Identification of a vaccine candidate from an...

Details Identification of a vaccine candidate from an... Identification of a vaccine candidate from an...

2000-09-22

2002-06-25

Smith, Lynette R. F. (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S069100, C435S071100, C435S252100, C435S252300, C435S252800

Reexamination Certificate

active

06410703

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to the field of vaccines for bacterial infections. More particularly, this invention identifies a gene that is expressed in extraintestinal isolates of
Escherichia coli
(
E. coli
), and can be used as an immunogen in vaccine formulations.
BACKGROUND OF THE INVENTION
E. coli
is part of the normal intestinal flora where it does not cause infections. However, infections can occur if the bacteria gain entrance to other tissues and organs. This group of
E. coli
strains has been designated herein as extraintestinal pathogenic
Escherichia coli
(ExPEC). Extraintestinal infections (EIs) due to
E. coli
are common in all age groups and can involve nearly any organ or anatomical site. Typical EIs include urinary tract infection (UTI), meningitis (mainly in neonates and following neurosurgery), diverse intra-abdominal infection, pneumonia (particularly in hospitalized and institutionalized patients), intra-vascular device infection, osteomyelitis, and soft tissue infection, which usually occurs in the setting of tissue compromise. Bacteremia can accompany infection at any of these sites.
Extraintestinal isolates of
E. coli
are responsible for the majority of urinary tract infections (UTI). Eighty to ninety percent of ambulatory UTI, 73% in individuals over 50, and 25% of nosocomial UTI are due to extraintestinal strains of
E. coli
. Uncomplicated urethritis or cystitis occurs most commonly. However, more severe sequelae of UTI includes pyelonephritis, intrarenal and perinephric abscess, and bacterimia with or without septic shock. Thus, it is clear that despite effective antimicrobial therapy, UTI due to
E. coli
causes considerable morbidity and mortality.
Although
E. coli
is considered to be a community-acquired pathogen, it also is the most frequently isolated gram-negative bacillus in long-term care facilities and hospitals. Severe illness and death can occur in otherwise healthy hosts, but adverse outcomes are considerably more likely in the presence of comorbid disease and impaired host defenses.
The scope and magnitude of infection caused by extraintestinal strains of
E. coli
is as great as any invasive bacterial pathogen. In fact, perhaps because disease due to extraintestinal isolates of
E. coli
is so common, the virulence and morbidity of this organism is often overlooked. As a result, extraintestinal strains of
E. coli
continue to be low profile “silent killers” imposing a medical-economic strain on the health care system.
Currently, no effective vaccine is available against ExPEC. To date, efforts to identify specific vaccine candidates against ExPEC have concentrated on virulence traits such as capsule, LPS, and pili. These studies have demonstrated that antibodies directed against these structures confer protection against homologous strains in vivo. However, the inherent marked antigenic variability of these components may limit their utility as vaccine candidates. Thus, there is an ongoing need for identification of novel effective strategies for the prevention and treatment of ExPEC infections including UTI.
SUMMARY OF THE INVENTION
The present invention is directed to identifying a gene, iroN
ec
, from an extraintestinal isolate of
E. coli
. This gene was identified by transposon mutagenesis. TnphoA mutagenesis was performed on the wild type isolate of
E. coli
, CP9, and a library of 527 mutants with active TnphoA fusions was generated and saved. This library was subsequently screened for mutant derivatives with increased PhoA activity in the presence of urine. By this method, the iroN
ec
gene was identified which encodes a protein of 725 amino acids. This protein has an extracytoplasmic location. DNA homology data and data demonstrating that its transcription is iron repressed supports its function as a siderophore receptor. High stringency Southern hybridization identified DNA sequences homologous to iroN
ec
in 80-93% of ExPEC strains.
This protein or antigenic epitopes thereof can be used for inducing an immune response against extraintestinal
E. coli
infections. Data is presented to show that IroN
ec
is strongly immunogenic (without adjuvant) in mice. Further, antibodies developed against IroN
ec
were observed to be protective in a mouse intra-peritoneal challenge model. Mice immunized with IroN
ec
had diminished mortality after intra-peritoneal challenge with the
E. coli
strain CP9. Additionally, there was diminished growth of the challenged strain in the liver and spleen in animals immunized with IroN
ec
compared to non-immunized controls.
Thus, it is an object of the present invention to identify a gene, designated herein as iroN
ec
in the extraintestinal isolates of
E. coli
with increased expression in human urine.
It is another object of the present invention to provide a polynucleotide that encodes the protein, IroN
ec
.
It is another object of the present invention to provide polynucleotides that hybridize, preferably under high stringency conditions, with a polynucleotide encoding IroN
ec
.
It is anther object of the present invention to provide peptides that are encoded by iroN
ec
and other polynucleotides of the present invention.
It is another object of the invention to provide antigenic compositions comprising the peptides or antigenic fragments thereof for the treatment or prevention of extraintestinal infections caused by ExPEC.


REFERENCES:
Baumler et al, Journal of Bacteriology, Mar. 1998, pp. 1446-1453.*
Johnson et al, Molecular Epidemiological and Phylogenetic Associations of Two Novel Putative Virulence Genes, iha and iroN E. coli′ amongEscherichia coliIsolates from Patients with Urospsis.; Infect. Immun. May 2000, vol. 68, No. 5, pp. 3040-3047.
Russo et al, Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes kpsD, kpsT, and kpsE from an Extraintestinal Isolate ofEscherichia coli(CP9, 04/K54/H5). J. Bacteriology, Jan. 1998, vol. 180, No. 2, pp. 338-349.
Russo et al, Identification of Genes in an Extraintestinal Isolate ofEscherichia coliwith Increased Expression after Exposure to Human Urine, Infect. Immun. Oct. 1999, vol. 67, No. 10, pp. 5306-5314.

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