Review of the Food-borne Zoonoses Research ... - ARCHIVE: Defra

Project code:
VTEC O157 Research Projects
OZ0703
Project title: Plant antibody delivery of passive immunisation
against E. coli O157:H7: a novel means of control in
the animal
Start date (dd/mm/yy): 01/09/1999
End date (dd/mm/yy): 01/06/2003
£354,108
Total cost:
Affiliation: Veterinary Laboratories Agency
Sub-contractor(s): ADAS Consulting
Abstract of research
Biology Department, University of Leicester
Intimin, TIR and EspA proteins are expressed by Enterohaemorrhagic E. coli O157:H7.
EspA proteins are part of the type three secretion system needle complex that delivers
TIR to the host epithelial cell whilst surface arrayed intimin docks the bacterium to the
translocated TIR. This process leads to intimate attachment resulting in attaching and
effacing lesions which are essential for colonisation and persistence in ruminants.
Recombinant forms of these effector proteins from E. coli O157:H7 were produced and
used to elicit immune responses in rabbits and immune phage-display antibody libraries
were produced. Screening of these immune libraries by phage-antibody panning and
colony filter screening produced a panel of antibodies with specificity for EspA or intimin.
Antibodies recognising different C-terminal epitopes on intimin bound specifically to the
gamma intimin of O157:H7 and not to other classes of intimin whereas antibodies raised
against EspA recognised EspA analogues from other serotypes also. Anti-intimin
antibodies were also produced as fusion proteins coupled to the reporter molecule
alkaline phosphatase, allowing the one-step detection of γ-intimin. The isolated
recombinant monoclonal antibodies were functional in a range of assay formats including
ELISA, Western blot and dot blots demonstrating diagnostic potential.
We assessed whether these antibody constructs could be used to disrupt the intimate
association of E. coli O157:H7 with the host cell but neither anti-γ intimin or anti-EspA
antibodies did so although the anti-EspA antibodies significantly reduced the extent of E.
coli O157:H7-induced host cell actin rearrangement. In tests with traditionally made
antibodies, both monoclonal and polyclonal antibodies completely blocked adherence
and cytoskeletal changes within the host cell. Both polyclonal and monoclonal antibodies
could be used to label E. coli O157 EspA filaments and these immunoreagents did not
inhibit the formation of such filaments. This is the first report of monoclonal antibodies to
EspA capable of disrupting the TTSS function of E. coli O157:H7.
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