HK1171249A

HK1171249A - Recombinant poxvirus expressing homologous genes inserted into the poxviral genome

Info

Publication number: HK1171249A
Application number: HK12112112.4A
Authority: HK
Inventors: 保罗．豪利; 桑加．莱勒
Original assignee: 巴法里安诺迪克有限公司
Priority date: 2002-05-16
Filing date: 2012-11-26
Publication date: 2013-03-22

Description

Recombinant poxvirus expressing homologous genes inserted in the poxvirus genome

The application is a divisional application of the invention named as 'recombinant poxvirus expressing homologous genes inserted into the genome of poxvirus', which is applied for 5/14/2003 and China application No. 03811106.3.

The present invention relates to recombinant poxviruses capable of expressing two or more homologous foreign genes, wherein said genes are heterologous to the viral genome but have homology to each other. Said genes are derived in particular from highly related variants or subtypes of microorganisms. The invention further relates to a method for preparing such recombinant poxvirus and to the use of such recombinant poxvirus as medicament or vaccine. Furthermore, the invention provides a method for influencing, preferably inducing, an immune response in a living animal, including a human.

Background

Each live infection or pathogenic agent such as bacteria, viruses, fungi or parasites. The so-called immune system of the organism prevents them from persistent infections, diseases or intoxications caused by these pathogenic agents.

The mammalian immune system can be divided into specific and non-specific parts, although these two parts are tightly cross-linked. Non-specific immune reactions can mediate instant defenses against a wide variety of pathogens or infectious agents (agents). The specific immune response develops after a lag period when the organism is challenged with the substance for the first time. The specific immune response is mainly due to the production of antigen-specific antibodies and the formation of macrophages and lymphocytes, such as cytotoxic T-Cells (CTLs). The so-called specific immune response is based on the fact that individuals with a specific infection are able to recover, but are still susceptible to other infections. Often, a second infection with the same or very similar infectious agent causes very mild symptoms or no symptoms at all. This so-called immunization lasts for a very long time, and in some cases even for life. This potential effect is commonly referred to as immunological memory, and can be used for vaccination.

The term vaccination describes a method in which an individual is challenged with a non-toxic, partial or inactivated form of an infectious agent to affect, preferably induce, an immune response in said individual resulting in prolonged, if not lifelong, immune resistance to the specific infectious agent.

Smallpox disease in humans is caused by smallpox (Variola) virus. Smallpox belongs to the family of Poxviridae (Poxviridae), a family of viruses with large complex DNA that replicate in the cytoplasm of vertebrate and invertebrate cells.

The poxviridae can be divided into two subfamilies, the Chordopoxvirinae (Chordopoxvirinae) and the entomopoxviridae (entomopoxxvirinae), depending on the host range of parasites on vertebrates and insects. The chordopoxvirae subfamily includes viruses of the genera orthopoxvirus (Orthopoxvirues) and fowlpox virus (Avipoxvirus) as well as some other genera (Fields Virology, ed. by Fields B.N., Lippincott-Raven Publishers,3rd edition 1996, ISBN: 0-7817-.

The genus orthopoxvirus includes variola virus, which is a pathogen causing variola in humans, and other very important viruses such as camelpox virus, vaccinia virus, sheep pox virus, goat pox virus, monkey pox virus and vaccinia virus. All members of this genus have genetic relatedness, similar morphology and host range. Restriction enzyme mapping has even shown up to 90% sequence identity between different members of the orthopoxvirus genus (Mackett & Archard, [1979], J Gen Virol,45: 683-701).

Vaccines that have been vaccinated against smallpox for at least 100 years are called Vaccinia Virus (VV). It is unclear whether VV is derived from vaccinia virus or vaccinia virus by prolonged serial passage-a live representative that has been eradicated at present or is the product of genetic recombination. Furthermore, during the history of VV, many vaccinia lines were formed. Different strains exhibit different immunogenicity and are involved in varying degrees of potential complications, the most severe of which can cause post-vaccination encephalitis. However, many of these strains were used for vaccination against smallpox. For example, NYCBOH, Western Reserve or Wyeth strains are used mainly in the United states, while the Ankara, Bern, Copenhagen, Lister and MVA strains are used in Europe. WHO implemented a global vaccination program of different strains of VV in 1980 eventually declared successful eradication of smallpox virus.

To date, VV has been used primarily in laboratory strains and, in addition, has been considered as a prototype of orthopoxvirus, which is why VV has become one of the most studied viruses (Fields Virology, ed. by Fields B.N., Lippincott-Raven Publishers,3rd edition 1996, ISBN: 0-7817-. VV and other recently used poxviruses have been used to insert and express foreign genes. The basic technique for inserting foreign genes into live infectious poxviruses involves recombination between poxvirus DNA sequences flanking the foreign genetic element on the donor plasmid and homologous sequences present in the rescue poxvirus. Genetic recombination is typically an exchange between homologous portions in two DNA strands. Some viral RNA may replace DNA. Homologous portions of nucleic acids may be nucleic acid sequences (DNA or RNA) having the same nucleotide base sequence. In infected host cells, genetic recombination may occur naturally during the production and replication of new viral genomes. Thus, when a host cell is infected with two or more different viruses or other genetic constructs, genetic recombination between the viral genes may occur during the viral replication cycle. The portion of the DNA sequence from the first genome is used interchangeably in the construction of a virus that co-infects with a second, wherein the DNA has homology to the DNA of the first viral genome.

Successful expression of an inserted DNA genetic sequence by modification of infectious virus requires two conditions. First, the insertion is in a non-essential region of the virus so that the modified virus is still viable. A second condition for expression of the inserted DNA is the presence of a promoter suitably associated with the inserted DNA. Typically, the promoter is located upstream of the DNA sequence to be expressed.

The use of recombinant VV expression, such as hepatitis B virus surface antigen (HBsAg), influenza virus hemagglutinin (InfHA), or Plasmodium falciparum sporozoite antigen (Plasmodium knowlesi spozoites) as an active vaccine to prevent infection with disease has been demonstrated and reviewed (Smith, et al, 1984 Biotechnology and Genetic Engineering Reviews 2, 383-.

Another advantage of VV is the ability of a single VV genome to carry multiple foreign sequences, genes or antigens (Smith & Moss [1983], Gene,25(1): 21-28). Furthermore, it has been reported that a single vaccination with a multivalent vaccine is capable of eliciting immunity to multiple heterologous infectious diseases (Perkus et al, [1985], Science, Vol.229, 981-984).

An example of expressing multiple antigens using a single VV is described by Bray et al. The recombinant VV is capable of expressing three different structural proteins of Dengue (Dengue) virus serotype 4, namely the capsid (C), pre-membrane (prm), envelope (E) proteins, and two different non-structural proteins of Dengue virus serotype 4, namely NS1 and NS2a, which protect mice against challenge with the homologous Dengue virus serotype 4 (Bray et al, [1989], Virology 2853-.

There are 4 serotypes of dengue virus, namely dengue virus serotype 1(Den-1) to dengue virus serotype 4 (Den-4). Dengue virus is one of the important members of the Flavivirus (Flavivirus) genus for being able to infect humans. Dengue Hemorrhagic Fever (DHF) of shock symptoms (DSS) can range from influenza-like symptomatic disease to severe or fatal disease. Dengue virus outbreaks remain a major public health problem in densely populated areas of tropical and subtropical regions, where there is a large number of transmission vectors, mosquitoes.

Since dengue virus infections and other diseases caused by mosquito-mediated flaviviruses are spread in many parts of the world, great efforts have been invested in the development of dengue virus vaccines that can prevent Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) and enable vaccinated individuals to fight against diseases caused by some or all mosquito-mediated flaviviruses.

Whereas most cases of DF are evident in a first infection with any of the 4 serotypes, a comparatively more DHF disease occurs in subjects infected with a second infection with a serotype different from the serotype of the first dengue virus infection. This phenomenon makes it possible to assume that individuals having antibodies to one dengue virus serotype are infected with different virus serotypes in succession at appropriate intervals, which may lead to the development of DHF in a considerable number of cases.

Thus, vaccination against one serotype does not yield complete protection against dengue virus infection, but only against infection by the same dengue virus strain. More importantly, humans vaccinated against one serotype are more likely to develop severe complications such as dengue hemorrhagic fever when infected with dengue viruses of a different serotype.

Thus, there is a need for multivalent vaccines against all 4 dengue virus serotypes.

It has been proposed to date to prepare multivalent vaccines by a panel of recombinant VVs, each of which encodes a different viral sequence (Moss, [1990] Immunology,2, 317-. However, such multivalent vaccines have several disadvantages. First, it is very cumbersome to obtain several independent recombinant VVs. In addition to the separate generation procedure, quality control and quality assurance are very time consuming. Secondly, infection with a mixture of recombinant viruses expressing different sequences always presents the problem that the infection is not particularly balanced. The main risk is that only a single recombinant, rather than all the different recombinants contained in the multivalent vaccine, infect the target cell. One reason for this may be the uneven distribution of the recombinant virus. Another reason is that there may be interference between different recombinant viruses when infecting single cells. This interference phenomenon is known as superinfection. In this case, only certain antigens, but not all the different antigens in the multivalent vaccine, are ultimately expressed by the infected cells and are thus presented to the patient's immune system. As a result, only immune protection against certain antigens is obtained, and complete immune protection against a variety of different antigens presented or presentable by multivalent vaccines is far from being provided.

In vaccines against dengue virus infection, the different sequences are expressed in different amounts or in an unknown manner, which also causes disadvantages for multivalent vaccines, such as the envelope protein of dengue virus 2 (Deuble et al, [1988], J.Virol 65:2853), which is very dangerous for the patient. Incomplete vaccination with the recombinant vaccinia virus group provides only immune protection against dengue virus of some, but not all, serotypes. Unfortunately also, incomplete vaccination in the case of dengue virus is highly unacceptable as it increases the chances of fatal complications such as dengue hemorrhagic fever.

Object of the Invention

It is therefore an object of the present invention to provide a stable, effective and reliable vaccine for immune protection against infectious diseases caused by pathogenic microorganisms of more than one strain, clade, variety, subtype or serotype.

It is a further object of the present invention to provide a stable, effective and reliable vaccine against dengue virus infection that provides reliable vaccination against all dengue virus serotypes.

Detailed description of the invention

The invention is based on the inclusion of infectious disease-causing microorganisms from different strains, clades, variants, subtypes or serotypes in the poxvirus homologous genes. As already mentioned above, for example, 4 groups, subtypes or serotypes of dengue virus all comprise the same type of gene, e.g.a gene encoding a capsid (C) protein, a gene encoding a pre-membrane (PrM) or envelope (E) protein. However, there is no complete identity or homology between the nucleic acid sequences of the same type of gene from 4 serotypes of the virus, for example, sequence comparison (Lasergene 4.05Magalign, Macintosh) between the PrM genes of dengue serotypes 1, 2, 3and 4(PrM1-4) reveals sequence identity of 66.5-72.9%, i.e., homology of 65-75%. It is envisaged that there will be differences and variations between the genes of different subtypes of infectious disease-causing microorganisms, which results in vaccination against one subtype not spontaneously providing protection against infection by other variants of the same microorganism. Thus, recombinant viruses can be produced that include very related or homologous genes from infectious disease-causing microorganisms of different strains, clades, varieties, subtypes or serotypes. However, as already mentioned above, homologous recombination between homologous sequences can occur in the viral life cycle, even between portions of DNA sequences that are not completely homologous. Thus, it can be expected that insertion of homologous genes into a single viral genome can cause homologous recombination, thereby generating a deletion in the inserted homologous genes.

However, when a recombinant vaccinia virus is produced that includes at least two foreign genes having at least 60% homology in its genome, the homologous genes are not expected to remain stable when inserted into the viral genome.

Even homologous genes, preferably at least 50% homologous, are inserted into the viral genome at different insertion sites, said homologous genes inserted in the viral genome still remaining stably inserted into the viral genome. In this case, it is expected that recombination events occurring between the homologous genes could additionally lead to the deletion of viral genes important for amplification of the virus or the viral life cycle, respectively, i.e. it is expected that the viral life cycle will be severely impaired. Thus, furthermore, the recombination frequency is proportional to the distance of two linked genes, it is expected that the recombination frequency between two or more homologous genes located at different insertion sites will be very high and thereby lead to deletion and/or severe interference of the genes. Accordingly, it was very surprising to find that no recombination events occurred, while the homologous genes inserted at different insertion sites in the viral genome remain stable.

According to the prior art, recombinant poxviruses comprising exogenous DNA from flaviviruses, such as Japanese Encephalitis Virus (JEV), Yellow Fever Virus (YFV) and dengue virus, are known (us 5514375). However, each gene from the flavivirus is only inserted a single time and at the same insertion site. Furthermore, sequence comparison with appropriate computer software (Lasergene 4.05Magalign, Macintosh) revealed homology of the genes inserted into the poxvirus genome, indicating that the genes from JEV have 20.2% to 29.6%, YFV have 29.2% to 45.3%, and dengue virus have 22.8% to 29.5%.

A similar disclosure is found in WO 98/13500, which describes the insertion of a dengue Virus antigen into the same insertion site, in particular into deletion site II, of a Modified Vaccinia Virus (Modified Vaccinia Virus) ankara (mva).

Us patent 5338683 describes the insertion of glycoprotein genes gp13 and 14 of herpes virus (herpesvirus) into two different sites of a single poxvirus, however, the two genes are only 25.2% homologous.

The sequence homology between influenza hemagglutinin and the nucleoprotein gene inserted into the same insertion site (deletion site III) of Modified Vaccinia Ankara (MVA) is 49.1% (U.S. Pat. No. 5,676,950; Sutter et al, [1994], Vaccine 12: 1032).

U.S. Pat. No. 5,891,442 discloses recombinant viruses comprising the coding sequences for the polyproteins VP2, VP 3and VP4 of the infectious burst disease. The genes are fused and inserted into a single insertion site, and the homology between the genes is 41.9% -50.3%.

Finally, U.S. Pat. No.6,217,882 describes a recombinant suipoxvirus vector comprising the pseudorabies antigens gp50 and gp63 inserted at the same site and having a homology of 52.7% with respect to each other.

In summary, it is considered from the prior art that homologous genes or sequences having at least 50% homology are all the same or a single insertion site into the viral genome.

According to the present invention, homologous genes or sequences having at least 50% homology means homology of 50 to 100%, i.e. at least 50% of the nucleotide bases are identical. Genes or sequences with less than 50% homology are considered to be heterologous. In the context of the present invention, the term "homologous" or "homology" is used to describe the situation when comparing genes or sequences, whereas the term "foreign" gene, "exogenous" or "heterologous" sequence is the situation when comparing genes or sequences to the genome of a poxvirus, i.e. the term means that DNA sequences are not normally associated in nature with the poxvirus used in the present invention. The present invention thus relates to a recombinant poxvirus comprising at least two genes which are heterologous in relation to the viral genome, but which have homology to each other. The term "gene" refers to a coding sequence that encodes, for example, a protein, polypeptide, peptide, antigen, etc. Proteins, polypeptides or peptides translated from homologous genes perform the same function (task) and exhibit the same functional properties. Homologous genes are usually from different but related sources or organisms. According to one embodiment of the invention, the homology of the coding sequences is preferably 70% to 80%, more preferably 80% to 90% or 90% to 100%. Most preferably, there is 65% to 75% homology.

Since the recombinant poxvirus according to the invention contains the relevant genetic information only in a single infectious unit or in the same viral particle, there is no problem of uneven infection and unbalanced expression of different homologous sequences. Thus, the recombinant poxvirus according to the invention, comprising and capable of expressing several closely or even very closely related genes or almost identical sequences in an infected cell, is particularly advantageous for the production of multivalent vaccines.

This benefit is of great interest in developing vaccines against diseases caused by several closely related strains or serotypes of viruses such as dengue virus. Recombinant poxviruses comprising homologous genes of different dengue virus serotypes are described in the examples.

The homologous genes or sequences according to the invention may be derived from any microorganism, such as any virus, any bacterium, fungus or parasite other than the vector virus. Preferably, the homologous genes or sequences are derived from infectious or pathogenic microorganisms, most preferably from different strains or clades, varieties, subtypes or serotypes of said microorganisms.

The term "strain" or "clade" is used as a technical term to describe the classification level of a microorganism and is well known to those skilled in the art. The classification system to date classifies all identified microorganisms into hierarchy order, family, genus, species, strain (Fields Virology, ed.by Fields b.n., Lippincott-Raven Publishers,4th edition 2001). The taxonomic criteria for the members of the family are their phylogenetic relationships, the genus includes all members with common traits, the species is defined as the polytopic class (polytopic class), constitutes the replication lineage and is present in a specific niche. The term "strain" or "clade" is used to describe the taxonomic hierarchy of microorganisms, i.e., viruses, that share common characteristics, such as basic morphology or genomic structure or organization, but differ in biological properties, such as host range, tissue tropism, geographic distribution, attenuation, or pathogenicity. The term "variant" or "serotype" is further used to distinguish between members of the same strain, also called "subtypes", between members exhibiting a single spectrum of infection or antigenic property that differs due to minor genomic variations.

According to a further embodiment of the invention, the homologous gene or sequence is selected from viruses, preferably viruses belonging to the genus flavivirus, such as preferably but not limited to dengue virus, West Nile (West Nile) virus or japanese encephalitis virus; viruses belonging to the genus of Retroviruses (Retroviruses), such as preferably but not limited to Human Immunodeficiency Virus (HIV); viruses belonging to the genus enterovirus (Enteroviruses), such as preferably but not limited to Hand (Hand), Foot (Foot) and oral (Mouth) diseases, EV 71; viruses belonging to the genus rotavirus (Rotaviruses) or viruses belonging to the genus Orthomyxoviruses (Orthomyxoviruses), such as preferably but not limited to influenza viruses. Most preferred are homologous genes derived from flavivirus.

According to a further embodiment of the invention, the homologous gene is selected from the group consisting of dengue virus genes, preferably C, NS1 and/or NS2, or preferably E, more preferably PrM. Most preferred are homologous genes derived from different serotypes of the virus, wherein the genes may be from 1, 2,3 or 4 serotypes of all 4 serotypes of the dengue virus.

According to a further embodiment of the invention, the homologous genes are selected from different strains of HIV or clades. Preferred homologous genes are selected from the gag/pol coding sequences, more preferably env coding sequences or even more preferably structural and/or regulatory coding sequences of HIV.

Suitable viral vectors for use in the present invention are selected from poxviruses which are readily cultured in selected host cells, such as avian host cells, but are highly replication deficient or virtually incapable of replication in human or human cells.

According to certain preferred embodiments, the poxvirus of the invention is selected from the group consisting of canarypox (canaryox) virus (Plotkin et al [1995] Dev Biol Standard.vol 84: pp 165-170.Taylor et al [1995] Vaccine, Vol 13.No.6: pp 539 549), fowlpox virus (Afonso et al [2000] J Virol, pp3815-3831.Fields Virology, ed.by Fields B.N., Lippincott-Raven Publishers,4th edition 2001, Chapter 85: page 2916), penguin pox (Stanngulin pox) (Stannard et al [1998] J Gen Virol,79, 1637-) or derivatives thereof. Since these viruses belong to the genus avipox virus (avipoxvirues), they are very easy to culture and propagate in avian cells. However, they are replication-defective in humans or human cells and produce essentially no or little infectious progeny virus.

According to a further embodiment of the invention, a vaccinia virus, preferably an attenuated vaccinia virus, is used to produce a recombinant poxvirus comprising two or more homologous genes.

Although Vaccinia Virus (VV) is known to undergo homologous recombination in the presence of short homologous sequences and to further delete the homologous sequences (Howley et al, [1996], Gene 172: 233-. This finding was particularly unexpected because, according to Howely et al, short sequences as long as 300 base pairs (bp) were sufficient to induce genomic rearrangements of vaccinia virus and deletions of homologous sequences. Thus, one skilled in the art can envision that longer sequences will have a greater likelihood of inducing recombination. However, according to the present invention, even a sequence containing a completely homologous gene can be stably inserted into the vaccinia virus genome.

An example of a vaccinia virus is a highly attenuated and host-limited vaccinia strain, i.e., modified vaccinia virus Ankara (MVA) (Sutter, G.et al. [1994], Vaccine 12:1032-40), but is not limited to this example. MVA was obtained by serial passages of vaccinia virus ankara (cva) strain in chicken embryonic fibroblasts about 570 passages (for review see Mayr, a., et al [1975], Infection 3, 6-14). Approximately 31kb of genomic sequence was deleted due to CVA in long-term passage. The resulting viral strain, i.e., MVA, is described as being highly host cell-restricted (Meyer, h.et al., j.gen.virol.72,1031-1038[1991 ]). A typical MVA strain is MVA575, deposited with the European Collection of Animal Cell Cultures under the accession number ECACC V00120707.

In another embodiment, the MVA-Vero strain or a derivative thereof may be used in the present invention. The MVA-Vero strain is deposited with the European Collection of animal cell cultures under accession numbers ECACCV99101431 and ECACC 01021411. The biological, chemical and physical properties of MVA-Vero reflect safety as described in International patent application PCT/EP 01/02703. Compared with the conventional MVA, the MVA-Vero genome has another deletion.

According to the invention, the term "derivative" of a virus means that the progeny virus has the same characteristics as the parent virus, but differs in one or more parts of its genome.

According to a further embodiment of the invention MVA-BN is used. MVA-BN is deposited at the European Collection of animal cell cultures under the accession number ECACC V00083008. By using MVA-BN or derivatives thereof, a particularly safe viral vaccine is created, since MVA-BN is derived from a modified vaccinia Ankara virus and shows an extremely attenuated virus. Thus, in a most preferred embodiment, the MVA-BN or derivatives thereof comprising two or more homologous genes of the present invention is used as a viral vector. The term "derivative of MVA-BN" refers to a virus having the same functional properties as MVA-BN. The characteristics of MVA-BN, biological analytical methods for assessing whether MVA is MVA-BN or a derivative thereof and methods for producing MVA-BN or a derivative thereof are described in WO02/42480 (which is incorporated herein by reference). A convenient way to measure the functional properties of MVA-BN or its derivatives is to measure its attenuating properties and lack of replication in human HaCat cells.

The recombinant poxvirus according to the invention preferably has its expression of the foreign sequence controlled by a poxvirus transcriptional regulatory element, more preferably by a MVA, canarypox, fowlpox or penguipox transcriptional regulatory element, or most preferably by a vaccinia virus promoter. The poxvirus transcriptional regulatory elements according to the present invention further comprise each transcriptional regulatory element that functions in the poxvirus system.

According to the invention, the foreign sequence is preferably inserted into a non-essential region of the viral genome. Non-essential regions are, for example, the loci (loci) or the Open Reading Frames (ORFs) of poxviruses, which are not essential for the life cycle of poxviruses. In the present invention, the intergenic sequence, i.e., the region between two ORFs, is also considered to be a non-essential region. In a further embodiment of the invention, the foreign sequence is inserted into the naturally occurring deletion site of the MVA genome (disclosed in PCT/EP96/02926, which is incorporated herein by reference).

The direction of the inserted DNA has no influence on the function or stability of the recombinant virus of the present invention.

Since the recombinant poxvirus according to the invention has a very limited growth and is thus highly attenuated, it is an ideal vaccine candidate for the treatment of mammals, including even immunocompromised humans. Accordingly, the present invention also provides a pharmaceutical composition, and a vaccine, for inducing an immune response in a living animal body, including a human.

The pharmaceutical compositions may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives (preservative), adjuvants, diluents and/or stabilizers. These auxiliary ingredients may be water, salt, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, etc. Suitable carriers are typically high molecular weight, slowly metabolised molecules such as proteins, polysaccharides, polylactic acids (polylactic acids), polyglycolic acids (polyglycolic acids), polymeric amino acids, amino acid copolymers, lipid aggregates (lipid aggregates) and the like.

For the preparation of vaccines, the recombinant poxviruses of the invention are converted into a physiologically acceptable form. This can be done based on the experience of preparing a poxvirus vaccine for use in vaccination against smallpox (see Stickl, H.et al [1974 ]]Dtsch.med.Wsch r.99, 2386-2392). For example, the virus is purified as 5X10E8TCID₅₀The titer was about 10mM Tris,140mM NaCl pH 7.4, and stored at-80 ℃. To prepare the vaccine particles (shot), 10E2-10E8 virus particles are frozen in 100ml sodium Phosphate Buffer (PBS) containing 2% peptone and 1% human blood protein, e.g., in each needle vial (ampoule), preferably a glass needle vial. Optionally, vaccine particles (shot) may also be obtained by stepwise freeze-drying of the virus in the formulation. Such formulations may contain additional additives suitable for in vivo administration, such as mannitol, dextran, sugars, glycine, lactose or polyvinylpyrrolidone, or other acids such as antioxidants or inert gases, stabilizers or recombinant proteins (e.g. human serum albumin). The glass needle cartridge is then sealed and can be stored for several months at 4 ℃ to room temperature. However, the syringe that is not required to be used is preferably stored at a temperature below-20 ℃.

For immunization or therapeutic use, the lyophilisate may be dissolved in 0.1 to 0.5ml of an aqueous solution, preferably in physiological saline or Tris buffer, administered systemically or topically, i.e., parenterally, subcutaneously, intravenously, intramuscularly, by dermabrasion (scarification) or by other means well known to those skilled in the art. The form, dosage and number of administrations can be optimized by means known in the art. However, the most common mode of vaccination is a second vaccination, which is performed about 1 month to 6 weeks after the first vaccination.

The recombinant viruses of the present invention can be used to introduce exogenous coding sequences into target cells. Exogenous coding sequences are introduced into target cells to produce in vitro proteins, polypeptides, peptides, antigens, and epitopes, respectively. Furthermore, the method of introducing homologous or heterologous sequences into cells may be applicable to both in vitro and in vivo therapy. For in vitro therapy, cells infected in vitro (ex vivo) with the recombinant poxvirus of the invention are isolated and administered to a living animal to induce an immune response. For in vivo treatment, the recombinant poxviruses of the invention are administered directly to living animals to induce an immune response. In this case, the cells around the inoculation site are directly infected with the virus of the present invention or its recombinant. Following infection, the cells synthesize proteins, polypeptides, peptides or antigens encoded by the foreign coding sequences, and these substances, or portions thereof, are then presented on the cell surface. The specific cells of the immune system recognize the presence of the above proteins, polypeptides, peptides, antigens and epitopes, and thus initiate specific immune responses.

Methods for obtaining recombinant poxviruses or for inserting exogenous coding sequences into the genome of poxviruses are well known to those skilled in the art. Furthermore, these methods are also described in the examples and can be derived or obtained entirely from the following references:

molecular Cloning, a Laboratory manual, second edition, by j.sambrook, e.f. fritsch and t.manitis, cold Spring Harbor Laboratory press.1989: standard molecular biology techniques and knowledge, such as DNA cloning, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR amplification techniques, are described.

Virology Methods manual. edited by Brian WJ major and Hillar o kango. academic press.1996: techniques for handling and manipulating viruses are described.

Molecular Virology A Practical Approach. edited by AJ Davison and RM Elliott. the Practical Approach series IRL Press at Oxford University Press. Oxford 1993, Chapter 9 expression of genes using poxvirus vectors.

Current Protocols in Molecular biology, public, John Wiley and Soninc.1998 Chapter 16, section IV, Expression of proteins in mammalian cells using a Vaccidia viral vector: processing, manipulation and genetic engineering techniques and knowledge of MVA are described.

To produce the recombinant poxvirus of the invention, different methods can be used: coli plasmid constructs into which DNA that is partially homologous to poxvirus DNA has been inserted. In addition, the DNA sequence to be inserted is linked to a promoter. The promoter-gene linkage is in the orientation in the plasmid construct such that the promoter-gene linkage is flanked by DNA sequences homologous to DNA sequences flanking a region containing a nonessential locus. The obtained plasmid construct was amplified and isolated by growth in E.coli. The isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, such as Chicken Embryo Fibroblasts (CEF), together with a poxvirus. Recombination occurs between homologous poxDNA in the plasmid and the viral genome to respectively obtain modified poxvirus containing exogenous DNA sequences.

According to a more preferred mode, cells of a suitable cell culture, such as CEF cells, are infected with a poxvirus. The infected cells are then transfected with a first plasmid vector comprising an exogenous gene, preferably under the transcriptional control of a poxvirus expression control element. As explained above, plasmid vectors also include sequences that direct the insertion of foreign sequences into selected sites in the poxvirus genome. Alternatively, the plasmid vector may also comprise an expression cassette comprising a marker and/or selection gene operably linked to a poxvirus promoter. Suitable marker or selection genes are, for example, genes encoding green fluorescent protein, beta-galactosidase, neomycin, phosphoribosyltransferase or other markers. The use of a selection or marker gene expression cassette can simplify the identification and isolation of the recombinant poxvirus produced. However, PCR techniques can also be used to identify recombinant poxviruses. Next, the cells were further infected with the recombinant poxvirus obtained above and transfected with a second vector containing a gene homologous to the gene included in the first vector. In this case, the gene should be included at a different insertion site in the poxvirus and the sequence of the second vector directing the integration of the homologous gene into the poxvirus genome is also different. After homologous recombination has occurred, a recombinant virus comprising two homologous genes can be isolated. The infection and transfection are repeated with the recombinant virus isolated in the previous infection step and another vector including another homologous gene for transfection, so that more than two homologous genes can be introduced into the recombinant virus.

Optionally, the infection and transfection steps described above may be interchanged, i.e., a suitable cell may first be transfected with a plasmid vector containing the foreign gene and then infected with the poxvirus.

Further alternatively, each of the homologous genes may be introduced into a different virus, and then cells are co-infected with all of the obtained recombinant viruses, and recombinants including all of the homologous genes are selected.

The invention further provides kits comprising two or more plasmid vector constructs that can integrate an expressible homologous gene into the poxvirus genome. In addition to having suitable cloning sites, such plasmid vectors include sequences capable of directing the insertion of exogenous sequences into selected portions of the poxvirus. Optionally, such vectors include a selection or marker gene expression cassette. The kit further comprises reagents and instructions for selecting viruses that are recombinants of one or more homologous genes, and optionally a selection or marker gene, inserted through the vector construct.

According to another embodiment of the invention, DNA sequences or parts thereof derived from or homologous to the recombinant poxvirus of the invention are included. Such sequences comprise at least the foreign sequence parts of the fragments of at least one homologous gene according to the invention and at least the fragments of the poxvirus genomic sequences according to the invention, which are preferably flanked by foreign sequences.

Such DNA sequences can be used for identifying or isolating viruses or derivatives thereof, for example for using them for generating PCR primers, hybridization probes or in matrix technology (array technologies)

Brief Description of Drawings

FIG. 1: schematic representation of the insertion sites of 4 PrM (= pre-membrane) genes (serotypes 1-4) in the MVA genome according to example 1.

FIGS. 2-9 and 12-17: the plasmid vector construct is shown inserted and the vector name, size and sequence position of interest are noted, such as AmpR = ampicillin resistance gene, bfp = blue fluorescent protein gene, dA = deletion A, dE = deletion E, d2= deletion 2, Ecogpt = e.coli guanine phosphoribosyltransferase gene, EGFP = enhanced green fluorescent protein gene, F1= flanking sequence 1, F2= flanking sequence 2, I4L spacer = I4L, IGR = spacer, NPT II = neomycin resistance gene, P = poxvirus promoter, pr7.5= vaccinia promoter 7.5, PrM = dengue virus pre-membrane gene, the number indicates which serotype from which, rpt = repetitive flanking sequence.

FIG. 10: PCR verification of the vector cloning technology for 4 different insertion vectors (pBN49, PBN50, PBN40, PBN 39). Each plasmid was tested with 4 different PCR primer combinations. Each combination is specific for a distinct PrM sequence integrated into a distinct insertion site.

FIG. 11: PCR validation of recombinant poxviruses comprising 4 PrM genes of homologous dengue viruses (example 1). The different PCR results for the recombinant virus are shown in the upper half of the gel and the results for the same PCR reaction for the control plasmid are shown in the lower half. The homologous sequences contained in the plasmids were designated pBN39, pBN49 or pBN50, respectively. PrM represents the inserted dengue virus pre-membrane gene, where the number indicates from which of the 4 serotypes it originated. dA = deletion A, dE = deletion E, d2= deletion 2, I4L = spacer I4L, used to describe the insertion site of the foreign DNA.

FIG. 18: according to example 2, a schematic of the insertion sites of 3 PrM genes (serotypes 2-4) in the MVA genome.

FIG. 19: PCR validation of recombinant poxviruses comprising 3 homologous dengue virus PrM genes inserted into the spacer (Example 2). The upper gel panel shows the results of a PCR reaction specific to PrM2, the middle gel panel shows the results of a PCR reaction specific to PrM3, and the bottom gel panel shows the results of a PCR reaction specific to PrM 4. Lane 8 shows the results of the same PCR reaction with the control plasmid. Lane 2 shows empty vector control MVA. PrM stands for inserted dengue virus pre-membrane gene, where the number indicates from which of the 4 serotypes it originated. M ═ molecular weight markers.

The following examples further illustrate the invention. Those skilled in the art will understand that the examples provided are not intended to limit the application of the invention to these examples in any way.

Example 1

Insertion vector

Insertion vector lacking A

To insert the foreign sequence at the corresponding genomic position 7608-7609 in the MVA genome at the site called deletion A or deletion 1, a plasmid vector was constructed comprising flanking sequences of about 600bp near the deletion site A. Suitable PCR primers were designed using a suitable computer program (DNAsis, Hitashi software engeening, San Bruno, USA) for the isolation of flanking sequences from the MVA-BN genome. Such primers include an extension (extension) with a restriction site for cloning the flanking sequence into the vector plasmid. Between the flanking sequences, a selection gene expression cassette is introduced, such as the NPT II gene (neomycin resistance gene) under the transcriptional control of the poxvirus promoter. In addition, a cloning site is provided for inserting additional genes or foreign sequences to be inserted into deletion site A. One such vector construct according to the present invention is shown in FIG. 2 (pBNX 10).

Insertion vector lacking E

To insert the foreign sequence at the corresponding genomic position 170480-170481 in the MVA genome at the site called deletion E or deletion 4, a vector was constructed comprising flanking sequences of about 600bp near the deletion site E. Vectors were designed and constructed in a similar manner as described above. An EGFP gene (green fluorescent protein, Clonetech) under the transcriptional control of a poxvirus promoter is placed between the flanking sequences. In addition, a cloning site is provided for inserting additional genes or sequences to be inserted into deletion site E. One such vector construct according to the present invention is shown in FIG. 3 (pBNX 32).

Insertion vector for deletion 2

In order to insert a foreign sequence at a site in the MVA genome corresponding to the so-called deletion 2 at genomic position 120718-20719, a vector was constructed comprising flanking sequences of about 600bp near the deletion site 2. Vectors were designed and constructed in a similar manner as described above. The hbfp gene (humanized blue fluorescent protein, Pavalkis GN et al.) was placed between the flanking sequences under the transcriptional control of the poxvirus promoter. In addition, a cloning site is provided for inserting additional genes or sequences to be inserted into deletion site 2. One such vector construct according to the present invention is shown in FIG. 4 (pBNX 36).

Insertion vector for spacer I4L

In order to insert a foreign sequence between the spacers in the MVA genome, i.e. between ORFs I3L and I4L at the position corresponding to the genome at 56760, a vector was constructed comprising flanking sequences of about 600bp near the spacer of the I4L locus. Vectors were designed and constructed in a similar manner as described above. Between the flanking sequences is placed the Ecogpt gene (or gpt, representing phosphoribosyltransferase isolated from E.coli) under the transcriptional control of the poxvirus promoter. In addition, it has a cloning site for insertion of additional genes or sequences to be inserted into the spacer region behind the I4L ORF. One such vector construct according to the present invention is shown in FIG. 5 (pBNX 39).

Construction of a recombinant poxvirus comprising several homologous genes integrated in its genome

Insertion vector

To insert 4 PrM genes from 4 serotypes of dengue virus into the MVA genome, 4 separate recombinant vectors were used.

As described in detail above, these vectors contain sequences homologous to the MVA genome in order to target the insertion by homologous recombination. In addition, each vector contains a selection or reporter gene expression cassette.

PrM sequences of 4 serotypes of dengue virus were synthesized by oligonucleotide (oligo) annealing and PCR amplification. The PrM sequence is cloned downstream of the poxvirus promoter elements to form an expression cassette. The expression cassette is then cloned into the relevant cloning site in the insertion vector construct.

As a result, the insertion vector construct deleted for A contained the PrM gene of dengue virus serotype 2 (FIG. 6-pBN 39). The insertion vector construct of deletion 2 comprises the PrM gene of dengue virus serotype 1 (fig. 7-pBN 49). The insertion vector for spacer I4L contained the PrM gene of dengue virus serotype 3 (fig. 8-pBN 50). The insertion vector for deletion E contained the PrM gene of dengue virus serotype 4 (FIG. 9-pBN40)

PCR validation of insert vectors

To check the success of the cloning strategy, a PCR analysis was performed. For PCR analysis, the primers were selected for the following primer combinations: a second primer that specifically binds to one of the highly homologous PrM genes of the dengue virus is specific for a primer that binds to a flanking sequence associated with the insertion site.

For insertion vectors containing deletion a of the PrM gene of dengue virus serotype 2, the selection primers used were: oBN93(CGCGGATCCATGCTGAACATCTTGAACAGGAGACGCAGA. SEQ ID NO.:1) and oBN477 (CATGATAAGATTGTATCAG. SEQ ID NO.: 2).

For an insertion vector containing deletion 2 of the PrM gene of dengue virus serotype 1, the screening primers used were: oBN194 (ATGTTGAACTAATGAACAGGAGGAAAAGATCTGTGACCATGCTCCTCATGCTGCCCACAGCCCTGCGTTCCATCT. SEQ ID NO. 3) and oBN476(GATTTT GCTATTCAGTGGACTGGATG. SEQ ID NO. 4).

For the insertion vector containing spacer I4L of the PrM gene of dengue virus serotype 3, the selection primers used were: oBN255 (CCTTAACGAATTCTCATGTCATGGATGGGTAACCAGCATTAAGT. SEQ ID NO: 5) and oBN479(GCTCCCATTCAATTCACATTGG. SEQ ID NO: 6).

For insertion vectors containing deletion E of the PrM gene of dengue virus serotype 4, the selection primers used were: oBN210 (ATCCATTCCTGAATGTGGTGTTAAGCTACTGAGCGCTTCTCTCTCCTCCGCTCTGGGTGCATGTCCATAC. SEQ ID NO: 7) and oBN478 (GTACATGGATGATATAGATG. SEQ ID NO: 8).

In a PCR instrument, Thermal cycler GeneAmp 9700(Perkin Elmer), with a PCR medium containing l0xPCR buffer, MgCl₂Buffer and Taq DNA polymerase kit (Roche, cat.no.201205) or equivalent for Taq DNA polymerase. In general, the PCR reaction requires 45ul of mastermix, sample DNA and ddH in a total reaction volume of 50. mu.l₂And O. Wherein the volume of the solution should be 30.75. mu.l DdH₂O, 5. mu.L of 10 XBuffer, 1. mu.L of dNTP mix (10 mM each), 2.5. mu.L of each primer (5 pmol/. mu.L), 3. mu.L of MgCl₂(25mM) and 0.25. mu.l Taq-DNA polymerase (5U/. mu.l) were prepared as mastermix.

The amplification reaction was performed with the following parameters:

1) denaturation: 94 ℃ for 4 minutes

2)30 cycles:

denaturation: 94 ℃ for 30 seconds

Annealing: 55 ℃ for 30 seconds

Extension: 72 ℃ for 1-3 min

3) Extension: 72 ℃ for 7 min

4) And (3) storage: 94 ℃;

depending on the size of the inserted gene, the extension time is at least 1 minute/kb.

The PCR results shown in fig. 10 indicate that the primer combinations used for the single insertions are specific.

oBN194/oBN476 primer combination is specific for deletion 2 and PrM1 inserted therein. The fragment PCR amplified for plasmid pBN49 was expected to be 678bp (shown in lane 3, top half of the gel).

The oBN255/oBN479 primer combination is specific for the spacer I4L and PrM3 inserted therein. The PCR amplified fragment for plasmid pBN50 was expected to be 825bp (shown in lane 9, top half of the gel).

The oBN210/oBN478 primer combination is specific for deletion E and PrM4 as an insert. The expected size of the fragment PCR amplified for plasmid pBN40 was 607bp (shown in lane 5, lower half of the gel).

The oBN93/oBN477 primer combination is specific for deletion A and PrM2 inserted therein. The PCR amplified fragment for plasmid pBN39 was expected to be 636bp (shown in lane 11, lower half of the gel).

Generation of recombinant MVA by homologous recombination

To express foreign genes by recombinant MVA, the gene to be expressed can be inserted into the viral genome by a method known as homologous recombination. For this purpose, the foreign gene of interest is cloned into an insertion vector as described above. The vector is then transfected into cells which have been infected with MVA-BN. Recombination occurs in the infected and transfected cytoplasm. Cells containing the recombinant virus can be identified and isolated using selection and/or reporter gene expression cassettes contained in the insertion vector.

Homologous recombination

For homologous recombination, BHK (Baby hamster kidney Kidney kidney) cells or CEF (native chick embryo fibrinogen) cells were seeded in 6-well plates in DMEM (Dulbecco's Modified Eagles Medium, Gibco BRL) +10% Fetal Calf Serum (FCS) or VP-SFM (Gibco BRL) +4mmol/l L-glutamic acid was used as the Medium for serum-free production procedures.

To keep the cells in the growth phase and therefore should reach 60-80% confluence the day of transfection, the cells were counted before performing the inoculation culture and the multiplicity of infection (moi) was determined as the known cell number.

For infection, MVA stock solution was diluted in DMEM/FCS or VP-SFM/L-glutamic acid to approximately the appropriate number of viruses contained in 500. mu.l of the dilution to obtain moi of 0.01. It is assumed that the cells divide once after the culture. The medium was removed and infected with 500. mu.l of diluted virus solution at room temperature for 1 hour. After removal of the inoculum, washing was performed with DMEM/VP-SFM. The infected cells were plated in 1.6ml DMEM/FCS and VP-SFM/L-glutamic acid for transfection (Qiagen Effect Kit).

Transfection was performed using the "Effectene" transfection kit (Qiagen). Transfection mix was prepared with 1-5. mu.g of linearized insert vector (total of multiple transfections) in a final volume of 150. mu.l of buffer EC. Add 8.0. mu.l Enhancer per. mu.g DNA, vortex and incubate at room temperature for 5 min. Then, after vortexing the mother liquor tube, 25. mu.l Effectene per. mu.g of DNA was added, the solution was thoroughly mixed by vortexing, and incubated at room temperature for 10 minutes. 600. mu.DMEM/FCS or VP-SFM/L-glutamic acid were added, respectively, and mixed well, and then the whole transfection mixture was added to the cells which had been coated with the medium. The plates were gently shaken and mixed to perform the transfection reaction. At 37 ℃ and 5% CO₂Incubate under conditions overnight. The following day the medium was removed and replaced with fresh DMEM/FCS or VP-SFM/L-glutamic acid. Incubation continued until day 3.

To collect the cells, the cells are scraped into a culture medium and suspended, and the cell suspension is transferred to an appropriate tube and cryopreserved at-20 ℃ for short term storage and-80 ℃ for long term storage.

Insertion of PrM4 into MVA

In the first round, cells were infected with MVA-BN following the procedure described above and infected with the insertion vector pBN40 comprising the PrM gene of dengue virus serotype 4 and EGPF as reporter gene. Since the transfection vector contains the reporter gene EGFP, the synthesized protein can be detected in cells infected with the recombinant virus at the latest on day 3. The obtained recombinant virus was purified by plaque purification.

Infected cells were isolated with pipette tips (fluorescent or stained), resuspended in 200 μ L PBS or culture medium and aspirated for plaque purification. Then, a new dish containing about 10E6 cells was infected with 100. mu.l of resuspended plaques. After 48 hours, the cells were suspended in 300. mu.l PBS. DNA was extracted from the suspension and screened by PCR analysis. Clones showing the expected bands were selected and fresh 6-well plates were infected with the virus at different amounts. The wells were covered with 1% agarose to prevent spreading of the virus. Infected cells containing the recombinant virus clones were isolated after 48 hours.

This procedure was repeated until no wild-type MVA-BN could be detected by PCR.

After 4 rounds of plaque purification, PCR analysis was performed to identify the recombinant virus, MVA-PrM4, using primer pairs capable of selectively amplifying the expected insertions (oBN 210 and OBN478 as described above), with control primer pairs specifically recognizing the insertion site deletion E (oBN 453: GTTGAAGGATTCACTTCCGTGGA, SEQ ID No.:9 and oBN454: GCATTCACAGATTCTATTGTGAGTC, SEQ ID No.: 10).

Insertion of PrM2 into MVA-PrM4

Cells were infected with MVA-PrM4 as described above and further infected with the insertion vector pBN39 containing the PrM gene of dengue virus serum 2 and NPT II (neomycin resistance gene) as selection genes. For purification of recombinant MVA expressing antibiotic resistance genes, it is recommended that the virus is preferably amplified for 3 rounds under selective conditions before performing a plaque purification step. Thus, G418 was added to the medium to select neomycin phosphotransferase. G418 is a neomycin derivative that inhibits protein synthesis by interfering with ribosome function. The activity of the NPT gene can inactivate G418 by phosphorylation.

After 16 rounds of plaque purification in the presence of neomycin selection, PCR analysis was performed to identify recombinant viruses, MVA-PrM4/PrM2, using primers capable of selectively amplifying the expected insertion (oBN 93 and oBN477 as described above), with a control primer pair specifically recognizing insertion site deletion a (oBN 477 as described above) and oBN452: GTTTCATCAGAAATGACTCCATGAAA, SEQ ID No.: 11). Furthermore, the insertion of PrM4 in deletion E was verified with the following primer pair: oBN210-oBN478 and oBN453-oBN 454.

Insertion of PrM1 into MVA

In the first round, cells were infected with MVA-BN following the procedure described above and transfected with the insertion vector pBN49 containing the PrM gene of dengue virus serotype 1 and hbfp as reporter gene (humanized blue fluorescent protein gene). The synthesized hbfp protein was detected on day 3 of infection of the cells with the recombinant virus. The obtained recombinant virus was purified by plaque purification.

After 10 rounds of plaque purification, PCR analysis was performed to identify the recombinant virus, MVA-PrM4, using a primer pair capable of selectively amplifying the expected insertions (oBN 194 and OBN476 as described above), with a control primer pair specifically recognizing insertion site deletion 2(oBN 54: CGGGGTACCCGACGAACAAGGAACTGTAGCAGAGGCATC, SEQ ID No.:12 and oBN56: AACTGCAGTTGTTCGTATGTCATAAATTCTTTAATTAT, SEQID No.: 13).

Insertion of PrM3 into MVA

In the first round, cells were infected with MVA-BN following the procedure described above and infected with the insertion vector pBN50 containing the PrM gene of dengue virus serotype 3and the Ecogpt gene (Ecogpt or abbreviated gpt, representing phosphoribosyltransferase gene) as reporter gene. The resulting recombinant virus was purified by 3 rounds of plaque purification, with selection conditions supplemented with mycophenolic acid, lutein and hypoxanthine to select for phosphoribosyltransferase metabolism. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, which leads to a hindrance of purine synthesis and thus to an inhibition of viral replication in most cell lines. This hindrance can be overcome by expression of Ecogpt through constitutive promoters and providing lutein and hypoxanthine as substrates.

PCR analysis was performed with primers capable of selectively amplifying the desired insertion (oBN 255 and oBN479 as described above) to identify the resulting recombinant virus, MVA-PrM3, wherein the control primer pair specifically recognized insertion site I4L (oBN499: CAACTCTCTTCTTGATTACC, SEQ ID No.:14 and oBN500: CGATCAAAGTCAATCTA TG, SEQ ID No.: 15).

Co-infection of MVA-PrM1 and MVA-PrM3

The cells were infected with equal amounts of MVA-PrMl and MVA-PrM3 obtained as described above. After selecting blue fluorescent clones of recombinant viruses for phosphoribosyltransferase metabolism, 3 rounds of plaque purification were performed, and PCR analysis was performed on the purified recombinant viruses using primer pairs (such as oBN255 and oBN479, oBN499 and oBN500, oBN194 and oBN476, oBN54 and oBN56, as described above). The resulting recombinant virus is designated MVA-PrM1/PrM 3.

Co-infection of MVA-PrM1/PrM 3and MVA-PrM2/PrM4

Cells were infected with equal amounts of MVA-PrM1/PrM 3and MVA-PrM2/PrM4 obtained as described above. Plaque purification was performed using phosphoribosyltransferase metabolism and neomycin selection. Recombinant viruses that produce green and blue fluorescence were isolated and subjected to PCR analysis using primer pairs (such as oBN255 and oBN479, oBN499 and oBN500, oBN194 and oBN476, oBN54 and oBN56, oBN93 and oBN477, oBN477 and oBN452, oBN210 and oBN478, oBN453 and oBN454, as described above).

As shown in FIG. 11, PCR analysis of the recombinant virus (Clone 20) revealed that all 4 PrM genes were contained. The upper half of the gel shows the results of different PCR reactions on recombinant virus and the lower half shows the results of the same PCR reaction on the control plasmid (as indicated). Lanes 1, 10 and 11 are 1kb and 100bp molecular weight markers.

Primer combination oBN210/oBN478 was specific for deletion E and PrM4 inserted therein. The PCR amplified fragment for the recombinant virus and plasmid pBN40 was expected to be 607bp (shown in lane 2).

Primer combination oBN453/oBN454 was specific for deletion E. The amplified fragment from PCR on the recombinant virus was expected to be 2.7kb and 2.3kb for the wild type virus (shown in lane 3). Specific bands for wild-type virus are also shown in the upper half of the gel. This indicates that the recombinant virus produced has not completely removed the wild-type virus. Further plaque purification was necessary.

Primer combination oBN93/oBN477 is specific for deletion A and PrM2 inserted therein. The amplified fragment of the PCR for the recombinant virus and plasmid pBN39 was expected to be 636bp (shown in lane 4).

Primer combination oBN477/oBN452 is specific for deletion A. The amplified fragment from PCR on the recombinant virus was expected to be 4.1kb and 2.7kb for the wild type virus (shown in lane 5). The gel fraction allowed identification of bands specific for wild-type virus.

The primer combination oBN255/oBN479 is specific for the spacer I4L and PrM3 inserted therein. The PCR amplified fragment for recombinant virus and plasmid pBN50 was expected to be 825bp (shown in lane 6).

The primer combination oBN499/oBN500 is specific for the spacer I4L. The amplified fragment from PCR on the recombinant virus was expected to be 1.0kb, 0.3kb for the wild type virus (shown in lane 7).

Primer combination oBN194/oBN476 is specific for deletion 2 and PrM1 inserted therein. The amplified fragment from PCR of recombinant virus and plasmid pBN49 was expected to be 678bp (shown in lane 8).

Primer combination oBN54/oBN56 is specific for deletion 2. The amplified fragment from PCR on the recombinant virus was expected to be 1.6 kb and 0.9kb for the wild type virus (shown in lane 9). Specific bands for the identified wild-type virus are also shown in the upper half of the gel.

Optionally, 4 different viruses can be produced, cells co-infected with these 4 viruses, and recombinants screened.

Recombinant vectors may also be improved to include additional selection or resistance markers.

Example 2

Insertion vector

Recombinant vector of spacer 136-137(IGR136-137)

In order to insert the foreign sequence into the MVA genome at what is referred to as the spacer (IGR)136-137 at the position corresponding to genomic position 129940, a plasmid vector was constructed comprising a flanking sequence of about 600bp near the insertion site. Suitable PCR primers are designed in order to isolate the flanking sequences in the MVA-BN genomic DNA. Such primers contain an overhang (extension) of the restriction enzyme site for cloning the flanking sequence into the vector plasmid. Between the flanking sequences, a selection gene expression cassette is introduced, such as the NPT II gene (neomycin resistance gene) under the transcriptional control of the poxvirus promoter (P). In addition, a cloning site was provided for insertion of additional genes or foreign sequences to be inserted into IGR136-137 (PacI). One such vector construct according to the invention is shown in FIG. 12 (pBNX 67).

Recombinant vector for spacer 07-08(IGR07-08)

In order to insert the foreign sequence into the MVA genome at a position corresponding to the genomic position 12800, called spacer (IGR)07-08, a plasmid vector was constructed comprising flanking sequences of about 600bp near the insertion site. Suitable PCR primers are designed in order to isolate the flanking sequences in the MVA-BN genomic DNA. Such primers are highlighted with restriction enzyme sites for cloning of the flanking sequences into the vector plasmid. Between the flanking sequences, a selection gene expression cassette is introduced, such as the Ecogpt gene (guanine phosphoribosyltransferase gene) under the transcriptional control of the poxvirus promoter (P). In addition, a cloning site (PacI) is provided for insertion of additional genes or foreign sequences to be inserted into IGR 07-08. One such vector construct according to the invention is shown in FIG. 13 (pBNX 88).

Recombinant vector for spacer 44-45(IGR 44-45)

In order to insert the foreign sequence into the MVA genome at what corresponds to the genomic position 37330 referred to as the spacer (IGR)44-45, a plasmid vector was constructed comprising flanking sequences of about 600bp near the insertion site. Suitable PCR primers are designed in order to isolate the flanking sequences in the MVA-BN genomic DNA. Such primers are highlighted with restriction enzyme sites for cloning of the flanking sequences into the vector plasmid. Between the flanking sequences, a selection gene expression cassette is introduced, such as the NPT II gene (neomycin resistance gene) under the transcriptional control of the poxvirus promoter (P). In addition, there is a cloning site (PacI) for insertion of additional genes or foreign sequences to be inserted into IGR 44-45. One such vector construct according to the invention is shown in FIG. 14 (pBNX 87).

Construction of recombinant poxviruses comprising integrated several homologous genes in the genome

Insertion vector

In order to insert 3 PrM genes from 3 serotypes of dengue virus, i.e. serotypes 2, 3and 4, respectively, into the MVA genome, 3 independent recombinant vectors were used.

PrM sequences of 3 serotypes of dengue virus were synthesized as described in example 1.

As a result, the insertion vector construct for IGR136-137 contained the PrM gene of dengue virus serotype 4 (FIG. 15-pBN 27). The insertion vector construct for IGR07-08 contains the PrM gene of dengue virus serotype 2 (FIG. 16-pBN 34). The insertion vector construct for IGR44-45 comprises the PrM gene of dengue virus serotype 3 (FIG. 17-pBN 47).

Generation of recombinant MVA by homologous recombination

The preparation of recombinant MVA by homologous recombination was carried out as described in example 1. The insertion sites of PrM4, PrM 3and PrM2 in the MVA genome are indicated in fig. 18.

Insertion of PrM4 into MVA

In the first round, cells were infected with MVA-BN following the procedure described above and transfected with the insertion vector pBN27 containing the PrM gene of dengue virus serotype 4 and EGFP as reporter gene. Since the transfected vector contains the reporter gene, EGFP, its synthesized protein can be detected in cells infected with the recombinant virus at the latest day 3. The resulting recombinant virus needs to be purified by plaque purification as described in example 1. After 4 rounds of plaque purification, the recombinant virus MVA-PrM4 was identified by PCR using primers capable of selectively amplifying the insertion site IGR136-137 (oBN1008: GATACCGGATCACGTTCTA. SEQ ID NO.:16 and oBN1009 ggatatgattgtagg. SEQ ID NO.: 17).

Insertion of PrM2 into MVA

Cells were infected with MVA-PrM4 following the procedure described above and transfected with the insertion vector pBN34 containing the PrM gene and BFP gene of dengue virus serotype 2 as reporter genes. Since the transfected vector contains the reporter gene BFP, its synthesized protein can be detected in cells infected with the recombinant virus at the latest on day 3. The resulting recombinant virus needs to be purified by plaque purification as described in example 1. After 6 rounds of plaque purification, the recombinant virus MVA-PrM4+ PrM2 was further passaged, propagated, and a crude stock solution was prepared. The recombinants were identified by PCR using primers capable of selectively amplifying the insertion site IGR07-08 (oBN 903: CTGGATAAATACGAGGACGT G.: SEQ ID NO. 18 and oBN904: GACAATTATCCGACGCACCG; SEQ ID NO. 19).

Insertion of PrM3 into MVA

Cells were infected with MVA-PrM2+4 following the procedure described above and transfected with the insertion vector pBN47 containing the PrM gene of dengue virus serotype 3and the EGFP gene as reporter genes. Since the transfected cells contained the reporter gene EGFP, the protein synthesized by them was detectable in cells infected with the recombinant virus at the latest day 3. The resulting recombinant virus needs to be purified by plaque purification as described in example 1. After 3 rounds of plaque purification, the recombinant virus MVA-PrM4+3+2 was identified by PCR with a primer pair (oBN904: CGTTAGACACACACACGATGAG. SEQ ID No.:20 and oBN905 CGGATGAAAAAATTT TTGGAAG. SEQ ID No.:21) capable of selectively amplifying the insertion sites IGR 44-45.

The results of PCR analysis of recombinant viruses containing 3 dengue virus PrM genes are shown in FIG. 19. The PCR experiment was carried out in the manner described in example 1. Primer combinations oBN1008 and oBN1009 are specific for IGR136-137, which contains an insertion of PrM4 (gel panel below FIG. 19). PCR was performed on the recombinant virus, and the expected amplified fragment was 1kb (shown in lanes 4, 5 and 6), using the plasmid as a positive control (lane 8). For the empty vector control without Prm4, the expected amplified fragment was 190bp (lane 2). Lane M is the molecular weight marker and lanes 1, 3and 7 are empty. Primer combinations oBN902 and oBN903 are specific for IGR07-08, which contains a PrM2 insertion (upper gel panel in FIG. 19). PCR was performed on the recombinant virus and the expected fragment was 960bp (shown in lanes 4-6), a plasmid positive control (lane 8). For the empty vector control without Prm2, the expected amplified fragment was 190bp (lane 2). Primer combinations oBN904 and oBN905 are specific for IGR44-45, which contains a Prm3 insertion (middle gel panel in FIG. 19). The expected PCR fragment size for the recombinant virus was 932bp (shown in lanes 4-6), the plasmid positive control (lane 8). For the empty vector control without Prm2, the expected fragment was 185bp (lane 2).

Applicant or attorney docket number BN46PCT

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Appendix 3

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Budapest treaty on International recognition of the deposit of microorganisms for patent procedures

International Bureau form

The receiver:

BAVARIAN NORDIC RESEARCH

INSTITUTE GMBH

FRAUNHOFERSTRASSE 18B

DI-82152MARTINSRIED

germany

Name and address of depositor

When applied to rule 6.4(d), the date on which the eligibility of the international depository was determined.

Table bp/4 (one page only)

Appendix 3

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International Bureau form

The receiver: proof of survival

International Bank specified by the next page for BAVARIAN NORDIC RESEAR

INSTITUTE GMBH

FRAUNHOFERSTRASSE 18B reservoir unit gives out the drug according to the rule 10.2

D-82152MARTINSRIED

Germany

Name and address of depositor

1 refers to the date of the initial deposit, or, when a new deposit or a change of deposit is made, to the most relevant date (the date of the new deposit or the date of the change).

2 for those cases represented by regulations 10.2(a) (ii) and (iii), refer to the most recent survivability test.

3 is selected with a cross.

Table BP/4 (page 1)

Appendix 3

Page 25

4 if this information is required and if the test result is negative, this item is filled in.

Certificate of analytical test

Product description MVA-575

Accession number 00120707

The approver: _____: ECACC quality supervisor

Date: _______

Certificate of analytical test

Product description MVA-575

Accession number 00120707

The approver: _____: ECACC quality supervisor

Date: ________

Applicant or attorney docket number BN46PCT

International application number

Instructions for the preservation of microorganisms

(the second of the design reside in 13)

Appendix 3

Page 14

International Bureau form

The receiver:

PROF DR ANTON MAYR

WEILHEIMER STR．1

D-82319STARNBERG

germany

Name and address of depositor

Form BP/4 (one page only)

Appendix 3

Page 24

International Bureau form

The receiver: proof of survival

PROF DR ANTON MAYR

Country indicated by next page

WEILHEIMER STR.1

D-82319STARNBERG depositary Committee under the Fine rule 10.2

German tool

Name and address of depositor

3 is selected with a cross.

Appendix 3

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BP/9 table (second page and last page)

Applicant or attorney docket number BN46PCT

International application number

Instructions for the preservation of microorganisms

(the second of the design reside in 13)

Appendix 3

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International Bureau form

The receiver:

PROF.DR.DR，H.C.MULT.ANTON

MAYR

WELHEIMER STR.1

D-82319STARNBERG

germany

Name and address of depositor

BP/4 table (one page only)

Appendix 3

Page 24

International Bureau form

The receiver:

demonstration of survival of pro, dr, h.c, mult

Country indicated by next page of MAYR

WELHEIMER STR.1

The international depository unit is according to rule 10.2

D-82319STARNBERG

German tool

Name and address of depositor

3 is selected with a cross.

Appendix 3

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BP/9 table (second page and last page)

Instructions for the preservation of microorganisms

(the second of the design reside in 13)

Appendix 3

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Appendix 3

Page 24

International Bureau form

The receiver:

BAVARIAN NORDIC RESEARCH

evidence of INSTITUTE GMBH survival

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Appendix 3

Page 14

The receiver: international Bureau form

BAVARIAN NORDIC RESEARCH

INSTITUTE GMBH

FRAUNHOFERSTRASSE 18B

D-82152MARTINSRIED

Germany

Name and address of depositor

Bp/4 table (one page only)

Certificate of analytical test

Description of the products MVA-BN

Deposit number 00083008

The approver: _____: ECACC quality supervisor

Date: ______

Certificate of analytical test

Description of the products MVA-BN

Deposit number 00083008

^***Is proved to be over^***

The approver: ________: ECACC quality director: _________ date: ________

Claims

1. A recombinant modified vaccinia Ankara virus (MVA) comprising at least two homologous, exogenous sequences having at least 50% homology, wherein each of said sequences is inserted into a different insertion site of the viral genome.

2. Recombinant MVA virus comprising at least two homologous foreign sequences having at least 60% homology.

3. The recombinant MVA virus according to claim 1 or 2, wherein the sequences have a homology of 65 to 75%.

4. The recombinant MVA virus of any of claims 1-3, wherein the sequence is a homologous gene.

5. The recombinant MVA virus of claim 4, wherein the homologous genes are from a flavivirus.

6. The recombinant MVA virus of claim 5, wherein the flavivirus is dengue virus.

7. The recombinant MVA virus according to claim 5 or 6, wherein the genes are at least two homologous genes derived from at least two different serotypes of the virus.

8. The recombinant MVA virus of any of claims 5-7, wherein the gene is at least two PrM genes.

9. The recombinant MVA virus of claim 8, wherein the genes are 4 PrM genes.

10. The recombinant MVA virus according to claim 1 or 2, wherein the homologous, exogenous sequences are identical.

11. The recombinant MVA virus according to claim 10, wherein the homologous exogenous sequence is a promoter.

12. The recombinant MVA virus according to claim 10 or 11, wherein the sequence is the vaccinia virus early/late promoter p 7.5.

13. The recombinant MVA virus according to any of claims 4 to 9 and 11, wherein the homologous genes are each under the transcriptional control of the same promoter.

14. The recombinant MVA virus according to claim 13, wherein the homologous genes are each under the transcriptional control of the vaccinia virus early/late promoter p 7.5.

15. The recombinant MVA virus according to any of claims 1 to 14, wherein the MVA used for the production of the recombinant virus is MVA-BN deposited at the european collection of animal cells (ECACC) under accession number V00083008.

16. The recombinant MVA virus according to any of claims 1 to 15, wherein the MVA is replication deficient or incapable of replication in a mammalian cell.

17. The recombinant MVA virus of claim 16, wherein the mammalian cells are human cells.

18. The recombinant MVA virus according to any of claims 1 to 17, wherein the sequence is inserted into a naturally occurring deletion site and/or spacer in the MVA virus genome.

19. A vaccine comprising a recombinant MVA virus according to any of claims 1 to 18.

20. A pharmaceutical composition comprising a recombinant MVA virus according to any of claims 1 to 18 and a pharmaceutically acceptable carrier, diluent, adjuvant and/or additive.

21. Use of a recombinant MVA virus according to any of claims 1 to 18 for the preparation of a medicament.

22. Use of a recombinant MVA virus according to any of claims 1 to 18, a vaccine according to claim 19 or a composition according to claim 20 for the preparation of a medicament for influencing the immune response of a living animal.

23. The use according to claim 22, wherein the living animal is a human.

24. Use of a recombinant MVA virus according to any of claims 1 to 18, a vaccine according to claim 19 or a composition according to claim 20 for the preparation of a medicament for inducing an immune response in a living animal.

25. The use according to claim 24, wherein the living animal is a human.

26. A cell comprising the recombinant MVA virus of any of claims 1 to 18.

27. A method of producing the recombinant MVA virus of any of claims 1 to 18, comprising the steps of:

-infecting cells with MVA virus;

-transfecting an infected cell with a first vector construct, wherein the first vector construct comprises a sequence to be introduced into the MVA virus genome and an integrated MVA virus genome sequence of said sequence capable of directing an insertion site to be inserted into the MVA virus genome;

-identifying and isolating the generated recombinant MVA virus;

-repeating the above steps with the recombinant MVA virus obtained in the above step of infecting the cells and a further vector construct comprising further sequences to be introduced into the MVA virus genome, which sequences are homologous to the sequences of the first vector construct.

28. The method of claim 27, further comprising the step of purifying the generated recombinant MVA virus.

29. A kit, comprising:

-two or more vector constructs, each construct comprising a stretch of sequence, wherein the sequences comprised in the different vectors are homologous sequences having at least 50% homology, and wherein each stretch of sequence is flanked by a MVA virus DNA sequence capable of directing the integration of the homologous sequences into the MVA virus genome; and

-agents for identifying and/or selecting a recombinant MVA virus into the genome of which said homologous sequences have integrated.

30. The kit according to claim 29, wherein each homologous sequence is flanked by MVA virus DNA sequences capable of directing the integration of said homologous sequence in each vector construct into a different insertion site in the MVA virus genome.

31. A DNA sequence of a recombinant MVA virus genome derived from the recombinant MVA virus of any of claims 1 to 18, wherein said DNA sequence comprises the at least two homologous sequences and at least part of the sequence of the MVA virus genome.

32. A method of detecting in vitro or in vitro a cell infected with the recombinant MVA virus of any of claims 1 to 18, comprising administering the DNA sequence of claim 31 to said cell.

33. A method for identifying the recombinant MVA virus of any of claims 1 to 18 in vitro or in vitro, comprising administering the DNA sequence of claim 31 to said virus.

34. A method of detecting a cell infected with the recombinant MVA virus of any of claims 4 to 18, comprising contacting the cell with DNA primers that selectively amplify the homologous exogenous sequence and/or flanking sequences associated with the insertion site of the exogenous sequence.

35. A method of identifying a recombinant MVA virus according to any of claims 4 to 18, comprising contacting a cell containing the recombinant MVA virus with DNA primers that selectively amplify the homologous exogenous sequence and/or flanking sequences associated with the insertion site of the exogenous sequence.