In-vitro and in-vivo expression analysis of pneumococcal vaccine candidates: pilus-1 components

Abstract

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive commensal of the nasopharyngeal tract of both children and healthy adults. However, S. pneumoniae is also a leading cause of morbidity and mortality worldwide, being responsible for non-invasive and invasive diseases such as acute otitis media, pneumonia, sepsis and meningitis. Despite the unquestionable efficacy of the available pneumococcal glycoconjugate vaccines, the limited coverage, along with the observed phenomenon of serotype replacement, could reduce their long-term effectiveness. For these reasons, the development of a serotype-independent vaccine relying on the use of surface-exposed protein antigens represents a valid alternative. In this context, pneumococcal pilus-1 components, and in particular the pilus backbone RrgB, demonstrated significant efficacy in protecting mice from lethal challenge. The S. pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. Since a combination of the three RrgB variants could broad the efficacy of a pilus-based vaccine, a fusion protein (RrgB321) containing the three RrgB variants in a head to tail organization was constructed. It was recently reported that RrgB321 elicites an antibody response against each of the variants and protectes mice against piliated pneumococcal strains of the three clades both by active and passive immunization, supporting the validity of this candidate as a potential antigen for the generation of a multi-component protein-based vaccine against S. pneumoniae. The data reported in this work contribute to the characterization of pilus-1 expression regulation in in vitro and in vivo experiments providing evidence that pilus expression is biphasic and demonstrating that the pilus expression level does not impair the protection induced by RrgB321 immunization in mouse models of infection. Analyzing the strains at the single-cell level, two phenotypically different sub-populations of bacteria (one that expresses the pilus, while the other does not) could be identified. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Moreover, evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria, was reported. Overall the in vitro data presented suggest that the observed biphasic pilus expression phenotype is an example of bistability in pneumococcus. Additionally, in this study, the ability of RrgB321 antibodies to kill both H and L S. pneumoniae populations in the opsonophagocytosis assay, as well as the ability of RrgB321 to confer protection in vivo against both populations were analyzed. The results obtained demonstrate that: i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine.