• Vaccine Ontology ID: VO_0004721
• Type: Recombinant vector vaccine
• Status: Research
• Host Species for Licensed Use: Baboon
• Preparation: Rabbit Enteropathogenic E. coli (REPEC), a member of the AEEC family. The C-terminal portion of intimin was delivered by the attenuated Vibrio cholerae vaccine strain CVD 103-HgR. To export intimin, a fusion was engineered with ClyA, a secreted protein from Salmonella enterica serovar Typhi (Keller et al., 2010).
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0000501
• Type: Subunit vaccine
• Antigen: The antigens for these vaccines are either verocytoxin 1 (VT1) or verocytoxin 2 (VT2). The prototype toxin VT1 is virtually identical to Shiga toxin produced by Shigella dysenteriae type 1. By using in vitro neutralization tests in Vero cells, VT1 has been shown to be serologically distinct from VT2 in that these toxins showed no cross-neutralization by heterologous antisera (Bielaszewska et al., 1997).
• Adjuvant:
  • VO ID: VO_0000142
  • Description: Freund’s incomplete adjuvant was used in the making of these vaccines (Bielaszewska et al., 1997).
• Preparation: VT1 was purified from JB28, an E. coli TB1 strain. VT2 was purified from E. coli R82pJES 120DH5a. The purity of these toxin preparations was established by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic analysis. The labeled toxins were characterized for specific activity (1.7 x 10^5 to 2.3 x 10^5 cpm/mg) and biological activity (3.8 x 10^5 and 1.9 x 10^3 50% cytotoxic doses [CD50s]/mg for VT1 and VT2, respectively). The Vero cell binding activities, which represent the percentage of the input activity bound to the 25-sq cm monolayers after 1h of incubation and three washes, were 48% for VT2 preparations and 75% for VT1 preparations. For the subunit immunizations, VT1 and VT2 were separated into the A and B subunit fractions by SDS-polyacrylamide gel electrophoresis. (Bielaszewska et al., 1997).

• 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:
  • VO ID: VO_0001263
  • Description: Quil‐A Saponin (Marcato et al., 2001)
• Adjuvant:
  • VO 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

• Vaccination Protocol: Three groups of six rabbits were primed on day 1 with one of three inocula: PBS, CVD 103-HgR (pSEC 91), or CVD 103-HgR (pInt248) and boosted on day 15 with the identical inoculum (Keller et al., 2010).
• Vaccine Immune Response Type: VO_0003057
• Challenge Protocol: Rabbits were challenged on day 29 with wild type REPEC strain E22 (6 × 10^7 CFU) (Keller et al., 2010).
• Efficacy: After immunization, antibodies specific to intimin from serum and bile samples were detected and moderate protection against challenge with a virulent REPEC strain was observed. Compared to animals immunized with vector alone, intimin-immunized rabbits exhibited reduced fecal bacterial shedding, milder diarrheal symptoms, lower weight loss, and reduced colonization of REPEC in the cecum. V. cholerae CVD 103-HgR shows promise as a vector to deliver antigens and confer protection against AEEC pathogens (Keller et al., 2010).

Rabbit Response

• Host Strain: New Zealand White
• Vaccination Protocol: Rabbits weighing approximately 2 kg were immunized subcutaneously with doses of 60 mg of toxoid mixed with equal volumes adjuvant in four sequential weekly intervals (Bielaszewska et al., 1997).
• Immune Response: Rabbits immunized with the VT A and B subunits developed NAb to the homologous toxin but not to the heterologous toxin. Rabbits immunized with the A subunits were reactive by ELISA with the homologous toxin but were less, if at all, reactive to the heterologous toxin. Immunization with the B subunits led to the appearance of ELISA antibodyto the homologous toxin in the case of VT1 but did not lead to a detectable level of antibody in the case of VT2. Immunization with VT1 and VT2 toxoids resulted in strong ELISA antibody responses to both homologous and heterologous toxins (Bielaszewska et al., 1997).
• Challenge Protocol: 125 I-labeled VT1 and VT2 were administered to rabbits which had been immunized, in groups of three, with either the A or B subunit of VT1, the A or B subunit of VT2, or with VT1 or VT2 holotoxoids and to nonimmune controls. Following administration of approximately 4 x 10^6 cpm of labeled VT through the ear vein, a 1- to 2-ml blood specimen was collected from the ear artery (Bielaszewska et al., 1997).
• Efficacy: Animals immunized by either the VT1 A subunit or the VT2 A subunit were protected from target tissue uptake of both the homologous and heterologous125 I-labeled holotoxins. In contrast, in animals immunized with the toxin B subunits, protection was extended only against challenge by the homologous toxin. Findings indicate that the in vivo cross-neutralization is a predominant function of antibodies directed to the VT A subunits. This suggests that the VT1 A or VT2 A subunit may be a suitable immunogen for immunizing humans against systemic VT-mediated disease (Bielaszewska et al., 1997).

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).