Pilus proteins from Streptococcus pyogenes stimulate innate immune responses through Toll-like receptor 2.

The group A Streptococcus (GAS) pilus is a long, flexible, hair-like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin-8. We further show that the surface-exposed backbone pilin and ancillary pilin 1 subunits are Toll-like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid-phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune-modulation agent.

Intranasal immunization with Ag85B peptide 25 displayed on Lactococcus lactis using the PilVax platform induces antigen-specific B- and T-cell responses.

Mycobacterium tuberculosis (Mtb) remains a global epidemic despite the widespread use of Bacillus Calmette-Guérin (BCG). Consequently, novel vaccines are required to facilitate a reduction in Mtb morbidity and mortality. PilVax is a peptide delivery strategy for the generation of highly specific mucosal immune responses and is based on the food-grade bacterium Lactococcus lactis that is used to express selected peptides engineered within the Streptococcus pyogenes M1T1 pilus, allowing for peptide amplification, stabilization and enhanced immunogenicity. In the present study, the dominant T-cell epitope from the Mtb protein Ag85B was genetically engineered into the pilus backbone subunit and expressed on the surface of L. lactis. Western blot and flow cytometry confirmed formation of pilus containing the peptide DNA sequence. B-cell responses in intranasally vaccinated mice were analyzed by ELISA while T-cell responses were analyzed by flow cytometry. Serum titers of peptide-specific immunoglobulin (Ig) G and IgA were detected, confirming that vaccination produced antibodies against the cognate peptide. Peptide-specific IgA was also detected across several mucosal sites sampled. Peptide-specific CD4+ T cells were detected at levels similar to those of mice immunized with BCG. PilVax immunization resulted in an unexpected increase in the numbers of CD3+ CD4- CD8- [double negative (DN)] T cells in the lungs of vaccinated mice. Analysis of cytokine production following stimulation with the cognate peptide showed the major cytokine producing cells to be CD4+ T cells and DN T cells. This study provides insight into the antibody and peptide-specific cellular immune responses generated by PilVax vaccination and demonstrates the suitability of this vaccine for conducting a protection study.

Publisher Correction: Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses.

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Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses.

The human pathogen Group A Streptococcus (GAS) produces pili that are involved in adhesion and colonisation of the host. These surface-exposed pili are immunogenic and therefore represent an attractive target for vaccine development. The pilus is encoded in the genomic region known as the fibronectin-collagen-T-antigen (FCT)-region, of which at least nine different types have been identified. In this study we investigate expressing two of the most common FCT-types (FCT-3 and FCT-4) in the food-grade bacteria Lactococcus lactis for use as a mucosal vaccine. We show that mucosally delivered L. lactis expressing GAS pili generates specific antibody responses in rabbits. Rabbit anti-pilus antibodies were shown to have both a neutralising effect on bacterial adhesion, and immunised rabbit antiserum was able to facilitate immune-mediated killing of bacteria via opsonophagocytosis. Furthermore, intranasal immunisation of mice improved clearance rates of GAS after nasopharyngeal challenge. These results demonstrate the potential for a novel, pilus-based vaccine to protect against GAS infections.