Development of an Aotus nancymaae model for Shigella Vaccine immunogenicity and efficacy studies.

Several animal models exist to evaluate the immunogenicity and protective efficacy of candidate Shigella vaccines. The two most widely used nonprimate models for vaccine development include a murine pulmonary challenge model and a guinea pig keratoconjunctivitis model. Nonhuman primate models exhibit clinical features and gross and microscopic colonic lesions that mimic those induced in human shigellosis. Challenge models for enterotoxigenic Escherichia coli (ETEC) and Campylobacter spp. have been successfully developed with Aotus nancymaae, and the addition of a Shigella-Aotus challenge model would facilitate the testing of combination vaccines. A series of experiments were designed to identify the dose of Shigella flexneri 2a strain 2457T that induces an attack rate of 75% in the Aotus monkey. After primary challenge, the dose required to induce an attack rate of 75% was calculated to be 1 × 10(11) CFU. Shigella-specific immune responses were low after primary challenge and subsequently boosted upon rechallenge. However, preexisting immunity derived from the primary challenge was insufficient to protect against the homologous Shigella serotype. A successive study in A. nancymaae evaluated the ability of multiple oral immunizations with live-attenuated Shigella vaccine strain SC602 to protect against challenge. After three oral immunizations, animals were challenged with S. flexneri 2a 2457T. A 70% attack rate was demonstrated in control animals, whereas animals immunized with vaccine strain SC602 were protected from challenge (efficacy of 80%; P = 0.05). The overall study results indicate that the Shigella-Aotus nancymaae challenge model may be a valuable tool for evaluating vaccine efficacy and investigating immune correlates of protection.

Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera.

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.

Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and brucella lumazine synthase confers total protection against Shiga toxins in mice.

The striking feature of enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs with anti-Stx-neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2 challenge, and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome-endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.

Selección de cepas de Shigella sonnei para el desarrollo de una vacuna efectiva contra la shigellosis / Selection of Shigella sonnei strains to develop an effective shigellosis vaccine

En el Instituto Finlay se desarrolló una metodología de trabajo que contribuyó a la selección de cepas de Shigellasonnei como posibles candidatos vacunales contra la shigellosis. Las cepas investigadas, donadas por el Centro Provincial de Higiene y Epidemiología de Ciudad de La Habana, se caracterizaron según los métodos convencionales. La identificación del serogrupo y serotipo se realizó por aglutinación en láminas portaobjetos con antisueros comerciales; mientras que para el estudio de la susceptibilidad antimicrobiana se utilizó el DIRAMIC 10, un equipo semiautomatizado que proporcionó los resultados 4 horas después de su realización. Se investigó también la presencia de plásmidos de virulencia, por el crecimiento de Shigella spp. en medio de agar Triptona Soya con Rojo Congo al 0,025 por ciento, así como la expresión de las proteínas de la membrana externa en SDS-PAGE; para las pruebas de virulencia y potencia se emplearon los modelos animales (modelo ratón-pulmón y Test de Sereny). Los resultados obtenidos conla metodología utilizada permitieron la selección de la cepa de S sonnei A-04 como la más adecuada para la obtencióndel posible candidato vacunal(AU)

A work methodology was developed at Finlay Institute that contributed to the selection of Shigella sonnei strains as possible vaccinal candidates against shigellosis. Strains under study , were donated by the provincial Centre of Hygiene and Epidemiology in the City of Havana and were characterized using traditional methods. The identification and of the serogroup and serotype was performed by agglutination in slides with commercial antisera. Whereas DIRAMIC 10, a semi automatedequipment was used to study antimicrobial susceptibility. Results were obtained four hours after. The presence of virulence plasmid for the growth of Shigella spp. In Agar Soy Triptone with Congo Red at 0,025 percent, as well as outer membrane protein expression in of SDS-PAGE. In addition, animal models mice-lung and the test of Sereny were used for virulence and potency tests. Results obtained allowed the selection of the strain S sonnei A-04 as the most adequate for the obtainment of a possible vaccinal candidate(AU)

Utilidad del modelo animal ratón-pulmón para evaluar la virulencia de posibles cepas vacunales de Shigella spp / Usefulness of lung-mouse animal model to evaluate the virulence of Shigella spp candidate vaccine straims

Shigella flexneri y Shigella sonnei, como cualquier otra especie del género Shigella, se sitúan entre los principalesagentes etiológicos de las enfermedades diarreicas agudas, sobre todo aquellas que ocurren en los países en vías dedesarrollo, aunque por la baja dosis infectante de este enteropatógeno no se excluyen los países desarrollados. Estasituación conlleva a la elaboración de vacunas para prevenir esta enfermedad y la necesidad de un modelo animal quepruebe la eficacia protectora e inmunogénica de posibles candidatos vacunales contra la shigellosis, situación que ha motivado numerosos estudios por la dificultad de demostrar la enteropatía intestinal en los monos y humanos. Lo anteriormente expuesto, más la capacidad de Shigella spp para mostrar resistencia a los ntimicrobianos, motivó la realización de este trabajo. En el mismo se constató la utilidad del modelo animal ratón-pulmón para evaluar la virulencia de candidatos vacunales a partir de este microorganismo. Se utilizó la técnica de inoculación intranasal con una concentración entre 107 y 109 UFC de cepas de Shigella flexneri y Shigella sonnei. Por todos los resultadosobtenidos con el modelo animal ratón-pulmón se concluyó que este modelo puede ser eficiente para los estudiospreclínicos de cualquier candidato vacunal a partir de Shigella spp(AU)

Shigella flexneri and Shigella sonnei, as other species of Shigella, are among the main etiological agents of acute diarrhoeal diseases worldwide, specially in developing countries, although we do not exclude developed ones, because of their low infective dose, between 10 and 100 colonies. This leads to the preparation of vaccines to prevent this illness and the need for an animal model to demonstrate the protective and immunogenic effectiveness of the shigellosis candidate vaccines. Many studies have been carried out since the intestinal enteropathy is difficult to demonstrate in monkeys and humans. The aforementioned and the capacity of Shigella sp to become resistant to antibiotic treatments motivated our study. In it we demonstrated theusefulness of the mouse-lung animal model to evaluate the virulence of vaccine candidates. Intranasal inoculation with Shigella flexneri and Shigella sonnei in concentration on between 107 and 109 CFU was used. The conclusion was that the lung-mouseanimal model can result efficient for preclinical studies of any Shigella spp vaccine candidate(AU)

Current aspects of Shigella pathogenesis.

Bacillary dysentery (shigellosis) is a severe human disease caused by Shigellae. In recent years, a large amount of information has been generated regarding the host, pathogen and environmental factors that impact the pathogenesis of shigellosis at the cellular and molecular level. This review summarizes what is currently known about Shigella, detailing those factors that contribute to pathogenesis and examining the current progress in the development of a vaccine.