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Soluble recombinant botulinum toxin proteins

a technology of botulinum toxin and soluble recombinant, which is applied in the field of soluble recombinant botulinum toxin proteins, can solve the problems of insufficient heating of food, flaccid paralysis, and association with very serious problems

Inactive Publication Date: 2005-10-27
ALLERGAN SALES ALLERGAN BOTOX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0112] The present invention further provides a

Problems solved by technology

Nonetheless, these species are associated with very serious diseases, including botulism, tetanus, an aerobic cellulitis, gas gangrene, bacteremia, pseudomembranous colitis, and clostridial gastroenteritis.
Botulinal toxin blocks nerve transmission to the muscles, resulting in flaccid paralysis.
When the toxin reaches airway and respiratory muscles, it results in respiratory failure that can cause death.
Food-borne botulism results from ingesting improperly preserved and inadequately heated food that contains botulinal toxin.
It is believed that infants are susceptible, due, in large part, to the absence of the full adult complement of intestinal microflora.
In this regard, some adults who have undergone antibiotic therapy which alters intestinal microflora become more susceptible to botulism.
Clinical symptoms of infant botulism range from mild paralysis, to moderate and severe paralysis requiring hospitalization, to fulminant paralysis, leading to sudden death.
First, extremely large dosages must be injected intravenously and / or intramuscularly.
Second, the antitoxin has serious side effects such as acute anaphylaxis which can lead to death, and serum sickness.
Finally, the efficacy of the antitoxin is uncertain and the treatment is costly.
The supply of this antitoxin is limited and cannot be expected to meet the needs of all individuals stricken with botulism disease.
However, this vaccine preparation has several disadvantages.
Second, immunization is painful (deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% of recipients upon initial injection; this number rises to approximately 11% of individuals who receive booster injections) [Informational Brochure for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control].
Third, preparation of the vaccine is dangerous as active toxin must be handled by laboratory workers.
As many of these patients are being treated with antimicrobials or other chemotherapeutic agents, such transmission of C. difficile represents a significant risk factor for disease.
This occasionally fatal disease is characterized by diarrhea, multiple small colonic plaques, and toxic megacolon.
Toxin B does not cause intestinal fluid accumulation, but it is 1000 times more toxic than toxin A to tissue culture cells and causes membrane damage.
Treatment of C. difficile disease is problematic, given the high resistance of the organism.
However there are problems associated with treatment utilizing these compounds.
Vancomycin is very expensive, some patients are unable to take oral medication, and the relapse rate is high (20-25%), although it may not occur for several weeks.

Method used

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  • Soluble recombinant botulinum toxin proteins
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  • Soluble recombinant botulinum toxin proteins

Examples

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Effect test

example 1

Production of High-Titer Antibodies to Clostridium difficile Organisms in a Hen

[0173] Antibodies to certain pathogenic organisms have been shown to be effective in treating diseases caused by those organisms. It has not been shown whether antibodies can be raised, against Clostridium difficile, which would be effective in treating infection by this organism. Accordingly, C. difficile was tested as immunogen for production of hen antibodies.

[0174] To determine the best course for raising high-titer egg antibodies against whole C. difficile organisms, different immunizing strains and different immunizing concentrations were examined. The example involved (a) preparation of the bacterial immunogen, (b) immunization, (c) purification of anti-bacterial chicken antibodies, and (d) detection of anti-bacterial antibodies in the purified IgY preparations.

[0175] a) Preparation of Bacterial Immunogen

[0176]C. difficile strains 43594 (serogroup A) and 43596 (serogroup C) were originally obta...

example 2

Treatment of C. difficile Infection with Anti-C. difficile Antibody

[0189] In order to determine whether the immune IgY antibodies raised against whole C. difficile organisms were capable of inhibiting the infection of hamsters by C. difficile, hamsters infected by these bacteria were utilized. [Lyerly et al., Infect. Immun., 59:2215-2218 (1991).] This example involved: (a) determination of the lethal dose of C. difficile organisms; and (b) treatment of infected animals with immune antibody or control antibody in nutritional solution.

[0190] a) Determination of the Lethal Dose Of C. difficile Organisms

[0191] Determination of the lethal dose of C. difficile organisms was carried out according to the model described by D. M. Lyerly et al., Infect. Immun., 59:2215-2218 (1991). C. difficile strain ATCC 43596 (serogroup C, ATCC) was plated on BHI agar and grown anaerobically (BBL Gas Pak 100 system) at 37° C. for 42 hours. Organisms were removed from the agar surface using a sterile dac...

example 3

Production of C. botulinum Type A Antitoxin in Hens

[0199] In order to determine whether antibodies could be raised against the toxin produced by clostridial pathogens, which would be effective in treating clostridial diseases, antitoxin to C. botulinum type A toxin was produced. This example involves: (a) toxin modification; (b) immunization; (c) antitoxin collection; (d) antigenicity assessment; and (e) assay of antitoxin titer.

[0200] a) Toxin Modification

[0201]C. botulinum type A toxoid was obtained from B. R. DasGupta. From this, the active type A neurotoxin (M. W. approximately 150 kD) was purified to greater than 99% purity, according to published methods. [B. R. DasGupta & V. Sathyarnoorthy, Toxicon, 22:415 (1984).] The neurotoxin was detoxified with formaldehyde according to published methods. [B. R. Singh & B. R. DasGupta, Toxicon, 27:403 (1989).]

[0202] b) Immunization

[0203]C. botulinum toxoid for immunization was dissolved in PBS (1 mg / ml) and was emulsified with an app...

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Abstract

The present invention includes recombinant proteins derived from Clostridium botulinum toxins. In particular, soluble recombinant Clostridium botulinum type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

Description

[0001] This application is a Continuation-In-Part of copending application Ser. No. 08 / 405,496, filed Mar. 16, 1995.FIELD OF THE INVENTION [0002] The present invention relates to the isolation of polypeptides derived from Clostridium botulinum neurotoxins and the use thereof as immunogens for the production of vaccines, including multivalent vaccines, and antitoxins. BACKGROUND OF THE INVENTION [0003] The genus Clostridium is comprised of gram-positive, anaerobic, spore-forming bacilli. The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous; they are commonly found in soil, dust, sewage, marine sediments, decaying vegetation, and mud. [See e.g., P. H. A. Sneath et al., “Clostridium,” Bergey's Manual® of Systematic Bacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986).] Despite the identification of approximately 100 species of Clostridium, only a small number have been recognized as ...

Claims

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Application Information

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IPC IPC(8): A61K39/00C07K14/245C07K14/33C07K16/02C07K16/12C12N1/21
CPCA61K39/00A61K2039/505C07K14/245C07K2319/00C07K16/02C07K16/1282C07K2317/34C07K14/33Y02A50/30
Inventor WILLIAMS, JAMES
Owner ALLERGAN SALES ALLERGAN BOTOX
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