Peptide fragments of cholera toxic B or enterotoxin B as vaccine adjuvants

A fragmented and disordered technology, used in peptides, depsipeptides, testing pharmaceutical preparations, etc.

Inactive Publication Date: 2001-10-10
UNIV OF BRISTOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Until now, there have been no reports of CtxB / EtxB mutants that retain GM-1 receptor binding but have no immunomodulatory effects

Method used

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  • Peptide fragments of cholera toxic B or enterotoxin B as vaccine adjuvants
  • Peptide fragments of cholera toxic B or enterotoxin B as vaccine adjuvants
  • Peptide fragments of cholera toxic B or enterotoxin B as vaccine adjuvants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Identification of residues in the Glu-51-Ile-58 loop that trigger immunomodulatory effects on leukocytes

[0242] NIH male mice were sacrificed, and the mesenteric lymph node tissues were taken and placed in Hanks balanced salt solution (without calcium and magnesium ions in HBSS, and 20 mM Hepes was added). Scatter lymphocytes from fibrous tissue by gently squeezing through nylon mesh, wash with HBSS three times, suspend lymph node cells with Eagle's modified medium (Gibco), this medium contains 20mM Hepes, 4mM L-glutamine, 100IU / ml Penicillin, 100 μg / ml streptomycin, 5×10 -5 M2-Mercaptoethanol, cell concentration controlled at 2*10 6 cells / ml. Then add or not add 3.45μM (40μg / ml) of wild-type EtxB or CtxB, or various B subunit mutants, such as EtxB(G33D), CtxB(E51A), CtxB(V52A), CtxB(P53A), CtxB (G54A), CtxB(S55A), CtxB(Q56A), CtxB(H57A) or CtxB(I58A) were incubated at 37°C for 96 hours. Then, with 0.4ml HBSS / 20mM Hepes / 0.1%NaN 3 / 10% mouse blood to wash and susp...

Embodiment 2

[0246] B subunit-deficient mutants at CD8 + T cell apoptosis maintains the ability to bind cell surface receptors

[0247] NIH male mice were sacrificed, and the mesenteric lymph node tissues were taken and placed in Hanks balanced salt solution (without calcium and magnesium ions in HBSS, and 20 mM Hepes was added). Lymphocytes were dispersed from the fibrous tissue by gently squeezing through a nylon mesh, washed three times with HBSS, and the lymphocytes were resuspended in 300ml pre-cooled and degassed MACS buffer (PBS, 5mM EDTA, 0.5% BSA, pH7.2) . Add 50ml of anti-CD4 and anti-B220MACS antibodies to the cells, and purify CD8 by magnetic MACS negative selection + T cells. CD8 + T cells were suspended in Eagle's modified medium (Gibco), which contained 20 mM Hepes, 4 mM L-glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin, 5×10 -5 M2-mercaptoethanol, the cell concentration was controlled at 2×10 6 cells / ml. Then add or not add 3.45μM (40μg / ml) of wild-type EtxB ...

Embodiment 3

[0251] The His-57 residue of EtxB is required for the induction of strong anti-EtxB responses

[0252] Eight groups of NIH female mice were immunized with 10ug EtxB or EtxB(H57S) 20μl nasally, and immunized three times within a week. 14 days after the third immunization, the rats were sacrificed and blood was collected by cardiac puncture. Anti-EtxB IgG levels were analyzed by GM-1-ELISA using 1 μg / ml EtxB-coated microtiter plates. Determined by end-point titration. (equivalent to a dilution with an absorbance of 0.1 above background)

[0253] result 3

[0254] Such as Figure 4 Indicated nasal immunization with EtxB produced high anti-EtxB IgG antibody titer sera (titer 5757+ / -785); whereas immunization with EtxB(H57S) induced a significantly (p=0.001) lower response (titer 1205 + / -222).

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PUM

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Abstract

A substance is described. The substance comprises any one or more of: an amino acid sequence comprising the sequence presented as SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to EtxB and / or CtxB; but wherein the substance does not exhibit GM-1 binding activity.

Description

field of invention [0001] The present invention relates to a substance. [0002] In particular, it relates to a substance exhibiting one or more properties useful in medicine. [0003] For example the substance can act as an immunomodulator and / or an adjuvant and / or an inhibitor of toxin-induced diarrhea. [0004] In particular, the present invention relates to the use of immunomodulatory substances for the modulation of immune responses, eg in relation to autoimmune diseases. [0005] Still more particularly, the invention relates to the use of this substance as an adjuvant together with a related or unrelated antigen. [0006] More specifically, the invention relates to the use of such substances for the suppression of toxin-induced diarrhea. [0007] The invention also relates to assays for the screening of reagents capable of interacting with the substance. Background of the invention [0008] Escherichia coli (E. coli) heat-labile enterotoxin (Etx) and its homologue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K39/00A61K39/39A61K45/00A61P1/12A61P37/02G01N33/50C07K14/245C07K14/28C12Q1/02G01N33/15G01N33/53
CPCC07K14/245C07K14/28A61K39/00A61P1/12A61P37/02
Inventor N·A·威廉斯T·R·希尔斯特
Owner UNIV OF BRISTOL
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