gene product, in an amount effective to induce the response.  
     
     
         2 . The method of  claim 1 , wherein the vector is selected from  Clostridium tetani, Clostridium botulinum, Clostridium butyricum, Clostridium baratii, Escherichia coli, Salmonella typhimurium, Bacillus anthracis , influenza virus, and yeasts that are rendered non-replicative by irradiation, antibiotics, fixatives, or gentle heat.  
     
     
         3 . The method of  claim 2 , wherein the vector is rendered non-replicative by γ-irradiation.  
     
     
         4 . The method of  claim 1 , wherein the gene products are botulinum neurotoxins, insulin, erythropoietin, tetanus toxin C-fragment, and growth hormone.  
     
     
         5 . The method of  claim 1  wherein the non-replicative vector is an Escherichia vector.  
     
     
         6 . The method of  claim 5  wherein the Escherichia vector is  Escherichia coli.    
     
     
         7 . The method of  claim 1  wherein the nucleic acid molecule is exogenous or heterologous to the vector.  
     
     
         8 . The method of  claim 1  wherein the response comprises a systemic immune response.  
     
     
         9 . The method of  claim 1  wherein the vector comprises and expresses an exogenous nucleic acid molecule encoding an epitope of interest.  
     
     
         10 . The method of  claim 1  wherein the vector comprises and expresses an antigen.  
     
     
         11 . The method of  claim 1  wherein the vector comprises and expresses a therapeutic product.  
     
     
         12 . The method of  claim 1  wherein the nucleic acid molecule encodes an epitope of interest and/or an antigen of interest and/or a nucleic acid molecule that stimulates and/or modulates an immunological response and/or stimulates and/or modulates expression comprising transcription and/or translation of an endogenous and/or exogenous nucleic acid molecule.  
     
     
         13 . The method of  claim 7  wherein the exogenous nucleic acid molecule encodes one or more of an antigen or portion thereof, or one or more of an epitope of interest, from a pathogen.  
     
     
         14 . The method of  claim 7  wherein the exogenous nucleic acid molecule encodes one or more of: influenza hemagglutinin, influenza nuclear protein, influenza M2, tetanus toxin C-fragment, anthrax protective antigen, anthrax lethal factor, anthrax germination factors, rabies glycoprotein, HBV surface antigen, HIV gp120, HIV gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg, botulinum toxin A, and mycobacterium tuberculosis HSP.  
     
     
         15 . The method of  claim 7  wherein the exogenous nucleic acid molecule encodes an immunomodulator.  
     
     
         16 . The method of  claim 1  wherein the response is induced by the vector expressing the nucleic acid molecule in the animal's cells.  
     
     
         17 . The method of  claim 16  wherein the cells comprise epidennal cells.  
     
     
         18 . The method of  claim 1  wherein the response comprises an immune response against a pathogen or a neoplasm.  
     
     
         19 . The method of  claim 1  wherein the animal is a vertebrate.  
     
     
         20 . The method of  claim 19  wherein the vertebrate is a bird or mammal.  
     
     
         21 . The method of  claim 20  wherein the bird or mammal is a human or a companion or domesticated or food-or feed-producing or livestock or game or racing or sport animal.  
     
     
         22 . The method of  claim 21  wherein the animal is a cow, a horse, a dog, a cat, a goat, a sheep, a pig, or a chicken, or a duck, or a turkey.  
     
     
         23 . The method of  claim 1  wherein the bacterium comprises an exogenous or heterologous nucleic acid molecule encoding the gene product for the response.  
     
     
         24 . The method of  claim 23  wherein the nucleic acid molecule is exogenous or heterologous and encodes an epitope of interest and the method is for inducing a systemic immunological response.  
     
     
         25 . The method of  claim 24  wherein the nucleic acid molecule is exogenous or heterologous and encodes one or more tetanus and anthrax epitopes of interest and/or one or more influenza antigens.  
     
     
         26 . The method of  claim 1  wherein the vector is matched to, or a natural pathogen of, the animal.  
     
     
         27 . The method of  claim 1  comprising application of a delivery device including the vector to the skin of the animal.  
     
     
         28 . The method of  claim 27  further comprising disposing the vector in and/or on the delivery device.  
     
     
         29 . The method of  claim 28  further comprising at least one application of the delivery device including the vector to the skin of the animal.  
     
     
         30 . The method of  claim 29  further comprising multiple applications of the delivery device including the vector to the skin of the animal.  
     
     
         31 . The method of  claim 1  wherein the vector induces an anti-tumor effect in the animal by expressing an oncogene, a tumor-suppressor gene, or a tumor-associated gene.  
     
     
         32 . The method of  claim 15 , wherein the immunomodulator comprises a co-stimulator and/or a cytokine.  
     
     
         33 . The method of  claim 7  wherein the response is against  Clostridium tetani  infection.  
     
     
         34 . The method of  claim 7  wherein the exogenous nucleic acid molecule encodes tetanus toxin C-fragment.  
     
     
         35 . The method of  claim 7  wherein the exogenous nucleic acid molecule encodes an antigen or epitope of tetanus toxin.  
     
     
         36 . The method of  claim 23  wherein the hair is not removed from the skin prior to applying the delivery device to the skin of the animal.  
     
     
         37 . The method of  claim 23  wherein the hair is removed from the skin prior to applying the delivery device to the skin of the animal.  
     
     
         38 . The method of  claim 14  wherein the exogenous nucleic acid encodes botulinum toxin A.  
     
     
         39 . The method of  claim 38 , wherein the systemic therapeutic response is used in cosmetic treatments or in the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles.  
     
     
         40 . The method of  claim 39 , wherein the cosmetic treatments include the reduction of facial or neck wrinkles.  
     
     
         41 . The method of  claim 40 , wherein the facial wrinkles are glabellar lines.  
     
     
         42 . The method of  claim 39 , wherein the neuromuscular disorder is selected from the group consisting of: migraine headaches, blepharospasm, strabismus spasm, hemifacial spasm, spasmodic dysphonia, dystonias in general, hyperhidrosis, and cerebal palsy.  
     
     
         43 . A method of non-invasive immunization in an animal and/or a method of inducing a systemic immune response or systemic therapeutic response to a gene product, in an animal, comprising contacting skin of the animal with cell-free extracts in an amount effective to induce the response, wherein the extracts are prepared by filtration of disrupted cells or vectors, wherein the cell or vector comprises and expresses a nucleic acid molecule encoding the gene product.  
     
     
         44 . The method of  claim 43 , wherein the cells or vectors are selected from the group consisting of bacterium, fungus, cultured animal cells and cultured plant cells.  
     
     
         45 . The method of  claim 43 , wherein the cells are disrupted by sonication.  
     
     
         46 . The method of  claim 45 , wherein the cells are not lysed by the sonication.  
     
     
         47 . The method of  claim 46 , wherein the cells remain viable after the sonication.  
     
     
         48 . The method of  claim 45 , wherein the cells are sonicated at a frequency of 20 kHz.  
     
     
         49 . The method of  claim 45 , wherein the sonication results in cavitation.  
     
     
         50 . The method of  claim 45 , wherein the sonication comprises fewer than 240 cycles, wherein each cycle comprises 15 seconds of sonication followed by 1 minute of no sonication.  
     
     
         51 . The method of  claim 45 , wherein the sonication comprises fewer than 240 cycles, wherein each cycle comprises 15 seconds of sonication followed by 1 minute of no sonication.  
     
     
         52 . The method of  claim 51 , wherein the sonication comprises fewer than 200 cycles.  
     
     
         53 . The method of  claim 52 , wherein the sonication comprises fewer than 150 cycles.  
     
     
         54 . The method of  claim 53 , wherein the sonication comprises fewer than 100 cycles.  
     
     
         55 . The method of  claim 54 , wherein the sonication comprises fewer than 75 cycles.  
     
     
         56 . The method of  claim 55 , wherein the sonication comprises fewer than 50 cycles.  
     
     
         57 . The method of  claim 56 , wherein the sonication comprises fewer than 40 cycles.  
     
     
         58 . The method of  claim 57 , wherein the sonication comprises fewer than 30 cycles.  
     
     
         59 . The method of  claim 58 , wherein the sonication comprises fewer than 20 cycles.  
     
     
         60 . The method of  claim 59 , wherein the sonication comprises 20 cycles.  
     
     
         61 . The method of  claim 44  wherein the cell is a bacterium.  
     
     
         62 . The method of  claim 61 , wherein the bacterium is selected from Clostridium, Escherichia, Salmonella, and Bacillus.  
     
     
         63 . The method of  claim 62 , wherein the bacterium is an Escherichia.  
     
     
         64 . The method of  claim 63 , wherein the bacterium is  Escherichia coli.    
     
     
         65 . The method of  claim 43 , wherein the gene products are botulinum neurotoxins, insulin, erythropoietin, tetanus toxin C-fragment, and growth hormone.  
     
     
         66 . The method of  claim 65 , wherein the nucleic acid molecule is exogenous or heterologous to the vector.  
     
     
         67 . The method of  claim 43 , wherein the response comprises a systemic immune response.  
     
     
         68 . The method of  claim 43 , wherein the cell comprises and expresses an antigen.  
     
     
         69 . The method of  claim 43 , wherein the cell comprises and expresses a therapeutic product.  
     
     
         70 . The method of  claim 43 , wherein the nucleic acid molecule encodes an epitope of interest and/or an antigen of interest and/or a nucleic acid molecule that stimulates and/or modulates an immunological response and/or stimulates and/or modulates expression comprising transcription and/or translation of an endogenous and/or exogenous nucleic acid molecule.  
     
     
         71 . The method of  claim 66 , wherein the exogenous nucleic acid molecule encodes one or more of an antigen or portion thereof, or one or more of an epitope of interest, from a pathogen.  
     
     
         72 . The method of  claim 66 , wherein the exogenous nucleic acid molecule encodes one or more of: influenza hemagglutinin, influenza nuclear protein, influenza M2, tetanus toxin C-fragment, anthrax protective antigen, anthrax lethal factor, anthrax germination factors, rabies glycoprotein, HBV surface antigen, HIV gp120, HIV gp160, human carcinoembryonic antigen, malaria CSP, malaria SSP, malaria MSP, malaria pfg, botulinum toxin A, and mycobacterium tuberculosis HSP.  
     
     
         73 . The method of  claim 66 , wherein the exogenous nucleic acid molecule encodes an immunomodulator.  
     
     
         74 . The method of  claim 43 , wherein the response comprises an immune response against a pathogen or a neoplasm.  
     
     
         75 . The method of  claim 43  wherein the animal is a vertebrate.  
     
     
         76 . The method of  claim 75  wherein the vertebrate is a bird or mammal.  
     
     
         77 . The method of  claim 76  wherein the bird or mammal is a human or a companion or domesticated or food- or feed-producing or livestock or game or racing or sport animal.  
     
     
         78 . The method of  claim 43  wherein the cell free extract contains the gene product.  
     
     
         79 . The method of  claim 43  comprising application of a delivery device including the extract to the skin of the animal.  
     
     
         80 . The method of  claim 79 , further comprising disposing the extract in and/or on the delivery device.  
     
     
         81 . The method of  claim 80 , further comprising at least one application of the delivery device including the extract to the skin of the animal.  
     
     
         82 . The method of  claim 81 , further comprising multiple applications of the delivery device including the extract to the skin of the animal.  
     
     
         83 . The method of  claim 79 , wherein the hair is not removed from the skin prior to applying the delivery device to the skin of the animal.  
     
     
         84 . The method of  claim 79 , wherein the hair is removed from the skin prior to applying the delivery device to the skin of the animal.  
     
     
         85 . A method of enhancing the immunogenicity and efficacy of an epicutaneous vaccine for inducing a systemic immune response to an antigen, in an animal, comprising contacting skin of the animal with a vaccine admixed with heat-shock protein 27, in an amount effective to induce the response.  
     
     
         86 . The method of  claim 85 , wherein the vaccines are vector-, nucleic acid-, or protein-based vaccines.  
     
     
         87 . The method of  claim 1 , further comprising admixing the non-replicative vector with heat-shock protein 27 prior to contacting the skin of the animal.  
     
     
         88 . The method of  claim 43 , further comprising admixing the cell-free extract with heat shock protein 27 prior to contacting the skin of the animal.  
     
     
         89 . The method of  claim 43 , further comprising admixing the cells with heat shock protein 27 prior to disruption of the cell.  
     
     
         90 . The method of  claim 87 , wherein the immunization or immune response or system therapeutic response is enhanced.  
     
     
         91 . The method of  claim 88 , wherein the immunization or immune response or system therapeutic response is enhanced.  
     
     
         92 . The method of  claim 89 , wherein the immunization or immune response or system therapeutic response is enhanced.