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Process for production of vaccines

A vaccine and toxin technology, applied in chemical instruments and methods, biochemical equipment and methods, pharmaceutical formulations, etc., can solve problems such as affecting the effectiveness of vaccines, costing, and loss of antigenic epitopes.

Inactive Publication Date: 2011-11-09
BOEHRINGER LNGELHEIM VETMEDICA GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all these measures result in the loss of epitopes of the pathogenic organism, which may affect the effectiveness of the vaccine, and / or be costly

Method used

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  • Process for production of vaccines
  • Process for production of vaccines
  • Process for production of vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Manufacture of Clostridium difficile vaccine

[0029] C. difficile samples are available from public collections such as the American Type Culture Center (ATCC), Manassas, VA, USA, numbers ATCC 9689, ATCC 43255. It was grown in the fermenter of BHI medium (brain heart infusion, Becton Dickinson, Heidelberg, Germany; see American Pharmaceutical Association, 1950, The national formulary, 9th edition, APA, Washington, D.C.) , fermented under anaerobic conditions at 37°C for 3 to 4 days. Two large cytotoxins, TcdA and TcdB, are released during the stable phase. At this time point, the culture was harvested and the bacteria were pelleted by centrifugation at 8000 xg for 10 minutes. The supernatant is used directly, or the toxin can be enriched by gel permeation chromatography (eg, on S300 Sephacryl), affinity chromatography, anion exchange chromatography and / or ultrafiltration. The reducing agent dithiothreitol is then added to the supernatant or toxin-enriched...

Embodiment 2

[0031] Embodiment 2: activity test

[0032] CHO cells (Chinese hamster ovary, e.g. DSMACC110, Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) are inoculated in e.g. Ham's F10 medium containing e.g. 5% FCS (fetal calf serum) into 96- or 24-well microplates (100 μl per well), and incubated overnight at 37°C in a humid environment until they reached confluence. Rinse with Ringer's solution without divalent ions such as magnesium or calcium, then add 100 μl (96 well plate) or 400 μl (24 well plate) Ringer's solution without Mg and Ca into the well. Draw the vaccine preparation of 100 or 400 μ l embodiment 1 respectively with pipette subsequently, and each dilution series (10 -1 to 10 -8 ) to the well, and each sample was repeated twice. The BHI medium was treated in the same way as the vaccine preparation as a negative control. Untreated C. difficile supernatant was used as a positive control. The plates were incubated for 3 to 24 hours in a...

Embodiment 3

[0034] Example 3: Vaccination of Animals

[0035] The Syrian golden hamster can be used as a standardized animal model for C. difficile infection. Animals weighing 60 to 100 g were used in the experiments. The animals received vaccine formulations at different concentrations (1 to 100 μg) by intraperitoneal or subcutaneous injection. The vaccine was adjuvanted or contained complete Freund's adjuvant (1:1 with the vaccine formulation) or Ribi (emulsion of monophosphoryl lipid A and trehalose dicorynomycolate) as adjuvants. BHI medium treated with the same method as the vaccine was used as a negative control. Two weeks after the last vaccination, the animals received 10 to 100 mg / kg clindamycin intraperitoneally or orally. After 24 hours, each animal was inoculated with at least 10 4 Viable Clostridium difficile or 100 c.f.u (colony forming units). The protective efficacy of the vaccine is determined by clinical monitoring of diarrhea or mortality.

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Abstract

The invention relates to a process for the production of a vaccine against bacterial pathogens which produce an AB toxin, like Clostridium, comprising (a) culturing the pathogen under conditions where the AB toxin is produced, and harvesting the culture (b) cleaving the AB toxin enzymatically in vitro, preferably using inositol hexaphosphate as a co-factor, and (c) combining the composition of step (b) with a pharmaceutically acceptable carrier.

Description

field of invention [0001] The present invention relates to a method for producing a vaccine, and the vaccine produced thereby. Background of the invention [0002] Clostridium difficile is a spore-forming gram-negative bacterium that causes 60% of antibiotic-associated diarrhea cases and almost 100% of patients with pseudomembranous colitis. The mechanism causing the outbreak is not well understood. It may be related to both host and strain factors, since not all patients infected with C. difficile develop disease. Clinical symptoms in infected patients range from asymptomatic to life-threatening toxic megacolon. [0003] C. difficile, like many other pathogens that cause disease in animals, including humans, produces toxins. Toxins are toxic substances produced by living cells or organisms that are active at very low concentrations. Toxins can be small molecules, peptides, or proteins that can cause disease when absorbed by human tissues by contacting or interacting wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/08C07K14/33
CPCA61K39/08A61P1/00A61P1/12A61P31/04A61P37/04C07K14/33C12N15/00
Inventor 杰西卡·赖内克
Owner BOEHRINGER LNGELHEIM VETMEDICA GMBH
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