ABSTRACT
SBA-15 ordered mesoporous silica can be considered a promising inorganic nanocarrier with emerging potential as an oral vaccine adjuvant. In this study, we investigated its application in the encapsulation of the diphtheria anatoxin (dANA). We observed a considerable preservation of dANA secondary and tertiary structures, even after the drying process by means of Circular Dichroism (CD) and fluorescence spectroscopies. Antigen loading was assessed at a number of different ratios of adjuvant-to-antigen using a combination of nitrogen adsorption porosimetry and Small Angle X-ray Scattering (SAXS). Our data showed that the mass ratio of 1:10 (dANA:SBA-15) is recommended for total encapsulation of dANA in the mesopores, considering that at this relative mass concentration antigen clustering was avoided, which is deleterious effect for immunization purposes. dANA release in mimetic intestine fluid, at a pH equal to 6.8, was followed by in-situ SAXS measurements and shown to be slow, being more pronounced after 6 h and continuous up to 35 h. Finally, the immunogenic complex was tested in isogenic Balb C mice by oral and subcutaneous immunization routes, including a comparison with the only permitted adjuvant for human use, aluminum hydroxide. A higher antibody titer was obtained by subcutaneous and oral administration routes using SBA-15 as the vehicle of dANA, compared with the conventional aluminum hydroxide, demonstrating the viability to use this ordered mesoporous silica in the formulation of oral vaccines, as already proved for the Virus Like Particles (VLP) Hepatitis B (HBsAg) case.
ABSTRACT
Enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea, is an important public health problem in Brazil and other developing countries. In vitro assays of bacterial adhesion to cultured cells are important tools for studying bacterial pathogenicity but do not reproduce all the events that occur in natural infections. In this study, the effects of oral infection with EPEC on mice selected for their minimal acute inflammatory response (AIR min) were evaluated. Mice were orally infected with EPEC and variations in body weight, bacterial shedding and antibody production observed. The infected animals developed seric and secretory anti-EPEC antibodies; however, neither mortality nor diarrhea was observed. Light microscopy of their intestines demonstrated histological modifications that were not present in controls. However, electron microscopy did not show bacteria attached to the intestinal epithelia to form attaching and effacing lesions, characteristic of EPEC in humans. The bacteria were detected in Peyer's patches and intestinal contents up to 5 hr post-infection. When human anti-EPEC secretory immunoglobulin A or avian immunoglobulin Y antibodies were administered to infected animals, they developed minor histological alterations compared with non-treated animals. In summary, it was found that EPEC triggers immune responses and intestinal histological alterations but does not produce evidence of diarrheal disease in mice infected by the oral route. This study of EPEC experimental infection provides a better understanding of the effects of antibodies on bacterial infections and may provide a suitable model for the design and testing of immunobiological products for active or passive immunization.
Subject(s)
Disease Models, Animal , Enteropathogenic Escherichia coli/immunology , Escherichia coli Infections/immunology , Escherichia coli Infections/pathology , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Bacterial Shedding , Body Weight , Female , Histocytochemistry , Male , Mice , Microscopy, ElectronABSTRACT
A Escherichia coli enteropatogênica EPEC é um importante patógeno humano, que ao aderir nas células epiteliais do intestino, provoca redução na capacidade de absorção da mucosa intestinal causando diarréia. Os trabalhos desenvolvidos in vitro não mostram toda a complexidade do mecanismo de infecção por EPEC. A transferência passiva de anticorpos para o recém-nascido é importante para sua proteção durante o amadurecimento do seu sistema imunológico. As linhagens estudadas neste trabalho são camundongos selecionados geneticamente de acordo com a intensidade inflamatória aguda (AIRmin e AIRmax). Objetivo: O objetivo do presente projeto foi avaliar a resposta imune de mucosas destas linhagens após a inoculação de Escherichia coli enteropatogênica (EPEC) por via oral e observar a possível transferência passiva de anticorpos para a prole. Métodos: Camundongos de 8 semanas de idade receberam doses de EPEC de 1010 ufc por via oral. Foram feitas avaliações clínicas, pesagem e coleta de fluídos (sangue, fezes e leite) para pesquisa de anticorpos séricos (IgG) e secretores (IgA) por ensaio de ELISA. Para avaliar a transferência de anticorpos entre mãe e prole, também foram coletados sangue e leite dos recém-nascidos para pesquisa de anticorpos anti-EPEC através de ELISA. Resultados e Discussão: Não houve variação de peso nos animais tratados com a bactéria em comparação com os controles. A inoculação não provocou diarréia e nenhum outro sintoma de doença, mas foi capaz de estimular a produção de anticorpos anti-EPEC nos animais adultos. Os recém-nascidos de mães tratadas com a bactéria apresentaram níveis de anticorpos mais elevados em relação aos grupos controles. Esse modelo de infecção oral se mostrou adequado para o estudo de transferência passiva de anticorpos.
