Patent Application
20090191157
The present invention relates generally to the fields of chemistry, biology, biochemistry, molecular biology, in particular animal and human therapeutics, more in particular human vaccination.
The invention provides for novel recombinant modified vaccinia virus Ankara for the prevention and/or treatment of type I hypersensitivity in a living animal and humans.
The invention also relates to the field of mammalian therapeutics and drug development, more in particularly to the field of vaccine therapeutics and vaccine drug development.
Almost 500 million individuals suffer from type I allergy, a genetically determined hypersensitivity disease, which is based on the formation of IgE antibodies against per se harmless antigens (allergens) (Kay, A. B. (1997), Allergy and Allergic Diseases, Blackwell Signs, Oxford, UK). The symptoms of type allergy (allergic rhinitis, conjunctivitis and allergic asthma) are mainly caused by the allergen-mediated cross-linking of cell bound specific IgE antibodies and the consecutive release of biological mediators (e.g. histamine, leukotriens) (Segal, D. M., Taurog, J. P., and Metzger, H. (1977). Dimeric Immunoglobulin E Serves as a Unit Signal for Mast Cell Degranulation. Proc. Natl. Acad. Sci. USA 42, 457-467). Allergens may be weeds, such as Cariophylales, Gentianales, Lamiales, Rosales, grasses, such as Poales, trees, such as Arezales, Fagales, Lamiales, Pinales, Plactanmarceae, mites such as American house dust mite, house dust mite, European house dust mite, storage mite, animals such as domestic cattle, dog, domestic horse, cat, guinea pig, mouse, rat, fungi such as Ascomyzota, Eurotiales, Hypocreales, Onygenales, insects, such as honeybee, German cockroach, American cockroach, foods such as cod, salmon, cattle, chicken, shrimp and other Crustaccea, squid, snail, mussle, oyster and other Mollusca, nuts such as cashew and fruits such as the papaya. In fact, an allergy may also be caused by rubbers, such as latex.
Grasses and corn are distributed worldwide, produce large amounts of pollen which become easily airborne and therefore belong to the most important allergen sources. More than 40% of allergic patients are sensitised against grass pollen allergens of which group two allergens represent one of the most frequently recognized allergens (Anvari, A. A., Shen Bagamurthi, P., and Marsch, D. G. (1989). Complete Primary Structure of a Lolium Perenneae (Perennial Grass) Pollen allergen, Lol p 3I: Comparison with known Lol p 1I and Lol p 2I sequences. Biochem. 28, 8665-8670).
Specific therapy of type I allergy can be achieved in principle by active and passive vaccination. Active vaccination is achieved by specific immunotherapy in order to induce unresponsiveness towards allergens. Although successfully practiced since 1911, the immunological mechanisms of specific immunotherapy are not completely understood (Noon, L. (1911). Prophylactic Innoculation against Hay Fever. Landset 1, 1572-1573; Bousquet, J., Lockay, R., Mulling, H. J., and the WHO Panel Members (1998). Allergen Immune Therapy: Therapeutic Vaccines for Allergic Diseases. A WHO position paper. J. Allergy Clin. Immunol. 102, 558-562). Induction of blocking antibodies of the IgG class which interfere with the IgE allergen interaction, modulation of T cell and effector cell responses and generation of tolerance are discussed as possible mechanisms (Durham, and S. R., Till, S. J. (1998). Immunological Changes associated with Allergen Immunotherapy. J. Allergy Clin. Immunol. 102, 157-164). In contrast to active vaccination, passive vaccination is based on the transfer of protective immunoglobulins and represents a routine treatment for many infectious diseases (e.g. hepatitis). The therapeutical efficacy of “passive vaccination” for the treatment of type I allergy has been demonstrated by classical experiments more than 60 years ago. 1935 Cooke and colleagues reported cure of a hay fever patient by transfer of blood from a patient who had been successfully treated by specific immunotherapy (Cooke, R. A., Bernhard, J. H., Hebald, S., and Stull, A. (1935). Serological Evidence of Immunity with Co-Existing Sensitization in Hay Fever Type of Human Allergy. J. Exp. Med. 62, 733-750).
Today, a method called specific immune therapy (SIT) is often applied in order to cope with type I allergies. Here, an extract comprising e.g. pollen allergens or individual allergen is given subcutaneously or sublingually to a patient. The problem with SIT is, that the composition of natural allergen extracts is bound to be subject to natural fluctuations. Thus, optimal dosing of the allergen molecule within the extract is a difficult task. Also, this type of immunotherapy often goes along with substantial side-effects. Therefore, this type of immunotherapy may not be used for particular allergies such as food allergies. Additionally, SIT requires a high number of immunizations.
Thus, it would be advantageous to have a medicament for the treatment of type I hypersensitivity with little or no side-effects and which would require only very few administrations.
SIT may not be applied to particular types of allergies such as mould allergies or food allergies. Thus, it would be advantageous to have a medicament for the treatment of type I hypersensitivities which in particular could be applied to mould or food allergies.
The production of natural allergen extracts is difficult. Thus, it would be advantageous to have a medicament for the treatment of type I hypersensitivities which would be easy to produce.
Traditional SIT usually leads initially to a TH2-response and consequently after further treatment to a switch from a TH2-response to a TH1-response. Thus, it would be advantageous to have available a medicament for the treatment of type I hypersensitivities resulting primarily in a TH1-response.
WO 2003/088994 discloses a modified vaccinia virus Ankara for the vaccination of neonates. EP 1 518 932 A1 is directed to a MVA mutant and its use in the immunotherapy and vaccination against numerous diseases, in particular in the prevention and therapy of cancer and infectious diseases.
WO 03/097844 A1 concerns recombinant modified vaccinia virus Ankara comprising a viral genome and expression cassette comprising the cow pox ATI promoter or a derivative thereof and a coding sequence, wherein the expression of the coding sequence is regulated by said promoter. The virus disclosed in WO 03/097844 A1 is supposedly useful as a vaccine or as part of a pharmaceutical composition.
U.S. Pat. No. 6,171,591 B1 discloses nodavirus related compositions. The chimeric virus particles are useful in therapeutic applications, such as vaccines and gene-delivery vectors, and in diagnostic applications, such as kits for the testing of body tissue or fluid samples.
WO 01/68820 A1 relates to new strains of the modified vaccinius virus Ankara (MVA) that have strongly reduced virulence for most mammals, especially humans, but nevertheless grow in cells of a continuous cell line approved for the production of a therapeutic agent such as a vaccine.
WO 02/18585 describes the use of recombinant modified vaccinia virus Ankara (MVA) for the expression of all of the RNA components necessary for the packaging of heterologous alphavirus replicon vectors and production of heterologous virus replicon particles, here specifically for the production of Venezuelian equine encephalitis (VEE) vectors/replicon particles. It is speculated that the produced replicon particles with packaged replicon vectors can then be used for application as experimental vaccines or immunotherapeutics. Thus, WO 02/18585 relates to MVA vector viruses that act as the production system for another viral vector (vaccines). This system has a number of drawbacks most importantly safety issues.
The inventors have been able to solve the above-identified problems by providing for a recombinant vaccinia virus Ankara (MVA).
The invention relates to the use of a recombinant modified vaccinia virus Ankara (MVA) comprising a heterologous nucleic acid for the production of a medicament for the prevention and/or treatment of type I hypersensitivity in a living animal including humans. Here, in contrast, to e.g. WO 02/18585 the inventive MVA vectors according to the invention can be directly used as recombinant vaccines.
The inventors have astonishingly found that a recombinant modified vaccinia virus Ankara (MVA) comprising a heterologous nucleic acid may be advantageously used for the treatment of type I hypersensitivity.
Such a recombinant modified vaccinia virus Ankara (MVA) according to the invention comprises a heterologous nucleic acid, wherein the heterologous nucleic acid is incorporated into a non-essential region of the genome of the MVA, the heterologous nucleic acid is under the control of a vaccinia virus-specific promoter, orthopox virus-specific promoter, poxvirus-specific promoter, and the heterologous nucleic acid is selected from the group of nucleic acids encoding an allergen selected from the group of weed pollens, grass pollens, tree pollens, mites, animals, fungi, insects, rubber, worms, human autoallergens, and foods.
The invention further relates to a method of introducing MVA according to the invention into a target cell comprising infection of the target cell with the inventive MVA.
The invention further relates to a method for immunization of a living animal body including a human, said method comprising administering to said living animal body including a human in need thereof a therapeutically effective amount of the MVA according to the invention.
The invention relates to the use of a recombinant modified vaccinia virus Ankara (MVA) comprising a heterologous nucleic acid for the production of a medicament for the prevention and/or treatment of type I hypersensitivity in a living animal including humans.
As outlined above, there are certain type I hypersensitivities, i.e. allergies which are very difficult to treat with the specific immunotherapy treatment (SIT). The inherent problem with SIT is that the mixture of natural allergen extract is bound to be subject to natural fluctuations. Also, SIT is known for substantial allergic side-effects. The recombinant modified vaccinia virus Ankara according to the present invention leads to an endogenous expression of the antigen, i.e. the heterologous nucleic acid, encoding the antigen in the host cell, which leads to a direct and preferred MHC I-restricted antigen presentation associated with a TH1-associated T helper cell response. Further, the inventors have found that for the strong immunogenicity of the inventive MVA as well as the strong immunomodulatory activity of the inventive MVA only few immunizations are necessary for a beneficial modulation of the immune response. Further, the inventors have astonishingly found that the MVA according to the present invention allows for preventive allergy vaccination which is so far not possible.
The MVA according to the present invention infects cells which produce the antigen. This leads to a predominantly MHC I restricted antigen presentation on the cell surface associated with a strong TH1 immune response against the allergen. Most advantageously, the MVA according to the invention may not replicate in animals or humans, which in turn leads to a lack of propagation and a high safety profile. Most advantageously, this effect makes it possible in a clinical environment to use the most efficient doses of the MVA according to the invention. Also, the MVA according to the invention may be used in a clinical and laboratory environment with the lowest safety rating (L1, S1).
In one embodiment the medicament according to the invention is a vaccine for immunotherapy and the immune response following application of said vaccine to said humans is primarily a TH1 response in humans. As outlined above, the inventors have astonishingly found, that the MVA leads to an advantageous TH1 response.
Proliferating helper T cells that develop into effector T cell differentiate into two major subtypes of cells known as TH1 and TH2 cells (also known as Type I and Type II helper T cells respectively). These subtypes are defined on a basis of the specific cytokines they produce. TH1 produce interferon-γ (or IFN-γ) and lymphotoxin (also known as tumor necrosis factors or TNF-β), while TH2 cells produce interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-13 (IL-13), among numerous other cytokines. Interleukin-2 is associated with TH1 cells, and its production by helper T cells is necessary for the proliferation of cytotoxic CD8+ T cells.
Given the relative specificity of the cytokines released by either response on particular sections of the immune system, it has been suggested, that both TH groups play separate roles during an immune response. That is, TH1 cells are necessary in maximizing the killing efficacy of the macrophages and in the proliferation of cytotoxic CD8+ T cells, therefore their primary role during an immune response is to activate and proliferate these cells.
The above-mentioned finding in a context of the MVA according to the invention is thus of great advantage.
According to a further aspect of the invention the heterologous nucleic acid of the MVA according to the invention is incorporated into a non-essential region of the genome of the MVA.
A modified vaccinia virus Ankara (MVA) is a chicken cell adapted strain of vaccinia virus. Because of its avirulence found upon inoculation of animals and its striking deficiency to produce substantial amounts of new viral progeny in most cells of mammalian origin, MVA can be used under laboratory conditions of biosafety level 1. MVA serves as an efficient vector virus for expression of recombinant genes (Sutter and Moss, 1992) and as candidate recombinant vaccine (Moss et al., 1996) with high safety profiles since MVA has been tested for preimmunization in over 100000 humans being vaccinated against smallpox without causing notable side-effects. Several MVA vector vaccines have already entered clinical evaluation (McConkey et al., 2003, Cosma et al., 2003). Most recently, MVA is reassessed as candidate second generation vaccine against smallpox. According to the invention, the heterologous nucleic acid according to the invention is incorporated into a non-essential region of the genome of the MVA. According to the present invention, any MVA strain may be used.
WO 03/097844 A1 discloses a number of MVA strains on page 4. One strain that may be used according to the present invention is the MVA-BN strain or a derivative thereof (WO 02/42480). Non-essential regions according to the present invention may be selected from (i) natural occurring deletion sides of the MVA genome with respect to the genome of the vaccinia virus strain Copenhagen or (ii) intergenic regions of the MVA genome. The term “intergenic region” refers preferably to those parts of the viral genome located between two adjacent genes that comprise neither coding nor regulatory sequences. However, the insertion sides for the incorporation of the heterologous nucleic acid according to the invention (non-essential region) are not restricted to these preferred insertion sides since it is within the scope of the present invention that the integration may be anywhere in the viral genome as long as it is possible to obtain recombinants that can amplified and propagated in at least one cell culture system, such as Chicken Embryo Fibroblasts (CEF cells). Thus, a non-essential region may also be a non-essential gene or genes, the functions of which may be supplemented by the cell system used for propagation of MVA.
In a particularly preferred embodiment the heterologous nucleic acid according to the invention is incorporated into the MVA genome at the side of deletion (III). Integration of the heterologous nucleic acid according to the invention is performed preferentially by homologous recombination of the flanking regions of a so-called transfer vector, which initially comprises the heterologous nucleic acid according to the invention prior to its integration into the MVA genome. Upon homologous integration selection of recombinant viruses is performed. One such method for selecting the recombinant viruses is expression of the host range gene K1L (see
According to one embodiment of the present invention the heterologous nucleic acid is under the control of a vaccinia virus-specific promoter, an orthopox virus-specific promoter or a pox virus-specific promoter. A number of promoters may be used for the present invention. For the expression of the heterologous nucleic acid according to the invention, several promoters, such the 30K and 40K promoters (U.S. Pat. No. 5,747,324, A Strong Synthetic Early-Late Promoter (Sutter, et al., Vaccine (1994), 12, 1032-1040)), the P7.5 promoter (Endo et al., J. Gen. Virol. (1991) 72, 699-703) and a promoter derived from the cow pox virus type A (inclusion ATI gene) (Lee et al., J. Gen. Virol. 1998), 79, 613) may be used. All of these promoters may be used according to the invention. In one embodiment it is desired that the antigen against which an immune response is to be induced is expressed in high amounts. WO 03/097844 A1 discloses such a promoter.
The heterologous nucleic acid according to the invention is selected from the group of nucleic acids encoding an allergen, a mutated allergen, or a fragment of an allergen selected from the group of weed pollens, grass pollens, tree pollens, mites, animals, fungi, insect, rubber, worms, human autoallergens and foods.
Rapid advances have been made in the past two years on allergen characterization in sequence determination by chemical and molecular biological approaches. This is indicated by the list of allergens with known partial or complete amino acid sequence. A useful source for allergen sequences is www.allergen.org. According to the invention, a heterologous nucleic acid of the invention is a nucleic acid encoding an allergen which may lead to a type I hypersensitivity reaction. Allergens are designated according to the accepted taxonomic name of their source as follows: The first three letters of the genus, space, the first letter of the species, space, an Arabic number. The numbers are assigned to the allergens in the order of their identification, and the same number is generally used to designate homologous allergens of related species. As to examples, Lol p 1 refers to the first pollen allergen identified from Lolium perenne, rye grass, and Cyn d 1 refers to homologous pollen allergen from Cynodon dactylon, Bermuda grass. In some instances, the above system of the first three letters of a genus and the first letter of the species has to be modified to include an additional letter for the designation of the exact genus or species.
An allergen from a single species may consist of several closely similar molecules. These similar molecules are designated as iso-allergens when they share the following biochemical properties: a) similar molecular size; b) identical biological function, if known e.g. enzymatic action; and c) >67% identity of amino acid sequences.
It is recognized that the recommended >67% sequence identity for two allergens to be assigned to the same group is only a guide. There are likely to be borderline cases. As an example, the rag weed allergens Amb a 1 and 2 share 65% amino acid sequence identity (Griffith, I. J., J. Pollock, D. G. Klapper, B. L. Rogers, and A. K. Nault. (1991) Sequence Polymorphism of Amb a 1 and Amb a 2, the major allergens in ambrosia artemisii folia (short rag weed) Int. Arch. Allergy Apple. Immunol. 96: 296-304).
cDNA cloning of allergens often show nucleic type mutations which are either silent or which can lead to single or multiple amino acid substitutions. In the revised system, members of allergen group which have >67 amino acid sequence identity are designated as iso-allergens. Each iso-allergen may have multiple forms of closely similar sequences, which are designated as variants. Table 1 shows some of the allergens according to the present invention.
In a preferred embodiment the heterologous nucleic acid of the present invention encodes an allergen which may not be treated with the specific immunotherapy (SIT). In a particularly preferred embodiment of the present invention the heterologous nucleic acid according to the invention encodes a food allergen such as the food allergen designated in table 1, in particular Gad c 1, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Pen a 1, Pen i 1, Bra j 1, Hor v 1 and Sin a 1. In a particularly preferred embodiment of the present invention the heterologous nucleic acid according to the present invention encodes Gal d 2.
In one aspect of the present invention the nucleic acid according to the present invention which is under the control of a vaccinia virus-specific promoter and incorporated into the MVA genome into a non-essential region, may in fact be the code for more than one allergen.
In one such embodiment such a nucleic acid encodes the most common food allergen, in another embodiment it encodes the most common weed pollens, grass pollens, tree pollens, mites, animals, fungi, insects and others. As known for mixtures used in the specific immunotherapy (SIT), it is thus also an object of this invention to combine allergens into one heterologous nucleic acid within the MVA according to the present invention. One skilled in the art will appreciate that various combinations in particular such combinations are useful, where numerous allergies are found in a majority of the population.
In a preferred embodiment the allergen according to the invention is selected from the group of allergens of table 1.
In a preferred embodiment the allergen according to the invention is selected from the group of hen's-egg ovalbumin (Gal d 2), ovomucoid (Gal d 1), milk, caseins (Bos d 8), β-lactoglubulin (Bos d 5), fish, parvalbumin (Gad c 1) from codfish and homologues from other species, shrimp, tropomyosin (Pen a 1) and homologues from other species including other Crustacea, arginine kinase (Pen m 2) and homologous from other species including other Crustacea, hazelnut, (Cor a 1.04, Cor a 8) and oleosins and 2S albumins from hazelnut and other kinds of tree nuts (e.g. brazil nut, cashew, almond, peanut in particular, Ara h 1, Ara h 2, Ara h 3) soybean in particular, Gly m 4, glycinin, β-conglycinin, Gly m bd 30 k, fruits in particular lipid transfer proteins and Bet v 1 homologous and rubber latex in particular Hev b 1 to Hev b 11.
In a further preferred embodiment the allergen is selected from the group of grass group I and group V allergens, tree pollen I in particular the Bet v I family, weed pollen in particular Amb a 1, 2, Art v 1, 2 and 3 and homolgues from other species, mites in particular Der p 1, Der f 1, Der p 2, Der f 2, Der p 10, Der f 10, animals in particular Fel d 1 and moulds in particular Alt a 1 to Alt a 13 and homologues from other species.
In one embodiment the nucleic acid according to the invention encodes two or more allergens, fragments or mutants from the group of allergens according to the invention. This means that the vector used for transfer into MVA as well as later, the MVA according to the invention actually comprises two or more allergens, fragments or mutants. That means that if the nucleic acid according to the invention is transcribed and expressed as a protein a multiple allergens, fragments or mutants may be produced.
In a preferred embodiment the nucleic acid according to the invention encodes two or more homologous allergens, fragments or mutants from different organisms or species.
In a particularly preferred embodiment the nucleic acid according to the invention encodes two or more homologous allergens, fragments or mutants all of which are Bet v 1 homologous comprising homologues such as those selected from the group of Bet v 1 from different pollen such as but not limited to hazel and alder, and Bet v 1 from foods such as but not limited to apple, hazelnut, carrot, celeriac and soybean.
In one embodiment according to the invention the nucleic acid according to the invention encodes two or more homologous allergens, fragments or mutants selected from the group of lipid transfer proteins such as but not limited to pollen, plant derived foods, mugwort pollen, ragweed pollen, plane tree pollen, Parietaria pollen, peach, hazelnut, peanut and wheat.
In a particularly preferred embodiment the allergen is ovalbumin.
The invention also relates to a recombinant modified vaccinia virus Ankara (MVA) comprising a heterologous nucleic acid, wherein the heterologous nucleic acid is incorporated into a non-essential region of the genome of the MVA, the heterologous nucleic acid is under the control of a vaccinia virus-specific promoter, orthopox virus-specific promoter, or a poxvirus-specific promoter and the heterologous nucleic acid is selected from the group of nucleic acids encoding an allergen selected from the group of weed pollens, grass pollens, tree pollens, mites, animals, funghi, insects, rubber, worms, human autoallergens and foods.
As already outlined above, in a preferred embodiment the recombinant modified vaccinia virus Ankara according to the invention comprises a nucleic acid encoding an allergen, fragment or mutant thereof which is selected from the group of allergens of table 1.
In a preferred embodiment the allergen of the recombinant modified vaccinia virus Ankara (MVA) according to the invention is selected from the group of hen's egg ovalbumin (Gal d 2), ovomucoid (Gal d 1), milk, caseins (Bos d 8), beta-lactoglubulin (Bos d 5), fish, parvalbumin (Gad c 1) from codfish and homologues from other species, shrimp tropomyosin (Pen a 1) and homologues from other species including other Crustacea and arthropods, arginine kinase (Pen m 2) and homologues from other species including other Crustacea and arthropods, hazelnut (Cor a 1.04, Cor a 8), and oleosins and 2S albumins from hazelnut and other kinds of tree nuts such as brazil nut, cashew, almond, peanut in particular, Ara h 1, Ara h 2, Ara h 3 soybean in particular Gly m 4, glycinin, beta-conglycinin, Gly m bd 30 k, fruits in particular lipid transfer proteins and Bet v 1 homologues and rubber latex in particular Hev b 1 to Hev b 11.
In a very preferred embodiment the allergen of the recombinant modified vaccinia virus Ankara (MVA) according to the invention is selected from the group of grass group I and group V allergens, tree pollen I in particular the Bet v 1 family, weed pollen in particular Amb a 1, 2, Art v 1, 2 and 3 and homologues from other species, mites in particular Der p 1, Der f 1, Der p 2, Der f 2, Der p 10, Der f 10, animals in particular Fel d 1 and moulds in particular Alt a 1 to Alt a 13 and homologues from other species.
The nucleic acid of the recombinant modified vaccinia virus Ankara (MVA) according to the invention may encodes two or more allergens, fragments or mutants.
In one embodiment the two or more allergens, fragments or mutants are from one organism or species.
In another embodiment the two or more homologous allergens, fragments or mutants are from different organisms or species.
In one embodiment the nucleic acid encodes two or more homologous allergens, fragments or mutants, all of which are Bet v 1 homologous comprising homologous such as those selected from the group of Bet v 1 from different pollen, such as but not limited to hazel and alder, and Bet v 1 from foods such as but not limited to apple, hazelnut, carrot, celeriac and soybean.
In one embodiment the nucleic acid of the recombinant modified vaccinia virus Ankara (MVA) according to the invention encodes two or more homologous allergens, fragments or mutants, selected from the group of lipid transfer proteins such as but not limited to from pollen, plant derived foods, mugwort pollen, ragweed pollen, plane tree pollen, parietaria pollen, peach, hazelnut, peanut and wheat.
In a particularly preferred embodiment the allergen is ovalbumin.
The invention also relates to a nucleic acid encoding a modified vaccinia virus Ankara (MVA).
In a further embodiment the invention relates to a method of introducing a MVA according to the invention into a target cell comprising infection of the target cell with an MVA according to the invention. Viruses are propagated and titered following standard methodology. To generate vaccine preparations, viruses may routinely be purified by ultracentrifugation through sucrose and reconstituted in, e.g. 1 mM Tris pH 9.0. CEF and rabbit kidney RK-13 (ATCC CCL-37) cells may be grown in minimal essential media (MEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37° C. and 5% CO2. CEF cells may be grown in 6 well tissue culture plates and infected with the multiplicity of 0.01 to 20 infectious units MVA.
The determined low or high multiplicity growth profiles, confluent CEF monolayers (grown on 6 well plates) may be infected with 0.05 infectious units (IU) or 10 IU MVA or MVA according to the invention per cell, respectively. After virus absorption for 60 min at 37° C. the inoculum may be removed. Cells may be washed twice with RPMI 1640 and incubated with fresh RPMI 1640 medium containing 10% FCS at 37° C. and 5% CO2. At multiple time points post infection (p.i.) infected cells may be harvested and virus may be released by freeze-drying and a brief sonication. Serial dilutions of the resulting virus suspension may be plated on confluent CEF monolayers grown in 6 well plates as replicates of two. At 48 hrs p.i. monolayers may be briefly fixed in acetone:methanol (1:1) and cells may be incubated for 60 min with polyclonal rabbit anti-vaccinia antibody (IgG fraction, Biogenesis Ltd., Pool, England), followed by an incubation for 45 min horseradish-peroxidase-conjugated polyclonal goat anti-rabbit antibody (Dianova, Hamburg). After washing with PBS, antibody-labeled cells may be developed using an O-Di-anisidine (Sigma, Taufkirchen, Germany) substrate solution, foci of stained cells may be counted, and virus titers may be calculated as IU/ml.
According to one embodiment of the present invention, the invention relates to a method for immunization of a living animal body including a human, said method comprising administering to said living animal body including a human in need of a therapeutically effective amount of the MVA according to the invention. Said method according to the invention may comprise a composition which may also contain (in addition to the ingredient and the carrier) diluents, common fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. It is necessary that these are pharmaceutically acceptable. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the MVA according to the invention. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to be applied for the method for immunization according to the invention to produce a synergistic effect or to minimize side-effects. Techniques for formulation and administration of the MVA according to the invention may be found in “Remington's Pharmaceutical Sciences”, (Muck Publishing Company, Easton, Pa., latest edition). The method for immunization according to the present invention will make use of a therapeutically effective amount of the MVA. A therapeutically effective dose further refers to that amount of the compound/ingredient sufficient to result in amelioration of symptoms, e.g. treatment, healing, prevention or amelioration of such conditions. To prepare vaccines, the MVA vaccinia virus generated according to the invention are converted into a physiologically acceptable form. This can be done based on the many years of experience in the preparation of vaccines used for vaccination against smallpox (Kaplan, B. R. Med. Bul. 25, 131-135 [1996]). Typically, about 106 to 107 particles of the recombinant MVA are freeze-dried in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule The lyophilisate can contain extenders, such as manitol, dextran, sugar, glycine, lactose or polydinylpyrolidole or other aids (such as anti-oxidants, stabilizers, etc.) suitable for parenteral administration. The glass ampoule is then sealed and can be stored, preferably at temperatures below −20° C. for several months. For vaccination the lyophilisate can be dissolved in 0.1 to 0.2 ml of aqueous solution, preferably physiological saline, and administered parentally, for example by intradermal inoculation. The vaccine according to the invention is preferably injected intracutaneously. Slight swelling and redness, sometimes also itching, may be found at the injection side. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner. It is expedient where appropriate to administer the vaccine several times over a lengthy period in order to obtain a high-level immune response against the foreign antigen. In a preferred embodiment the immunizing may be both prophylactic and/or therapeutic. In a further preferred embodiment an allergen may be administered after administration of the MVA according to the invention. In a particularly preferred embodiment the allergen to be administered after the MVA is the same allergen as encoded by the heterologous nucleic acid of the MVA.
After introduction of the OVA-gene into the MVA transfer vector pIIIdHR under the control of the vaccinia virus-specific promoter P7.5 the OVA expression cassette was inserted into deletion III of the MVA genome by means of homologous recombination of the flanking regions (flank 1 and flank 2). Selection of recombinant viruses was performed by expression of the vaccinia virus host range gene K1L.
Female BALB/c mice were vaccinated twice with 106, 108 MVA-OVA or with saline. Sensitizing occurred by intraperitoneal injection of OVA (0.1 μg, 1 μg or 10 μg) or PBS with Al(OH)3 as adjuvant. In one preferred embodiment of the invention PBS with Al(OH)3 is used as an adjuvant. The sera taken upon sensitization were tested by means of ELISA and RBL-testing of IgG subclasses and IgE response.
Upon incubation of mouse sera, the RBL cells bind IgE by means of the FcεRezeptor on the cell surface. If the respective allergen is added, cross-linking of the antibodies occurs which leads to a degranulation of the cells. The mediator release is quantified by photometric measurement of the β-hexosamidase activity.
Vaccinated mice sensitized with 10 μg OVA or analyzed by means of IgG subclass ELISA. The respective ratios of IgG 2A/IgG 1 are shown in the diagram over the bars.
Cytoplasmic lysates of baby hamster kidney (BHK) cells and mouse fibroblast cells (3T3) infected with rMVA-Cor a 8 were analysed by Western blot analysis at different times after infection. Expression of rCor a 8 was detected with a polyclonal rabbit-anti-Cor a 8 serum. Uninfected cells served as controls.
Cytoplasmic lysates of baby hamster kidney (BHK) cells and mouse fibroblast cells (3T3) infected with rMVA-Pen a 1 were analysed by Western blot analysis at different times after infection. Expression of rPen a 1 was detected with a polyclonal rabbit-anti-Pen a 1 serum as well as with a anti-myc antibody, since the expressed Pen a 1 carried a myc-tag. Uninfected cells served as controls.
The growth properties of rMVA-Pen a 1 compared to wtMVA was investigated on permissive chicken embryo fibroblast (CEF) cells. CEF cells were infected with different doses (multiplicity of infection (moi) 5 and 0.05) and the titers of the viruses after different times post infection were determined.
Human (HaCat) and mouse (3T3) cells were infected with rMVA-Pen a 1, wtMVA and vaccinia virus Western Reserve strain (WR). Titers 0 h and 72 h post infection were determined to investigate the growth capacity on mammalian cells.
The ovalbumin (OVA gene) was inserted under the control of the vaccinia virus-specific P7.5 promoter into the MVA genome (
The vaccination of the mice with MVA-OVA led to a reduced or deferred production of OVA specific IgE antibodies in comparison to non-vaccinated mice. Additionally, higher doses of MVA-OVA (108 IU) led to a stronger immune modulation which persisted for a longer period of time (
Also this effect is dose-dependent. The IgG2A/IgG1 ratio of the mice immunized with 108 IU MVA-OVA decreases slower than the mice immunized with 106 IU MVA-OVA. Thus, the vaccination with MVA-OVA leads to an immune response against OVA which protect the mice long before sensitization with the allergen. The IgE response against OVA is much lower in vaccinated mice than in non-vaccinated mice. Most advantageously, the ratio of IgG2A to IgG1 in vaccinated mice speaks for a TH1 induced response and thus not for an allergic immune response.
Successful generation of recombinant MVA vectors encoding for two major food allergens The gene sequence of Cor a 8, a major hazelnut allergen causing anaphylactic reactions, has been stably inserted in the genome of MVA, allowing for efficient expression of the recombinant allergen in MVA-Cor a 8 infected cells. Production of recombinant Cor a 8 upon infection was ascertained by western blot analysis of infected hamster (BHK) and mouse cells (3T3) at different time points after infection using polyclonal rabbit anti-Cor a 8 antiserum. Strong rCor a 8 expression was detectable in both cell types (
The same is true for a recombinant MVA containing the gene sequence encoding for a major shrimp allergen (Pen a 1) (
MVA-Pen a 1 was further characterized regarding its growth capacity and safety aspects to facilitate application in mice under biosafety-level 1 conditions. Compared to wtMVA, replication of rMVA-Pena a 1 in chicken cells (CEF) is not impaired (
Growth of rMVA-Pen a 1 in human (HaCat) and mouse (3T3) cells is almost completely abrogated, as was described for the wtMVA (
Despite the fact that rPen a 1 is growth deficient in mouse cells, there is an efficient recombinant protein expression, demonstrating the advantages of recombinant MVA as safe but efficient vectors for in vivo application in mouse models of food allergy.
Asterales
Ambrosia artemisiifolia-
Ambrosia trifida
Artemisia vulgaris
Helianthus annuus
Mercurialis annua
Chenopodium album
Salsola kali
Catharanthus roseus
Rosy periwinkle
Plantago lanceolata
Humulus japonicus
Parietaria Judaica
Parietaria officinalis
Cynodon dactylon
Dactylis glomerata
Festuca pratensis
Holcus lanatus
Lolium perenne
Phalaris aquatica
Phleum pratense
Poa pratensis
Sorghum halepense
Phoenix dactylifera
Alnus glutinosa
Betula verrucosa
Carpinus betulus
Castanea sativa
Corylus avellana
Quercus alba
Fraxinus excelsior
Ligustrum vulgare
Olea europea
Syringa vulgaris
Cryptomeria japonica
Cupressus arizonica
Cupressus sempervirens
Juniperus ashei
Juniperus oxycedrus
Juniperus sabinoides
Juniperus virginiana
Platanus acerifolia
Acarus siro
Blomia tropicalis
Dermatophagoides farinae
Dermatophagoides microceras
Dermatophagoides pteronyssinus
Euroglyphus maynei
Glycyphagus domesticus
Lepidoglyphus destructor
Tyrophagus putrescentiae
Bos domesticus
Canis familiaris
Equus caballus
Felis domesticus
Cavia porcellus
Mus musculus
Rattus norvegius
Alternaria alternata
Cladosporium herbarum
Aspergillus flavus
Aspergillus fumigatus
Aspergillus niger
Aspergillus oryzae
Penicillium brevicompactum
Penicillium chrysogenum
Penicillium citrinum
Penicillium oxalicum
Fusarium culmorum
Trichophyton rubrum
Trichophyton tonsurans
Candida albicans
Psilocybe cubensis
Coprinus comatus
Rhodotorula
Malassezia furfur
Malassezia sympodialis
Epicoccum purpurascens
Aedes aegyptii
Apis mellifera
Bombus pennsylvanicus
Blattella germanica
Periplaneta americana
Chironomus kiiensis
Chironomus thummi thummi
Ctenocephalides felis felis
Thaumetopoea pityocampa
Lepisma saccharina
Dolichovespula maculata
Dolichovespula arenaria
Polistes annularies
Polistes dominulus
Mediterranean paper
Polistes exclamans
Polistes fuscatus
Polistes gallicus
Polistes metricus
Vespa crabo
Vespa mandarina
Vespula flavopilosa
Vespula germanica
Vespula maculifrons
Vespula pennsylvanica
Vespula squamosa
Vespula vidua
Vespula vulgaris
Myrmecia pilosula
Solenopsis geminata
Solenopsis invicta
Solenopsis saevissima
Triatoma protracta
Gadus callarias
Salmo salar
Bos domesticus
Gallus domesticus
Metapenaeus ensis
Penaeus aztecus
Penaeus indicus
Penaeus monodon
Todarodes pacificus
Helix aspersa
Haliotis midae
Rana esculenta
Brassica juncea
Brassica napus
Brassica oleracea
Brassica rapa
Hordeum vulgare
Secale cereale
Triticum aestivum
Zea mays
Oryza sativa
Apium graveolens
Daucus carota
Corylus avellana
Fragaria ananassa
Malus domestica
Pyrus communis
Persea americana
Prunus armeniaca
Prunus avium
Prunus domestica
Prunus dulcis
Prunus persica
Asparagus officinalis
Crocus sativus
Lactuca sativa
Vitis vinifera
Musa x paradisiaca
Ananas comosus
Citrus limon
Citrus sinensis
Litchi chinensis
Sinapis alba
Glycine max
Vigna radiata
Arachis hypogaea
Lens culinaris
Pisum savitum
Actinidia chinensis
Capsicum annuum
Lycopersicon esculentum
Solanum tuberosum
Bertholletia excelsa
Juglans nigra
Juglans regia
Anacardium occidentale
Ricinus communis
Sesamum indicum
Cucumis melo
Ziziphus mauritiana
Anisakis simplex
Argas reflexus
Ascaris suum
Carica papaya
Dendronephthya nipponica
Hevea brasiliensis
Homo sapiens
Triplochiton scleroxylon
| Number | Date | Country | Kind |
|---|---|---|---|
| 06005164.6 | Mar 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP07/02427 | 3/14/2007 | WO | 00 | 12/17/2008 |
