• Vaccine Name: E. coli vaccine using intimin polypeptide
• Target Pathogen: Escherichia coli
• Target Disease: Hemorrhagic colitis
• Tradename: None
• Vaccine Ontology ID: VO_0000478
• Type: Subunit vaccine
• Antigen: E. coli Intimin polypeptide(van et al., 2007).
• eae gene engineering:
  • Type: Preparation of recombinant protein
  • Description: The portion of the eae gene that encodes the carboxyl-terminal 280 amino acids of intimin was amplified by polymerase chain reaction from EHEC O26:H- strain 193 (Int280-β) and EHEC O157:H7 strain EDL933 (Int280-γ) using a conserved forward primer (Int-LIC-for: 5′-GAC GAC GAC AAG ATT ACT GAG ATT AAG GCT G-3′) and subtype-specific reverse primers (O26Int-LIC-rev: 5′-GAG GAG AAG CCC GGT TTA TTT TAC ACA AAC AG-3′ and O157Int-LIC-rev: 5′-GAG GAG AAG CCC GGT TTA TTC TAC ACA AAC CG-3′). The products were cloned in pET30-Ek/Lic (Novagen®) by a ligation-independent method as amino-terminal 6×His-S-tag fusions. Proteins were expressed in E. coli K-12 strain BL21 (DE3) Star cells which lack RNaseE to stabilise mRNA. The Overnight Express™ Autoinduction System I (Novagen®) was used to induce Int280-γ and Int280-β expression. Cell extracts were prepared using BugBuster® (Novagen®) and the supernatant fraction mixed with His-Mag™ Agarose Beads (Novagen®) for affinity purification of the Int280 proteins as described by the manufacturer
    (van et al., 2007).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0000127
  • Description: Aluminium hydroxide oil-based adjuvant (Alu-Oil; Intervet International BV, Boxmeer, The Netherlands) (van et al., 2007).
• Preparation: Proteins were expressed in E. coli K-12 strain BL21 (DE3) Star cells which lack RNaseE to stabilise mRNA. The Overnight Express™ Autoinduction System I (Novagen®) was used to induce Int280-γ and Int280-β expression. Cell extracts were prepared using BugBuster® (Novagen®) and the supernatant fraction mixed with His-Mag™ Agarose Beads (Novagen®) for affinity purification of the Int280 proteins as described by the manufacturer (van et al., 2007).
• Virulence: Not noted.
• Description: Enterohaemorrhagic Escherichia coli (EHEC) are zoonotic enteric pathogens of worldwide importance. EHEC strains produce intimin, an outer membrane adhesin encoded by the eae gene located in a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin mediates intimate bacterial attachment to enterocytes by binding to Tir, a bacterial protein which is translocated into host cells by a LEE-encoded type III secretion system. Intimin can also bind in vitro to β1-integrins and cell-surface localised nucleolin and these proteins can be detected proximal to adherent EHEC O157:H7 in vivo. Intimin is a key colonisation factor for EHEC O157:H7 in neonatal calves, young and weaned calves, and adult cattle and sheep. In addition, intimin influences the carriage and virulence of EHEC O157:H7 in streptomycin pre-treated mice, infant rabbits, and gnotobiotic and neonatal piglets (van et al., 2007).

Cattle Response

• Host Strain: Calves
• Vaccination Protocol: In Trial 1, on day 0 and day 28 calves were vaccinated i.m. with Int280-γ. In Trial 2, calves were vaccinated with Int280-β on days 0 and 28 (van et al., 2007).
• Persistence: Not noted.
• Side Effects: Not noted.
• Challenge Protocol: In Trial 1, on day 42 oral challenge was administered with 2.9 ± 0.78 × 10^10 colony forming units (CFU) of EHEC O157:H7 strain EDL933 nalR. In Trial 2, on day 42 oral challenge was performed using 2.8 ± 0.67 × 10^10 CFU EHEC O26:H- strain STM2H2 (van et al., 2007).
• Efficacy: Subunit vaccines based on intimin polypeptides induced serum IgG and variable salivary IgA responses following parenteral immunisation of cattle. However, such responses did not confer significant resistance to intestinal colonisation by EHEC strains expressing the homologous antigens, even after boosting of such animals by the mucosal route (van et al., 2007).
• Description: While it has been shown that i.n. immunisation of cattle with a carboxyl-terminal 64 kDa intimin polypeptide adjuvated with a low-toxicity derivative of E. coli heat-labile toxin induces antigen-specific serum IgG1 and salivary IgA, the protective efficacy of intimin-based subunit vaccines in cattle has yet to be tested. The present study assessed the protective efficacy of subunit vaccines comprising of intimin polypeptides against intestinal colonisation of cattle by EHEC strains of serotypes O157:H7 and O26:H- following parenteral and mucosal immunisation (van et al., 2007).

Mouse Response

• Host Strain: Female BALB/c mice of 16 to 18 g (Charles River Laboratories, Inc.).
• Vaccination Protocol: NT-1 cells or transgenic NT-1 cell clones that expressed Int261 were grown in 40-ml suspension cultures to confluence. Five grams of NT-1 cell material was divided into aliquots, and 0.5 g of sucrose was added to each sample. A 7.5-µg dose of purified cholera toxin (CT) (Sigma) was also added to appropriate samples to serve as an oral adjuvant. Mice were made to fast overnight before they were allowed to eat the plant material ad libitum. Mice immunized i.p. with purified His-tagged Int261 plus TiterMax served as the positive control (Judge et al., 2004).
• Persistence: Not noted.
• Side Effects: Not noted.
• Challenge Protocol: Mice were made to fast overnight and fed a total inoculum of 10^8 to 10^9 CFU of E. coli O157:H7 strain 86-24 Strr or 86-24 Strr eae10 in each of two doses administered 4 h apart (Judge et al., 2004).
• Efficacy: Parenteral priming of mice with intimin purified from transgenic plant cells can assist in the development of an intimin-specific fecal immune response when these mice are subsequently boosted with oral feeding of the same intimin-expressing transgenic plant material. Mice that were parenterally primed and then given an oral booster showed a statistically significant decrease in the duration of colonization by wild-type E. coli O157:H7 upon challenge. Mice immunized entirely by oral feeding did exhibit a reduction in the duration of colonization versus unimmunized mice, but the reduction was not statistically significant. These results suggest that a combination of vaccination strategies with a vaccine antigen produced in and delivered by transgenic plants can function in inducing beneficial, specific immune responses (Judge et al., 2004).
• Description: An oral inoculation system was sought to facilitate induction of mucosal antibodies and for ease of administration. A transgenic plant cell system for intimin expression was used, with the ultimate goal of moving the antigen into whole-plant expression and delivery systems. Transgenic plants offer the flexibility to function as low-cost, efficient, and practical vaccine antigen oral delivery systems to stimulate mucosal immunity or to boost and shift initial immunity to a mucosal antibody response. Transgenic plants have already been used as successful vaccine antigen production and delivery systems. Carboxy-terminal third of intimin-expressing plant cells were created. Capacity of this transgenic material to induce adherence-blocking antibodies and to reduce levels and/or time of E. coli O157:H7 fecal shedding in a mouse model of intimin-dependent colonization were then evaluated (Judge et al., 2004).