Vaccine against Clostridium difficile containing recombinant toxin

A technology of binary toxin and composition, applied in the field of Clostridium difficile vaccine, can solve problems such as failure to eliminate CDAD

Inactive Publication Date: 2018-02-09
MERCK & CO INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing treatments often fail to eliminate CDAD or lead to recurrent disease, necessitating new approaches to preventive or therapeutic management

Method used

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  • Vaccine against Clostridium difficile containing recombinant toxin
  • Vaccine against Clostridium difficile containing recombinant toxin
  • Vaccine against Clostridium difficile containing recombinant toxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Expression of Clostridium difficile TcdA and TcdB mutants in E. coli.

[0187] The C. difficile native A and toxin B encoding nucleotide sequences derived from strain VPI10463 were subjected to mutagenesis using the Quick Change® XL Site-Directed Mutagenesis Kit (Stratagene Corp., La Jolla, CA). As shown in Figure 3, several mutant toxins with different combinations of mutations were prepared. Mutants were expressed and purified from E. coli. In addition, the codon-optimized nucleotide sequence for the E. coli expression system was obtained from GenScript (GenScript USA, Inc., Piscataway, NJ). Of the mutants shown in Figure 3, only the E. coli codon-optimized triple mutant TcdB (W102A, D288A, E515Q) was expressed in full-length form. Additional constructs consisting only of the catalytic domain expressed in E. coli were tested.

[0188] Construct expression in E. coli was assessed by SDS-PAGE. Such as Figure 4 As shown in , expression analysis of the Tcd mutant en...

Embodiment 2

[0190] TcdA and TcdB enzyme activity assays with RhoA glucosylation assay.

[0191] Toxicity of recombinant TcdA and TcdB enzyme domains was compared in vitro using the RhoA glucosylation activity assay. Briefly, the protein was mixed at two different concentration levels (1 and 10 µg / mL) with a reaction buffer containing substrate (recombinant RhoA-GTPase), cofactor (MnII) and radiolabeled co-substrate (UDP-14C-glucose). Reactions were incubated at 37°C for 90 minutes, and ice-cold 10% trifluoroacetic acid was used to precipitate the protein along with any incorporated 14C. Precipitated material was recovered by filtration and the filtrate was quantified in a liquid scintillation counter.

[0192] The measurement results about TcdB are shown in Figure 5 middle. The triple mutant W102A, D288A, E515Q catalytic domain appeared to retain about 10% of the glucosylation activity of the native catalytic domain, an activity comparable to that of the W102 deletion mutant. No det...

Embodiment 3

[0195] Expression of TcdA and TcdB in baculovirus expression system.

[0196] To overcome the difficulties encountered in the expression of recombinant TcdA and TcdB, several expression platforms were evaluated. E. coli was initially tested; however, the inventors were unable to express 3mTcdA using this system, and although 3mTcdB was successfully expressed, it was partially degraded. The present inventors also evaluated the expression of 4mTcdA / B in E. coli, and again, 4mTcdA could not be successfully produced in this host system, but 4mTcdB was produced at high levels (~1.6 g / L). The Bacillus megaterium expression system was also evaluated and successfully produced low to moderate amounts of 5mTcdA (~75mg / L) and 5mTcdB (~200-700mg / L).

[0197] Based on previous examples of expressing full-length TcdA and TcdB in this organism (Burger et al. Biochem Biophys Res.Commun . 307(3): 584-8(2003); Yang et al., BMC Microbiol. 8:192(2008)), evaluating Bacillus megaterium as an...

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Abstract

The present invention relates to recombinant Clostridium difficile toxin A (TcdA) and toxin B (TcdB) and binary toxin A (CDTa) proteins comprising specifically defined mutations relative to the native toxin sequence that substantially reduce or eliminate toxicity. The present invention also relates to vaccines and immunogenic compositions comprising these recombinant toxins, as well as combinations of these toxins with binary toxin B (CDTb), capable of conferring protection against C. difficile infection and/or its effects. The invention also relates to methods of inducing an immune response against C. difficile comprising administering to a patient the vaccines and immunogenic compositions described herein. The present invention also includes methods for expressing recombinant C. difficile toxin A and toxin B and CDTa mutants and CDTb in a recombinant expression system. In an exemplary embodiment, TcdA, TcdB, and CDTa mutant toxins are expressed in a baculovirus/insect cell expression system, which contain sufficient mutations to substantially reduce or eliminate toxicity.

Description

field of invention [0001] The present invention relates to the use of selected from Clostridium difficile ( Clostridium difficile A method of inducing an immune response against Clostridium difficile of at least one recombinant toxin protein of the toxin A (TcdA), toxin B (TcdB) and binary toxin (CDTa and CDTb) proteins of ) and in combination with an additional immunogen. The present invention also relates to immunogenic compositions, in particular Clostridium difficile vaccines, comprising CDTa and / or CDTb proteins alone or in combination with one or more additional immunogens such as TcdA and TcdB, capable of inducing a protective immune response . [0002] Cross References to Related Applications [0003] This application claims U.S. Provisional Application No. 61 / 591,631, filed January 27, 2012, U.S. Provisional Application No. 61 / 596,419, filed February 8, 2012, and U.S. Provisional Application No. 61 / 596,419, filed September 20, 2012 61 / 703,754, the contents of which...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/33A61K39/00
CPCA61K39/08C07K14/33A61K38/00A61K2039/55505A61K2039/55577C12N9/1077C12N9/52C12N9/1051A61P31/04A61P37/04
Inventor J.H.海恩里希斯J-L.博默S.L.塞科尔A.R.格尔克I.卡罗亚圭拉M-P.金泰尔M.S.霍尔顿M.R.米翟夫斯基J.M.斯金纳P.J.A.森德梅耶S.苏布拉马尼安K.H.A.v.d.海登利夫肯斯S.王J.谢R.F.埃克索科诺斯特尔J.K.佐尔曼
Owner MERCK & CO INC
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