Development of FAcE (Formulated Alhydrogel competitive ELISA) method for direct quantification of OAg present in Shigella sonnei GMMA-based vaccine and its optimization using Design of Experiments approach.

Many formulated vaccines, including 1790GAHB Shigella sonnei GMMA-based vaccine, contain Alhydrogel (aluminum hydroxide), consequently the antigen content must be determined in the formulated final vaccine product, as required by regulatory authorities. The direct quantification of antigens adsorbed on aluminum salts is difficult, and antigens may need to be extracted using laborious and often ineffective desorption procedures. To directly quantify the sugar vaccine target in the LPS of 1790GAHB, we have developed a new FAcE (Formulated Alhydrogel competitive ELISA) method. FAcE is an immunoassay based on the competition between S. sonnei LPS, coated on the ELISA plate, and the LPS in formulated S. sonnei GMMA, in binding a specific monoclonal antibody. To optimize the method, which is as easy to perform as a standard ELISA, we have applied a Design of Experiments (DOE) approach. A model was found to define the significant assay variables and to predict their impact on the output responses. Results obtained using the DOE optimized FAcE assay showed that the method is sensitive (0.02 µg/mL lower detection limit), precise, reproducible and can accurately quantify independently formulated drug products, making it a useful tool in routine tests of Alhydrogel-based vaccines. We are currently using this method to determine S. sonnei vaccine potency, stability and lot-to-lot variations, and are broadening its applicability to quantify active ingredients of other Alhydrogel GMMA-vaccines and in multivalent vaccines formulations.

A Phase I trial to evaluate the safety and immunogenicity of WRSs2 and WRSs3; two live oral candidate vaccines against Shigella sonnei.

Effective vaccines are needed to combat diarrheal diseases due to Shigella. Two live oral S. sonnei vaccine candidates, WRSs2 and WRSs3, attenuated principally by the lack of spreading ability, as well as the loss of enterotoxin and acyl transferase genes, were tested for safety and immunogenicity. Healthy adults 18-45 years of age, assigned to 5 cohorts of 18 subjects each (WRSs2 (n = 8), WRSs3 (n = 8) or placebo (n = 2)) were housed in an inpatient facility and administered a single oral dose of study agent 5 min after ingestion of oral bicarbonate. Ascending dosages of vaccine (from 103 CFU to 107 CFU) were evaluated. On day 8, treatment with ciprofloxacin (500 mg BID for 3 days) was initiated and subjects were discharged home 2 days after completing antibiotics. Subjects returned for outpatient visits on day 14, 28 and 56 post-vaccination for monitoring and collection of stool and blood samples. Both WRSs2 and WRSs3 were generally well tolerated and safe over the entire dose range. Among the 80 vaccinees, 11 subjects developed diarrhea, 8 of which were mild and did not affect daily activities. At the 107 CFU dose, moderate diarrhea occurred in one WRSs2 subject while at the same dose of WRSs3, 2 subjects had moderate or severe diarrhea. Vaccinees mounted dose-dependent mucosal and systemic immune responses that appeared to correlate with fecal shedding. S. sonnei vaccine candidates WRSs2 and WRSs3 are safe and immunogenic over a wide dose range. Future steps will be to select the most promising candidate and move to human challenge models for efficacy of the vaccine.

Status of vaccine research and development for Shigella.

Shigella are gram-negative bacteria that cause severe diarrhea and dysentery. In 2013, Shigella infections caused an estimated 34,400 deaths in children less than five years old and, in 2010, an estimated 40,000 deaths in persons older than five years globally. New disease burden estimates from newly deployed molecular diagnostic assays with increased sensitivity suggest that Shigella-associated morbidity may be much greater than previous disease estimates from culture-based methods. Primary prevention of this disease should be based on universal provision of potable water and sanitation methods and improved personal and food hygiene. However, an efficacious and low-cost vaccine would complement and accelerate disease reduction while waiting for universal access to water, sanitation, and hygiene improvements. This review article provides a landscape of Shigella vaccine development efforts. No vaccine is yet available, but human and animal challenge-rechallenge trials with virulent Shigella as well as observational studies in Shigella-endemic areas have shown that the incidence of disease decreases following Shigella infection, pointing to biological feasibility of a vaccine. Immunity to Shigella appears to be strain-specific, so a vaccine that covers the most commonly detected strains (i.e., S. flexneri 2a, 3a, 6, and S. sonnei) or a vaccine using cross-species conserved antigens would likely be most effective. Vaccine development and testing may be accelerated by use of animal models, such as the guinea pig keratoconjunctivitis or murine pneumonia models. Because there is no correlate of protection, however, human studies will be necessary to evaluate vaccine efficacy prior to deployment. A diversity of Shigella vaccine constructs are under development, including live attenuated, formalin-killed whole-cell, glycoconjugate, subunit, and novel antigen vaccines (e.g., Type III secretion system and outer membrane proteins).

Vaccines for the prevention of diarrhea due to cholera, shigella, ETEC and rotavirus.

BACKGROUND: Diarrhea is a leading cause of mortality in children under 5 years along with its long-term impact on growth and cognitive development. Despite advances in the understanding of diarrheal disorders and management strategies, globally nearly 750,000 children die annually as a consequence of diarrhea. METHODS: We conducted a systematic review of the efficacy and effectiveness studies. We used a standardized abstraction and grading format and performed meta-analyses for all outcomes. The estimated effect of cholera, shigella, Enterotoxigenic Escherichia coli (ETEC) and rotavirus vaccines was determined by applying the standard Child Health Epidemiology Reference Group (CHERG) rules. RESULTS: A total of 24 papers were selected and analyzed for all the four vaccines. Based on the evidence, we propose a 74% mortality reduction in rotavirus specific mortality, 52% reduction in cholera incidence due to their respective vaccines. We did not find sufficient evidence and a suitable outcome to project mortality reductions for cholera, ETEC and shigella in children under 5 years. CONCLUSION: Vaccines for rotavirus and cholera have the potential to reduce diarrhea morbidity and mortality burden. But there is no substantial evidence of efficacy for ETEC and shigella vaccines, although several promising vaccine concepts are moving from the development and testing pipeline towards efficacy and Phase 3 trials.

O-specific [corrected] polysaccharide conjugate vaccine-induced [corrected] antibodies prevent invasion of Shigella into Caco-2 cells and may be curative.

The O-specific polysaccharide (O-SP) domain of Shigella LPS is both an essential virulence factor and a protective antigen for this genus. A critical level of serum IgG anti-O-SP was shown to confer immunity to shigellosis, likely by complement-mediated bacteriolysis of the inoculum. Conjugate Shigella O-SP vaccines were shown to be safe and immunogenic in children, and, in a preliminary study, Shigella sonnei vaccine was protective in young adults. Characteristic of shigellosis is bacterial invasion of intestinal cells. Incubation of shigellae with postimmunization but not preimmunization sera of children vaccinated with S. sonnei or Shigella flexneri 2a O-SP conjugate vaccines inhibited in a type-specific and dose-dependent manner in vitro invasion of intestinal epithelial cells (Caco-2) and the infection-associated increases in IL-1beta and IL-8 mRNA and extracellular cytokine levels. Pretreatment of these sera or of Caco-2 cells with O-SP abrogated these effects also in a type-specific and dose-dependent manner. Confocal microscopy demonstrated antibody-specific inhibition of bacterial adhesion to HeLa cells. These protective effects were duplicated by IgG purified from these sera. These results suggest a dual role for IgG anti-O-SP. In addition to lysis of the inoculum in immune individuals, the newly synthesized IgG anti-O-SP in patients may terminate an established infection by inhibiting shigellae released from epithelial cells from invading new ones. A critical level of IgG anti-O-SP could, therefore, have a protective as well as a curative role in shigellosis.

Vaccines against cholera, typhoid fever and shigellosis for developing countries.

Enteric diseases, such as cholera, typhoid fever and shigellosis, still produce a significant burden, especially among the poor in countries where these illnesses are endemic. Older-generation, parenteral, whole-cell vaccines against cholera and typhoid fever were abandoned in many countries as public health tools because of problems with insufficient protection and/or inadequate safety profiles. Modern-generation licensed vaccines are available for cholera and typhoid fever, but are not widely used by those in greatest need. A number of experimental candidates exist for all three diseases. Future research should focus on generating the evidence necessary to obtain a consensus on the deployment of existing vaccines against cholera and typhoid fever, and on clinical evaluation of pipeline vaccine candidates against all three diseases.

Prospective cohort studies of shigellosis during military field training.

Epidemiological and clinical features of shigellosis occurring among cohorts of Israeli recruits followed-up for 3-6 months during the summer field training of years 1993-1997 were studied. The incidence rate of culture-proven shigellosis was the highest (78 cases per 1,000 recruits) in 1996 and the lowest (13 cases per 1,000 recruits) in 1995. Shigella sonnei (152 isolates) and Shigella flexneri (151 isolates) were the most common species. Fifty percent of the patients with shigellosis had fever (>37.5 degrees C), compared to only 18% of the subjects with other diarrheal diseases (P < 0.001). The duration of illness was longer among subjects with shigellosis than among those with other diarrheal diseases (P < 0.001). Illness due to Shigella flexneri was more severe than illness caused by Shigella sonnei.

Safety and immunogenicity of improved Shigella O-specific polysaccharide-protein conjugate vaccines in adults in Israel.

Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) of Shigella species is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP of Shigella species were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity of Shigella conjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates of Shigella sonnei and Shigella flexneri 2a bound to succinylated mutant Pseudomonas aeruginosa exotoxin A (rEPAsucc) or native or succinylated Corynebacterium diphtheriae toxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic. S. sonnei-CRM9, S. sonnei-CRM9succ, and S. sonnei-rEPAsucc elicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P < 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P < 0.0001). GM IgG anti-LPS levels elicited by S. flexneri 2a-rEPAsucc were significantly higher than those elicited by S. flexneri 2a-rCRM9succ at all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P < 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these five Shigella conjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678-3687, 1993). Based on these data, we chose S. sonnei-CRM9 and S. flexneri 2a-rEPAsucc for evaluation in children.