MXPA06008005A - Monoparaimmunity inducers based on attenuated rabbit myxoma viruses - Google Patents

Abstract

The invention relates to monoparaimmunity inducers that are based on paraimmunizing viruses or viral components of a myxoma virus strain of diseased rabbits that show the general symptoms of the disease. The invention also relates to a method for producing the inventive inducers and to their use as drugs for the regulatory optimization of the paraimmunizing activities for use in the prophylaxis and therapy of various human and animal dysfunctions.

Description

INOPERS OF MONOPARAINMUNITY BASED ON MIXED RABBIT MIXOMA VIRUS DESCRIPTION OF THE INVENTION The invention relates to inducers of monoparaimmunity based on viruses or paraimmunizing viral components, characterized in that viruses or viral components are derived from an attenuated strain of myxoma virus. rabbit, to a method to produce the inducers of monoparamunity and to the use of these as drugs. The endogenous immune system of highly developed organisms, especially that of mammals and birds, includes a specific part of antigen and a non-specific part of antigen. Both parts of the immune system are linked and also interact with each other. Antigen-specific mechanisms are responsible for forming immunity, and antigen-specific ones are responsible for forming paramunity. Paraimmunity refers to the condition of a well-regulated, non-specific, well-functioning defensive system, linked to increased protection that develops rapidly over a limited time against a large number of different pathogens, antigens and other harmful agents. The basis for the development of paramunity are, for historical and functional reasons, the mechanisms of nonselective and conditionally selective paraspecific defense, which from the phylogenetic point of view are old and considered primitive. The paraspecific activities of the non-specific antigen immune system (also: "innate immune system") include non-selective protective elements such as, for example, organelles that consume foreign material, and conditionally selective protective elements such as, for example, micro and macrophages, cells natural destroyers, dendritic cells and soluble factors such as cytokines, which show non-specific reactions to pathogens or non-specific to antigens, according to their origin. Para-specific activities can be observed in the relevant organism immediately after contact with the antigen, while the effects of the antigen-specific immune system only occur after days or weeks. In higher organizational life forms, additional time is gained in order to create specific defense systems against harmful agents that have not yet been eliminated and that have antigenic properties. The benefits of para-immunization, that is, of the paraspecific activities of the immune system for Prophylaxis and therapy in a patient have become increasingly evident since their development (Antón Ayr, "Paramunisierung: Empirie oder issenschaft", Biol. Med. edition 26 (6): 256-261, 1997). Paraphysical defenses allow the organism to defend itself immediately when confronted with a wide variety of foreign materials, infectious pathogens, toxins and transformed endogenous cells. There are close interactions between the specific and specific activities of the immune system, being that the flow of information usually comes from the paraspecific part, which reacts initially, to the specific part of later functioning of the immune system (for example, with antigen mediation). In the event of infections with particularly virulent pathogens, the paraspecific defenses of the organism in this way are able to cover the time until the specific immunity develops (eg, antibodies, immune cells). Immune-specific defenses are a physiological process and can be defined as "primary control" in the confrontation with the environment. Not only are they indispensable for lower organisms, but also in particular for more highly developed and highly developed vertebrates. The congenital defects Primary in this biological defense system lead to situations that endanger life. An example that can be mentioned is the "Chediak-Steinbrinck-Higashi syndrome" in humans, which is characterized by granulocyte deficiencies and dysfunction of natural killer cells (NK cells), and in most cases causes the death of the patient at the end of the tenth year of life. The para-immunity condition is characterized by an increased phagocytosis regime, an increased function of cell-mediated spontaneous cytotoxicity (NK cells), and increased activity of other lymphoreticular cells. At the same time there is the release of particular cytokines that have stimulant and / or inhibitory effects (for example, by means of repressive mechanisms) both with cellular elements and between them. In addition, this biological system of closely linked paraimmunity that responds in stages with its various acceptor, effector and target cells and signaling cytokines is completely connected to the hormonal and nervous systems. In this way it is an important constituent of the communication, interaction and regulation network. Paraimmunity is induced by paramunization. This implies the pharmacological activation of cellular elements of the paraspecific part of the immune system and, associated with it, the production of cytokines in order to eliminate dysfunctions, rapidly increasing the specific non-pathogenic and non-antigenic protection of an individual (optimal bioregulation), eliminating an immunosuppression or immunodeficiency that originated as a result of stress or other conditions (for example, in pharmacological form), repairing deficiencies and / or acting as a regulator between the immune, hormonal and nervous systems. This means that certain endogenous non-specific defense processes can be increased, supplemented or depressed, depending on the type of para-immunization and the capacity to respond, such as, for example, the patient's state of health. Paraimmunity inducers are used for para-immunization and must meet certain safety and efficacy criteria, which is why they differ from immunostimulants. The paramunity inducer by itself is not comparable to an antibody or a chemical, an antibiotic, a vitamin or a hormone. On the contrary, by means of a mechanism by stages it activates the paraspecific immune system, so that the latter mobilizes sufficiently cellular and humoral defense mechanisms. The paramunity inducer has in this case both regulatory and reparative effects on the immune defenses. With respect to the mode of action of the parainmunity inducers, it is known that they are absorbed by the phagocytic cells (acceptor cells) which in this way activate and release mediators that in turn mobilize the effector cells. The latter finally ignite the regulatory mechanisms of the paraspecific defenses. Multiple paramunity inducers based on combinations in two or more poxvirus viral components derived from different poxvirus strains with paraimmunizing properties are described in European Patent EP 0 669 133 Bl. The present invention is based on the paramuizing properties of attenuated myxoma virus and / or its viral constituents. Attenuation refers to the modification of the properties of an infectious pathogen that leads to the weakening of the pathogen ("attenuate" = weaken, mitigate). Alterations in infectious pathogens often occur spontaneously in nature, and it is possible that the periods of time extend for several centuries. The ability of infectious pathogens to change in order to adapt to environmental changes is it can be used experimentally, and the period of time necessary for attenuation can be shortened drastically, for example, through extended passes in certain host systems. Attenuation has been used to date to obtain innocuous parasite-induction strains and antiviral inoculation. Attenuation usually leads to loss of virulence and infection, reduction of immunizing properties and host range, and small modifications in the pathogen genome with the occurrence of deletions, preferably in the terminal regions. There is usually a parallel increase in the para-immunizing activities of the modified pathogen. In rare cases, an attenuation can also lead to an increase in virulence and infection, especially if an attempt is made to obtain an experimental attenuation through genetic manipulations. Myxoma viruses are the pathogens of myxomatosis, a contagious systemic viral disease of wild and domesticated rabbits that progresses in cycles and is characterized by generalized subcutaneous edema, in some cases hemorrhagic, in the head and especially in the body, preferably in the region Anal, vulva and tuba, very different from any other infectious disease. The introduction of myxomatosis in a country previously free of the disease results in a rapid and fatal progression. After the virus becomes endemic, the character of the disease changes until the infections are not clinically apparent (Mayr A.: Medizinische Mikrobiologie, Infections- und Seuchenlehre, 7. ed., Enke Publishing, Stuttgart, 2002). The disease is widespread among the cottontail rabbits of the Sylvilagus species that populate the new world exclusively. These wild rabbits are the only natural reserve of the disease. The infection takes a mild form on them. In contrast, the disease causes a mortality of almost 100% in European wild and domestic rabbits of the species Oryctolagus, which are also naturalized in Australia, when the pathogen is introduced. The natural host range of the myxoma virus (Leporipoxvirus species) has narrow limits. In general, the virus only replicates in American cottontail rabbits and domestic and wild European rabbits. However, a few infections in European wild hares have also been observed. Attempts to transmit to other species and humans had negative results. The present invention is based on the object of provide new inducers of monoparamunity for human medicine and veterinary medicine. Another object of the present invention is to provide a method for producing these monoparamunity inducers. It is a further object of the present invention to provide pharmaceutical compositions for use as medicaments based on monoparamunity inducers. Therefore, the present invention relates to inducers of monoparaimmunity based on viruses or paraimmunizing viral components, characterized in that viruses or viral components are derived from an attenuated strain of rabbit myxoma virus. Viral components preferably include viral envelopes or aberrant forms of paraimmunizing viral envelopes of an attenuated strain of myxoma virus. Preferred strains having the paraimmunizing properties of the invention are strains M-2, M-7, Lausanne, Aust / Uriarra / Verg-86/1. Strains M-7, Lausanne, Aust / Uriarra / Verg-86/1 are also suitable for producing live vaccines by virtue of being only subject to partial attenuation of their virulence in order to have an adequate immunizing effect. Particularly preferred is a monoparamunity inducer based on the myxoma virus strain M-2. An attenuated strain of myxoma virus produced by the method of the invention described below is deposited in the Public Health Laboratory Service (PHLS = Public Health Laboratory Service), Center for Applied Microbiology & Research (Center for Applied Microbiology and Research), European Collection of Animáis Cell Cultures (ECACC = European Collection of Animal Cell Cultures), Salisbury, Wiltshire, United Kingdom, with deposit number 03121801. The invention also relates to a method for produce monoparamunity inducers based on an attenuated strain of rabbit myxoma virus. For this purpose the myxoma viruses are initially isolated from the infected tissue of a rabbit that typically suffers from generalized myxomatosis. Subsequently, the virus adapts to a permissive cellular system, that is to say, a cellular material that allows the replication of the virus, such as, for example, cell cultures, incubated chicken eggs or, in any case, experimental animals. In particular it is possible to use cells of the natural host or of a species closely related to the host for adaptation. Examples of permissive cellular systems suitable for myxoma virus are chicken embryo fibroblasts (CEF) as well as cell cultures produced from kidneys or rabbit testis. According to the invention, it is preferred adapting the isolated myxoma virus to the chorioallantoic membrane (CAM) of chicken eggs incubated through one or several passes, preferably through two to six passes, and particularly preferably through three passes. For this adaptation, the isolated viruses are inoculated in the CAM and replicated through passages through the CAM. Myxoma viruses are preferably initially isolated from infected tissue by replication in a permissive cellular system. For this purpose it is possible to inoculate a permissive cellular system, for example, with homogenate of infected tissue that is obtained by disruption. Subsequently, the viruses obtained by the initial replication either are additionally adapted to the same permissive cellular system that was already used for the isolation, or another additional permissive cellular system is used. The adaptation of the virus to the same type of permissive cellular system that is also used for isolation is preferred. Therefore, preferably the myxoma viruses of infected tissue are isolated by replication in a permissive cellular system and subsequently adapted to the permissive cellular system through further passes. Particularly preferred is the culture and initial isolation of the myxoma viruses by replication in the CAM of incubated chicken eggs and the subsequent adaptation of the virus in the CAM through additional passes, preferably through 2 additional passes. However alternatively it is also possible to use the allantoic fluid from chicken eggs incubated for culture and / or adaptation. Then the current attenuation takes place through prolonged passes through one or several permissive cell cultures until an attenuation or the desired degree of attenuation of the virus is reached. For this purpose it is possible to first test several permissive cellular systems for virus replication, and subsequently select for the additional passes one or more cellular systems in which the highest infection titers are reached. For the attenuation through prolonged passages both primary and secondary cell cultures as well as permanent and continuous cell lines are suitable. Therefore, attenuation can be effected by replication in primary or secondary cultures of chicken embryo fibroblast (CEF) or in permanent CEF cell cultures. For the attenuation of myxoma virus it is preferred according to the invention to pass or replicate the virus in a permanent cell culture, in particular in a "vero" cell culture, preferably through .80 to 150 passed, and particularly preferably through 120 passes. Alternatively or additionally, according to the invention the virus is passed through a binary permanent cell line, in which case AVIVER cells are preferably used. These cells or cell cultures were obtained by cell fusion between chicken embryo fibroblasts (CEF) and monkey kidney vero cells. For the attenuation of myxoma viruses of the invention, the isolated and adapted viruses are preferably passed in a first step by vero cell cultures, then the viruses are transferred to a binary AVIVER cell culture and replicated in this preferably through 10 to 50 passes, in particular through 20 to 30 passes, and particularly preferably through 25 passes. Then the attenuated myxoma virus can be replicated further through additional attenuation passes. Additional replication of the viruses is preferably carried out through additional passes in monkey kidney vero cells, in particular through 100 to 200 passages. An additional step of the method that is particularly preferred is the further deactivation of the attenuated myxoma virus. The deactivation can be carried out by chemical treatment, radiation, thermal action or pH, in particular by chemical treatment with beta- propiolactone. The treatment of myxoma viruses attenuated with beta-propiolactone further increases the paraspecific activities, while the immunizing properties which, if appropriate, are still present after attenuation are lost. A particularly preferred embodiment of the method of the invention includes the following steps: isolation of the myxoma virus from infected tissue of a rabbit that typically suffers from generalized myxomatosis by chorioallantic membrane (CAM) replication of incubated chicken eggs and subsequent adaptation of the virus to the CAM through another 2 passes; - attenuation of viruses isolated through passages in vero cell cultures, preferably through 120 passages; - transfer of viruses to a binary AVIVER cell culture, where AVIVER cells were obtained by cell fusion between chicken embryo fibroblasts (CEF) and monkey kidney vero cells, and attenuation of the virus in this cell culture through 10 to 50, preferably 25 passes, - subsequent return transfer of the virus on monkey kidney vero cells, and replication of the viruses through additional attenuation passes in the vero cells, preferably through approximately 150 past and optionally deactivating attenuated myxoma viruses by treatment with beta-propiolactone, whereby this treatment increases the paraspecific activities, while the immunizing properties still present after attenuation are lost. Attenuation through prolonged passes is usually concluded by 3 to 5 final dilutions of plaque. After several clones have been obtained and tested, the clones selected for additional replication are those with which the highest infectious titers are obtained and with which a high paramunizing activity is detected - for example, in the VSV challenge test in the baby mouse . This procedure is intended in particular to ensure that genetically uniform virus material is provided for the subsequent use. The term "attenuation" as used in connection with this invention refers to the experimental modification of the originally virulent myxoma virus to the modified form with a simultaneous increase in paramunizing properties. Attenuation can be detected by one or more of the following properties: - reduction or weakening or loss of virulence for European domestic and wild rabbits (genus Oryctolagus caniculus csp.), weakening or loss of infection, restriction of host range in cell cultures, alteration of immunizing properties, - acquisition of protective paramunizing activities in the short term. An attenuation may cause deletions in the terminal region of the myxoma virus genome. It is frequently observed that an increase in the degree of attenuation is associated with an increase in the number of deletions in the viral genome. The degree of attenuation can be verified and monitored during the passages by appropriate tests of suitable activity as are known in the art (compare for example, US 6,805,870, column 12, and the additional references cited therein) and by cloning. The present invention further relates to attenuated myxoma viruses which are obtained by the "method of the invention, to pharmaceutical compositions that include the attenuated myxoma virus or the inducer of myxoma virus monoparamunity of the invention, and to the use of the monoparamemunity inducers of myxoma virus to activate the paraspecific immune system in a mammal, and to the use of the virus attenuated to produce a corresponding medicine. Due to the surprising paramunizing properties, the myxoma virus monoparamunity inducers of the invention are suitable for the treatment and / or prophylaxis of immune system dysfunctions, immunosuppression, immunodeficiency disorders, homeostasis dysfunction between systems hormonal, circulatory, metabolic and nervous, of threat of neonatal infection, of neoplastic diseases, of viral diseases, of microbial diseases, of diseases of infectious factor resistant to therapy, of mixed viral and microbial infections, of chronic manifestations of infectious processes , liver disorders of various etiologies, chronic skin disorders, herpes diseases, chronic hepatitis, influenza infections, endotoxin damage. The paramunity inducers of the invention are generally harmless to the environment and effective in the sense of paramunization for mammals such as, for example, humans, horses, dogs, cats, pigs, for birds and also for reptiles such as, for example, lizards, snakes, Chelonians. Therefore they are particularly suitable for use in human and veterinary medicine.

Additionally, the monoparamunity inducers of the invention show a very good paramunizing activity with high potency. They can be produced in a suitable manner by the method of the invention and are safe for use in the medical sector. The attenuation of the myxoma viruses reduces the immunizing properties of the myxoma viruses while the paraspecific activities are increased. Thus, the monoparamunity inducers of the invention have paramunizing rather than immunizing properties, thereby allowing multiple and continuous use. These paramunizing properties of the rabbit myxoma virus and its viral paraimmunizing components were surprising and unpredictable. The term "para-immunization" as used in connection with this invention refers to the pharmacological activation of the cellular elements of the paraspecific immune system and the production or release of cytokines associated therewith for the purpose of eliminating dysfunctions, rapidly increasing non-specific protection of pathogen and non-specific antigen of an individual and that have a regulatory effect between the immune, hormonal, nervous and vascular systems. Parainmunization leads to the protected state of paramunity. The term "paraimmunity" as used in Connection with this invention refers to the actively acquired condition of a functional and optimally regulated paraspecific defense system which is associated with a limited time protection that develops rapidly against a large number of pathogens, antigens and other harmful agents. The rate of phagocytosis, the function of NK cells (natural killer cells) and the activity of other lymphoreticular cells (e.g., dendritic cells) rise to the physiological optimum. The term "paramunity inducer" as used in connection with this invention refers to a pyrogen-free, non-toxic drug intended to be used in humans and animals to generate and regulate defense mechanisms and endogenous protectors in the sense of parainmunization. The term "myxoma virus paramunity inducer" as used in connection with this invention refers to a medicament based on attenuated rabbit myxoma virus or an attenuated strain of myxoma virus. including the viral components paraimmunizantés and its constituents that produce the condition of paraimmunity in an organism, preferably in a mammal (for example, human). The term "myxoma virus" as used in Connection with this invention refers to the myxomatosis virus species of the leporipoxvirus genus. The myxoma virus belongs to the Chordopoxviridiae subfamily and to the family of Poxviridae (varicella virus). The term "viral paramunizing components" as used in connection with this invention includes a large number of viral structures that are derived from a myxoma virus having paramunizing properties, for example, viable or inactivated myxoma virus finishes being isolated, viable recombinant myxoma virus or inactivated derivatives of myxoma viruses that have just been isolated, viral envelopes, the envelopes removed and the cleavage products and aberrant forms of these envelopes, polypeptides, native or recombinant proteins, in particular membrane and surface receptors that occur in myxoma viruses that have just been isolated or which are expressed recombinantly by a genetically modified myxoma virus or a part of their genetic information. Tables 1 to 4 summarize the clinical results with the myxoma virus monoparaimmunity inducer PIND-MYXO in humans. Table 1 shows the clinical results on the prophylactic use of PINPAR-MYXO myxoma virus monoparamunism inducer in humans. Table 2 shows the clinical results on the therapeutic use of the PINPAR-MYXO myxoma virus monoparamunity inducer in humans. Table 3 shows the effect of paraimmunization with the inducer of myxoma virus monoparaimmunity PIND-MYXO in patients with low immunity parameters (7 days after the administration of PIND-MYXO). Table 4 shows the effect of paraimmunization with the inducer of myxoma virus monoparaimmunity PIND-MYXO in patients with high immunity parameters (7 days after the administration of PIND-MYXO). The invention is based on the surprising discovery that attenuated rabbit myxoma viruses or their para-immunizing constituents are capable of inducing very good paramunizing properties in a recipient organism, which lead to the protected state of paramunity. The basis of this invention was the first successful attenuation of rabbit myxoma virus in cell cultures. The paramunity inducers of the invention are preferably based on myxoma virus or its lyophilized, attenuated and inactivated paraimmune viral components. The infected myxoma viruses or their viral components of the invention are preferably derived from a myxoma virus strain or from a plurality of different attenuated myxoma virus strains. In this context, you prefer that the monoparamunity inducers of the invention include combinations of one or more strains of myxoma viruses or their viral paramunizing components. The paramunizing properties induced in a mammal like, for example, in humans by administration of myxoma virus monoparamunity inducer are particularly beneficial to eliminate dysfunctions, to increase the non-specific protection of an individual's antigen, to eliminate an immunosuppression or immunodeficiency that originated as a result of stress or of other causes (for example, pharmacological) and in order to have regulatory effects between the immune, hormonal and vascular systems. The invention is further based on the successful attenuation of a strain of myxoma virus through passages through cell cultures, whereby the virulent and / or immunizing properties of the myxoma virus strain are reduced or lost. Further deactivation of the myxoma viruses can be carried out further by radiation, thermal action or pH, or, particularly preferably by chemical treatment with beta-propiolactone. The paramunity inducers are based on lyophilized attenuated myxoma viruses, the invention also comprising the individual viral components of a myxoma virus which are suitable for inducing activities parainmunizantes in an organism. It is then desired to describe an embodiment of the production method of the invention for monoparaimmunity inducers based on a strain of rabbit myxoma virus isolated and attenuated by passage through cell cultures. In addition, the production method is not restricted to this preferred strain, but can be applied in the same manner to other strains of rabbit myxoma virus. The present invention also encompasses the recombinant forms of a myxoma virus strain that were produced by genetic modification. In this context preference is given to recombinant myxoma virus strains in which one or more segments of the genome encoding cytokine receptors was modified by a modification in the form of an addition, substitution or deletion, the receptor properties of the Cytokine receptor are lost by modification. These are preferably the gene segments encoding the interferon (IFN), interleukin (IL) and tumor necrosis factor (TFN) receptors, in particular for IFN-aR, IFNα-R, TNF-R, IL-1R , IL-2-R, IL-6-R and IL-12-R. Additionally, the numerical values presented in this document that refer to the incubation time or the number of passes through cell cultures are not They intend to be considered restrictive. This invention also comprises the slight modifications of these parameters and the modifications evident for the expert in the field and which also lead to the preparation of attenuated myxoma virus. A preferred embodiment of the present invention relates to the successful attenuation of the myxoma virus strain M-2. The M-2 strain of myxoma virus was isolated from European wild rabbits with myxomatosis (Herrlich A., Mayr A. and Munz E .: "Die Pocken", 2nd edition, Georg Thieme Editorial, Stuttgart, 1967). The altered skin cells obtained from the subcutaneous tissue of the diseased rabbit are inoculated after disruption in a chorioallantic (CAM) membrane of chicken eggs that were preferably incubated for 10-12 days. Myxoma viruses replicate further and adapt through 2 to 6 passages, preferably through 3 passages through the chorioallantoic membrane (CAM passes, for the method see Herrlich A., Mayr A. and Munz E .: Die Pocken ", 2nd edition, Editorial Georg Thieme, Stuttgart, 1967). The second through sixth pass, preferably the third CAM pass, serves as the initial material for the additional attenuation of the myxoma virus in cell cultures. The attenuation takes place in 3 stages after the adaptation of the viruses in the chorioallantoic membrane (obvious to typical foci on the chorioallantoic membrane). In step 1 80 to 150, preferably 120 passes of continuous passes called final dilution in vero cells (vero cells, ATCC CCL-81) are performed. The virulence of the myxoma virus that underwent these passes is weakened. In a 2. step after 80 passes to 150 passes, preferably after 120 passes in vero cells, the viral suspension is transferred to the so-called AVIVER cells and is continued through 10 to 50 passes, preferably through 20 to 30, in particular through 25 passes. AVIVER cells are obtained by cell fusion between chicken embryo fibroblasts (CEF) and vero cells and are called continuous binary cell culture. The last passage through AVIVER cells, preferably passage 25, is transferred back to vero cells and continued in the third attenuation step through 100 to 200 additional passes, preferably through approximately 157 passes in vero cells. In this way the myxoma virus replicates through a total of more than 300 passages through cell cultures. After the third stage of replication of the myxoma virus in cell cultures, the virus-myxoma is sufficiently attenuated. The cultures of vero cells and AVIVER cells preferably they are grown using a fully synthetic medium, particularly preferably the MEM medium (minimal essential medium) plus 5 to 20%, preferably 10% BMS (serum substitute medium) and 5 to 20, preferably 10% lactalbumin hydrolyzate. . The virus medium used after exchange with the culture medium is preferably MEM medium with 5 to 20%, preferably with 10% lactalbumin hydrolyzate without BMS or without calf fetus serum and without antibiotics. All production methods are preferably carried out at pH values of 7.0 to 8.0, preferably at a pH of 7.25. As the starting material for producing the PIND-MYXO monoparamunity inducer of the invention, preferably virus harvests with a titer of 105-0 to 107-5, preferably at least 106".5 TCID50 / ml (TCID50 = 50% Infectious dose of unwound culture.) The replication of the myxoma virus in vero cells leads to a typical cytopathic effect (cpE) that is finally characterized by a destruction of the infected cells (lysis) .The inoculation of approximately 10 MOI (multiplicity of infection) It results, after a short finishing phase (1-2 days), in cellular structures reticulated for approximately 3 days and in lysis of the cells after approximately 5 days. The past 301 in vero cells had an infectious titer. approximately 106"5 TCID50 / ml The attenuated myxoma virus is deactivated by chemical treatment with beta-propiolactone at a concentration of 0.01-1% beta-propiolactone, preferably at a 0.05% concentration of beta-propiolactone. effect a complete loss of the immunizing properties that remain present where appropriate, while the paraspecific activities are not only retained but actually increase significantly For the further processing of attenuated and deactivated myxoma viruses. inducer of myxoma virus monoparamunity (P1ND-MYX0), in viral starting material used for virus inactivation should have a viral titer of approximately 105'0 to 107-0, preferably at least 106-5, TCID5o / ml. The purification is preferably carried out by spinning at low revolutions (eg 1000 rpm), after centrifuging, 0.5-10% gela is added. succinylated tub (for example, polygeline, for example, from Co. Hausmann, St. Gallen / Switzerland), preferably 5% succinylated gelatin. The resulting mixture can be subsequently lyophilized in 1.5 ml portions in appropriate sterile glass tubes or ampoules and, if required, dissolved in distilled water. A volume of 0.5-2 ml, preferably 1.0 ml of the lyophilisate dissolved in distilled water corresponds to a dose of inoculation for humans in intramuscular administration (see also Mayr A. and Mayr B.: "Von der Empirie zur Wissenschaft", Tierárztl. Umschau, edition 56: 583 -587, 2002). The attenuation can be detected clinically due to the loss of virulence of European domestic and wild rabbits (genus Oryctolagus caniculus csp.), By a loss of the contagious, by a virtually complete restriction of the range of hosts in cell cultures and by alteration of immunizing properties. The lyophilized product can be stored at temperatures of, preferably, around + 4 ° C at lower temperatures, preferably around -60 ° C with stability for unlimited time. It was demonstrated by genetic technology investigations of attenuated myxoma viruses that multiple deletions had occurred in the myxoma virus genome. In the case of the initial M-2 strain, the myxoma virus genome consists of a single linear deoxyribonucleic acid (DNA) with a total length of approximately 160 kilobases (kb), which codes for several hundred proteins (Herrlich A., Mayr A. and Munz E .: "Die Pocken", 2nd edition, Editorial Georg Thieme, Stuttgart, 1967). The sequences of the terminally inverted inverted repeats (TIR = inverted terminal repeats) are located at approximately 11 kb of the genome segment (McFadden, G. and Graham K.: "Modulation of cytokine networks by pox virus", Virology, edition 5: 421-429, 1994). Therefore it was found that the attenuation of myxoma viruses along continuous passes of vero cells led to a loss of the gene segments encoding the interferon α and α receptors. (IFN to IFN?), For the tumor necrosis factor (TNF) and for the interleukins (IL) 1, 2, 6 and 12. It is interesting that these cytokines belong to the paraspecific defense factors of the non-specific immune system. The cytokines are neutralized by binding to the corresponding viral receptors, so that the virus is able to replicate without hindrance. The deletions of the gene segments encoding the cytokine receptors mentioned above relate mainly to the terminal regions of the DNA. However, it was possible to additionally detect deletions that occurred during the passage of AVIVER cells in the conserved part of the DNA. These deletions are related to two genes that code for an immune epitope and virulent gene. These genetic modifications are presumably one of the reasons for the reduction in immunizing activities, ie, antigen-specific, and the simultaneous increase of the paraspecific activities of attenuated myxoma virus. Immunizing epitopes and paraspecific and non-specific epitopes are in competition. Therefore, a reduction in the aforementioned peptides or proteins leads to an increase in the effect of the paraspecific activities. Residues of immunizing and magnifying virulence proteins are eliminated in the preparation of the monoparaimmunity inducers by the method described above to deactivate attenuated myxoma viruses by the method described above to deactivate attenuated myxoma viruses. The monoparamunity inducer of the invention, also called PIND-MYXO, is based on the use of attenuated myxoma virus or its constituent paraimmunizing parts, and is suitable on the basis of its paramunizing properties for the following prophylactic or therapeutic indications in a patient: diseases of infectious factor and mixed infections, chronic manifestations of infectious processes, recurrent refractory infections and viral and bacteriological infections resistant to chemotherapy - weakened defenses and deregulations in the defensive system of an organism - neonatal infection threatening - auxiliary therapy for certain '' neoplc diseases, for example, prevention of metsis, reduction of side effects of virus to chyme and radioterampia - regulation of homeostasis between the hormonal, circulatory, metabolic and nervous. The paramunity inducers of the invention can be administered parenterally or locally to mammals, including humans, birds and reptiles. Local administration of para-immunity inducers specifically stimulates paraspecific defense mechanisms on mucous membranes and on the skin. However, there is also a certain systemic effect. In contr para-immunizations applied parenterally have little influence on local defense mechanisms in the skin and mucous membrane, preferably having a systemic effect. No side effects are present even applying continuously numerous parenteral administrations in humans and animals. The indications for the use of PIND-MYXO are the same for animals and for humans. At the same time, in problem operations, specifically in the administration of horses, pigs, dogs and cats, parainmunization of neonates is recommended immediately on the day of birth and preferably in the first and possibly also the second day after birth. The individual dose is approximately 0.5 to 5 ml of the dissolved lyophilisate, in horses and pigs the individual dose is preferably 2 ml and in dogs and cats preferably 0.5 ml with parenteral administration. In accordance with the invention it is advisable to administer PIND-MYXO parenterally one day before and / or at the same time as specific protective inoculations in order to avoid side reactions and assist immunization when administering vaccines. One embodiment of the invention relates to the production of a pharmaceutical composition for local administration to induce paramunity in the skin and mucous membranes. The pharmaceutical composition preferably relates to a bucal or sucking tablet based on constitutive components of an attenuated and deactivated myxoma virus cell culture. The buccal tablets of the invention are preferably produced with the addition of sorbitol, polyethylene glycol 6.00, potassium bisphosphate, essence for tyrospirol tablets, kollidon 25 and magnesium stearate. However, PIND-MYXO can also be administer nasally, rectally or vaginally with suitable vehicles. The following examples are preferred embodiments of the invention and serve to further explain the object of the invention. EXAMPLE 1 The myxoma virus of the edematous subcutis of a European wild rabbit (genus Oryctolagus) suffering from myxomatosis in a typical manner was isolated as starting material to produce the PINPAR-MYXO monoparamunity inducer of the invention by culture in the chorioallantoic membrane. (CAM) of chicken eggs (Valo eggs) incubated for 10 days, and adapted three times by the method of Herrlich et al. with passages through the CAM (Herrlich A., Mayr A. and Munz E .: "Die Pocken", 2nd edition, Editorial Georg Thieme, Stuttgart, 1967). The third passage through CAM was adapted in a first stage in vero cells through 120 passages (ATCC CCL-81, WHO, American Type Culture Collection), it was replicated in a second stage through 24 intermediate passages in AVIVER cell cultures , and was further cultured in a third phase in vero cells. In total, about 300 passes were carried out with the aim of attenuation. After these continuous final dilution runs, the originally virulent myxoma virus will Dimmed. The attenuated myxoma virus replicates in vero cells. The vero cell cultures are grown. using a completely synthetic medium consisting of MEM (minimal essential medium) plus 10% BMS (serum substitute medium) and 10% lactalbumin hydrolyzate. The virus medium used after exchange with the culture medium is only MEN with 10% lactalbumin hydrolyzate without BMS or without fetal calf serum and without antibiotics. All production methods were carried out at pH values above 7.25. Viral crops with titres greater than 106.5 TCID50 / ml serve as starting material for producing the PINP-MYXO monoparamunity inducer of the invention. The deactivation of the viral crops with 0.05% beta-propiolactoma and centrifugation at low speed are followed by the addition of 5% succinylated gelatin (polygeline) to the viral material prior to lyophilization. The lyophilized product is stable at ambient temperatures and at temperatures of about 4 ° C to -80 ° C, and can be maintained without time restriction preferably at about 4 ° C or otherwise at about -60 ° C. A volume of 1 ml of the lyophilisate dissolved in sterile distilled water corresponds to the inoculation dose. An administration takes place deep or local intramuscular (see examples 3, 4 and 5). EXAMPLE 2 The PIND-MYXO inducer of the invention is administered in a manner analogous to the description of Example 1 in anhydrous form (freeze-dried undissolved) locally on the mucous membranes of the upper respiratory tract, preferably in nasal form, three times a day for prophylaxis or therapy (1 ml per application) of ultifactorial infections (eg, influenza infections). EXAMPLE 3 The PIND-MYXO inducer in liquid form produced as in Example 1 is analogously coated in the cutaneous form to improve perfusion of the skin, to accelerate wound healing and to treat varicose veins and chronic venous insufficiency (ulcer of leg) in humans. For this purpose, the lyophilisate can be absorbed, for example, in a greasy cream (for example Bepanthen, linoleic fat), in which case the pH should be slightly alkaline. This preparation must be prepared fresh for each use. The administration is carried out several times a day by manually spraying on undamaged skin. Open wounds can be treated by drip application of the finished product to be dissolved over the wound regions. The treatment must be done daily until heal. EXAMPLE 4 The PINP-MYXO monoparamunity inducer produced as in Example 1 is administered analogously in a parenteral manner to prevent side reactions and to improve the inoculation result one day before and simultaneously with a protective inoculation with conventional specific vaccines. Example 5 The PINP-MYXO monoparamunity inducer produced as in Example 1 is processed in an analogous manner to obtain buccal or oral tablets. suck. The production and use of suction tablets for local parainmunization of mucous membranes of the ear, the nose, throat and mouth are novel and are constitutive of the invention. Via the activated mucosal membranes of the mouth, there is not only an effect back to the starting point (migration of the defense cells to the mucous membranes of other organ systems), but also a partial parenteral paraimmunization. The following production method was accredited as suitable for the production of buccal and sucking tablets: Freeze-dried from PIND-MYXO 65 mg Urea 50 mg Sorbitol 267 mg Polyethylene glycol 6000 118 mg Magnesium stearate 0.5 mg Tablet weight 500.5 mg The patient should take 4-6 tablets at regular intervals daily for optimal para-immunization. The following formula of a pharmaceutical composition was accredited as suitable for producing buccal tablets: Lyophilized from PIND-MYXO 155 mg Sorbitol 360 mg Polyethylene glycol 6000 300 mg Potassium dihydrophosphate (KH2P04), anhydrous 2 mg Disodium hydrophosphate (Na2HP04), anhydrous 8 mg Tablets of essence of tyrospirol 0. 8 mg Magnesium stearate 20 mg Tablet weight 805.8 mg Tablets dissolve slowly in the patient's mouth and can be swallowed after dissolving. The clinical test results with the monoparamunity inducer of the invention based on the lyophilisate of the myxoma virus strain M-2, which are compiled in Tables 1 to 4 demonstrate the very good para-immunizing activities of lyophilized myxoma viruses in Humans. These data apply equally to other mammals as well as to birds and reptiles.

Table 1 Clinical results with an inducer of monoparamunity of cell cultures of attenuated myxoma virus in humans - prophylactic uses - (lyophilized 1 OP Inductor (1 ml) intramuscular) Table 1 Table 2 Clinical results with an inducer of monoparamunity of cell cultures of attenuated myxoma virus in humans - therapeutic uses - (lyophilized Inductor 1 OP (1 ml) intramuscular) Indications Appropriate administration methods Herpes diseases 1 daily injection during (zoster, mononucleosis 3-5 days or until the infectious, herpes simplex, disappearance of the etc.) symptoms; then one injection every second or third day until full recovery Chronic hepatitis One "course" every month: 3 injections at 24-hour intervals each Infections due to influenza 1 daily administration Mixed viral infections and even the bacterial ones disappear (in combination symptoms, then 1 injection with antibiotics or every second day until chemotherapy) complete recovery Table 3 Effect of paraimmunization with myxoma inducer (PIND-MYXO) in patients with low immunity parameters (7 days after the administration of PIND-MYXO) Table 4 Effect of paraimmunization with myxoma inducer (PIND-MYXO) in patients with high immunity parameters (7 days after the administration of PIND-MYXO)

Claims (45)

1. CLAIMS 1. Attenu myxoma virus or components thereof, characterized in that it is used as an inducer of monoparamunity.
2. 2. Paramunity inducer as claimed in claim 1, characterized in that the attenu myxoma virus strain has a modification in the form of an addition, substitution or deletion in one or more segments of the gene coding for the production of cytokine receptors. , being that the receptor properties of the cytokine are lost by modification.
3. 3. Paraimmunity inducer as claimed in claim 2, characterized in that the cytokine receptors are selected from the group of receptors for interferons, interleukins and tumor necrosis factor.
4. Paramunity inducer as claimed in claim 3, characterized in that the cytokine receptors are selected from the group of receptors for IFNα-R, IFNα-R, TNF-R, IL-1R, IL-2-R, IL -6-R and IL-12-R.
5. 5. Paramunity inducer-as claimed in any of claims 1 to 4, characterized in that the viral components include viral envelopes or aberrant forms of viral envelopes of an attenu myxoma virus strain.
6. 6. Paramunity inducer as claimed in any of claims 1 to 5, characterized in that the myxoma virus strain is selected from the group consisting of M2, M
7. 7, Lausanne, Aust / Uriarra / Verg-86/1. Parammunity inducer as claimed in claim 6, characterized in that the strain of myxoma virus is strain M-2 with deposit number ECACC 03121801.
8. 8. Parammunity inducer as claimed in any of claims 1 to 7, characterized in that the myxoma virus strain was attenu in cell cultures.
9. 9. Paramunity inducer as claimed in claim 8, characterized in that the cell cultures comprise monkey kidney vero cells and / or AVIVER cells.
10. 10. Paraimmunity inducer as claimed in any of claims 1 to 9, characterized in that the attenu myxoma viruses are deactiv.
11. 11. Paraimmunity inducer as claimed in claim 10, characterized in that myxoma attenu viruses were deactiv with beta-propiolactone.
12. 12. Paraimmunity inducer as claimed in claim 11, characterized in that the concentration of beta-propiolactone is 0.01-1%.
13. 13. Paraimmunity inducer as claimed in claim 12, characterized in that the concentration of beta-propiolactone is 0.05%.
14. 14. Paraimmunity inducer as claimed in any of claims 1 to 13, characterized in that the myxoma viruses were lyophilized.
15. 15. Use of the paramunity inducer as claimed in any of claims 1 to 14, characterized in that it is for producing a pharmaceutical composition for activating the paraspecific immune system in humans and animals.
16. 16. Use of the paramunity inducer as claimed in any of claims 1 to 14, characterized in that it is for producing a pharmaceutical composition for the parenteral treatment and / or for the prophylaxis of dysfunctions of the immune system, immunosuppression, immunodeficiency disorders, dysfunctions of the homeostasis between the hormonal, circulatory, metabolic and nervous systems, danger of neonatal infection, neoplastic diseases, viral diseases, microbial diseases, diseases of infectious factor resistant to therapy, mixed viral and bacterial diseases, chronic manifestations of infectious processes, liver disorders of various etiologies, chronic skin disorders, herpes diseases, chronic hepatitis, influenza diseases, damage by endotoxins
17. 17. Use as claimed in any of claims 15 to 16, characterized in that the treatment is carried out by local administration of the pharmaceutical composition via the patient's skin or mucous membranes.
18. 18. Pharmaceutical composition including a paramunity inducer as claimed in any of claims 1 to 14, characterized in that it includes an acceptable pharmaceutical vehicle.
19. 19. Pharmaceutical composition as claimed in claim 18, characterized in that it is for local or parenteral administration.
20. 20. Pharmaceutical composition as claimed in claim 18, characterized in that the composition is in the form of buccal tablets or to be sucked.
21. 21. Method for producing a paramunity inducer based on a strain of rabbit myxoma virus attenuated, characterized in that it comprises the steps of: - isolating myxoma virus from infected tissue of a rabbit that typically suffers from generalized myxomatosis; - adapt the virus to a permissive cellular system; - passing the virus 5 isolated in a culture of vero and binary cells until the attenuation of the virus is achieved.
22. 22. Method as claimed in claim 21, characterized in that for the adaptation, isolated viruses are inoculated in the membrane 10. chorioallantic chicken eggs incubated.
23. 23. Method as claimed in claim 22, characterized in that the virus replicates through 2 to 6 passages, preferably through three passages in the chorioallantoic membrane.
24. 24. Method as claimed in claim 21, characterized in that the myxoma viruses present in the infected tissue are isolated for replication in a permissive cellular system.
25. 25. Method as claimed in claim 24, characterized in that the virus is isolated by culturing it in the chorioallantoic membrane of incubated chicken eggs.
26. 26. Method as claimed in claim 25, characterized in that subsequently the virus adapts to the chorioallantic membrane through additional passes, preferably through two additional passes.
27. 27. Method as claimed in any of claims 21 to 26, characterized in that the attenuation of the myxoma viruses is carried out through 80 to 150 passes, preferably through 120 passes in vero cell cultures.
28. 28. Method as claimed in any of claims 21 to 27, characterized in that the virus is passed through a culture of AVIVER cells.
29. 29. Method as claimed in claim 28, characterized in that the step is carried out through 10 to 50 passes, preferably through 25 passes.
30. 30. Method as claimed in any of claims 21 to 29, characterized in that attenuated myxoma viruses are further replicated through additional attenuation passes.
31. 31. Method as claimed in claim 30, characterized in that the replication is performed on monkey kidney cells.
32. 32. Method as claimed in claim 31, characterized in that the additional growth is effected in vero cells through 100 to 200 passes.
33. 33. Method as claimed in any of claims 30 to 32, characterized in that the viral cultures of the cell culture have an infectious titer of 105'0 to 107'5, preferably of at least 106'5, TCID5o / ml.
34. 34. Method as claimed in any of claims 21 to 33, characterized in that the attenuated myxoma viruses are further deactivated.
35. 35. Method as claimed in claim 34, characterized in that attenuated myxoma viruses are treated with beta-propiolactone for deactivation.
36. 36. Method as claimed in claim 35, characterized in that the concentration of beta-propiolactone is 0.01-1%.
37. 37. Method as claimed in claim 36, characterized in that the concentration of beta-propiolactone is 0.05%.
38. 38. Method as claimed in any of claims 21 to 37, characterized in that it includes the steps of isolating myxoma virus from the infected tissue of a rabbit suffering from myxomatosis by replication in the chorioallantoic membrane of incubated and subsequent chicken eggs. adaptation of the isolated myxoma virus to the chorioallantic membrane through 2 additional passages, - attenuation of viruses isolated by - passed in cultures of vero cells, preferably through 120 passages, - transfer of the viruses to the binary culture of AVIVER cells with additional attenuation of the virus, preferably through 24 intermediate passages in this cell culture, subsequent transfer of the virus back to the monkey kidney vero cells, and replication of the attenuated myxoma viruses by additional attenuation passages in the vero cells., preferably through 150 passes.
39. 39. Attenuated myxoma virus, characterized in that it is obtained by the method as claimed in any of claims 21 to 38.
40. 40. Myxoma virus as claimed in claim 39, characterized in that it is deposited in the European Collection of Cell Cultures with deposit number 03121801.
41. 41. Myxoma virus as claimed in claim 39 or 40, characterized in that the myxoma virus is genetically modified.
42. 42. Myxoma virus as claimed in claim 41, characterized in that the myxoma viruses have a modification in the form of an addition, substitution or deletion in one or several gene segments that encode for the production of cytokine receptors, whereby the receptor properties of the cytokine are lost through the modifications.
43. 43. Myxoma virus as claimed in claim 42, characterized in that the cytokine receptors are selected from the group of receptors for interferons, interleukins and tumor necrosis factors.
44. 44. Myxoma virus as claimed in claim 43, characterized in that the cytokine receptors are selected from the group of receptors for IFNα-R, IFNα-R, TNF-R, IL-1R, IL-2-R, IL- 6-R and IL-12-R.
45. 45. M2 strain of attenuated myxoma virus characterized by deposit number ECACC 03121801.