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Recombinant proteins of parapoxvirus ovis and pharmaceutical compositions prepared therefrom

A technology of recombinant proteins and drugs, applied in the direction of drug combinations, viruses, viral peptides, etc., can solve the problems of inability to treat chronic and persistent latent virus infections, difficult problems, etc.

Inactive Publication Date: 2010-01-06
AICURIS GMBH & CO KG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is also known that chronic persistent latent viral infections are rarely or not treated with conventional low molecular weight antiviral substances alone

Method used

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  • Recombinant proteins of parapoxvirus ovis and pharmaceutical compositions prepared therefrom
  • Recombinant proteins of parapoxvirus ovis and pharmaceutical compositions prepared therefrom
  • Recombinant proteins of parapoxvirus ovis and pharmaceutical compositions prepared therefrom

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Determination of integrated PPVO fragments in active VVOVs.

[0085] DNA was prepared from Listeria vaccinia virus / PPVO recombinants as follows:

[0086] Make BK-KL 3A cells at 175cm 2 Flasks (Becton Dickson Labware, Heidclberg, Germany) were grown to confluence. Cells were infected with recombinant Listeria vaccinia virus / PPVO virus (VVOV) of Mercer et al. (1997, Virology, 229: 193-200) at an MOI (multiplicity of infection) of 0.01-0.32 and incubated at 37°C until 100% CPE (cytopathic effect). Infected cells were frozen at -80°C, thawed and processed as described below: Modifications of the RNA extraction method of Vilcek et al. (1994, J. Clin. Microbiol. 32:2225-2231). A 0.5 ml aliquot of the cell suspension was mixed with 100 μg proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) and 50 μl SDS (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany) using 2 ml PLG Heavy Eppendorf tubes (Eppendorf, Hamburg, Germany). Germany) were incubated togethe...

Embodiment 2

[0097] Example 2: Induction of interferon gamma and tumor necrosis factor alpha by the PPVO gene product

[0098] The 16 recombinants were tested for their ability to induce tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in whole blood cultures.

[0099] Whole blood cultures containing blood and RPMI medium (LifeTechnologies GmbH, Karlsruhe, Germany) in a ratio of 1:5 were stimulated with the recombinant virus. Pure Listeria vaccinia virus and complete PPVO preparations were used as controls. All formulations were used at a final 1:10 dilution. Since the virus alone was unable to induce IFN-γ, co-stimulation with Concanavalin A (SIGMA, St. Louis, MO) was performed to determine IFN-γ. Cells were then incubated for 24 hours (TNF-α) and / or 72 hours (IFN-γ). The cytokine concentrations in cell culture supernatants were determined by TNF-α or IFN-γ specific ELISA. These time points were found to be optimal when the experimental conditions were determin...

Embodiment 3

[0107] Example 3: Local immunomodulation by the PPVO gene product in liver sinusoidal endothelial cells (LSEC)

[0108] We have established a new cell-based assay system capable of examining the hepatoprotective properties of recombinant PPVO proteins expressed in different systems (eg, vaccinia virus). These assay systems use primary murine hepatocytes, LSEC, which play a central role in determining whether immunity or tolerance is induced in the liver. The unique ability of LSEC to present exogenous antigens to CD8+ T cells on MHC class I molecules enables immunosurveillance of hepatocytes since viral antigens released by the infected hepatocytes may be acquired and presented by LSEC to the cells of the immune system. The new assay enables measurement of the ability of LSEC to specifically interact with the CD8+ T cell antigen responsible for tissue destruction in necroinflammatory hepatitis.

[0109] Pure LSEC populations were isolated from murine liver by a stepwise pr...

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PUM

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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.

Description

Field of Invention [0001] 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 production of pharmaceutical compositions. Background of the Invention [0002] Latent and chronic persistent viral infections are known to be activated or reactivated by immunosuppression, or conversely suppress the immune system in acute illnesses that may be caused by latent viruses (eg in the context of stress or cortisone administration, immunosuppression leads to latent of herpesvirus infection recurs in the form of lip vesicles). Chronic persistent latent viral infections are also known to be difficult or impossible to treat with conventional low molecular weight antiviral substances alone. [0003] Class I-restricted cytotoxic T cells have been shown to suppress hepatocyte HBV gene expression in HBV transgenic mice, and this process was shown to be caused by TNF-α and IFN-γ...

Claims

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Application Information

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IPC IPC(8): A61K39/275C07K14/065C12N5/10C12N15/39C12N15/863A61P31/00A61K38/00
CPCC12N2710/24143C12N15/86C07K2319/00C12N2710/24043A61K2039/525C07K14/005A61K38/00C12N2710/24222C12N2710/24243A61P1/04A61P1/16A61P11/06A61P17/02A61P17/12A61P25/00A61P29/00A61P31/00A61P31/12A61P31/20A61P31/22A61P35/00A61P37/00A61P37/08C12N7/00C12N2710/24221C12N2710/24233
Inventor O·韦伯S·M·弗里德里希斯A·西格林T·施拉普A·A·默瑟S·B·弗勒明H·-D·沃尔克
Owner AICURIS GMBH & CO KG
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