Enterohemorrhagic escherichia coli vaccine

a technology of escherichia coli and vaccine, which is applied in the direction of antibacterial agents, immunological disorders, antibody medical ingredients, etc., can solve the problems of difficult and expensive production and purification of recombinant proteins in sufficient quantities for use as antigens, and prevent ehec infection. , the effect of reducing the colonization of ehec in the mammal is easy and relatively inexpensive to prepar

Inactive Publication Date: 2009-03-12
FINLAY B BRETT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present invention satisfies the above need by providing such compositions and methods. In particular, the methods of the present invention make use of a composition comprising a cell culture supernatant (hereinafter “CCS”) derived from an EHEC culture to elicit an immune response against one or more EHEC secreted antigens, thereby treating and / or preventing EHEC infection and / or reducing EHEC colonization of the mammal. The compositions can be delivered with or without a coadministered adjuvant. In certain embodiments, EspA and Tir comprise at least 20% of the cell culture supernatant protein. The EHEC culture supernatant may be derived from any EHEC serotype, but is preferably obtained from a culture of EHEC O157:H7 and / or EHEC 0157:NM (non-motile). The cell culture supernatant of the present invention is easy and relatively inexpensive to prepare and is effective at dose regimens that have minimal toxicity.

Problems solved by technology

Because of the bulk processing of slaughtered cattle and the low number of EHEC O157:H7 (10-100) necessary to infect a human, EHEC O157:H7 colonization of healthy cattle remains a serious health problem.
However, production and purification of recombinant proteins in amounts sufficient for use as antigens is both difficult and expensive.

Method used

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  • Enterohemorrhagic escherichia coli vaccine
  • Enterohemorrhagic escherichia coli vaccine
  • Enterohemorrhagic escherichia coli vaccine

Examples

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example 1

Preparation of Cell Culture Supernatant (CCS)

[0103]Wild type EHEC O157:H7 were grown under conditions to maximize the synthesis of CCS proteins (Li et al., Infect. Immun. (2000) 68:5090). Briefly, an overnight standing culture of EHEC O157:H7 was grown in Luria-Bertani (LB) medium overnight at 37° C. (±5% CO2). The culture was diluted 1:10 in M-9 minimal medium supplemented with 0.1% Casamino Acids, 0.4% glucose, 8 mM MgSO4 and 44 mM NaHCO3. Cultures were grown standing at 37° C. in 5% CO2 to an optical density at 600 nm of 0.7 to 0.8 (6-8 h). Bacteria were removed by centrifugation at 8000 rpm for 20 min at 4° C. The supernatant was concentrated 100 fold by ultrafiltration and total protein was determined by the bicinchoninic acid protein assay method.

[0104]FIG. 1 shows molecular weight markers (lane 1) and a typical CCS protein profile obtained by electrophoresis of CCS in a SDS-10% polyacrylamide gel (SDS-PAGE) followed by Coomassie blue staining (lane 2). The positions of EspA (...

example 2

Preparation of Recombinant Proteins

[0105]The genes coding for EspA, EspB, Intimin and Tir were isolated (Li et al., Infect. Immun. (2000) 68:5090). A clinical isolate of EHEC O157:H7 was used as the source of DNA. EspA, EspB, Tir, and the region of eae encoding the 280 carboxyl-terminal amino acids of Intimin were amplified from chromosomal DNA using PCR to introduce unique restriction sites, followed by cloning into appropriate plasmids. The resulting plasmids were cleaved and ligated to create histidine-tagged fusions. Plasmids were electrocuted into an expression strain of E. coli and the E. coli were propagated (Ngeleka et al., Infect. Immun. (1996) 64:3118). Gene expression was driven using the Tac promoter following IPTG (isopropyl-β-D-thiogalactopyranoside) induction. Bacteria were pelleted, resuspended in Tris-buffered saline and lysed by sonication. The lysate was centrifuged to remove insoluble material and the histidine-tagged proteins were purified by passage through a s...

example 3

Vaccine Formulation and Delivery

[0107]Vaccines were formulated by mixing CCS or rEspA+rTir in 2 ml of a carrier containing from 30 to 40% of an adjuvant. Vaccines were delivered subcutaneously. Animals were immunized on day 1 and again at a 3-4 week intervals (boost). Serum samples were obtained prior to the first immunization, at the time of each boost and at the end of the experiment.

[0108]The serological response to immunization was determined using an enzyme-linked immunosorbent assay (ELISA). One hundred p1 of rEspA (0.16 μg / well), rTir (0.1 μg / well), rEspB (0.24 μg / well) and rIntimin (0.187 μg / well) were used to coat the wells in microtiter plates and the plates were incubated overnight at 4° C. The wells were washed 3×, blocked with 0.5% nonfat dried milk in phosphate-buffered saline. Serial dilutions of sera were added to each well and incubated for 2 h at 37° C. The wells were washed and blocked and 100 μl of peroxidaseconjugated rabbit anti-bovine immunoglobulin G antibodi...

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Abstract

Compositions and methods for stimulating an immune response against a secreted enterohemorragic Escherichia coli (EHEC) antigen are disclosed. The compositions comprise EHEC cell culture supernatants.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. application Ser. No. 10 / 039,760 filed Jan. 3, 2002 which claims benefit U.S. provisional application No. 60 / 259,818 filed Jan. 4, 2001, the contents of which are incorporated herein by reference in their entirety.FIELD[0002]The present invention relates to compositions and methods for eliciting an immune response in mammals against enterohemorragic Escherichia coli. In particular, the invention relates to the use of cell culture supernatants for treating and preventing enterohemorragic E. coli colonization of mammals.BACKGROUND[0003]Enterohemorragic Escherichia coli (EHEC), also called Shiga toxin E. coli (STEC) and vertotoxigenic E. coli (VTEC) are pathogenic bacteria that cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, kidney failure and death in humans. While many Shiga-like toxinproducing EHEC strains are capable of causing disease in humans, those of serotype O157:H7 cause the ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/108A61P31/06A61P37/04A61K35/74A61K39/00A61K39/02A61K39/39A61P31/00A61P31/04C07K14/245
CPCA61K39/0258C07K14/245A61K2039/55566A61P31/00A61P31/04A61P31/06A61P37/04Y02A50/30
Inventor FINLAY, B. BRETTPOTTER, ANDREW A.
Owner FINLAY B BRETT
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