Intradermal Immunization of Mice with Cholera Toxin B-Pneumococcal Surface Protein A Fusion Protein Is Protective against Intraperitoneal Challenge with Streptococcus pneumoniae

Abstract

Cholera toxin B subunit is known for its adjuvant properties when associated with different antigens. In this work, we have fused the ctxB gene to the pneumococcal surface protein (pspA) gene from Streptococcus pneumoniae. Intradermal administration of the fusion protein in mice induced anti-PspA antibodies and protection against pneumococcus in a sepsis model.Streptococcus pneumoniae is the major cause of bacterial pneumonia, meningitis, and otitis media cases around the world, leading to up to 1 million deaths per year. Pneumococcal surface protein A (PspA) is a virulence factor partially conserved among the Streptococcus pneumoniae isolates that plays a role in complement inactivation during infection (18). Based on amino acid sequence diversity, PspAs can be classified into three families and six clades (8) that also display immunological cross-reactivity among themselves (17). In several pneumoccocal infection challenge models, PspA has proved to be a good vaccine candidate when administered either in protein-adjuvant formulations or in DNA-based vaccines (4,11,12). PspA was also shown to bind and to prevent the bactericidal activity of apolactoferrin (15), present in saliva and other mucosal secretions. In addition, anti-PspA antibodies were shown to enhance pneumococcal killing by lactoferrin, suggesting a mechanism for the reduction of pneumococcal carriage by these antibodies (15). In a recent work, we tested the immunogenic potential of a fusion protein composed of the cholera toxin B subunit (CTB) and pneumococcal surface antigen A from Streptococcus pneumoniae by intranasal inoculation of mice (3). In this work, we have amplified the 5Ј terminus region (which encodes the ␣-helix region plus the prolinerich domain) of the pspA clade 3 gene from pTG-pspAЈ3 (11) (pspAЈ3 accession number, AY082389), which carried the gene isolated from S. pneumoniae St 259/98 strain (Instituto Adolpho Lutz, São Paulo, SP, Brazil). The pspAЈ gene was then cloned downstream of the ctxB gene in the pAE-CTB plasmid (2) in order to express a CTB-PspAЈ fusion protein in Escherichia coli. Using this expression system, the recombinant protein contains an N-terminal six-His tag that allows purification through Ni 2ϩ charged columns. This system was used for the expression and purification of the fusion protein CTB-PspAЈ and CTB, as previously described (2, 3), and PspAЈ, with the observation that the last protein did not adsorb very well, and it was released from the column with 20 mmol · liter Ϫ1 imidazole.Protein characterization. The recombinant CTB-PspAЈ purified from E. coli BL21-SI extracts was able to form pentamers, as evaluated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unboiled samples (data not shown). Since the adjuvant effect of CTB is dependent on the binding of the pentameric form to the GM1 gangliosides present on cellular surfaces (14), we performed an enzyme-linked immunosorbent assay (ELISA) experiment, using 10 g · ml Ϫ1 GM1 in phosphate-buffered saline for an overnight coating and incr...

Intradermal immunization in the ear with cholera toxin and its non‐toxic β subunit promotes efficient Th1 and Th17 differentiation dependent on migrating DCs

“…Interestingly, we also observed IFN-g and IL-17 CD4 1 T-cell differentiation after immunization with the CTB subunit, which argues against previous data that indicated that the adjuvant role of CT is mediated by CT a subunit activity [21]. However, the use of CT as an adjuvant has yielded conflicting data [24,29,30]. In our experiments, both CT and the CTB subunit induced the expression of TGF-b in dermal skin cells and had a similar adjuvant effect in CD4 1 T-cell priming.…”

“…However, the use of CT as an adjuvant has yielded conflicting data [24,29,30]. In our experiments, both CT and the CTB subunit induced the expression of TGF-b in dermal skin cells and had a similar adjuvant effect in CD4 1 T-cell priming.…”

Streptococcus pneumoniae: proteomics of surface proteins for vaccine development

“…Animals were challenged with 0.5 mL of a 5.5 · 10 6 CFU ⁄ mL suspension, i.e., >1000-fold the LD50. Unfortunately, it was not possible to compare the ACE antigens with the existing Pneumovax vaccine in this animal model of sepsis, as, in contrast to published results [23,24], reliable protection of BALB ⁄ c mice against S. pneumoniae D39 was not achieved using Pneumovax (data not shown).…”

Desenvolvimento e caracterização de uma vacina antiesquistossomose composta pela proteína Sm14 de Schistosoma mansoni utilizando a subunidade B da toxina colérica (CTB) com adjuvante

“…Na verdade, a afinidade de CTB a GM1 quando em fusão com outras proteínas pode ser reduzida de forma significativa, a depender da proteína a ser fusionada (Liljeqvist, Stahl et al, 1997;Bublin, Hoflehner et al, 2007). (Fonseca, Brito et al, 2004) (Areas, Oliveira et al, 2005 Sm29GST e CTB (Sun, Mielcarek et al, 1999;Lebens, Sun et al, 2003). (Holmgren, Adamsson et al, 2005;Bublin, Hoflehner et al, 2007;Takagi, Hiroi et al, 2008).…”