Recombinant Orf virus vector

Abstract

A nucleic acid molecule can code for an Orf virus vector promoter. A recombinant Orf virus vector can be included in a cell. The nucleic acid molecule, the vector and/or the cell can be included in a composition. The recombinant Orf virus vector can be used for the production of a foreign gene.

Claims

1. A recombinant Orf virus (ORFV) vector, which comprises: (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, wherein: the nucleotide sequence is localized in at least one of insertion loci (IL) 1, 2 and 3, which are localized in the ORFV genome in a region selected from the group consisting of the following regions: TABLE-US-00004 IL 1 IL 2 IL 3 Restriction HindIII HindIII fragment I/ HindIII fragment G/ fragment fragment C, J, KpnI fragment B, D, KpnI KpnI fragment BamHI fragment A, fragment B, BamHI G, BamHI EcoRI fragment A/E fragment A, fragment C/G, EcoRI fragment D EcoRI fragment B Gene/ORF 006, 102, 114, 007 (dUTPase), 103 115, 008 (G1L-Ank), 116, 009 (G2L) 117 (GIF) and Nucleotide nt 500 ± 100 to nt 5,210 ± 100 to nt 15,660 ± 100 to position nt 2,400 ± 600 nt 7,730 ± 100 nt 17,850 ± 100, and wherein the OFFV is of the strain D1701.

2. The recombinant ORFV vector of claim 1, wherein the at least one ORFV promoter is an early ORF promoter.

3. The recombinant ORFV vector of claim 2, wherein the early ORF promoter comprises a nucleotide sequence which is selected from the group consisting of: SEQ ID No. 1 (P1), SEQ ID No. 2 (P2), SEQ ID No. 3 (“optimized early”), SEQ ID No. 4 (7.5 kD promoter), and SEQ ID No. 5 (VEGF).

4. The recombinant ORFV vector of claim 1, wherein the at least one 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.

5. The recombinant ORFV vector of claim 1, wherein at least in one of the IL 1, 2 or 3 there is inserted more than one nucleotide sequence encoding and expressing a foreign gene.

6. The recombinant ORFV vector of claim 5, wherein the number of the inserted nucleotide sequences encoding and expressing a foreign gene is selected from the group consisting of: 2, 3, 4 and more than 4.

7. The recombinant ORFV vector of claim 1, further comprising an additional nucleotide sequence encoding and expressing a foreign gene, which is under the control of an early ORFV promoter, and is inserted into an insertion locus which in the ORFV genome is located in the vegf E gene.

8. The recombinant ORFV vector of claim 1, wherein the foreign gene is selected from the group consisting of the following antigens: a viral antigen; a tumor antigen; a tumor associated antigen; a parasitic antigen; and a cytokine.

9. A cell containing the recombinant ORFV vector of claim 1.

10. The cell of claim 9, which is a mammalian cell.

11. The cell of claim 9, which is a Vero cell.

12. A pharmaceutical composition containing the recombinant ORFV vector of claim 1.

13. The pharmaceutical composition of claim 12, which is a vaccine.

14. A pharmaceutical composition containing the cell of claim 9.

15. The pharmaceutical composition of claim 14, which is a vaccine.

16. The recombinant ORFV vector of claim 8, wherein the viral antigen is: a rabies virus antigen; or an influenza A antigen selected from the group consisting of nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA).

17. The recombinant ORFV vector of claim 8, wherein the tumor antigen is a viral tumor associated antigen.

18. The recombinant ORFV vector of claim 8, wherein the viral tumor associated antigen is HPV selective viral tumor-associated antigen.

19. The recombinant ORFV vector of claim 8, wherein the parasitic antigen is plasmodium antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the map of the Hind III restriction fragments of the ORFV D1701-V DNA genome. The hatched boxes represent the insertion positions IL1, IL2, IL3 and vegf. ITRL stands for the inverted terminal repeats of the ends of the genome.

(2) FIG. 2 shows a schematic representation of the transfer plasmid pD1-GFP-D2Cherry. The vector shows the AcGFP gene (line hatching) which stands under the control of the artificial early promoter P1 (black arrow, top), and the mCherry gene (box hatching) which stands under the control of the artificial early promoter P2 (black arrow, right). Following both fluorescence genes there are pox-virus specific early transcription stop motives T5NT (black). The genes are separated from each other via a spacer (Sp). Several multiple cloning sites (MCS 1-6) allow the exchange of the fluorescence marker gene by the desired foreign genes. The genes enclose flanking regions which are downstream homologous to the ORFV genome region ORF117/118, upstream homologous to the ORFV genome region ORF114, and allow a targeted integration into the IL 2 locus of the D1701-V genome via homologous recombination.

(3) FIG. 3 shows an expression analysis of different fluorescence recombinants. (A,a) Fluorescence microscopic image of a 6-well plate with D1701-V-D2Cherry infected Vero cells. For the selection of the recombinants Cherry fluorescent plaques were picked and the virus was grown from the plaques. After four plaque purifications the homogenity of D1701-V-D2Cherry was ensured via PCR analyses. (A,b) Determination of the Cherry expression by means of flow cytometry. The figure shows exemplarily the expression of D1701-V-D2Cherry infected Vero cells (MOI=1.0) in the flow cytometer. After 48 hours approx. 45% of all living single cells express Cherry. Non-infected Vero cells serve as negative control. (B) Fluorescence expression of the recombinant D1701-VGFP-D2Cherry Vero cells were infected with D1701-V-GFP-D2Cherry (MOI=0.5). In the top row a fluorescence image 48 hours after the infection is shown (magnification: 20×). The lower row shows the fluorescence expression after 24 hours (magnification: 63×). The fluorescence microscopy allows the imaging of the AcGFP-(GFP), the mCherry expression (mCherry), and of both fluorescences in one cell (merged). In addition, the cells were imaged in the microscope 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 the expression was determined in the flow cytometer. After 24 hours approx. 25% of all living single cells express both mCherry and also GFP. Non-infected Vero cells were used as negative control.

(4) FIG. 4 shows the determination of the fluorescence intensities of the various recombinants. (A) Vero cells were infected with GFP expressing recombinants (MOI approx. 1.5) and 24 hours later the average fluorescence intensity was determined by flow cytometer. Non-infected Vero cells serve as negative control. M1 describes the region where 99.39% of all non-infected cells (front first curve) can be detected. In contrast, in the region M2 the GFP-positive cells can be found. The population of GFP-positive cells after the infection was comparable with the GFP-expressing recombinants (38.2%-40.0%). It was apparent that the GFP intensity in D1701-V-D1GFP-infected cells (continuous line) was the lowest, in D1701-V-D2GFP-infected cells (⋅-⋅-⋅-⋅-) was the strongest. (B) Vero cells were infected with mCherry-expressing recombinants (MOI approx. 3.0) and 24 hours later the average fluorescence intensity was measured by flow cytometry. Non-infected Vero cells serve as negative control. M1 describes the area where 99.47% of all non-infected cells (front first curve) can be detected. In contrast, in the area M2 there are Cherry-positive cells. The population of mCherry-positive cells after the infection was comparable 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 of the fluorescence intensity of various fluorescence recombinants in relation to D1701-V-GFP (C) or to D1701-V-Cherry (D). The data represent mean values from at least 3 independent experiments.

EXAMPLES

(5) 1. The ORFV genome

(6) The ORFV genome consists of a linear double-stranded DNA and has a length of about 138 kB, a GC content of about 64% and comprises 130-132 genes. The construction of the ORFV genome is similar to that of other pox viruses. It consists of a central region with essential genes which comprise a high degree of conservation within the pox viridae. In the ORFV genome there are 88 genes which are conserved in all Chordopoxvirinae. In the terminal regions viral genes are localized which are non-essential for the growth in vitro, however which are relevant for the pathogenicity and the tropism of the virus.

(7) In comparison to other Orf viruses the D1701 virus which is adapted to the replication in cell culture shows a significant enlargement of the inverted terminal repeats (ITR). These alterations do not only cause a loss but also a duplication of several genes, including the vegf-e gene. The adaptation of D1701-B amplified in bovine BKKL3A cells to the growth in Vero cells resulted in three additional insertion loci IL 1, IL 2 and IL 3 in the virus genome which is now referred to as D1701-V. They are illustrated in the FIG. 1.

(8) 2. Poxvirus Promoters

(9) Poxviruses comprise “early”, “intermediate” and “late” promoters. These different promoters comprise several characteristic sequence features, which is further explained in the following with the example of VACV. The “early” promoter of the VACV consists of a critical region with the length of 16 or 15 nucleotides, respectively, which are spaced by an initiator region with a length of 7 nucleotides by a spacer region of 11 nucleotides. This critical region is rather adenine rich, whereas the spacer region is rather rich in thymine. The initiation of the transcription always takes place at a purine, with rare exceptions. Nucleotide substitutions in the critical region may have a dramatically negative effect on the promoter activity, even a complete loss of the activity is possible. Substitution analysis of the early VACV 7.5 kDa promoter showed an optimized critical region, in addition it was succeeded in deriving a consensus sequence for the “early” poxvirus promoter which is shown in the table 1. The “intermediate” promoters consist of an AT rich core sequence which is about 14 nucleotides long, followed by a spacer region of 10-11 nucleotides, which is followed by a short initiator region. The structure of the “late” promoters consists of an upstream AT-rich region of about 20 nucleotides which is separated from the transcription start position by a spacer region of about 6 nucleotides which includes the highly conserved sequence-1 TAAAT+4.

(10) TABLE-US-00002 TABLE 1 Used promoters; P1 and P2 were newly developed by the inventors. critical region −28 −27 −26 −25 −24 −23 −22 −21 −20 −19 −18 optimized A A A A A T T G A A A “early” 7.5 kDa A A A A G T A G A A A promoter consensus A A A A A A T G A A A “early” VEGF C A Â A A T G T A A A P1 A A A A A T T G A A A P2 A A A A A T T G A A A % identity critical region SEQ with opt. −17 −16 −15 −14 −13 ID “early” optimized A A C/T T A 3 Spacer Initiator “early” region Region 7.5 kDa A T T A 4 75 Thymine Start promoter rich −12 mostly at consensus A A A A/T A 5 87.5 to −2 purine-1 “early” to 6 VEGF T T A T A 6 62.5 P1 A A T T A 1 100 P2 T T C T A 2 87.5
3. Production of the Recombinant ORFV Vector

(11) The inventors have searched for a new strategy for the production of a recombinant polyvalent ORFV vector. During the adaption of ORFV to Vero culture cells several deletions in the viral genome have occurred. It was examined whether the regions of the deletions are suited for the integration of foreign genes (FIG. 1A).

(12) For this reason, along with further plasmids the transfer plasmid pDel2 was designed which includes the homologous sections of the IL 2 region (FIG. 2). The cloning of foreign genes into the plasmid was enabled by using several MCS (multiple cloning sites). Additionally, the plasmid was designed in such a way that it allows the simultaneous integration of multiple foreign genes which are each under the control of artificial early ORFV promoters and which are bounded by pox-virus specific T5NT early transcription stop motives (FIG. 2).

(13) Nucleotide sequences of the new artificial early ORFV promoters P1 and P2 were designed.

(14) In a first experiment it should be examined whether the IL 2 locus is appropriate for the stabile integration of foreign genes. For this purpose the mCherry fluorescence marker gene was cloned under the control of the promoter P2 into the pDel2 transfer plasmid. In the following the plasmid was transfected into Vero cells which were infected by D1701-VrV, and new recombinant viruses were visually selected after the identification of red-shiny cells by means of fluorescence microscopy and the homogenous recombinant D1701-V-D2-Cherry obtained via several plaque purifications was cultivated (FIG. 3A,a).

(15) The correct integration of the mCherry gene into the IL 2 locus of D1701-VrV was ensured by specific PCR analyses and Southern blot hybridizations, the correct expression could be demonstrated by fluorescence and Western blot analyses but also by means of flow cytometry (FIG. 3A,b).

(16) In this context a strong expression could be detected early after the infection. By multiple passages in vitro of the recombinant it could be demonstrated that the integration of the foreign gene into the ORFV genome was stable.

(17) It could be also demonstrated by means of the generated recombinant D1701-V-GFP-D2-Cherry where the AcGFP gene is integrated in the vegf-e gene and the mCherry gene is integrated into the IL 2 locus that two fluorescence genes were simultaneously early expressed in different insertion loci (FIG. 3B).

(18) Furthermore it should be examined whether at the same time a second foreign gene can be stably integrated into the IL 2 locus. For this purpose, in addition to the P2-controlled mCherry gene the AcGFP gene under the control of the P1 promoter was cloned into the pDel2 transfer plasmid. The selection and purification of the homologous recombinant D1701-V-D1-GFP-D2-Cherry was realized in analogy to the previously described D1701-V-P2-Cherry selection.

(19) Again, the correct integration of both of the foreign genes into the IL 2 locus was demonstrated via PCR and Southern blot analysis. The detection of the expression was made via fluorescence microscopy and flow cytometry (FIG. 3C).

(20) The strength of the promoter P1 and P2 was compared among each other and with the promoter P.sub.vegf in expression analyses. It could be shown that the promoter P2 induced the strongest and the promoter P1 included the weakest gene expression (FIG. 4A+4C). This was surprising because P1 corresponds by 100% to the consensus sequence from the vaccinia virus, not P2.

(21) It could also be demonstrated that the integration of a second regulated foreign gene under the control of an individual promoter has no effect on the expression strength 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 region could be analyzed in comparison with the recombinant which had the P2-regulated mCherry gene integrated into the IL 2 locus. It could be shown that the gene expression in the VEGF locus was about two times stronger than in the IL 2 locus (FIG. 4B+4D).

(22) To summarize, it could shown that the Orf virus vector D1701-V is very well suited for the production of polyvalent recombinants. Several foreign genes could be stably integrated into the viral genome, e.g. via the newly discovered insertion loci IL 1, 2 and 3, or via the known insertion locus VEGF. The strength of the foreign gene expression depends both on the promoter but also on the insertion locus. The strongest gene expression was achieved after the integration of a P2-controlled foreign gene into the VEGF locus.

(23) The inventors have generated further various vectors which can be distinguished from the kind and constellation of the different marker foreign genes, insertion regions and promoters (Tab. 2).

(24) TABLE-US-00003 TABLE 2 Tabular overview on the newly generated fluorescent ORFV vectors. Locus Foreign Gene Recombinant VEGF IL2 Expression D1701-V-Cherry P.sub.vegf: mCherry — — +++ D1701-V-Cherry-D1GFP P.sub.vegf: mCherry P1: AcGFP — +++/+ D1701-V-Cherry-D2GFP P.sub.vegf: mCherry — P2: AcGFP +++/++++ D1701-V12-Cherry P2: mCherry — — ++++ D1701-V12-Cherry-D2GFP P2: mCherry — P2: AcGFP ++++/++++ D1701-V-GFP P.sub.vegf: AcGFP — — +++ D1701-V-GFP-D2Cherry P.sub.vegf: AcGFP — P2: mCherry +++/++++ D1701-V-GFP-D2CD4 P.sub.vegf: AcGFP — P2: hCD4 +++/++++ D1701-V-D1GFP P.sub.vegf: LacZ P1: AcGFP — +++/+ D1701-V-D1GFP-D2Cherry P.sub.vegf: LacZ P1: AcGFP P2: mCherry +++/+/++++ D1701-V-D2GFP P.sub.vegf: LacZ — P2: AcGFP +++/++++ D1701-V-D2Cherry P.sub.vegf: LacZ — P2: mCherry +++/++++ D1701-V-D2Orange P.sub.vegf: LacZ — P2: mOrange +++/++++ D1701-V-CD4-D2Cherry P.sub.vegf: hCD4 — P2: mCherry +++/++++

(25) The table gives an overview on the fluorescent recombinant ORFV vectors which were generated during the works resulting in the invention. The region of insertion (locus) and the promoters used for controlling the foreign gene expression (P.sub.vegf, P1, P2) are indicated in the table for each of the recombinants. In addition, the strength of the foreign gene expression is indicated (very strong=++++ to weak=+).

(26) The invention creates a variety of options for the development of new recombinant ORFV based vaccines. Recombinants can be generated which simultaneously express multiple antigenes. This could be a significant advantage in the production of a universal vaccine of combination vaccines or of therapeutic tumor vaccines which are directed against multiple tumor antigens. In addition, the immune response can be influenced by a simultaneous insertion of antigene and cytokines in a targeted manner.