Indian Patents. 228892:ALTERED STRAIN OF THE MODIFIED VACCINIA VIRUS ANKARA (MVA)

Title of Invention	ALTERED STRAIN OF THE MODIFIED VACCINIA VIRUS ANKARA (MVA)
Abstract	A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, wherein said cell line is a Vero cell line.

Full Text	FORM 2 THE PATENTS ACT 1970 [39 OF 1970] & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION [See Section 10; rule 13] 'ALTERED STRAIN OF THE MODIFIED VACCINIA VIRUS ANKARA (MVA)" BAVARIAN NORDIC A/S, a Danish company of Boegeskowej 9, DK-3490 Kvistgaard, Denmark, The following specification particularly describes the invefttion and the manner in which it is to be performed: WO 01/68820 PCT/EP01/02703 Altered Strain of the Modified Vaccinia Virus Ankara (IWVA) The present invention relates to new strains of the Modified Vaccinia virus Ankara (MVA) that have a strongly reduced virulence for most mammals, 5 especially humans, but nevertheless grows In cells of a continuous cell line approved for the production of a therapeutic agent such as a vaccine. The invention also relates to ^ method for producing said adapted MVA strains. The MVA can be used e.g. for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator 10 of the unspecific components of the immune system. Background of the invention is An organism is constantly challenged by infectious agents like bacteria, ■ viruses, fungi or parasites. The immune system prevents the organism from permanent infection caused by these agents by the destruction and elimination of these infectious agents and any toic molecules produced by them. The immune system can be divided into a specific and an unspecific 20 part although both parts are closely cross linked. The unspecific immune response enables an immediate defense against a wide variety of foreign substances and infectious agents. In contrast, the specific immune response is raised after a lag phase, when the organism is challenged with a substance for the first time. However, the specific immune response 25 is highly efficient. The specific immune response is responsible for the fact that an individual who recovers from a specific infection is protected against this specific infection but still susceptible for other infectious diseases. In general, a second infection with the same or a very similar infectious agent causes much milder symptoms or no symptoms at all. The immunity 30 persists for a long time, in some cases even lifelong. This immunological memory is used for vaccination, where the organism is challenged with a harmless or inactivated form of the infectious agent to induce a specific WO 01/68820 2 immunity. Sometimes adjuvants are incorporated into vaccines to enhance the specific immune response. Much of the knowledge about infectious diseases -and immunity is 5 contributed by studies of smallpox. The disease is caused by the variola virus, a member of the genus of Orthopox viruses. Nearly two centuries ago, prophylactic inoculations with cowpox was initiated resulting >m the immunization against smallpox. Later immunization was performed with the Vaccinia virus. In the early 1950s, many of the industrialized countries to had eliminated endemic smallpox by using vaccination with Vaccinia virus. However, smallpox vaccination with this Vaccinia virus resulted occasionally in serious complications, such as postvaccinal encephalitis, generalized Vaccinia or contact infection. 15 A new vaccine that does not show these complications was developed by Anton Mayr. The pox vaccine consists of the pox virus Modified Vaccinia Virus Ankara (MVA) and was used for parenteral vaccination against smallpox in about 150 000 vaccinations without causing any complications related to the vaccination. Even children with immunologic deficiencies did 20 not show serious side effects. The MvA was obtained by mutation and selection of the original vaccina virus Ankara after 575 passages in chicken embryo fibroblast cultures. The safety of this MVA is reflected by biological, chemical and physical characteristics. MvA has a reduced molecular weight, six deletions in the genome, and is highly attenuated for 25 mammalian cells, i.e. DNA and protein is synthesized but virtually no viral particles are produced. The Modified Vaccina virus Ankara developed by Anton Mayr was deposited at the European Collection of Cell Cultures (ECACC), Salisbury, UK, under depository No. V 94012707. 30 The vaccination against smallpox was highly successful. In 1979, the World Health Organization declared the eradication of smallpox. Accordingly, the 3 mass vaccination of children was discontinued and only laboratory workers and members of the armed forces of some countries are vaccinated. With the eradication of smallpox, the predominant cause of pox infection in 5 humans was removed- However, some non-human poxviruses have reduced host specificity, i.e. they cause infections not only in their typical host (e.g. for cowpox the cow), but also in other animals, (e.g. rats and cats). Humans can be infected by this route as well. Since parts of the population are no longer immune against smallpox, orthopox infections of 10 animal species can be dangerous for them. Domestic animals are the main source of infection for humans. Accordingly, the vaccination of domestic animals against orthopoxviruses is of increasing importance. In addition, the MVA may be of significance as a vector for gene therapy, i.e. to transfer nucleic acid sequences into a target cell where they are expressed. 15 For a logarithmic reproduction of the MVA, cell cultures of primary or secondary chicken embryo fibroblasts are needed. The cells are obtained from chicken eggs that are incubated for 10 to 12 days. Since eggs are subjected to a biological variability, the cells obtained for the cell culture 20 system are variable on a cellular level as well. In addition, in a chicken embryo "fibroblast culture" often other cell types such as epithelial cells are found. This variation of the cells also results in variation of viruses produced in chicken embryo fibroblasts. It is therefore difficult to standardize and validate the cell culture system to guarantee a constantly 25 high quality of the MVA produced. Furthermore, contamination of the cell culture system by microorganisms or viruses already present in the incubated eggs can not be completely excluded. When the MVA is grown in virus-contaminated cells, the MVA may recombine with the contaminating virus. Thereby an MVA with new and unpredictable characteristics may be 30 generated. For the production of the virus in large scale in a suspension culture, primary or secondary chicken embryo fibroblasts are also not highly suitable. In addition, the purification and concentration of MVA by ultra gradient centrifugation would be advantageous. However, such purification is difficult, when MVA is cultivated on primary or secondary chicken embryo fibroblast Finally, an increasing number of patients have developed allergies against chicken egg's albumen. Although the in vitro conditions of 5 the cultivation strongly reduce the allergenic potential, a hazard of an allergic reaction can not be completely excluded. In conclusion, on the one hand the MVA can only be efficiently grown in primary or secondary chicken embryo fibroblasts causing a number of 10 disadvantages, however, on the other hand the save application of the M/A in humans has been shown by its large-scale application as a vaccine. Object of the invention 15 ft is an object of the present invention to provide conditions for the production of homogeneous virus particles of the MVA Additionally, said conditions should allow an easy and large-scale production of the MVA. Detailed description of the invention , 20 To achieve the foregoing and other objects, the present invention provides an MVA strain that is adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic agent. 25 According to the present invention, for the first time an efficient and large-scale production of MVA is possible. Since cells of a continuous cell line are homogeneous and their characteristics are stable the MVA harvested from these cell lines is also homogeneous with highly predictable characteristics. Furthermore, the risk of contamination by microorganisms 30 can be controlled and contamination of the MVA preparation by proteins of the chicken egg - as found when cultivating MVA on chicken embryo 5 fibroblasts - can be excluded. The handling of a permanent cell line is convenient and thus highly suitable for industrial application. In a preferred embodiment of the invention, the MVA is adapted for growing 5 in cells of a mammalian cell line, which is approved for the production of a vaccine. It has been surprisingly found that the MVA adapted to a mammalian cell line such as the Vera cell line still has a reduced virulence for humans and also for a wide range of other mammals. Accordingly, the MVA is highly attenuated i.e. DNA and protein is synthesized but virtually no 10 viral particles are produced, resulting in a virtually eliminated disease-causing capacity. Hence, the MVA according to the present invention is also highly suitable as a vaccine for humans and for a wide range of mammals. Accordingly, the MVA is especially applicable in the veterinary field. 15 Furthermore, a method to obtain an MVA strain according to the present invention is provided. According to this embodiment of the invention, cells of a cell line that is approved for the production of a therapeutic substance, are infected with the wild-type MVA. Preferably a high multiplicity of infection (MOI), i.e. a high number of viruses per cell is used for this infection. Then, 20 the viruses are harvested and fresh cells of the same cell line are infected with the newly produced viruses. Said process is repeated (serial passaging) until the MVA is adapted to said cell line. Adaptation is reached, when 72h post infection, the virus titer is at least 1- to 9-fold, preferably 10-to 99-fold, more preferably 100- to 106-fold, and most preferably more than 25 107-to 1010-foid increased compared to the input virus titer. The adaptation is reached after a limited number of passages. "Adapted for growing" means that the amount of virus produced from an infection (Output) is increased compared to the amount of virus originally 30 used to infect the cells (Input). In this case the Output/Input ratio is greater than 1. "Derivative" of the MVA deposited at ECACC, Salisbury, UK, under the depository number 99101431 and/or provisional accession number 01021411 means an MVA which is adapted for growing in Vero cells at a rate, which is essentially the same as the growth rate of the deposited 5 strain but carries at least one difference in its genome compared to the deposited strain. The term "immune system" basically describes a complex involved in the defence of the organism against foreign substances and microorganisms. 10 It is divided into a cellular part comprising several cell types, such as e.g. f lymphocytes and other cells derived from white blood cells, and a humoral part comprising peptides and proteins such as antibodies, complement factors, and cytokins. 15 The term "immune response" describes the reaction of the immune system, when a foreign substance or microorganism enters the organism. Generally, the immune response is divided into a specific and an unspecific reaction although both are closely cross linked. The unspecific immune response is regarded as the immediate defence against a wide 20 variety of foreign substances and infectious agents. The specific immune response can be characterised as a highly efficient defence mechanism of ( the organism against a foreign substance which is raised against said substance after a lag phase and highly specific for said substance. The specific immune response is responsible for the phenomenon that an 25 individual who has recovered from a specific infection is protected against this specific infection in future. "Activator of the immune system" means any substance capable of provoking or enhancing an immune response. 30 "Suppressor of the immune system" means any substance capable of reducing or inhibiting an immune response. "Stabilizer of the immune system" means any substance capable of keeping the immune response on a constant level. The inventors provide two preferred MVA strains that are adapted to an 5 African green monkey cell line, called Vera cell line (ATCC No. CCL-81). The MVA-strain, which was passaged 100-times in Vero cells was called "Vero-MVA" and deposited at the European Collection of Cell Cultures, Salisbury, UK under depositary No. 99101431. The MvA strain after 200 passages in Vero cells was called uVero-MVA-200' and deposited at 10 ECACC under provisional accession number 01021411. The MVA obtained as described above is further amplified by cultivating the cells of the approved cell line under suitable conditions, infecting cells with the MVA and harvesting the viral particles produced by said cells. Hence the 15 MVA can efficiently and easily be amplified in large-scale. Surprisingly, the MVA of the invention does not show increased virulence in cells other than Vero cells such as human cell lines including HL, HEP-2 or HeLA. In another embodiment of the invention, the MVA contains at least one 20 heterologous nucleic acid sequence i.e. a nucleic acid sequence that is not naturally found in the MVA genome (recombinant MVA). Preferably, the heterologous nucleic acid sequence is a gene, more preferably a gene encoding an immunizing protein, and most preferably encoding a protein immunizing against malaria, rabies and/or hepatitis. The expression of 25 said heterologous nucleic acid sequence is preferably under the transciptionaf control of a vaccinia virus promoter, more preferably of an MVA-own promoter. In a further preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at a naturally occurring deletion site in the MVA genome (disclosed in PCT/EP96/02926). 30 The recombinant MVA is used for the introduction of a nucleic acid sequence into a target cell, said nucleic acid sequence being homologous or heterologous to the target cell. The introduction of a heterologous nucleic > acid sequence into a target cell may be used to produce heterologous nucleic acids, peptides and/or polypeptides and/or proteins encoded by said nucleic acid sequence in vitro. This method comprises the infection of 5 a host cell with the recombinant MVA cultivation of the infected host cell under suitable conditions, and optionally isolation and/or enrichment of the peptide and/or protein produced by said host cell. Furthermore, the introduction of a homologous or of a heterologous 10 sequence may be applied for in vitro and preferably in vivo gene therapy. , For in vitro and ex vivo gene therapy respectively, cells are isolated from the individual to be treated, transformed with the recombinant MVA and reintroduced into the individual the cells were taken from. For in vivo gene therapy, the recombinant MVA is directly administered to the living animal is body Including the human body. In a preferred embodiment of the invention, the recombinant MVA expresses an antigen or an antigenic epitope. Most preferably, said vector expresses an antigenic determinant from Plasmodium falciparum, Mycobacteria, Herpes virus, Influenza virus, hepatitis, or a human Immunodeficiency virus. 20 Since the MVA according to the invention is - surprisingly - still highly { attenuated, the MVA is ideal to immunize a wide range of mammals including humans. Hence, the present invention also provides a vaccine comprising the MVA for the immunization of a living animal body including a 25 human against pox infections, preferably orthopox infections. The vaccine may contain in addition to the MVA one or more additives such as an antibiotic, a preservative, or a stabilizer. The vaccine is especially applicable m the veterinary field, e.g. for the Immunization of animals against orthopox infections such as cats against cat pox, mice against so ectromelia or camels against camelpox. The immunization is preferably performed parenterally. 9 The immunizing effect of an antigenic determinant in a vaccine is often enhanced by the addition of a so-called adjuvant. An adjuvant co-stimulates the immune system in an unspecific manner causing a stronger specific immune reaction against the antigenic determinant of the vaccine. 5 According to another embodiment of the invention, the MVA is used as an adjuvant, to co-stimulate the immune response against the antigenic determinant of a vaccine. In this case it is preferred that the MVA is inactivated. The inactivation of the MVA may be performed e.g. by heat or chemicals. Preferably, the MVA is inactivated by 0-propiolacton. According to 10 this embodiment of the invention, the inactivated MVA may be added to vaccines against, numerous infectious diseases to increase the immunity against this disease. In case of an infection, the immune, the nervous, the hormonal and the 15 vascular system of an individual work closely together. These interactions can be regulated by elements of the unspecific immune system e.g. cytokines such as interferons and interleukins. Pox viruses can influence the regulation of the immune system (Swiss Vet 11/99, 13-17). Hence, in a further embodiment of the invention, the MVA and preferably the inactivated 20 MVA is used in mammals including humans to regulate the cellular and humoral elements of the unspecific (innate) immune system. Preferably the MVA is used as a bioregulator, wherein dysfunctions of the immune system are eliminated and the body's own defence mechanisms are activated, stabilized and/or suppressed. Most preferably, the MVA is used as a 25 bioregulator in case of a viral infection e.g. with herpes, hepatitis B or C virus, in case of a chronic inflammatory disease and/or to support tumor therapy. The MVA may also be used to stabilize the immune system in a situation of increased susceptibility against infections such as in the case of stress or in neonatals. The active and/or preferably the inactivated IWA 30 can be applied systemically e.g. intramuscularly and/or locally e.g. through mucous membranes and/or skin. In conclusion, the present invention provides MVA strains that can in general be used for the same applications as the wifd-type MVA but eliminate the problems caused by the amplification of the wild-type MVA in chicken embryo fibroblasts. 5 Summary of the invention The invention inter alia comprises the following, alone or m combination: 10 A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic substance. The MVA as above adapted for growing in cells of a mammalian cell line. 15 The MVA as above, wherein the cell line is approved for the production of a vaccine. The MVA as above, wherein said approved cell line is a Vero cell line. The MVA as above, wherein said approved cell line is the Vero cell line ATCCNo.CCL-81. 20 The MVA as above, deposited at the European Collection of Cell Cultures, Salisbury, UK under depositary Mo. 99101431 and/or a derivative thereof. The MVA as above, deposited at the ECACC, Salisbury, UK, under provisional accession number 01021411 and/or a derivative thereof. The MVA as above, comprising at least one heterologous nucleic acid 25 sequence. 1 II The MVA as above comprising -a heterologous nucleic acid sequence coding e.g. for a therapeutic protein and/or an antigenic determinant such as a peptide immunizing against malaria, hepatitis and/or rabies infection. A host cell infected by the above described MVA. 5 A composition, preferably a pharmaceutical composition, comprising the. above described MVA and/or the DNA of the MVA. The pharmaceutical composition described above, wherein the pharmaceutical composition is a vaccine. The vaccine described above for the immunization of a living animal body 10 including a human. The vaccine as above for the immunization against an Orthopox infection. The vaccine as above for the immunization of cats against a cat pox infection, mice against ectromelia infection and/or camels against cameJpox infection. 15 The pharmaceutical composition described above, wherein the MVA is an activator, suppressor and/or stabilizer of the unspecific immune system. A pharmaceutical composition comprising the above described MVA and/or the DNA of the MVA as an adjuvant. A pharmaceutical composition comprising the above described 20 recombinant MVA and/or the DNA of the recombinant MVA. The pharmaceutical composition as described above for use in gene therapy. A method for introducing a homologous and/or heterologous nucleic acid sequence into a target cell comprising infection of the target cell with the 25 above described MVA. A method for obtaining an MVA strain as described above, comprising a) infection of ceils of an approved cell line with a wiid-type MVA preferably the MVA deposited at ECACC under depository No. V 94012707, b) harvesting 5 of the viruses, c) infection of fresh cells of the same ceil line with the newly produced viruses, and, optionally, d) repetition of b) and c) untii the virus is adapted to growth in cells of said cell line. A method for producing viral particles of the above described MvA, 10 comprising cultivating the cells of an approved cell line under suitable conditions, infecting said cell line with said MvA. and harvesting the viral particles produced by said cells. The method as described above, wherein said cell line is infected with the MVA deposited at the ECACC under depositary No. 99101431 and/or the 15 MVA deposited at the ECACC under provisional accession number 01021411 or a derivative of one of those strains. A method for producing a nucleic acid sequence, a peptide polypeptide and/or protein, comprising infection of a host cell with the above described recombinant MVA, cultivation of the infected host cell under suitable 20 conditions, and, optionally, isolation and/or enrichment of the nucleic acid sequence, peptide and/or protein produced by said host cell. Use of the above described MVA for producing a pharmaceutical composition for the treatment or prevention of a disease or disorder responsive to said MVA 25 Use of the above described MVA for producing a vaccine for the immunization of a living animal body including a human. Use of the above described MVA for producing an activator, suppressor and/or stabilizer of the unspecific immune system. The use as described above for the manufacture of an adjuvant. Use of the above described MVA as a vaccine. Use of the above described MVA as an adjuvant. Use of the above described MVA as an activator, suppressor andVor stabilizer of the unspecific immune system. 5 A method for immunization of a living animal body including a human said method comprising administering to a person in need thereof a therapeutically effective amount of an above described pharmaceutical composition. A method for introducing a homologous and/or heterologous nucleic acid 10 sequence into a target cell comprising infecting the target cell with the above described MVA and/or the DNA of the MVA. A method for the activation, suppression and/or stabilization of the immune system of a living animal body including a human said method comprising administration of the above described pharmaceutical composition to a 15 living animal body including a human. A method for enhancing a specific immune response against an antigenic determinant in a vaccine comprising administration of the above described MVA as an adjuvant to a Jiving animal body including a humanA Modified Vaccinia virus Ankara adapted for growing in cells of a continuous cell line obtainable by a process 20 comprising the following steps:infecting cells of a cell line being approved for the production of a therapeutic substance, harvesting the viral particles produced by said cell lines and optionally, repeating the above steps until the desired growth characteristics of said MVA are obtained in said cells. 25 Examples The following examples will further illustrate the present invention, ft wiJJ be well understood by a person skilled in the art that the provided examples in no way may be interpreted in a way that limits the applicability of the technology provided by the present invention to these examples. 5 Example 1: Adaptation of the MVA to Vero cells and characterization of said MVA strain 1. Adaptation of the MVA to Vero cells io The by Anton Mayr developed wild-type MVA that is a modified Vaccina virus Ankara was deposited at ECACC under depository No. V 94012707. The wild-type MVA was adapted to grow in Vero-cells by serial passaging of the virus in Vero cells (Table 1). The cell clone ATCC-No. CCL-81 of the stationary Vero cell line (WHO seed stock ECACC No. 88020401) was is used in the passage No. 148 to 165 (WHO seed lot, Master and Working Bank). The cells were propagated in a medium consisting of Earle's MEM (ICN), pH 7,4 - 7,6, and 5% of the serum substitute BMS (Biochrom). According to a technique known by people skilled in the art, always the same cells of the working bank were seeded by splitting the cells 1:2 to 1:4. 20 The medium contained approximately 250 000 cells per ml. The cells were respectively propagated in tubes (2ml), Roux dishes (100ml), and plastic (^ dishes (6 and 40ml respectively). In general, the cells formed a confluent monolayer after 16 to 24h. Afterwards, the medium was replaced by plain Earle's MEM without any additives. 25 For the adaptation of the wild-type MVA a tube culture system was used. The results of the passages are summarized in Table 1 and 2. The Vero cells were infected by 10 MOI (multiplicity of infection) of the wild-type MVA i.e. in average, 10 viral particles per Vero cell. The wild-type MVA to start with 30 was a genetically homogeneous, plaque-purified MVA after 575 passages in chicken embryo fibroblasts (titer: 107,75 KID50/mI). After 24h, 90% of the Vero cells of the confluent monolayer were destroyed by toxic processes (50% by toxicity, 40% by lysis). The medium plus the cell dedritus after freezing and thawing of the cells, containing the produced viruses, was harvested and 0,2ml of this mixture were seeded on the monolayer of Vero cells in the. culture tubes (2nd passage). This procedure1 was repeated 200 5 times. After the third passage, no toxic effect was observed any more, whereas a mild cytopathic effect (CPE) characterized by rounding of the. cells and lysis in a period of 4 to 6 days post infection (p.inf.) was seen. The virus titer was 101,0 KlD50/ml. It was concluded that the proliferation of the MVA in Vero cells had started although very inefficiently. After the fifth 10 passage, a typical CPE was observed which was completed after 4 to 5 days p.inf. The virus titer increased from 101,0 KlD50/ml after the third passage to 104,0 KID50/ml after the fifth passage. Hence, the virus amplified more efficiently in Vero cells. In the passages No. 5 to 11, a complete CPE was observed more and more early and the virus titer increased with every 15 passage. At passage No. 11, a plateau was reached at 10' KIDa/ml. Accordingly, after eleven passages the adaptation of the MVA to Vero cells was achieved. In the following 30 additional passages, the results were for all passages the same and highly reproducible: The CPE began already 24h p.inf. and all cells were affected after three days p. inf. At that time, 20% 20 of the Vero cells were rounded and 80% were lysed. After three days p.inf., the virus titer was always about 107'75 KJD50/ml. After the fifteenth passage, the viruses were always harvested after two to three days p.inf., and only 1 MOI instead of 10 MOI were used to infect the cells (Table 2). In the following additional passages the growth characteristics of the MVA 25 changed only slightly. Remarkably, the optimum virus titer further increased and reached 1010 Klp50/ml at passage 200. In conclusion, the virus grows reproducibly in an exponential manner in Vero cells. Said growth characteristic is surprisingly different to the 30 characteristics of the wild-type MVA. Accordingly, a new strain of the MVA was obtained by the serial passaging. Said new strain was called "Vero-MVA" and after passage 200 in vero cells "Vero-MVA-200". The Vero-MVA and Vero-MVA-200 were cultivated in larger quantities. For storage, the Vero-MVA was concentrated by centrifugation, resuspended in 2,5 % polygeline and lyophilized in vials of 2ml. The titer after lyophilization 5 was still at least 108,5 KJD50/ml. The lyophilized Vero-MVA and Vero-MVA-200 was checked for contamination and toxicity and stored at +4°C. 2. Characterization of the biological properties of the Vero-MVA 10 The biological characteristics of the Vero-MVA (passage 100) and Vero- r MVA-200 (passage 200) were compared with the characteristics of the wild-type MvA (Table 3 and Table 5). Thereby, the techniques known by the skilled practitioner were applied. The inventors showed that neither the host range of the virus was changed except for the Vero cells, nor the is virulence for humans or animals was increased. The Vero-MVA is still characterized by the abortive propagation in non-permissive host cells. The principal identity of the viral particles of the Vero-MVA compared to the viral particles of the Elstree strain of the Vaccinia virus was shown by cross 20 reactivity of antibodies raised against the Elstree strain. The Elstree strain is a Vaccinia strain recommended by the WHO for the smallpox vaccination. C The polyclonal hyperimmune serum of rabbits raised against the Elstree strain was added to the Vero-MVA. 100 KID50/ml of the Vero MVA were completely neutralized at a dilution of the serum of 1:512. A twofold dilution 25 of the serum was necessary to neutralize the same amount of Vaccinia Elstree strain (1:256). Accordingly, the Vero-MVA can still be efficiently neutralized by Vaccinia immune serum. The Vero-MVA, the Vero-MVA-200 and the wild-type MVA were compared by 30 a number of additional tests as indicated in Table 3, 4 and 5. The inventors showed that the virulence of Vero-MVA and Vero-MVA-200 for mammals • including humans was not increased compared to the wild-type MVA. It was also shown that the Vero-MVA and Vero-MVA-200 are not contagious or toxic for mammals including humans. Surprisingly, the cell specificity of the Vero-MVA was more or less identical to the specificity of the wild-type MvA except for the Vera cells: The Vero-MVA amplifies nearly as inefficiently in 5 cells of human cell fines (see table 4: HL-, HEP-2-, and HeLa-cefls) as the wild-type MVA does. Accordingly, although human cells and cells of African green monkeys are phylogenetically closely related, the Vero-MVA did not gain the ability to amplify in human cells. In other tests, no significant difference were seen either. 10 Furthermore, the physical, chemical, and biological characteristics of the wild-type MVA and the Vero-MVA-200 were compared (Table 5). Whereas the wild-type MVA growing in chicken embryo fibroblast cell cultures has three deletions in the left inverted terminal region, the Vero-MVA-200 has 15 four deletions in the left terminal region compared to the genome of the pox virus as originally isolated in Ankara. Hence, passaging of the wild-type MVA in Vero cells resulted in an additional deletion. The Vero-MVA was used to immunize domestic animals against Orthopox 20 infections. The serum of the animals was collected and a neutralization test was performed. The inventors showed that the animals produced antibodies in high titers. The antibody titers were stable over a period of at least 111 days. It was also shown that the antibodies were able to neutralize in vitro viral particles of the MVA in a plaque-reduction test. In 25 conclusion, the Vero-MVA can be used as a vaccine against Orthopox infections in domestic animals and in humans. Table 1: Adaptation of the MVA to Vero cells Passage No. Cell culture Highest virus titer [log10/mrj Result Conclusion 1 toxic effect after 24h 2,0 Rests of the virus seeded Blind passages Phenomenon of zones and cytokine production 3 No toxicity, moderate CPE after 4-6 days 1,0 Rests of the virus seeded? Begin of the virus reproduction 5 typical CPE completed after 4-5 days 4,0 Increasing virus reproduction 11 CPE completed after 3 days 7,5 Logarithmic virus reproduction Adaptation successful 12-42* CPE begins after 24h, completed after 3 days 7,75 Reproducible virus reproduction Vero-MVA 43-100* CPE begins after 24h, completed after 3 days 8,0 Repoducible virus reproduction Vero MVA 100-200* CPE begins after 24h, completed after 3 days 10,0 Repoducible virus reproduction _ — Results in Vero-MVA-200 * Only 1 MOI instead of 10 MOI are seeded after the eleventh passage. Table 2: Change of the virus titers during the adaptation of the MVA to Vero cells Passage No. Harvested after [days Titer per ml [log10/ml] p.inf. ] 1 1 2 3 2,0 3 5 1,0 5 5 4,0 8 4 6,5 11 , 3 7,5 18 2 8,0 19 2 7,75 20 3 8,0 25 2 7,75 29 2 7,75 30 3 7,75 31 3 8,0 45 2 7,75 51 3 7,75 60 v 2 8,0 66 2 7,75 68 2 8,0 75 3 8,0 100 2 8,0 200 2 10,0 Table 3: Comparison of the biological characteristics of the wild-type MVA and Vero-MVA Marker Wild-type MVA Vero-MVA (100. passage) Vero-MVA-200 CPE in monolayer ceil cultures (1 MOI seeded) Titer of the optimal harvest Abortive virus reproduction in non-permissive ceil systems Reduced virulence for humans and animals Contagiousness Character of the primary plaques on the chorion allantois membrane Hemagglutination (chicken erythrocytes) Rounding and lysis of the cells after day 5 (90% CPE) 108'0 KIDso/m! Yes Yes No No proliferative nodes without necrosis Negative Rounding and iysis of the cells after day 5 (100% CPE) 107'75 KID50/mI Yes Yes No No proliferative nodes without necrosis Negative Rounding and lysis of the cells after day 3 to 5 (100% CPE) 10i0,0KlD50/ml Yes Yes: not virulent at all No No proliferative nodes without necrosis Negative Inactivatlon by ft- Kinetic of first order for Kinetic of first order for Kinetic of first order for 0,05% propiolactone Protective effect in VSV- Yes baby-mouse challenge test Toxicity for humans and No animals Cytokine stimulation Interferon a, IL-2, and 12, CSA Actuation of phagocytosis, Yes natural killer cells, and T-[ymphocytes 0,05% 0,04-0,05% ~~ § Yes Yes S 00 00 o No No Interferon a, IL-2, and 12, Interferon a and y, IL-1,2, CSA and 12, CSA Yes Yes, increased M 3 -4 O 22 Table 4: Reproduction rate in KlD50/mI of Vero-MVA and the wild-fype MVA in different cell culture systems [log10/ml] Cell culture system Vero-MVA Wild-type MVA (575. (31 .Vero-passage) passage in primary chicken embryo fibroblasts) 1) Vero (African green 8,0 4,5 monkey kidney cells) Primary chicken 4,5 8,5 embryo fibroblasts 1'2) HL (human lung) 3,0 2,5 1'2) HEP-2 (human 3,0 2,5 epidermoid carcinoma) 1,2)HeLA (human cervix 2,75 2,75 carcinoma 1>2) BHK (hamster 5,75 5,25 kidney cells) ^MDBK (bovine 3,5 3,5 kidney cells) 1'2) PK-15"(porcine 3,25 3,5 kidney cells) , 1) Continuous cell line derived from the tissue and species indicated in brackets. 2) Cell lines obtained from the collection of the institute of medical microbiology in Munich, Germany. 23 rTabIe 5: Comparison of the wild-type IWVA (572. passage in chicken embryo fibroblasts (CEF)) with Vero-MVA-200 (200. passage in vero cells) Marker Wild-type MVA Verc-MVA-200 Genetic markers 3 deletions in the left (comparison with terminal region (inverted pox virus strain as terminal repeat) isolated in Ankara) Genome size reduced from 208 to 178 kb Loss of 15% of the molecular weight of the original genome Cellular markers Cytokine Virus titer Immune system Loss of the interferon receptor Activation of T-helper cells (CD4, CD8, CD25) Activation of NK cells Abortive reproduction in mammalian cells (except BHK cells) Interferon a, IL-2, IL-12 CEF:109'6KlD50/ml Vero cells: 104,0KID50/ml Reduction of activity of specific immune system 4 deletions in the left terminal region Further reduction of the genome size to 172kb Loss of 20% of the molecular weight of the original genome Additional Joss of receptors e.g. for 1L-1J5 Increased activation of cytotoxic T-lymphocytes Increased activation of NK cells Further narrowing of the host spectrum in cell culture systems interferon a and y, JL-1,2, 12 CEF:104,5KID5o/ml Vero cells: 109^KID50/ml Inhanced activity of the unspecific immune system Virulence for humans and animals low none WO 01/68820 PCT/EP01/02703 24 Applicant's or agent's file. reference number : BN 33 PCT International appticatioii No. '■ .'PCT/EP 01/02703 INDICATIONS RELATING TO ADEPOSTED MICROORGANISM (PCT Rule libis) A. TheindicadorismadebelowiebtetotherniCTOorganismrefem^toipthedescription onpage 6,7,10,12,24 tUne 3,10,23,16,25 B. IDENTlFICATIONOFDEPOSrr Furtherdepositsareidentifiedonanadditionalsheet ) j Nameofdepositaryinstitution ECACC Centre for Applied Microbiology and Research & European Collection of Ceil Cultures Address of depositary institution (including postal code and country) Salisbury Wiltshire SP40JG United Kingdom Date of deposit AccessionNumber February 15,2001 (Provisional) 01021411 C ADI>lTlONALINDlCATIONS(Uave blank if not applicable) This information is continued on an additional sheet Q In respect of all designated States to which such action Is possible and to the extent that it is legally permissible under the law of the designated State, it is requested that a sample of the deposited microorganism be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g., EPC Rule 28(4); UK Patent Rules 1995, Schedule 2, Paragraph 3; Australian Regulation 3.25(3); Danish Patents Act Sections 22 and 33(3) and generally similar provisions mutatis mutandis for any other designated State. D. DESIGNATEDSTATESFORW^CnmDICATLONSAJCEWa)E(ifthemdicaiionsaTenoiforattd^gnatedStates) £. SEPAI^TEFURNISHINGOFINDICATIONSf/MveWanJtifnoi^pIicaWe) The indications listed below will be submitted to the International Bureau later (spec%ytoegenerdnature qj the indications '«$, "Accession Number of Deposit") Copy of the final Certificate of Deposit Forreceiving Office use only For International Bureauuse only This sheet was received with the international application I \| ThissheetwasreceivedbythelntemationalBureauon: Authorizedofiicer CLAUDIA ARAGO! Only 1992) -RTMI DTT/DA/I'M. Authorized officer We Claim 1. A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, wherein said cell line is a Vero cell line. 2. The MVA as claimed in claim 1, wherein said cell line is Vero cell line ATCC No.CCL-81. 3. The MVA as claimed in claim 2, deposited at the European Collection of Cell Culture (ECACC) Salisbury, UK, under depository No. 99101431 and/or derivative thereof. 4. The MVA as claimed in claim 2, deposited at the European Collction of Cell Cultures (ECACC), Salisbury, UK, under depository No. 01021411 and/or derivative thereof. 5. The MVA as claimed in any of the preceding claims 1 to 4 comprising at least one heterologous nucleic acid sequence. 6. The MVA as claimed in claim 5, wherein the heterologous nucleic acid sequence codes for a therapeutic protein and/or antigenic determinant. 27 7. A composition preferably a pharmaceutical composition, comprising the MVA and/or DNA of the MVA as claimed in any of the preceding claims 1 to 6 and the conventional additives such as stabilizer. 8. The composition as claimed in claim 7, wherein the composition is a vaccine. 9. The vaccine as claimed in claim 8 is applicable for the immunization of a living animal body including a human. 10. The vaccine as claimed in claim 8 or 9 is applicable for the immunization against an Orthopox infection. 11. The vaccine as claimed in claim 8 to 10 is applicable for the immunization of cats against a cat pox infection, mice against ectromelia infection and/or camels against camel pox infection. 12. The composition as claimed in claim 7 wherein the MVA is an activator, suppressor and/or stabilizer of the immune systems 28 13. A composition, preferably a pharmaceutical composition, comprising the MVA and/or DNA of the MVA as claimed in any of the preceding claims 1 to 6 as an adjuvant and the conventional additives such as stabilizer. 14. A composition preferably a pharmaceutical composition, comprising the recombinant MVA and/or DNA of the recombinant MVA as claimed in claim 5 or 6. Dated this the 12th day of September 2002 (R.P. Bhattadharya) Of DEPENNING & DEPENNING Agent for the Applicants

Full Text

FORM 2
THE PATENTS ACT 1970
[39 OF 1970]
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See Section 10; rule 13]
'ALTERED STRAIN OF THE MODIFIED VACCINIA VIRUS ANKARA
(MVA)"
BAVARIAN NORDIC A/S, a Danish company of Boegeskowej 9, DK-3490 Kvistgaard, Denmark,
The following specification particularly describes the invefttion and the manner in which it is to be performed:

WO 01/68820

PCT/EP01/02703

Altered Strain of the Modified Vaccinia Virus Ankara (IWVA)
The present invention relates to new strains of the Modified Vaccinia virus Ankara (MVA) that have a strongly reduced virulence for most mammals, 5 especially humans, but nevertheless grows In cells of a continuous cell line approved for the production of a therapeutic agent such as a vaccine. The invention also relates to ^ method for producing said adapted MVA strains. The MVA can be used e.g. for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator 10 of the unspecific components of the immune system.
Background of the invention
is An organism is constantly challenged by infectious agents like bacteria,
■ viruses, fungi or parasites. The immune system prevents the organism
from permanent infection caused by these agents by the destruction and
elimination of these infectious agents and any to*ic molecules produced by
them. The immune system can be divided into a specific and an unspecific
20 part although both parts are closely cross linked. The unspecific immune response enables an immediate defense against a wide variety of foreign substances and infectious agents. In contrast, the specific immune response is raised after a lag phase, when the organism is challenged with a substance for the first time. However, the specific immune response
25 is highly efficient. The specific immune response is responsible for the fact that an individual who recovers from a specific infection is protected against this specific infection but still susceptible for other infectious diseases. In general, a second infection with the same or a very similar infectious agent causes much milder symptoms or no symptoms at all. The immunity
30 persists for a long time, in some cases even lifelong. This immunological memory is used for vaccination, where the organism is challenged with a harmless or inactivated form of the infectious agent to induce a specific

WO 01/68820
2
immunity. Sometimes adjuvants are incorporated into vaccines to enhance the specific immune response.
Much of the knowledge about infectious diseases -and immunity is 5 contributed by studies of smallpox. The disease is caused by the variola virus, a member of the genus of Orthopox viruses. Nearly two centuries ago, prophylactic inoculations with cowpox was initiated resulting >m the immunization against smallpox. Later immunization was performed with the Vaccinia virus. In the early 1950s, many of the industrialized countries to had eliminated endemic smallpox by using vaccination with Vaccinia virus. However, smallpox vaccination with this Vaccinia virus resulted occasionally in serious complications, such as postvaccinal encephalitis, generalized Vaccinia or contact infection.
15 A new vaccine that does not show these complications was developed by Anton Mayr. The pox vaccine consists of the pox virus Modified Vaccinia Virus Ankara (MVA) and was used for parenteral vaccination against smallpox in about 150 000 vaccinations without causing any complications related to the vaccination. Even children with immunologic deficiencies did
20 not show serious side effects. The MvA was obtained by mutation and selection of the original vaccina virus Ankara after 575 passages in chicken embryo fibroblast cultures. The safety of this MVA is reflected by biological, chemical and physical characteristics. MvA has a reduced molecular weight, six deletions in the genome, and is highly attenuated for
25 mammalian cells, i.e. DNA and protein is synthesized but virtually no viral particles are produced. The Modified Vaccina virus Ankara developed by Anton Mayr was deposited at the European Collection of Cell Cultures (ECACC), Salisbury, UK, under depository No. V 94012707.
30 The vaccination against smallpox was highly successful. In 1979, the World Health Organization declared the eradication of smallpox. Accordingly, the

3
mass vaccination of children was discontinued and only laboratory workers and members of the armed forces of some countries are vaccinated.
With the eradication of smallpox, the predominant cause of pox infection in 5 humans was removed- However, some non-human poxviruses have reduced host specificity, i.e. they cause infections not only in their typical host (e.g. for cowpox the cow), but also in other animals, (e.g. rats and cats). Humans can be infected by this route as well. Since parts of the population are no longer immune against smallpox, orthopox infections of 10 animal species can be dangerous for them. Domestic animals are the main source of infection for humans. Accordingly, the vaccination of domestic animals against orthopoxviruses is of increasing importance. In addition, the MVA may be of significance as a vector for gene therapy, i.e. to transfer nucleic acid sequences into a target cell where they are expressed.
15
For a logarithmic reproduction of the MVA, cell cultures of primary or secondary chicken embryo fibroblasts are needed. The cells are obtained from chicken eggs that are incubated for 10 to 12 days. Since eggs are subjected to a biological variability, the cells obtained for the cell culture
20 system are variable on a cellular level as well. In addition, in a chicken embryo "fibroblast culture" often other cell types such as epithelial cells are found. This variation of the cells also results in variation of viruses produced in chicken embryo fibroblasts. It is therefore difficult to standardize and validate the cell culture system to guarantee a constantly
25 high quality of the MVA produced. Furthermore, contamination of the cell culture system by microorganisms or viruses already present in the incubated eggs can not be completely excluded. When the MVA is grown in virus-contaminated cells, the MVA may recombine with the contaminating virus. Thereby an MVA with new and unpredictable characteristics may be
30 generated. For the production of the virus in large scale in a suspension culture, primary or secondary chicken embryo fibroblasts are also not highly suitable. In addition, the purification and concentration of MVA by ultra

gradient centrifugation would be advantageous. However, such purification is difficult, when MVA is cultivated on primary or secondary chicken embryo fibroblast Finally, an increasing number of patients have developed allergies against chicken egg's albumen. Although the in vitro conditions of 5 the cultivation strongly reduce the allergenic potential, a hazard of an allergic reaction can not be completely excluded.
In conclusion, on the one hand the MVA can only be efficiently grown in primary or secondary chicken embryo fibroblasts causing a number of 10 disadvantages, however, on the other hand the save application of the M/A in humans has been shown by its large-scale application as a vaccine.
Object of the invention
15 ft is an object of the present invention to provide conditions for the production of homogeneous virus particles of the MVA Additionally, said conditions should allow an easy and large-scale production of the MVA.
Detailed description of the invention ,
20
To achieve the foregoing and other objects, the present invention provides an MVA strain that is adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic agent.
25 According to the present invention, for the first time an efficient and large-scale production of MVA is possible. Since cells of a continuous cell line are homogeneous and their characteristics are stable the MVA harvested from these cell lines is also homogeneous with highly predictable characteristics. Furthermore, the risk of contamination by microorganisms
30 can be controlled and contamination of the MVA preparation by proteins of the chicken egg - as found when cultivating MVA on chicken embryo

5
fibroblasts - can be excluded. The handling of a permanent cell line is convenient and thus highly suitable for industrial application.
In a preferred embodiment of the invention, the MVA is adapted for growing 5 in cells of a mammalian cell line, which is approved for the production of a vaccine. It has been surprisingly found that the MVA adapted to a mammalian cell line such as the Vera cell line still has a reduced virulence for humans and also for a wide range of other mammals. Accordingly, the MVA is highly attenuated i.e. DNA and protein is synthesized but virtually no 10 viral particles are produced, resulting in a virtually eliminated disease-causing capacity. Hence, the MVA according to the present invention is also highly suitable as a vaccine for humans and for a wide range of mammals. Accordingly, the MVA is especially applicable in the veterinary field.
15 Furthermore, a method to obtain an MVA strain according to the present invention is provided. According to this embodiment of the invention, cells of a cell line that is approved for the production of a therapeutic substance, are infected with the wild-type MVA. Preferably a high multiplicity of infection (MOI), i.e. a high number of viruses per cell is used for this infection. Then,
20 the viruses are harvested and fresh cells of the same cell line are infected with the newly produced viruses. Said process is repeated (serial passaging) until the MVA is adapted to said cell line. Adaptation is reached, when 72h post infection, the virus titer is at least 1- to 9-fold, preferably 10-to 99-fold, more preferably 100- to 106-fold, and most preferably more than
25 107-to 1010-foid increased compared to the input virus titer. The adaptation is reached after a limited number of passages.
"Adapted for growing" means that the amount of virus produced from an infection (Output) is increased compared to the amount of virus originally 30 used to infect the cells (Input). In this case the Output/Input ratio is greater than 1.

"Derivative" of the MVA deposited at ECACC, Salisbury, UK, under the depository number 99101431 and/or provisional accession number 01021411 means an MVA which is adapted for growing in Vero cells at a rate, which is essentially the same as the growth rate of the deposited 5 strain but carries at least one difference in its genome compared to the deposited strain.
The term "immune system" basically describes a complex involved in the defence of the organism against foreign substances and microorganisms. 10 It is divided into a cellular part comprising several cell types, such as e.g. f lymphocytes and other cells derived from white blood cells, and a humoral part comprising peptides and proteins such as antibodies, complement factors, and cytokins.
15 The term "immune response" describes the reaction of the immune system, when a foreign substance or microorganism enters the organism. Generally, the immune response is divided into a specific and an unspecific reaction although both are closely cross linked. The unspecific immune response is regarded as the immediate defence against a wide
20 variety of foreign substances and infectious agents. The specific immune response can be characterised as a highly efficient defence mechanism of ( the organism against a foreign substance which is raised against said substance after a lag phase and highly specific for said substance. The specific immune response is responsible for the phenomenon that an
25 individual who has recovered from a specific infection is protected against this specific infection in future.
"Activator of the immune system" means any substance capable of provoking or enhancing an immune response.
30 "Suppressor of the immune system" means any substance capable of reducing or inhibiting an immune response.

"Stabilizer of the immune system" means any substance capable of keeping the immune response on a constant level.
The inventors provide two preferred MVA strains that are adapted to an 5 African green monkey cell line, called Vera cell line (ATCC No. CCL-81). The MVA-strain, which was passaged 100-times in Vero cells was called "Vero-MVA" and deposited at the European Collection of Cell Cultures, Salisbury, UK under depositary No. 99101431. The MvA strain after 200 passages in Vero cells was called uVero-MVA-200' and deposited at 10 ECACC under provisional accession number 01021411.
The MVA obtained as described above is further amplified by cultivating the cells of the approved cell line under suitable conditions, infecting cells with the MVA and harvesting the viral particles produced by said cells. Hence the 15 MVA can efficiently and easily be amplified in large-scale. Surprisingly, the MVA of the invention does not show increased virulence in cells other than Vero cells such as human cell lines including HL, HEP-2 or HeLA.
In another embodiment of the invention, the MVA contains at least one 20 heterologous nucleic acid sequence i.e. a nucleic acid sequence that is not naturally found in the MVA genome (recombinant MVA). Preferably, the heterologous nucleic acid sequence is a gene, more preferably a gene encoding an immunizing protein, and most preferably encoding a protein immunizing against malaria, rabies and/or hepatitis. The expression of 25 said heterologous nucleic acid sequence is preferably under the transciptionaf control of a vaccinia virus promoter, more preferably of an MVA-own promoter. In a further preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted at a naturally occurring deletion site in the MVA genome (disclosed in PCT/EP96/02926).
30
The recombinant MVA is used for the introduction of a nucleic acid sequence into a target cell, said nucleic acid sequence being homologous

or heterologous to the target cell. The introduction of a heterologous nucleic > acid sequence into a target cell may be used to produce heterologous nucleic acids, peptides and/or polypeptides and/or proteins encoded by said nucleic acid sequence in vitro. This method comprises the infection of 5 a host cell with the recombinant MVA cultivation of the infected host cell under suitable conditions, and optionally isolation and/or enrichment of the peptide and/or protein produced by said host cell.
Furthermore, the introduction of a homologous or of a heterologous 10 sequence may be applied for in vitro and preferably in vivo gene therapy. , For in vitro and ex vivo gene therapy respectively, cells are isolated from the individual to be treated, transformed with the recombinant MVA and reintroduced into the individual the cells were taken from. For in vivo gene therapy, the recombinant MVA is directly administered to the living animal is body Including the human body. In a preferred embodiment of the invention, the recombinant MVA expresses an antigen or an antigenic epitope. Most preferably, said vector expresses an antigenic determinant from Plasmodium falciparum, Mycobacteria, Herpes virus, Influenza virus, hepatitis, or a human Immunodeficiency virus.
20
Since the MVA according to the invention is - surprisingly - still highly { attenuated, the MVA is ideal to immunize a wide range of mammals including humans. Hence, the present invention also provides a vaccine comprising the MVA for the immunization of a living animal body including a
25 human against pox infections, preferably orthopox infections. The vaccine may contain in addition to the MVA one or more additives such as an antibiotic, a preservative, or a stabilizer. The vaccine is especially applicable m the veterinary field, e.g. for the Immunization of animals against orthopox infections such as cats against cat pox, mice against
so ectromelia or camels against camelpox. The immunization is preferably performed parenterally.

9
The immunizing effect of an antigenic determinant in a vaccine is often enhanced by the addition of a so-called adjuvant. An adjuvant co-stimulates the immune system in an unspecific manner causing a stronger specific immune reaction against the antigenic determinant of the vaccine. 5 According to another embodiment of the invention, the MVA is used as an adjuvant, to co-stimulate the immune response against the antigenic determinant of a vaccine. In this case it is preferred that the MVA is inactivated. The inactivation of the MVA may be performed e.g. by heat or chemicals. Preferably, the MVA is inactivated by 0-propiolacton. According to 10 this embodiment of the invention, the inactivated MVA may be added to vaccines against, numerous infectious diseases to increase the immunity against this disease.
In case of an infection, the immune, the nervous, the hormonal and the
15 vascular system of an individual work closely together. These interactions can be regulated by elements of the unspecific immune system e.g. cytokines such as interferons and interleukins. Pox viruses can influence the regulation of the immune system (Swiss Vet 11/99, 13-17). Hence, in a further embodiment of the invention, the MVA and preferably the inactivated
20 MVA is used in mammals including humans to regulate the cellular and humoral elements of the unspecific (innate) immune system. Preferably the MVA is used as a bioregulator, wherein dysfunctions of the immune system are eliminated and the body's own defence mechanisms are activated, stabilized and/or suppressed. Most preferably, the MVA is used as a
25 bioregulator in case of a viral infection e.g. with herpes, hepatitis B or C virus, in case of a chronic inflammatory disease and/or to support tumor therapy. The MVA may also be used to stabilize the immune system in a situation of increased susceptibility against infections such as in the case of stress or in neonatals. The active and/or preferably the inactivated IWA
30 can be applied systemically e.g. intramuscularly and/or locally e.g. through mucous membranes and/or skin.

In conclusion, the present invention provides MVA strains that can in general be used for the same applications as the wifd-type MVA but eliminate the problems caused by the amplification of the wild-type MVA in chicken embryo fibroblasts.
5
Summary of the invention
The invention inter alia comprises the following, alone or m combination: 10
A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, said cell line being approved for the production of a therapeutic substance.
The MVA as above adapted for growing in cells of a mammalian cell line.
15 The MVA as above, wherein the cell line is approved for the production of a vaccine.
The MVA as above, wherein said approved cell line is a Vero cell line.
The MVA as above, wherein said approved cell line is the Vero cell line ATCCNo.CCL-81.
20 The MVA as above, deposited at the European Collection of Cell Cultures, Salisbury, UK under depositary Mo. 99101431 and/or a derivative thereof.
The MVA as above, deposited at the ECACC, Salisbury, UK, under provisional accession number 01021411 and/or a derivative thereof.
The MVA as above, comprising at least one heterologous nucleic acid 25 sequence.

1
II
The MVA as above comprising -a heterologous nucleic acid sequence coding e.g. for a therapeutic protein and/or an antigenic determinant such as a peptide immunizing against malaria, hepatitis and/or rabies infection.
A host cell infected by the above described MVA.
5 A composition, preferably a pharmaceutical composition, comprising the. above described MVA and/or the DNA of the MVA.
The pharmaceutical composition described above, wherein the pharmaceutical composition is a vaccine.
The vaccine described above for the immunization of a living animal body 10 including a human.
The vaccine as above for the immunization against an Orthopox infection.
The vaccine as above for the immunization of cats against a cat pox infection, mice against ectromelia infection and/or camels against cameJpox infection.
15 The pharmaceutical composition described above, wherein the MVA is an activator, suppressor and/or stabilizer of the unspecific immune system.
A pharmaceutical composition comprising the above described MVA and/or the DNA of the MVA as an adjuvant.
A pharmaceutical composition comprising the above described 20 recombinant MVA and/or the DNA of the recombinant MVA.
The pharmaceutical composition as described above for use in gene therapy.
A method for introducing a homologous and/or heterologous nucleic acid sequence into a target cell comprising infection of the target cell with the 25 above described MVA.

A method for obtaining an MVA strain as described above, comprising a) infection of ceils of an approved cell line with a wiid-type MVA preferably the MVA deposited at ECACC under depository No. V 94012707, b) harvesting 5 of the viruses, c) infection of fresh cells of the same ceil line with the newly produced viruses, and, optionally, d) repetition of b) and c) untii the virus is adapted to growth in cells of said cell line.
A method for producing viral particles of the above described MvA, 10 comprising cultivating the cells of an approved cell line under suitable conditions, infecting said cell line with said MvA. and harvesting the viral particles produced by said cells.
The method as described above, wherein said cell line is infected with the MVA deposited at the ECACC under depositary No. 99101431 and/or the 15 MVA deposited at the ECACC under provisional accession number 01021411 or a derivative of one of those strains.
A method for producing a nucleic acid sequence, a peptide polypeptide and/or protein, comprising infection of a host cell with the above described recombinant MVA, cultivation of the infected host cell under suitable 20 conditions, and, optionally, isolation and/or enrichment of the nucleic acid sequence, peptide and/or protein produced by said host cell.
Use of the above described MVA for producing a pharmaceutical composition for the treatment or prevention of a disease or disorder responsive to said MVA
25 Use of the above described MVA for producing a vaccine for the immunization of a living animal body including a human.
Use of the above described MVA for producing an activator, suppressor and/or stabilizer of the unspecific immune system.
The use as described above for the manufacture of an adjuvant.

Use of the above described MVA as a vaccine.
Use of the above described MVA as an adjuvant.
Use of the above described MVA as an activator, suppressor andVor stabilizer of the unspecific immune system.
5 A method for immunization of a living animal body including a human said method comprising administering to a person in need thereof a therapeutically effective amount of an above described pharmaceutical composition.
A method for introducing a homologous and/or heterologous nucleic acid 10 sequence into a target cell comprising infecting the target cell with the above described MVA and/or the DNA of the MVA.
A method for the activation, suppression and/or stabilization of the immune system of a living animal body including a human said method comprising administration of the above described pharmaceutical composition to a 15 living animal body including a human.
A method for enhancing a specific immune response against an antigenic determinant in a vaccine comprising administration of the above described MVA as an adjuvant to a Jiving animal body including a humanA Modified Vaccinia virus Ankara adapted for growing in cells of a continuous cell line obtainable by a process 20 comprising the following steps:infecting cells of a cell line being approved for the production of a therapeutic substance, harvesting the viral particles produced by said cell lines and optionally, repeating the above steps until the desired growth characteristics of said MVA are obtained in said cells.
25
Examples
The following examples will further illustrate the present invention, ft wiJJ be well understood by a person skilled in the art that the provided examples in

no way may be interpreted in a way that limits the applicability of the technology provided by the present invention to these examples.
5 Example 1: Adaptation of the MVA to Vero cells and characterization of said MVA strain
1. Adaptation of the MVA to Vero cells
io The by Anton Mayr developed wild-type MVA that is a modified Vaccina virus * Ankara was deposited at ECACC under depository No. V 94012707. The wild-type MVA was adapted to grow in Vero-cells by serial passaging of the virus in Vero cells (Table 1). The cell clone ATCC-No. CCL-81 of the stationary Vero cell line (WHO seed stock ECACC No. 88020401) was
is used in the passage No. 148 to 165 (WHO seed lot, Master and Working Bank). The cells were propagated in a medium consisting of Earle's MEM (ICN), pH 7,4 - 7,6, and 5% of the serum substitute BMS (Biochrom). According to a technique known by people skilled in the art, always the same cells of the working bank were seeded by splitting the cells 1:2 to 1:4. 20 The medium contained approximately 250 000 cells per ml. The cells were
respectively propagated in tubes (2ml), Roux dishes (100ml), and plastic (^ dishes (6 and 40ml respectively). In general, the cells formed a confluent monolayer after 16 to 24h. Afterwards, the medium was replaced by plain Earle's MEM without any additives.
25
For the adaptation of the wild-type MVA a tube culture system was used. The results of the passages are summarized in Table 1 and 2. The Vero cells were infected by 10 MOI (multiplicity of infection) of the wild-type MVA i.e. in average, 10 viral particles per Vero cell. The wild-type MVA to start with 30 was a genetically homogeneous, plaque-purified MVA after 575 passages in chicken embryo fibroblasts (titer: 107,75 KID50/mI). After 24h, 90% of the Vero cells of the confluent monolayer were destroyed by toxic processes

(50% by toxicity, 40% by lysis). The medium plus the cell dedritus after freezing and thawing of the cells, containing the produced viruses, was harvested and 0,2ml of this mixture were seeded on the monolayer of Vero cells in the. culture tubes (2nd passage). This procedure1 was repeated 200 5 times. After the third passage, no toxic effect was observed any more, whereas a mild cytopathic effect (CPE) characterized by rounding of the. cells and lysis in a period of 4 to 6 days post infection (p.inf.) was seen. The virus titer was 101,0 KlD50/ml. It was concluded that the proliferation of the MVA in Vero cells had started although very inefficiently. After the fifth
10 passage, a typical CPE was observed which was completed after 4 to 5 days p.inf. The virus titer increased from 101,0 KlD50/ml after the third passage to 104,0 KID50/ml after the fifth passage. Hence, the virus amplified more efficiently in Vero cells. In the passages No. 5 to 11, a complete CPE was observed more and more early and the virus titer increased with every
15 passage. At passage No. 11, a plateau was reached at 10' KIDa/ml. Accordingly, after eleven passages the adaptation of the MVA to Vero cells was achieved. In the following 30 additional passages, the results were for all passages the same and highly reproducible: The CPE began already 24h p.inf. and all cells were affected after three days p. inf. At that time, 20%
20 of the Vero cells were rounded and 80% were lysed. After three days p.inf., the virus titer was always about 107'75 KJD50/ml. After the fifteenth passage, the viruses were always harvested after two to three days p.inf., and only 1 MOI instead of 10 MOI were used to infect the cells (Table 2). In the following additional passages the growth characteristics of the MVA
25 changed only slightly. Remarkably, the optimum virus titer further increased and reached 1010 Klp50/ml at passage 200.
In conclusion, the virus grows reproducibly in an exponential manner in Vero cells. Said growth characteristic is surprisingly different to the 30 characteristics of the wild-type MVA. Accordingly, a new strain of the MVA was obtained by the serial passaging. Said new strain was called "Vero-MVA" and after passage 200 in vero cells "Vero-MVA-200".

The Vero-MVA and Vero-MVA-200 were cultivated in larger quantities. For storage, the Vero-MVA was concentrated by centrifugation, resuspended in 2,5 % polygeline and lyophilized in vials of 2ml. The titer after lyophilization 5 was still at least 108,5 KJD50/ml. The lyophilized Vero-MVA and Vero-MVA-200 was checked for contamination and toxicity and stored at +4°C.
2. Characterization of the biological properties of the Vero-MVA
10 The biological characteristics of the Vero-MVA (passage 100) and Vero- r MVA-200 (passage 200) were compared with the characteristics of the wild-type MvA (Table 3 and Table 5). Thereby, the techniques known by the skilled practitioner were applied. The inventors showed that neither the host range of the virus was changed except for the Vero cells, nor the
is virulence for humans or animals was increased. The Vero-MVA is still characterized by the abortive propagation in non-permissive host cells.
The principal identity of the viral particles of the Vero-MVA compared to the viral particles of the Elstree strain of the Vaccinia virus was shown by cross
20 reactivity of antibodies raised against the Elstree strain. The Elstree strain
is a Vaccinia strain recommended by the WHO for the smallpox vaccination. C The polyclonal hyperimmune serum of rabbits raised against the Elstree strain was added to the Vero-MVA. 100 KID50/ml of the Vero MVA were completely neutralized at a dilution of the serum of 1:512. A twofold dilution
25 of the serum was necessary to neutralize the same amount of Vaccinia Elstree strain (1:256). Accordingly, the Vero-MVA can still be efficiently neutralized by Vaccinia immune serum.
The Vero-MVA, the Vero-MVA-200 and the wild-type MVA were compared by 30 a number of additional tests as indicated in Table 3, 4 and 5. The inventors showed that the virulence of Vero-MVA and Vero-MVA-200 for mammals • including humans was not increased compared to the wild-type MVA. It was

also shown that the Vero-MVA and Vero-MVA-200 are not contagious or toxic for mammals including humans. Surprisingly, the cell specificity of the Vero-MVA was more or less identical to the specificity of the wild-type MvA except for the Vera cells: The Vero-MVA amplifies nearly as inefficiently in 5 cells of human cell fines (see table 4: HL-, HEP-2-, and HeLa-cefls) as the wild-type MVA does. Accordingly, although human cells and cells of African green monkeys are phylogenetically closely related, the Vero-MVA did not gain the ability to amplify in human cells. In other tests, no significant difference were seen either.
10
Furthermore, the physical, chemical, and biological characteristics of the wild-type MVA and the Vero-MVA-200 were compared (Table 5). Whereas the wild-type MVA growing in chicken embryo fibroblast cell cultures has three deletions in the left inverted terminal region, the Vero-MVA-200 has 15 four deletions in the left terminal region compared to the genome of the pox virus as originally isolated in Ankara. Hence, passaging of the wild-type MVA in Vero cells resulted in an additional deletion.
The Vero-MVA was used to immunize domestic animals against Orthopox 20 infections. The serum of the animals was collected and a neutralization test was performed. The inventors showed that the animals produced antibodies in high titers. The antibody titers were stable over a period of at least 111 days. It was also shown that the antibodies were able to neutralize in vitro viral particles of the MVA in a plaque-reduction test. In 25 conclusion, the Vero-MVA can be used as a vaccine against Orthopox infections in domestic animals and in humans.

Table 1: Adaptation of the MVA to Vero cells

Passage No. Cell culture Highest virus titer [log10/mrj Result Conclusion
1 toxic effect after 24h 2,0 Rests of the virus seeded Blind passages
Phenomenon of zones and cytokine production
3 No toxicity, moderate CPE after 4-6 days 1,0 Rests of the virus seeded?
Begin of the
virus
reproduction

5 typical CPE
completed after 4-5 days 4,0 Increasing virus reproduction

11 CPE
completed after 3 days 7,5 Logarithmic
virus
reproduction Adaptation successful
12-42* CPE begins after 24h, completed after 3 days 7,75 Reproducible
virus
reproduction Vero-MVA
43-100* CPE begins after 24h, completed after 3 days 8,0 Repoducible
virus
reproduction Vero MVA
100-200* CPE begins after 24h, completed after 3 days 10,0 Repoducible
virus
reproduction
_ — Results in Vero-MVA-200
* Only 1 MOI instead of 10 MOI are seeded after the eleventh passage.

Table 2: Change of the virus titers during the adaptation of the MVA to Vero cells
Passage No. Harvested after [days Titer per ml [log10/ml]
p.inf. ]

1 1 2 3 2,0
3 5 1,0
5 5 4,0
8 4 6,5
11 , 3 7,5
18 2 8,0
19 2 7,75
20 3 8,0
25 2 7,75
29 2 7,75
30 3 7,75
31 3 8,0
45 2 7,75
51 3 7,75
60 v 2 8,0
66 2 7,75
68 2 8,0
75 3 8,0
100 2 8,0
200 2 10,0

Table 3: Comparison of the biological characteristics of the wild-type MVA and Vero-MVA

Marker

Wild-type MVA

Vero-MVA (100. passage) Vero-MVA-200

CPE in monolayer ceil cultures (1 MOI seeded)
Titer of the optimal harvest
Abortive virus reproduction in non-permissive ceil systems
Reduced virulence for humans and animals
Contagiousness
Character of the primary plaques on the chorion allantois membrane
Hemagglutination (chicken erythrocytes)

Rounding and lysis of the cells after day 5 (90% CPE)
108'0 KIDso/m!
Yes
Yes
No
No proliferative nodes without necrosis
Negative

Rounding and iysis of the cells after day 5 (100% CPE)
107'75 KID50/mI
Yes
Yes
No
No proliferative nodes without necrosis
Negative

Rounding and lysis of the cells after day 3 to 5 (100% CPE)
10i0,0KlD50/ml
Yes
Yes: not virulent at all
No
No proliferative nodes without necrosis
Negative

Inactivatlon by ft-

Kinetic of first order for

Kinetic of first order for

Kinetic of first order for

0,05%
propiolactone
Protective effect in VSV- Yes
baby-mouse challenge
test
Toxicity for humans and No animals
Cytokine stimulation Interferon a, IL-2, and 12,
CSA
Actuation of phagocytosis, Yes natural killer cells, and T-[ymphocytes

0,05% 0,04-0,05% ~~ §
Yes Yes S
00 00
o
No No
Interferon a, IL-2, and 12, Interferon a and y, IL-1,2,
CSA and 12, CSA
Yes Yes, increased

M

3
-4
O

22
Table 4: Reproduction rate in KlD50/mI of Vero-MVA and the wild-fype MVA in different cell culture systems [log10/ml]
Cell culture system Vero-MVA Wild-type MVA (575.
(31 .Vero-passage) passage in primary
chicken embryo fibroblasts)
1) Vero (African green 8,0 4,5
monkey kidney cells)
Primary chicken 4,5 8,5
embryo fibroblasts
1'2) HL (human lung) 3,0 2,5
1'2) HEP-2 (human 3,0 2,5
epidermoid carcinoma)
1,2)HeLA (human cervix 2,75 2,75
carcinoma
1>2) BHK (hamster 5,75 5,25
kidney cells)
^MDBK (bovine 3,5 3,5
kidney cells)
1'2) PK-15"(porcine 3,25 3,5
kidney cells) ,
1) Continuous cell line derived from the tissue and species indicated in brackets.
2) Cell lines obtained from the collection of the institute of medical microbiology in Munich, Germany.

23
rTabIe 5: Comparison of the wild-type IWVA (572. passage in chicken embryo fibroblasts (CEF)) with Vero-MVA-200 (200. passage in vero cells)

Marker

Wild-type MVA

Verc-MVA-200

Genetic markers 3 deletions in the left (comparison with terminal region (inverted pox virus strain as terminal repeat) isolated in
Ankara) Genome size reduced
from 208 to 178 kb
Loss of 15% of the molecular weight of the original genome
Cellular markers
Cytokine Virus titer
Immune system

Loss of the interferon receptor
Activation of T-helper cells (CD4, CD8, CD25)
Activation of NK cells
Abortive reproduction in mammalian cells (except BHK cells)
Interferon a, IL-2, IL-12
CEF:109'6KlD50/ml Vero cells: 104,0KID50/ml
Reduction of activity of specific immune system

4 deletions in the left terminal region
Further reduction of the genome size to 172kb
Loss of 20% of the molecular weight of the original genome
Additional Joss of receptors e.g. for 1L-1J5
Increased activation of cytotoxic T-lymphocytes
Increased activation of NK cells
Further narrowing of the host spectrum in cell culture systems
interferon a and y, JL-1,2, 12
CEF:104,5KID5o/ml Vero cells: 109^KID50/ml
Inhanced activity of the unspecific immune system

Virulence for humans and animals

low none

WO 01/68820

PCT/EP01/02703

24

Applicant's or agent's file. reference number

: BN 33 PCT

International appticatioii No. '■

.'PCT/EP 01/02703

INDICATIONS RELATING TO ADEPOSTED MICROORGANISM
(PCT Rule libis)
A. TheindicadorismadebelowiebtetotherniCTOorganismrefem^toipthedescription
onpage 6,7,10,12,24 tUne 3,10,23,16,25

B. IDENTlFICATIONOFDEPOSrr

Furtherdepositsareidentifiedonanadditionalsheet ) j

Nameofdepositaryinstitution ECACC
Centre for Applied Microbiology and Research & European Collection of Ceil Cultures
Address of depositary institution (including postal code and country)
Salisbury
Wiltshire SP40JG United Kingdom

Date of deposit

AccessionNumber

February 15,2001

(Provisional) 01021411

C ADI>lTlONALINDlCATIONS(Uave blank if not applicable) This information is continued on an additional sheet Q
In respect of all designated States to which such action Is possible and to the extent that it is legally permissible under the law of the designated State, it is requested that a sample of the deposited microorganism be made available only by the issue thereof to an independent expert, in accordance with the relevant patent legislation, e.g., EPC Rule 28(4); UK Patent Rules 1995, Schedule 2, Paragraph 3; Australian Regulation 3.25(3); Danish Patents Act Sections 22 and 33(3) and generally similar provisions mutatis mutandis for any other designated State.
D. DESIGNATEDSTATESFORW^CnmDICATLONSAJCEWa)E(ifthemdicaiionsaTenoiforattd^gnatedStates)
£. SEPAI^TEFURNISHINGOFINDICATIONSf/MveWanJtifnoi^pIicaWe)
The indications listed below will be submitted to the International Bureau later (spec%ytoegenerdnature qj the indications '«$, "Accession Number of Deposit")
Copy of the final Certificate of Deposit

Forreceiving Office use only

For International Bureauuse only

This sheet was received with the international application

I | ThissheetwasreceivedbythelntemationalBureauon:

Authorizedofiicer
CLAUDIA ARAGO!
Only 1992)
-RTMI DTT/DA/I'M.

Authorized officer

We Claim
1. A modified vaccinia virus Ankara (MVA) adapted for growing in cells of a continuous cell line, wherein said cell line is a Vero cell line.
2. The MVA as claimed in claim 1, wherein said cell line is Vero cell line ATCC No.CCL-81.
3. The MVA as claimed in claim 2, deposited at the European Collection of Cell Culture (ECACC) Salisbury, UK, under depository No. 99101431 and/or derivative thereof.
4. The MVA as claimed in claim 2, deposited at the European Collction of Cell Cultures (ECACC), Salisbury, UK, under depository No. 01021411 and/or derivative thereof.
5. The MVA as claimed in any of the preceding claims 1 to 4 comprising at least one heterologous nucleic acid sequence.
6. The MVA as claimed in claim 5, wherein the heterologous nucleic acid sequence codes for a therapeutic protein and/or antigenic determinant.
27

7. A composition preferably a pharmaceutical composition, comprising the MVA and/or DNA of the MVA as claimed in any of the preceding claims 1 to 6 and the conventional additives such as stabilizer.
8. The composition as claimed in claim 7, wherein the composition is a vaccine.
9. The vaccine as claimed in claim 8 is applicable for the immunization of a living animal body including a human.

10. The vaccine as claimed in claim 8 or 9 is applicable for the immunization against an Orthopox infection.
11. The vaccine as claimed in claim 8 to 10 is applicable for the immunization of cats against a cat pox infection, mice against ectromelia infection and/or camels against camel pox infection.
12. The composition as claimed in claim 7 wherein the MVA is an activator, suppressor and/or stabilizer of the immune systems
28

13. A composition, preferably a pharmaceutical composition, comprising the MVA and/or DNA of the MVA as claimed in any of the preceding claims 1 to 6 as an adjuvant and the conventional additives such as stabilizer.
14. A composition preferably a pharmaceutical composition, comprising the recombinant MVA and/or DNA of the recombinant MVA as claimed in claim 5 or 6.
Dated this the 12th day of September 2002
(R.P. Bhattadharya) Of DEPENNING & DEPENNING Agent for the Applicants

ALTERED STRAIN OF THE MODIFIED VACCINIA VIRUS ANKARA (MVA)

Documents:

Inventors:

PCT Conventions: