| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | July 13, 2004 |
| PATENT TITLE |
Modified vaccinia ankara virus variant |
| PATENT ABSTRACT |
The present invention provides an attenuated virus, which is derived from Modified Vaccinia Ankara virus and characterized by the loss of its capability to reproductively replicate in human cell lines. It further describes recombinant viruses derived from this virus and the use of the virus, or its recombinants, as a medicament or vaccine. A method is provided for inducing an immune response in individuals who may be immune-compromised, receiving antiviral therapy, or have a pre-existing immunity to the vaccine virus. In addition, a method is provided for the administration of a therapeutically effective amount of the virus, or its recombinants, in a vaccinia virus prime/vaccinia virus boost innoculation regimen. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 16, 2003 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Antoine et al, Virology 244:365-96, 1998.* Schieflinger et al, PNAS 89:9977-81, 1992.* Merchlinsky et al, Virology 190:522-6, 1992.* Gilbert et al, Biol. Chem. 380:299-303, 1999.* Mayr, A., Zbl Vet B 23, 417-430 (1976). Blanchard, J.T., et al., Journal of General Virology, 79, 1159-1167 (1998). Caroll, W.M. and Moss, B., Virology 238, 1998-211 (1997). Meyer, H., et al., Journal of general virology, 72, 1031-1038 (1991). Bender, et al., Journal of Virology, vol. 70, No. 9, pp. 6418-6424 (Sep. 1, 1996). Schneider, et al. (1998), Nature Medicine 4, 397-402. Sutter, et al. (1994), Vaccine 12, 1032-1040. |
| PATENT CLAIMS |
What is claimed is: 1. A vaccinia virus which is that virus deposited at the European Collection of Cell Cultures (ECACC), Salisbury (UK) under number V00083008 and derivatives thereof. 2. The vaccinia virus of the claim 1, comprising at least one heterologous nucleic acid sequence. 3. The vaccinia virus of claim 2, wherein the heterologous nucleic acid sequence codes for at least one antigen, antigenic epitope, or a therapeutic compound. 4. A pharmaceutical composition comprising the vaccinia virus of claim 1 and pharmaceutically acceptable carrier, diluent and/or additive. 5. A vaccine comprising the vaccinia virus of claim 1. 6. A method for affecting a specific immune response in a living animal body, comprising administering an amount of a vaccinia preparation which includes an effective amount of a vaccinia virus of claim 1. 7. The method of claim 6, wherein a specific immune response is induced. 8. The method of claim 6, wherein the animal is a human. 9. The method of claim 8, wherein the human is immune compromised. 10. A method of claim 6, wherein the specific immune response is against an orthopox virus. 11. A method of claim 10, wherein the specific immune response is against smallpox. 12. A method of claim 6, wherein the specific immune response is against a HIV. 13. A method of claim 6, wherein the animal is immune compromised. 14. A method of claim 6, wherein the preparation is administered in therapeutically effective amounts in a first inoculation of "priming inoculation" and in a second inoculation or "boosting inoculation". 15. A method of claim 14, wherein the specific immune response is against an orthopox virus. 16. A method of claim 15, wherein the specific immune response is against smallpox. 17. A method of claim 14, wherein the specific immune response is against HIV. 18. A vaccinia virus of claim 3, wherein the heterologous nucleic acid codes for an HIV epitope. 19. A method for affecting an immune response against HIV in a living animal body, comprising administering an amount of a vaccinia preparation which includes an effective amount of a vaccinia virus of claim 18. 20. The method of claim 19, wherein a specific immune response is induced. 21. The method of claim 19, wherein the animal is a human. 22. A method for affecting a specific immune response in a living animal body, comprising administering an amount of a vaccinia preparation which includes an effective amount of a vaccinia virus of claim 18, wherein the preparation is administered in therapeutically effective amounts in a first inoculation or "priming inoculation" and in a second inoculation or "boosting inoculation". 23. The method of claim 22, wherein a specific immune response is induced. 24. The method of claim 22, wherein the animal is a human. 25. A method of claim 14 or 22, wherein the animal is immune compromised. 26. The method of claim 25, wherein the immune compromised animal is a human. 27. A method of affecting a specific immune response in a living animal body, comprising administering an amount of a vaccine preparation which includes an effective amount of a vaccinia virus of claim 1. 28. The method of claim 27, wherein a specific immune response is induced. 29. The method of claim 27, wherein the animal is a human. 30. A method of affecting a specific immune response in a living animal body, wherein a vaccine preparation which includes an effective amount of a vaccinia virus of claim 1 is administered in a first incoulation or "priming inoculation" and in a second incoculation or "boosting inoculation". 31. The method of claim 30, wherein a specific immune response is induced. 32. The method of claim 30, wherein the animal is a human. 33. A method of enhancing a specific immune response against an antigen, comprising including the virus of claim 1 as an adjuvant in an immunogenic composition. 34. A genome of the vaccinia virus of claim 1. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
The present invention provides an attenuated virus which is derived from Modified Vaccinia Ankara virus and which is characterized by the loss of its capability to reproductively replicate in human cell lines. It further describes recombinant viruses derived from this virus and the use of the virus or its recombinants as a medicament or vaccine. Additionally, a method is provided for inducing an immune response even in immune-compromised patients, patients with pre-existing immunity to the vaccine virus, or patients undergoing antiviral therapy. BACKGROUND OF THE INVENTION Modified Vaccinia Ankara (MVA) virus is related to vaccinia virus, a member of the genera Orthopoxvirus in the family of Poxviridae. MVA was generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., et al. Infection 3, 6-14 [1975]). As a consequence of these long-term passages, the resulting MVA virus deleted about 31 kilobases of its genomic sequence and, therefore, was described as highly host cell restricted to avian cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 [1991]). It was shown in a variety of animal models that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K. [1978] Dev. Biol. Stand. 41: 225-34). Additionally, this MVA strain has been tested in clinical trials as a vaccine to immunize against the human smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]). These studies involved over 120,000 humans, including high-risk patients, and proved that compared to vaccinia based vaccines, MVA had diminished virulence or infectiousness while it induced a good specific immune response. In the following decades, MVA was engineered for use as a viral vector for recombinant gene expression or as a recombinant vaccine (Sutter, G. et al. [1994], Vaccine 12: 1032-40). In this respect, it is most astonishing that even though Mayr et al. demonstrated during the 1970s that MVA is highly attenuated and avirulent in humans and mammals, some recently reported observations (Blanchard et al., 1998, J Gen Virol 79, 1159-1167; Carroll & Moss, 1997, Virology 238, 198-211; Altenberger, U.S. Pat. No. 5,185,146; Ambrosini et al., 1999, J Neurosci Res 55(5), 569) have shown that MVA is not fully attenuated in mammalian and human cell lines since residual replication might occur in these cells. It is assumed that the results reported in these publications have been obtained with various known strains of MVA since the viruses used essentially differ in their properties, particularly in their growth behavior in various cell lines. Growth behavior is recognized as an indicator for virus attenuation. Generally, a virus strain is regarded as attenuated if it has lost its capacity or only has reduced capacity to reproductively replicate in host cells. The above-mentioned observation, that MVA is not completely replication incompetent in human and mammalian cells, brings into question the absolute safety of known MVA as a human vaccine or a vector for recombinant vaccines. Particularly for a vaccine, as well as for a recombinant vaccine, the balance between the efficacy and the safety of the vaccine vector virus is extremely important. OBJECT OF THE INVENTION Thus, an object of the invention is to provide novel virus strains having enhanced safety for the development of safer products, such as vaccines or pharmaceuticals. Moreover, a further object is to provide a means for improving an existing vaccination regimen. DETAILED DESCRIPTION OF THE INVENTION To achieve the foregoing objectives, according to a preferred embodiment of the present invention, new vaccinia viruses are provided which are capable of reproductive replication in non-human cells and cell lines, especially in chicken embryo fibroblasts (CEF), but not capable of reproductive replication in a human cell line known to permit replication with known vaccinia strains. Known vaccinia strains reproductively replicate in at least some human cell lines, in particular the human keratinocyte cell line HaCat (Boukamp et al. 1988, J Cell Biol 106(3): 761-71). Replication in the HaCat cell line is predictive for replication in vivo, in particular for in vivo replication in humans. It is demonstrated in the example section that all known vaccinia strains tested that show a residual reproductive replication in HaCat also replicate in vivo. Thus, the invention preferably relates to vaccinia viruses that do not reproductively replicate in the human cell line HaCat. Most preferably, the invention concerns vaccinia virus strains that are not capable of reproductive replication in any of the following human cell lines: human cervix adenocarcinoma cell line HeLa (ATCC No. CCL-2), human embryo kidney cell line 293 (ECACC No. 85120602), human bone osteosarcoma cell line 143B (ECACC No. 91112502) and the HaCat cell line. The growth behaviour or amplification/replication of a virus is normally expressed by the ratio of virus produced from an infected cell (Output) to the amount originally used to infect the cell in the first place (Input) ("amplification ratio). A ratio of "1" between Output and Input defines an amplification status wherein the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells. This ratio is understood to mean that the infected cells are permissive for virus infection and virus reproduction. An amplification ratio of less than 1, i.e., a decrease of the amplification below input level, indicates a lack of reproductive replication and thus, attenuation of the virus. Therefore, it was of particular interest for the inventors to identify and isolate a strain that exhibits an amplification ratio of less than 1 in several human cell lines, in particular all of the human cell lines 143B, HeLa, 293, and HaCat. Thus, the term "not capable of reproductive replication" means that the virus of the present invention exhibits an amplification ratio of less than 1 in human cell lines, such as 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 outlined in Example 1 of the present specification. Preferably, the amplification ratio of the virus of the invention is 0.8 or less in each of the above human cell lines, i.e., HeLa, HaCat, and 143B. Viruses of the invention are demonstrated in Example 1 and Table 1 not to reproductively replicate in cell lines 143B, HeLa and HaCat. The particular strain of the invention that has been used in the examples was deposited on Aug. 30, 2000 at the European Collection of Cell Cultures (ECACC) under number V00083008. This strain is referred to as "MVA-BN" throughout the Specification. It has already been noted that the known MVA strains show residual replication in at least one of the human cell lines tested (FIG. 1, Example 1). All known vaccinia strains show at least some replication in the cell line HaCat, whereas the MVA strains of the invention, in particular MVA-BN, do not reproductively replicate in HaCat cells. In particular, MVA-BN exhibits an amplification ratio of 0.05 to 0.2 in the human embryo kidney cell line 293 (ECACC No. 85120602). In the human bone osteosarcoma cell line 143B (ECACC No. 91112502), the ratio is in the range of 0.0 to 0.6. For the human cervix adenocarcinoma cell line HeLa (ATCC No. CCL-2) and the human keratinocyte cell line HaCat (Boukamp et al. 1988, J Cell Biol 106(3): 761-71), the amplification ratio is in the range of 0.04 to 0.8 and of 0.02 to 0.8, respectively. MVA-BN has an amplification ratio of 0.01 to 0.06 in African green monkey kidney cells (CV1: ATCC No. CCL-70). Thus, MVA-BN, which is a representative strain of the invention, does not reproductively replicate in any of the human cell lines tested. The amplification ratio of MVA-BN is clearly above 1 in chicken embryo fibroblasts (CEF: primary cultures). As outlined above, a ratio of more than "1" indicates reproductive replication since the amount of virus produced from the infected cells is increased compared to the amount of virus that was used to infect the cells. Therefore, the virus can be easily propagated and amplified in CEF primary cultures with a ratio above 500. In a particular embodiment of the present invention, the invention concerns derivatives of the virus as deposited under ECACC V0083008. "Derivatives" of the viruses as deposited under ECACC V00083008 refer to viruses exhibiting essentially the same replication characteristics as the deposited strain but exhibiting differences in one or more parts of its genome. Viruses having the same "replication characteristics" as the deposited virus are viruses that replicate with similar amplification ratios as the deposited strain in CEF cells and the cell lines HeLa, HaCat and 143B; and that show a similar replication in vivo, as determined in the AGR129 transgenic mouse model (see below). In a further preferred embodiment, the vaccinia virus strains of the invention, in particular MVA-BN and its derivatives, are characterized by a failure to replicate in vivo. In the context of the present invention, "failure to replicate in vivo" refers to viruses that do not replicate in humans and in the mouse model described below. The "failure to replicate in vivo" can be preferably determined in mice that are incapable of producing mature B and T cells. An example of such mice is the transgenic mouse model AGR129 (obtained from Mark Sutter, Institute of Virology, University of Zurich, Zurich, Switzerland). This mouse strain has targeted gene disruptions in the IFN receptor type I (IFN-.alpha./.beta.) and type II (IFN-.gamma.) genes, and in RAG. Due to these disruptions, the mice have no IFN system and are incapable of producing mature B and T cells, and as such, are severely immune-compromised and highly susceptible to a replicating virus. In addition to the AGR129 mice, any other mouse strain can be used that is incapable of producing mature B and T cells, and as such, is severely immune-compromised and highly susceptible to a replicating virus. In particular, the viruses of the present invention do not kill AGR129 mice within a time period of at least 45 days, more preferably within at least 60 days, and most preferably within 90 days post infection of the mice with 10.sup.7 pfu virus administered via intra-peritoneal injection. Preferably, the viruses that exhibit "failure to replicate in vivo" are further characterized in that no virus can be recovered from organs or tissues of the AGR129 mice 45 days, preferably 60 days, and most preferably 90 days after infection of the mice with 10.sup.7 pfu virus administered via intra-peritoneal injection. Detailed information regarding the infection assays using AGR129 mice and the assays used to determine whether virus can be recovered from organs and tissues of infected mice can be found in the example section. In a further preferred embodiment, the vaccinia virus strains of the invention, in particular MVA-BN and its derivatives, are characterized as inducing a higher specific immune response compared to the strain MVA 575, as determined in a lethal challenge mouse model. Details of this experiment are outlined in Example 2, shown below. Briefly, in such a model unvaccinated mice die after infection with replication competent vaccinia strains such as the Western Reserve strain L929 TK+ or IHD-J. Infection with replication competent vaccinia viruses is referred to as "challenge" in the context of description of the lethal challenge model. Four days after the challenge, the mice are usually killed and the viral titer in the ovaries is determined by standard plaque assays using VERO cells (for more details see example section). The viral titer is determined for unvaccinated mice and for mice vaccinated with vaccina viruses of the present invention. More specifically, the viruses of the present invention are characterized in that, in this test after the vaccination with 10.sup.2 TCID.sub.50 /ml of virus of the present invention, the ovarian virus titers are reduced by at least 70%, preferably by at least 80%, and more preferably by at least 90%, compared to unvaccinated mice. In a further preferred embodiment, the vaccinia viruses of the present invention, in particular MVA-BN and its derivatives, are useful for immunization with prime/boost administration of the vaccine. There have been numerous reports suggesting that prime/boost regimes using a known MVA as a delivery vector induce poor immune responses and are inferior to DNA-prime/MVA-boost regimes (Schneider et al., 1998, Nat. Med. 4; 397-402). In all of those studies the MVA strains that have been used are different from the vaccinia viruses of the present invention. To explain the poor immune response if MVA was used for prime and boost administration it has been hypothesized that antibodies generated to MVA during the prime-administration neutralize the MVA administered in the second immunization, thereby preventing an effective boost of the immune response. In contrast, DNA-prime/MVA-boost regimes are reported to be superior at generating high avidity responses because this regime combines the ability of DNA to effectively prime the immune response with the properties of MVA to boost the response in the absence of a pre-existing immunity to MVA. Clearly, if a pre-existing immunity to MVA and/or vaccinia prevents boosting of the immune response, then the use of MVA as a vaccine or therapeutic would have limited efficacy, particularly in the individuals that have been previously vaccinated against smallpox. However, according to a further embodiment, the vaccinia virus of the present invention, in particular MVA-BN and its derivatives, as well as corresponding recombinant viruses harboring heterologous sequences, can be used to efficiently first prime and then boost immune responses in naive animals, as well as animals with a pre-existing immunity to poxviruses. Thus, the vaccinia virus of the present invention induces at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes compared to DNA-prime/vaccinia virus boost regimes. The term "animal" as used in the present description is intended to also include human beings. Thus, the virus of the present invention is also useful for prime/boost regimes in human beings. If the virus is a non-recombinant virus such as MVA-BN or a derivative thereof, the virus may be used as a smallpox vaccine in humans, wherein the same virus can be used in both the priming and boosting vaccination. If the virus is a recombinant virus such as MVA-BN or a derivative thereof that encodes a heterologous antigen, the virus may be used in humans as a vaccine against the agent from which the heterologous antigen is derived, wherein the same virus can be used in both the priming and boosting vaccination. A vaccinia virus is regarded as inducing at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes if, when compared to DNA-prime/vaccinia virus boost regimes, the CTL response, as measured in one of the following two assays ("assay 1" and "assay 2"), preferably in both assays, is at least substantially the same in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. More preferably, the CTL response after vaccinia virus prime/vaccinia virus boost administration is higher in at least one of the assays, when compared to DNA-prime/vaccinia virus boost regimes. Most preferably, the CTL response is higher in both of the following assays. Assay 1 For vaccinia virus prime/vaccinia virus boost administrations, 6-8 week old BALB/c (H-2d) mice are prime-immunized by intravenous administration with 10.sup.7 TCID.sub.50 vaccinia virus of the invention expressing the murine polytope as described in Thomson et al., 1988, J. Immunol. 160, 1717 and then boost-immunized with the same amount of the same virus, administered in the same manner three weeks later. To this end, it is necessary to construct a recombinant vaccinia virus expressing the polytope. Methods to construct such recombinant viruses are known to a person skilled in the art and are described in more detail below. In DNA prime/vaccinia virus boost regimes the prime vaccination is done by intra muscular injection of the mice with 50 .mu.g DNA expressing the same antigen as the vaccinia virus. The boost administration with the vaccinia virus is done in exactly the same way as for the vaccinia virus prime/vaccinia virus boost administration. The DNA plasmid expressing the polytope is also described in the publication referenced above, i.e., Thomson, et al. In both regimes, the development of a CTL response against the epitopes is determined two weeks after the boost administration. The determination of the CTL response is preferably done using the ELISPOT analysis as described by Schneider, et al., 1998, Nat. Med. 4, 397-402, and as outlined in the examples section below for a specific virus of the invention. The virus of the invention is characterized in this experiment in that the CTL immune response against the epitopes mentioned above, which is induced by the vaccinia virus prime/vaccinia virus boost administration, is substantially the same, preferably at least the same, as that induced by DNA prime/vaccinia virus boost administration, as assessed by the number of IFN-.gamma. producing cells/10.sup.6 spleen cells (see also experimental section). Assay 2 This assay basically corresponds to assay 1. However, instead of using 10.sup.7 TCID.sub.50 vaccinia virus administered i.v., as in Assay 1; in Assay 2, 10.sup.8 TCID.sub.50 vaccinia virus of the present invention is administered by subcutaneous injection for both prime and boost immunization. The virus of the present invention is characterized in this experiment in that the CTL immune response against the epitopes mentioned above, which is induced by the vaccinia virus prime/vaccinia virus boost administration, is substantially the same, preferably at least the same, as that induced by DNA prime/vaccinia virus boost administration, as assessed by the number of IFN-.gamma. producing cells/10.sup.6 spleen cells (see also experimental section). The strength of a CTL response as measured in one of the assays shown above corresponds to the level of protection. Thus, the viruses of the present invention are particularly suitable for vaccination purposes. In summary, a representative vaccinia virus of the present invention is characterized by having at least one of the following properties: (i) capability of reproductive replication in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in a human cell line known to permit replication with known vaccinia strains, (ii) failure to replicate in vivo in those animals, including humans, in which the virus is used as a vaccine or active ingredient of a pharmaceutical composition, (iii) induction of a higher specific immune response compared to a known vaccinia strain and/or (iv) induction of at least substantially the same level of a specific immune response in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. Preferably, the vaccinia virus of the present invention has at least two of the above properties, and more preferably at least three of the above properties. Most preferred are vaccinia viruses having all of the above properties. Representative vaccinia virus strains are MVA 575 deposited on Dec. 7, 2000 at the European Collection of Animal Cell Cultures (ECACC) with the deposition number V00120707; and MVA-BN, deposited on Aug. 30, 2000, at ECACC with the deposition number V000083008, and derivatives thereof, in particular if it is intended to vaccinate/treat humans. MVA-BN and its derivatives are most preferred for humans. In a further embodiment, the invention concerns a kit for vaccination comprising a virus of the present invention for the first vaccination ("priming") in a first vial/container and for a second vaccination ("boosting") in a second vial/container. The virus may be a non-recombinant vaccinia virus, i.e., a vaccinia virus that does not contain heterologous nucleotide sequences. An example of such a vaccinia virus is MVA-BN and its derivatives. Alternatively, the virus may be a recombinant vaccinia virus that contains additional nucleotide sequences that are heterologous to the vaccinia virus. As outlined in other sections of the description, the heterologous sequences may code for epitopes that induce a response by the immune system. Thus, it is possible to use the recombinant vaccinia virus to vaccinate against the proteins or agents comprising the epitope. The viruses may be formulated as shown below in more detail. The amount of virus that may be used for each vaccination has been defined above. A process to obtain a virus of the instant invention may comprise the following steps: (i) introducing a vaccinia virus strain, preferably MVA 574 or MVA 575 (ECACC V00120707) into non-human cells in which the virus is able to reproductively replicate, wherein the non-human cells are preferably selected from CEF cells, (ii) isolating/enriching virus particles from these cells and (iii) analyzing whether the obtained virus has at least one of the desired biological properties as previously defined above, wherein the above steps can optionally be repeated until a virus with the desired replication characteristics is obtained. The invention further relates to the viruses obtained by the method of the instant invention. Methods for determining the expression of the desired biological properties are explained in other parts of this description. In applying this method, the inventors identified and isolated in several rounds of clone purification a strain of the present invention starting with the MVA isolate passage 575 (MVA 575). This new strain corresponds to the strain with the accession number ECACC V0083008, mentioned above. The growth behavior of the vaccinia viruses of the present invention, in particular the growth behavior of MVA-BN, indicates that the strains of the present invention are far superior to any other characterized MVA isolates in terms of attenuation in human cell lines and failure to replicate in vivo. The strains of the present invention are therefore ideal candidates for the development of safer products such as vaccines or pharmaceuticals, as described below. In one further embodiment, the virus of the present invention, in particular MVA-BN and its derivatives, is used as a vaccine against human poxvirus diseases, such as smallpox. In a further embodiment, the virus of the present invention may be recombinant, i.e., may express heterologous genes as, e.g., antigens or epitopes heterologous to the virus, and may thus be useful as a vaccine to induce an immune response against heterologous antigens or epitopes. The term "immune response" means the reaction of the immune system when a foreign substance or microorganism enters the organism. By definition, the immune response is divided into a specific and an unspecific reaction although both are closely related. The unspecific immune response is the immediate defense against a wide variety of foreign substances and infectious agents. The specific immune response is the defense raised after a lag phase, when the organism is challenged with a substance for the first time. The specific immune response is highly efficient and is responsible for the fact that an individual who recovers from a specific infection is protected against this specific infection. Thus, a second infection with the same or a very similar infectious agent causes much milder symptoms or no symptoms at all, since there is already a "pre-existing immunity" to this agent. Such immunity and immunological memory persist for a long time, in some cases even lifelong. Accordingly, the induction of an immunological memory can be used for vaccination. The "immune system" means a complex organ involved in the defense of the organism against foreign substances and microorganisms. The immune system comprises a cellular component, comprising several cell types, such as, e.g., lymphocytes and other cells derived from white blood cells, and a humoral component, comprising small peptides and complement factors. "Vaccination" means that an organism is challenged with an infectious agent, e.g., an attenuated or inactivated form of the infectious agent, to induce a specific immunity. The term vaccination also covers the challenge of an organism with recombinant vaccinia viruses of the present invention, in particular recombinant MVA-BN and its derivatives, expressing antigens or epitopes that are heterologous to the virus. Examples of such epitopes are provided elsewhere in the description and include e.g., epitopes from proteins derived from other viruses, such as the Dengue virus, Hepatitis C virus, HIV, or epitopes derived from proteins that are associated with the development of tumors and cancer. Following administration of the recombinant vaccinia virus, the epitopes are expressed and presented to the immune system. A specific immune response against these epitopes may be induced. The organism, thus, is immunized against the agent/protein containing the epitope that is encoded by the recombinant vaccinia virus. "Immunity" means partial or complete protection of an organism against diseases caused by an infectious agent due to a successful elimination of a preceding infection with the infectious agent or a characteristic part thereof. Immunity is based on the existence, induction, and activation of specialized cells of the immune system. As indicated above, in one embodiment of the invention the recombinant viruses of the present invention, in particular recombinant MVA-BN and its derivatives, contain at least one heterologous nucleic acid sequence. The term "heterologous" is used hereinafter for any combination of nucleic acid sequences that is not normally found intimately associated with the virus in nature; such virus is also called a "recombinant virus". According to a further embodiment of the present invention, the heterologous sequences are preferably antigenic epitopes that are selected from any non-vaccinia source. Most preferably, the recombinant virus expresses one or more antigenic epitopes from: Plasmodium falciparum, mycobacteria, influenza virus, viruses of the family of flaviviruses, paramyxoviruses, hepatitis viruses, human immunodeficiency viruses, or from viruses causing hemorrhagic fever, such as hantaviruses or filoviruses, i.e., ebola or marburg virus. According to still a further embodiment, but also in addition to the above-mentioned selection of antigenic epitopes, the heterologous sequences can be selected from another poxviral or a vaccinia source. These viral sequences can be used to modify the host spectrum or the immunogenicity of the virus. In a further embodiment the virus of the present invention may code for a heterologous gene/nucleic acid expressing a therapeutic compound. A "therapeutic compound" encoded by the heterologous nucleic acid in the virus can be, e.g., a therapeutic nucleic acid, such as an antisense nucleic acid or a peptide or protein with desired biological activity. According to a further preferred embodiment, the expression of a heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter, more preferably of a vaccinia virus promoter. According to still a further embodiment, the heterologous nucleic acid sequence is preferably inserted into a non-essential region of the virus genome. In another preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at a naturally occurring deletion site of the MVA genome as disclosed in PCT/EP96/02926. Methods for inserting heterologous sequences into the poxviral genome are known to a person skilled in the art. According to yet another preferred embodiment, the invention also includes the genome of the virus, its recombinants, or functional parts thereof. Such viral sequences can be used to identify or isolate the virus or its recombinants, e.g., by using PCR, hybridization technologies, or by establishing ELISA assays. Furthermore, such viral sequences can be expressed from an expression vector to produce the encoded protein or peptide that then may supplement deletion mutants of a virus that lacks the viral sequence contained in the expression vector. "Functional part" of the viral genome means a part of the complete genomic sequence that encodes a physical entity, such as a protein, protein domain, or an epitope of a protein. Functional part of the viral genome also describes parts of the complete genomic sequence that code for regulatory elements or parts of such elements with individualized activity, such as promoter, enhancer, cis- or trans-acting elements. The recombinant virus of the present invention may be used for the introduction of a heterologous nucleic acid sequence into a target cell, the sequence being either homologous or heterologous to the target cell. The introduction of a heterologous nucleic acid sequence into a target cell may be used to produce in vitro heterologous peptides or polypeptides, and/or complete viruses encoded by the sequence. This method comprises the infection of a host cell with the recombinant MVA; cultivation of the infected host cell under suitable conditions; and isolation and/or enrichment of the peptide, protein and/or virus produced by the host cell. Furthermore, the method for introduction of a homologous or heterologous sequence into cells may be applied for in vitro and preferably in vivo therapy. For in vitro therapy, isolated cells that have been previously (ex vivo) infected with the virus are administered to a living animal body for inducing an immune response. For in vivo therapy, the virus or its recombinants are directly administered to a living animal body to induce an immune response. In this case, the cells surrounding the site of inoculation are directly infected in vivo by the virus, or its recombinants, of the present invention. Since the virus of the invention is highly growth restricted in human and monkey cells and thus, highly attenuated, it is ideal to treat a wide range of mammals, including humans. Hence, the present invention also provides a pharmaceutical composition and a vaccine, e.g., for inducing an immune response in a living animal body, including a human. The virus of the invention is also safe in any other gene therapy protocol. The pharmaceutical composition may generally include one or more pharmaceutical acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. For the preparation of vaccines, the virus or a recombinant of the present invention, is converted into a physiologically acceptable form. This can be done based on experience in the preparation of poxvirus vaccines used for vaccination against smallpox (as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386-2392). For example, the purified virus is stored at -80.degree. C. with a titre of 5.times.10.sup.8 TCID.sub.50 /ml formulated in about 10 mM Tris, 140 mM NaCl, pH 7.4. For the preparation of vaccine shots, e.g., 10.sup.2 -10.sup.8 particles of the virus are 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, the vaccine shots can be produced by stepwise, freeze-drying of the virus in a formulation. This formulation can contain additional additives such as mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, or other additives, such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 4.degree. C. and room temperature for several months. However, as long as no need exists the ampoule is stored preferably at temperatures below -20.degree. 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., by parenteral, intramuscular, or any other path of administration know to a skilled practitioner. The mode of administration, dose, and number of administrations can be optimized by those skilled in the art in a known manner. Additionally according to a further embodiment, the virus of the present invention is particularly useful to induce immune responses in immune-compromised animals, e.g., monkeys (CD4<400/.mu.l of blood) infected with SIV, or immune-compromised humans. The term "immune-compromised" describes the status of the immune system of an individual that exhibits only incomplete immune responses or has a reduced efficiency in the defense against infectious agents. Even more interesting and according to still a further embodiment, the virus of the present invention can boost immune responses in immune-compromised animals or humans even in the presence of a pre-existing immunity to poxvirus in these animals or humans. Of particular interest, the virus of the present invention can also boost immune responses in animals or humans receiving an antiviral, e.g., antiretroviral therapy. "Antiviral therapy" includes therapeutic concepts in order to eliminate or suppress viral infection including, e.g., (i) the administration of nucleotide analogs, (ii) the administration of inhibitors for viral enzymatic activity or viral assembling, or (iii) the administration of cytokines to influence immune responses of the host. According to still a further embodiment, the vaccine is especially, but not exclusively, applicable in the veterinary field, e.g., immunization against animal pox infection. In small animals, the immunizing inoculation is preferably administered by nasal or parenteral administration, whereas in larger animals or humans, a subcutaneous, oral, or intramuscular inoculation is preferred. The inventors have found that a vaccine shot containing an effective dose of only 10.sup.2 TCID.sub.50 (tissue culture infectious dose) of the virus of the present invention is sufficient to induce complete immunity against a wild type vaccinia virus challenge in mice. This is particularly surprising since such a high degree of attenuation of the virus of the present invention would be expected to negatively influence and thereby, reduce its immunogenicity. Such expectation is based on the understanding that for induction of an immune response, the antigenic epitopes must be presented to the immune system in sufficient quantity. A virus that is highly attenuated and thus, not replicating, can only present a very small amount of antigenic epitopes, i.e., as much as the virus itself incorporates. The amount of antigen carried by viral particles is not considered to be sufficient for induction of a potent immune response. However, the virus of the invention stimulates, even with a very low effective dose of only 10.sup.2 TCID.sub.50, a potent and protective immune response in a mouse/vaccinia challenge model. Thus, the virus of the present invention exhibits an unexpected and increased induction of specific immunity compared to other characterized MVA strains. This makes the virus of the present invention and any vaccine derived thereof, especially useful for application in immune-compromised animals or humans. According to still another embodiment of the invention, the virus is used as an adjuvant. An "adjuvant" in the context of the present description refers to an enhancer of the specific immune response in vaccines. "Using the virus as adjuvant" means including the virus in a pre-existing vaccine to additionally stimulate the immune system of the patient who receives the vaccine. The immunizing effect of an antigenic epitope in most vaccines is often enhanced by the addition of a so-called adjuvant. An adjuvant co-stimulates the immune system by causing a stronger specific immune reaction against an antigenic epitope of a vaccine. This stimulation can be regulated by factors of the unspecific immune system, such as interferon and interleukin. Hence, in a further embodiment of the invention, the virus is used in mammals, including humans, to activate, support, or suppress the immune system, and preferably to activate the immune response against any antigenic determinant. The virus may also be used to support the immune system in a situation of increased susceptibility to infection, such as in the case of stress. The virus used as an adjuvant may be a 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 an adjuvant is a recombinant virus containing in its genome heterologous DNA sequences that are not naturally present in the viral genome. For use as an adjuvant, the recombinant viral DNA preferably contains and expresses genes that code for immune stimulatory peptides or proteins such as interleukins. According to a further embodiment, it is preferred that the virus is inactivated when used as an adjuvant or added to another vaccine. The inactivation of the virus may be performed by e.g., heat or chemicals, as known in the art. Preferably, the virus is inactivated by .beta.-propriolacton. According to this embodiment of the invention, the inactivated virus may be added to vaccines against numerous infectious or proliferative diseases to increase the immune response of the patient to this disease. SUMMARY OF THE INVENTION The invention inter alia comprises the following, alone or in combination: A method for inducing a specific immune response in a living animal body, including a human, comprising administering an amount of a vaccinia preparation which includes an effective amount of a vaccinia virus having at least one of the following advantageous properties: capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in a human cell line known to permit replication with known vaccinia strains, failure to replicate in vivo in those animals including humans in which the virus is used as a vaccine or active ingredient of a pharmaceutical composition, induction of a higher specific immune response compared to a known vaccinia strain, and/or induction of at least the same level of a specific immune response in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes, wherein the preparation is administered in therapeutically effective amounts in a first inoculation or "priming inoculation" and in a second inoculation or "boosting inoculation", such a method, wherein the vaccinia virus has at least two (2) of the advantageous properties, such a method, wherein the vaccinia virus has at least three (3) of the advantageous properties, such a method, wherein the vaccinia virus has all four (4) of the advantageous properties, such a method, wherein the vaccinia virus is not capable of reproductive replication in the human keratinocyte cell line (HaCat), such a method, wherein the vaccinia virus is not capable of reproductive replication in any of the following human cell lines: the human keratinocyte cell line (HaCat), the human embryo kidney cell line (293), the human bone osteosarcoma cell line (143B), and the human cervix adenocarcinoma cell line (HeLa), such a method, wherein the vaccinia virus is capable of a replication amplification ratio of greater than 500 in CEF cells, such a method, wherein the vaccinia virus is not capable of replication in mammals, such a method, wherein the vaccinia virus is not capable of replication in humans, such a method, wherein the known vaccinia strain is a Modified Vaccinia Ankara virus (MVA), such a method, wherein the known vaccinia strain is MVA 572, such a method, wherein the known vaccinia strain is MVA 575, such a method, in which the vaccinia virus is that virus deposited at the European Collection of Cell Cultures (ECACC), Salisbury (UK) under number V00083008 and derivatives thereof, such a method, wherein the vaccinia virus is monoclonal, such a method, wherein the vaccinia virus is not capable of replicating in immune compromised animals, including humans, such a method, wherein the vaccinia virus comprises at least one heterologous nucleic acid sequence, such a method, wherein the vaccinia virus comprises a heterologous nucleic acid sequence selected from a sequence coding for at least one antigen, antigenic epitope, or a therapeutic compound, such a method, wherein the specific immune response is directed to the vaccinia virus, such a method, wherein the specific immune response is directed to heterologous material encoded in the vaccinia virus, such a method, wherein the specific immune response is directed to HIV, such a method, wherein the specific immune response is two (2) fold, such a method, wherein the specific immune response is a specific immunity to an orthopox, such a method, wherein the specific immune response is a specific immunity to smallpox, such a method, comprising the administration of at least 10.sup.2 tissue culture infectious dose (TCID.sub.50) of the vaccinia virus, such a method, comprising the administration of a genome or functional parts of the vaccinia virus, to induce a specific immune response, such a method, wherein the vaccinia virus, genome, or functional part thereof, is administered as adjuvant. A vaccinia virus having at least one of the following properties: capability of reproductive replication in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in the human cell line HaCat, failure to replicate in vivo, in those animals, including humans, in which the virus is used as a vaccine or active ingredient of a pharmaceutical composition, induction of a higher specific immune response compared to the strain MVA 575 (ECACC V00120707) in a lethal challenge model and/or induction of at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. The virus as above, wherein the virus is not capable of reproductively replicating in any of the following human cell lines: the human embryo kidney cell line 293, the human bone osteosarcoma cell line 143B and the human cervix adenocarcinoma cell line HeLa. The virus as above, being deposited at the European Collection of Cell Cultures (ECACC), Salisbury (UK) under number V00083008 and derivatives thereof. The virus as above, comprising at least one heterologous nucleic acid sequence. The virus as above, wherein the heterologous nucleic acid sequence is a sequence coding for at least one antigen, antigenic epitope, and/or a therapeutic compound. A genome or functional parts thereof derived from the virus as defined above. A pharmaceutical composition comprising the virus as above, and/or the genome and/or functional part thereof as defined above, and a pharmaceutically acceptable carrier, diluent and/or additive. A vaccine comprising the virus as above, and/or the genome and/or functional part thereof, as defined above. The virus as above, the genome and/or functional part thereof as defined above, the composition as defined above or the vaccine as defined above as a medicament for affecting, preferably inducing, an immune response in a living animal, including a human. The virus as above, the pharmaceutical composition as defined above, the vaccine as defined above or the virus as defined above, wherein the virus, the composition or the vaccine is administered in therapeutically effective amounts in a first inoculation ("priming inoculation") and in a second inoculation ("boosting inoculation"). The use of the virus as above, and/or the genome as defined above, for the preparation of a medicament or a vaccine. A method for introducing homologous and/or heterologous nucleic acid sequences into target cells comprising the infection of the target cells with the virus comprising heterologous sequences as defined above, or the transfection of the target cell with the genome as defined above. A method for producing a peptide, protein and/or virus comprising Infection of a host cell with the virus as above, Cultivation of the infected host cell under suitable conditions, and Isolation and/or enrichment of the peptide and/or protein and/or viruses produced by said host cell. A method for affecting, preferably inducing an immune response in a living animal body, including a human, comprising administering the virus as above, the genome and/or functional part thereof as defined above, the composition as defined above or the vaccine as defined above to the animal or human to be treated. The method as above, comprising the administration of at least 10.sup.2 TCID.sub.50 (tissue culture infectious dose) of the virus. The method as above, wherein the virus, the composition, or the vaccine is administered in therapeutically effective amounts in a first inoculation ("priming inoculation") and in a second inoculation ("boosting inoculation"). The method as above, wherein the animal is immune-compromised. The method as above, wherein the animal has a pre-existing immunity to poxviruses. The method as above, wherein the animal is undergoing an antiviral therapy. The method wherein the animal is undergoing an antiviral therapy, characterized in that the antiviral therapy is an antiretroviral therapy The use of the virus as above, the genome and/or functional part thereof as defined above, as an adjuvant. A method for enhancing a specific immune response against an antigen and/or an antigenic epitope included in a vaccine, comprising administration of the virus as above or the genome as defined above, as an adjuvant to a living animal body including a human to be treated with the vaccine. The virus as above or the genome as defined above, as adjuvant. A cell, preferably a human cell containing the virus as above or the genome or functional part thereof as defined above. A method for obtaining the vaccinia virus as above comprising the following steps: introducing a vaccinia virus strain, preferably MVA 575 into non human cells in which the virus is able to reproductively replicate, wherein the non-human cells are preferably selected from CEF cells, isolating/enriching virus particles from these cells and analyzing whether the obtained virus has at least one of the biological properties as defined above, wherein the above steps can optionally be repeated until a virus with the desired replication characteristics is obtained A kit for prime/boost immunization comprising a virus as above, a vaccine as above, or the virus as drug as defined above for a first inoculation ("priming inoculation") in a first vial/container and for a second inoculation ("boosting inoculation") in a second vial/container. The use of the virus as above, the composition as defined above and/or of the vaccine as defined above, for the preparation of a vaccine wherein the virus, the composition or the vaccine is administered in a prime inoculation and wherein the same virus or vaccine is administered in a boost inoculation. BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Growth kinetics of different strains of MVA in different cell lines. In 1A, the results are grouped according to the MVA strains tested; whereas in 1B, the results are grouped according to the cell lines tested. In 1B, the amount of virus recovered from a cell line after four days (D4) of culture was determined by plaque assay and expressed as the ratio of virus recovered after 4 days to the initial inoculum on day 1 (D1). FIG. 2: Protection provided against a lethal challenge of vaccinia following vaccinations with either MVA-BN or MVA 575. The protection is measured by the reduction in ovarian titres determined 4 days post challenge by standard plaque assay. FIG. 3: Induction of CTL and protection provided against an influenza challenge using different prime/boost regimes. 3A: Induction of CTL responses to 4 different H-2d restricted epitopes following vaccination with different combinations of DNA or MVA-BN vaccines encoding a murine polytope. BALB/c mice (5 per group) were vaccinated with either DNA (intramuscular) or MVA-BN (subcutaneous) and received booster immunizations three weeks later. CTL responses to 4 different epitopes encoded by the vaccines (TYQRTRALV, infuenza; SYIPSAEKI, P. Berghei; YPHFMPTNL, cytomegalovirus; RPQASGVYM, LCV) were determined using an ELISPOT assay 2 weeks post booster immunizations. 3B: Induction of CTL responses to 4 different epitopes following vaccination with different combinations of DNA or MVA-BN vaccines encoding a murine polytope. BALB/c mice (5 per group) were vaccinated with either DNA (intramuscular) or MVA-BN (intraveneous) and received booster immunizations three weeks later. CTL responses to 4 different epitopes encoded by the vaccines (TYQ, influenza; SYI, P. Berghei; cytomegalovirus; RPQ, LCV) were determined using an ELISPOT assay 2 weeks post booster immunizations. 3C: Frequency of peptide and MVA specific T cells following homologous prime/boost using an optimal dose (1.times.10.sup.8 TCID.sub.50) of recombinant MVA-BN, administered subcutaneous. Groups of 8 mice were vaccinated with two shots of the combinations as indicated in the figure. Two weeks after the final vaccination, peptide-specific splenocytes were enumerated using an IFN-gamma ELISPOT assay. The bars represent the mean number of specific spots plus/minus the standard deviation from the mean. FIG. 4: SIV load of monkeys vaccinated with MVA-BN nef or MVA-BN. FIG. 5: Survival of vaccinated monkeys following infection with SIV. FIG. 6: Monkey serum antibody titres to MVA-BN. The antibody titres for each animal are shown as different shapes, whereas the mean titre is illustrated as a solid rectangle. FIG. 7: Levels of SIV in immune-compromised monkeys (CD4<400 ml blood) following vaccinations with MVA-BN encoding tat. Monkeys had previously received three vaccinations with either MVA-BN or MVA-BN nef (week 0, 8, 16) and had been infected with a pathogenic isolate of SIV (week 22). At week 100, 102 and 106 (indicated by arrows) the monkeys were vaccinated with MVA-BN tat. FIG. 8: SIV levels in monkeys undergoing anti-retroviral therapy and therapeutic vaccination using MVA-BN. Three groups of monkeys (n=6) were infected with SIV and treated daily with PMPA (indicated by black line). At week 10 and 16 the animals were vaccinated (indicated by arrows) with either mixtures of recombinant MVA or saline. FIG. 9: Humoral response generated to SIV following infection and vaccination with recombinant MVA. Three groups (n=6) of monkeys were infected with a pathogenic isolate of SIV (week 0) and then treated with the anti-retroviral therapy (PMPA; indicated by bold line). Monkeys were vaccinated with mixtures of recombinant MVA or saline at week 10 and 16. Antibodies to SIV were determined using infected T cell lysates as antigen in a standard ELISA. FIG. 10: Humoral response generated to MVA in SIV infected monkeys undergoing anti-retroviral therapy. Three groups (n=6) of monkeys were infected with a pathogenic isolate of SIV (week 0) and then treated with the anti-retroviral therapy (PMPA; indicated by bold line). Monkeys were vaccinated with mixtures of recombinant MVA or saline at week 10 and 16. Antibodies to MVA were determined using a standard capture ELISA for MVA. FIG. 11: Induction of antibodies to MVA following vaccination of mice with different smallpox vaccines. The levels of antibodies generated to MVA following vaccination with MVA-BN (week 0 and 4), was compared to conventional vaccinia strains, Elstree and Wyeth, given via tail scarification (week 0), MVA 572 (week 0 and 4), and MVA-BN and MVA 572 given as a pre-Elstree vaccine. MVA 572 has been deposited at the European Collection of Animal Cell Cultures as ECACC V94012707. The titres were determined using a capture ELISA and calculated by linear regression using the linear part of the graph and defined as the dilution that resulted in an optical density of 0.3. * MVA-BN: MVA-BN is significantly (p>0.05) different to MVA 572: MVA 572. |
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