Vaccines against clostridium difficile comprising recombinant toxins

A technology of Clostridium difficile, Clostridium difficile toxin, applied in the field of Clostridium difficile vaccine

Inactive Publication Date: 2014-09-10
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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  • Vaccines against clostridium difficile comprising recombinant toxins
  • Vaccines against clostridium difficile comprising recombinant toxins
  • Vaccines against clostridium difficile comprising recombinant toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0190] Expression of Clostridium difficile TcdA and TcdB mutants in E. coli.

[0191] The C. difficile native A and toxin B encoding nucleotide sequences derived from strain VPI10463 were mutagenized 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.

[0192] Construct expression in E. coli was assessed by SDS-PAGE. Such as Figure 4 As shown in , expression analysis of the Tcd mutant enzyme domain (...

Embodiment 2

[0194] TcdA and TcdB enzyme activity assays with RhoA glucosylation assay.

[0195] 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.

[0196] 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

[0199] Expression of TcdA and TcdB in baculovirus expression system.

[0200] 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).

[0201] 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 C. 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 invention also relates to vaccines and immunogenic compositions comprising these recombinant toxins, as well as combinations of these toxins with binary toxin B (CDTb), which are capable of providing protection against C. difficile infection and / or the effects thereof. The invention also relates to methods of inducing an immune response to C. difficile comprising administering the vaccines and immunogenic compositions described herein to a patient. The invention also encompasses methods of expressing recombinant C. difficile toxin A and toxin B and CDTa mutants and CDTb in recombinant expression systems. In exemplary embodiments, TcdA, TcdB, and CDTa mutant toxins comprising sufficient mutations to substantially reduce or eliminate toxicity are expressed in the baculovirus / insect cell expression system.

Description

Area of invention [0001] The present invention involves the use of hardclavacia ( CLOSTRIDIUM Difficile ) Toxins A (TCDA), toxin B (TCDB), and binary toxins (CDTA and CDTB) proteins at least one restructuring toxin protein and combination with other immunoglobinism induces immune response to difficultylobia.The present invention also involves an immunogenic combination that contains CDTA and / or CDTB protein, which contains a separate or or more immune such as TCDA and TCDB.Essence [0002] Cross reference for related applications [0003] This application requirement for temporary application number 61 / 591,631, which submitted on January 27, 2012, US temporary application number 61 / 596,419 submitted on February 8, 2012, and temporary US temporary application submitted on September 20, 2012The equity of 61 / 703,754, the content of the US patent application is incorporated into this article as a whole. [0004] Referred to the serial list of electronics [0005] The serial list of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/33A61K39/00
CPCA61K38/00C12N9/1077C12N9/1051A61K2039/55577A61K2039/55505C07K14/33A61K39/08C12N9/52A61P31/04A61P37/04
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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