HK1060063A - Use of strains of the parapox ovis virus for producing antiviral pharmaceuticals and anticancer pharmaceuticals - Google Patents

Description

Use of parapoxvirus ovis strains for the production of antiviral and anticancer drugs

no marking

The present invention relates to the use of Parapoxvirus ovis strain as an immunotherapeutic agent for infectious or non-infectious immune attenuation, and the use of said Parapoxvirus strain for the treatment of neoplastic diseases, viral infections and diseases caused thereby. The invention also relates to the use of strains of parapox ovis virus for the preparation of a medicament for humans and animals.

Furthermore, the present invention relates to the use of strains of parapox ovis virus and dosage forms prepared therefrom as immunotherapeutics or immunoprophylaxis agents for preventing or preventing infectious diseases in stress-conditioning following stress (e.g., surgery); to the use in infection prevention for the prevention or prophylaxis of infectious diseases by administration prior to surgery or intervention (e.g. prior to implantation of a prosthesis or prior to dental surgery), to the use in prevention or treatment after infection by acute or chronic viral infections such as respiratory tract infections, papilloma virus infections, herpes virus infections, HIV infections and viral infections of internal organs such as hepatitis virus infections, to the use in wound healing for promoting wound healing and to the use in promoting wound healing that does not heal well or does not heal at all (e.g. leg ulcers), to the use in diseases such as multiple sclerosis, asthma, warts and other skin neoplasms, to the use in many allergic diseases, to the use in preventing the occurrence of systemic allergies, to the use in local allergies and to the use in, for example, improving the health of elderly people, among the strains of ovine parajaundice used in the present invention are NZ2, NZ-7, NZ-10 and orf-11.

Progeny of these strains, or portions or fragments of viruses derived from these strains or from these progeny, obtained by passaging and/or adapting to specific cells, such as WI-38, MRC-5 or Vero cells, may also be used. By parts is understood a genomic or subgenomic fragment which is expressed in a suitable system, for example in fibroblast cell culture, by means of a suitable vector, for example vaccinia. Fragments are understood to be fractions obtained by biochemical purification of particles by physical disruption, e.g. by sonication, e.g. using chromatography.

The invention further relates to the use of said parapoxvirus ovis strain for the production of a medicament and a pharmaceutical preparation. In addition, the invention relates to the use of said parapoxvirus ovis strain in combination with other drugs for the production of a medicament and a pharmaceutical preparation for antiviral or cancer therapy.

It is known that latent and chronic persistent viral infections can be activated or reactivated by immunosuppression or, conversely, that the immune system suppresses acute diseases which latent viruses can induce (e.g. recurrent latent herpes virus infections associated with immunosuppression: herpes labialis associated with stress or administration of cortisone). It is also known that the treatment of chronic persistent and latent viral infections using conventional antiviral substances based on low molecular weight is difficult or even impossible.

The reason for this is that there is no viral enzyme activity in such an infection (e.g. no viral polymerase activity that first inserts a nucleoside inhibitor into the viral nucleic acid such that the inhibitor is capable of, for example, causing the viral DNA chain to be terminated; e.g. no viral thymidine kinase activity that first phosphorylates an antiviral compound such that the compound is capable of becoming active), or no recognition thereof by infected or degenerated cells such as cancer cells or viral antigens by the host immune system.

It is also known that in connection with chronic persistent viral infection, reinfection with another virus can bring about an antiviral effect against this chronic persistent virus¹⁾. Authors refer to¹⁾This effect of interferons, such as IFN-. gamma.and TNF-. alpha.secreted by T cells, by natural killer cells and macrophages can be demonstrated.

The results obtained by these authors confirm another early study demonstrating that class I-restricted cytotoxic T cells can inhibit HBV gene expression in hepatocytes in HBV-transgenic mice, the process occurs without any disruption of hepatocytes, and TNF- α and IFN- γ trigger the process²⁾。

In veterinary practice, products which induce "class-specific immunity", so-called immune-like inducers, have long been used for therapy, for postconditioning and for prophylaxis. An immunity-like inducer consists, for example, of the chemically inactivated parapoxvirus ovis strain D1701(DE 3504940). BAYPAMUN^Was prepared on the basis of this virus (parapox ovis virus strain D1701).

In animals, inactivated parapoxviruses induce nonspecific protection against infection by a wide variety of pathogens. It is hypothesized that this protective effect is mediated by various mechanisms of the body's intrinsic defense system.

These mechanisms include induction of interferon, activation of natural killer cells, induction of "colony stimulating activity" (CSA),and stimulation of lymphocyte proliferation. Early studies of the mechanism of action demonstrated the stimulatory effects of interleukin2 and interferon-alpha³⁾。

Therefore, in this context, the present invention aims to further improve the therapeutic use of the excellent effect of parapoxvirus ovis, so that the general induction of the class-specific immune response of the aforementioned parapoxvirus ovis strain D1701 is qualitatively enhanced, and to improve it so that a better antiviral or antitumor effect can be achieved with a low dose, as described below. And it is also expected that such therapeutic effect has less side effects.

Therefore, it is an object of the present invention to increase the immune effect of parapoxviruses. This object was achieved by using the abovementioned strains of parapox ovis virus instead of the conventionally used strain D1701.

The present invention relates to the use of a virus taxonomically belonging to the parapoxvirus ovis strains NZ2, NZ-7, NZ-10 or orf-11 for the preparation of a medicament against viral infections in humans and animals.

The invention further relates to the use of the progeny of the strains according to the invention obtained by passaging or adaptation to suitable cell lines, such as human cells, for example WI-38, MRC-5, monkey cells, for example Vero cells, bovine cells, for example BK-K13A47/Reg or MDBK, and sheep cells, for example MDOK, for the preparation of a medicament against viral infections and cancer in humans and animals, and also to the use of parts or fragments of said strains and their passaged and adapted variants, which parts are understood as genomic or subgenomic fragments expressed with the aid of suitable vectors, such as vaccinia viruses in suitable systems, such as fibroblast cultures, which fragments are understood as parts obtained by biochemical purification, for example chromatography, of expressed or physically disrupted virus particles, and also to the use of one of said strains of parapox ovis virus and derivatives derived as described above for the production of a medicament and pharmaceutical preparations for use as an immunotherapeutic or immunoprophylactic medicament in autoimmune diseases and for acute or chronic viral infections of the respiratory tract and internal organs, to the use of one of said strains of disease and derivatives derived for the production of a medicament and pharmaceutical preparations for the poststress recuperation and for the prevention or alleviation of poststress infectious diseases and for the prophylaxis in surgical and dental surgery, and to the use of one of said strains of disease and derivatives derived for the production of a medicament for the postinfection recuperation or treatment of acute and chronic viral infections such as respiratory tract infections, papilloma virus infections, herpes virus infections, HIV infections, or infections of internal organs, such as hepatitis virus infections, and for the associated diseases such as multiple sclerosis, the use of a medicament and a pharmaceutical preparation for asthma, warts and other skin neoplasms, and to the use of one of said pathogenic strains and derived derivatives for the preparation of a medicament and a pharmaceutical preparation for use on wounds to promote the wound healing process, and for promoting healing of wounds and leg ulcers that do not heal well or do not heal at all, and to the use of one of said pathogenic strains and derived derivatives for the preparation of a medicament and a pharmaceutical preparation for use in broad-spectrum allergic diseases, psoriasis, neurodermatitis and other autoimmune diseases such as lupus erythematosus, and for improving the health of e.g. elderly patients, and to the use of one of said strains and derived derivatives for the preparation of a medicament and a pharmaceutical preparation for combating inflammatory, degenerative and proliferative diseases of internal organs, skin, blood, central nervous system and its accessory organs including the eye, such as crohn's disease, including cancer, it also relates to the use of one of said strains and derived derivatives in combination with other drugs for the preparation of a medicament and a pharmaceutical preparation for antiviral or cancer treatment in humans and animals.

The invention preferably relates to the use of one of said strains of parapox ovis virus in combination with other drugs for the preparation of a medicament and a pharmaceutical preparation for oral administration, and/or a gastric juice resistant preparation for oral administration.

The parapoxvirus ovis NZ-2 mentioned here by way of example was deposited at 10.7.2001 in the center for cell cultures Europe, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP 40 JG, United Kingdom. The accession number is ____.

The following examples illustrate:

1 demonstration of therapeutic Activity of hepatitis B Virus-transgenic mice

Hepatitis B virus-transgenic mice (HBV1.3Xtg species) were used for "proof of concept" experiments. 7 males, 8-10 weeks old, were used per group. Various doses in 0.15 ml volumes were administered intraperitoneally on the first day (start of experiment) and the fourth day.

Upon administration, the following dilutions of each virus strain were prepared for each dose group:

first dose 1.5X 10⁶TCID₅₀

Second dose 5X 10⁵TCID₅₀

Third dose 1.5X 10⁵TCID₅₀

Fourth dose 5X 10⁴TCID₅₀

Sterilized pyrogen-free PBS was used as a blank control.

The virus was concentrated by the following method: from parapoxvirus ovis strain NZ2 (titer approximately 2X 10)⁵TCID₅₀Perml) and parapox ovis virus strain D1701 (titer approximately 1X 10)⁷ TCID₅₀Per ml) was adjusted to the same virus titer. Beckmann ultracentrifugation (SW28 rotor at 28000RPM and 4 ℃ for 3 hours) was used for this purpose. After centrifugation, the sediment was adjusted to 1X 10 using an appropriate volume of dilution medium⁷ TCID₅₀The titer per ml.

Control back-drops were performed using corresponding dose aliquots and the titers of these doses were confirmed. After removal of the control aliquot, the corresponding dose was inactivated for 1 hour at 56 ℃.

Dilution medium: 10 ml of EMEM10x

2.7 ml of 2 g/l bicarbonate

1 ml of 1% glutamine

86.3 ml of double distilled water

On day 5, animals were killed painlessly and the liver and blood were removed. Approximately 20 mg of liver from each animal was treated with QIAamp tissue kit (Qiagen, Hilden) and the DNA concentration was determined photometrically while checking its integrity by electrophoresis in a 1% agarose gel. In the dot blot hybridization method, the DNA is hybridized with HBV-specific probes. To exclude signals caused by RNA, the DNA was previously treated with RNAse A (Qiagen, Hilden). 20 microliters of DNA (10 micrograms) were loaded onto a nylon membrane (Boehringer Mannheim) and treated 4 times with Soak I (0.5N NaOH; 1M NaCl) for 3 minutes each and 2 times with Soak II (3M NaCl; 0.5N tris-HCl, pH 7.4); the DNA was then oven-dried at 120 ℃ for half an hour, followed by prehybridization at 60 ℃ for 30 minutes with standard hybridization buffer (5XSSC, N-lauroyl sarcosine, 0.1% w/v; SDS, 0.02%; blocking reagent, 1X and 100. mu.g of protamine DNA/ml) without any probe contained therein. After this step, the DNA was hybridized with random oligonucleotide-sensitized probes (20-40ng/ml hybridization buffer) containing the entire HBV genome. Thereafter, the filter was rinsed in 4 XSSC/0.1% SDS, 2 XSSC/0.1% SDS and 1 XSSC/0.1% SDS at 64 ℃ for 10 minutes.

Using CDP-Star^The system (Boehringer Mannheim) performs the immunoassay according to the manufacturer's instructions. Lumi Imager was used for evaluation^(Boehringer Mannheim). Quantitative determination of HBV-specific DNA in blood by means of quantitative PCR. Plasma was first separated by centrifugation of EDTA blood. DNA was purified using the HighPure16 System Virus nucleic acid kit (Boehringer Mannheim) and using quantitative PCR with ABI PRI SM^TM7700 the sequence System (PE Applied Biosystems) detects HBV-specific signals. The following primers and probes were used:

ayw-570f (sense) 5'-CTGTACCAAACCTTCGGACGG-3'

ayw-670r (antisense) 5'-AGGAGAAACGGGCTGAGGC-3'

And (3) probe:

ayw-613t 5′-CCATCATCCTGGGCTTTCGGAAAATT-3′。

the DNA was amplified in a 50. mu.l reaction volume (the reaction contained 1.4mM of each dNTP, 4.75mM MgCl₂15pmol of each primer and probe, 5 μ l of 10-fold PCR buffer [ all PCR reagents from TaqMan core kit; perkin Elmer/Roche molecular Systems Inc.]And 1.25U Taq DNA polymerase and 0.25U Amperase. After the initial denaturation step (95 ℃, 10 minutes), the samples were subjected to 40 cycles of denaturation (95 ℃, 30 seconds) and quenching/extension (56 ℃, 1 minute). Use of ABI PRISM^TM7700 Standard software of sequencing System analyzes the product.

Histochemical analysis was performed using antibodies against hepatitis B virus core antigen (Dako). For this purpose, a portion of one liver lobe was fixed overnight in 4% formaldehyde, embedded in paraffin and sectioned (5 μm). After paraffin removal and rehydration, 3% H was used₂O₂Endogenous peroxidase activity was quenched for 20 min and non-specific binding blocked with normal goat serum. Then, the ratio of 1: the sections were incubated with 500 diluted antibodies for 30 minutes at room temperature. All following steps were performed using the Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions.

The immunoreactions were observed using 3, 3' -diaminobenzidine tetrachloride and hydrogen peroxide. Sections were counterstained with hematoxylin/eosin.

Results were statistically analyzed using analysis of variance and post hoc comparisons.

The results unexpectedly found that the antiviral effect obtained when using the NZ2 strain was enhanced compared to that obtained with the known parapoxvirus ovis strain D1701. This makes it possible for the first time to use parapoxvirus ovis to induce a complex capacity with a strong immune system that differs significantly in intensity from the effects achieved with previously known paraimmune inducers.

The following results were surprisingly obtained:

liver: a significantly greater reduction in HBV-specific DNA was observed in animals treated with NZ2 compared to animals treated with D1701. The antiviral activity of NZ2 was more potent than that of D1701: in the highest dose group, administration of NZ2 reduced HBV-specific DNA more effectively, more than 45-fold, than the same amount of D1701, while in the lowest dose group, the effect was 57-fold better. In plasma, administration of NZ2 reduced HBV-specific DNA, more than 10-fold more effective in the lowest dose group than administration of the same amount of D1701. This demonstrates that parapox ovis virus strain NZ2 is much more superior in therapeutic efficacy than strain D1701.

In the figure, the reduction of HBV-specific signal is given compared to the placebo group (equal to 100%).

FIG. 1 of the drawingsResults of treatment of HBV-transgenic mice with D1701 or NZ2 strains are illustrated. With both strains in all dose groups, HBV-specific DNA was significantly reduced in the liver compared to the placebo group, more when NZ2 strain was used. In the two lowest NZ2 dose groups, the reduction in HBV-specific DNA was significantly greater than that of the equivalent D1701 dose group.

FIG. 2 of the drawingsIllustrating the results obtained in plasma from HBV-transgenic mice treated with the D1701 or NZ2 strains. With both strains, there was a significant reduction of HBV-specific DNA in plasma compared to the placebo group in all dose groups, which was more reduced when NZ2 strain was used. The lowest dose of the D1701 strain no longer had a significant antiviral effect compared to the lowest dose of NZ 2.

2. Induction of cytokines: female Balb/c mice, 7 to 8 weeks old, were maintained under sterile conditions and used for the experiments. Animals were randomly divided into groups, each group consisting of 6 animals. The following treatment protocol was carried out:

group 1: placebo group

Group 2: parapoxvirus ovis strain D1701; 5X 10⁴ TCID₅₀Dosage of

Group 3: parapoxvirus ovis strain NZ 2; 5X 10⁴ TCID₅₀Dosage of

Group 4: placebo group

Group 5: parapoxvirus ovis strain D1701; 5X 10⁴ TCID₅₀Dosage of

Group 6: parapoxvirus ovis strain NZ 2; 5X 10⁴ TCID₅₀Dosage of

The volume of administration was 10 ml/kg, administered intraperitoneally.

Animals from groups 1-3 were sacrificed 6 hours after dosing, while animals from groups 4-6 were sacrificed 12 hours after dosing. Peritoneal cells were obtained by washing with ice-cold PBS, phylum and mesenteric lymph nodes were isolated.

The peritoneal cells were concentrated using a centrifugation step (centrifugation at 3000rpm for 5 minutes at room temperature in an Eppendorf-benchtop centrifuge), then dissolved in 0.2ml of lysis buffer (lysis buffer: 25mM sodium citrate, 4M guanidine isothiocyanide, 0.5% N-lauroyl-creatinine), snap frozen and stored at-75 ℃ until they were used for RNA preparation.

The whole RNA was prepared by acidic phenol/chloroform extraction. For this purpose, the samples frozen in the lysis buffer were thawed at room temperature and extracted by treatment with the following solutions: 1/10 lysis buffer volumes of 2M sodium acetate (pH4.0), 1 lysis buffer volume of water saturated phenol, and 1/5 lysis buffer volumes of chloroform/isoamyl alcohol (24: 1). The ingredients were mixed in a vortexer for 10 seconds and then kept on ice at a constant temperature for 10 minutes. The phases were separated by centrifugation at 15365g for 30 min at 4 ℃. The aqueous phase is then transferred to a new container and, in order to separate the RNA present in this phase, the RNA from Rnaid is added^TM8ml of RNA-MATRIX from Plus's kit (DIANOVA) and incubated for 15 minutes at room temperature. The resulting RNA/RNA-MATRIX complex was deposited by centrifugation at 7000g and the supernatant was discarded. The sediment was then washed twice with 250ml each time of RNA-WASH (DIANOVA), and after the last wash, dried by centrifugation in vacuo. RNA was eluted by adding 20-30ml of distilled water without RNA and heating all at 55 ℃ for 15 minutes. At 7000g andafter centrifugation at room temperature for 1 minute, the matrix was separated by transferring the RNA solution to a new container.

The quality of the RNA was checked by gel electrophoresis. RNA was stored at-70 ℃.

cDNA was synthesized by reverse transcription of mRNA using oligo (dT) -primers as the initiator molecule for polymerization. The following ingredients were present in the synthesis mixture: 200 ng-2. mu.g total RNA, 2. mu.l M-MLV reverse transcriptase (200U/ul) (GIBCO/BRL), 8. mu.l of the appurtenant 5 XRT-buffer (GIBCO/BRL), 1. mu.l DTT (0.1M) (GIBCO/BRL), 4. mu.l dNTP (2.5mM) (SIGMA), 2. mu.l oligo (dT)_12-18-primers (100 microgram/ml) (PROMEGA), 1 microliter of human placental RNAse inhibitor (10000U/ml) (GIBCO/BRL) and a total volume of water to 40 microliter. The mixture was left at room temperature for 10 minutes and incubated at 37 ℃ for 45 minutes; then heated at 95 ℃ for 3 minutes and immediately cooled on ice. The cDNA synthesized in this way was stored at-20 ℃.

The amount of cDNAs was normalized using the "housekeeping" gene (β -actin). Application of ABI PRISM^TM7700 the sequence detection System (PE Applied Biosystems) performs quantitative PCR. The following primers were used: β -actin sense: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'

Antisense: 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3' IFN-. gamma.sense: 5'-AGC GGC TGA CTG AAC TCA GAT TGT AG-3'

Antisense: 5'-GTC ACA GTT TTC AGC TGT ATA GGG-3' TNF- α sense: 5'-GGC AGG TCT ACT TTG GAG TCA TTG C-3'

Antisense: 5'-ACA TTC GAG GCT CCA GTG AAT TCG G-3' IL-15 sense: 5'-GCC AAC TGG ATA GAT GTA AGA TAT GAC CT-3'

Antisense: 5' -CGT GTT GAT GAA CAT TTG GAC AAT GCG

TAT-3′

The DNA was amplified in a 25. mu.l reaction volume (the reaction contained 1.4mM each dNTP, 4mM MgCl)₂0.3 micromolar of each primer and probe, 2.5 microliters of 10-fold PCR buffer containing SYBR Green [ all PCR reagents were from SYBR Green PCR core kit; perkin Elmer/Roche Molecular Systems Inc.]And 1.25U Taq DNA polymerase and 0.25U Amp Erase. After the initial denaturation step (95 ℃, 10 minutes), the samples were subjected to 40 cycles of denaturation (95 ℃, 30 seconds) and quenching/extension (60 ℃, 1.3 minutes). Use of ABI PRISM^TM7700 Standard software of sequencing System analyzes the product.

Results were statistically analyzed using analysis of variance and post hoc comparisons.

The following results were unexpectedly obtained:

1. induction of interferon gamma expression 6 and 12 hours after administration after treatment with strain D1701 or strain NZ2 ((FIG. 3). In the case of strain NZ2, the induction was significantly higher than in the case of the blank control and in the case of strain D1701. The observed values for interferon gamma expression after D1701 administration were not significantly different from those of the blank control. The figure illustrates the values determined in the cells obtained by peritoneal washing.

2. After treatment with strain D1701, expression of TNF α was induced 12 hours after administration, whereas after treatment with strain NZ2, expression was induced 6 and 12 hours after administration (a decrease already observed after 12 hours compared to the 6 hour value;FIG. 4). In the case of strain NZ2, the induction was much higher 6 hours after administration than that observed in the case of strain D1701. The figure illustrates the values determined in the cells obtained by peritoneal washing.

3. Induction of IL-5 expression 6 and 12 hours after administration following treatment with Strain D1701 or Strain NZ2 ((FIG. 5). In the case of strain NZ2, the induction was significantly higher 6 hours after administration than in the case of strain D1701 or the blank control. IL-5 observed after D1701 administrationThe values expressed were not significantly different from the placebo. The figure illustrates the values determined in the cells obtained by peritoneal washing.

3. Demonstration of therapeutic efficacy in tumor-bearing nude mice

37℃、5％CO₂Next, MDA-MB231 cells (ATCC # HTB26) were cultured in complete medium (885DMEM, 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine (each Gibco Life technologies)) in an incubator. On the day of transplantation, cells were approximately 70% confluent. Cells were trypsinized, washed with HBSS, counted and adjusted to 2.5X 10 with pre-chilled PBS⁷Cells/ml. Female NCr nude mice (taconic) were used. These mice are 8-10 weeks old and weigh about 22 g. All manipulations of the animals were performed under sterile conditions. Subcutaneous injection of 5X 10 to the lateral costal region in a total volume of 0.2ml⁶And (4) cells. Thereafter, the mice were reared for 7 more days until the tumors reached an average mass of approximately 80 mg. Tumors were measured and mice were randomized into three subgroups of 10 animals each. The following were given to each panel:

group 1: blank control (PBS)

Group 2: parapoxvirus ovis, Strain D1701

Group 3: parapoxvirus ovis, strain NZ2

The dose for D1701 administration was 2.5X 10⁵ TCID₅₀And NZ2 is administered at a dose of 1X 10⁵TCID₅₀(ii) a These doses were applied four times in each case at three day intervals in each case. Tumors were measured twice weekly. Statistical significance was determined using the Student's test.

FIG. 6Mean tumor size (mg) in animals of groups 1-3 during the experiment (days) is indicated.

(symbol: group 1, circle; group 2, triangle; group 3, square)

Surprisingly, a statistically significant (p < 0.05) antitumor activity was found in the experimental system compared to the control group. The NZ2 strain was found to be much more potent than strain D1701 in this regard, and only about half the amount of NZ2 was required to achieve the same effect as D1701.

This finding provides a clear demonstration of the therapeutic efficacy of the viral formulations according to the invention in tumor bearing nude mice.

Nude mice are immunodeficient and do not have any functional T cells. In experimental systems, it is hypothesized that the antitumor activity is due to natural killer cells (NK), to other cells and to the direct effects of cytokines/chemokines. It is expected that the superior efficacy of NZ2 will be more pronounced in the case of a complete and intact immune system.

4. Other biological differences between NZ2 and D1701

In contrast to strain D1701, parapoxvirus ovis NZ2 was found to be passable serially on a human cell line. This demonstrates a substantial difference between NZ2 and D1701 in replication behavior and/or viral receptors.

Adaptation to human cell lines is an important prerequisite for the production of viral strains on human cell lines.

A) ability to serial passage on human MRC-5 cells.

FIG. 7An attempt to adapt strains NZ2 and D1701 to the human diploid cell line MRC-5 is illustrated. MRC-5 is suitable for the production of biologically active compounds and vaccines. The same starting titer was used for both strains and the corresponding cell culture supernatants were serially passaged for 5 passages in MRC-5 cells. The figure illustrates the results from infection in each case with TCID₅₀The titers of the indicated samples of the supernatant of MRC-5 cells are plotted against the passage number. Only strain NZ2, but not D1701, was countertitre to bovine kidney cell cultures (BK clone 3 a). This indicates that there are fundamental biological differences between the two strains in their infection and replication behavior. Also, the results indicate that NZ2 can replicate in a wider range of cells than D1701, thereby giving rise to human thin linesPossibility of cell-based production methods.

B) ability of NZ2 and D1701 to passage on WI-38 cells and BK clone 3a cells

FIG. 8AndFIG. 9The experiments described in (a) also clearly demonstrate the fundamental biological difference between NZ2 and D1701.FIG. 8Illustrates that the virus titers obtained in the attempt to passage D1701 on bovine BK clone 3a cells and human WI-38 cells are plotted against the passage number. It can be seen that D1701 could be passaged on BK clone 3a cells but not on human WI-38 cells.

This is different from the NZ2 strain. The strain can be continuously passaged for several days on BK clone 3a cells and human WI-38 cells (FIG. 9)。

These results also indicate significant differences in NZ2 and D1701 infection and replication behavior.

C) dose-dependent Effect of NZ2 in herpes Virus challenge experiments after WI-38 passages

To investigate the immunostimulatory performance of the NZ2 strain passaged on WI-38 cells, a herpes virus challenge experiment was performed on mice. Respectively using 1X 10⁴ TCID₅₀，5×10⁴ TCID₅₀And 1X 10⁵TCID5₀Three groups of 10 test animals were treated, while a placebo was administered to the control group. Figure 10 illustrates the survival of the four experimental groups over time after herpes virus infection. It was surprisingly found that NZ2 did not lose its immunostimulatory properties by passage in WI-38 cells.

All experimental results in this example indicate basic biological differences in infection and replication behavior of NZ2 and D1701. It was surprisingly found that the use of the preparation of NZ2 as an immunomodulator, in contrast to the case of D1701, is not limited to bovine kidney cells as a producer cell line.

Latent and chronic persistent viral infection based on Th1 immune response^4，5)And proliferative diseases such as cancer^8，9)And the fact that the immunomodulating properties of parapoxvirus ovis strain NZ2 are superior to those of parapoxvirus ovis strain D1701, the use of an immunomodulator based on parapoxvirus ovis strain NZ2 or one of the above mentioned strains as a monotherapy in humans and animals or in combination with a biological activity such as an antiviral low molecular weight compound, and also for the infection of hepatitis B or hepatitis C viruses or other pathogens from the group of hepatitis viruses, and other viral infections of internal organs, as well as infections accompanied by other diseases and various types of Herpes Simplex Virus (HSV), various types of Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV), varicella-zoster virus, Human Cytomegalovirus (HCMV), and the corresponding therapeutic use in antiviral therapy of animal viral diseases.

Furthermore, on the basis of the proven mechanism of action, the abovementioned parapoxvirus ovis strains can be used effectively for the following preventive or therapeutic treatments, in particular: prevention of recurrence associated with herpes virus infection, post-disease maintenance, i.e. prevention of the formation of viral infections (e.g. HIV) when treated with the method immediately after exposure⁷⁾. On the basis of this mechanism of action, it is likewise possible to treat cancer^8，9)。

Depending on the nature of the clinical problem, either systemic use, i.e. for example intramuscular, subcutaneous, intraperitoneal, intravenous or oral administration, or also local administration of a therapeutic drug based on parapoxvirus ovis. In this connection, the parapoxvirus ovis is either present in a purified and lyophilized state and/or is suspended in a suitable solvent only prior to administration, or is present in another suitable dosage form or is present in a gastric juice-resistant administration form or another form for oral administration.

Suitable formulations may also be prepared from progeny of NZ2 obtained by passaging and/or adapting to particular cells, for example WI-38, MRC-5 or Vero cells, or other such strains or parts or fragments of NZ2 and other such strains or their progeny. Part is understood to mean a genomic or subgenomic fragment which is expressed in a suitable system, for example in fibroblast cell culture, with the aid of a suitable vector, for example vaccinia. Fragments are understood to be fractions obtained by biochemical purification of particles by physical disruption, for example by ultrasonication, for example by means of chromatography.

In this connection, several administrations according to a sequential schedule meeting the requirements of clinical problems, or long-term treatment may be necessary.

Thus, applications such as (but not limited to) the following protocols have proven particularly suitable in cancer therapy: intramuscular administration of 10 every three days in each case⁶To 10⁷ TCID₅₀(tissue culture infectious dose) for four weeks followed by two weeks off; intramuscular administration of 10 every three days was repeated in each case⁶To 10⁷ TCID₅₀The medicine is taken for four weeks, and the medicine is stopped for two weeks; intramuscular administration of 10 every three days was repeated in each case⁶To 10⁷ TCID₅₀Four weeks following dosing, two weeks following discontinuation; depending on the severity of the disease and the effectiveness of the treatment, these cycles may be supplemented by several cycles; alternatively, the dosing regimen may be such that the formulation is administered 4-5 days each for at least 3 months.

For example, in the case of chronic viral infection, 10 may be administered every three days, subcutaneously in the abdomen or intramuscularly in the deltoid or quadriceps muscle⁶To 10⁷ TCID₅₀The formulation of (1), administered a total of 5 times. This dosing regimen may be varied as required by the disease. For the prevention of cold, the formulation must be used to rinse the mouth and repeatedly administered daily as long as there is a risk of infection.

To prevent infection after surgery (e.g., dental surgery) in the oral region, the formulation may be rinsed for 1-2 minutes in the evening prior to surgery.

Reference documents:

guidotti, l.g., Borrow, p., Hobbs, m.v., Matzke, b., Gresser, i., Oldstone, m.b.a., and Chisari, F.V, (1996): virus crossing: intracellular inactivation of hepatitis B virus during liver-unrelated Viral infection (Viral secretion: Intracellular inactivation of the hepatitis B virus infection of the liver), Proc. Natl. Acad. Sci. USA.93: 4589-4594.

Guidotti, l.g., Ando, k., Hobbs, m.v., Ishikawa, t., Runkel, l., Schreiber, r.d., and Chisari, F.V. (1994): cytotoxic T cells inhibit hepatitis B virus gene expression in transgenic mice by a non-cytolytic mechanism (Cytotoxic T lymphocytes inhibition hepatitis B virus genexpression by a non Cytotoxic hepatitis B cytokine metabolism in transgenic mice, proc. natl. acad. sci. usa.91: 3764-3768.

Steimasll, g., g.wolf (1990): bildung von Interleukin2 und Interferon durch mononukle Leukozyten des Schweinesnach in vitro-Stimulation mit verschieden Virusparaten (production of Interleukin2 and Interferon by porcine mononuclear leukocytes after in vitro Stimulation with various virus preparations) J.Vet.Med.B37, 5, 321-.

P.lucin, s.jonjic, m.messerle, b.polar, h.hengel, u.h.koszynowski (1994): synergistic effects of gamma interferon and alpha tumor necrosis factor inhibit murine cytomegalovirus replication at an advanced stage (Late-Phase inhibition of murine cytomegalovirus replication by synergistic interaction of interferon gamma and tumor necrosis factor alpha), j.gen.virol 75: 101-110; P.M are provided.

Smith, r.m.wolcott, r.chervenak, s.r.jennings (1994): control of acute cutaneous herpes simplex virus Infection-T cell-mediated viral clearance depends on gamma interferon (Control of acute cutaneous herpes simplex virus-Infection-T-cell mediated viral clearance is dependent on interferon gamma.) Virology 202 (1): 76-88.

Y.kawanashi, n.hayashi, k.katayama, k.ueda, t.takehara, e.miyoshi, e.mi ta, a.kasahara, h.fusamoto, t.kamada (1995): alpha Tumor necrosis factor and interferon gamma synergistically inhibit viral replication in hepatitis B virus replicating cells J.medical Virology 47 (3): 272-277.

Dhawan, s., l.m.wahl, a.heredia, y.h.zhang, j.s.epstein, m.s.meltzer, i.k.hewlett (1995): interferon gamma inhibits HIV-induced monocyte invasion (Interferon gamma inhibitors HIV-induced invasion of Monocytes) j. leukocyte Biology, 58 (6): 713-716.

J.f.bromberg, c.m.horvath, z.l.wen, r.d.schreiber, j.e.darnell (1996): transcriptional activation state 1 is essential for the antiproliferative effects of alpha-and gamma-interferons (transcriptional active site requiredfor the antiproliferative effects of both interferon alpha-interferon.) PNAS93 (15): 7673-7678.

M.klouche, h.kirchner, f.holzel (1994): anti-proliferative effects of gamma interferon binding to alpha-difluoromethyloornithine on human cancer cell cultures (anti-proliferative effects of inter-feron gamma-incorporation with alpha-difluoromethylonitrile on human cancer cell cultures J. cancer Research and clinical Oncology120 (12): 706. sequence listing

Use of <130> parapoxvirus ovis strain NZ2<140> <141> <160>11<170> PatentIn Ver.2.1<210>1<211>25<212> DNA <213> Mus sp. <220> 223> beta-actin primer, antisense <400>1taaaacgcag ctcagtaaca gtccg <210>2<211>25<212> DNA <213> Mus sp. <220> beta-actin primer, sense <400>2 3925 <210>3<211>26<212> DNA <213> hepatitis B virus <220> <223> HBV probe, ayw-613t <400>3ccatcatcct gggctttcgg aaaatt <210>4<211>19 > DNA <213> 220> 223> HBV primer, ayw-670 <120> HBV <210>4<211> HBV <212> HBV <220> HBV <223> HBV primer, antisense ayw-600 <210> aggagaaacg ggctgaggc > 210>5<21 > HBV <212> HBV <220> HBV <213>,223 > HBV primer, ayw-570f (sense) <400>5 3621 <210>6<211>24<212> DNA <213> Mus sp <220> <223> γ -IFN primer (antisense) <400>6gtcacagttt tcagctgtat aggg <210>7<211>26<212> DNA <213> Mus sp <220> <223> γ -IFN primer (sense) <400>7agcggctgac tgaactcaga ttgtag <210>8<211>30<212> DNA <213> Mus sp <220> <223> IL-15 primer (antisense) <400>8cgtgttgatg aacatttgga caatgcgtat <210>9<211>29<212> DNA <213> Mus sp <220> <223> IL-15 primer (sense) <400>9 > gccaactgga tagatgtaag atatgacct <210>10 >25 <213> TNF <213> m sp <220> mu sp <223> α -TNF <210> antisense <400> 25<210> TNF <11 > TNF <210> α sp <220> 223> 3> TNF <220> α -223 >2< 210> TNF <210> 2> TNF <220> Sense) <400>11ggcaggtcta ctttggagtc attgc 25

Claims (11)

1. Use of a strain NZ2, NZ-7, NZ-10 or orf-11, which belongs taxonomically to parapoxvirus ovis, for the preparation of a medicament against viral infections and tumors in humans and animals.

2. Use of the virus according to claim 1, progeny obtained by passaging or adapting to suitable cell lines such as human cells, e.g. WI-38 and MRC-5, vero cells, bovine cells, e.g. BK-K13a47/Reg or MDBK, and ovine cells, e.g. MDOK, for the preparation of a medicament against viral infections and cancer in humans and animals.

3. Use of parts or fragments of a virus according to claims 1 and 2, understood as being genomic or subgenomic fragments expressed in a suitable system, such as fibroblast cell culture, by means of a suitable vector, such as vaccinia virus, understood as being the parts obtained by biochemical purification, such as chromatography, of the expressed or physically disrupted viral particles, for the preparation of a medicament against viral infections and cancer in humans and animals.

4. Use of one of the parapoxvirus ovis strains according to claims 1 to 3 for the preparation of medicaments and pharmaceutical preparations as immunotherapeutic or immunoprophylactic medicaments for autoimmune diseases and for acute or chronic viral infections of the respiratory tract and internal organs.

5. Use of one of the strains according to claims 1 to 3 for the preparation of medicaments and pharmaceutical preparations for post-stress maintenance and for the prevention or alleviation of infectious diseases after stress and in connection with infection prevention in surgery and dental surgery.

6. Use of one of the strains according to claims 1 to 3 for the preparation of medicaments and pharmaceutical preparations for the postinfectious maintenance or treatment of acute viral infections such as respiratory tract infections, papilloma virus infections, herpes virus infections, HIV infections, or viral infections of internal organs such as hepatitis virus infections, and for related diseases such as multiple sclerosis, asthma, warts and other skin neoplasms.

7. Use of one of the strains according to claims 1-3 for the preparation of medicaments and pharmaceutical preparations for use on wounds to promote the wound healing process and for the promotion of healing of wounds and leg ulcers that do not heal well or do not heal at all.

8. Use of one of the strains according to claims 1-3 for the preparation of a medicament and pharmaceutical preparations for various allergic diseases, psoriasis, neurodermatitis and other autoimmune diseases, such as lupus erythematosus, as well as for improving the health of, for example, elderly patients.

9. Use of one of the strains according to claims 1-3 for the preparation of a medicament and pharmaceutical preparations for combating inflammatory, degenerative and proliferative diseases of internal organs, skin, blood, the central nervous system and its accessory organs including the eye (e.g. crohn's disease), including cancer.

10. Use of one of the strains of parapox ovis virus according to claims 1 to 3 in combination with other drugs for the preparation of a medicament and a pharmaceutical preparation for antiviral or cancer therapy in humans and animals.

11. Use of one of the strains of parapox ovis virus according to claims 1 to 3 in combination with other drugs for the preparation of a medicament and a pharmaceutical preparation for oral administration and/or for oral administration of gastric juice resistant preparations.