• Vaccine Name: S. flexneri 2a strain CVD 1205
• Target Pathogen: Shigella
• Target Disease: Shigellosis
• Vaccine Ontology ID: VO_0004143
• Type: Recombinant vector vaccine
• Antigen: The antigen for this vaccine is S. flexneri 2a strain CVD 1205, which carries deletion mutations in the guaB-A operon and in the virG gene (also called icsA) (Noriega et al., 1996).
  
• GuaB gene engineering:
  • Type: Recombinant protein preparation
  • Description: CVD 1205 is made through deletion of virG from CVD 1204. Producing strain CVD 1205 took many steps and began with construction of the guaB-A deletion cassette pFM726A. In the construction of the guaB-A deletion cassette, DNA segments that included the 59 terminus of guaB and the 39 terminus of guaA were amplified (from S. flexneri 2a strain 2457T genomic DNA) and fused by PCR, originating the guaB-A allele. With the internal primers (primers 2 and 3) was introduced an in-frame stop sign upstream of two unique restriction sites that were added for the future introduction of foreign genes into the chromosomal DguaB-A allele. The external primers (primers 1 and 4) were designed to introduce unique restriction sites that were used to clone the guaB-A allele into the temperature-sensitive, pSC101-based suicide plasmid pFM307A, originating pFM726A. The same external primers were used to amplify the wild-type guaB-A operon (from strain 2457T), which was subsequently cloned in pGEM-T, yielding pGEM::gua, and in pFM307A, yielding pFM215A. Suicide cassette-driven deletion mutations and repair of the same. Deletion cassette pFM726A was used to introduce the deletion mutation into wild-type S.flexneri 2a strain 2457T by homologous recombination as described in previously published method, yielding strain CVD 1204. Plasmid FM215A was used to repair the deletion mutation by homologous recombination of the chromosomal guaB-A allele in strain CVD 1204 for the wild-type operon contained in the suicide plasmid.
    A second deletion mutation on the virulence gene virG was performed with a previously described suicide deletion cassette (pDvirG) and methods (24), yielding strain CVD 1205. The deletion mutation corresponds to 900 bases representing amino acids 341 to 640 of the 120-kDa VirG protein. The specific engineered site for this deletion in the protein represents a highly hydrophobic, poorly antigenic portion of the molecule genic index (Noriega et al., 1996).
  • Detailed Gene Information: Click Here.
• Preparation: Overnight cultures of guaB-A virG S. flexneri 2a strain CVD 1205 and HS strains were harvested and resuspended PBS to an optical density at 600 nm of 0.5 (equivalent to 5 3 10^8 CFU/ml) and concentrated by centrifugation to the desired concentration (Noriega et al., 1996).
• Virulence: Upon Sereny test challenge with wild-type S. flexneri 2a, CVD 1205-vaccinated animals were significantly protected against keratoconjunctivitis (Noriega et al., 1996).

Guinea pig Response

• Host Strain: Hartley
• Vaccination Protocol: Randomized, nonpreconditioned Hartley guinea pigs were given intranasally 100 ml of bacterial suspension containing 10^9 CFU as described previously. A booster dose was administered 14 days later in the identical manner (Noriega et al., 1996).
  
• Persistence: At 72 h postinoculation, the (blinded) observer grading the inflammatory response in the guinea pigs could not distinguish the inoculated eye from the noninoculated one in any of the animals that received the attenuated mutant CVD 1205, while all animals that received wild-type strain 2457T had full-blown purulent keratoconjunctivitis (Noriega et al., 1996).
  
• Immune Response: The serum antibody response was more delayed, since no serum IgG or IgA anti-Shigella LPS was detected after the first immunization. However, by day 2 animals immunized with CVD 1205 had specific anti-S. flexneri 2a LPS IgA (i.e., 78-fold rise in GMT) and IgG (i.e., 60-fold rise in GMT) titers that were highly significant with respect to those obtained at day 0 in the same guinea pigs or at days 0 and 28 in the strain HS controls (Noriega et al., 1996).
  
• Side Effects: No side effects were noted.
• Challenge Protocol: Protection of guinea pigs against wild-type challenge. On day 28 after the first immunization, the 16 guinea pigs that had received CVD 1205 or placebo were challenged with 3 x 10^7 CFU of wild-type S. flexneri 2a strain 2457T in 10 ml of PBS (Noriega et al., 1996).
• Efficacy: Full-blown purulent keratoconjunctivitis developed in five of seven control animals vaccinated with placebo (71% attack rate) versus none of the eight guinea pigs immunized with two spaced intranasal doses of CVD 1205 (Noriega et al., 1996).