Modified variant of ankara vaccina virus

FIELD: biotechnology, virology, medicine.

SUBSTANCE: invention relates to attenuated virus derived from modified Ankara vaccina virus. Said virus are not able for reproduction by replication in human cell lines. Also disclosed are application of virus or recombinant variants thereof as drug or vaccine, as well as method for inducing of immune response in patients with defected immunity, in patients having immunity to vaccine virus, or in patient during antiviral therapy.

EFFECT: variant of Ankara vaccina virus effective in medicine and veterinary.

86 cl, 15 dwg, 1 tbl, 2 ex

 

In the present invention proposes an attenuated virus, which is derived from modified vaccinia virus Ankara and which is characterized by loss of ability to its reproduction by replication in human cell lines. It additionally describes recombinant viruses that occur from this virus, and the use of virus or recombinant variants as drugs or vaccines. Further provides a method of inducing an immune response even in patients with compromised immunity, patients with baseline existing immunity to the vaccine virus or patients during antiviral therapy.

Prior art

Modified vaccinia virus Ankara (MVA) refers to the cowpox virus, a member of the kind classified in the genus orthopoxvirus family Poxiviridae. MVA was created as a result of 516 serial passages of the virus, vaccinia virus (CVA) in embryonic fibroblasts of chicken lines Ankara (as a review see Mayr, A., et al. Infection 3, 6-14 [1975]). From these numerous passages the resulting virus MVA has lost approximately 31 thousand bases its genomic sequence and, therefore, has been described as a virus with very limited cell-owners relating to the caged birds (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). In various animal models it was shown that the obtained MA was almost avirulent (Mayr, A. & Danner, K. [1978], Dev. Biol. Stand. 41:225-34). In addition, this strain MVA has been tested in clinical trials as a vaccine for immunization against smallpox (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [19974]). In this study involving more than 120,000 people, including patients with high risk disease, and it was proved that, compared with vaccines based on vaccinia virus, MVA has attenuated virulence or infectivity, while at the same time good immunogenicity.

In subsequent decades were created genetically engineered designs MVA for use as a viral vector for the expression of recombinant genes or as a recombinant vaccine (Sutter, G. et al. [1994], Vaccine 12:1032-40).

In this respect, the most amazing is that even though Mayr et al. in 1970-ies have shown that MVA is a highly attenuated and avirulent in humans and mammals, currently presents some works (Blandchard et al., 1998, J Gen Virol 79, 1159-1167; Carroll & Moss, 1997, Virology 238, 198-211; Altenberger, U.S. patent 5185146; Ambrosini et al., 1999, J Neurosci Res 55(5), 569) have shown that in human cell lines and mammalian MVA is not completely weakened, as these cells can occur residual replication. It is assumed that the results presented in d is the R publications obtained with different strains MVA, as used viruses vary considerably in their properties, especially in terms of its growth in different cell lines. Character growth is considered as an indicator of the weakness of the virus. Typically, the strain of the virus is weakened if he lost his ability or only decreased their ability to their reproduction by replicating in the cells of the host. As mentioned above, the fact that MVA is not completely incapable of replication in human cells and mammals, raises the question of how the absolute safety of MVA as a vaccine for humans or vector for recombinant vaccines.

Especially for vaccines and recombinant vaccines balance between efficiency and security of viral vector vaccines is extremely important.

The PURPOSE of the INVENTION

Thus, the aim of the invention is the provision of new strains, characterized by increased security, to develop safer products, such as vaccines or drugs. Moreover, an additional objective is the provision of means to improve the existing regime of vaccination.

DETAILED description of the INVENTION

To achieve the above objectives, in accordance with the preferred implementation of the present invention offer the new viruses are stated cowpox, which are able to reproduce by replication in cells and in cell lines, animals, non-human, especially in embryonic fibroblasts of chicken (CEF) and in the line of kidney cells baby hamster BHK (ECACC 85011433), but not able to reproduce by replication in human cell lines.

Known strains of cowpox viruses are able to reproduce by replication in at least some human cell lines, in particular in a cell line of human keratinocytes HaCat (Boukamp et al. 1988, J Cell Biol 106(3): 761-71). Replication in a cell line HaCat allows replication in vivo, in particular replication in vivo in humans. Of course, in the examples section shows that all tested known strains of cowpox virus, which demonstrate ability to residual reproduce by replication in HaCat are also replicated in vivo. Thus, the invention preferably relates to the cowpox viruses that are not able to reproduce by replication in a cell line HaCat human. Most preferably the invention relates to strains of vaccinia virus, which is not able to reproduce by replication in any of the following cell lines human: cell lines adenocarcinoma human cervical HeLa (ATCC no CCL-2), cell line kidney of a human embryo 293 (ECACC No. 85120602), cell line osteo ramy human bone 143B (ECACC No. 91112502) and cell line HaCat.

Character growth or amplification/replication of the virus is usually expressed by the ratio of the amount of virus produced by an infected cell (output), quantity, originally used for the initial infection of cells (input) ("amplification ratio"). The relationship between output and input is equal to "1", is determined such amplication status in which the amount of virus produced by infected cells is the same as the number, the source used to infect cells. This status indicates the fact that infected cells are permissive for viral infection and reproduction of the virus. Amplication ratio which is less than 1, i.e. reducing the amplification below the entry level is an indicator of the failure of replication and, thus, an indicator of the weakness of the virus. Therefore, inventors of particular interest is the identification and, ultimately, the selection of the strain, which is characterized by the amplification ratio is less than 1 in several human cell lines, in particular in all human cell lines 143B, HeLa, 293 and HaCat.

Thus, the term "not able to reproduce by replication" means that the virus in accordance with the invention is characterized by the amplification ratio is less than 1 in cleto the data lines man, such as cell line 293 (ECACC NO. 85120602), 143B (ECACC No. 91112502), HeLa (ATCC no CCL-2) and HaCat (Boukamp et al. 1988, J Cell Biol 106(3): 761-71), under the conditions described in example 1 of the present description, some specific strains MVA. Preferably, the amplication ratio for the virus in accordance with the invention was 0.8 or less in each of the above lines HeLa, HaCat and 143B.

In example 1, and table 1 shows that the viruses in accordance with the present invention is not able to reproduce by replication in any of the cell lines 143B, HeLa and HaCat. Special strain in accordance with the present invention, which was used in the examples was put in storage in the European collection of cell cultures under number V00083008. This strain is indicated throughout the description as "MVA-BN".

Known strains MVA demonstrate ability to residual replication in at least one of the tested cell lines of human (figure 1, example 1). All known strains of the virus cowpox demonstrate ability to at least some replication in cell line HaCat, while strains MVA in accordance with the present invention, in particular MVA-BN does not show the ability to reproduce by replication in HaCat cells. In a more detailed presentation of MVA-BN are characterized by amplification ratio from 0.05 to 0.2 in cell line SMOS and human embryo 293 (ECACC No. 85120602). In cell lines of human bone osteosarcoma 143B (ECACC No. 91112502) ratio is in the range from 0.0 to 0.6. For cell lines adenocarcinoma human cervical HeLa (ATCC no CCL-2) and cell line of human keratinocytes HaCat (Boukamp et al. 1988, J Cell Biol 106(3): 761-71) amplication ratio is in the range from 0.04 to 0.8 and from 0.02 to 0.8, respectively. MVA-BN is characterized by the amplification ratio of from 0.01 to 0.06 in the kidney cells of the African green monkey (CV1: ATCC no CCL-70). Thus, MVA-BN, which is indicative of the strain in accordance with the present invention, is not able to reproduce by replication in any of the tested cell lines.

Amplication ratio of MVA-BN is equal to the value, certainly above 1 in embryonic fibroblasts of chicken (CEF: primary culture or cell line kidney cubs hamster BHK (ATCC no CRL-1632). As noted above, the ratio of more than "1"indicates a reproduction by replication, because the amount of virus produced by infected cells, is increased in comparison with the amount of virus used to infect cells. Therefore, the virus can easily multiply and amplificates in primary cultures of CEF with the attitude above 500 or BHK cells with the ratio above 50.

In a particular implementation of the present invention from Britanie relates to derivatives of the virus, deposited under the number ECACC V0083008. "Derivatives" of viruses, deposited under the number ECACC V00083008 indicate viruses expressing essentially the same replicating the characteristics of the deposited strain, but differing in one or more parts of their genome. Viruses that have the same "replication features that deposited the virus are viruses that replicate with similar amplification relations, and that the deposited strain, in CEF cells and cell lines, BHK, HeLa, HaCat and 143B, and who show a similar replication in vivo when determining on the model of transgenic mice AGR129 (see below).

In the preferred implementation of the strains of cowpox virus in accordance with the present invention, in particular MVA-BN and its derivatives, are characterized by lack of ability to replicate in vivo. In the context of the present invention "lack of ability to replicate in vivo" refers to viruses that do not replicate in human cells and mouse models described below. "The lack of ability to replicate in vivo can be determined preferably in mice, which are unable to produce Mature B - and T-cells. An example of such mice are transgenic mouse model AGR129 (obtained from Mark Sutter, Institute of Virology, University of Zurich, Switzerland). This line of mice is characterized by the directional genetic damage in the genes of the IFN receptor type I (IFN-α /β) and type II (IFN-γ) and RAG. Because of these injuries mouse does not have the IFN system and is not capable of producing Mature B and T cells and are characterized by significantly impaired immunity and high susceptibility to viral replication. Instead AGR129 mice can be used in any other line of mice, which are unable to produce Mature B - and T-cells and is characterized by significantly impaired immunity and high susceptibility to viral replication. In particular, the viruses in accordance with the present invention does not kill the AGR129 mice over a period of time, at least 45 days, more preferably for at least 60 days, and most preferably within 90 days after infection of mice by intraperitoneal administration of 107The FIGHT of the virus. Preferably, the viruses that show a lack of ability to replicate in vivo", was additionally characterized by the fact that no virus could not be detected in the organs or tissues of the AGR129 mice after 45 days, preferably 60 days, and most preferably 90 days after infection of mice by intraperitoneal administration of 107The FIGHT of the virus. Detailed information about the tests for infection AGR129 mice and tests that are used to determine whether the virus detection is taken in the organs or tissues of infected mice, can be found in the examples section.

In the preferred implementation of the strains of cowpox virus in accordance with the present invention, in particular MVA-BN and its derivatives, are characterized by a higher immunogenicity compared with the known strain MVA-575 when determining the model of mice from lethal infection. Details of the experiment described in example 2 below. Briefly, in this model, unvaccinated mice die after infection replicative components of strains of cowpox viruses, such as strain L929 TK+ Western Reserve or IHD-J. In the context of a model with a lethal infection, "infection" refers to an infection of replicative components of cowpox viruses. Four days after infection, mice usually score and determine the titer of virus in the ovaries using standard tests plaque formation using VERO cells (for a more detailed description see examples). The titer of the virus determines the unvaccinated mice and mice vaccinated with virus vaccine in accordance with the present invention. More specifically, the viruses in accordance with the present invention are characterized by the fact that in this test after vaccination 102TCID50/ml of virus in accordance with the present invention the viral titres in the ovary reduced the, at least 70%, preferably at least 80%, more preferably at least 90% compared to unvaccinated mice.

In the preferred implementation of the cowpox viruses in accordance with the present invention, in particular MVA-BN and its derivatives, suitable for immunization by primary/re-introduction of the vaccine. There are numerous reports suggesting that the modes of the primary/secondary immunization with the use of MVA as a vector for delivery to induce a weak immune responses and inferior to primary immunization with DNA and re-immunization with MVA (Schneider et al., 1998, Nat. Med. 4; 397-402). In all these studies used the MVA strains, which differ from viruses cowpox in accordance with the present invention. To explain the weak immune response when using MVA for primary and re-introduction has been hypothesized that antibodies produced against MVA during the initial introduction, neutralize MVA entered upon repeated immunization, preventing an effective increase of the immune response. In contrast, the modes with the primary DNA immunization and re-immunization with MVA, as reported, are excellent in terms of generating antibodies with high avidity, because in this mode, the ability of DNA efficiency is but premirovat immune response combined with the properties of MVA to boost the response when re-immunization in the absence of pre-existing immunity with respect to the MVA. It is clear that, if pre-existing immunity to MVA and/or the cowpox virus prevents amplification of the immune response after the second immunization, the use of MVA as a vaccine or drug should be limited, especially in patients who have been vaccinated against smallpox. However, in accordance with the additional implementation of vaccinia virus in accordance with the present invention, in particular MVA-BN and its derivatives, as well as the corresponding recombinant virus comprising a heterologous sequence, can be used for effective primary premirovannogo and subsequent amplifying immune responses in intact animals and in animals with preexisting immunity against the virus of smallpox. Thus, vaccinia virus in accordance with the present invention induces at least essentially the same level of immunity in the modes of primary immunization with vaccinia virus/re-immunization with vaccinia virus in comparison with the modes of the primary immunization with DNA/re-immunization with vaccinia virus.

Vaccinia virus is considered as inducing at least essentially the same level of immunity in the modes of primary immunization with vaccinia virus/re-immunization with vaccinia OS the s compared to the modes of the primary immunization with DNA/re-immunization with vaccinia virus, if CTL (CTL) response in the measurement in one of the following two tests ("test 1" and "test 2"), preferably in both tests is at least essentially the same when the modes of the primary immunization with vaccinia virus/re-immunization with vaccinia virus in comparison with the modes of the primary immunization with DNA/re-immunization with vaccinia virus. More preferably, the CTL response after primary immunization with vaccinia virus/re-immunization with vaccinia virus was higher in at least one of the tests compared with the modes of the primary immunization with DNA/re-immunization with vaccinia virus. Most preferably, the CTL response was higher in both of the following tests.

Test 1:for the primary introduction of cowpox virus/re-introduction of cowpox virus conduct primary immunization 6-8 week BALB/c mice (H-2d) by intravenous injection of 107TCID50cowpox virus in accordance with the present invention, expressing the murine polytope as described in Thomson et al., 1988, J. Immunol. 160, 1717, and again subjected to immunization with the same amount of the same virus entered the same way in three weeks. For this purpose it is necessary to create the design of recombinant vaccinia virus expressing the specified polytope. How to create con is trucci such recombinant viruses known to the specialist in the art and are described in more detail below. In the case of the modes of the primary immunization with DNA/re-immunization with vaccinia virus primary vaccination is carried out by intramuscular injection of mice with 50 μg DNA expressing the same antigen as the cowpox virus; with the reintroduction of the injected cowpox virus in exactly the same way as in the case of primary immunization with vaccinia virus/re-immunization with vaccinia virus. Plasmid DNA expressing polytope, also described in the above publication, Thomson et al. In both modes the development of CTL responses against epitopes SYIPSAEKI, RPQASGVYM and/or YPHFMPTNL define two weeks after re-introduction. The definition of a CTL response is preferably carried out by using ELISPOT analysis as described by Schneider et al., 1998, Nat. Med. 4, 397-402, and are presented in the examples section below for a specific virus in accordance with the present invention. The virus in accordance with the present invention is characterized in this experiment, the fact that CTL immune response against the above epitopes, which is induced by the primary introduction of cowpox virus/re-introduction of cowpox virus, is essentially the same, preferably at least the same, and that induced by the primary introduction of DNA/re-introduction of cowpox virus in assessing the number of CL is current, producing IFN-γ/106the spleen cells (see also experimental section).

Test 2this test meets test 1. However, instead of using the/in the 107TCID50cowpox virus, as in the case of test 1 in this test 108TCID50cowpox virus in accordance with the present invention is administered subcutaneously for the primary immunization and re-immunization. The virus in accordance with the present invention is characterized in this experiment, the fact that CTL immune response against the epitopes mentioned above, which is induced by the primary introduction of cowpox virus/re-introduction of cowpox virus, is essentially the same, preferably at least the same, and that induced by the primary introduction of DNA/re-introduction of cowpox virus in assessing the number of cells producing IFN-γ/106the spleen cells (see also experimental section).

The strength of the CTL response measured in one of the tests presented above corresponds to the level of protection.

Thus, the viruses in accordance with the present invention are particularly suitable for the purposes of vaccination.

In General, the virus in accordance with the present invention is characterized by the presence of at least one of the following properties:

(i) ---the ability to reproduce by replication in embryonic fibroblasts of chicken (CEF) and in cell lines BHK, but the lack of ability to reproduce by replication in a cell line HaCat person,(ii) lack of ability to replicate in vivo

(iii) induction of a higher immunity in comparison with the known strain MVA-575 (ECACC V00120707) in a model of lethal infection and/or

(iv) by induction, at least, essentially the same level of immunity in the modes of primary immunization with vaccinia virus/re-immunization with vaccinia virus in comparison with the modes of the primary immunization with DNA/re-immunization with vaccinia virus.

Preferably vaccinia virus in accordance with the present invention has at least two of the above properties, more preferably, at least three of the above properties. Most preferred are the cowpox viruses that has all the above properties.

In an additional implementation, the invention concerns a kit for vaccination, including the virus in accordance with the present invention for the primary vaccination ("premirovany") in a single vial/container and for re-inoculation ("boosting") in the second vial/container. The virus can be non-recombinant vaccinia virus, i.e. the cowpox virus, which does not contain heterologous nucleotide sequences. Example VI the USA cowpox virus is MVA-BN and its derivatives. Alternatively, the virus may be a recombinant vaccinia virus that contains additional nucleotide sequences that are heterologous in relation to the cowpox virus. As indicated in other sections of the description, the heterologous sequence can encode epitopes that induce a response of the immune system. Thus, it is possible to use recombinant vaccinia virus for vaccination against proteins or agents, including the specified epitope. Viruses can be included in the compositions as shown in more detail below. The amount of virus that can be used for each vaccination, above.

Specialist in the art will know how to get the virus cowpox, having at least one of the following properties:

- the ability to reproduce by replication in embryonic fibroblasts of chicken (CEF) and in the line of kidney cells baby hamster BHK, but the lack of ability to reproduce by replication in a cell line of human keratinocytes HaCat,

- lack of ability to replicate in vivo

the higher induction of immunity in comparison with the known strain MVA-575 model of lethal infection and/or

- induction, at least, essentially the same level of immunity in the modes PE the primary immunization with vaccinia virus/re-immunization with vaccinia virus in comparison with the modes of the primary immunization with DNA/re-immunization with vaccinia virus.

The method of obtaining such a virus may include the following stages:

(i) the introduction of a known strain of vaccinia virus, preferably MVA-574 or MVA-575 (ECACC V00120707) in cells than human cells in which the virus is able to reproduce by replication, where cells than human cells, preferably selected from CEF cells and cell lines BHK,

(ii) selection/increase in concentration of viral particles from these cells and

(iii) an analysis of whether the received virus having at least one desired biological properties, as indicated above,

where the above stages may not necessarily be repeated until then, until there is a virus with the desired replicative characteristics. The invention additionally relates to the viruses obtained in this way in accordance with the present invention. How how can be defined the desired biological properties, as explained in other parts of the present description.

Applying this method, the inventors have identified and isolated as a result of several cycles of cleaning clone strain in accordance with the present invention, since the passage 575 isolate MVA (MVA-575). This new strain corresponds to a strain with deponents number ECACC V00883008 above.

The nature of the growth of the virus cowpox in accordance with this from what rutenium, in particular, the nature of growth of MVA-BN, indicates that the strains in accordance with the present invention significantly ahead of any other characterized to date, isolate MVA in relation to the weakness of actions in human cell lines and lack of ability to replicate in vivo. Strains in accordance with the present invention are, therefore, ideal candidates for the development of safer products, such as vaccines or drugs, as described below.

In one implementation, the virus in accordance with the present invention, in particular MVA-BN and its derivatives, used as vaccines against viral diseases of man, accompanied by a rash, such as smallpox. In an additional implementation of the virus in accordance with the present invention can be recombinant, i.e. can Express heterologous genes, such as, for example, antigens or epitopes, heterologous with respect to the virus, and may thus be suitable as a vaccine for the induction of immune responses against heterologous antigens or epitopes.

The term "immune response" refers to a reaction of the immune system when entering the body of foreign substances or organisms. By definition, the immune response is divided into specific and nonspecific reactions, although both is neither closely intertwined. Nonspecific immune response is an immediate protection from a wide variety of foreign substances and infectious agents. The specific immune response is a defense that may occur after a lag period after the first infection of the body substance. The specific immune response is a highly effective and defines the fact that the individual who had a specific infection, is secured against the specific infection. Thus, the re-infection with the same or very close infectious agent causes much smoother symptoms or does not cause symptoms because you already have pre-existing immunity" with respect to this agent. Such immunity and immunological memory, respectively, is stored for a long time, in some cases, even throughout life. Accordingly, the induction of immunological memory can be used for vaccination.

"The immune system" refers to a complex of organs involved in the body's defense against foreign substances and microorganisms. The immune system includes a cellular component, including several types of cells, such as lymphocytes and other cells derived from white blood cells, and humoral component, including small pepti the s and the complement factors.

"Vaccination" means that the body burden of infectious agent, for example, attenuated or inactivated form of the infectious agent, for the induction of specific immunity. The term vaccination also covers infection with recombinant virus cowpox in accordance with the present invention, in particular, recombinant MVA-BN and its derivatives, expressing antigens or epitopes that are heterologous in relation to the virus. Examples of such epitopes are given in other parts of the description and cover, for example, epitopes of proteins derived from other viruses, such as Dengue virus, hepatitis C virus, HIV or epitopes derived from proteins that are associated with the development of tumors and cancer. After the introduction of recombinant vaccinia virus in the body epitopes expressed and the immune system and may be the induction of specific immune responses against these epitopes. The body, therefore, is immunized against the agent/protein containing the epitope, which is encoded by recombinant vaccinia virus.

"Immunity" refers to partial or complete protection of the body from diseases caused by infectious agent, due to the successful liquidation of prior infection with specified infectious and entom or its significant part. Immunity is based on the existence of induction and activation of specialized cells of the immune system.

As stressed above, in one implementation of the invention the recombinant viruses in accordance with the present invention, in particular, recombinant MVA-BN and its derivatives contain at least one heterologous sequence is a nucleic acid. The term "heterologous" is used hereafter for any combination of sequences of nucleic acid that is not normally found in close connection with the virus in nature, this virus is also called "recombinant virus".

In accordance with the additional implementation of the present invention, the heterologous sequences are preferably antigenic epitopes selected from any source that is not a cowpox virus. It is most preferable that the recombinant virus expressed one or more antigenic epitopes from Plasmodium falciparum, Mycobacteria, Influenza virus, virus, selected from the family of Flaviviruses, Paramyxoviruses, hepatitis viruses, the human immunodeficiency viruses, or viruses causing hemorrhagic fever, such as Hantaviruses or Filoviruses, i.e. Ebola or Marburg.

In accordance with another implementation, but also in addition to the above choice is antigenic epitopes, heterologous sequences can be selected from other sources smallpox and cowpox. Data viral sequences can be used to modify the spectrum of the owners or immunogenicity of the virus.

In an additional implementation of the virus in accordance with the present invention may encode heterologous gene/nucleic acid expressing therapeutic connection. "Therapeutic compound"encoded by a heterologous nucleic acid in the virus, can represent, for example, a therapeutic nucleic acid such as an antisense nucleic acid, or a peptide or protein with a desired biological activity.

In accordance with additional preferred implementation of the expression of the heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of the promoter of the smallpox virus, more preferably the promoter of the vaccinia virus.

In accordance with another implementation of the inserted heterologous nucleic acid sequence is in a region of the viral genome, which is not necessary. In another preferred implementation of the invention the heterologous nucleic acid sequence is inserted into existing natural site DeleteItem MVA (disclosed in the patent application PCT/EP96/02926). Methods for insertion of heterologous sequences into the genome of variola virus known to specialists in this field of technology.

In accordance with another additional preferred implementation of the invention also includes the genome of the virus and its recombinant variants or functional parts. These viral sequences can be used to identify or highlight virus or recombinant variants, for example, using PCR, hybridization methods or by conventional ELISA tests. Moreover, these viral sequences can be expressed expression vector with the receipt of the encoded protein or peptide, which can then complement the deletion mutants of the virus that have no viral sequence contained in the expression vector.

"Functional part" of the viral genome refers to the part of the complete genomic sequence that encodes a physical object, such as a protein, a domain of the protein, the epitope of the protein. The functional part of the viral genome also describes part of the complete genomic sequences that encode regulatory elements or parts of such elements with an individualized activity, such as promoter, enhancer, CIS - or TRANS-acting elements.

The recombinant virus according to Nast is Asim invention can be applied for the introduction of a heterologous nucleic acid sequence in the target cell, moreover, the specified sequence is either homologous or heterologous with respect to the target cell. The introduction of a heterologous nucleic acid sequence in the target cell can be used to obtain in vitro heterologous peptides or polypeptides, and/or full of viruses encoded by the specified sequence. This method involves infection of a host cell recombinant MVA, the cultivation of the infected host cell in suitable conditions, and selecting and/or increasing the concentration of peptide, protein and/or virus produced by the specified cell of the host.

Moreover, the method of introduction of homologous or heterologous sequences into cells can be used for therapy in vitro and preferably in vivo. For the treatment of in vitro selected cells that were pre - (ex vivo) were infected with a virus, injected into the body of a living animal for the induction of immune responses. For therapy in vivo virus or recombinant variants directly injected into the body of a living animal for the induction of immune responses. In this case, the cells around the site of inoculation become infected with a virus or recombinant variants directly in vivo.

As the virus in accordance with the present invention is characterized by extremely limited growth in human cells and obese is n and thus, it is weakened, it is an ideal tool for the treatment of a wide range of mammals, including humans. Therefore, in the present invention it is also proposed pharmaceutical composition or a vaccine, for example, for the induction of immune response in the body of the living animal, including humans. The virus of the invention is also safe for any other therapy protocols.

The pharmaceutical composition may typically include one or more pharmaceutically acceptable and/or acceptable carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such excipients may be water, saline, glycerol, ethanol, moisturizing or emulsifying agents, substances, buferiruemoi pH, or the like. Suitable carriers are typically large, slowly metabolized molecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids, aggregates of lipids or the like.

To obtain the vaccine virus or recombinant variants in accordance with the invention is transferred in a physiologically acceptable form. This can be done based on the experience with receiving the vaccine the smallpox virus for vaccination against smallpox (campisano Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, the purified virus with a titer of 5 x 108TCID50/ml in approximately 10 mm Tris, 140 mm NaCl, pH 7,4 stored at -80°C. For the preparation of vaccines for injections, for example, 102-108viral particles lyophilized in 100 ml of phosphate buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule, preferably a glass ampoule. Alternatively, a vaccine for injections can be obtained using a phased lyophilized virus composition. This composition may contain additional additives such as mannitol, dextran, sugar, glycine, lactose, or polyvinylpyrrolidone, or other additives, such as antioxidants or inert gas, stabilizers and / or recombinant proteins (e.g. serum albumin human), which are suitable for administration in vivo. The glass ampoule is then sealed and can be stored at temperatures between 4°and With room for several months. However, when there is no need to use capsules, it is stored preferably at a temperature below -20°C.

For vaccination or therapy, the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e. using parenteral, intramuscular or any who go the other route of administration, well-known specialist in this field of technology. The route of administration, dose and number of injections can be optimized by a person skilled in the technical field in a known manner.

Additionally in accordance with another implementation of the virus according to the present invention is particularly useful for the induction of immune responses in animals with impaired immunity, for example, in monkeys infected with SIV (CD4 < 400/µl of blood), or in people with impaired immunity. The term "immunocompromised" describes the state of the immune system of the individual, which occurs only incomplete immune responses, or which is characterized by a reduced efficiency of protection against infectious agents. Even more interesting, and in accordance with another implementation is the fact that the virus is in accordance with the present invention may enhance immune responses in animals or people with impaired immunity even in the presence of these animals or humans preexisting immunity to the smallpox virus. Especially interesting was the fact that the virus is in accordance with the present invention may enhance immune responses in the animals or people undergoing antiviral therapy such as therapy against retroviruses. "Antiviral therapy" includes the concept of treatment aimed at the destruction of the sludge is suppression of viral infection, including, for example, (i) the use of nucleotide analogues, (ii) inhibitors of the enzymatic activity of viruses or Assembly, or (iii) the use of cytokines to influence immune responses of the host.

In accordance with another implementation of the vaccine is especially, but not exclusively, useful in the field of veterinary medicine, for example, for immunization against infection caused by a virus of smallpox animals. Small animal inoculation for immunization is preferably parenterally or intranasally, while in larger animals or humans is preferred subcutaneous, intramuscular or oral inoculation.

The inventors have found that the injection of a vaccine containing an effective dose of only 102TCID50(the dose of infection in tissue culture) virus in accordance with the present invention, is sufficient for full induction of immunity against vaccinia virus wild-type control mice infection with it. This is particularly unexpected, since such a high degree of weakness of the virus in accordance with the present invention has, as expected, to influence negatively and, as a result, to reduce its immunogenicity. This assumption is based on the idea that for the induction of immune responses antigenic epitopes is wideouts in the presentation to the immune system in sufficient quantity. The virus, which is extremely weak, and thus not replicated, can present only a very small number of antigenic epitopes, that is, as long as he includes. This amount of antigen, which are viral particles, are not considered as sufficient for the induction of a strong immune response. However, the virus in accordance with the invention stimulates, even at very low effective dose of only 102TCID50,strong and protective immune response in a model of mouse infection with cowpox virus. The virus in accordance with the present invention, therefore, is characterized by unexpected and even increased immunogenicity compared to other characterized to date, strains MVA. This high immunogenicity makes the virus in accordance with the present invention and any vaccine related, especially suitable for use in animals and people with compromised immune systems.

In accordance with another implementation of the invention, the virus is used as adjuvant. "Adjuvant" in the context of the present description denotes an amplifier specific immune response in vaccines. "The use of the virus as adjuvant" refers to the inclusion of the virus in already existing vaccine for additional stimulation of the immune system of the patient, the which receives the vaccine. Immunizing effect antigenic epitope in most vaccines are often enhanced by the addition of so-called adjuvant. Adjuvant will costimulate the immune system by inducing more strong specific immune response against the antigenic epitope vaccines. This stimulation can be regulated by factors of non-specific immunity, such as interferon and interleukin. Therefore, in further implementation of the invention, the virus used in mammals, including humans, to activate, support or suppress the immune system and it is preferable to activate the immune response against any antigenic determinants. The virus can also be applied to support the immune system in the case of increased susceptibility to infection, as in the case of stress.

The virus used in the adjuvant may be non-recombinant virus, i.e. a virus that does not contain heterologous DNA in its genome. An example of this type of virus is MVA-BN. Alternatively, the virus used as the adjuvant, is a recombinant virus containing in their genome sequences of heterologous DNA that are not present in the viral genome in nature. For use as adjuvant recombinant viral DNA virus preferably contain and Express what induces genes which encode immunostimulatory peptides or proteins such as interleukins.

In accordance with the additional implementation it is preferable that the virus was inaktivirovannye when used as adjuvant or add to another vaccine. Inactivation of the virus can be carried out, for example, by heating or chemical agents, as is known in the art. Preferably, the virus inaktivirovanie using β-propriolactone. In accordance with this implementation of the invention inactivated virus may be added to vaccines against many infectious diseases or proliferative diseases to increase the immune response of the patient to the disease.

A BRIEF STATEMENT of the substance of the INVENTION

The invention inter alia includes the following individually or in combination:

vaccinia virus having at least one of the following properties is the ability to reproduce by replication in embryonic fibroblasts of chicken (CEF) and in the line of kidney cells baby hamster BHK, but the lack of ability to reproduce by replication in a cell line of human keratinocytes HaCat,

- lack of ability to replicate in vivo

the higher induction of immunity in comparison with the known strain MVA-575 model of lethal infection and/or/p>

- induction, at least, essentially the same level of immunity in the modes of primary immunization with vaccinia virus/re-immunization with vaccinia virus in comparison with the modes of the primary immunization with DNA/re-immunization with vaccinia virus.

The virus, as described above, where the virus is unable to reproduce by replication in any of the following cell lines human: cell line kidney 293 human embryo, cell lines of human bone osteosarcoma 143B cell line adenocarcinoma of the cervix HeLa human.

The virus, as described above, due to be deposited in the European collection of cell cultures (ECACC), Salisbury (UK) under number V00083008 and its derivatives.

The virus, as described above, comprising at least one sequence of a heterologous nucleic acid.

The virus, as described above, where the specified sequence heterologous nucleic acid selected from the sequence that encodes at least one antigen, antigenic epitope, and/or therapeutic connection.

Gene or a functional part, derived from the virus, as described above. Pharmaceutical composition comprising the virus, as indicated above, and/or gene and/or its functional part, as described above, and a pharmaceutically acceptable carrier, diluent and/or add to the U.

A vaccine comprising the virus, as indicated above, and/or gene and/or its functional part, as indicated above.

The virus, as indicated above, the genome and/or a functional part, as indicated above, the composition as described above, or vaccine as described above, as drugs to influence the immune response, preferably for its induction, from a living animal, including humans.

The virus, as indicated above, the pharmaceutical composition, as described above, the vaccine, as described above, or a virus, as described above, where the virus, the composition or vaccine is administered in therapeutically effective amounts in a first inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation").

The use of virus as indicated above, and/or genome, as described above, to obtain drugs or vaccines.

The method of introduction of homologous and/or heterologous sequences of nucleic acids into target cells, including infection of target cells with a virus comprising a heterologous sequence, as described above, or transfection of the target cell genome, as described above.

A method of obtaining a peptide, protein and/or virus, including

infection of the host cell by the virus, as indicated above,

- the cultivation of the infected host cell in suitable conditions, and

- allocation and/or increased the f concentration of the peptide, and/or protein and/or virus produced by the specified cell of the host.

The way to influence the immune response, preferably induction, in the body of the living animal, including humans, including the introduction of the virus, as indicated above, genome and/or a functional part, as indicated above, the compositions, as described above, or vaccine as described above, the animal or person being treated.

The method as described above, comprising the introduction of at least 102TCID50(dose infection of tissue culture) of the virus.

The method as described above, in which the virus, the composition or vaccine is administered in therapeutically effective amounts in a first inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation").

The method as above, where the animal is immunocompromised.

The method as above, where the animal has preexisting immunity against smallpox.

The method as above, where the animal is subjected to antiviral therapy.

Way, where the animal is subjected to antiviral therapy, wherein the antiviral therapy is a therapy against retroviruses.

The use of virus as indicated above, its genome and/or a functional part, as stated above, the adjuvant.

The way to increase specifications the ical immune response against the antigen and/or antigenic epitope, included in the vaccine, including the introduction of adjuvant virus, as described above, or genome, as described above, in the body of the living animal, including humans, being treated with the vaccine.

The virus, as indicated above, or genome, as described above, as an adjuvant.

Cell, preferably a cell that contains a virus, as described above, or gene, or a functional part, as indicated above.

A method of obtaining a virus, vaccinia virus, as described above, comprising the following stages:

introduction usually available strain of vaccinia virus, preferably MVA-575 in cells than human cells in which the virus is able to reproduce by replication, where cells than human cells, preferably selected from CEF cells and cell lines BHK,

- select/increase in the concentration of viral particles from these cells and

analysis whether the received virus to have at least one of the biological properties, as indicated above,

where the above stages may not necessarily be repeated until then, until there is a virus with the desired replicative characteristics.

Set for premirovanii/booster immunization, including the virus, as indicated above, the vaccine, as described above, or a virus as medicine, as stated above, for the first inoculation ("emeruwa inoculation") in a first vial/container and for a second inoculation ("boosting inoculation") in a first vial/container.

The use of virus as indicated above, the compositions, as described above, and/or vaccine as described above, to obtain the vaccine, where the virus, the composition or vaccine is administered during premirovanii inoculation and where the same virus or the vaccine is administered during the booster inoculation.

BRIEF DESCRIPTION of FIGURES

Figure 1: Kinetics of growth of different strains MVA in different cell lines. In part (A) results grouped by the tested strains MVA, whereas in part (B) results grouped by tested the cell lines. B) the Number of viruses isolated from cell line after four days (D4) cultivation, was determined using the test plaques and expressed as the ratio of the viruses isolated after 4 days, to the source of grafting material on day 1 (D1).

Figure 2: Protection from lethal infection of the cow pox, provided by vaccination with MVA-BN or MVA-575. Protection was measured by reduction of ovarian titers determined using the standard test plaques 4 days after control of infection.

Figure 3: Induction of CTL and protection against influenza infection, provided by the use of different modes of premirovany repeated immunization.

3A: Induction of CTL responses to 4 different H-2drestricted epitope after vaccination with different combinations of DNA or vaccines, MVA-BN-coding polytope mouse. BALB/c mice (5 per group) WAC who was inerval or DNA (intramuscular), either MVA-BN (subcutaneously), and mice were subjected to booster immunization after three weeks. CTL responses to 4 different epitope encoded by the vaccine (TYQRTRALV, influenza; SYIPSAEKI, P. Berghei; YPHFMPTNL, Cytomegalovirus; PRQASGVYM, LCV) were determined using ELISPOT analysis 2 weeks after booster immunization.

3B: Induction of CTL responses to 4 different epitope after vaccination with different combinations of DNA or vaccines, MVA-BN-coding polytope mouse. BALB/c mice (5 per group) were vaccinated with either DNA (intramuscularly)or MVA-BN (intravenous), and mice were subjected to booster immunization after three weeks. CTL responses to 4 different epitope encoded by the vaccine (TYQRTRALV, influenza; SYIPSAEKI, P. Berghei; YPHFMPTNL, Cytomegalovirus; PRQASGVYM, LCV) were determined using ELISPOT analysis 2 weeks after booster immunization.

3C: the Frequency of T cells specific peptide and MVA, after homologous primary immunization using optimal dose (1 x 108TCID50) recombinant MVA-BN, administered subcutaneously. Groups of 8 mice were vaccinated with two injections of combinations, as indicated in the figure. Two weeks after the last vaccination was measured by the number of specific peptide splenocytes using analysis of INF-gamma ELISPOT. Bars represent the average number of specific spots plus/minus the standard deviation from the mean.

Figure 4: Infection with SIV monkey, vacciner the bathrooms MVA-BN nef or MVA-BN.

Figure 5: Survival of vaccinated monkeys after injection SIV.

Figure 6: the antibody Titers monkeys against MVA-BN. Antibody titers for each animal shown in the form of various icons, and the average titer is shown as a dark rectangle.

Figure 7: Levels of SIV in monkeys with impaired immunity (CD4<400 ml of blood) after vaccination with MVA-BN coding for tat. Monkeys had previously received three vaccinations or MVA-BN or MVA-BN nef (0, 8, 16 weeks), and monkeys infected with pathogenic SIV isolate (22 week). 100, 102 and 106 week (indicated by arrows) monkeys were vaccinated with MVA-BN tat.

Figure 8: Levels of SIV in monkeys exposed to antiretroviral therapy and therapeutic vaccination with the use of MVA-BN. Three groups of monkeys (n=6) SIV infected and treated daily PMPA (marked with a black line). 10 and 16 weeks, animals were vaccinated (marked by arrows), or mixtures of recombinant MVA or saline solution.

Figure 9: the Humoral response induced against SIV, after infection and vaccination with recombinant MVA. Three groups (n=6) monkeys infected with pathogenic SIV isolate (week 0) and then subjected to antiretroviral therapy (PMPA, marked with a bold line). Monkeys were vaccinated with mixtures of recombinant MVA or saline at 10 and 16 weeks. Antibodies against SIV was determined using lysates of infected kletok as antigen using a standard ELISA.

Figure 10. The humoral response induced against MVA SIV infected monkeys subjected to antiretroviral therapy. Three groups (n=6) monkeys infected with pathogenic SIV isolate (week 0) and then subjected to antiretroviral therapy (PMPA, marked with a bold line). Monkeys were vaccinated with mixtures of recombinant MVA or saline at 10 and 16 weeks. Antibodies against MVA was determined using a standard ELISA with absorption for MVA.

Figure 11. Induction of antibodies against MVA after vaccination of mice of different vaccines against smallpox. The level of antibodies produced against MVA after vaccination with MVA-BN (0 and 4 weeks), compared with the traditional strains of cowpox, Elstree and Wyeth, administered by tail scarification (0 week), MVA-572 (0 and 4 weeks) and MVA-BN and MVA-572, administered in the form of pre-Elstree vaccine. MVA-572 was deposited in the European collection of cell cultures, animals as ECACC V94012707. Titers were determined using ELISA with absorption, was calculated by linear regression using linear part of the graph was defined as the dilution that gave an optical density of 0.3. * MVA-BN: MVA-BN significantly (p>0,05) different from MVA-572: MVA-572.

EXAMPLES

The following examples will further illustrate the present invention. Specialist in the art should be well understood that the proposed clause is emery in no way can be interpreted as limiting the applicability of the technology, proposed by the present invention to these examples.

Example 1

The kinetics of growth of a new strain of MVA in a separate cell lines and replication in vivo

(i) the Kinetics of growth in cell lines:

For the characterization of new isolates according to the present invention (hereinafter referred to as MVA-BN) compared the kinetics of growth of this new strain with such other MVA strains that have already been characterized.

The experiment was carried out by comparing the kinetics of growth of the following viruses in the following primary cells and cell lines:

MVA-BN (Viral storage #23, 18. 02. 99 raw, titrated at concentrations of 2,0x107TCID50/ml);

MVA, characterized Altenburger (U.S. patent 5185146) and hereinafter referred to as MVA-HLR;

MVA (passage 575), characterized by Anton Mayr (Mayr, A., et al. [1975] Infection 3; 6-14) and hereinafter referred to as MVA-575 (ECACC V00120707); and

MVA-Vero described in International patent application PCT/EP01/02703 (WO 01/68820)(Viral storage, passage 49, #20, 22.03.99 raw, titrated at concentrations of 4,2x107TCID50/ml).

Used the following primary cells and cell lines:

CEFFibroblasts embryo Chicks (their eggs SPF);
HeLaAdenocarcinoma (epithelial) human cervical, ATCC no CCL-2;
143BThe human bone osteosarcoma TK-, ECACC No. 91112502;
HaCaTCell line of human keratinocytes, Boukamp et al. 1988, J Cell Biol 106(3): 761-771;
BHKBuds baby hamster, ECACC 85011433;
VeroFibroblasts kidney of the African green monkey, ECACC 85020299;
CV1Fibroblasts kidney of the African green monkey, ECACC 87032605.

To infect various cells were sown in 6-hole tablets at a concentration of 5x105cells/well and incubated over night at 37°C, 5% CO2in DMEM (Gibco, Cat. No. 61965-026) plus 2% FCS. The cell culture medium was removed, and cells were infected with approximately 0.05 moi for one hour at 37°C, 5% CO2(for infection was the number of cells was doubled over night). The number of viruses used for each infection different types of cells, was 5x104TCID50and it will be denoted as the input. The cells were then washed 3 times with DMEM, and then added 1 ml of DMEM, 2% FCS, and the tablets were kept incubated for 96 hours (4 days) at 37°C, 5 % CO2. This infection was stopped by freezing the plate at -80°C, ready for analysis by titration.

Analysis by titration (immunocyte specific in from the Oseni cowpox virus antibody)

For the titration of the amount of virus cells for testing were sown in 96-well plates in RPMI (Gibco, Cat. No. 61870-010), 7% FCS, 1% antibiotic/antifungal agent (Gibco, Cat. No. 15240-062) at a concentration of 1x104cells/well and incubated over night at 37°C, 5% CO2. 6-Hole tablets containing material experiments of infection were subjected to freezing/thawing 3 times, and got cultivation from 10-1up to 10-12using the growth medium RPMI. Cultivation of the virus was introduced to the cells for testing, and incubated for five days at 37°C, 5% CO2to ensure the development of CPE (cytopathic effect). Cells for testing recorded (a mixture of acetone/methanol 1:1) for 10 min, washed with PBS, and incubated with a polyclonal antibody specific against cowpox virus (Quartett Berlin, Cat. No. 9503-2057) at a dilution of 1:1000 in buffer for incubation for one hour at room temp. After washing twice with PBS (Gibco, Cat. No. 20012-019) was added HPR conjugated to the antibody against rabbit (Promega Mannheim, Cat. No. W4011) at a dilution of 1:1000 in buffer for incubation (PBS containing 3% FCS) for one hour at room temp. The cells are again washed twice PBS and incubated with staining solution (freshly prepared 10 ml of PBS, 200 ál of a saturated solution of o-dianisidine in 100% ethanol+15 ál of H2O2) to proyavlena the brown spots (two hours). The dye solution was removed, and PBS was added to stop the staining reaction. Each well showed a brown stain was noted as positive in relation to the CPE, and the expected titer by the formula Cerberus (based on the analysis of TCID50) (Kaerber, G. 1931. Arch. Exp. Pathol. Pharmakol. 162, 480).

The viruses were used to infect duplicate rows, on the one hand, CEF and BHK, which, as expected, are permissive for MVA, and, on the other hand, CV-1, Hela, 143B and HaCat, which, as expected, are supermassive for MVA, at low multiplicity of infection (moi), i.e. 0,05 infectious units per cell (5x104TCID50). After that viral grafting material was removed, and the cells three times washed to remove any remaining unabsorbed virus. Infected cells were left in total for four days, when she got viral extracts and then was titrated on CEF cells. In table 1 and figure 1 shows the test results of the titration, where the values are presented as the total number of viruses, formed after 4 days after infection.

It has been shown that in cells CEF (chicken embryo fibroblasts) well, it provided amplification of all viruses that could be expected, since CEF is the permissive cell line for all MVA. In addition, it was shown that all viruses well, it provided amplification in BHK (cell line kidney is omacka). MVA acted in the best way, because BHK is the permissive cell line.

As for replication in Vero cells (a cell line of monkey kidney), MVA-Vero, as expected, was amplificatoare well, namely 1000-fold compared to the input. MVA-HRL and MVA-575 was amplificadores well, with a 33-fold and 10-fold increase relative to the input, respectively. Only MVA-BN, as it was discovered, not amplificates as well in these cells, like the others, but it was observed only a 2-fold excess of the input.

As for replication in CV1 cells (cell line of monkey kidney), it was found that MVA-BN is very strongly suppressed in this cell line. It was shown 200-fold lower compared to the input. Similarly, MVA-575 was not amplificatoare higher level of input and has also been shown to slightly negative amplification, namely 16-fold reduction compared to the input. Best amplificatoare MVA-HLR 30-fold increase compared with the input, and is followed by MVA-Vero with a 5-fold increase compared with the input.

Most interesting was the comparison of the kinetics of growth of various viruses in human cell lines. Reproductive replication in 143B cells (cell line cancer of the bone man) it was shown that MVA-Vero is only showing amplification compared to the input (3-Crat the increase). All other viruses were not amplificadores in relation to the input, but there was a big difference between MVA-HLR and MVA-BN and MVA-575. MVA-HLR was "borderline" (1-fold reduction relative to the input), and in respect of MVA-BN was shown to be greatest inhibition (30-fold reduction relative to the input), followed by MVA-575 (59-fold reduction relative to the input). In the end, MVA-BN is higher in relation to suppression in 143B cells of the person.

Moreover, in relation to replication in HeLa cells (cells of cervical cancer man), it was shown that in this cell line MVA-HLR well amplificada, even better than in permissive BHK cells (HeLa = 125-fold increase compared with the input; BHK = 88-fold increase compared to the input). MVA-Vero was also amplificatoare in this cell line (27-fold increase compared to the input). However, MVA-BN, and, to a lesser extent, MVA-575 was suppressed in these cell lines (MVA-BN = 29-fold lower compared with the input and MVA-575 = 6-fold lower compared to the input).

As for replication in HaCat cells (a cell line of human keratinocytes), it was shown that MVA-HLR well amplificates in this cell line (55-fold increase compared to the input). MVA-Vero adapted and MVA-575 showed amplification in this cell line (1.2 and 1.1-fold increase compared with the input, respectively). Though the MVA-BN was the only which showed inhibition (5-fold lower compared to the input).

In conclusion, it can be argued that MVA-BN is the most suppressed viral strain of this group of viruses: MVA-BN shows maximum inhibition in human cell lines, showing the relationship of amplification from 0.05 to 0.2 in kidney cells of a human embryo (293: ECACC No. 85120602) (data not included in table 1), it gives forth the ratio of amplification of approximately 0.0 V 143B cells; the ratio of amplification of approximately 0.04 in HeLa cells; the ratio of amplification of approximately 0,22 in HaCat cells. In addition, MVA-BN shows the ratio of the amplification of approximately 0.0 V CV1 cells. Only in Vero cells can be observed amplification (ratio of 2.33), but not to the same extent as in permissive cell lines such as BHK and CEF (compare with table 1). Thus, MVA-BN is the only strain MVA showing the amplification ratio below 1 in all human cell lines 143B, Hela, HaCat and 293.

MVA-575 exhibits a profile similar to MVA-BN, but not suppressed, as MVA-BN.

MVA-HLR well amplificates in all tested cell lines (with the exception of 143B cells), thus, it can be regarded as competent for replication in all tested cell lines, with the exception of 143B cells. In one case he amplificates in the line of human cells (HeLa) even the better than in permissive cell lines (BHK).

MVA-Vero shows amplification in all cell lines, but to a lesser extent than it shows MVA-HLR (while ignoring the result with 143B). However, from the viewpoint of suppressing it cannot be considered as belonging to the same "class"as MVA-BN and MVA-575.

2. Replication in vivo

Taking into account the fact that some strains MVA explicitly replicated in vitro, tested the ability of different strains MVA to replicate in vivo using transgenic model AGR129 mice. In these mice are aimed damaged genes receptor type I IFN (IFN-α/β) and type II (IFN-γ) and in the gene RAG. Due to data damage in mice lacking the IFN system, and they are unable to produce Mature B - and T-cells, and essentially have a severe impairment of the immune system and are highly sensitive to the replicated virus. Groups of six mice were immunized (b) 107The BATTLE of MVA-BN, MVA-HLR or MVA-572 (used at 120,000 in Germany) and monitored daily on the subject of clinical manifestations. All mice vaccinated with MVA-HLR or MVA-572, died within 28 and 60 days, respectively. At autopsy were found common signs of severe infection in most organs, and, using standard test plaques in the ovaries was detected MVA (108The COMBAT). In contrast, the ISI, vaccinated with the same dose of MVA-BN, corresponding to the stored strain ECACC V00083008) survived for more than 90 days, and the MVA was not possible to extract from organs or tissues.

Taken together, these in vitro and in vivo studies clearly show that MVA-BN is suppressed to a much greater extent than the original and a commercially available strain MVA-HLR.

Example 2

Immunological data and in vivo

These experiments were undertaken to compare different doses and modes of vaccination with MVA-BN with other MVAs.

2.1. Different MVA strains differ in their ability to stimulate immune response

Replication competent strains of cowpox to induce strong immune responses in mice and in high doses are lethal. Although MVA is a highly attenuated and have reduced ability to replicate in mammalian cells, between different strains MVA there are differences in the degree of attenuation. Indeed, MVA-BN, apparently, is more weak compared to other MVA strains, even in comparison with the original strain MVA-575. To determine whether this difference in degree of weakening the effectiveness of MVA in the induction of protective immune responses, made a comparison of different doses of MVA-BN and MVA-575 model of lethal infection control cow pox. The degree of protection was measured by reduction credits Corot is gay virus in the ovaries 4 days after control of infection, because it provided a quantitative assessment of the impact of different doses and strains MVA.

Model of lethal infection control

Free from this pathogen 6-8-week-old female mice BALB/c (H-2d) (n=5) were immunized (b) with different doses (102, 104or 106TCID50/ml) or MVA-BN or MVA-575. MVA-BN and MVA-575 were propagated on CEF cells, was purified in sucrose and prepared in Tris, pH of 7.4. Three weeks later, mice received repeated injections of the same dose of the same strain MVA followed two weeks later with a lethal infection control (/b) is capable of replicating strain of cowpox. As replication competent strain of vaccinia virus (referred to as "rVV") used or strain WR-L929 TK+or strain IHD-j Control mice received the vaccine placebo. Protection was measured by reduction of titers in the ovaries, certain 4 days after control of infection using standard test spots. For this purpose, mice were scored on day 4 after control of infection, the ovaries were isolated, homogenized in PBS (1 ml), and the titres were determined using standard test plaques using VERO cells (Thomson et al., 1998, J. Immunol. 160: 1717).

Mice vaccinated with two immunizations 104or 106TCID50/ml MVA-BN or MVA-575, were fully protected, judging by the 100% reduction credits rVV in the ovaries 4 days after control of Lenogo infection (Fig. 2). Virus control contamination was removed. However, at lower doses, observed differences in the degree of protection provided by MVA-BN or MVA-575. Mice that received two immunizations 102TCID50/ml MVA-575, were not protected, judging by the high titers rVV in the ovaries (an average of 3.7 x 107FIGHT +/- 2,11 x 107). In contrast, mice vaccinated with the same dose of MVA-BN, were induced a significant reduction (96%) of the title rVV in the ovaries (average of 0.21 x 107FIGHT +/- 0,287 x 107). The control mice treated with vaccine-placebo, the average titer of the virus was 5,11 x 107The FIGHT(+/- 3,59 x 107) (Fig. 2).

Both strain MVA induce protective immune responses in mice against lethal infection control rVV. Although both strains MVA equally effective at higher doses of MVA-BN is more effective than its parent strain MVA-575 in the induction of protective immune response against lethal control rVV infection that may be associated with an increased weakening of MVA-BN compared with MVA-575.

2.2. MVA-BN modes Primerose repeated vaccination

2.2.1.: Induction of antibodies against MVA after vaccination of mice of different vaccines against smallpox

The efficacy of MVA-BN compared with other strains MVA and cowpox that was previously used to suppress the virus. The study consisted of a single immunization with the use of strains cow the Spa Elstree and Wyeth, grown in CEF cells and injected by tail scarification, and immunization with the use of MVA-572, which was previously used in the program suppression of smallpox in Germany. In addition, comparisons were made with MVA-BN and MVA-572 as pre-vaccine followed by vaccination Elstree by tail scarification. For each group used eight BALB/c mice, and all of MVA vaccination (1x107TCID50produced subcutaneously at 0 and 3 weeks. Two weeks after the second immunization, the mice were subjected to control contamination of cow pox (IHD-J), and 4 days after control of infection was determined titers in the ovaries. All vaccines and modes provided 100% protection.

Immune responses induced by the application of these various vaccines or modes, measured in animals prior to infection control. Used tests to measure levels of neutralizing antibodies, proliferation of T-cells, production of cytokines (IFN-γ against IL-4) and production of IFN-γ T-cells. The level of response of T-cells induced by MVA-BN, the results of measurement using the Elispot was generally equivalent to those that were called other MVA and cowpox viruses that shows biological equivalence. Weekly analysis of antibody titers against MVA after application of different vaccination regimens showed that vaccination with MVA-BN significantly increased the rate and in the guise of antibody responses compared with other modes of vaccination (Fig. 11). Indeed, the antibody titers against MVA were significantly higher (p>0,05) at 2, 4 and 5 weeks (1 week after re-reimmunization in week 4) after vaccination with MVA-BN compared with vaccination of mice with MVA-572. After booster vaccination in week 4, the antibody titers were also significantly higher in the group of MVA-BN compared with mice receiving a single vaccination strains of cowpox Elstree or Wyeth. These results clearly show that 2 vaccination with MVA-BN causes greater antibody responses compared to the classical single vaccination traditional strains of cowpox (Elstree and Wyeth), and confirm the data section 1.5 that MVA-BN is more immunogenic than other strains MVA.

2.2.2.: Modes premirovany and MVA boosting cause the same level of protection as the modes premirovany DNA and boosting with MVA on the model of the control of influenza infection.

Evaluate the effectiveness of modes premirovany-boosting-MVA induction vysokoavidnyh CTL responses and compared with the modes premirovany DNA/MVA boosting, which, as reported, are the best. Various modes were evaluated using murine polytope design, coded or vector DNA or MVA-BN, and compared the levels of CTL induction using ELISPOT, and the avidity of the response was measured as the degree of attainable protection after the control of influenza infection.

DESIGN

<> Plasmid DNA encoding a polytope mouse (10 CTL epitopes, including influenza, ovalbumin), was previously described (Thomson et al., 1998, J. Immunol. 160: 1717). This polytope is the mouse was inserted into the deletion site II MVA-BN, propagated in CEF cells, purified in sucrose and presented in Tris, pH 7,4.

METHODS of VACCINATION

In this study, specific pathogen used 6-8-week-old female mice BALB/c (H-2d). For ELISPOT analysis used a group of 5 mice, and in experiments with control flu was applied to 6 mice per group. Mice were vaccinated with the use of different modes premirovany-boosting using MVA or DNA that encodes a polytope mouse, as described in detail in the results. For DNA immunization mice were anestesiology and then were administered a single injection of 50 μg free from endotoxin plasmid DNA (50 μl PBS) in the quadriceps muscle under General anesthesia. Primerose immunization using MVA carried out either by intravenous injection of 107The BATTLE of MVA-BN on the mouse, or by subcutaneous injection of 107The FIGHT or 108The BATTLE of MVA-BN on the mouse. Bustamove immunization was performed three weeks after Primerose immunizations. Boosting plasmid DNA was produced in the same manner as Primerose DNA immunization (see above). To establish CTL responses was performed by standard ELISPOT analyses (Schneider et al., 1998, Nat. Med. 4; 397-402) splenocyte is 2 weeks after the last busterboy immunization using epitope peptide CTL influenza (TYQRTRALV), epitope peptide of P. Berghei (SYIPSAEKI), epitope peptide Cytomegalovirus (YPHFMPTNL) and/or epitope peptide LCV (PRQASGVYM).

For experiments with control infected mice were anestesiology and infected and/n sublethal dose of common influenza virus, Mem71 (4.5 x 105The BATTLE in 50 ml PBS). On day 5 after infection, lungs were isolated, and the titres were determined in two Parallels on line kidney cell dogs Madin-Darby using standard test plaques flu.

The RESULTS:

When applying one of a DNA vaccine CTL induction at 4 H 2depitope encoded by the polytope mouse was bad, and only two epitope of P. Berghei (SYIPSAEKI) and virus lymphocytic choriomeningitis (PRQASGVYM) it was possible to identify weak answers. In contrast, when using mode premirovany DNA and boosting with MVA (107The BATTLE of MVA-BN, injected subcutaneously) was observed significantly higher CTL induction on the SLY (8-fold increase) and PRQ (3-fold increase), and responses were also observed with respect to the third epitope, mouse cytomegalovirus (YPHFMPTNL) (Fig. 3A). However, the application of 107The BATTLE of MVA-BN, injected subcutaneously, at homologous mode premirovany-boosting induced the same level of responses that DNA with subsequent MVA-BN (Fig. 3A). It was unexpectedly found that when using a single immunization with MVA-BN (107TCID50) there is no significant difference in the number of CTL, inducir the bathrooms are against three epitopes, that indicates that the secondary immunization with MVA-BN does not increase significantly CTL responses.

Previously it was shown that subcutaneous administration of 107The BATTLE MVA is the most inefficient way and the concentration of virus for vaccination using other MVA strains, especially compared with intravenous immunization (Schneider et al 1998). To determine the optimal modes of immunization the above experiment was repeated with a change in either the amount of virus, or a way of introduction. In one experiment vaccination 107The BATTLE of MVA-BN were carried out intravenously (Fig. 3B). In another experiment, 108The BATTLE of MVA-BN was administered subcutaneously (Fig. 3C). In these experiments, immunization premirovany-boosting with MVA-BN induced large average numbers of CTL against all three CTL epitopes compared with modes premirovany DNA and boosting with MVA. Also in contrast to the 107The BATTLE of MVA-BN, introduced subcutaneously, immunization, 107The BATTLE of MVA-BN, injected, and immunization 108The BATTLE of MVA-BN, introduced subcutaneously, significantly increased CTL responses, clearly showing that MVA-BN can be used to enhance CTL responses in the presence of pre-existing immunity against the vector.

2.2.3.: The efficacy of the vaccine MVA-BN nef in rhesus monkeys infected with SIV.

Determine the effectiveness of the vaccine MVA-BN nef carried out by estimating the amount of virus and is eriki disease after controlling infection virulent primary SIV isolate. In addition, the study had to be established whether MVA-BN can be used to safely enhanced immune responses in monkeys with immune insufficiency in the presence of preexisting immunity against MVA.

METHODS of VACCINATION

Two groups (n=6) rhesus monkeys (Macaca malalta) were vaccinated intramuscular bolus injection or one of MVA-BN or a recombinant MVA-BN nef at 0, 8 and 16 weeks. 22 week all monkeys were subjected to intravenous infection control MID 5050pathogens associated with cell material SIV (1XC) of the primary rekultivirovannyh PBMC rhesus monkeys. The clinical condition of the animals was monitored with high frequency, and regularly took blood samples to measure viremia, immunological parameters and a full range of haematological parameters and parameters of clinical chemistry of blood. The animals, which AIDS developed in the form of illness, scored, and surviving monkeys were monitored for 99 weeks after vaccination. 100 week surviving monkeys were immunized/m MVA-BN tat, and animals received supplemental immunization same MVA-BN tat 102 and 106 weeks.

No observed adverse effects after any vaccination or MVA-BN or MVA-BN nef. After infection of monkey SIV sharp increase in the level of viremia with a maximum of two weeks after infection (Fig. 4). Because of the large mill is artnik variance within groups there was no significant difference between the mean levels of SIV in groups, vaccinated with MVA-BN nef or MVA-BN. However, the number of SIV in the group vaccinated with MVA-BN nef, was generally 10 times lower compared to the control (MVA-BN) group. Moreover, after 35 weeks after infection (the beginning of the observation period), only 1 of the six monkeys vaccinated with MVA-BN nef, had to kill in connection with disease severity compared with 4 out of 6 animals in the control group (Fig. 5). The disease is clearly correlated with a large amount of virus, and therefore animals were additionally observed for 29 weeks after infection. Vaccine MVA-BN nef, obviously, slowed the progression of the disease compared with the control group, and even on a 46 week after infection 5 out of 6 animals with MVA-BN nef were alive (Fig. 5). However, to 59 week after infection two animals from group vaccinated with nef, were killed, resulting survived five animals (three from group MVA-BN nef and two vaccinated with MVA-BN). Determination of titers of antibodies produced against MVA-BN in 12 monkeys, clearly showed that MVA-BN may enhance the immune response even in the presence of preexisting immunity against MVA (Fig. 6). After Primerose immunization with MVA-BN or MVA-BN nef all monkeys were induced antibody responses against MVA with an average titer of 1000. This antibody responses were significantly increased after the second immunization, which is clearly what has, that MVA can be used for the induction of immune responses by premirovanii-boosting healthy monkeys. These antibody titers gradually reduced, although to a 49 week after immunization titers were on the plateau, resulting in the average titers against MVA 99 week was equal to 2000.

Five surviving monkeys were infected SIV and had HIV with CD4 counts below 400/µl of blood. To study the influence of applied MVA-BN in monkeys with compromised immune five animals were vaccinated three times with MVA-BN tat 100, 102 and 106 weeks after the initial vaccination. The first immunization with MVA-BN tat significantly increased antibody responses against MVA these monkeys with impaired immunity, which additionally increased the third immunization six weeks later (Fig 6). These results additionally show that MVA-BN may enhance immune responses in the presence of significant preexisting immunity to MVA, even in monkeys with compromised immune systems. Although the immune response of the monkeys was increased after immunization with MVA-BN tat, the levels of SIV remained stable, indicating that immunization with MVA-BN are safe and do not affect the level of SIV in monkeys with compromised immune systems (Fig. 7).

This study showed that MVA-BN can premirovat to enhance immune responses in rhesus monkeys with compromised immune systems and that immunizes and MVA-BN are safe and do not affect the level of viremia in animals infected with SIV. The delay in the development of AIDS-like disease in animals vaccinated with vaccine MVA-BN nef, indicates the successful formation of the immune response to nef.

2.2.4.: Therapeutic vaccination with SIV infected monkeys subjected to antiretroviral treatment

Therapeutic HIV vaccine based on MVA-BN, apparently, can be used in individuals exposed to antiretroviral treatment. In this regard, the present study investigated safety (action level SIV) and the efficiency of recombinant MVA encoding a lot of SIV antigens (gag, pol, env, rev, tat and nef) in SIV infected monkeys treated with PMPA. PMPA is a nucleoside analogue and is effective against HIV and SIV (Rosenwirth, B. et al., 2000, J Virol 74, 1704-11).

DESIGN

All recombinant constructs MVA were propagated on CEF cells, was purified in sucrose and was in Tris pH 7,4.

The TECHNIQUE of VACCINATION

Three groups (n=6) rhesus monkeys (Macaca mulatta) infected MID 5050pathogenic primary isolate SIV (1XC) and then were daily treated with PMPA (60 mg/kg, injected s/C) for 19 weeks. 10 week animals were vaccinated with recombinant MVA-BN (V/m) or saline, the animals received identical vaccination after 6 weeks. Group 1 received a mixture of MVA gag-pol and MVA-env, group 2 received MVA-tat, MVA-rev and MVA-nef, and group 3 received physiologist the ical solution. The clinical condition of the animals was often tested, and blood samples were regularly taken for measurement of viremia, immunological parameters and a full range of haematological parameters and parameters of clinical chemistry of blood.

All animals were set high levels of SIV with a maximum of 2 weeks after infection (Fig. 8). After daily treatment with PMPA levels of SIV decreased and stabilized at a low level for week 9. As in the previous study, vaccination with MVA at 10 and 16 weeks did not affect the levels of SIV, suggesting that MVA-BN is a safe vaccine vector for animals with compromised immune systems. After discontinuation of the treatment of animals PMPA (21 week) levels of SIV increase. Although three animals in group 1 the levels of SIV were reduced compared with the control group 3, significant differences of the average number SIV between any of the groups after the treatment PMPA was not (Fig. 8). Using ELISA for lysates infected with SIV T-cells has been shown that in animals of all groups occurs antibody responses against SIV to 4 weeks after infection (Fig. 9). Titers of antibodies against SIV in the control group (saline solution) was decreased during treatment PMPA, and grew rapidly after cessation of treatment PMPA, which reflects the decrease and subsequent increase in the level of SIV in the period protivovetrovye therapy (Fig. 9). A similar pattern of titer antibodies against SIV was observed in group 2, treated with MVA-tat, MVA-rev and MVA-nef, which possibly reflects the reduced expression data of regulatory proteins in the lysates infected with SIV T-cells, shown by ELISA. In contrast, however, the titers of antibodies against SIV in group 1 were increased after vaccination with MVA gag-pol and MVA-env at 10 weeks, which indicates that MVA-BN may enhance the immune response against SIV infected (SIV) animals subjected to antiretroviral therapy. It is important that the titers of antibodies against SIV increased after the second immunization in the 16th week, which again shows that MVA may enhance immune responses in animals with compromised immune systems, even in the presence of preexisting immunity against MVA (Fig. 8). Antibody titers against MVA in group 1 also reflect this pattern with the formation of antibody responses after primary immunization, and this response was significantly increased after the second vaccination (Fig. 10).

MVA-575
Table 1
CEFHelaHaCat143BBHKVeroCV-1
MVA-BN579,730,040,220,0065,882,330,00
796,530,151,170,02131,2210,660,06
MVA-HLR86,68124,9759,090,8387,8634,9729,70
MVA-Vero251,8927,411,282,91702,771416,464,48

The amplification of the virus above the level of the input 4 days after infection

The amplification ratio=output TCID50enter TCID50.

Values are expressed in TCID50.

Sources of information

1. Schneider, J., Gilbert, SC., Blanchard, TJ., Hanke, T., Robson, KJ., Hannan, CM., Becker, M., Sinden, R., Smith, GL., and Hill, AVS. 1998. Enhanced immunogenicity for CD8+ T cell induction and complete efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat. Med. 4; 397-402.

2. Thomson, SA., Sherritt, MA., Medveczky, J., Elliott, SL, Moss, DJ., Fernando GJP., Brown, LE., and Suhrbier, A. 1998. Delivery of multiple CDS cytotoxic T cell epitopes by DNA vaccination. J. Immunol.160: 1717.

1. Vaccinia virus having at least one of the following properties:

(i) the ability to reproduce by replication in embryonic fibroblasts of chicken (CEF) and in the line of kidney cells baby hamster KSS with the lack of ability to reproduce by replication in human cell lines, and

(ii) the lack of ability to replicate in vivo in mice with significant violated the em immunity, where a mouse with a material breach of the immunity are mice that are unable to produce Mature b and T cells,

as pharmaceutical agents for expression in the animal, including humans, therapeutic nucleic acid, peptide or protein, as pharmaceutical agents for influencing the immune response and/or for inducing an immune response in an animal, including humans, as a means acting as adjuvant in relation to an animal, including humans, as a means of activating, supporting or suppressing the immune system of an animal, including humans, or as a vaccine to an animal, including humans.

2. The virus according to claim 1, where the lines of human cells are a cell line of human bone osteosarcoma V, cell line of human keratinocytes HaCat and lines adenocarcinoma cells human cervical HeLa.

3. The virus according to any one of claims 1 and 2, where the virus purified by cloning.

4. The virus according to any one of claims 1 to 3, where the virus is a modified vaccinia virus Ankara (MVA).

5. The virus according to claim 4, where MVA is an MVA-BN deposited at the European collection of cell cultures (ESAS) Salisbury (UK) under number V00083008.

6. The virus according to any one of claims 1 to 5, comprising at least one sequence of a heterologous nucleic acid,/p>

7. The virus according to claim 6, where the sequence of the heterologous nucleic acid is selected from a sequence that encodes at least one antigen, antigenic epitope, and/or therapeutic connection.

8. The virus according to claim 7, where the antigenic epitopes are derived from viruses selected from the family of Influenza virus, Flaviviruses, Paramyxovirus, hepatitis viruses, the human immunodeficiency viruses or viruses that cause hemorrhagic fever.

9. The virus according to any one of PP 8, wherein the virus is a virus MVA-BN containing the nef gene.

10. The virus according to any one of claims 1 to 9, where the virus is administered in therapeutically effective amounts in a first inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation") in therapeutically effective amounts.

11. The virus according to any one of claims 1 to 9 for immunization of a living animal, including humans, against disease caused by the Poxvirus.

12. The virus according to claim 11, where the human disease caused by the Poxvirus is a pox.

13. The virus according to any one of claims 1 and 2, where the animal, including man, has immunity.

14. The virus according to any one of claims 1 to 13, where the animal, including man, already has immunity against Poxvirus.

15. The virus according to any one of claims 1 to 14, where an animal, including humans, exposed to antiviral therapy.

16. The virus according to any one of claims 1 to 12, where antiviral who erapy is a therapy against retroviruses.

17. The virus according to any one of p-9 for the introduction of homologous and/or heterologous nucleic acid in the target cell.

18. Virus genome according to any one of claims 1 to 9 as pharmaceutical agents for expression in the animal, including humans, therapeutic nucleic acid, peptide or protein, as pharmaceutical agents for influencing the immune response and/or for inducing an immune response in an animal, including humans, as a means acting as adjuvant in relation to an animal, including humans, as a means of activating, supporting or suppressing the immune system of an animal, including humans, or as a vaccine to an animal, including humans.

19. The genome by p for immunization of a living animal, including humans, against disease caused by the Poxvirus.

20. Genome according to claim 19, where the human disease caused by the Poxvirus is a pox.

21. The gene according to any one of p-20, where the animal, including man, has immunity.

22. The gene according to any one of p-21, where the animal, including man, already has immunity against Poxvirus.

23. The gene according to any one of p-22, where an animal, including humans, exposed to antiviral therapy.

24. Genome according to item 23, where antiviral therapy is a therapy against retroviruses.

25. The genome by p for the introduction of analogichnoi and/or heterologous nucleic acid in the target cell.

26. Pharmaceutical composition comprising a virus according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier, diluent and/or additive (i) for expression in an animal, including humans, therapeutic nucleic acid, peptide or protein, (ii) to influence the immune response and/or for inducing an immune response in an animal, including humans, (iii) as an adjuvant in relation to an animal, including humans, (iv) to enable, support or suppress the immune system of an animal, including humans, or (v) for vaccination of an animal, including person.

27. The pharmaceutical composition according p, where the composition is administered during the first inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation") in therapeutically effective amounts.

28. The pharmaceutical composition according to any one of p and 27, where the composition comprises at least 102TCID50(cytopathic infective dose for tissue culture) of the virus.

29. The pharmaceutical composition according to any one of p-28 for immunization of a living animal, including humans, against disease caused by the Poxvirus.

30. The pharmaceutical composition according to clause 29, where the human disease caused by the Poxvirus is a pox.

31. The pharmaceutical composition according to any one of p-30, where the animal, including man, has immunity.

32. farmacevticheskaja composition according to any one of p-31, where an animal, including humans, already has immunity against Poxvirus.

33. The pharmaceutical composition according to any one of p-32, where an animal, including humans, exposed to antiviral therapy.

34. The pharmaceutical composition according p where antiviral therapy is a therapy against retroviruses.

35. The pharmaceutical composition according to any one of p-34 for the introduction of homologous and/or heterologous nucleic acid in the target cell.

36. Pharmaceutical composition comprising the gene according p and a pharmaceutically acceptable carrier, diluent and/or additive (i) for expression in an animal, including humans, therapeutic nucleic acid, peptide or protein, (ii) to influence the immune response and/or for inducing an immune response in an animal, including humans, (iii) as an adjuvant in relation to an animal, including humans, (iv) to enable, support or suppress the immune system of an animal, including humans, or (v) for vaccination of an animal, including humans.

37. The pharmaceutical composition according p for immunization of a living animal, including humans, against disease caused by the Poxvirus.

38. The pharmaceutical composition according to clause 29, where the human disease caused by the Poxvirus is a pox.

39. The pharmaceutical composition according to any one of p-38, where the animal, including the human, has immunity.

40. The pharmaceutical composition according to any one of p-39, where the animal, including man, already has immunity against Poxvirus.

41. The pharmaceutical composition according to any one of p-40, where an animal, including humans, exposed to antiviral therapy.

42. The pharmaceutical composition according to paragraph 41, where antiviral therapy is a therapy against retroviruses.

43. The pharmaceutical composition according to any one of p-42 for introducing homologous and/or heterologous nucleic acid in the target cell.

44. Vaccine against poxviruses (Poxvirus) person, in particular smallpox, against antigens or epitopes expressed from the recombinant virus and/or against the infection caused by a poxvirus of the animal comprising a virus according to any one of claims 1 to 9.

45. The vaccine according to item 44, where the vaccine is administered during the first inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation") in therapeutically effective amounts.

46. The vaccine according to item 44 or 45, where the vaccine contains at least 102TCID50(cytopathic infective dose for tissue culture) of the virus.

47. The vaccine according to any one of paragraphs 44-46 for immunization of a living animal, including humans, against disease caused by the Poxvirus.

48. The vaccine p, where the human disease caused by Poxvirus, is the Wallpaper smallpox.

49. The vaccine according to any one of paragraphs 44 to 48, where the animal, including man, has immunity.

50. The vaccine according to any one of paragraphs 44 to 49, where the animal, including man, already has immunity against Poxvirus.

51. The vaccine according to any one of paragraphs 44 to 50, where an animal, including humans, exposed to antiviral therapy.

52. The vaccine according to § 51, where antiviral therapy is a therapy against retroviruses.

53. The vaccine according to any one of paragraphs 44-52 for introducing homologous and/or heterologous nucleic acid in the target cell.

54. Vaccine against poxviruses (Poxvirus) person, in particular smallpox, against antigens or epitopes expressed from the recombinant virus and/or against infections caused by animal poxviruses, including the gene for p.

55. The vaccine according to item 54 for immunization of a living animal, including humans, against disease caused by the Poxvirus.

56. The vaccine according to § 55, where the human disease caused by the Poxvirus is a pox.

57. The vaccine according to any one of p-56, where the animal, including man, has immunity.

58. The vaccine according to any one of p-57, where the animal, including man, already has immunity against Poxvirus.

59. The vaccine according to any one of p-58, where an animal, including humans, exposed to antiviral therapy.

60. The vaccine p where antiviral therapy, not only is no a therapy against retroviruses.

61. The vaccine according to any one of p-60 for introducing homologous and/or heterologous nucleic acid in the target cell.

62. The use of a virus according to any one of claims 1 to 9 to obtain (i) a pharmaceutical for expression in the animal, including humans, therapeutic nucleic acid, peptide or protein, (ii) pharmaceutical agents to influence the immune response and/or for inducing an immune response in an animal, including humans, (iii) funds, acting as adjuvant in relation to an animal, including humans, (iv) means, energizing, supporting or suppressing the immune system of an animal, including humans, or (v) for the vaccine to an animal, including humans.

63. The application of item 62, where the pharmaceutical agent or the vaccine is administered to the living animal, including humans, for the induction of an immune response.

64. The use according to any one of p and 63, where the pharmaceutical agent or a vaccine designed against human diseases caused by Poxvirus.

65. Use p, where the human disease caused by the Poxvirus is a pox.

66. The use according to any one of p and 63, where the pharmaceutical agent or the vaccine contains at least 102TCID50(cytopathic infective dose for tissue culture) of the virus.

67. The use according to any one of PP-66, where the virus the vaccine is injected at what erway inoculation ("primaryusage inoculation") and in a second inoculation ("boosting inoculation") in therapeutically effective amounts.

68. The use according to any one of p-67, where the animal, including man, has immunity.

69. The use according to any one of p-68, where the animal, including man, already has immunity against Poxvirus.

70. The use according to any one of p-69, where the animal, including humans, exposed to antiviral therapy.

71. The application of item 70, where antiviral therapy is a therapy against retroviruses.

72. The application of genome on p to obtain (i) a pharmaceutical for expression in the animal, including humans, therapeutic nucleic acid, peptide or protein, (ii) pharmaceutical agents to influence the immune response and/or for inducing an immune response in an animal, including humans, (iii) funds, acting as adjuvant in relation to an animal, including humans, (iv) means, energizing, supporting or suppressing the immune system of an animal, including humans, or (v) for the vaccine to an animal, including humans.

73. The application of item 72, where the pharmaceutical agent or the vaccine is administered to the living animal, including humans, for the induction of an immune response.

74. The use according to any one of p and 73, where the pharmaceutical agent or a vaccine designed against human diseases caused by Poxvirus.

75. Use p, where the human disease caused by Poxvrus, represents the smallpox.

76. The use according to any one of p-75, where the animal, including man, has immunity.

77. The use according to any one of p-76, where the animal, including man, already has immunity against Poxvirus.

78. The use according to any one of p-77, where the animal, including humans, exposed to antiviral therapy.

79. Use p where antiviral therapy is a therapy against retroviruses.

80. Set for Primerose/booster immunization comprising a virus according to any one of claims 1 to 9, for the first inoculation ("primaryusage inoculation") in a first vial/container and for a second inoculation ("boosting inoculation") in a first vial/container.

81. Set for Primerose/booster immunization, including composition according to any one of p-43, for the first inoculation ("primaryusage inoculation") in a first vial/container and for a second inoculation ("boosting inoculation") in a first vial/container.

82. Set for Primerose/booster immunization, including vaccine according to any one of paragraphs 44 to 61, for the first inoculation ("primaryusage inoculation") in a first vial/container and for a second inoculation ("boosting inoculation") in a first vial/container.

83. The method of introduction of homologous and/or heterologous nucleic acid sequence into target cells in vitro, VK is uchumi infection of target cells with the virus according to any one of p-9.

84. The method of introduction of homologous and/or heterologous nucleic acid sequence in the target cell in vitro, comprising transferowania of target cells by genome on p.

85. The method of producing a peptide, protein and/or virus, including:

(a) infecting a host cell with a virus according to any one of claims 1-9,

(b) culturing the infected host cell in suitable conditions, and

(c) isolation and/or enrichment of peptide, protein and/or virus produced by the specified cell of the host.

86. A method of obtaining a virus according to any one of claims 1 to 3, comprising the following stages:

introduction available to normal strain of vaccinia virus, preferably MVA 575, cells other than human cells in which the virus is able to reproduce by replication, where cells than human cells, preferably selected from CEF cells and cell lines VNK,

selection/enrichment of viral particles from these cells and

analysis whether the received virus to have biological properties as described in any one of claims 1 and 2,

where the above stages may be repeated until then, until there is a virus with the desired replicative characteristics.



 

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FIELD: genetic engineering, virology, pharmacy.

SUBSTANCE: invention proposes the recombinant modified virus OF VACCINE Ankara able to express structural antigens of hepatitis C virus. Virus comprises DNA sequences encoding structural antigens of hepatitis C virus or their functional regions or epitopes of hepatitis C virus structural antigens. Also, invention proposes a pharmaceutical composition comprising such virus, eucaryotic cell infected with such virus, a method for preparing such virus and a method for preparing hepatitis C virus structural polypeptides. Invention can be used in virology and medicine for preparing hepatitis C virus antigen.

EFFECT: valuable properties of virus.

20 cl, 14 dwg, 1 tbl

The invention relates to genetic engineering and can be used to produce medicines new generation to fight with severe human diseases associated with overproduction of tumor necrosis factor-alpha (TNF-alpha)

FIELD: medicine, in particular, oncology and urology, possible use for treatment of surface cancer of urinary bladder.

SUBSTANCE: in accordance to method, tumor is irradiated by laser with output power 0,5-2 Wt with wave length 662 nm and light energy of 300-600 J/cm2 during 10-30 minutes in presence of specified photosensitizer injected intravenously in volume of 0,8-1 mg/kg. Then a silicon vessel is inserted into urinary bladder with fibro-optical filament with cylindrical diffuser positioned therein. Vessel is filled with distilled water and laser irradiation of whole mucous tunic of urinary bladder by light energy at 30-40 J/cm2 is continued during 40-60 minutes.

EFFECT: possible decrease of frequency of relapses and collateral reactions.

1 ex

FIELD: pharmacy, chemical technology, medicine.

SUBSTANCE: invention proposes a composition that comprises 6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone as an active component, antioxidant, non-ionogenic surface-active substance, preserving agent, lipid-soluble stabilizing agent of emulsion, water-soluble stabilizing of emulsion and water. Method for preparing the indicated composition involves mixing the definite amounts of non-ionogenic surface-active substance with antioxidant, heating to temperature 40-120°C, dissolving the necessary amounts of lipid-soluble stabilizing agent of emulsion and 6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone are dissolved in the prepared solution. The prepared mixture is added at intensive stirring to the mixed solution of water-soluble stabilizing agent of emulsion and preserving agent in water preliminary heated to temperature 30-100°C. Invention provides improving bioavailability and increasing storage time of the composition. Proposed composition is used in prophylaxis and treatment of different diseases and for recreating the working ability.

EFFECT: improved preparing method, valuable medicinal properties of composition.

2 cl, 1 tbl, 5 ex

FIELD: medicine, traumatology, resuscitation, field surgery, medicine of catastrophes.

SUBSTANCE: the present innovation deals with helping patients at any variant of limb's detachment at its preparing to replantation. Into soft tissues of detached limb one should introduce oxygenated perfluorane at the dosage of 30 mg/kg of detached limb, and then it should be placed into replantation sac. The innovation enables to hold back the development of ischemic toxicosis due to creating the stock of oxygen carrier, temporarily, up to the moment of replanting, improve tissue structures, up to the moment of replantation, improve the safety of tissue structures and increase the quality of replant's functioning.

EFFECT: higher efficiency.

1 ex

FIELD: medicine, cosmetology.

SUBSTANCE: claimed method includes administration of dispersed biological material diluted in physiological solution or topical anesthetic agent solution in amount of 10-50 mg biological material per 1-15 ml of solution, in dermal and subdermal skin layers. Solution is injected in single dose of 0.1-5 ml and number of injections is 1-20 per course and number of courses is 1-5 with distance between courses from 2 days to 3-4 weeks.

EFFECT: method with increased effectiveness due to activation of base structures of dermal matrix.

2 ex

FIELD: medicine, cosmetology.

SUBSTANCE: claimed method includes application of mask based on natural ion exchanger minerals or sorbents with addition of sterile fly larva ground to paste-like state in ratio of 1:2.5, wherein mineral part size is not more than 1 mm. Before application composition is agitated up to full paste absorption and dries for 30-40 min followed by application on skin surface.

EFFECT: mask with prolonged hemolymph storage time and increased effectiveness.

1 dwg, 4 cl

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to a method for treatment of vascular proliferation in a patient. Method involves administration of agonist of somatostatin receptor type 1 in the therapeutically effective dose to a patient wherein indicated agonist of somatostatin receptor type 1 show the inhibition constant value (Ki) lower 5 nM for somatostatin receptor type 1 and its Ki value for somatostatin receptor type 1 at least by 10 times lower as compared with Ki values for each somatostatin receptors type 2, type 3, type 4 and type 5. Invention provides the maximal inhibition of unfavorable vascular proliferation at minimal symptoms of adverse effects based on the selective anti-angiogenic effect of agonist of somatostatin receptor type 1.

EFFECT: valuable medicinal properties of compositions.

12 cl, 3 tbl, 16 dwg

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