US Government As Represented by the Secretary of the Army

Other

Biotherapeutics, Inc.

Leonard A Smith

Michael P Byrne

Douglas R Brown

Michael A Clayton

John L Middlebrook

Hugh Lapenotiere

Melody Jensen

Peter M Pushko

Jonathan F Smith

Michael D Parker

Istvan J Enyedy

David D Mundschenk

John S Lee

Mark T Dertzbaugh

David C H Yang

Nizamettin Gul

Sivanesan Dakshanamurthy

John H Cardellina, Ii

Salimuddin Shah

Rebecca C Vieira

Virginia I Roxas-Duncan

Susan M Ensel

The invention provides potent quinolinol-based BoNT/A small-molecule inhibitors of botulinum neurotoxins, in particular ofserotype A neurotoxins. The invention also provides methods of using these small-molecule inhibitors to inhibit infections by, as well as, methods of preventing infections bythrough materials that may be ingested.

Small molecule inhibitors of botulinum neurotoxins

The present invention relates to the construction, expression, and purification of synthetic or recombinant light chain (LC) botulinum neurotoxin genes from all botulinum neurotoxin serotypes. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product is stable and proteolytically active. Methods of using the products of the invention are described.

Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy

This invention is directed to preparation and expression of synthetic genes encoding polypeptides containing protective epitopes of botulinum neurotoxin (BoNT). The invention is also directed to production of immunogenic peptides encoded by the synthetic genes, as well as recovery and purification of the immunogenic peptides from recombinant organisms. The invention is also directed to methods of vaccination against botulism using the expressed peptides.

Recombinant vaccine against botulinum neurotoxin

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates to the construction, expression, purification, and use of synthetic or recombinant botulinum neutoroxin genes. For example, a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A) was constructed and overexpressed in. The gene product was purified from inclusion bodies. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product was stable in solution at 4&#xb0; C. for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency k/Kwas higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Znto a Zn-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing.

This invention is directed to preparation and expression of synthetic genes encoding polypeptides containing protective epitopes of botulinum neurotoxin (BoNT). The invention is also directed to production of immunogenic peptides encoded by the synthetic genes, as weel as recovery and purification of the immunogenic peptides from recombinant organisms. The invention is also directed to methods of vaccination against botulism using the expressed peptides.

neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates to the construction, expression, purification, and use of synthetic or recombinantneutoroxin genes. For example, a synthetic gene for the LC of theneurotoxin serotype A (BoNT/A) was constructed and overexpressed in. The gene product was purified from inclusion bodies. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product was stable in solution at 4&#xb0; C. for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency k/Kwas higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Znto a Zn-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing.

[object Object]

A method of preparing a bioactive polypeptide in a stable, inactivated form, the method comprising the step of treating the polypeptide with ozonated water in order to oxidize and/or stabilize the cysteine residues, and in turn, prevent the formation of disulfide bridges necessary for bioactivity. The method can involve the use of ozonated water to both oxidize the disulfide bridges in a bioactive polypeptide, and to then stabilize the resultant cysteine residues. Optionally, and preferably, the method can involve the use of ozonated water to stabilize the cysteine residues, and thereby prevent the formation of disulfide bridges, in a polypeptide produced by recombinant means in a manner that allows the polypeptide to be recovered with the disulfide bridges unformed.

Compositions comprising bioactive peptides prepared without formation of native disulfide bonds

Using the nontoxic heavy chain fragment from botulinum neurotoxins A-G, compositions and methods of use in inducing an immune response which is protective against intoxication with botulinum in subjects is described.

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Clarksburg, MD, United States of America

Leonard A Smith