# Patent

## Title
Recombinant proteins of parapdxvirus ovis and pharmaceutical compositions therefrom

## Abstract

The invention relates to polynucleotides coding for the PPVO viral genome, to fragments of the polynucleotides coding for the PPVO genome and to polynucleotides coding for individual open reading frames (ORFs) of the PPVO viral genome. The invention also relates to recombinant proteins expressed from the above mentioned polynucleotides and to fragments of said recombinant proteins, and to the use of said recombinant proteins or fragments for the preparation of pharmaceutical compositions.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/717,640, filed 17 Dec. 2012, now allowed, which is a continuation of U.S. patent application Ser. No. 10/481,112, filed 11 Jun. 2004, now patented as U.S. Pat. No. 8,357,363, which is a U.S. National Phase Application of International Patent Application No. PCT/EP2002/006440, having an international filing date of 12 Jun. 2002, which claims priority to New Zealand Patent Application No. 512341 filed 13 Jun. 2001. The contents of each of these applications are incorporated herein by reference in their entirety.


SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 595282000303SeqList.txt, date recorded: 5 Sep. 2014, size: 234,776 bytes).
FIELD OF THE INVENTION
The present invention relates to polynucleotides and recombinant proteins of Parapoxvirus ovis (PPVO) and their use, alone or in combination with other substances, for the manufacture of pharmaceutical compositions.
BACKGROUND OF THE INVENTION
It is known that latent and chronically persistent viral infections can be activated or reactivated by immunosuppression, or conversely that the immune system suppresses acute diseases which may be caused by a latent virus (for example a latent herpes virus infection recurs as a result of immunosuppression in the form of lip vesicles in cases of stress or the administration of cortisone). It is also known that chronically persistent latent viral infections can only be treated with difficulty or not at all using conventional low-molecular-weight antiviral substances.
It was demonstrated that class I restricted cytotoxic T cells were capable of inhibiting hepatocellular HBV gene expression in HBV-transgenic mice, and that this process was caused by TNF-α and IFN-γ.
It is also known that in the case of chronically persistent viral infections a superinfection with another virus can produce antiviral effects against the chronically persistent virus. The dependence of this effect on interferons such as IFN-γ, as well as other cytokines and chemokines, such as TNF-α, which are secreted by T cells, natural killer cells and macrophages, has been demonstrated.
BAYPAMUN®, a pharmaceutical product for inducing “paraspecific immunity”, i.e., a pharmaceutical product for inducing the unspecific immune system, is used therapeutically, metaphylactically and prophylactically for the treatment of animals in need. BAYPAMUN® is manufactured from chemically inactivated PPVO strain D1701 (see German Patent DE3504940). The inactivated PPVO induces in animals non-specific protection against infections with the most diverse types of pathogens. It is assumed that this protection is mediated via various mechanisms in the body's own defense system. These mechanisms include the induction of interferons, the activation of natural killer cells, the induction of “colony-stimulating activity” (CSA) and the stimulation of lymphocyte proliferation. Earlier investigations of the mechanism of action demonstrated the stimulation of interleukin-2 and interferon-α.
The processes for the production of the above-mentioned pharmaceutical compositions are based on the replication of the virus in cultures of suitable host cells.
One aspect of the invention relates to the use of particle-like structures comprising recombinant proteins of the invention. These particle-like structures can be, e.g., fusion proteins, protein-coated particles or virus-like particles.
Methods to produce fusion proteins, protein-coated particles or virus-like particles comprising recombinant proteins of the invention are well known to persons skilled in the art: Casal (Biotechnol. Genet. Eng. Rev. (2001) 18:73-87) describes the use of baculovirus expression systems for the generation of virus-like particles. Ellis (Curr. Opin. Biotechnol. (1996) 7(6):646-652) presents methods to produce virus-like particles and the application of suitable adjuvants. Roy (Intervirology (1996) 39(1-2):62-71) presents genetically engineered particulate virus-like structures and their use as vaccine delivery systems. Methods to produce fusion proteins are also well known to the person skilled in the art (Gaudin, et al., Gen. Virol. (1995) 76:1541-1556; Hughson, Curr. Biol. (1995) 5(3):365-374; Uhlen, et al., Curr. Opin. Biotechnol. (1992) 3(4):363-369). Known to the person skilled in the art is also the preparation of protein-coated micro- and nanospheres (Arshady, Biomaterials (1993) 14(1):5-15). Proteins can be attached to biodegradable microspheres (Cleland, Pharm Biotechnol. (1997) 10:1-43) or attached to other polymer microspheres (Hanes, et al., Pharm. Biotechnol. (1995) 6:389-412) such as, e.g., polysaccharides (Janes, et al., Adv. Drug Deliv. Rev. (2001) 47(1):83-97).
PPVO NZ2 is another Parapoxvirus strain that exhibits immunostimulatory effects when administered in inactivated form to mammals.
The closest prior art describes the construction of an expression library representing about 95% of the PPVO NZ2 genome using the Vaccina lister virus to create recombinant viruses comprising the complete Vaccina lister genome and various fragments of the PPVO genome (Mercer, et al., Virology, (1997) 229:193-200). For the construction of the library, 16 PPVO DNA fragments with an average size of 11.4 kb were inserted into the Vaccinia lister genome. Each fragment was mapped relative to the PPVO restriction endonuclease maps but was otherwise uncharacterized (FIG. 1). It was found that a major portion of the PPVO genes were expressed in cells infected by the recombinant virus. The authors also showed that the entirety of all PPVO proteins expressed by some of the recombinant viruses of the expression library was able to provide protection against challenge with virulent PPVO. Expression of PPVO genes of the individual recombinant viruses has been demonstrated by immunofluorescence and immune precipitation (Mercer, et al., Virology (1997) 229:193-200).
To identify components of PPVO responsible for the vaccinating activity of PPVO, the Vaccinia lister/PPVO NZ2 expression library was applied.
Based on the above background it was desirable to develop PPVO based pharmaceutical compositions with antiviral and anti-tumor efficacy as well as with efficacy in paraimmunization and other desirable therapeutic effects. It was also desirable to obtain a pharmaceutical composition that exerts its full therapeutic effect while showing fewer side effects. It was furthermore desirable to find methods to produce PPVO based pharmaceutical compositions in large quantities and in economically advantageous manners.
These desirable effects have been achieved by the systematic use of selected recombinant proteins of PPVO alone or in combination with other recombinant proteins from PPVO for the preparation of pharmaceutical compositions for the treatment of objects in need.
SUMMARY OF THE INVENTION
The invention relates to polynucleotides coding for the PPVO viral genome, to fragments of the polynucleotides coding for the PPVO genome and to polynucleotides coding for individual open reading frames (ORFs) of the PPVO viral genome. The invention also relates to fragments of said polynucleotides of at least 15 or 30 or 100 base pairs in length. The invention also relates to recombinant proteins expressed from the above mentioned polynucleotides and to fragments of said recombinant proteins of at least 5 or 10 or 30 amino acids, and to the use of recombinant proteins or fragments for the preparation of pharmaceutical compositions.
“Fragments” of a polynucleotide, within the meaning of the invention, shall be understood as polynucleotides that have the same nucleotide sequence as contiguous parts of the full length (the original) polynucleotide.
“Active fragments”, within the meaning of the invention, shall be those fragments of the PPVO genome the expression products of which have demonstrated to be pharmacologically active according to the invention, when inserted into the Vaccina lister genome and expressed in a suitable host.
Whereas the use of the complete PPVO virus for the manufacture of vaccines against PPVO challenge has been described, the present invention relates to the use of polynucleotides coding for the PPVO viral genome and selected fragments of the PPVO viral genome and of selected PPVO expression products, alone or in combination with others, for the preparation of improved pharmaceutical compositions for the treatment of various diseases.
The systematic use of selected genomic fragments of PPVO and their recombinant expression products makes it possible to produce pharmaceutical compositions which contain fewer (and may not contain any) inactive components (i.e., polynucleotides and proteins of PPVO) in addition to the active components.
These pharmaceutical compositions which contain less, or do not contain any additional inactive components are generally preferred by doctors and patients compared to the less well defined biological preparations of inactivated virus material. Furthermore, the possibility of producing the recombinant product in fermentation processes allows an economically advantageous mode of production. It is well known to persons skilled in the art that an economically advantageous mode of production can be achieved, e.g., by using rapidly growing production organisms (host organisms) which might also place low demands on the culture medium employed. Microorganisms which can advantageously be used as hosts for the production of recombinant proteins include, e.g., but are not limited to, Escherichia coli, Bacillus spec., Corynebacterium spec., Streptomyces spec., as well as yeasts, e.g., Saccharomyces cerevisiae, Candida spec., Pichia spec., Hansenula spec., and filamentous fungi, e.g., Aspergillus spec., Penicillium spec. and other suitable microorganisms.
Recombinant proteins of the invention can also be produced from cell lines expressing the proteins of interest. These cell lines can be recombinant mammalian cell lines, recombinant insect cell lines (e.g., using the baculovirus transfection system) or other suitable expression systems. Transfection can be achieved by various techniques known to the skilled person, one of which is the use or recombinant viruses such as the Vaccinia virus/PPVO recombinants (VVOVs) described in the examples.
DESCRIPTION OF THE INVENTION
The invention relates to fragments of the PPVO genome of at least 15 or 30 or 100 base pairs in length, and recombinant proteins expressed therefrom and to the use of said fragments and recombinant proteins for the preparation of pharmaceutical compositions. The invention also relates to individual genes (ORFs) of PPVO and their expression products, and their use, alone or in combination with others, for the preparation of pharmaceutical compositions.
A protein, within the meaning of the invention, is any polypeptide of at least five amino acids. A recombinant protein, within the meaning of the invention, is any protein that is expressed in a cell, to which the coding polynucleotide was introduced using recombinant DNA technology.
A polynucleotide, within the meaning of the invention, is meant to comprise, polyribonucleotides and/or polydesoxyribonucleotides.
Pharmaceutical compositions of the invention can be used as immunotherapeutic or immunoprophylactic agents for the treatment of infectious and non-infectious immunodeficiencies. They can also be used for the treatment of tumor diseases, cancer, viral infections and diseases associated therewith, such as, e.g., hepatitis, papillomatosis, herpes virus infections, liver fibrosis, for the prevention or prophylaxis of infectious diseases after stress (e.g., operations), for the prevention and prophylaxis of infectious diseases by administration prior to operations or procedures (e.g., preceding implantations of artificial limbs or dental procedures), for the prophylactic and metaphylactic treatment of non-viral infections, for the healing of wounds, and in particular for accelerating wound-healing processes and for promoting the healing of poorly healing or non-healing wounds (e.g., Ulcus cruris), for diseases such as multiple sclerosis, warts and other skin neoplasms, for allergic diseases, for preventing the onset of systemic allergies and for topical allergies and for improving well-being, e.g., in old age, for autoimmune diseases, chronic inflammatory diseases, such as, e.g., Crohn's disease, COPD and asthma. It is an object of the invention to use of polynucleotides and recombinant proteins of PPVO for the production of pharmaceutical compositions for the treatment of the above mentioned conditions and diseases in humans and animals.
The viral strains of the invention are PPVO NZ2 and homologues, such as D1701, NZ7, NZ10 and orf-11 strains. It is also possible to use polynucleotides and recombinant proteins of the progeny of these strains obtained by passaging and/or adaptation using specific cells, such as, e.g., WI-38, MRC-5 or Vero cells.
We have found that the identified recombinant proteins are effective for the treatment of viral diseases, cancer and other diseases or conditions in which a Th1 type immune response is of benefit. The results obtained also imply that PPVO gene products or parts thereof protect hepatitis virus-expressing hepatocytes (e.g., hepatitis B virus, HBV, or hepatitis C virus, HCV) from immune attack through HBV or HCV specific cytotoxic CD8+ T cells circulating in the blood because T cells will not leave the blood stream if their specific antigen is not presented by liver sinus endothelial cells (LSEC, that anatomically separate hepatocytes from T cells passing the liver with the blood). Therefore, we expect to have a recombinant protein that is derived from the ORFs 120-R3 (base pairs 122616-136025 Bp, recombinant virus VVOV82) that is able to down-modulate or prevent side effects such as necroinflammatory liver disease when immunostimulants, e.g., cytokines or any others including the proteins described above administered to, e.g., hepatitis patients.
Considering the knowledge about the influence of a Th1 type immune induction in conditions and diseases such as latent and or chronic viral infections, proliferative diseases such as cancer and the capability of recombinant proteins that contain gene products of PPVO or parts thereof to induce a Th1 immune response or a local immune response selectively, we claim the use of polynucleotides and recombinant polypeptides of PPVO and recombinant proteins that contain gene products of PPVO or parts thereof for the manufacture of pharmaceutical compositions for use in humans and animals. The recombinant proteins are made from products or parts thereof of the following open reading frames (ORFs) of PPVO NZ2: 64r-96r (recombinants VVOV 285 and VVOV 330 as well as VVOV 243 and VVOV 283), 18r-57 (recombinants VVOV 97, VVOV 96 and VVOV 245), 4r-14r (recombinant VVOV 215). The recombinant protein may also be made from gene products or parts thereof of ORFs 120-R3 (recombinant VVOV 82). The proteins may be prepared and used in any combination.
Recombinant proteins of PPVO within the meaning of the invention shall be understood as proteins that derive from PPVO and are expressed in homologous or heterologous systems other than the systems in which PPVO is naturally produced. Examples for recombinant proteins of PPVO are proteins of PPVO which are expressed using Vaccinia virus vectors and fibroblasts as host cells or baculovirus vectors and insect cells as host cells. Recombinant proteins, within the meaning of the invention, could also be produced in bacterial cells (e.g., Escherichia coli, Bacillus spec., Streptomyces spec.) or in yeast (e.g., Saccharomyces cerevisiae, Candida spec., Pichia pastoris, Hansenula spec.) systems. In these cases, polynucleotides of the PPVO genome would typically be brought into the respective host genome so that PPVO genes are expressed by the host. Recombinant proteins of PPVO could also be expressed by the object in need in the sense of a gene therapy.
Recombinant proteins, within the meaning of the invention, could also be recombinant virus particles that contain PPVO derived proteins. Recombinant proteins, within the meaning of the invention, could also be in form of viral-like particles that are formed or assembled from PPVO derived proteins. Recombinant proteins, within the meaning of the invention, could also be chimeric proteins that contain PPVO gene products.
In a preferred embodiment of the invention the recombinant proteins are attached to particle-like structures or be part of particle-like structures.
In another preferred embodiment of the invention the recombinant proteins are attached to, or part of, fusion proteins.
In another preferred embodiment of the invention the recombinant proteins are attached to, or part of, protein-coated particles.
In another preferred embodiment of the invention the recombinant proteins are attached to, or part of, virus-like particles.
Particle-like structures, such as particle-like fusion proteins, protein-coated particles or virus-like particles can be phagocytosed and processed by monocytes or macrophages. The process of phagocytosis enhances the efficacy of recombinant proteins of the invention in uses within the meaning of the invention.
A particle-like structure, within the meaning of the invention, is particulate matter in particle-like form of which the average particle size and other characteristics are suitable for medical application. Preferred particle-like structures are, e.g., fusion proteins, protein-coated particles, or virus-like particles.
Immunomodulating activity is defined as local or systemic suppression and/or stimulation and/or induction of any Th-1 or Th-2 type cytokine response or of any effector function of these cytokines, (e.g., cytolytic or antiviral activity or humoral response) or the modulation of MHC cross-presentation. Immunomodulating activity could also be the induction of apoptosis in antigen presenting cells or recruiting of antigen presenting cells.
Nucleotides and recombinant proteins of the invention can be administered at the same time or sequentially, administered with other agents and drugs, e.g., with drugs that treat the disease or are supportive, e.g., in the case of cancer therapy with antineoplastic or other anti-cancer agents or/and anti-coagulants or vitamins, pain relief and others.
The nucleotides and recombinant proteins can be administered systemically (e.g., intravenously, subcutaneously, intramuscularly, intracutaneously, intraperitoneally), locally (e.g., into a tumor) or orally (per os). The recombinant proteins or products thereof should be formulated appropriately, e.g., in a non-pyrogenic solution or suspension for i.v. use or in capsules for implantation or in capsules for per os use. Pharmaceutical compositions of the invention can be administered, e.g., oral, nasal, anal, vaginal etc., as well as parenteral administration. Pharmaceutical compositions of the invention can be in the form of suspensions, solutions, syrups, elixirs or appropriate formulations in polymers as well as liposomes.
Recombinant proteins of the invention can also be prepared with suitable recombinant cell lines and other cell lines. Alternatively, non-recombinant cell lines, such as WI-38, MRC-5, Vero cells could be infected with recombinant viruses that carry the recombinant genes using viral vectors such as, but not limited to, the Vaccina virus (e.g., Vaccina lister). In addition, other suitable viruses can be used in combination with other suitable cells (e.g., using Vaccinia virus vectors and fibroblasts as host cells or baculovirus vectors and insect cells as host cells). It is advantageous to cultivate the recombinant cell cultures in high-cell-density fermentations to achieve favorable productivity and a good overall process performance.
The invention relates to purified and isolated polynucleotides with the sequence of SEQ ID NO:1. The invention also relates to purified and isolated polynucleotides of at least 15 or 30 or 100 nucleotides which bind under stringent conditions to the polynucleotide of SEQ ID NO:1 or its complementary sequences.
Stringent conditions, within the meaning of the invention are 65° C. in a buffer containing 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% (w/v) SDS.
The invention also relates to purified and isolated polynucleotides which comprise the polynucleotide sequence of SEQ ID NO:1 or polynucleotide sequences encoding the same amino acid sequence and fragments of at least 15 or 30 or 100 nucleotides thereof. The invention also relates to recombinant proteins of five and more amino acids encoded by these polynucleotides.
The invention also relates to purified and isolated polynucleotides which show at least 99%, 95% or 90% or 80% sequence homology to the polynucleotides of the previous paragraph.
Homology of biological sequences, within the meaning of the invention, shall be understood as the homology between two biological sequences as calculated by the algorithm of Needleman and Wunsch. (J. Mol. Biol. (1970) 48:443-453) using the BLOSUM62 substitution matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA (1992) 89:10915-10919) for proteins and penalties of +4 and −3 for identical and non-identical bases, respectively, when comparing polynucleotide sequences. For comparison of protein sequences the gap creation penalty and the gap extension penalty are 8 and 2, respectively. For comparison of polynucleotide sequences the gap creation penalty and the gap extension penalty are 20 and 3, respectively.
The invention also relates to purified and isolated polynucleotides which are active fragments of the PPVO genome, with a sequence selected from a group of sequences consisting of nucleotides 122616-136025 of SEQ ID NO:1 (PPVO insert of VVOV 82), 31003-46845 of SEQ ID NO:1 (PPVO insert of VVOV 96), 24056-33789 of SEQ ID NO:1 (PPVO insert of VVOV 97), 10264-20003 of SEQ ID NO:1 (PPVO insert of VVOV 215), 82324-92502 of SEQ ID NO:1 (PPVO insert of VVOV 243), 47952-66263 of SEQ ID NO:1 (PPVO insert of VVOV 245), 89400-103483 of SEQ ID NO:1 (PPVO insert of VVOV 283), 74804-88576 of SEQ ID NO:1 (PPVO insert of VVOV 285), and 102490-108393 of SEQ ID NO:1 (PPVO insert of VVOV 330).
The invention also relates to purified and isolated polynucleotide which encode for the same amino acid sequence as the active fragments of the PPVO genome of the previous paragraph and to polynucleotides of at least 15 or 30 or 100 nucleotides binding under stringent conditions to the above mentioned active fragments of the PPVO genome or its complementary sequence.
The invention also relates to polynucleotides with 99%, 95%, or 90%, or 80% sequence homology to sequences consisting of nucleotides 122616-136025 of SEQ ID NO:1 (PPVO insert of VVOV 82), 31003-46845 of SEQ ID NO:1 (PPVO insert of VVOV 96), 24056-33789 of SEQ ID NO:1 (PPVO insert of VVOV 97), 10264-20003 of SEQ ID NO:1 (PPVO insert of VVOV 215), 82324-92502 of SEQ ID NO:1 (PPVO insert of VVOV 243), 47952-66263 of SEQ ID NO:1 (PPVO insert of VVOV 245), 89400-103483 of SEQ ID NO:1 (PPVO insert of VVOV 283), 74804-88576 of SEQ ID NO:1 (PPVO insert of VVOV 285), and 102490-108393 of SEQ ID NO:1 (PPVO insert of VVOV 330) or the respective complementary sequences.
The invention also relates to purified and isolated polynucleotide, with a sequence of nucleotides 3 to 539 (ORF L1), 781 to 449 (ORF L2r), 1933 to 1664 (ORF L3r), 3269 to 2790 (ORF L4r), 2799 to 3851 (ORF L5), 2962 to 3753 (ORF L6), 3784 to 3122 (ORF L7r), 4341 to 4129 (ORF L8r), 4904 to 4428 (ORF 1ar), 6517 to 4970 (ORF 1r), 8042 to 6684 (ORF 2r), 9989 to 8070 (ORF 3r), 11195 to 10062 ORF 4r), 11493 to 11227 (ORF 5r), 11802 to 12038 (ORF 6), 12358 to 12080 (ORF 7r), 13980 to 12364 (ORF 8r), 14826 to 14053 (ORF 9ar), 15080 to 15394 (ORF 10), 16838 to 15423 (ORF 11r), 19021 to 16847 (ORF 12r), 19704 to 19156 (ORF 13r), 20314 to 19736 (ORF 14r), 20401 to 22101 (ORF 15), 22125 to 22940 (ORF 6), 23003 to 23866 (ORF 17), 26908 to 23873 (ORF 18r), 26926 to 27213 (ORF 19), 27626 to 27216 (ORF 20r), 29754 to 27616 (ORF 21r), 32217 to 29800 (ORF 22r), 33380 to 32418 (ORF 23r), 33602 to 33393 (ORF 24r), 34466 to 33612 (ORF 25r), 34735 to 34502 (ORF 26r), 35905 to 34739 (ORF 27r), 37194 to 35905 (ORF 28r), 37200 to 39248 (ORF 29), 41037 to 39229 (ORF 30r), 41374 to 42066 (ORF 31), 42336 to 41731 (ORF 32r), 42407 to 41997 (ORF 33r), 42410 to 43765 (ORF 34), 43770 to 43958 (ORF 35), 43980 to 44534 (ORF 36), 45727 to 44537 (ORF 37r), 45760 to 46557 (ORF 38), 46567 to 47568 (ORF 39), 47572 to 48303 (ORF 40), 48352 to 48621 (ORF 41), 49887 to 48634 (ORF 42r), 49917 to 50693 (ORF 43), 50719 to 51102 (ORF 44), 51059 to 51511 (ORF 44a), 51584 to 52591 (ORF 45), 52509 to 53066 (ORF 46), 53523 to 53023 (ORF 47r), 53607 to 57473 (ORF 48), 58070 to 57528 (ORF 49r), 57700 to 58662 (ORF 50), 59674 to 58673 (ORF 51r), 62089 to 59678 (ORF 52r), 62198 to 62881 (ORF 53), 62909 to 63862 (ORF 55), 63858 to 64271 (ORF 56), 64309 to 66831 (ORF 57), 67266 to 66799 (ORF 58r), 67803 to 67273 (ORF 58ar), 67915 to 68607 (ORF 59), 68624 to 70984 (ORF 60), 70994 to 72898 (ORF 61), 72938 to 73507 (ORF 62), 73540 to 74211 (ORF 63), 76120 to 74207 (ORF 64r), 76749 to 76186 (ORF 65r), 77698 to 76799 (ORF 66r), 79343 to 77709 (ORF 67r), 79816 to 79367 (ORF 68r), 80529 to 79858 (ORF 69r), 80774 to 80529 (ORF 70r), 82815 to 80788 (ORF 71r), 83835 to 82834 (ORF 72r), 83874 to 85583 (ORF 73), 85535 to 84402 (ORF 74r), 88096 to 85574 (ORF 75r), 87759 to 88667 (ORF 76), 88920 to 88642 (ORF 77r), 91652 to 88938 (ORF 78r), 91667 to 92674 (ORF 79), 93466 to 92681 (ORF 80r), 93761 to 93486 (ORF 81r), 94060 to 93788 (ORF 82r), 94238 to 94080 (ORF 83r), 94508 to 94242 (ORF 84r), 95571 to 94498 (ORF 85r), 96187 to 95600 (ORF 86r), 96202 to 97665 (ORF 87), 97915 to 97643 (ORF 88r), 98251 to 99537 (ORF 89), 99537 to 99974 (ORF 90), 100001 to 101140 (ORF 91), 101168 to 104650 (ORF 92), 106354 to 104795 (ORF 93r), 107947 to 106400 (ORF 94r), 108256 to 107990 ORF 95r), 108719 to 108300 (ORF 96r), 109679 to 108738 (ORF 97r), 109861 to 109682 (ORF 98r), 110830 to 10033 (ORF 99r), 110208 to 110417 (ORF 100), 110469 to 110651 (ORF 100a), 110915 to 111397 (ORF 101), 111419 to 111913 (ORF 102), 111949 to 112485 (ORF 103), 112593 to 113450 (ORF 104), 113323 to 112967 ORF 105r), 113526 to 114152 (ORF 106), 114199 to 115236 (ORF 107), 115353 to 115787 (ORF 108), 115859 o 116551 (ORF 109), 116729 to 117523 (ORF 110), 117572 to 117114 (ORF 111r), 117423 to 118085 (ORF 12), 118968 to 118375 (ORF 114r), 118508 to 119119 (ORF 115), 119588 to 120202 (ORF 116), 120314 to 21231 (ORF 117), 121380 to 123920 (ORF 118), 121288 to 122256 (ORF 119), 122350 to 123924 (ORF 120), 123962 to 125566 (ORF 121), 125193 to 124591 (ORF 122r), 125689 to 123935 (ORF 123r), 123839 to 123297 ORF 123ar), 125652 to 126170 (ORF 124), 126121 to 125699 (ORF 125r), 126279 to 127769 (ORF 126), 127851 to 128408 (ORF 127), 128520 to 130076 (ORF 128), 130105 to 131700 (ORF 129), 131790 to 133283 (ORF 130), 133246 to 133920 (ORF 131), 133972 to 134370 (ORF 132), 134418 to 134693 (ORF 133a), 134402 to 134992 (ORF R1), 134853 to 134419 (ORF R2r), 135628 to 135897 (ORF R3), 136780 to 137112 ORF R4), and 137558 to 137022 (ORF R5r) of SEQ ID NO:1, which encode for the identified open reading frames (ORFs) listed in Table 7. ORFs of this paragraph of which the start position is a larger number than the stop position are coded by the complementary sequence of SEQ ID NO:1. The names of these ORFs end with the letter “r”. The invention also relates to the complementary sequences of the sequences of this paragraph.
The invention also relates to polynucleotides which encode for the same amino acid sequence as encoded by the identified ORFs of the previous paragraph. The invention also relates to polynucleotides of at least 15, 30 or 100 nucleotides binding under stringent conditions to the identified ORFs. The invention also relates to polynucleotides which show at least 99%, 95% or 90% or 80% sequence homology to the sequences of the previous paragraph or which are functional variants a sequence of the previous paragraph.
A functional variant of a gene, within the meaning of the invention, shall be defined as a gene which is at least 99%, or 95%, or 90%, or 80% homologous to the first gene and which has a similar biological function as the first gene. A functional variant of a gene can also be a second gene encoding the same amino acid sequence as does the first gene (or as does a functional variant thereof), employing the degeneration of the genetic code. A functional variant of a gene can also be a polynucleotide comprising the same sequence as has said gene, however said polynucleotide being shorter (i.e., by means of deletions of one or several nucleotides at one or both ends of the polynucleotide) or said polynucleotide having additional nucleotides at one or both ends of the identical part of the polynucleotide.
A functional variant of a protein, within the meaning of the invention, shall be defined as another protein which is at least 99%, or 95%, or 90%, or 80% homologous to the first protein and which has a similar biological function as has the original protein.
The invention also relates to recombinant proteins encoded by nucleotides of the invention and parts and fragments of said proteins which are at least 5 or 7 or 10 or 30 amino acids long.
The invention also relates to recombinant proteins encoded by nucleotides of the invention and parts and fragments of said proteins which are at least 5 or 7 or 10 or 30 amino acids long, said recombinant proteins being attached to a carrier protein or to another carrier. Attaching a protein to a carrier protein can improve or strengthen the immune response to said protein, thereby enhancing the therapeutic or prophylactic effect of administering said protein to a subject.
The invention also relates to vectors containing polynucleotides of the invention and cells containing these vectors or polynucleotides of the invention.
The invention also relates to the use of recombinant proteins and polynucleotides of the invention, alone or in combination with at least one other recombinant protein or polynucleotide of the invention for the manufacture of pharmaceutical compositions.
Combinations of recombinant proteins (or polynucleotides) according to the invention, comprise

    
    
        combinations of at least two recombinant proteins encoded by SEQ ID NO:1 (or combinations of at least two fragments of a polynucleotide of SEQ ID NO:1),
        combinations of at least two recombinant proteins encoded by the same active fragment of the PPVO genome, i.e., two or more recombinant proteins encoded by the same VVOV of
        Table 3, Table 4, Table 5, and Table 6 (or combinations of at least two fragments of the same active fragment (VVOV) of the PPVO genome),
        combinations of at least two recombinant proteins, encoded by at least two distinct active fragments of the PPVO genome, i.e., from distinct VVOVs of
        Table 3, Table 4, Table 5, and Table 6 (or combinations of at least two fragments of at least two distinct active fragments (VVOVs) of the PPVO genome), or
        combinations of at least two distinct recombinant proteins encoded by ORFs of Table 7 (or combinations of at least two polynucleotides with the sequence of any of the ORFs listed in Table 7).
    
    


The invention also relates to the use of recombinant viruses comprising the Vaccina lister genome and selected fragments of the PPVO genome for the manufacture of pharmaceutical compositions.
The invention also relates to the use of recombinant proteins and polynucleotides of the invention for the manufacture of pharmaceutical compositions for the treatment of virus related diseases, viral infections, non-viral infections, proliferative diseases, inflammatory diseases, allergic diseases, and autoimmune diseases.
Viral infections, within the meaning of the invention, shell be understood as diseases associated with viral infections of the human or animal body, such as hepatitis, papillomatosis, herpes virus infections, liver fibrosis, HIV infections, AIDS, and influenza.
Non-viral infections, within the meaning of the invention, shell be understood as diseases associated with non-viral infections of the human or animal body, such as infections with mycobacteria, mycoplasma, amoeba, and plasmodia.
Proliferative diseases, within the meaning of the invention, shell be understood as diseases associated with proliferative disorders, such as cancer, leukemia, warts, tumor diseases, and other skin neoplasms.
Inflammatory diseases, within the meaning of the invention, shell be understood as diseases associated with acute or chronic inflammatory conditions, such as inflammation of the skin or organs, Crohn's disease, COPD, asthma, but also conditions related to the healing of wounds, e.g., Ulcus cruris, and others.
Allergic diseases, within the meaning of the invention, shell be understood as comprising both systemic and topical allergies.
Autoimmune diseases within the meaning of the invention, shell be understood as comprising systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, rheumatoid arthritis, and juvenile diabetes mellitus, and other autoimmune diseases.
The invention also relates to the use of recombinant viruses comprising a Vaccinia lister genome and fragments of a PPVO genome for the manufacture of pharmaceutical compositions.
The invention also relates to the use of recombinant viruses comprising a Vaccinia lister genome and at least one heterologous gene to express at least one heterologous gene in a subject, e.g., for prophylactic and/or therapeutic purposes.
The invention also relates to the use of a recombinant viruses comprising a Vaccinia lister genome and at least one heterologous gene for gene therapy.
“Gene therapy”, within the meaning of the invention, shall be understood as the act of administering to a subject polynucleotides (and, if necessary, suitable adjuvants or suitable carriers) for the purpose of obtaining a prophylactic or therapeutic effect in said subject. Typically, the polynucleotides administered are expressed in the subject and the expressed gene products exert a prophylactic or therapeutic effect.
The invention also relates to

    (a) a particle-like structure comprising a recombinant polypeptide encoded by an open reading frame (ORF) of the polynucleotide of SEQ ID NO:1 or functional variants of said polypeptides,
    (b) the use of a particle-like structure of (a) for the preparation of a medicament,
    (c) the use of a particle-like structure of (a) for the preparation of a medicament for the treatment of virus related diseases, viral infections, non-viral infections, proliferative diseases, inflammatory diseases, allergic diseases, and/or autoimmune diseases,
    (d) pharmaceutical compositions comprising a particle-like structure of (a), and to
    (e) pharmaceutical compositions comprising a particle-like structure of (a) for the treatment of virus related diseases, viral infections, non-viral infections, proliferative diseases, inflammatory diseases, allergic diseases, and/or autoimmune diseases.





BRIEF DESCRIPTION OF THE FIGURE
FIG. 1 shows the genomic locations of the DNA fragments constituting the insertion library. The position of each DNA fragment is shown against the KpnI map of PPVO NZ2 (Mercer, et al., Virology (1997) 229:193-200).



EXAMPLES
Example 1: Determination of the Integrated PPVO Fragments in the Active VVOVs
DNA Preparation from Vaccinia lister/PPVO Recombinants was Performed as Follows:
BK-KL 3A cells were grown to confluency in 175 cm2 flasks (Becton Dickson Labware, Heidelberg, Germany). Cells were infected with a recombinant Vaccina lister/PPVO virus (VVOV) of Mercer, et al. (Virology (1997) 229:193-200) at a MOI (multiplicity of infection) of 0.01-0.32 and incubated at 37° C. until 100% CPE (cytopathic effect) had been reached. The infected cells were frozen at −80° C., thawed and processed as follows, with modification to the RNA extraction method of Vilcek, et al. (J. Clin. Microbiol. (1994) 32:2225-2231). Using 2 ml PLG Heavy Eppendorf tubes (Eppendorf, Hamburg, Germany) 0.5 ml aliquots of cellular suspension were incubated with 100 μg Proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) and 50 μl SDS (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany) at 56° C. for 25 min 0.5 ml Roti®-Phenol/Chloroform (Carl Roth GmbH, Karlsruhe, Germany) was added and the tubes were inverted for several times. After centrifugation at 12000×g for 10 min, the upper phase was transferred into a fresh tube and two volumes of ethanol (Merck Eurolab GmbH, Darmstadt, Germany) and 1/10 volume of sodium acetate (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany) was added. The reagents were mixed several times and stored at −80° C. for 3 h. The tubes were centrifuged at 14000×g for 30 min, the supernatant was decanted and the pellet was air-dried for 5-10 min Finally the DNA pellet was resuspended in 30 μl nuclease free water and stored at −20° C. until used.
DNA concentration was measured spectrophotometrically on a BioPhotometer 6131 (Eppendorf, Hamburg, Germany) at 260/280 nm. The DNA yield of different sample preparations spanned from 100 ng/ml up to 1 μg/ml.
Polymerase Chain Reaction (PCR) of Terminal Flanking Regions of the Integrated Fragments in the Vaccinia Lister/PPVO Recombinants was Performed as Follows:
Three different PCR amplification systems were used for amplifying the terminal flanking regions. Each reaction mixture of 50 μl contained 100 ng-1 μg resuspended DNA and primers (Table 1)) were added in a final concentration of 300 nM. Amplifications were carried out on a Mastercycler® gradient (Eppendorf, Hamburg, Germany).
The 3-prime flanking region of recombinant VVOV 285 had been analyzed using 2× Ready-MixTM PCR Master Mix (1.5 mM MgCl2) (AB Gene, Hamburg, Germany). 1 μl BSA (MBI Fermentas GmbH, St. Leon-Rot, Germany) was added to each reaction. Denaturation was performed at 94° C. for 3 min, followed by 30 cycles (94° C. for 30 s, 58.7° C.-65.3° C. for 30 s, 72° C. for 1 mM) and 72° C. for 5 min.
The 5-prime flanking region of the PPVO insert of recombinant VVOV 285, the 3-prime flanking region of VVOV 97, and both terminal flanking regions of VVOV 215, VVOV 243, VVOV 245 were amplified using PfuTurbo® DNA Polymerase (Stratagene, Amsterdam, Netherlands). The reactions were setup with 2.5 U of enzyme, 1.5 mM MgCl2 and 200 μM of each dNTP. Denaturation was performed at 94° C. for 3 mM, followed by 30 cycles (94° C. for 30 s, 58.7° C.-65.3° C. for 30 s, 72° C. for 1 mM) and 72° C. for 5 min.
The amplification of the 5-prime flanking region of VVOV 97 and VVOV 82, the 3-prime flanking region of VVOV 96 and VVOV 283 were performed with Platinium® Pfx DNA Polymerase (Life Technologies GmbH, Karlsruhe, Germany). A reaction of 50 μl contained 1.25 U polymerase, 1-1.5 mM MgCl2 and 300 μM of each dNTP. Additional use of PCRx Enhancer Solution was necessary for amplification of the 5-prime flanking regions of VVOV 96 (lx concentrated) and the 3-prime flanking regions of VVOV 82 (2× concentrated). Denaturation was performed at 94° C. for 2 mM, followed by 30 cycles (94° C. for 15 s, 54.6° C.-60.7° C. for 30 s, 68° C. for 1-2 mM) and 68° C. for 5-7 min.
18 μl of each amplification product was analyzed by agarose gel electrophoresis on 1.5-2% SeaKem LE agarose (Biozym, Hessisch Oldendorf, Germany). After staining in a ethidium bromide solution for 20 mM the DNA fragments were visualized on an UV transilluminator UVT-20 M/W (Herolab, Wiesloch, Germany).
The sequence of the amplified DNA-fragments were determined by standard sequencing procedures and compared to the published Vaccinia lister thymidine kinase-sequence and the genome sequence of PPVO NZ2 to determine exactly the integrated PPVO NZ2 sequences.







TABLE 1







PCR-primers, amplification and sequencing  


of the terminal flanking regions of  


the integrated fragments in the  


Vaccinia lister/PPVO NZ2 recombinants













Ampli-



Length



fied



of












terminal
Primers used

amplifi-



region
for amplification
SEQ
cation












VVO
of NZ2
Primer 
Sequence
ID
product


V
insert
name
5′ → 3′
NO:
[bp]















VVO
5′
VAC-
ATTACAGTGATG
 2
 264


V 215

P11-1
CCTACATGCCG






PPVO 
GCTGTAGTCGT
 3





14r-1
GGTCCGGC





3′
PPVO 
CTTCCTAGGCT
 4
 402




4r-2
TCTACCGCACG






VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT




 


VVO
5′
VAC-
ATTACAGTGATG
 2
 553


V 245

P11-1
CCTACATGCCG






PPVO 
CTGGCCAACG
 6





57-1
ACGCCTTC





3′
PPVO 
TCTGGTACCCC
 7
 321




40-1
TTGCCGG






VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT




 


VVO
5′
VAC-
ATTACAGTGAT
 2
 241


V 285

P11-1
GCCTACATGCCG






PPVO 
GAACCCGCTCT
 8





78r-5
CGCTCGA





3′
PPVO 
GCCGGGCAAGT
 9
 320




64r-1
GTCTGGTC






VAC-
CGGTTTACGTTG
 5





TK-1
AAATGTCCCAT




 


VVO
5′
VAC-
ATTACAGTGAT
 2
 392


V 330

P11-1
GCCTACATGCCG






PPVO 
CTCGAAGTAGC
10





92-1
TGATGTCGCG





3′
PPVO 
AGAGCTTTAC
11
 462




96r-1
GTAGACTCT







CCAAGTGTC






VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT




 


VVO
5′
VAC-
ATACGGAACGGG
12
 239


V 96

TK-fwd
ACTATGGACG






PPVO  
GCGGTGGCCATG
13





22r-3
TACGTG





3′
PPVO  
GGTTGTGGCGA
14
1055




22r-4
TGGTCGG






VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT




 


VVO
5′
VAC- 
ATACGGAACGGG
12
 309


V 97

TK-fwd
ACTATGGACG






PPVO  
CTTGATGAGCCG
15





18r-1
GACGCA





3′
PPVO 
CCGAGTTGGAG
16
 318




25r-1
AGGAAGGAGC




 

VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT




 


VVO
5′
VAC-
ATTACAGTGAT
 2
 478


V 243

P11-1
GCCTACATGCCG






PPVO 
CTGTTGGAGGAT
17





79-1
GAGGTCAAGGA





3′
PPVO 
CGTGCTCATGC
18
 269




71r-1
CTGTGGAC






VAC-
CGGTTTACGTT
5



 

TK-1
GAAATGTCCCAT




 


VVO
5′






V 283
3′
PPVO 
CGACATCCTCA
19
 234




92-4
CCTGCAAGAAG






VAC-
CGGTTTACGTTG
 5





TK-1
AAATGTCCCAT




 


VVO
5′
VAC-
ATACGGAACGG
12
 275


V 82

TK-fwd
GACTATGGACG




 

PPVO 
TACAGGCAGCC
20





120-1
CGTGACC





3′
PPVO 
GCCGTGTGTC
21
1960




R3R4-3
ACGTTGATGC




 

VAC-
CGGTTTACGTT
 5





TK-1
GAAATGTCCCAT









Example 2: Induction of Interferon Gamma and Tumor Necrosis Factor Alpha by PPVO Gene Products
The 16 recombinants were tested of their ability to induce tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in whole blood cultures.
Whole blood cultures containing blood and RPMI medium (Life Technologies GmbH, Karlsruhe, Germany) in the ratio of 1:5 were stimulated with the recombinant viruses. A pure Vaccinia lister and a whole PPVO preparation served as controls. All preparations were used at a final dilution of 1:10. The stimulation for the IFN-7 determination was done together with Concanavalin A (SIGMA, St. Louis, Mo.), because the virus alone does not induce IFN-γ. Then the cells were incubated for 24 h (TNF-α) and or 72 h (IFN-γ). The cytokine concentration was then determined in the cell culture supernatants by TNF-α or IFN-γ specific ELISA. These time points were found to be optimal when the experimental conditions were determined using whole PPVO as a control.
It was possible to identify 5 active recombinant viruses (VVOV 96, VVOV 97, VVOV 243, VVOV 285, and VVOV 330) that induced both TNF-α and IFN-γ secretion and, thus, could mimic the effect of the whole PPVO. The results are depicted in Table 2.







TABLE 2







TNF-α was determined after 24 h stimulation of blood cells with


the recombinant virus or the controls, respectively. IFN-γ was


determined after 72 h stimulation of blood cells with the recombinant


virus or the controls. Stimulation was performed in the presence of


the mitogen ConA. The relative induction in percent of the Vaccinia


virus control is shown. Therefore, values greater than 100% are due


to the activity of the PPVO fragments. Active PPVO fragments are in


bold. The data represent mean values of three different blood donors.









Recombinant Virus Clone or
Interferon
TNF


control
Induction (%)
Induction (%)












Vaccinia virus control
100
100


NZ-2 control
2224
264


VVOV 80
200
66


VVOV 82
173
65


VVOV 85
209
94


VVOV 86
138
73


VVOV 96
1638
1016


VVOV 97
1713
1285


VVOV 212
94
62


VVOV 213
192
38


VVOV 215
97
82


VVOV 216
197
71


VVOV 243
1446
933


VVOV 245
98
45


VVOV 247
85
74


VVOV 283
115
78


VVOV 285
1128
1127


VVOV 330
1762
2135
















TABLE 3







The recombinant Vaccinia lister/PPVO viruses that induce both


interferon gamma and TNF-α expression are listed in column


1, the corresponding PPVO sequence in column 2 and all open


reading frames (ORFs) that are completely or partially contained


in the recombinant are depicted in column 3.










PPVO NZ2 Sequence
PPVO NZ2 ORFs


Active recombinant
[Bp] that is contained in
that are contained in


PPVO Vaccinia virus
the recombinant
the recombinant





VVOV 97
24056-33789
 18r-25r


VVOV 96
31003-46845
22r-39


VVOV 285
74804-88576
64r-76


VVOV 243
82324-92502
71r-79


VVOV 330
102490-108393
  92-96r









Example 3: Local Immunomodulation by PPVO Gene Products in Liver Sinus Endothelial Cells (LSEC)
We have established a new cell-based assay system that allows testing of hepatoprotective properties of recombinant PPVO proteins expressed in different systems (e.g., Vaccinia virus). This assay system uses primary murine liver cells, which play the central role in deciding whether immunity or tolerance is induced in the liver, the LSEC. The unique ability of LSEC to present exogenous antigens to CD8+ T cells on MHC class I molecules allows immune surveillance of hepatocytes as viral antigens released by infected hepatocytes are likely to be taken by LSEC and presented to cells of the immune system. The new assay allows to measure the ability of LSEC to interact antigen-specifically with CD8+ T cells, that are responsible for tissue destruction in necroinflammatory hepatitis.
Pure populations of LSEC are isolated from murine liver by a stepwise procedure of portal-vein perfusion with collagenase A (0.05%), mechanical dispersion and further enzymatic digestion in a rotatory waterbath for 40 min at 37° C. (245 rpm), gradient centrifugation (metrizamide 1.089 g/cm3) and centrifugal elutriation using a Beckman Avanti J25I centrifuge equipped with a JE-6B rotor and a standard elutriation chamber. LSEC cell populations isolated by this method are typically around 95-99% pure as measured by uptake of endothelial cell specific substrate (acetylated low density lipoprotein). LSEC were seeded onto collagen type I coated 24 well tissue culture plates at a density of 100.000 cells per well and were further cultured in Dulbecco's modified Eagle Medium supplemented with 5% fetal calf serum (specially tested not to interfere with the assay system) and 2% glutamine. Three days after isolation, when LSEC gained a post mitotic and quiescent state, we tested for the ability of LSEC to present soluble ovalbumin to (ovalbumin-specific) CD8+ T cells. LSEC were incubated with 1 μM of ovalbumin for three hours (antigen dose and time were previously shown to be optimal for testing of substances suspected to influence antigen-presentation), washed and incubated with a CD8+ T cell hybridoma (200.000 cells/well) that recognizes the peptide SIINFEKL (SEQ ID NO:320). SIINFEKL (SEQ ID NO:320) is recognized in a H2b context and directly binds on the MHC-I molecules. Therefore, it has not to be processed by the cell. This allows to differentiate between accessory functions of LSEC (such as MHC-I expression) and antigen-processing function.
The extent of CD8+ T cell activation was measured by determining the extent of IL-2 release from T cells by specific sandwich ELISA.
Using Vaccinia virus expressed recombinant proteins derived from PPVO we have been able to attribute hepatoprotective activity to individual clones. To be able to compare different clones directly with respect to their ability to influence cross-presentation by LSEC, we used equal amounts of “infectious units”.
We found that LSEC cross-present exogenous ovalbumin very efficiently on MHC class I molecules (kb) to CD8+ T cells. To our surprise we found if LSEC were incubated with several recombinant PPVO proteins we observed subsequently a potent downregulation of cross-presentation by more than 60% compared to the mock-treated control that includes all but the active ingredient. Several regions within the genome of PPVO have immunoregulatory properties. Especially the region termed 82 (43% reduction) which is located at the 3′ end of the genome appears to be responsible for the overall effect of PPVO on cross-presentation by LSEC. Further regions (VVOV 215, VVOV 212, VVOV 247 and VVOV 86) bear further immunoregulatory potential, although to a lesser degree (around 30% reduction in cross-presentation). It further appears that genes coding for proteins that downregulate cross-presentation are arranged in clusters. It is of interest to note that we identified two gene clusters coding for proteins that improved cross-presentation (VVOV 330, VVOV 283, VVOV 285, VVOV 97, and VVOV 96). However, for unknown reasons the downregulatory effect of the proteins mentioned above is dominant in the activity of PPVO on cross-presentation.
Our results strongly suggest that PPVO contains a mixture of different proteins that in a complementary way work to eliminate hepatocytes from hepatitis B virus while conserving hepatic integrity and avoiding long lasting damage secondary to hepatic fibrosis. As PPVO contains a gene with high homology to the anti-inflammatory agent IL-10 (located in the 5-prime region of the genome) we wondered whether the potent downregulatory effect of the clone 82 was due to expression of ovine IL-10. This assumes that there is cross-reactivity between murine and ovine IL-10 at the level of receptor recognition. We have been unable to demonstrate involvement of ovine IL-10 on the immunoregulatory potential of PPVO. Recombinant murine IL-10 did not show any influence on cross-presentation through LSEC and several monoclonal antibodies to murine and human IL-10 did not influence PPVO mediated downregulation of cross-presentation. We conclude that the immunoregulatory component of PPVO is probably not IL-10 but a new, so far not identified mediator. The data for the MHC-I cross-presentation—down-modulating recombinant virus are depicted in Table 4, those for the MHC-I cross-presentation—stimulating recombinant viruses in Table 5.







TABLE 4







The recombinant Vaccinia lister/PPVO virus that down-modulates


the MHC-I cross presentation is designated in column 1,


the corresponding PPVO sequence in column 2 and all open


reading frames (ORFs) that are completely or partially contained


in the recombinant are depicted in column 3.










PPVO NZ2 Sequence
PPVO NZ2 ORFs


Active recombinant
[Bp] that is contained in
that are contained in


PPVO Vaccinia virus
the recombinant
the recombinant





VVOV 82
122616-136025
120-R3
















TABLE 5







The recombinant Vaccinia lister/PPVO viruses that stimulate


the MHC-I cross presentation are designated in column 1,


the corresponding PPVO sequence in column 2 and all open


reading frames (ORFs) that are completely or partially contained


in the recombinant are depicted in column 3.










PPVO NZ2-Sequence
PPVO NZ2-ORFs


Active recombinant
[Bp] that is contained in
that are contained in


PPVO Vaccinia virus
the recombinant
the recombinant





VVOV 97
24056-33789
 18r-25r


VVOV 96
31003-46845
22r-39


VVOV 285
74804-88576
64r-76


VVOV 283
  89,4-103483
78r-92


VVOV 330
102490-108393
  92-96r









Example 4: Determination of the Immunostimulatory Activity of the Vaccinia Lister/PPVO Recombinants in the Aujeszky Mouse Model
We also tested the activity of recombinant Vaccinia lister/PPVO NZ2-viruses in the Aujeszky mouse model, a lethal challenge model of acute Suid Herpesvirus 1 disease for determining the activity of various immunostimulators (e.g., Baypamun®, CpG oligonucleotides).
a) Conditions Employed for the Mice
The NMRI mice (outbreed strain HdsWin:NMRI; female; weight: 18-20 g; obtained via Harlan/Winkelmann, Borchen, Germany) were kept in autoclavable polycarbonate crates lined with sawdust in an S2 isolation stall at 20-22° C. (atmospheric humidity: 50-60%) and subjected to an artificial day/night rhythm (illumination from 6:30 h to 18:30 h and darkness from 18:30 h to 6:30 h). They had free access to feed and water.
b) Challenge Model
Groups of mice consisting of 10 mice per group were used for the tests. All of the animals in one group were given the same test substance.
After the mice were supplied they were kept in the animal stall for 2-3 days. Then the Vaccinia lister/PPVO NZ2 recombinants were diluted with PBS (Life Technologies GmbH, Karlsruhe, Germany) to a titer equivalent of approx. 108 TCID50/ml and thermally inactivated (twice for one hour at 58° C.). Of these solutions 0.2 ml was administered per mouse intraperitoneally.
24 hours after the treatment the mice were infected with the pseudorabies virus of the Hannover H2 strain by intraperitoneal administration. For this purpose the virus was diluted in PBS to a test titer of approx. 104 TCID50/ml and 0.2 ml of this suspension was administered.
As a negative control one group of mice was treated with PBS and then infected. The mice in this group died 3-8 days after infection. A large proportion of the mice treated the Vaccinia lister/PPVO NZ2 recombinants VVOV 215, VVOV 245, VVOV 285 or VVOV 330 survived infection with the pseudorabies virus. 10 days after the infection with the virus the test was ended.
The level of induced immunostimulation was determined by comparing the number of dead mice in the PBS control group with the number of dead mice in the test groups and was quantified by the efficacy index (EI). This index indicates the percentage proportion of mice protected against the lethal effects of the Aujeszky virus infection through immune stimulation by the substance to be tested. It is calculated by means of the following formula:

EI=(b−a)/b×100,

where b is the percentage proportion of the dead mice in the control group and a the percentage proportion of the dead mice in the test group.

A chi-square test was used for the statistical evaluation. This test reveals the minimum activity indices indicating a significant difference between the mortality rate of those mice treated with the test substance and those treated with PBS. Activity indices of ≧60% are significant where at least 5 of the mice used in tests with n=6 mice per group in the PBS control group and at least 7 of the mice used in tests with n=10 in the PBS control group do not survive the infection with the Aujeszky virus.
Altogether 3 separate tests were carried out in each case. The testing of Vaccinia lister/PPVO NZ2 recombinants in the Aujeszky mouse model shows the following:
Surprisingly, after the treatment of the mice with the Vaccinia lister/PPVO NZ2 recombinants VVOV 215, VVOV 245, VVOV 285 or VVOV 330 the average activity indices of higher than 60% demonstrated immunostimulation. By contrast all of the other Vaccinia lister/PPVO NZ2 recombinants were ineffective. The data is summarized in Table 6.







TABLE 6







The recombinant Vaccinia lister/PPVO viruses that protected


mice from herpesvirus induced death are designated in column


1, the corresponding PPVO sequence in column 2 and all open


reading frames (ORFs) that are completely or partially contained


in the recombinant are depicted in column 3.










PPVO NZ2-Sequence
PPVO NZ2 ORFs


Active recombinant
[Bp] that is contained in
that are contained in


PPVO Vaccinia virus
the recombinant
the recombinant





VVOV 215
10264-20003
 4r-14r


VVOV 245
47952-66263
40r-57


VVOV 285
74804-88576
64r-76


VVOV 330
102490-108393
  92-96r
















TABLE 7







Sequences of the Parapox ovis open reading frames. ORFs the


names of which end with “r” are encoded on the complementary


DNA strand. Base pair positions in the “from” and “to”


column are relative to SEQ ID NO: 1.


















SEQ

SEQ







ID

ID



ORF
from
to
N-term
NO
C-term
NO
Comment





 L1
     3
   539
IRGFAG
 22
PQKVFRL
 23
long termal repeat 









(LTR)-protein,









retroviral 









pseudoprotease


 


 L2r
   781
   449
MSEGGRL
 24 
LLGLLFP
 25
LTR-protein, 









retroviral









pseudoprotease


 


 L3r
  1933
  1664
MTVHPPK
 26
VLPPNSL
 27 
LTR-protein, 









retroviral









pseudoprotease


 


 L4r
  3269
  2790 
MHPSPRR
 28
PVSHPFL 
 29 
LTR-protein, 









retroviral









pseudoprotease


 


 L5
  2799
  3851
MGDREGE
 30 
FEDGVKC
 31
LTR-protein, 









retroviral









pseudoprotease


 


 L6
  2962
  3753 
MCTVATF
 32
GAPRAGW
 33 
LTR-protein, 









similar to 134r,









retroviral 









pseudoprotease


 


 L7r
  3784
  3122
MTPTSRE
 34 
ARTAPPR
 35
LTR-protein, 









retroviral









pseudoprotease


 


 L8r
  4341
  4129 
MPGEGQY
 36 
NGGLGKI
 37
LTR-protein, 









retroviral









pseudoprotease


 


  1ar
  4904
  4428
MEFCHTE
 38
DTAWYIS
 39
dUTPase


 


  1r
  6517
  4970
MLSRESV
 40
RAMLTRP
 41 
homolog of G1L in NZ2, 









Ankyrin-repeats


 


  2r
  8042
  6684 
MFFWFWC
 42 
SGEGVPV
 43



 


  3r
  9989
  8070 
MLGFWGK
 44 
VLPSVSR
 45 
involved in maturation 









of EEV (Extracellular









Enveloped Virions)


 


  4r
 11195
 10062
MWPFSSI
 46
EFCKPIN
 47 
Phospholipase D-type enzyme


 


  5r
 11493
 11227
MLIYGPR
 48
RLLKDFP
 49
homolog of B3L in NZ2


 


  6
 11802
 12038
MGVVMCG
 50
APAGVTE
 51



 


  7r
 12358
 12080
MPVKVKQ
 52 
ASREFIV
 53
ubiquitination 









protein with RING-









finger-motiv (related









to yeast proteins APC11









 and HRT1)


 


  8r
 13980
 12364 
MEEELTR
 54 
SPMVVFN
 55
no Vaccinia 









virus homolog


 


  9ar
 14826
 14053 
MIRIGGG
 56 
DNMRVDD
 57



 


 10
 15080
 15394
MDGGVHK
 58
EQMCRRQ
 59
virion core









DNA-binding









phosphoprotein


 


 11r
 16838
 15423
MAPPVIE
 60
AKNVITH
 61
polyA polymerase


 


 12r
 19021
 16847 
MLQLLKR
 62
NNRGFRK
 63



 


 13r
 19704
 19156
MACECAS
 64
NNCGISF 
 65
interferon resistance 









protein, homology to









mammalian PACT (protein









activator of the









interferon-induced 









protein kinase) also









called PRKRA (dsRNA









dependent activator of









Interferon-induced 









protein kinase),









13r-protein contains a 









dsRBD motiv (double-









stranded RNA binding









domain) and a ′DRADA′-









domain that is typical









for RNA-editing enzymes)


 


 14r
 20314
 19736
MDEDRLR
 66
KKGKPKS
 67
RNA polymerase


 


 15
 20401
 22101
MDFVRRK
 68
VVLQGRA
 69



 


 16
 22125
 22940
MVDSGTH
 70
PENVVLL 
 71



 


 17
 23003
 23866
MASYISG
 72
RTHTVYV
 73



 


 18r
 26908
 23873
MLFEMEL
 74
SKPVFTG 
 75
DNA polymerase


 


 19
 26926
 27213
MEPRFWG
 76
AKVRPLV 
 77
distant homolog of 









the ERV1/ALR-protein-









family (ERV1: yeast 









protein, Essential for









Respiration and









Vegatative growth, ALR:









mammalian protein, Aug-









menter of Liver









Regeneration)


 


 20r
 27626
 27216
MEAINVF
 78
RAYEGML 
 79



 


 21r
 29754
 27616
MLLYPKK
 80
LLGDGGD
 81
related to 12r


 


 22r
 32217
 29800
MLIRTTD
 82
EAQNMQN 
 83



 


 23r
 33380
 32418
MEDERLI
 84
PSPCGGE
 85



 


 24r
 33602
 33393
MDKLYTG
 86
FHYLKLV
 87



 


 25r
 34466
 33612
MKRAVSK
 88
LEAPFNI 
 89
DNA binding 









phosphoprotein


 


 26r
 34735
 34502
MESRDLG
 90
LNARRQN 
 91



 


 27r
 35905
 34739
MNHFFKQ
 92
RSLYTVL
 93



 


 28r
 37194
 35905
MDKYTDL
 94
PEKPAAP
 95
core protein


 


 29
 37200
 39248
MENHLPD
 96
IEAEPPF 
 97
RNA helicase


 


 30r
 41037
 39229
MIVLENG
 98
RMGARPR
 99
Zn-protease,  









involved in virion









morphogenesis


 


 31
 41374
 42066
MTFRELI
100
DSMASRS
101
late transcription 









factor


 


 32r
 42336
 41731
MRGHPAH
102
VAPREEL
103



 


 33r
 42407
 41997
MASDASP
104
QPSSSRR
105
Glutaredoxin-









like enzyme


 


 34
 42410
 43765
MGIKNLK
106
PRLLKLR
107



 


 35
 43770
 43958
MVFPIVC
108
LPMLDIS
109
RNA polymerase


 


 36
 43980
 44534
MREFGLA
110
AEPPWLV 
111



 


 37r
 45727
 44537
MESSKQA
112
TRAPPLF 
113
core virion 









protein precursor


 


 38
 45760
 46557
MTLRIKL
114
DRSLSCD 
115
late transcription 









factor


 


 39
 46567 
 47568
MGGSVSL
116
YLLIVWL
117



 


 40
 47572
 48303
MGAAASI 
118
TEFPPSV 
119
virion protein, 









related to vaccinia F9L


 


 41
 48352
 48621
MVRRVLL
120
LCLFSMD
121



 


 42r
 49887
 48634
MEEKRGR
122
ARAMVCL 
123



 


 43
 49917
 50693
MTNLLSL
124
TGAEAAP
125
core protein, 









DNA binding domain


 


 44
 50719
 51102
MAAPTTP
126
VDVLGGR
127



 


 44a
 51059
 51511
MDHEKYV
128
ATLSPGL
129



 


 45
 51584
 52591
MEGVEMD
130
RPLRGGK
131
polyA polymerase


 


 46
 52509
 53066
MNRHNTR
132
SVSVVLD
133
RNA polymerase


 


 47r
 53523
 53023
MFFRRRA
134
GRRPPRP
135



 


 48
 53607
 57473
MSVVARV
136
EAAEEEF
137
RNA polymerase 









chain 1


 


 49r
 58070
 57528
MGDKSEW
138
FVCDSPS
139
tyrosine phosphatase


 


 50
 57700 
 58662
MAAAPLR
140
ATSGVLT 
141



 


 51r
 59674
 58673
MDPPEIT
142
LLVTAIV 
143
immunodominant 









envelope protein


 


 52r
 62089
 59678
MDSRESI
144
YMINFNN 
145
RNA polymerase-









associated trans-









cription specificity 









factor (also called 









RAP94)


 


 53
 62198
 62881
MSSWRLK
146
KAAACKK
147
late transcription 









factor


 


 55
 62909
 63862
MRALHLS
148
NSEQVNG
149
topoisomerase I


 


 56
 63858 
 64271
MDEALRV
150
FIRAAVA
151



 


 57
 64309 
 66831
MDAPSLD
152
LYVFSKR
153
mRNA capping 









enzyme


 


 58r
 67266
 66799
MEPSAMR
154
DVQHVDL
155
virion protein


 


 58ar 
 67803
 67273
MAGFSQS
156
TTCVPPQ
157



 


 59
 67915
 68607
MATPANA
158
FSFYSEN 
159
Uracil DNA 









glycosylase


 


 60
 68624
 70984
MAAPICD
160
IEDVENK
161
ATPase, involved 









in DNA









replication


 


 61
 70994
 72898
MNSDVIK
162
EVSVVNI
163
early transcription 









factor


 


 62
 72938 
 73507
MSTFRQT
164
ASPAAKN 
165
RNA polymerase


 


 63
 73540
 74211
MRTYTSL
166
WGAAVTR
167
NTP pyrophos-









phohydrolase


 


 64r
 76120 
 74207
MTSAHAA
168
VDPASIA
169
virion NTPase


 


 65r
 76749
 76186
MEGRARF
170
RFCNYCP
171



 


 66r
 77698
 76799
MKTDCAS
172
KLKLLLQ
173
mRNA capping enzyme


 


 67r
 79343 
 77709
MNNSVVS
174
AEKVTAQ
175
rifampicin resistance, 









virion membrane


 


 68r
 79816
 79367
MKRIALS
176
MALKSLI
177
late transactivator 









protein


 


 69r
 80529 
 79858
MNLRMCG
178
AACSLDL
179
late transactivator 









protein


 


 70r
 80774 
 80529
MGDNVWF 
180
VLGLEQA
181
thioredoxin-like 









protein


 


 71r
 82815 
 80788
MESPACA 
182 
NMCDVLC
183
major core protein


 


 72r
 83835
 82834
MDLRRRF 
184 
VDNTGTS
185
core protein


 


 73
 83874
 85583
MEESVAV 
186
LLNYGCG
187
RNA-polymerase


 


 74r
 85535
 84402
MDRLRTC 
188
AEAAESA 
189



 


 75r
 88096 
 85574
MVSVMRK 
190
QEFYPQP
191
early transcription 









factor


 


 76
 87759
 88667
MFQPVPD 
192
SACRASP
193



 


 77r
 88920
 88642
MRPCYVT 
194
TRGTQTG
195



 


 78r
 91652
 88938
MTAPNVH 
196
AVSFDSE
197
major core protein


 


 79
 91667
 92674
MTAVPVT 
198
VRKLNLI
199



 


 80r
 93466
 92681
MASEKMA
200
DLDGGMC
201
virion protein


 


 81r
 93761 
 93486
MGLLDAL 
202
RFSAASS
203
virion membrane 









protein


 


 82r
 94060
 93788
MDIFETL 
204
DIELTAR
205
virion membrane 









protein


 


 83r
 94238
 94080
MVSDYDP 
206
HFVHSVI
207



 


 84r
 94508
 94242
MFLDSDT
208
DMPFSVV
209



 


 85r
 95571
 94498
MGDTVSK
210
KTINVSR
211



 


 86r
 96187
 95600
MESYFSY 
212
EDLFFAE 
213
virion membrane 









protein


 


 87
 96202
 97665
MFGGVQV 
214
GRDLAAV
215
RNA helicase


 


 88r
 97915
 97643
MSAVKAK 
216
PLRDLAR
217
Zn-finger protein


 


 89
 98251
 99537
MTSESDL 
218
AIARAQP
219
DNA polymerase 









processivity factor


 


 90
 99537 
 99974
MIVAAFD
220 
NYVLRTN 
221



 


 91
100001
101140
MLALFEF
222
LKELLGP
223
intermediate 









transcription factor


 


 92
101168
104650
MEQALGY
224
SLFSPED
225
RNA polymerase









b-chain


 


 93r
106354
104795
MESDNAL
226
GQHAAIW
227
A-type inclusion 









body/Fusion









peptide


 


 94r
107947
106400
MEKLVSD
228
GRSGAIW 
229
A-type inclusion









body/Fusion









peptide


 


 95r
108256
107990
MDENDGE
230
QTGYSRY
231
viral fusion 









protein


 


 96r
108719
108300
MDAVSAL
232
LFLKSIL 
233
 


 


 97r
109679
108738
MADAPLV
234
RELRANE
235
RNA polymerase 









subunit


 


 98r
109861
1109682
MEEDLNE
236
MGQASSA
237



 


 99r
110830
110033
MDVVQEV
238
ADSDGGN
239
ATPase


 


100
110208
110417
MRSWFWQ 
240
PLTGMCL
241



 


100a 
110469
110651
MRPKSVG
242
SGHTKPS
243



 


101
110915
111397
MAHNTFE
244
KYFCVSD
245
enveloped virion 









glycoprotein


 


102
111419
111913
MGCCKVP
246
CMKEMHG
247
enveloped virion 









glycoprotein


 


103
111949
112485
MSRLQIL
248
RKLDVPI
249



 


104
112593
113450
MKAVLLL
250
LNLNPGN
251
GM-CSF/IL-2 









inhibition factor


 


105r 
113323
112967
MHASLSS
252
DETLTYR
253



 


106
113526
114152
MEVLVII
254
GEFFYDE
255



 


107
114199
115236
MPLFRKL
256
RDALDGL
257



 


108
115353
115787
MACFIEL
258
TTFSSSE
259



 


109
115859
116551
MSSSSSE
260
TTGTSTS
261






TT






 


110
116729
117523
MACLRVF
262
CSMQTAR
263
GM-CSF/IL-2 









inhibition factor


 


111r 
117572
117114
MAIAHTT
264
FRFRTPG
265



 


112
117423
118085
MAATIQI
266
KRDGYSR
267



 


114r 
118968
118375
MEGLMPK
268
RPISVQK
269



 


115
118508
119119
MDSRRLA
270
LGDSDSD
271



 


116
119588
120202
MRLILAL
272
PQMMRIG
273



 


117
120314
121231
MAGFLGA
274
CKVEEVL
275



 


118
121380
123920
MHLHKDP
276
LAFPSLA
277



 


119
121288
122256
MANRLVF
278
RPMEIDG
279



 


120
122350
123924
MENNDGN
280
RFLPSHK
281
related to 1r/G1L 









with Ankyrin-









repeats


 


121
123962
125566
MDPAGQR
282
CSETDRW
283



122r 
125193
124591
MSSSAAA
284
IAPDSRM
285



123r 
125689
123935
MTAEASI
286
DPVYHKK
287



123ar
123839
123297
MPRTTSG
288
REQTEGL
289



124
125652
126170
MANREEI
290
VRVLRRT
291



125r 
126121
125699
MTAPTPR
292
AAYSLAR
293



126
126279
127769
MADEREA
294
LACAMRK
295
related to 1r/G1L 









with Ankyrin-repeats


 


127
127851
128408
MSKNKIL
296
SYMTTKM
297
sheep-like 









Interleukin 10


 


128
128520
130076
MLTRCYI
298
RASGLAE
299
related to 1r/G1L 









wih Ankyrin-repeats


 


129
130105
131700
MVGFDRR
300
CGRRAPE
301
related to 1r/G1L, 









with Ankyrin-repeats









(NT slightly changed)


 


130
131790
133283
MILARAG
302
PDAAALS
303
Kinase


 


131
133246
133920
MPPRTPP
304
RPAALRA
305



 


132
133972
134370
MKLLVGI 
306
RPPRRRR
307
homolog to the 









sheep VEGF









(Vascular Endothelial









Growth Factor)


 


133a 
134418
134693
MRKKAPR
308 
ARTAPPR
309
corresponds to L7r


 


R1
134402
134992
MMRSGHA
310
RMHRSEL 
311
LTR-protein (corresponds 









to L4r), retroviral









pseudoprotease


 


R2r
134853
134419
MCTVATF 
312 
SVAPSSA
313
LTR-protein (corresponds 









to L6, 134r), retroviral









pseudoprotease


 


R3
135628
135897
MTVHPPK 
314
VLPPNSL
315
LTR-protein (corresponds 









to L3r), retroviral









pseudoprotease


 


R4
136780
137112
MSEGGRL 
316
LLGLLFP
317
LTR-protein (corresponds 









to L2r), retroviral









pseudoprotease


 


R5r
137558
137022
IRGFAGG
318
PQKVFRL
319
LTR-protein (corresponds









to L1r), retroviral









pseudoprotease

## Claims

1. A method for down-regulating cross-presentation of an antigen with a MHC Class I molecule on a cell in a subject, comprising administering to the subject a recombinant protein encoded by a polynucleotide selected from the group consisting of:
a polynucleotide comprising the sequence of nucleotide residues 122616 to 136025 of SEQ ID NO:1 (PPVO insert of VVOV 82), or a complementary sequence thereof; and
a polynucleotide comprising the sequence of nucleotide residues 10264 to 20003 of SEQ ID NO:1 (PPVO insert of VVOV 215), or a complementary sequence thereof,
thereby down-regulating MHC Class I cross-presentation of an antigen on a cell in the subject,
wherein the subject has hepatitis, papillomatosis, herpes virus infection, liver fibrosis, HIV infection, AIDS, or influenza.

2. The method of claim 1, wherein the recombinant protein is attached to or is a part of a structure selected from the group consisting of a particle-like structure, a fusion protein, a protein coated particle, and a virus-like particle.

3. The method of claim 1, wherein the cell is an antigen presenting cell, a liver sinus endothelial cell, or a macrophage in the subject.

4. The method of claim 1, wherein the down-regulation of MHC Class I cross-presentation of an antigen reduces tolerance to the antigen.

5. The method of claim 1, wherein the antigen is a viral antigen.

6. The method of claim 1, wherein the subject has hepatitis or liver fibrosis.

7. A method for down-regulating cross-presentation of an antigen with a MHC Class I molecule on a cell in a subject, comprising administering to the subject a polynucleotide or a vector, a virus, or a host cell comprising the polynucleotide, wherein the polynucleotide is selected from the group consisting of:
a polynucleotide comprising the sequence of nucleotide residues 122616 to 136025 of SEQ ID NO:1 (PPVO insert of VVOV 82), or a complementary sequence thereof; and
a polynucleotide comprising the sequence of nucleotide residues 10264 to 20003 of SEQ ID NO:1 (PPVO insert of VVOV 215), or a complementary sequence thereof,
thereby down-regulating MHC Class I cross-presentation of an antigen on a cell in the subject,
wherein the subject has hepatitis, papillomatosis, herpes virus infection, liver fibrosis, HIV infection, AIDS, or influenza.

8. The method of claim 7, wherein the cell is an antigen presenting cell, a liver sinus endothelial cell, or a macrophage in the subject.

9. The method of claim 7, wherein the down-regulation of MHC Class I cross-presentation of an antigen reduces tolerance to the antigen.

10. The method of claim 7, wherein the antigen is a viral antigen.

11. The method of claim 7, wherein the subject has hepatitis or liver fibrosis.