Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

Gynecological cancers are a leading cause of mortality in women. CD8+ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8+ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8+ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8+ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8+ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8+ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8+ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers

T Lymphocytes from a Subset of Patients with Pemphigus Vulgaris Respond to Both Desmoglein-3 and Desmoglein-1

Pemphigus vulgaris and pemphigus foliaceus are cutaneous autoimmune diseases characterized by intraepithelial blisters and autoantibodies to desmosomal glycoproteins. The antigens recognized by pemphigus vulgaris and pemphigus foliaceus autoantibodies are desmoglein- 3 (Dsg3) and desmoglein-1 (Dsg1), respectively. Dsg3 and Dsg1 are members of the desmoglein subfamily of the cadherin supergene family of cell adhesion molecules. It has been well documented that a subset of pemphigus vulgaris sera have IgG reactivity to both Dsg1 and Dsg3, suggesting that Dsg1 may also participate in the autoimmune response of these patients. The cellular mechanisms of T cell autoimmunity in these patients, however, are completely unknown. In this study, we tested the proliferative responses of T lymphocytes from eight pemphigus vulgaris patients after incubation with Dsg3 and Dsg1 fusion proteins. The sera of four of these PV patients showed reactivity with both Dsg1 and Dsg3, whereas the remaining four reacted only with Dsg3. We found that T cells obtained from those patients that exhibited the combined Dsg1/Dsg3 autoantibody reactivity showed a proliferative response after exposure to either Dsg1 or Dsg3 fusion proteins. The cellular responses to both of these recombinant proteins were highly specific and restricted to the CD4-positive T cell population. T cells from pemphigus vulgaris patients with no anti-Dsg1 serum reactivity showed a proliferative response to Dsg3, but not to Dsg1. The Dsg1 fusion protein used in this study has minimal sequence homology with Dsg3. Thus, this study provides the first evidence that T cells from a subset of pemphigus vulgaris patients respond to both Dsg1 and Dsg3

Engineered hydrogen-bonded glycopolymer capsules and their interactions with antigen presenting cells

Hollow glycopolymer microcapsules were fabricated by hydrogen-bonded layer-by-layer (LbL) assembly, and their interactions with a set of antigen presenting cells (APCs), including dendritic cells (DCs), macrophages (MACs), and myeloid derived suppressor cells (MDSCs), were investigated. The glycopolymers were obtained by cascade postpolymerization modifications of poly(oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate) involving the modification of the glycidyl groups with propargylamine and the subsequent attachment of mannose azide by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC). Multilayer assembly of the hydrogen-bonding pair (glycopolymer/poly(methacrylic acid) (PMA)) onto planar and particulate supports (SiO2 particles, d = 1.16 μm) yielded stable glycopolymer films upon cross-linking by CuAAC. The silica (SiO2) particle templates were removed yielding hollow monodisperse capsules, as demonstrated by fluorescence and scanning electron microscopy. Cellular uptake studies using flow cytometry revealed the preferential uptake of the capsules by DCs when compared to MACs or MDSCs. Mannosylated capsules showed a cytokine independent cis-upregulation of CD80 specifically on DCs and a trans-downregulation of PDL-1 on MDSCs. Thus, the glycopolymer capsules may have potential as vaccine carriers, as they are able to upregulate costimulatory molecules for immune cell stimulation on DCs and at the same time downregulate immune inhibitory receptors on suppressor APC such as MDSCs

Post-translational modification directs nuclear and hyphal tip localization of Candida albicans mRNA-binding protein Slr1

The morphological transition of the opportunistic fungal pathogen Candida albicans from budding to hyphal growth has been implicated in its ability to cause disease in animal models. Absence of SR-like RNA-binding protein Slr1 slows hyphal formation and decreases virulence in a systemic candidiasis model, suggesting a role for post-transcriptional regulation in these processes. SR (serine–arginine)-rich proteins influence multiple steps in mRNA metabolism and their localization and function are frequently controlled by modification. We now demonstrate that Slr1 binds to polyadenylated RNA and that its intracellular localization is modulated by phosphorylation and methylation. Wildtype Slr1-GFP is predominantly nuclear, but also co-fractionates with translating ribosomes. The non-phosphorylatable slr1-6SA-GFP protein, in which six serines in SR/RS clusters are substituted with alanines, primarily localizes to the cytoplasm in budding cells. Intriguingly, hyphal cells display a slr1-6SA-GFP focus at the tip near the Spitzenkörper, a vesicular structure involved in molecular trafficking to the tip. The presence of slr1-6SA-GFP hyphal tip foci is reduced in the absence of the mRNA-transport protein She3, suggesting that unphosphorylated Slr1 associates with mRNA–protein complexes transported to the tip. The impact of SLR1 deletion on hyphal formation and function thus may be partially due to a role in hyphal mRNA transport

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films

Daily assessment of the percentage of erythrocytes that are infected ('percent-parasitaemia') across a time-course is a necessary step in many experimental studies of malaria, but represents a time-consuming and unpopular task among researchers. The most common method is extensive microscopic examination of Giemsa-stained thin blood-films. This study explored a method for the assessment of percent-parasitaemia that does not require extended periods of microscopy and results in a descriptive and permanent record of parasitaemia data that is highly amenable to subsequent 'data-mining'. Digital photography was utilized in conjunction with a basic purpose-written computer programme to test the viability of the concept. Partial automation of the determination of percent parasitaemia was then explored, resulting in the successful customization of commercially available broad-spectrum image analysis software towards this aim. Lastly, automated discrimination between infected and uninfected RBCs based on analysis of digital parameters of individual cell images was explored in an effort to completely automate the calculation of an accurate percent-parasitaemia

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50–80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant

Integrated analysis of carbon dioxide and oxygen concentrations as a quality control of ocean float data

The distributions of dissolved O2 and CO2 have not previously been systematically compared across the global surface ocean, despite their significance for life and climate. Here we analyze carbon dioxide and oxygen concentrations relative to saturation (equilibrium with the atmosphere) in surface waters, using two large datasets (ship-collected and float-collected data). When applied to a high-quality global ship-collected dataset, CO2 and O2 concentrations relative to saturation exhibit large seasonal and geographic variations. However, linear fits of CO2 and O2 deviations from saturation (ΔCO2 against ΔO2) yield y-intercepts close to zero, which suggests a requirement for data validity. We utilize this finding to investigate the accuracy of carbonate system data from biogeochemical-Argo floats. We find significant discrepancies in ΔCO2-ΔO2 y-intercepts compared to the global reference, implying overestimations of float-based CO2 release in the Southern Ocean. We conclude that this technique can be applied to data from autonomous platforms for quality assessment

Agrobacterium tumefaciens-Mediated Transformation of Maize Embryos Using a Standard Binary Vector System

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciensstandard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L−1 l-cysteine. Inclusion of l-cysteine in cocultivation medium lead to an improvement in transient β-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar andgustransgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciensstandard binary vector system

The Inhibition of Polo Kinase by Matrimony Maintains G2 Arrest in the Meiotic Cell Cycle

Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function