For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens.

Summary. Background: Endothelial cell protein C receptor (EPCR) binds protein C through its c-carboxyglutamic acid (Gla) domain and enhances its thrombin-thrombomodulin complex-dependent activation. So far, only protein C/ activated protein C has been shown to interact with EPCR. Factor VII (FVII), the coagulation trigger upon tissue factor (TF) interaction, is a serine protease whose Gla domain is highly homologous to the Gla domain of protein C. Objectives: To characterize the binding of FVII/FVIIa to EPCR and its functional consequences. Methods and results:We demonstrated by surface plasmon resonance (SPR) that FVII/FVIIa binds to EPCR through its Gla domain. At therapeutic concentrations, FVIIa reduced the activation of protein C by 40%. Soluble EPCR (sEPCR) was also able to prolong dose-dependently the clotting time induced by the FVIIa-TF complex. SPR and amidolytic experiments showed that FVIIa is able to interact simultaneously with TF and EPCR, thus ruling out the possibility that the effect of EPCR on clotting time was due to the inhibition of the binding between FVIIa and TF. sEPCR inhibited dose-dependently the activation of FX by the FVIIa-TF complex. Notably, blocking the binding site of EPCR on the endothelial surface increased the generation of FXa 2-fold. Conclusions: EPCR binds to FVII/ FVIIa and inhibits the procoagulant activity of the FVIIa-TF complex.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, crossspecies reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αβ TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR β-chain contacts are mostly through the variable CDR3β loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/ MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αβ T-cell recognition of MHC and MHC-like ligands.M ucosal-associated invariant T (MAIT) cells are a highly conserved T-cell subset found in most mammalian species (1-4). In humans, they can constitute up to 10% of circulating double-negative T cells, although they are much less frequent in mice (1,5,6). Most MAIT cells lack expression of the CD4 or CD8 coreceptors, although many MAIT cells express the αα form of the CD8 coreceptor (1). In humans, these cells are found at moderate frequency in the intestine and represent up to ∼50% of T cells in the liver (7). The cells exhibit an effector-memory phenotype and express the CD161 receptor (6). Their presence as mature effector cells in the periphery is dependent on B cells and the gut commensal flora (6, 8). Stimulated human MAIT cells can express both proinflammatory cytokines (IFN-γ, TNF-α, and IL-17) and cytolytic effectors (granzyme B) (7, 9, 10). MAIT cells are known best for their reactivity against various microorganisms from both bacterial and fungal origin (9, 10). These microorganisms include several important human pathogens, such as Mycobacterium tuberculosis, Salmonella typhimurium, and Staphylococcus aureus. Indeed, a significant proportion of the nonclassically restricted responding T cells in M. tuberculosisinfected individuals were determined to be of the MAIT lineage (9). MAIT cells have also demonstrated autoreactivity and have been associated with various autoimmune disorders (11, 12); they have also been found in both k...

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Invariant Natural Killer T (iNKT) cells use highly restricted αβ T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted α1 helix resulting in an open A' pocket. Binding of the iNKT TCR requires a 7-Å displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3α loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3β and Jβ segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

MR1-restricted Mucosal Associated Invariant T (MAIT) cells represent a sub-population of αβ T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest due to their reactivity against bacterial and yeast species, suggesting they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-antigen complexes is unclear. Here we present our structural and biochemical characterization of MAIT TCR engagement of MR1 presenting an E. coli-derived stimulatory ligand, rRL-6-CH2OH previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved binding orientation, with a clear role for both the CDR3α and CDR3β loops in recognition of the rRL-6-CH2OH stimulatory ligand. We also present two additional xeno-reactive MAIT TCR/MR1 complexes that recapitulate the docking orientation documented previously despite having variation in the CDR2β and CDR3β loop sequences. Our data supports a model by which MAIT TCRs engage MR1 in a conserved fashion, their binding affinities modulated by the nature of the MR1 presented antigen or diversity introduced by alternate Vβ usage or CDR3β sequences.

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-β1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-β1 and TGF-β2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-β may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.