Structural vaccinology is an emerging strategy for the rational design of vaccine candidates. We successfully applied structural vaccinology to design a fully synthetic protein with multivalent protection activity. In Group B Streptococcus, cell-surface pili have aroused great interest because of their direct roles in virulence and importance as protective antigens. The backbone subunit of type 2a pilus (BP-2a) is present in six immunogenically different but structurally similar variants. We determined the 3D structure of one of the variants, and experimentally demonstrated that protective antibodies specifically recognize one of the four domains that comprise the protein. We therefore constructed a synthetic protein constituted by the protective domain of each one of the six variants and showed that the chimeric protein protects mice against the challenge with all of the type 2a pilus-carrying strains. This work demonstrates the power of structural vaccinology and will facilitate the development of an optimized, broadly protective pilus-based vaccine against Group B Streptococcus by combining the uniquely generated chimeric protein with protective pilin subunits from two other previously identified pilus types. In addition, this work describes a template procedure that can be followed to develop vaccines against other bacterial pathogens.backbone protein | isopeptide bonds | homology modeling

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics. IntroductionDendritic cells (DCs) are arrayed with diverse pathogen sensors (eg, Toll-like receptors (TLR)) and reside in tissues throughout the body, rendering them uniquely poised to detect invading pathogens. 1,2 During the initiation and amplification of the immune response, DCs rally other cells of both the innate and adaptive immune systems for the elimination of infections. 3,4 In the context of different infections, DC populations are also critical in determining the quality of the response through the efficient and rapid production of discrete subsets of cytokines, chemokines, and interferons (IFNs), which selectively direct the recruitment and activation of other immune effectors. 3,4 Because DCs are key antigen-presenting cells (APCs), the instructive role of DC soluble factors shapes adaptive immunity in various ways, resulting in focused and optimized antigen-specific responses to different pathogen classes (eg, viruses vs bacteria). 5,6 There are numerous distinct DC populations that vary in their tissue distribution, cytokine/chemokine secretion, and/or their interactions with infectious agents and other cells of the host. [7][8][9][10] Of these, blood myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) represent 2 well-characterized populations that differ in their morphology, phenotype, TLR expression, and cytokine, chemokine, and type I IFN production. [10][11][12][13][14] These differences imply that mDCs and pDCs have evolved to sense distinct classes of pathogens and selectively steer subsequent innate and adaptive immunity. Even though both DC types are considered effective APCs, 11,15 the nonoverlapping distribution of TLRs and the pattern of cytokine production in human mDCs and pDCs suggest specialized and perhaps complementary functi...

Group B Streptococcus (GBS) is cause of neonatal invasive diseases as well as of severe infections in the elderly and immune-compromised patients. Despite significant advances in the prevention and treatment of neonatal disease, sepsis and meningitis caused by GBS still represent a significant public health care concern globally and additional prevention and therapeutic strategies against infection are highly desirable. The introduction of national recommended guidelines in several countries to screen pregnant women for GBS carriage and the use of antibiotics during delivery significantly reduced disease occurring within the first hours of life (early-onset disease), but it has had no effect on the late-onset diseases occurring after the first week and is not feasible in most countries. Availability of an effective vaccine against GBS would provide an effective means of controlling GBS disease. This review provides an overview of the burden of invasive disease caused by GBS in infants and adults, and highlights the strategies for the development of an effective vaccine against GBS infections.

In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.