Study of the adjuvant effect of CTB in fusions with PsaA and PspA pneumococcal proteins in the development of proten-based vaccines against Streptococcus pneumoniae

Author(s)

Ana Paula de Mattos Arêas

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2005-05-18

Examining board members

Paulo Lee Ho; Ises de Almeida Abrahamsohn; Luís Carlos de Souza Ferreira; Frederico José Gueiros Filho; Marilis do Valle Marques

• Advisor: Paulo Lee Ho

Abstract

The colonization of the respiratory mucosa is the first step in the pathogenesis of Streptococcus pneumoniae, bacterium that causes pneumonia, meningitis and otitis media. It is responsible for more than one million deaths per year worldwide. The surface proteins PsaA and PspA have been investigated as vaccine candidates against these diseases. CTB is the non-toxic portion of cholera toxin (CT), responsible for the toxin binding to the cellular receptor GM1 and described as mucosal adjuvant. In this study, these genes from S. pneumoniae were cloned in pAE, an E. coli expression vector that uses T7 promoter, or downstream to ctxB gene in the pAE-ctxB plasmid. The recombinant proteins CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 and CTB-PspA3 were expressed in E. coli BL21 (SI) and purified through a chelating resin charged with nickel. GM1 binding assays showed that CTB and CTB portion of the fusion proteins were functional. Intranasal immunization with CTB-PsaA and, intranasal and intradermal administration of CTB-PspA1 and CTB-PspA3 induced IgG production in the serum. On the other hand, only CTB-PsaA fusion protein induced IgA in the mucosal secretions. Nasopharyngeal colonization assays in BALB/C and C57BL/6 mice showed that intranasal immunization with CTB-PsaA results in a decrease of colonization by S. pneumoniae. Lethal challenges with S. pneumoniae virulent strains indicated that intradermal immunization with CTB-PspA3, in contrast to the intranasal immunization, is able to protect mice. Since the fusion proteins induced a specific immune response, they should be further investigated as components of a new vaccine against infections caused by S. pneumoniae. (AU)