Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes β or γ via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int280-β) or EHEC O157:H7 (Int280-γ) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1′) from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1′) or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int280-γ and Efa-1′ or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

Efa-1/LifA mediates intestinal colonization of calves by enterohaemorrhagic Escherichia coli O26 : H– in a manner independent of glycosyltransferase and cysteine protease motifs or effects on type III secretion

“…Efa-1/LifA, but not internal glycosyltransferase or cysteine protease motifs, is required for intestinal colonization of calves by EHEC O26 : HWe created a panel of efa-1/lifA mutants in an EHEC O26 : H-strain proven to efficiently colonize the intestines of calves (van Diemen et al, 2005(van Diemen et al, , 2007. This strain (193 nal R ) was first rendered incapable of producing Shiga toxin, as Stx1 inhibits proliferation of bovine lymphocytes (Menge et al, 1999) and may mask lymphostatin activity in EHEC (Stevens et al, 2002b;Abu-Median et al, 2006).…”

“…This strain (193 nal R ) was first rendered incapable of producing Shiga toxin, as Stx1 inhibits proliferation of bovine lymphocytes (Menge et al, 1999) and may mask lymphostatin activity in EHEC (Stevens et al, 2002b;Abu-Median et al, 2006). Stx has been reported to influence intestinal colonization of mice by EHEC O157 : H7 (Robinson et al, 2006); however, the course of faecal excretion of 193 nal R Dstx1 following oral inoculation of calves did not reveal obvious attenuation relative to datasets accrued for the parent strain (van Diemen et al, 2005(van Diemen et al, , 2007. A null efa-1/lifA : : kan R mutation from EHEC O111 : H-strain EBK4 (Nicholls et al, 2000) was transferred to strain 193 nal R Dstx1 by lRed recombinase-mediated integration of a PCR amplicon.…”

“…Although we have established an important role for Efa-1/ LifA in colonization of the reservoir host by EHEC O26 : H-and other non-O157 EHEC (Stevens et al, 2002b), we were previously unable to protect calves against challenge with the 193 nal R strain by intramuscular immunization with the N-terminal and central thirds of Efa-1 expressed in E. coli (van Diemen et al, 2007). Antigen-specific humoral responses were detected and it is conceivable that they may mediate protection against natural exposure but not the high experimental challenge doses used (van Diemen et al, 2007).…”

“…Antigen-specific humoral responses were detected and it is conceivable that they may mediate protection against natural exposure but not the high experimental challenge doses used (van Diemen et al, 2007). Given the important role of Efa-1/LifA in calf colonization, the high degree of conservation of Efa-1/LifA in non-O157 strains and the emergence of such serogroups in cattle and humans (Bettelheim 2007), further studies using multivalent vaccines or other vaccination formulae or regimens may be warranted.…”

“…Oligonucleotides used to create and validate bacterial mutants are listed in Table 2. EHEC O26 : H-strain 193 nal R was selected for this study as it efficiently colonizes the intestines of calves (van Diemen et al, 2005(van Diemen et al, , 2007. A deletion spanning the stx1AB genes was first created using the positive-selection suicide replicon pCVDDstx1, as described previously (Stevens et al, 2002a).…”