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More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus, resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N. americanus and A. ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N. americanus and A. ceylanicum, respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis, and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease. Author summary: Human hookworm infections constitute a significant global health burden, and a vaccine is urgently needed to overcome limitations in current management strategies. No current proteomic study on the secretome of hookworm infective larvae exists and this stage represents a distinct and crucial window where the parasite first encounters the immune system during infection. Identifying excretory/secretory proteins associated with this stage of the lifecycle uncovers novel targets for vaccines and diagnostics. This study comprehensively characterizes and compares excretory/secretory proteins from Ancylostoma ceylanicum and Necator americanus adult hookworms and infective larvae using mass spectrometry. We found that A. ceylanicum and N. americanus infective larvae predominantly express excretory/secretory proteins that are unique to that developmental stage (i.e. not detected in the adult stage secretome) and are likely tailored to the skin and lung environments through which the larvae traverse. This proteome offers a new set of vaccine and diagnostic targets unique to infective hookworm larvae. We also identify several conserved and diversified proteins across both the adult and infective larvae stage of either hookworm. Conserved proteins could represent proteins essential to common functions and may be ideal candidates for a pan-hookworm vaccine. [ABSTRACT FROM AUTHOR]

Spirulina is an edible cyanobacterium that increasingly gaining recognition for it untapped potential in the biomanufacturing of pharmaceuticals. Despite the rapidly accumulating information on extracellular vesicles (EVs) from most other bacteria, nothing is known about Spirulina extracellular vesicles (SPEVs). This study reports the successful isolation, characterization and visualization of SPEVs for the first time and it further investigates the potential therapeutic benefits of SPEVs using a mouse model. SPEVs were isolated using ultracentrifugation and size‐exclusion‐chromatography. Cryo‐Transmission Electron Microscopy revealed pleomorphic outer‐membrane‐vesicles and outer‐inner‐membrane‐vesicles displaying diverse shapes, sizes and corona densities. To assess short‐ and long‐term immune responses, mice were injected intraperitoneally with SPEVs, which demonstrated a significant increase in neutrophils and M1 macrophages at the injection site, indicating a pro‐inflammatory effect induced by SPEVs without clinical signs of toxicity or hypersensitivity. Furthermore, SPEVs demonstrated potent adjuvanticity by enhancing antigen‐specific IgG responses in mice by over 100‐fold compared to an unadjuvanted model vaccine antigen. Mass‐spectrometry identified 54 proteins within SPEVs, including three protein superfamily members linked to the observed pro‐inflammatory effects. Our findings highlight the potential of SPEVs as a new class of vaccine adjuvant and warrant additional studies to further characterize the nature of the immune response. [ABSTRACT FROM AUTHOR]

Introduction [CD8.sup.+] T cells recognize short peptide fragments presented by MHC class I (MHC-I) molecules on the surface of nucleated cells (1-3). These peptide-MHC-I (pMHC-I) molecular arrays are scanned by [...], Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial [CD8.sup.+] T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific [CD8.sup.+] T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.

Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34 + hematopoietic stem cells. These "humanized" mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs) in mice are categorized into cDC1, which mediate T helper (Th)1 and CD8 + T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141 + DC and CD1c + DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141 + DC and CD1c + DC develop in humanized mice, to further explore their equivalency in vivo . Global transcriptome analysis of CD141 + DC and CD1c + DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo , with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c + DC, while poly I:C upregulated IFN-λ genes specifically by CD141 + DC. MYCL expression, known to be essential for CD8 + T cell priming by mouse DC, was specifically induced in CD141 + DC after activation. Concomitantly, CD141 + DC were superior to CD1c + DC in their ability to prime naïve antigen-specific CD8 + T cells. Thus, CD141 + DC and CD1c + DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8 + T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study human DC in vitro and in vivo .