ABSTRACT
Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.
Subject(s)
Female , Humans , Mice , Adjuvants, Immunologic/administration & dosage , Administration, Intranasal , Animals , Bacterial Toxins/immunology , Bacterial Toxins/genetics , Bacterial Toxins/administration & dosage , Enterotoxins/immunology , Enterotoxins/genetics , Enterotoxins/administration & dosage , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Feces , Gastric Mucosa/microbiology , Gastric Mucosa/immunology , Helicobacter pylori , Immunoglobulin A, Secretory/immunology , Immunoglobulin G/immunology , Mice, Inbred BALB C , Mutagenesis, Site-Directed , ADP Ribose Transferases/immunology , ADP Ribose Transferases/genetics , Nasal Mucosa/immunology , Point Mutation , Urease/immunology , Urease/administration & dosage , VaccinationABSTRACT
Se empleó el método de ELISA Ureasa indirecto para detectar anticuerpos monoclonares obtenidos a partir de ratones inmunizados con merozoitos libres de Plasmodium falciparum. Se seleccionaron los anticuerpos monoclonales que mostraron ser muy estables en posteriores ensayos de caracterización. Los anticuerpos obtenidos no fueron específicos contra antígeno de Plasmodium falciparum pero permitieron identificar la proteína espectrina del citoesqueleto de eritrocito humano. En el ensayo fue empleado un antígeno de esquizontes concentrados y solubilizados. Se obtuvo la determinación del punto final de la reacción a través de lectura visual semicuantitativa. A partir de la enzima Ureasa de alta pureza que se encuentra disponible comercialmente y, empleando métodos muy sencillos se prepararon los reactivos utilizados en el ensayo