• Vaccine Ontology ID: VO_0000668
• Type: Live, attenuated vaccine
• Antigen: The antigen for this vaccine is CVD 1208s, which is Shigella flexneri containing deletions in sen, set, and guaBA (Kotloff et al., 2007).
• Preparation: The inocula were derived from frozen master cell banks containing prepared strains of CVD 1208S. Frozen master seed was plated onto SP agar. After incubation, several Congo Red-dyed colonies proven to be Shigella were suspended in sterile saline. This saline was used to inoculate SP plates for the heavy bacterial growth. These resulting colonies were harvested into PBS and diluted to the appropriate bacterial count, which was between 1 x 10^8 and 1 x 10^9 CFU per ml (Kotloff et al., 2007).
• Description: CVD 1208S is the same thing as CVD 1208, but CVD 1208S was prepared using animal-free media (Kotloff et al., 2007).

• Vaccine Ontology ID: VO_0000670
• Type: Recombinant vector vaccine
• Antigen: This vaccine is an aro-D mutant derivative of EcSf2a-1 (Kotloff et al., 1992).
• Preparation: The vaccine was suspended in 20 ml of BHI broth and contained 1.5 x 10^11 CFU of bacteria (Kotloff et al., 1992).
• Virulence: Vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (Kotloff et al., 1992).

• Vaccine Ontology ID: VO_0000677
• Type: Recombinant vector vaccine
• IcsA/VirG gene engineering:
  • Type: Recombinant protein preparation
  • Description: CVD 1207 was constructed from wild-type S. flexneri 2a strain 2457T by a series of double homologous recombinations using suicide plasmid deletion cassette technology as described in detail elsewhere. In brief, a specific, in-frame deletion mutation in the guaBA operon was first introduced, followed by a second in-frame deletion mutation in the plasmid virulence gene virG. The chromosomal mutation set was accomplished with
    deletion of 85% of subunit A of set. Finally, a sen cassette was constructed by fusing two 700-bp segments that include the N and C termini of sen minus 300 bp corresponding to the putative active site in the N-terminal region. The ars operon, conferring resistance to arsenite, was cloned into the sen locus to allow facile transfer of the double-deletion mutation (virG and sen) virulence plasmid to candidate Shigella vaccine strains and as a marker to distinguish CVD 1207 in the field. As previously described, CVD 1207 does not grow in minimum medium unless supplemented with guanine. The lack of enterotoxic activity has been confirmed in Ussing chambers. CVD 1207 is significantly less invasive for HeLa cells than its wild-type parent strain 2457T (approximately 1 log unit fewer intracellular CFU detected) but does not differ from its single-mutant strain progenitor guaBA CVD 1204 (unpublished observations). CVD 1207 undergoes fewer intracellular generations in HeLa cells than either CVD 204 (10-fold; 4.5 doublings in 4 h) or 2457T (30-fold; 5 doublings in 4 h) (unpublished observations) (Kotloff et al., 2000).
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• Preparation: Shigella flexneri strain CVD 1207 carries deletions of the plasmid gene virG (also known as icsA), which encodes a protein responsible for cell-to-cell spread of Shigella in the intestinal epithelium; (ii) the chromosomal gene set encoding Shigella enterotoxin 1 (ShET1), which is present almost exclusively in S. flexneri 2a; (iii) the plasmid gene sen, encoding Shigella enterotoxin 2 (ShET2), which is present in virtually all serotypes of Shigella; and (iv) the guaBA chromosomal operon that regulates synthesis of IMP dehydrogenase (encoded by guaB) and GMP synthetase (encoded by guaA), two enzymes employed in the distal de novo purine biosynthesis pathway. CVD 1207 thus expresses type-specific O-polysaccharide and invades epithelial cells (albeit less competently than the wild type) but undergoes only limited intracellular proliferation and intercellular spread and has no detectable enterotoxic activity (Kotloff et al., 2000).
• Virulence: Protective efficacy against shigellosis following rechallenge was 70% (Kotloff et al., 2000).

• Vaccine Ontology ID: VO_0000678
• Type: Recombinant vector vaccine
• AroA gene engineering:
  • Type: Gene mutation
  • Description: WRSS1 was constructed from the Mosely strain of S. sonnei. A parent strain was selected that exhibited stability of the form I colonial phenotype, then sacB suicide vector pCVD422 was used to replace the wild-type virG allele with virG possessing a 212-bp deletion. In preclinical experiments (Kotloff et al., 2002).
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• Preparation: WRSS1 is a stable S. sonnei mutant with a deletion in virG (Kotloff et al., 2002). The final composition of the vaccine consisted of 3.7 x 10^10 CFU of WRSS1 per vial in PBS containing 7.5% dextran T10, 2% sucrose, and 1.5% glycerol as a cryopreservative (Kotloff et al., 2002).
• Virulence: WRSS1 vaccine is remarkably immunogenic in doses ranging from 10^3 to 10^6 CFU (Kotloff et al., 2002).

• Vaccine Ontology ID: VO_0000663
• Type: Live, attenuated vaccine
• Antigen: The antigen for this vaccine is Shigella flexneri 2a strain SC602 (Coster et al., 1999).
• IcsA/VirG gene engineering:
  • Type: Recombinant protein preparation
  • Description: This SC602 vaccine was constructed with S. flexneri 2a strain 454 as the progenitor. The iuc mutation neccessary for producing SC602 was generated by recombination of iuc::Tn10 into the chromosome by using phage P1 transduction. Spontaneous excision of the tetracycline resistance gene, and its flanking regions including the iuc locus, was selected by growth on fusaric acid medium. The icsA gene was inactivated by double recombination with a kanamycin resistance-sucrose sensitivity cartridge carrying flanking regions of icsA. Deletion of the Kmr-sacB cartridge was selected by growth on sucrose, and the resistant clones were screened for retention of the invasive phenotype in HeLa cells. An isolate designated SC602 had suffered a deletion of the entire icsA gene along with substantial flanking sequences.This SC602 isolate was expanded into a master cell bank and was used in the vaccines (Coster et al., 1999).
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• Preparation: The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA (mediating intra- and intercellular spread) and the chromosomal locus iuc (encoding aerobactin).
• Virulence: Attenuated.

Human Response

• Vaccination Protocol: 16 volunteers were assigned to receive between 1 x 10^8 and 1 x 10^9 CFU per ml of CVD 1208S or a placebo. Volunteers ingested a buffer solution, then ingested 1 ml of vaccine or placebo suspended in 30 ml of buffer solution. Vaccine excretion was monitered through the culture of all stools and through collection of blood samples (Kotloff et al., 2007).
• Persistence: Vaccine was detected in the stool of all volunteers that received the 1 x 10^9 CFU dosage for at least one day. The same was true for six of the volunteers that received 1 x 10^8 CFU, with no vaccine detected in the volunteers that received the placebo. Peak excretion occurred on Day 1 and total excretion did not exceed the dosage levels (Kotloff et al., 2007).
• Immune Response: Following vaccination, all subjects mounted an anti-LPS IgA ASC response and 5 exhibited an anti-LPS IgG ASC response. Anti-LPS responses typically reached a peak on Day 14. Some volunteers exhibited a serum antibody response to Ipa and IpaB. All recipients of the higher dose (1 x 10^9 CFU) demonstrated a response in two or more of the immunologic assays (Kotloff et al., 2007).
• Side Effects: Side effects included mucoid or loose stools and fever, in some cases (Kotloff et al., 2007).

Human Response

• Vaccination Protocol: 19 volunteers ingested three doses of either ca. 5.0 x 10^6, 5.0 x 10^7, or 2.0 x 10^9 CFU of bacteria (Kotloff et al., 1992).
• Persistence: All 46 vaccine recipients excreted the vaccine strain on at least one occasion. Recipients of three spaced doses of ca. 2.0 x 10^9 CFU shed ca. 10^5 organisms per gram of stool for an average of 7 days. Duodenal colonization was detected in five subjects, all recipients of ca. 2.0 x 10^9 CFU (Kotloff et al., 1992).
• Immune Response: A fourfold increase in IgA or IgG antibody titers recognizing purified LPS was detected in 61% of those tested, and a seroresponse to IPA was detected in 44% of the 38 recipients of ca. 2.0 x 10^9 CFU. The IgM response was meager. Circulating IgA ASC specific for S. flexneri 2a LPS and for IPA were each detected in 97% and 60%, respectively, of the 30 subjects tested (Kotloff et al., 1992).
• Side Effects: Diarrhea, dysentery, and fever were observed in vaccine recipients (Kotloff et al., 1992).
• Challenge Protocol: Challenge doses of 1.6 x 10^9 and 1.8 x 10^9 CFU of ECSf2a-2 were given to the volunteers (Kotloff et al., 1992) .
• Efficacy: Vaccine efficacy was only 9% in the challenge study, in which 30% of recipients of three doses of ca. 2.0 x 10^9 CFU developed illness, compared 33% of 9 unvaccinated control subjects. Overall, the vaccine efficacy was 36%. Neither class-specific titers in prechallenge sera nor fourfold rises in antibody titer after vaccination could be correlated with protection against challenge (Kotloff et al., 1992).

Human Response

• Vaccination Protocol: Groups of 3 to 7 outpatient volunteers were assigned, in an incremental fashion, to receive a single oral dose of CVD 1207 at a desired inoculum (the actual inocula administered are in parentheses) of either 10^6,10^7, 10^8, 10^9, or 10^10 CFU. Fasting volunteers swallowed the vaccine suspended in a solution of NaHCO3 buffer, as previously described (Kotloff et al., 2000).
• Persistence: Fecal excretion of the vaccine strain in the volunteer’s stools was measured on days 1, 2, 3, 7, 10, 14, and 21 after ingestion of the vaccine. The duration of excretion was 1 to 3 days except for two recipients of ca. 10^9 CFU, who each had one additional positive stool culture 2 weeks after vaccination (Kotloff et al., 2000).
• Immune Response: A dose-related, immunoglobulin A antibody-secreting cell (ASC) response to S. flexneri 2a O-specific lipopolysaccharide was seen, with geometric mean peak values of 6.1 to 35.2 ASCs/10^6 peripheral blood mononuclear cells (PBMC) among recipients of 10^7 to 10^10 CFU. The cytokine response to Shigella-specific antigens observed in volunteers’ PBMC following vaccination suggested a Th1 pattern with stimulation of gamma interferon and absence of interleukin 4 (IL-4) or interleukin 5(Kotloff et al., 2000).
• Side Effects: Some test subjects experienced diarrhea and vomiting. No subjects experienced fever or dysentery (Kotloff et al., 2000).

Human Response

• Vaccination Protocol: Fasting volunteers ingested 2g of sodium bicarbonate buffer dissolved in 150 ml of water, followed 1 min later by 30 ml of water containing the assigned vaccine dose or no vaccine (placebo) (Kotloff et al., 2002).
• Immune Response: Vaccination elicited vigorous IgA ASC anti-LPS responses in all of the groups. ASC responses were less common and smaller in magnitude in the IgG anti-LPS assay and in both anti-Ipa assays. Geometric mean peak postvaccination anti-LPS serum IgG and fecal IgA titers were also robust. Most of the subjects exhibited a fourfold rise in serum and/or fecal anti-LPS antibody titers. Whereas the anti-LPS IgA ASC and fecal antibody responses tended to increase with the dose, a similar trend was not apparent in serum antibody responses. Postvaccination antigen-specific proliferative responses and increases in IL-10 production were not seen (Kotloff et al., 2002).
• Side Effects: Side effects included fever, loose stools or aysmptomatic diarrhea, and mild cramps (Kotloff et al., 2002).
• Description: This is a Phase I study.

Human Response

• Vaccination Protocol: Volunteers fasted for 90 minutes before and after vaccination. The inoculum was ingested by each volunteer 2 min after ingestion of 120 ml of the sodium bicarbonate solution. Placebo controls received sodium bicarbonate buffer with no added bacteria. SC602 dose selection studies. Thirty-three subjects were enrolled in the initial, placebo-controlled dose selection trial: eighteen subjects received the SC602 vaccine and fifteen received sodium bicarbonate placebo (Coster et al., 1999).
• Persistence: Robust and prolonged intestinal colonization by S. flexneri 2a was observed in all volunteers who had ingested the SC602 vaccine. The peak excretion of vaccine was 10^4 to 10^6 CFU/g of stool regardless of the dose ingested (Coster et al., 1999).
• Immune Response: Immune correlates of vaccine efficacy against diarrhea and severe shigellosis included a significant IgA ASC response and a threefold or greater rise in serum IgA antibody against S. flexneri 2a LPS. Other correlates of protection against all symptoms included urinary sIgA responses against 2a LPS in addition to IgG ASC and IgG serum responses. ASC levels peaked on day 7 and ELISA titers peaked on day 14 for vaccination(Coster et al., 1999).
  
• Side Effects: Reportable intestinal symptoms included abdominal cramps, nausea, emesis, tenesmus, and gas. Constitutional symptoms in-
  cluded headache, myalgia, arthralgia, loss of appetite, and fatigue (Coster et al., 1999).
• Challenge Protocol: The challenge inoculum, containing approximately 10^3 CFU of virulent S. flexneri 2a strain 2457T, was prepared and administered with sodium bicarbonate as described previously. All subjects
  who were vaccinated or challenged with S. flexneri were treated with ciprofloxacin (Coster et al., 1999).
• Efficacy: SC602 gave significant protection against fever and severe shigellosis (Coster et al., 1999).
• Host human IgA response
  • Description: Immune correlates of vaccine efficacy against diarrhea and severe shigellosis included a significant IgA ASC response and a threefold or greater rise in serum IgA antibody against S. flexneri 2a LPS as compared to titers on day 0. Four of 12 vaccinees experienced a significant increase in IgA titers (Coster et al., 1999).
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• Host IgG response
  • Description: Correlates of protection against all symptoms included IgG ASC and IgG serum responses. A majority of volunteers had IgG anti-Ipa responses. Antibody titers were compared to day 0 titers (Coster et al., 1999).
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Guinea pig Response

• Vaccination Protocol: The protective efficacy and immunogenicity of WRSS1 were measured with the guinea pig keratoconjunctivitis model. Ocular immunization with 3 × 10^8 to 4 × 10^8 CFU of WRSS1/eye on days 0 and 14.
• Challenge Protocol: Four weeks after the last immunization, both the immunized animals and the unimmunized control animals were challenged with 4 × 10^8 CFU of virulent S. sonnei 53G/eye.
• Efficacy: In animals immunized with WRSS1 grown from overnight plate cultures, 13 of 16 eyes showed no signs of disease (83% complete protection), while 3 eyes showed mild conjunctivitis (17% partial protection). When reconstituted lyophilized cultures were used, 10 of 16 eyes did not develop disease (63% complete protection), while 4 eyes developed mild disease (25% partial protection). In both cases, protection against challenge was significant by the Fisher exact test (P < 0.001), and there was no significant difference in the levels of protection conferred by the two formulations.
• Description: WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model (Hartman and Venkatesan, 1998).
• Host human IgA response
  • Description: WRSS1 elicits vigorous anti-LPS IgA ASC and serum IgA and IgG antibody responses that are similar in magnitude to those elicited by other strains that prevented illness. IgA responses were significantly greater 7 to 10 days post inoculation in those that received WRSS1 as opposed to the placebo (Kotloff et al., 2002).
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• Host IgG response
  • Description: WRSS1 elicits vigorous anti-LPS IgA ASC and serum IgA and IgG antibody responses that are similar in magnitude to those elicited by other strains that prevented illness. IgG responses were significantly greater 7 to 10 days post inoculation in those that received WRSS1 as opposed to the placebo (Kotloff et al., 2002).
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