A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens.

Background: Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination.

A semi high-throughput whole blood-based flow cytometry assay to detect and monitor -specific Th1, Th2 and Th17 responses.

Introduction: The characterization of (Bp) antigen-specific CD4 T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays.

Methods: To overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation).

Uncovering Distinct Primary Vaccination-Dependent Profiles in Human Specific CD4+ T-Cell Responses Using a Novel Whole Blood Assay.

To advance research and development of improved pertussis vaccines, new immunoassays are needed to qualify the outcome of (Bp) specific CD4+ T-cell differentiation. Here, we applied a recently developed whole blood assay to evaluate Bp specific CD4+ T-cell responses. The assay is based on intracellular cytokine detection after overnight Bp antigen stimulation of diluted whole blood.

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube.

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models-monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID).

Identification of Naturally Processed Mumps Virus Epitopes by Mass Spectrometry: Confirmation of Multiple CD8+ T-Cell Responses in Mumps Patients.

Background: The re-emergence of mumps among vaccinated young adults has become a global issue. Besides waning of antibody responses, suboptimal induction of T-cell responses may reduce protection. In a recent study, we observed a dominant polyfunctional CD8+ T-cell response after natural mumps virus (MuV) infection that was not present after vaccination.

Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07.

Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail.

The Use of Innovative Two-Component Cluster Analysis and Serodiagnostic Cut-Off Methods to Estimate Prevalence of Pertussis Reinfections.

Bordetella pertussis circulates even in highly vaccinated countries affecting all age groups. Insight into the scale of concealed reinfections is important as they may contribute to transmission. We therefore investigated whether current single-point serodiagnostic methods are suitable to estimate the prevalence of pertussis reinfection.

Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity.

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4(+)T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides,i.

Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation.

Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8(+) cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8(+) CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules.

Comprehensive Analysis of the Naturally Processed Peptide Repertoire: Differences between HLA-A and B in the Immunopeptidome.

The cytotoxic T cell (CTL) response is determined by the peptide repertoire presented by the HLA class I molecules of an individual. We performed an in-depth analysis of the peptide repertoire presented by a broad panel of common HLA class I molecules on four B lymphoblastoid cell-lines (BLCL). Peptide elution and mass spectrometry analysis were utilised to investigate the number and abundance of self-peptides.

Extended O-GlcNAc on HLA Class-I-Bound Peptides.

We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc's rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane and is hence exposed to glycosyltransferases.

Differential B-cell memory around the 11-month booster in children vaccinated with a 10- or 13-valent pneumococcal conjugate vaccine.

Background: Both the 10- and 13-valent pneumococcal conjugate vaccines (PCV10 and PCV13) induce immunological memory against Streptococcus pneumoniae infections caused by vaccine serotypes. In addition to comparing serum antibody levels, we investigated frequencies of serotype-specific plasma cells (PCs) and memory B-cells (Bmems) as potential predictors of long-term immunity around the booster vaccination at 11 months of age.

Methods: Infants were immunized with PCV10 or PCV13 at 2, 3, 4, and 11 months of age.

Toward Understanding the Essence of Post-Translational Modifications for the Mycobacterium tuberculosis Immunoproteome.

CD4(+) T cells are prominent effector cells in controlling Mycobacterium tuberculosis (Mtb) infection but may also contribute to immunopathology. Studies probing the CD4(+) T cell response from individuals latently infected with Mtb or patients with active tuberculosis using either small or proteome-wide antigen screens so far revealed a multi-antigenic, yet mostly invariable repertoire of immunogenic Mtb proteins. Recent developments in mass spectrometry-based proteomics have highlighted the occurrence of numerous types of post-translational modifications (PTMs) in proteomes of prokaryotes, including Mtb.

Bordetella pertussis proteins dominating the major histocompatibility complex class II-presented epitope repertoire in human monocyte-derived dendritic cells.

Knowledge of naturally processed Bordetella pertussis-specific T cell epitopes may help to increase our understanding of the basis of cell-mediated immune mechanisms to control this reemerging pathogen. Here, we elucidate for the first time the dominant major histocompatibility complex (MHC) class II-presented B. pertussis CD4(+) T cell epitopes, expressed on human monocyte-derived dendritic cells (MDDC) after the processing of whole bacterial cells by use of a platform of immunoproteomics technology.

Age related differences in dynamics of specific memory B cell populations after clinical pertussis infection.

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture.

Hyperproliferation of B cells specific for a weakly immunogenic PorA in a meningococcal vaccine model.

Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity against Neisseria meningitidis group B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes.

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag.

Dynamics of measles virus protein expression are reflected in the MHC class I epitope display.

Following measles virus (MV) infection, viral peptides are presented to CTL by MHC class I molecules on infected antigen presenting cells at widely different epitope densities. Whereas three MV epitopes (MV-M(211-219), MV-F(438-446) and MV-H(30-38)) derived from different structural proteins occur at regular densities, one peptide derived from the non-structural C protein (MV-C(84-92)) fully dominates the MV peptide display in HLA class I molecules on end-stage-infected human B cells. Here we demonstrate that this hierarchy in MV epitope density is not a constant, but varies with progression of infection.

The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1.

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1(vhs-) deletion mutant.