• Vaccine Name: Shiga Toxin 2 B Subunit Vaccine
• Target Pathogen: Escherichia coli
• Target Disease: Hemorrhagic colitis
• Vaccine Ontology ID: VO_0004145
• Type: Subunit vaccine
• Antigen: The antigen for this vaccine is Shiga Toxin 2 B subunit (Marcato et al., 2001).
• CS1 gene engineering:
  • Type: Recombinant protein preparation
  • Description: The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned. This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit. The construct was expressed in the TOPP3 E. coli strain. The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue. The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitt's lymphoma cells (Moravec et al., 2007).
  • Detailed Gene Information: Click Here.
• StxB2 gene engineering:
  • Type: Recombinant protein preparation
  • Description: Shiga toxin 2 subunit B was used in the formation of a construct (Marcato et al., 2001).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001263
  • Description: Quil‐A Saponin (Marcato et al., 2001)
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0001267
  • Description: Quil‐A Saponin (Marcato et al., 2001).
• Preparation: The vaccine contained cloned low endotoxin Stx2 B subunit preparation homogenized in an equal volume of adjuvant. The sham vaccine (used for a control) contained a 1:1 homogenate of Quil‐A and pyrogen‐free 0.9% NaCl irrigation solution (Marcato et al., 2001).

Rabbit Response

• Host Strain: New Zealand White
• Vaccination Protocol: Eight female rabbits, weighing 2 kg each, in 2 groups of 4 were immunized. The rabbits in 1 group were injected in the subscapular region with the cloned low endotoxin Stx2 B subunit preparation homogenized inadjuvant. The rabbits in the second group were sham immunized. The rabbits were injected 3 times, on a monthly schedule, the first time with 150 μg of antigen and each subsequent time with 100 μg of antigen. The 8 rabbits then were subgrouped for a second round of immunization. Four rabbits, 2 from the low endotoxin Stx2 B subunit–immunized group and 2 from the sham‐immunized control group, were given 2 additional 100‐μg injections of a cloned Stx2 B subunit preparation, in which the endotoxin concentration had only been reduced to 2000 endotoxin U/mL (high endotoxin Stx2 B subunit preparation). The remaining 4 rabbits, 2 previously immunized with the low endotoxin Stx2 B subunit preparation and 2 from the sham‐immunized control group, received 2 additional 100‐μg injections of the low endotoxin Stx2 B subunit preparation (Marcato et al., 2001).
  
• Immune Response: As anticipated, none of the preimmunization serum samples from the 8 rabbits nor any serum samples from the 4 sham‐immunized control animals contained evidence of Stx2‐reactive antibodies by ELISA, immunoblot, or Vero cytotoxicity neutralizing assays. Rabbits which were primed with 3 injections of the low endotoxin Stx2 B subunit preparation and then were injected twice with high endotoxin (2000 U/mL) Stx2 B subunit, developed a specific antibody response to the immunogen after the first of the 2 additional injections. In addition, after receiving 2 injections of the low endotoxin Stx2 B preparation, 1 of the first round, sham‐immunized control rabbits, K103, produced a specific antibody response to the Stx2 B subunit. A rabbit of the first round sham‐immunized animals produced a weak antibody response to the Stx2 B subunit after 2 injections with the high endotoxin Stx2 B subunit preparation (Marcato et al., 2001).
  
• Challenge Protocol: Rabbits were challenged with 5 μg of Stx2 holotoxin per kilogram of body weight. The purified Stx2 holotoxin preparations were homogenized with an equal volume of Quil‐A adjuvant and were injected into the subscapular region of each rabbit. The rabbits then were monitored every 4 h for 1 week and thereafter once daily for 3 weeks. The rabbits were killed as soon as toxic effects (anterior ataxia or paralysis) were observed. At the end of the 1‐month study, asymptomatic surviving rabbits were also killed for postmortem examination (Moravec et al., 2007).
  
• Efficacy: All the Stx2 holotoxin‐challenged rabbits that failed to display Western immunoblot evidence of Stx2 B subunit–specific antibodies developed Stx2‐related symptoms between postchallenge days 2 and 4 and were killed. One rabbit, which developed a weak Western immunoblot response to the Stx2 B subunit, also developed Stx2‐related symptoms on postchallenge day 2 and was killed. In contrast, three other rabbits, which produced Western immunoblot‐positive Stx2 B subunit antibodies, remained asymptomatic throughout the 1‐month study. At postmortem examination, all the unprotected rabbits displayed various degrees of Stx‐mediated organ and tissue damage. In contrast, all tissues and organs in each of the three protected rabbits appeared to be normal (Moravec et al., 2007).