Antibodies to intimin-like proteins of E. coli

Details Antibodies to intimin-like proteins of E. coli Antibodies to intimin-like proteins of E. coli

1998-04-02

2003-09-23

Graser, Jennifer E. (Department: 1645)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

Binds antigen or epitope whose amino acid sequence is...

C424S130100, C424S133100, C424S134100, C424S139100, C424S141100, C424S150100, C424S163100, C424S164100, C424S178100, C424S184100, C435S006120, C435S007370, C435S091100, C435S320100, C530S350000, C530S825000, C536S023700, C536S024320

Reexamination Certificate

active

06623737

ABSTRACT:

The present invention relates to antibodies, particularly monoclonal antibodies which recognise particular epitopes of the intimin protein of enteropathogenic
E. coli
and enterohemorrhagic
E. coli
, their use in the detection of such enterophathogenic
E. coli
, and kits containing such antibody for such use.
Enteropathogenic
Escherichia coli
(EPEC) were the first
E. coli
linked with diarrhoea in humans. They are a sub-group of pathogenic
E. coli
that are a major cause of infant diarrhoea. Enterohemorrhagic
E. coli
(EHEC) are another such sub-group. EPEC normally fall into several distinct serotype groups based on the type of lipopolysaccharide which is expressed at their cell surface. The most common serogroups that are associated with EPEC are O26, O55, O111, O114, O126, O127, O128, O142 and O157. Although EPEC and EHEC infections are still a serious problem world-wide, no specific treatment or vaccine is as yet available for treating these diseases and furthermore, no relatively simply and/or accurate tests are as yet available for diagnosis.
One major problem in diagnosing EPEC infections is that, apart from the link with several O serotypes, EPEC are difficult to distinguish from commensal, non-pathogenic
E. coli
which are usually resident in the intestines of healthy individuals. Ultra-structural studies of intestinal biopsy specimens from children with EPEC-induced diarrhoea have shown that EPEC attach to the intestinal epithelium in a characteristic fashion, which is central to the pathogenesis of the disease.
Wherever bacteria bind to the brush border of the intestine, microvilli are destroyed, or “effaced”. The apical cell membrane of the bare enterocytes form cup-like pedestals upon which the bacteria intimately adhere. EHEC,
Hafnia alvei
and
Citrobacter freundii
biotype 4280 have also been shown to induce similar lesions, termed effacement and attachment lesions, to the intestinal brush border of infected hosts.
Pedestal formation is associated with accumulation of polymerised actin below the surface of the eukaryotic cell directly engulfing the bacteria. This polymerised actin can be detected by staining with a specific dye called phalloidin. A diagnostic test, based on the use of this stain with cultured mammalian cells incubated with pathogenic bacteria has been developed. However, this test is not EPEC-specific, and is expensive and time consuming, which renders its routine use difficult.
The EPEC or EHEC gene product that mediates intimate attachment to epithelial cells is a protein called intimin (Int
EPEC
and Int
EHEC
respectively), which is a 94 kdal outer membrane protein encoded by the chromosomal eaeA gene. The amino-terminis of Int
EPEC
and Int
EHEC
have a high degree of homology with the eae gene products of
Citrobacter freundii
biotype 4280 (Int
CF
) and also with the amino terminus of invasin (Inv), the product of inv gene of
Yersinia pseudotuberculosis
(Inv
YP
) and
Yersinia enterocolitica
(Inv
YE
).
The DNA and protein sequences of various intimins were disclosed by Kaper (
Proc. Natl. Acad. Sci. USA,
87: 7839-7843 (1990) and
Mol. Microbiol.,
6: 411-417 (1992)). However, the proteins themselves were not purified. Frankel et al. (
Infec. Immun.,
62: 1835-1842 (1994)) have isolated the intimin proteins from various sources and have generated Maltose-binding protein (MBP), fusion proteins carrying particular domains of the intimin proteins.
The cell binding activity of Inv
YP
resides predominantly in the 192 carboxy-terminus amino acids of the protein. Previously, Frankel et al. (supra) used fusions to maltose binding protein (MBP) to show that, like Inv, an Int domain which mediates receptor binding resides in the 280 (or 275 in the case of EHEC) amino acids of the carboxy-terminus of the protein (Int
EPEC280
, Int
EHEC275
, Int
HA280
, Int
CF280
).
However, unlike Inv, which by itself can convert an
E. coli
K12 strain to an organism capable of attaching and then invading mammalian cells, Int requires the cooperation of other EPEC proteins to induce the effacement and attachment lesion. Notwithstanding that, however, Int
EPEC
is a virulent factor that induces seroconversion in human volunteers (Donnenberg et al,
J. Clin. Invest.,
92: 1412-1417 (1993)). The carboxy-terminal domain of this protein is able to bind to target cells and thus it is likely to be surface exposed, thus making it accessible, on whole bacteria, to immune reagents.
We have now generated further fusion proteins in addition to MBP-Int
EPEC280
(amino acids 660-939). These fusion proteins include different amino acid sequences derived from the 280 binding domain. MBP-Int
EPEC150
(amino acids 790-939) was found to be the smallest fusion protein that had the ability to mediate cell binding. In addition the cysteine residue at position 937 of Int seems to be required for binding activity, as substitution with serine results in a loss in biological activity. Using the MBP expression system we have obtained high yields of MBP
IntEPEC280
, MBP-Int
EHEC275
, MBP-Int
CF280
and MBP-Inv
YP280
fusion proteins. These proteins can be used to immunise mice in order to generate specific monoclonal antibodies. We have now identified monoclonal antibodies which specifically recognise the antigen MBP-Int
EPEC280
and which do not recognise MBP, MBP-Int
EHEC275
, MBP-Int
CF280
or MBP-Inv
YP280
. These antibodies are therefore specific for the 280 amino acid domain of Int
EPEC
and will therefore be useful in both the detection and/or treatment of EPEC infection.
Similarly, MBP-Int
EHEC275
can be used to generate monclonal antibodies which recognise MBP-Int
EHEC275
but not MBP-Int
EPEC280
.
Thus, in a first aspect, the present invention provides an antibody which recognises a region within either the carboxy-terminus 280 amino acid domain of the enteropathogenic
E. coli
intimin protein (Int
EPEC280
) or the enterohemorrhagic
E. coli
intimin protein (Int
EPEC280
).
In the context of the present invention, “antibody” refers to conventional polyclonal and monoclonal antibodies and includes antigen binding portions or fragments thereof, e.g. Fab and/or fragments of Fab.
In a preferred embodiment, the antibody is a monoclonal antibody. In particularly preferred embodiments, the antibody is a monoclonal antibody which recognises a region within the following carboxy-terminus 280 amino acid sequence:
[SEQ. ID. NO:1]
ITEIKADKTT AVANGQDAIT YTVKVMKGDK PVSNQEVTFT

TTLGKLSNST EKTDTNGYAK VTLTSTTPGK SLVSARVSDV

AVDVKAPEVE FFTTLTIDDG NIEIVGTGVK GKLPTVWLQY

GQVNLKASGG NGKYTWRSAN PAIASVDASS GQVTLKEKGT

TTISVISSDN QTATYTIATP NSLIVPNMSK RVTYNDAVNT

CKNFGGKLPS SQNELENVFK AWGAANKYEY YKSSQTIISW

VQQTAQDAKS GVASTYDLVK QNPLNNIKAS ESNAYATCVK
or
a region within the following carboxy-terminus 275 amino acid sequence:
[SEQ. ID. NO:2]
ITEIKADKTT AVANGKDAIK YTVKVMKNGQ PVNNQSVTFS

TNFGMFNGKS QTQATTGNDG RATITLTSSS AGKATVSATV

SDGAEVKATE VTFFDELKID NKVKIIGNNV RGELPNIWLQ

YGQFKLKASG GDGTYSWYSE NTSIATVDAS GKVTLNGKGS

VVIKATSGDK QTVSYTIKAP SYMIKVDKQA YYADAMSICK

NLLPSTQTVL SKIYDSWGAA NKYSHYSSMN SITAWIKQTS

SEQRSGVSST YNLITQNPLP GVNVNTPNVY AVCVE.
Preferably, a monoclonal antibody recognizing Int
EPEC280
does not recognise the carboxy-terminus 275 amino acid domain of the intimin protein from enterohemorragic
E. coli
(Int
EHEC
275) or the carboxy-terminus 280 amino a id domain of
Citrobacter freundii
biotype 4280 Int
CF
280 or the invasin protein from
Yersinia pseudotuberculosis (Inv
YP
280).
More preferably, the monoclonal antibody recognising Int
EPEC280
recognises an epitope in the 660-790 amino acid region of the enterophathogenic
E. coli
intimin protein.
Hybridomas producing monoclonal antibodies of the invention are also included within the scope of the invention.
The properties of the monoclonal antibodies of the invention render them useful for detecting enterophathogenic
E. coli
and enterohemorrhagic
E. coli
. Thus, the invention also provides the use of such monoclonal antibodies in detecting enteropathogenic and enterohemorrhagic
E. coli

Affiliated with

Also associated with

Rating

Antibodies to intimin-like proteins of E. coli does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Antibodies to intimin-like proteins of E. coli, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibodies to intimin-like proteins of E. coli will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3006608