Soluble recombinant botulinum toxin proteins

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 ...

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[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.

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