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  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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  • A61K2039/5254—Virus avirulent or attenuated
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  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/24011—Poxviridae
  • C12N2710/24061—Methods of inactivation or attenuation
  • C12N2710/24064—Methods of inactivation or attenuation by serial passage

Definitions

• the present invention relates to highly attenuated animal poxviruses, paramunity inducers derived therefrom, and vector vaccines based on highly attenuated animal pox virus strains.
• the highly attenuated animal poxviruses according to the invention have no virulent and immunizing properties by the process of high attenuation.
• Another aspect of the invention relates to methods of producing such highly attenuated animal pox virus strains and their use as paramunity inducers to induce paramunity, i. for activation of the nonspecific (paraspecific) immune system in humans and animals or as a vector vaccine for immunizing a mammal or human.
• the highly attenuated animal pox viruses according to the invention are also suitable for the prophylaxis and treatment of multifactorial, usually chronic diseases.
• Preferred embodiments of the invention relate to animal pox virus strains isolated from infected animals and highly attenuated by serial passages of all genera of the family Poxviridae.
• the animal pox strains according to the invention have excellent paramunizing properties, the virulent and immunizing properties having been lost by the method of high attenuation according to the invention.
• the body's immune system of mammals can be divided into an antigen-specific and an antigen-unspecific (paraspecific) part.
• the antigen-specific part of the immune system includes, for example, antibodies or specific immune cells.
• the antigen-specific mechanisms are responsible for the establishment of a specific immunity, the antigen-specific for the building of paramunity.
• the parasitic activities of the antigen-immune system include nonselective cellular and soluble protective elements, such as the complement lysozyme system and the regulatory cytokine cascade, as well as cellular protective elements, such as granulocyte, micro and macrophages, natural killer cells, non-antigenic T-lymphocytes, dendritic cells and others
• nonselective cellular and soluble protective elements such as the complement lysozyme system and the regulatory cytokine cascade
• cellular protective elements such as granulocyte, micro and macrophages, natural killer cells, non-antigenic T-lymphocytes, dendritic cells and others
• Paramunity is the state of a well-regulated and optimally functioning nonspecific defense system, which gives the organism a rapid, temporally limited, increased protection against a multiplicity of different pathogens, antigens and other noxae.
• Paraspecific activities are present in the organism immediately after contact with noxa, i. after about 2 to 6 hours, while the effects of the antigen-specific immune system occur only after 5-8 days (cellular specific immunity) or even weeks (antibodies). This provides additional time to build up specific defense responses to the antigens that could not be neutralized by the paramunizing activities.
• the paraspecific defense therefore allows the organism to encounter immediately upon confrontation with a wide variety of foreign substances, infectious agents, toxins and transformed endogenous cells, i. to put up with no loss of time (Anton Mayr, "Paramunization: Empiricism or Science", Biol. Med., Aufl. 26 (6): 256-261, 1997).
• the paraspecific immune defense is thus a physiological process and can be defined as a "primary barrier” in dealing with a pollutant-containing environment. This form of defense is irreplaceable not only for the lower organisms, but especially for the higher and highly developed organisms It can be shown that primary congenital defects in this biological defense system can lead to life-threatening situations, for example the "Chediak-Steinbrinck-Higashi-syndrome" of humans, caused by granulocyte defects
• NK cells natural killer cells
• the state of paramunity is characterized by increased phagocytosis rate, increased function of spontaneous cell-mediated cytotoxicity (NK cells), and increased activity of other non-antigen-specific lymphoreticular cells.
• certain cytokines release, which stimulate and / or suppress (eg via repressor mechanisms) both with the cellular elements and with one another, ie have an optimally regulating effect.
• This tightly networked and stepwise reactive biological System of paramunity with its different acceptor, effector and target cells and the signal-transmitting molecular messengers (cytokines) is also intensively linked to the hormone and nervous system, sometimes even with the vascular and metabolic system.
• a drug-based paramunization can be achieved by activation of the cellular elements of the paras-specific part of the immune system and the associated formation of cytokines, with the aim to eliminate dysfunctions, to quickly increase the pathogen and antigen-unspecific protection of an individual (optimal bioregulation) to eliminate immunosuppression or immunodeficiency, to repair deficits and / or to act as regulator between immune, hormone and nervous system (Anton Mayr, "Paramunization: Empiricism or Science", Biol.
• the paramunity inducer per se is a protein, i. it is not comparable to an antibody, a chemical, an antibiotic, vitamin or hormone. Rather, like a catalyst, it activates the paraspecific mechanism via a stepwise mechanism
• Paramunity inducers act both in regulating and repairing the immune defense.
• phagocytic cells acceptor cells
• mediators e.g. Release cytokines
• the animal poxvirus strains underlying these paramunity inducers have been attenuated in a conventional manner, i. they are in a reduced state in which the virulent and especially the immunizing properties of the virus are attenuated but not completely lost.
• Such high attenuation of poxviruses is in the present invention for the first time on the basis of the Orthopoxviruses Camelpox virus (Orthopoxvirus cameli) and Ektromelievirus (Orthopoxvirus muris), the leporipox virus Myxomatosevirus (Leporipoxvirus myxomatosis), avipoxvirus chickenpox virus (Avipoxvirus gallinae) and canarypox virus (Avipoxvirus serinis) and the Parapox virus Ecthyma (Parapoxvirus ovis).
• Orthopoxviruses Camelpox virus (Orthopoxvirus cameli) and Ektromelievirus (Orthopoxvirus muris)
• the leporipox virus Myxomatosevirus Leporipoxvirus myxomatosis
• avipoxvirus chickenpox virus Avipoxvirus gallinae
• Exemplary embodiments of the invention relate to the high attenuation of the Orthopoxvirus strain.
• Other preferred embodiments relate to other strains of orthopoxvirus, as well as strains of the parapoxviruses and avipoxviruses (see below).
• the Camelpocken are the causative agents of a dangerous, cyclic-systemic virus disease of the camelids, which is characterized by an exanthema of the skin and mucosa of the head, neck and neck, as well as the extremities and Inguinalministerd (Münz, E., 1999: " pox and pox-like diseases in cameis "Proc.l st Int. Camel Conf. 1, 43-46). the disease occurs cyclically every 2 to 3 years if a sufficiently large sensitive population is present. of the Camelpox viruses are preferably infested with two genera (Lama and Camelus) of the family Camelidae (Mayr, A.
• Camelus comprises the single-humped dromedary ⁇ Camelus dromedarius) and the two-humped camouflage ⁇ Camelus ferus bactrianus), mainly of dromedaries and bactids in the countries of the so-called "Old World” (deserts, steppes of North Africa, Arabia, Mongolia), while the Lama is favored in South America.
• the camelpox virus ⁇ Orthopoxvirus camel ⁇ is a particularly close relative of the variola virus, the causative agent of human smallpox (variola). For humans, camelpox virus is not pathogenic. Like all classical pox viruses, the camelpox virus is cuboid and has characteristic surface proteins that are responsible for the immunizing and paramunizing properties of the virus or its constituents.
• the average size is 280 nm in the longitudinal direction, depending on the genus or strain, and about 180 nm in the transverse direction (Otterbein, CK., 1994: “Pheno- and genotypic studies of two camelpox virus isolates before and after attenuation by cell culture passages ", Vet.Med.Diss, Kunststoff.)
• the camelpox virus genome consists of a linear, double-stranded DNA, the two DNA strands being covalently bound together at the genome ends, such that the viral DNA forms a continuous polynucleotide chain.
• Myxomaviruses are the causative agents of myxomatosis, a cyclical, contagious viral disease of the wild and domestic rabbits, characterized by generalized, partial hemorrhagic subcutaneous edema of the head and the whole body, with preference of the anal site, the vulva and the tube, like no other infectious disease is. If myxomatosis is reintroduced into a previously disease-free country, it will be rapid and fatal. After the virus has settled, the epidemic character changes to clinically inapparent infections (Mayr A .: Medical Microbiology, Infection and Epidemiology, 7th ed., Erike Verlag, Stuttgart, 2002).
• the disease is widespread among American cottontail rabbits of the genus Sylvilagus, which colonize exclusively the New World. These wild rabbits are the only natural reservoir of the disease. The infection is mild in their form. In contrast, the disease has in European wild and domestic rabbits of the genus Oryctolagus, which are also native to Australia, a nearly 100% mortality at Neueinschleppung the pathogen.
• the natural host range of the myxomavirus (genus Leporipoxvirus) is very limited. In general, the virus only reproduces in American cottontail rabbits and European domestic and wild rabbits. Occasionally, however, infections were observed in European wild hares. Transmission attempts to other species and to humans were negative.
• Chickenpox comes from Asia and has been known for millennia. They are worldwide and very resistant. Transmission occurs through penetration via skin lesions. It can also be piercing Insects involved in the transmission. The incubation period for the disease is 4 to 14 days. There are two forms of progression, whereby a distinction is made between the so-called skin shape and the mucous membrane form.
• the skin's shape is characterized by vesicles or scabrous nodes on the head, crest, neck and feet.
• the mucous membrane shows yellowish-white deposits on the tongue, the mucous membranes of the beak, the larynx, the trachea and the eyes.
• the incubation period for canarypox virus infection is 3 to 16 days. After the outbreak of the disease, a large part of the stock dies within just a few hours. The infected animals show nodules on the horn parts and at the beak angles. It comes to massive respiratory problems and the birds stifle quite quickly to the caused by the virus cheesy deposits in the airways.
• Attenuated strains of avipoxviruses were obtained by successive passages in cell cultures from chicken embryo fibroblasts and used to vaccinate chickens.
• the best studied and available strain is Strain HP-I (A. Mayr and K. Malicki, 1966: "Attenuation of Virulent Chickenpox Virus in Cell Cultures and Properties of the Attenuated Virus", Zbl.Vet.Med.B 13, 1-13
• More than 200 passages in chick embryo fibroblasts result in an attenuated but still replicable virus, but with residual pathogenicity to chickens on intravenous or Aersol administration, viruses more than 400 times passaged are considered to be non-pathogenic and are considered to be efficacious and extremely safe Vectors were considered for use in mammals Immunization could be achieved without complete productive replication of the virus.
• the object of the present invention is therefore to provide animal pox strains which are stable, have a high degree of attenuation and in which the poxviruses are modified in such a way that they have completely lost their virulent and immunizing properties and thus can be used as harmless paramunity inducers and vector vaccines.
• the highly attenuated by the inventive method animal pox strains are outstandingly suitable as Paramunticiansinducer or for the production of vector vaccines.
• the invention relates to highly attenuated animal pox virus strains and their use as paramunity inducers or for the production of vector vaccines.
• Particular embodiments of highly attenuated animal pox strains are strains of myxomatosis and camelpox virus. Particularly preferred is the Camel pollen virus strain hM 27 with accession number 05040602 and myxomatosis virus strain hM 2 with accession number 05040601. The virus was deposited at the Public Health Laboratory Service (PHLS) Center for Applied Microbiology & Research (CAMR), European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, Wiltshire, United Kingdom.
• PHLS Public Health Laboratory Service
• AMR Public Health Laboratory Service
• ECACC European Collection of Animal Cell Cultures
• Porton Down Salisbury, Wiltshire, United Kingdom.
• inventions relate to the high attenuation of the canarypox virus (Avipoxvirus serinae), preferably of the strain KP1, of the ectomelie virus (Orthopoxvirus muris), preferably of the strain Mü 1 and chickenpox virus (Avipoxvirus gallinae), preferably of the strain HPI and of the parapoxvirus (Parapoxvirus ovis) ,
• the virulence of the virus strains and their immunizing properties are completely lost in comparison to conventionally attenuated animal pox strains.
• the highly attenuated animal pox viruses according to the invention therefore no longer have any residual virulence or immunity.
• the highly attenuated animal pox strains are particularly suitable for use as paramunity inducers or for the production of vector vaccines.
• the invention further relates to processes for the preparation of the highly attenuated poxvirus strains according to the invention.
• Preferred poxvirus strains are strains belonging to the genus orthopoxvirus, avipoxvirus, leporipoxvirus and parapoxvirus.
• High attenuation of poxvirus strains is achieved by additional plaque final dilution passages (ie, inoculation and propagation) of conventionally attenuated virus strains in optimized, selected, permanent cell lines (eg, VERO cells) in primary cell cultures (eg, chicken embryo fibroblast (FHE) cell cultures). incubated chicken eggs or reached in experimental animals. It was surprisingly found that this virginity and immunogenicity of the animal poxviruses and their constituents is lost as compared to conventionally attenuated strains by this additional passaging.
• optimized, selected, permanent cell lines eg, VERO cells
• primary cell cultures eg, chicken embryo fibroblast (FHE) cell cultures
• the passage in the selected cell systems or cultures is continued until the desired properties are achieved, ie until the animal poxviruses no longer exhibit any virulence or immunogenicity and instead have an increased activity of the unspecific immune system (paramunity).
• this can be achieved by at least 300-500 passages in optimized cell systems, such as cell cultures of VERO-cell passages (ATCC CCL-81, WHO, American Type Culture Collection).
• optimized cell systems give the required high infectivity titer.
• the other desirable biological, genetic and immunological properties that result from high attenuation of animal pox strains are listed in Table 3.
• the method according to the invention for the production of highly attenuated animal poxviruses can be defined by the following steps:
• Infectivity titers in particular AVIVER or VERO cells
• the highly attenuated orthopoxvirus strain is a highly attenuated camelopardal virus, in particular strain h-M 27.
• a preferred method for high attenuation of camelpox viruses comprises the steps of: (a) culturing the isolated camelpox virus for about 2 to 4 passages in
• Cells preferably plaque final dilution passages.
• the animal poxviruses thus produced can be used for the production of paramunity inducers and vector vaccines.
• For the production of the reproducible virus harvest is used.
• a further embodiment of the present invention relates to the highly attenuated leporipoxvirus strain Myxomatosevirus ⁇ Leporipoxvirus myxomatosis), strain h-M 2.
• a preferred method for high attenuation of such a myxomatosis virus strain comprises the following steps:
• the virus harvests are inactivated by treatment with beta-propiolactone.
• the invention also relates to pharmaceutical compositions which contain one or more highly attenuated smallpox strains of different origin in combination and to which, if appropriate, a pharmaceutical carrier is added.
• a further aspect of the invention therefore relates to the use of one or more highly attenuated animal pox strains according to the invention (for example in combination) or components of the highly attenuated animal pox strains for activating the paraspecific immune system in a mammal or in humans for prophylaxis and therapy.
• the highly attenuated animal poxviruses are used for the production of vector vaccines, for this purpose the replicable virus harvests are used.
• a nucleic acid coding for a foreign antigen is thereby incorporated into one of the deletions of the nucleic acid of the vector (animal poxvirus), which is the result of the high attenuation, so that the foreign gene can be expressed by the vector.
• the resulting foreign proteins provide immunizing epitopes and thereby stimulate the body's own specific defense system.
• Attenuation (English: attenuate) of an infectious agent (e.g., viruses, bacteria, fungi) is basically understood the reduction of its virulent and immunizing properties.
• infectious agent e.g., viruses, bacteria, fungi
• High attenuation means the further reduction of simply attenuated but still partially virulent and immunizing pathogens, up to a complete loss of virulence and the immunizing potential, whereby the high attenuation leads to an extreme narrowing of the host range paramunizing potential greatly increased Reactivation of their lost virulent and immunizing properties, the highly attenuated poxviruses are more stable than conventionally attenuated strains, ie reconversion is not possible.
• the highly attenuated animal pox strains are genetically engineered to differ from the conventionally attenuated animal pox strains by further decreasing the molecular weight of the virus nucleic acid and increasing deletions in the nucleic acid; biologically by the complete loss of virulence and contagiousness, these strains simultaneously achieving in the permissive host system an optimal and compared to conventionally attenuated strains high infectivity titers; immunologically by the total loss of immunogenicity, and molecular biologically by the loss of cytokine receptors, e.g. Receptors for interferon and certain interleukins.
• cytokine receptors e.g. Receptors for interferon and certain interleukins.
• Virulence refers to the degree of pathogenic properties of a particular strain of a pathogenic type of pathogen in a given host under defined infection conditions. The degree of virulence can vary considerably within the strains of a species. There are strong, weak and non-virulent (avirulent) strains. If host and environmental conditions are changed, the virulence of the strain may change as well, but it may also remain unchanged. Thus, the host's defenses, the anatomical and physiological conditions of the host flora, the environmental temperature, the humidity, etc., can be synergistic or antagonistic.
• the presence of virulence or the loss of virulence can be evaluated in the test system known to the person skilled in the art and relevant for the particular animal pox virus.
• the animal poxviruses of the present invention show no virulence in human hosts.
• pathogenicity refers to the property of an infectious agent or metazoan parasite, after penetration, adherence and identical multiplication in a host to lead to a local or general impairment of performance (functio laesa) and create an infectious disease Since the emergence of an infectious disease depends on the pathogen and the host the pathogenicity not only the pathogen, but the pathogen-host system.
• the pathogenicity is related to the species of a pathogen, not to a variant, a strain or a colony. It is a basic property, a potency that can work but does not have to. A pathogenic species related to a particular host-host system can not become non-pathogenic in nature, since this basic ability of an entire species is not lost.
• Immunogenicity of an animal pox virus refers to the ability of the animal pox virus in a vertebrate, preferably in the natural host of the virus or in humans, to elicit a cellular specific and / or humoral immune response, e.g. Stimulate T-cell proliferation and / or the generation of antibodies.
• the loss of immunogenicity of the highly attenuated animal pox strains of the present invention is associated with the loss of this ability. The immunogenicity or the loss of these can be tested in test system, which are known in the art.
• Vector vaccines are understood to be vaccines consisting of two components: a microbial carrier (vector) and an immunizing antigen whose coding nucleic acid is incorporated into the vector Nucleic acid deletions and their paramunizing properties are preferably (highly) attenuated animal poxviruses. [Fremd.] The introduced foreign gene nucleic acid is expressed in the vaccine by the vector, resulting in the formation of specific immunizing reactions.
• Paraspecific vaccines refers to bioregulative preparations of attenuated, avirulent and inactivated animal poxviruses, depending on Attenu réellesgrad only residues of (conventional attenuated) or no (highly attenuated) immunizing
• the invention is based on the surprising discovery that the virulence and immunizing properties of conventionally attenuated animal poxvirus strains can be reduced to complete loss by additional plaque final dilution passages in permissive cell cultures, incubated chicken eggs or experimental animals. In contrast to a potential reactivation of these properties, such highly attenuated strains are stable.
• high attenuation This process, which goes beyond simple, conventional attenuation, is referred to in the present invention as "high attenuation.”
• These highly attenuated animal poxvirus strains are markedly improved over conventionally attenuated pathogens
• the highly attenuated animal pox strains as summarized in Table 3, are genetically engineered from the conventionally attenuated animal pox strains by the decrease of the molecular weight of the virus nucleic acid and the increase of deletions in the nucleic acid, biologically by the loss of virulence and contagiousness, immunologically by the loss of immunogenicity, and molecular biologically by the loss of cytokine receptors.
• Avipoxviren shown, where it is not limited to this genera.
• a high attenuation with a myxomatosis virus and a camelpox virus is described in the present invention.
• Highly attenuated poxvirus strains also lose their specific immunizing capacity through the high attenuation procedure described below, while their parasite specificity is purposefully enhanced. Therefore, the highly attenuated poxvirus strains are suitable as paramunity inducers or for the production of vector vaccines.
• the enhancement of paraspecific properties is probably due to the fact that immunizing and paramunizing proteins of animal poxviruses interfere with each other.
• the loss of immunizing proteins caused by high attenuation activates additional paramagnetizing proteins which significantly increase the paramunizing efficacy of these strains. In this way, one obtains highly effective and harmless paramunity inducers, which cause no allergies or other immunogenic side effects even in short-term repeated and frequent applications.
• the present invention relates to a highly attenuated animal poxvirus based on an animal poxvirus strain of the family Poxviridae, characterized in that the animal poxvirus no longer possesses virulent and immunizing properties and the highly attenuated animal poxvirus has a lower molecular weight of the virus nucleic acid, more frequent deletions in the terminal region and has an increased loss of cytokine receptors compared to conventionally attenuated animal pox strains.
• Known attenuated animal poxviruses have between 0 and 3 deletions in one or both terminal regions of the virus genome.
• the number of deletions in different strains of merely attenuated animal poxviruses varies so that the highly attenuated animal poxviruses of the present invention taken together have 1, 2, 3, 4, 5 or more deletions in the terminal regions of the virus genome than those in the merely attenuated animal poxviruses.
• the highly attenuated animal poxviruses of the present invention have a total of 5, 6, 7, 8, 9, 10 or more deletions in the terminal regions. preferably have more frequent deletions, preferably at least 2 deletions in the right and at least 2 deletions in the left.
• the virus genome has a loss of cytokine receptors for interferon ⁇ and ⁇ . It is particularly preferred that the Tiepockenvirus also has a loss of receptors for IL-1 ß and / or TH 1 cells.
• the virus genome of the animal pox virus is 16%, 17%, 18%, 19%, and more preferably about 20% smaller than the wild-type virus genome.
• the deletions are in one or both terminal regions of the animal poxvirus genome.
• the highly attenuated animal pox virus of the present invention is obtainable by the following method:
• at least 100, preferably at least 110, at least 120, at least 130 or more VERO cell passages are performed followed by at least 20, preferably at least 24 intermediate passages in AVIVER cell cultures and other VERO cell passages;
• AVIVER cells especially for 200 to 300 passages.
• inoculation and continuation of the animal poxviruses in VERO cells for at least 90 passages, preferably for at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more passages.
• plaque purified passages are performed.
• Attenuation begins with the adaptation of isolated animal poxviruses in homologous or heterologous permissive cell systems, e.g. Cell cultures, incubated chicken eggs or in experimental animals. This is followed by attenuation through continuous passages in various permissive cell systems.
• the permissive cell systems appropriate for each virus strain are specifically selected for each species of animal pox virus. The selection depends on the infectivity titre of the viruses in the respective cell system.
• the cell system is selected for passenger, which gives the highest In Stammiösticianstiter for each virus species.
• Such a cell system, in particular cell line can be determined by those skilled in the art by methods known in the art.
• This also corresponds to the optimal virus titer.
• the attenuation is continued by removing the animal pox viruses for about 100-300, in particular 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 , 290 or 300 passages in these optimal cell systems.
• This is followed by a final phase characterized by 3-5 plaque final dilution passages. This material can be further processed according to further use.
• a high attenuation of poxviruses by at least 60 to 300 passages depending on the respective virus strain in VERO cell cultures (for example, 150 or 260 passages).
• the strain Leporipoxvirus myxomatosis is highly attenuated by approximately at least an additional 150 to 300 passages in MA and VERO cell cultures, preferably 290 passages.
• the strain Avipoxvirus gallinae is highly attenuated by about 100 to 150 additional passages, preferably by 98 passages, in FHE cell cultures.
• the parapoxvirus strain is highly attenuated by an additional 100 to 160, preferably 164 passages (Tables 3 and 4). It is preferred that in the passage of the virus strains (ie in the transfer and inoculation) the so-called plaque final dilution method is used.
• a fully synthetic medium is preferably used, particularly preferably the medium MEM, which is 5% to 20%, preferably 10% BMS (serum replacement medium) and 5% to 20% %, preferably 10% lactalalbumin hydrolyzate is used as the virus medium after exchange with the culture medium, preferably MEM medium, with 5% to 20%, preferably 10% lactalalbumin hydrolyzate, without BMS or without fetal calf serum and without antibiotics
• Production processes are preferably carried out at pH values of 7.0 to 8.0, preferably at a pH of 7.25.
• Virus crops with titers of 10 s to 10 8 TCID 50 / ml, preferably of at least 10 7 5 TCID 5 0 / ml are preferred as starting material for the production of highly attenuated animal pox strains.
• the proliferation of poxviruses in VERO cells leads to a typical cytopathic effect, which leads to the destruction of the infected cells (lysis).
• a primary vaccine dose of about 10 MOI ("multiplicity of infection") after a short period of inactivation (1-2 days), reticulated cell structures are formed for about 3 days and, after about 5 days, the cells are lysed.
• the virus harvests obtained from the last pass can be further processed according to their use.
• the nucleic acids contained in the viruses for the production of vector vaccines can be recombinantly cloned.
• the highly attenuated virus crops can be lyophilized and stored for example by the addition of 2.5% gelatin at 4 ° C for further use, for example as Paramunticiansinducer.
• the lyophilizate can be tested for its harmlessness and effectiveness.
• the orthopoxviruses can be highly attenuated by the method described by way of example with camelpox viruses:
• Orthopoxyirus cameli, hM27 camelpox viruses isolated from the pustular material of diseased animals, such as strain M27, are grown in embryonic lamb kidney cell cultures for about 2 passages.
• the thus bred animal poxviruses are transferred by a suitable method, preferably by the plaque final dilution method in VERO cells and continued there for about 5 passages.
• the last cell culture passage is adapted to MA cells (monkey kidney MA-104 cells) and continued approximately 114 times in passages.
• the resulting 121st plaque-purified MA passage (total 284 passages) has been found to be simply attenuated.
• the highly attenuated camelpox virus, strain hM 27, obtained by the method of the invention has a total loss of virulence and contagiousness for the homologous host and a high infectivity titer in VERO cells (10 7> 25 KID 50 / ml). It is therefore particularly suitable for use as a paraspecific vaccine (paramunity inducer).
• the paramunity inducers based on highly attenuated animal poxviruses can be used in both replicable and inactivated form. In inactivated form, the highly attenuated virus is treated with beta-propiolactone as described below (V. frainger, T. Schlapp, W. Strube, N. Schmeer and A.
• the genome length of the virus is already significantly reduced by occurring deletions.
• the genome length of the parent virus wild-type
• the genome length of the attenuated M27 strain is about 172,400 bp.
• Conventional attenuation thus results in a significant loss of nucleotides in the DNA.
• Restriction digestion with the restriction enzyme HindIII shows that in the genome four restriction fragments are present less in the analysis gel (Otterbein CK., 1994, Vet.Med.Diss.Munich).
• the central, conserved region of the viral genome remains unchanged (C. Gubser, S. Hue, P.
• Leporipoxy virus mvxomatosis, Mvxomatosevirus, h-M 2 was carried out with the myxomatosis virus strain M 2. Again, a pass in CAM cells was followed by several passages of VERO and AVIVER cells and finally further passages in VERO cells.
• the method of producing paramunity inducers from highly attenuated myxoma viruses comprises the steps of:
• the myxomatosis virus used for attenuation from the edematous subcutis (left ear) of a typical European wild rabbit (genus Oryctolagus) suffering from myxomatosis was infected with myxoma virus by culturing on the chorioallantoic membrane (CAM) 10 days of incubated chicken eggs (VALO). Eggs) and three times by the method of Herrlich et al. Adapted on the CAM in passages (Herrlich A., Mayr A.
• the third CAM passage was in a 1st stage on VERO cells were adapted over 120 passages (ATCC CCL-81, WHO, American Type Culture Collection), amplified in a second step through 24 intermediate passages in AVIVER cell cultures, and further bred in VERO cells in the 3rd phase Passages with the aim of attenuation were carried out after these continuous final dilution passages, the originally virulent myxomavirus was attenuated.
• High attenuation of the myxomavirus strain M 2 is achieved by the continuation of the attenuated strain in VERO cells. For this purpose, at least another 250 to 350 passages, preferably 300 plaque-cleaned passages in VERO cells must be performed.
• Table 3 and Table 4 shows an overview of the biological and genetic engineering differences between a conventional attenuation and a high attenuation according to the invention using the example of the vaccinia virus, strain MVA.
• deletions occur more frequently in the terminal regions of the virus genome (inverted terminal repeat) and the molecular weight is reduced due to lower base pairs.
• Highly attenuated animal poxviruses lack about 20% of the original genome (which makes them attractive as vector vaccines, see below).
• receptors eg for IL-1 ⁇ and TH 1 cells
• an enhancement of NK cell activation and the formation of haematopoietic stem cells as well as a further narrowing of the host range in cell cultures.
• interferon is ⁇ and ⁇ , IL-I, 2, 6, 12, and GM-CSA, TNF amplified.
• highly attenuated animal pox strains have no specific immunogenicity, but an increased activity of the unspecific immune system (paramunity). A virulence towards humans or animals is completely missing. Further processing of highly attenuated parapoxyiren to paramunity inducers
• Concentration of 0.01% -l% beta-propiolactone can be performed. Particularly preferred here is a concentration of 0.05% beta-propiolactone. Ideally, one will
• the highly attenuated virus particles are preferably purified by means of low-speed centrifugation (eg 1000 rpm). After centrifugation, 0.5-10% succinylated gelatin (eg, polygeline, available from, for example, Hausmann Company, St. Gallen / Switzerland), preferably 5% succinylated gelatin, may be added.
• succinylated gelatin eg, polygeline, available from, for example, Hausmann Company, St. Gallen / Switzerland
• the resulting mixture can then be lyophilized in portions of, for example, 1.5 ml in corresponding sterile glass vials or ampoules and, if necessary, dissolved with distilled water (distilled water). A volume of 0.5-2 ml, preferably 1.0 ml, of the distilled water.
• dissolved lyophilisate corresponds to a vaccine dose for humans in intramuscular administration (see also Mayr A. and Mayr B .: “From Empiricism to Science", Veterinary Surgery, Aufl., 57: 583-587, 2002.)
• the lyophilized preparation can be stored at temperatures of about + 4 ° C to +8 0 C or at lower temperatures (eg -60 ° C) stable indefinitely.
• a further aspect of the invention relates to the use of highly attenuated animal pox virus strains or components of highly attenuated animal pox strains individually or combinations as paramunity inducers.
• Examples are replicable or inactivated freshly isolated animal poxviruses, replicable or inactivated recombinant animal poxviruses derived from freshly isolated animal poxviruses, viral envelopes, separated envelopes, and cleavage products and aberrant forms of these envelopes, single native or recombinant polypeptides or proteins, especially membrane and surface receptors occur in isolated animal poxviruses or from one genetically modified smallpox virus or part of its genetic information can be recombinantly expressed.
• Another aspect of the present invention is therefore to combine various highly attenuated pox strains of the same or a different genus for use as paramunity inducers.
• the highly attenuated animal poxviruses are suitable for the following prophylactic or therapeutic indications in humans and animals:
• Deficiencies or dysregulations in the immune system of an organism Deficiencies or dysregulations in the immune system of an organism; neonatal infection threat; adjuvant therapy for certain tumor diseases, e.g. Prevention of
• the paramunity inducers based on highly attenuated animal pox strains according to the invention are thus suitable for inducing the paras-specific immune system and / or for the prophylaxis or treatment of defects or multicausal infectious diseases.
• diseases are dysfunctions of the immune system, immunosuppression, immunodeficiency diseases, dysfunctions of homeostasis between hormone, circulatory, metabolic and nervous system, neonatal Infection threat, tumor diseases, viral diseases, bacterial diseases, therapy-resistant infectious factor diseases, viral and bacterial mixed infections, chronic manifestations of infectious processes, liver diseases of various origins, chronic skin diseases, herpes diseases, chronic hepatitis, influenza infections, endotoxin damage, improvement of wound healing with prevention of secondary infections.
• the highly attenuated animal pox strains described herein can be local or parenteral.
• the local administration of paramunity inducers specifically stimulates the paraspecific defense mechanisms in the mucous membranes and in the skin.
• parenterally applied paramunizations hardly influence the local defense mechanisms in the skin and the mucous membrane.
• a pharmaceutical composition comprising one or more of the highly attenuated animal pox strains of the invention and optionally a pharmaceutically acceptable carrier.
• Such carrier or additives are e.g. Polyethylene glycol, dextrose, sorbitol, mannitol, polyvinyl pyrrolidone, gelatin, magnesium stearate, carboxyl polymethylene, carboxymethyl cellulose, cellulose acetate phthalate or polyvinyl acetate.
• a further aspect of the invention relates to the use of the highly attenuated poxvirus strains for the production of vector vaccines (review: Pastoret, P. -P. and Vanderplasschen, A., 2003).
• highly attenuated animal poxvirus strains are even better suited as vectors for the production of vector vaccines, since they have completely lost their immunizing properties due to high attenuation. Because the viruses are passaged due to their infectious titre, the deletions are located in areas that are not required for viral replication.
• the highly attenuated animal poxviruses provide sufficient space for the insertion of a foreign nucleic acid (DNA) or a foreign immunogen to be expressed.
• the foreign nucleic acid may encode a peptide or protein that provides immunizing epitopes.
• the invention is not intended to a particular peptide or Be restricted protein.
• the foreign genes can be cloned into the appropriate deletion region of the virus according to their size.
• the expression of the entrained peptide or protein can be controlled by control elements such as a promoter and, if necessary, enhancer elements.
• control elements such as a promoter and, if necessary, enhancer elements.
• the cloning of recombinant animal poxviruses as vector vaccines occurs after the last plaque final dilution passage.
• the virus nucleic acid can be cleaved for cloning with suitable restriction endonucleases and ligated to the foreign nucleic acid sequences by standard ligation techniques.
• the vector vaccines according to the invention have the advantage that they do not have an allergic effect and offer the eluted specific antigen an optimally regulated immune system, which contributes to optimal vaccination success. Also, the vector vaccines of the invention are free of local or systemic negative side effects. Since they take advantage of the immunological gap until the full development of immunity, they are particularly suitable for emergency vaccination (for example, acute danger of infection, unexpected travel).
• vector poxvirus The observation that the excellent parasite-specific activity of the vector poxvirus can significantly increase the vaccination success and the harmlessness of the vaccine is novel and makes the strains attractive for the production of vector vaccines.
• Vector vaccines based on highly attenuated animal pox strains are therefore superior in their efficacy and safety to conventional vector vaccines.
• Table 2 System of orthopoxviruses (genus orthopoxvirus, OVP) Table 3: Differences between conventional attenuation and high attenuation Table 4: Passenger numbers of a conventional attenuation compared to a high attenuation.
• Table 5 Application scheme for treatment with paramunity inducers
• Table 6 Indications for paramunization with the highly attenuated myxomavirus h-M 2.
• myxoma paramunity inducers h-PIND-Myxo
• the VERO virus harvests of the highly attenuated leporipox virus myxomatosis hM 2 have a titer of at least 10 > 75 KID 50 / ml.
• the highly attenuated leporipox viruses thus obtained showed no virulent or immunizing properties and were further processed as paramunity inducers as follows:
• the virus harvests were inactivated with beta-propiolactone 0.05% (pH 7.8, 1 h at + 4 ° C (stirred), stirred for 4 hours at a temperature of + 37 ° C at a pH of 7.8 (pH - Check the value and if necessary adjust to pH 7.8), incubate overnight (about 12 hours stationary at a temperature of + 4 ° C) and then purified by coarse-speed centrifugation (15 min, about 4000g) virus material polygeline (pH 7.8) for a total gelatin concentration was added by 2.5%.
• the thus prepared viral material was dissolved in sterile vials ä ml bottled 1.5 and lyophilized.
• the lyophilized compositions were stored at a temperature of + 4 0 C Before use, the lyophilizates were dissolved with ImI sterile distilled water per inj and administered deeply intramuscularly.
• the myxoma paramunity inducer is useful for, for example, the adjunctive treatment of Herpes zoster (mode 4) by means of paramunization. Hiebei leads the treatment to cure the disease-typical pustules after 3-4 days. In the case of pre-flu infections, a complete disappearance of the symptoms (fever, fatigue, headache and pain in the limbs) was observed when using the paramunity inducers according to the invention. In patients with wound injuries (eg after operations) h-PIND-Myxo showed an unusually rapid wound healing without secondary infections. Stomatitis and the lesions during a visit to the dentist caused the disappearance of the apthene or lesions after rubbing in of the lyophilisate after 1-2 hours.
• Attenuated canarypox virus (Avipox serinae, KPI, 535th FHE passage) was highly attenuated by another 67 passages in FHE (see Table 4).
• the 602nd FHE passage served as a highly attenuated canarypox virus in an analogous manner to Examples 1 and 2 for the production of paramunity inducers.
• the recovered viruses showed no virulent or immunizing properties due to the Hochattenu réelle.
• Attenuated chickenpox virus (Avipoxvirus gallinum, HPI, 444.FHE passage) was highly attenuated by further 98th FHE passages. From the 542nd FHE pass, the chicken pox virus HPI proved to be highly attenuated and was used in an analogous manner as in Example 1 for the preparation of paramunity inducers. The recovered viruses showed due to the Hochattenu réelle no virulent or immunizing properties.
• Example 5 Analogous to the methods described in the preceding examples, paramunity inducers based on myxomavirus (OPV muris) and parapoxviruses were also prepared.
• Example 6 Highly attenuated animal pox strains are used to produce vector vaccines.
• the pH should be about 7.8.
• the attenuation and high attenuation of the viruses used for vector production are carried out as described in Example 1.
• all common genetic engineering methods for the insertion of nucleic acid segments encoding specific antigens against which a specific vaccination reaction, i. Immunity formation is to be achieved.
• the foreign gene is inserted by default by means of suitable restriction enzymes into the deleted nucleic acid regions of the animal pox strains according to the invention produced by the high attenuation. Standard restriction digestion and cloning techniques are used.
• any (selection) marker genes or selection cassettes may be used, such as the ⁇ -galactosidase gene, which are under the control of appropriate control sequences.
• Poxvirus genomes a phylogenetic analysis. J. Gen.. Virol. 85, 105-117.

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