RECOMBINANT ORF-VIRUS-VECTOR

Description

[0001] The present invention relates to a recombinant Orf virus vector, a cell containing the recombinant Orf virus vector, a composition containing the recombinant Orf virus vector according to the invention and / or the cell according to the invention, the use of the recombinant Orf virus vector according to the invention for the expression of a foreign gene and a nucleic acid molecule encoding an Orf virus vector promoter.

[0002] Viral vectors are used in biotechnology to deliver genetic material into target cells. The introduced genetic material often encodes foreign genes used to produce a recombinant protein. Viral vectors therefore represent an important platform technology, particularly for the production of recombinant vaccines, which, in addition to the traditional prevention of infectious diseases, are increasingly being used to develop new, innovative therapeutic concepts, such as therapeutic tumor immunization.

[0003] The Orf virus (ORFV) belongs to the poxvirus family and has a variety of properties that make it interesting for the production of recombinant vaccines and preferable to other technologies. It represents the prototype of the parapoxvirus genus and belongs to the family of Poxviridae ORFVs are enveloped, complex dsDNA viruses with a wool-ball-like morphology and an average size of approximately 260 x 160 nm. They possess a linear, GC-rich, approximately 130 to 150 kbp DNA genome, the central region of which is bordered on both sides by ITR regions ("inverted terminal repeats") and terminates in a hairpin structure that covalently links both single DNA strands. The central region of the genome predominantly contains genes essential for viral replication and morphogenesis and highly conserved within poxviruses. In contrast, the ITR regions contain so-called non-conserved virulence genes, which significantly determine the host spectrum, pathogenicity, and immune modulation, thus characterizing the virus.

[0004] Viral replication in poxviruses is restricted to the cytoplasm and begins with the virus binding to the host cell surface. After fusion, the so-called viral core, a protein core containing the viral genome, early viral mRNA, and viral transcription factors (TFs), is released into the cytoplasm. Early viral gene expression then begins, in which viral mRNA is synthesized under the control of poxvirus-specific early promoters. After the core structure is broken down, viral DNA is released into the cytoplasm. In contrast to the transcription of early genes, which occurs exclusively under the control of viral TFs, the subsequent intermediate and late gene transcription depends on the assistance of cellular TFs. Thus, the poxviral expression of early genes, unlike the expression of intermediate and late genes, requires neither the replication of viral DNA nor virus production.

[0005] ORFV is characterized by a very narrow natural host range, which includes sheep and goats. Infections occur via skin lesions that allow the virus to penetrate. Replication of the dermatotropic virus is then limited to regenerative keratinocytes, causing contagious dermatitis or Ecthyma contagiosum This generally mild, self-limiting infection manifests as a locally confined skin or mucous membrane lesion, with pustules caused by massive infiltration of polymorphonuclear lymphocytes occurring primarily in the mouth and udder. The lesion heals without scarring after approximately 4 to 6 weeks. Despite the strong immune response, long-lasting immune protection is not developed, so reinfection is possible after just a few weeks, although clinical symptoms and virus production are significantly reduced. Systemic spread or viremia has not been observed for ORFV, even after experimental intravenous injection of high doses of infectious virus.

[0006] ORFV is considered a zoonotic pathogen and, in rare cases, can be transmitted to humans through broken skin. Following infection, localized modular swelling occurs, usually confined to the fingers and hands, as well as occasional swelling of the lymph nodes and fever. The course is usually harmless, free of complications, and resolves completely within three to eight weeks without any clinical aftereffects. More severe cases of infection have also been observed in immunosuppressed individuals, but these cases resolved completely after treatment with antiviral drugs such as cidofovir or imiquimod.

[0007] ORFV is of interest for the production of recombinant vaccines. Compared to orthopoxviruses, ORFV is characterized by a very narrow natural host tropism, encompassing sheep and goats. This virtually eliminates the possibility of inhibitory "preimmunity" against the vector caused by natural infection in humans, as observed with the most common viral vectors of vaccinia and adenoviruses. Furthermore, the exceptionally weak and short-lived ORFV-specific vector immunity enables highly effective booster and / or booster vaccinations or immunizations with ORFV-based vaccines directed against additional pathogens.

[0008] Administration of ORFV leads to a strong immunostimulatory response in both permissive and non-permissive hosts, characterized by a pronounced induction of innate immune mechanisms and the release of interferons, cytokines, and chemokines. Shortly after immunization, dendritic cells accumulate at the injection site and subsequently initiate a specific adaptive immune response by activating T and B cells. In contrast to vaccines based on inactivated viruses or live vectors, which usually induce a humoral immune response, the balanced induction of the cellular and humoral immune response following immunization with recombinant ORFV is a decisive advantage. At the same time, it eliminates the need for adjuvants, which can lead to undesirable side effects and inflammatory reactions.Further advantages include the possibility of standardized production of recombinant vaccines in permanent cell lines without the use of antibiotics, as well as the elimination of production in chicken eggs (increasing number of protein and antibiotic intolerances).

[0009] An attenuated ORFV vaccine approved for veterinary use is designated D1701, corresponding to the ORFV strain of the same name. This vaccine, known in inactivated form under the trade names Baypamun (Bayer) or Zylexis (Pfizer), was originally obtained by isolating a wild-type virus from sheep and subsequently adapting it through multiple passaging in bovine kidney culture cells. This was followed by further adaptation in Vero cells (African green monkey kidney cells), resulting in the ORFV vector D1701-V. D1701-V is further attenuated and causes only asymptotic infections, even in immunosuppressed sheep.

[0010] Previous recombinant ORFV vectors select the VEGF locus as the insertion site. According to current knowledge, this offers the perceived advantage of further attenuation of the vector by eliminating the vegf-e gene, which is under the control of a poxvirus-specific "early" promoter and is considered a virulence factor. Several ORFV-based recombinant vaccines have now been produced and tested in animal models. Recombinant ORFV vaccines are currently being used against various infectious diseases, such as Aujeszky's disease, rabies, Borna disease, influenza, and classical swine fever.

[0011] The effective immunostimulatory and prophylactic effects of recombinant ORF viruses have been demonstrated in recent years through the establishment of a series of vaccines against various viral infectious diseases. An overview of the use of recombinant poxviruses, including ORFV, for the production of recombinant proteins can be found in Rziha et al. (2000), "Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes," Journal of Biotechnology, Vol. 83, pages 137-145, and Büttner and Rziha (2002), "Parapoxviruses: From the Lesion to the Viral Genome," J. Vet. Med. B., Vol. 49, pages 7-16.

[0012] The limited use of existing recombinant ORFV vectors is primarily due to the lengthy selection process. In particular, the production of polyvalent vaccines has not been possible to date. Furthermore, the known recombinant ORFV vectors do not allow for the specifically regulated expression of foreign genes.

[0013] Ning et al., 2011 describe a rapid method for the production of recombinant Orf viruses (ORFV) based on the enhanced green fluorescent protein (EGFP) reporter gene as a selective marker (Ning et al., BMC Veterinary Research, Generation of recombinant Orf virus using an enhanced green fluorescent protein reporter gene as a selectable marker, 2011, 7:80, 1-13). Tan et al., 2011 investigate strategies for expression of the immunogenic Echinococcus granulosus Peptide EG95 by ORFV with the aim of developing a recombinant bivalent vaccine (Tan et al., Vaccine, Development of orf virus as a bifunctional recombinant Vaccine: Surface display of Echinococcus granulosus antigen EG95 by fusion to membrane structural proteins, 2011, 30, 398-406).

[0014] Against this background, it is an object underlying the invention to provide a novel recombinant ORFV vector, as defined in the claims, with which the disadvantages of the known ORFV vectors and vector systems are reduced or avoided.

[0015] The insertion loci (IL) 1, 2 and 3 described here are located in the ORFV genome in the following regions: IL 1 IL 2 IL 3 Restriktionsfragment Hind III -Fragment C, Hind III -Fragment I / J, Hind III -Fragment G / D, Kpn I -Fragment G, Kpn I -Fragment B, Kpn I -Fragment B, Bam HI -Fragment C / G, Bam HI -Fragment A, Bam HI -Fragment A, Eco RI -Fragment B Eca RI -Fragment A / E Eca RI -Fragment D und / oder Gen / OLR 006, 102, 114, 007 (dUTPase), 103 115, 008 (G1L-Ank), 116, 009 (G2L) 117 (GIF) und / oder Nukleotidposition nt 500 ± 100 bis nt 5.210 ± 100 bis nt 15.660 ± 100 bis nt 2.400 ± 600 nt 7.730 ± 100 nt 17.850 ± 100

[0016] In general, an Orf virus (ORFV) refers to all viruses belonging to the species Parapoxvirus ovis viruses and virus strains. According to the invention, the claimed recombinant vector is based on strain D1701.

[0017] According to the invention, a recombinant ORFV vector is understood to be a vector based on the ORFV genome which is designed for the transport and / or expression of a foreign gene in biological cells.

[0018] According to the invention, a foreign gene is understood to be a gene or open reading frame (OLR) that does not originate from the ORFV genome.

[0019] According to the invention, a promoter is understood to be a nucleic acid segment that enables the regulated expression of the foreign gene in the ORFV vector according to the invention. It is preferably an ORF promoter, i.e., a promoter present in the wild-type ORFV genome or a promoter derived therefrom and possibly artificial, such as a poxvirus promoter, CMV promoter, etc.

[0020] In the recombinant ORFV vector, the position of the insertion loci IL 2 and 3 according to the invention in the ORFV genome can be determined in various ways: by means of restriction fragments, the ORFV genes or open reading frames (OLR) or nucleotide positions in the ORFV genome.

[0021] The traditional description of the localization of IL 1 (not claimed), 2, and 3 is based on restriction maps and the specification of restriction fragments on which the insertion regions are located. The restriction map of ORFV is given as an example for strain D1701 in Cottone et al. (1998), Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701, Virus Research, Vol. 56, pages 53-67. According to the invention, for example, for IL 1, the specification Hind III- Fragment C, Kpn / Fragment G, Bam HI -Fragment C / G, Eco RI -Fragment B, that this insertion locus differs from the Hind III -Fragment C up to the Eco RI -Fragment B. IL 2 extends from the Hind III -Fragment IJ up to the Eco RI -Fragment A / E. IL 3 extends from the Hind III -Fragment G / D up to the Eco RI -Fragment D.

[0022] The designation 006, 007 (dUTPase), 008 (G1L-Ank), 009 (G2L) for IL-1 means that the insertion locus extends from gene or OLR 006 to gene or OLR 009. The information in parentheses refers to the coding products or encoded enzymatic activities, as far as they are currently known.

[0023] According to the invention, IL 1 lies in a range that begins at nucleotide 400 to 600 (500 ± 100) and ends at nucleotide 1800 to 3000 (2,400 ± 600).

[0024] What has been said for IL 1 applies to IL 2 and IL 3 as shown in the table above.

[0025] The inventors were able to determine the indicated nucleotide positions (nt) from numerous determinations on various ORFV strains. The investigations revealed the following positions for strain D1701 and its variants: nt 496 − nt 2.750 ; nt 496 − nt 1.912 und nt 511 − 2.750 nt 5.210 − 7.736 nt 15.656 − 17.849 .

[0026] The inventors were able to determine that foreign genes can be stably integrated into the ORFV genome at the newly identified insertion loci. This was surprising. Previously, it was assumed that the regions of the genome affected by the insertion loci were required for virus replication or unsuitable for the expression of foreign genes.

[0027] A particular advantage of the ORFV vector according to the invention has been found to be that gene expression can be controlled by selecting the insertion locus. For example, the expression of foreign genes in IL-2 is reduced by a factor of 2 compared to the previously used VEGF locus. Furthermore, the strength and timing of expression of the foreign gene can be specifically influenced by selecting the promoter.

[0028] The problem underlying the invention is hereby completely solved.

[0029] According to the inventive design of the recombinant ORFV vector, ORFV is a vector of strain D1701.

[0030] This measure has the advantage of using a vector that is attenuated and causes only asymptomatic infections in the host. All variants of D1701, including D1701-B and D1701-V, are hereby disclosed.

[0031] According to a further preferred embodiment of the recombinant ORFV vector according to the invention, the promoter is an ORFV promoter, more preferably an early ORFV promoter, which more preferably has a nucleotide sequence selected from: SEQ ID No. 1 (P1 promoter), SEQ ID No. 2 (P2 promoter), SEQ ID No. 3 (optimized "early" promoter), SEQ ID No. 4 (7.5 kDa promoter), SEQ ID No. 5 (VEGF promoter) and SEQ ID No. 6 (consensus "early" promoter).

[0032] This approach has the advantage of using promoters that enable high levels of expression of the foreign gene and targeted control of expression. The P1 and P2 promoters were newly developed by the inventors. The remaining promoters originate from the vaccinia virus and are described in other contexts in Davidson and Moss (1989), Structure of vaccinia virus late promoters, J. Mol. Biol., Vol. 210, pages 771 to 784, and Yang et al. (2011), Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts, J. Virology, Vol. 85, No. 12, pp. 5897-5909, Broyles (2003), Vaccinia virus transcription, J. Gen. Virol., Vol. 84, No. 9, pp. 2293-2303. According to the inventors' findings, P2 results in a significantly higher expression level than P1.This was surprising, as the P1 promoter corresponds 100% to the consensus sequence from the vaccinia virus, but not P2. The low expression of the "optimal" vaccinia virus promoter (Orthopox) in the ORFV (Parapox) is a contradiction and surprising. It is also surprising that the P2 promoter leads to very strong expression.

[0033] According to a further preferred embodiment of the recombinant ORFV vector according to the invention, the promoter is arranged at a position of nt 28 ± 10 to nt - 13 ± 10 upstream with respect to the nucleotide sequence coding for the foreign gene.

[0034] This measure has the advantage that the promoter is located at a position that allows high expression strength and controlled expression of the foreign gene.

[0035] According to a further preferred embodiment of the recombinant ORFV vector according to the invention, more than one, preferably 2, 3, 4 or more, nucleotide sequence coding for and expressing a foreign gene is used in at least one of the IL 1, 2 or 3.

[0036] This measure has the advantage that multiple foreign genes can be expressed using a recombinant ORFV vector according to the invention. This configuration is particularly suitable for the production of polyvalent vaccines that simultaneously target multiple antigenic structures. Several foreign genes, preferably 2, 3, 4, or more, can be expressed at each insertion locus.

[0037] According to a preferred embodiment of the invention, the recombinant ORFV vector has a further nucleotide sequence coding for and expressing a foreign gene, which is under the control of a preferably early ORFV promoter, and which is inserted into an insertion locus located in the vegf-E gene in the ORFV genome.

[0038] This approach has the advantage of using a well-characterized insertion locus that has already been described in the state of the art. The use of the vegf locus can be used for targeted control of gene expression. Thus, the expression of the foreign gene can be increased in the vegf locus compared to one of the new expression locuses, e.g., IL-2.

[0039] According to a preferred embodiment of the invention, the foreign gene of the recombinant ORFV vector is selected from the groups of the following antigens: Viral antigen, preferably rabies virus antigen, including glycoprotein (RabG); influenza A antigen, including nucleoprotein (NP), hemaglutinin (HA), neuraminidase (NA); tumor antigen, preferably viral tumor antigen, including HPV-selective viral tumor antigen; tumor-associated antigen, including viral tumor-associated antigen, including HPV-selective viral tumor-associated antigen; parasitic antigen, preferably Plasmodium antigen; cytokine.

[0040] This measure has the advantage that particularly important antigens, in particular for the production of vaccines, can be expressed via the recombinant ORFV virus according to the invention.

[0041] A further subject matter of the present invention relates to a biological cell, preferably a mammalian cell, more preferably a Vero cell, containing the ORFV vector according to the invention.

[0042] The present invention further relates to a composition, preferably a pharmaceutical composition, containing the ORFV vector according to the invention and / or the cell according to the invention. The pharmaceutical composition can preferably be a vaccine, more preferably a polyvalent vaccine.

[0043] The properties, advantages, features and developments of the recombinant ORFV vector according to the invention apply accordingly to the cell according to the invention and the composition according to the invention.

[0044] A further object of the present invention relates to the use of the recombinant ORFV vector according to the invention for the expression of at least one foreign gene, more preferably for the expression of at least one vaccine containing a foreign gene product (monovalent vaccine), more preferably of a vaccine containing at least two foreign gene products (polyvalent vaccine).

[0045] In this context, "at least" means that 2, 3, 4, 5, 6, 7, 8, 9, etc. foreign genes are included.

[0046] The features, advantages, properties and developments of the recombinant ORFV vector according to the invention apply accordingly to the use according to the invention.

[0047] A further object of the present invention relates to a nucleic acid molecule encoding an ORFV promoter, preferably an early ORFV promoter, which has a nucleotide sequence selected from the nucleotide sequences SEQ ID No. 1 (P1) and SEQ ID No. 2 (P2).

[0048] The nucleic acid molecule of the invention encodes novel ORFV promoters that are particularly suitable for the expression of foreign genes in the recombinant ORFV vector of the invention. The promoters result in very strong, early gene expression.

[0049] It is understood that the features mentioned above and those to be explained below can be used not only in the combination specified, but also in other combinations or on their own.

[0050] The invention will now be explained in more detail using exemplary embodiments, from which further features, advantages, and characteristics of the invention will emerge. Reference is made to the accompanying figures, in which the following is shown: Fig. 1 shows the map of the Hind III Restriction fragments of the ORFV D1701-V DNA genome. The hatched boxes represent the insertion sites IL1, IL2, IL3, and vegf. ITRL stands for the inverted terminal repeats of the genome ends. Figure 2 shows a schematic representation of the transfer plasmid pD1-GFP-D2Cherry. The vector displays the AcGFP gene (hatched lines), which is under the control of the artificial early promoter P1 (black arrow, top), and the mCherry gene (hatched boxes), which is under the control of the artificial early promoter P2 (black arrow, right). Behind both fluorescent genes are poxvirus-specific early transcription stop motifs T5NT (black). The genes are separated by a spacer (Sp). Several multiple cloning sites (MCS 1-6) allow the replacement of the fluorescent marker genes with desired foreign genes.The flanking regions containing genes are downstream homologous to the ORFV genome region ORF117 / 118 and upstream homologous to the ORFV genome region ORF114, ensuring targeted integration into the IL-3 locus of the D1701-V genome via homologous recombination. Figure 3 shows an expression analysis of different fluorescent recombinants. (A,a) Fluorescence micrograph of a 6-well plate containing D1701-V-D2Cherry-infected Vero cells. To select the recombinants, Cherry-fluorescent plaques were picked, and the virus was grown from the plaques. After four plaque purifications, the homogeneity of D1701-V-D2Cherry was ensured by PCR analysis. (A,b) Determination of Cherry expression by flow cytometry. The figure shows an example of D1701-V-D2Cherry expression in infected Vero cells (MOI = 1.0) in a flow cytometer. After 48 hours, approximately 45% of all living single cells express Cherry.Uninfected Vero cells served as a negative control. (B) Fluorescence expression of the recombinant D1701-V-GFP-D2Cherry. Vero cells were infected with D1701-V-GFP-D2Cherry (MOI = 0.5). The top row shows a fluorescence image 48 hours after infection (magnification: 20X). The bottom row shows the fluorescence expression after 24 hours (magnification: 63X). Fluorescence microscopy allowed the visualization of AcGFP (GFP), mCherry (mCherry), and both fluorescences in one cell (merged). In addition, the cells were imaged under transmitted light (transmitted light). (C) Fluorescence expression of the recombinant D1701-V-D1GFP-D2Cherry. Vero cells were infected with D1701-V-D1GFP-P2Cherry (MOI = 1.0), and expression was determined using a flow cytometer. After 24 hours, approximately 25% of all viable single cells expressed both mCherry and GFP. Uninfected Vero cells served as a negative control. Fig.Figure 4 shows the determination of the fluorescence intensity of different recombinants. (A) Vero cells were infected with GFP-expressing recombinants (MOI approx. 1.5), and 24 hours later, the mean fluorescence intensity was determined by flow cytometry. Uninfected Vero cells served as a negative control. M1 describes the area in which 99.39% of all uninfected cells (front first curve) are detected. In contrast, area M2 contains GFP-positive cells. The population of GFP-positive cells was comparable to the GFP-expressing recombinants after infection (38.2% - 40.0%). It was evident that the GFP intensity was lowest in D1701-V-D1GFP-infected cells (solid line) and highest in D1701-V-D2GFP-infected cells (----). (B) Vero cells were infected with mCherry-expressing recombinants (MOI approximately 3.0) and 24 hours later the mean fluorescence intensity was determined by flow cytometry.Uninfected Vero cells served as a negative control. M1 describes the area in which 99.47% of all uninfected cells (front first curve) are detected. In contrast, the area M2 contains Cherry-positive cells. The population of mCherry-positive cells was comparable after infection with the GFP-expressing recombinants (62.5% - 63.3%). The mCherry intensity in D1701-V-Cherry-infected cells (- - - - -) was significantly lower than in D1701-V-D2Cherry- (blue line) or in D1701-V2Cherry-infected cells (red line). (C+D) The graphs show the percentage fluorescence intensity of different fluorescence recombinants in relation to D1701-V-GFP (C) and D1701-V-Cherry (D). The data represent averages from at least three independent experiments. Ausführungsbeispiele 1. The ORFV genome

[0051] The ORFV genome consists of a linear double-stranded DNA and has a length of approximately 138 kB, a GC content of approximately 64%, and contains 130-132 genes. The structure of the ORFV genome is similar to that of other poxviruses. It consists of a central region with essential genes that display a high degree of conservation within the Poxviridae. Thus, the ORFV genome contains 88 genes that are conserved in all Chordopoxvirinae. Viral genes that are responsible for the in vitro Growth is not essential, but is relevant for the pathogenicity and tropism of the virus.

[0052] Compared to other Orf viruses, the D1701 virus, which has been adapted for cell culture growth, exhibits a significant increase in the number of inverted terminal repeats (ITRs). These changes led not only to the loss but also to the duplication of some genes, such as the vegf-e gene. Adaptation of D1701-B, propagated in bovine BK-KL3A cells, to growth in Vero cells generated three additional insertion loci, IL 1, IL 2, and IL 3, in the viral genome of the virus now designated D1701-V. These are shown in the Fig. 1 illustrated. 2. Poxvirus promoters

[0053] Poxviruses possess "early," "intermediate," and "late" promoters. These different promoters exhibit several characteristic sequence properties, which are explained below using VACV as an example. The "early" promoter of VACV consists of a 16- or 15-nucleotide critical region, separated from a 7-nucleotide initiator region by an 11-nucleotide spacer region. The critical region is more adenine-rich, whereas the spacer region is more thymine-rich. With rare exceptions, transcription initiation always occurs at a purine. Nucleotide substitutions in the critical region can have a drastically negative effect on promoter activity; even a complete loss of activity is possible. Substitution analyses of early VACV 7.5 kDa promoters provided an optimized critical region, and a consensus sequence for the "early" poxvirus promoter was derived, shown in Table 1. The "intermediate" promoters consist of an AT-rich core sequence approximately 14 nucleotides long, followed by a 10-11 nucleotide spacer region, followed by a short initiator region. The "late" promoters consist of an approximately 20 nucleotide-long AT-rich region, separated by a spacer region of approximately 6 nucleotides before the transcription start site, which contains the highly conserved sequence -1 TAAAT +4. 3. Production of the recombinant ORFV vector

[0054] The inventors sought a new strategy for producing a recombinant polyvalent ORFV vector. During the adaptation of ORFV to Vero culture cells, several deletions in the viral genome occurred. It was investigated whether the deletion regions were suitable for the integration of foreign genes ( Fig. 1A ).

[0055] Therefore, in addition to other plasmids, the transfer plasmid pDel2 was designed, which includes the homologous regions of the IL 3 region ( Fig. 2 ). The cloning of foreign genes into the plasmid was enabled by the use of multiple MCS (multiple cloning sites). In addition, the plasmid was constructed to allow the simultaneous integration of several foreign genes, each under the control of artificial early ORFV promoters and delimited by poxvirus-specific T5NT early transcription stop motifs ( Fig. 2 ).

[0056] Nucleotide sequences of the new artificial early ORFV promoters P1 and P2 were designed.

[0057] In a first experiment, the suitability of the IL-3 locus for the stable integration of foreign genes was investigated. For this purpose, the mCherry fluorescent marker gene was cloned into the pDel2 transfer plasmid under the control of the P2 promoter. The plasmid was then transfected into D1701-VrV-infected Vero cells. New recombinant viruses were visually selected after identifying red-glowing cells using fluorescence microscopy. The homogeneous recombinant D1701-V-D2-Cherry, obtained through multiple plaque purifications, was grown. (Fig. 3A,a ).

[0058] The correct integration of the mCherry gene into the IL 3 locus of D1701-VrV was ensured by specific PCR analyses and Southern blot hybridizations. The correct expression was demonstrated by fluorescence and Western blot analyses as well as flow cytometry ( Fig. 3A,b ).

[0059] Strong expression was detected early after infection. By repeated in vitro Passengers of the recombinant showed that the foreign gene integration into the ORFV genome was stable.

[0060] Furthermore, the simultaneous early expression of two fluorescent genes in different insertion locuses was successfully demonstrated using the recombinant D1701-V-GFP-D2-Cherry, in which the AcGFP gene is integrated into the vegf-e and the mCherry gene into the IL 3 locus ( Fig. 3B ).

[0061] Furthermore, it was investigated whether a second foreign gene could be stably integrated into the IL-3 locus at the same time. For this purpose, the AcGFP gene under the control of the P1 promoter was cloned into the pDel2 transfer plasmid, alongside the P2-controlled mCherry gene. The selection and purification of the homologous recombinants D1701-V-D1-GFP-D2-Cherry was carried out analogously to the previously described D1701-V-P2-Cherry selection.

[0062] Here, too, PCR and Southern blot analyses demonstrated the correct integration of the two foreign genes into the IL-3 locus. Expression was detected by fluorescence microscopy and flow cytometry ( Fig. 3C ).

[0063] The strength of the P1 and P2 promoters was compared with each other and with the P vegf promoter in expression analyses. It was found that the P2 promoter induced the strongest gene expression, while the P1 promoter induced the weakest ( Fig. 4A + 4C). This was surprising, since P1 corresponds 100% to the consensus sequence from the vaccinia virus, but not P2.

[0064] Furthermore, it was demonstrated that the integration of a second foreign gene, regulated under the control of an independent promoter, had no effect on the expression level of the first foreign gene. It was irrelevant whether the second gene was integrated into the same or a different insertion locus. After the insertion of a P2-controlled mCherry gene into the VEGF locus, the influence of the insertion site could be investigated by comparing it with the recombinant that had integrated the P2-regulated mCherry gene into the IL-3 locus. This showed that gene expression in the VEGF locus was approximately twice as strong as in the IL-3 locus ( Fig. 4B + 4D).

[0065] In summary, it was shown that the Orf virus vector D1701-V is highly suitable for the production of polyvalent recombinants. Several foreign genes could be stably integrated into the viral genome, for example, via the newly discovered insertion locus IL 1, 2, and 3, or the known VEGF insertion locus. The strength of foreign gene expression depends on both the promoter and the insertion locus. The strongest gene expression was achieved after integration of a P2-driven foreign gene into the VEGF locus.

[0066] The inventors produced further different vectors that differ from each other in the type and constellation of the various marker foreign genes, insertion sites and promoters (Table 2). Tab. 2: Tabular overview of the newly produced fluorescent ORFV vectors. Rekombinante Lokus Fremdgenexpression VEGF IL3 D1701-V-Cherry P vegf : mCherry - - +++ D1701-V-Cherry-D1GFP P vegf : mCherry P1:AcGFP - +++ / + D1701-V-Cherry-D2GFP P vegf : mCherry - P2: AcGFP +++ / ++++ D1701-V12-Cherry P2: mCherry - - ++++ D1701-V12-Cherry-D2GFP P2: mCherry - P2: AcGFP ++++ / ++++ D1701-V-GFP P vegf : AcGFP - - +++ D1701-V-GFP-D2Cherry P vegf : AcGFP - P2: mCherry +++ / ++++ D1701-V-GFP-D2CD4 P vegf : AcGFP - P2: hCD4 +++ / ++++ D1701-V-D1GFP P vegf : LacZ P1: AcGFP - +++ / + D1701-V-D1GFP-D2Cherry P vegf : LacZ P1: AcGFP P2: mCherry +++ / + / ++++ D1701-V-D2GFP P vegf : LacZ - P2: AcGFP +++ / ++++ D1701-V-D2Cherry P vegf : LacZ - P2: mCherry +++ / ++++ D1701-V-D2Orange P vegf : LacZ - P2: mOrange +++ / ++++ D1701-V-CD4-D2Cherry P vegf : hCD4 - P2: mCherry +++ / ++++

[0067] The table provides an overview of the fluorescent recombinant ORFV vectors produced during the work leading to the invention. The insertion site (locus) and the promoters used to control foreign gene expression (P vegf , P1, P2) are shown for the respective recombinants in the table. The strength of foreign gene expression is also indicated (very strong = ++++ to weak = +).

[0068] The invention opens up a wide range of options for the development of new recombinant ORFV-based vaccines. For example, recombinants can be produced that express multiple antigens simultaneously. This could be a significant advantage in the production of a universal vaccine, combination vaccines, or therapeutic tumor vaccines targeting multiple tumor antigens. Furthermore, the immune response could be specifically influenced by the simultaneous insertion of antigen and cytokines.

Claims

1. Recombinant Orf virus (ORFV) vector comprising: (1) at least one nucleotide sequence encoding and expressing a foreign gene, and (2) at least one promoter controlling the expression of the nucleotide sequence, characterized in that: the nucleotide sequence is localized in at least one of insertion loci (IL) 2 and 3, which are localized in the ORFV genome in the regions below: IL 2IL 3Gene / ORF102,114,103115,116,117 (GIF) characterized in that the ORFV is of the strain D1701.

2. Recombinant ORFV vector of claim 1, characterized in that the ORFV promoter is an early ORF promoter, preferably comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 (P1 promoter), SEQ ID NO: 2 (P2 promoter), SEQ ID NO: 3 (optimized "early" promoter), SEQ ID NO: 4 (7.5 kDa promoter), SEQ ID NO: 5 (VEGF promoter) and SEQ ID NO: 6 (Consensus "early" promoter).

3. Recombinant ORFV vector of any one of the preceding claims, characterized in that the promoter is located at a position of nt -28 ± 10 to nt -13 ± 10 upstream in relation to the nucleotide sequence encoding the foreign gene.

4. Recombinant ORFV vector of any one of the preceding claims, characterized in that in at least one of IL 2 or 3 more than one nucleotide sequence encoding and expressing a foreign gene is inserted, preferably 2, 3, 4 or more nucleotide sequences.

5. Recombinant ORFV vector of any one of the preceding claims, characterized in that the recombinant ORFV vector comprises a further nucleotide sequence encoding and expressing a foreign gene, which is under the control of a, preferably, early ORFV promoter, and which is inserted into an insertion locus, which is located in the vegf-E-gene in the ORFV genome.

6. Recombinant ORFV vector of any one of the preceding claims, characterized in that the foreign gene is selected from the groups of the following antigens: - a viral antigen, preferably Rabies virus antigen, including glykoprotein (RabG); Influenza A antigen, including nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA); - tumor antigen; preferably viral tumor antigen, including HPV- selective viral tumor antigen; - tumor associated antigen; including viral tumor associated antigen, including HPV-selective viral tumor associated antigen; - parasitic antigen; preferably plasmodium antigen; - cytokine.

7. Cell, preferably a mammalian cell, further preferably a Vero cell, containing the recombinant ORFV vector according to any one of the preceding claims.

8. Composition, preferably pharmaceutical composition, containing the recombinant ORFV vector of any one of claims 1 to 6 and / or the cell of claim 7.

9. Composition of claim 8, characterized in that the pharmaceutical composition is a vaccine, preferably a polyvalent vaccine.

10. Use of the recombinant ORFV vector of any one of the preceding claims 1 to 6 for the expression of at least one foreign gene, excluding for the expression in vivo.

11. Use of claim 10 for producing a vaccine comprising at least one foreign gene product, which is a monovalent vaccine, preferably of a vaccine comprising at least two foreign gene products, which is a polyvalent vaccine.