Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells.

Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r2>0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs. 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs. 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood.

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

Introduction: The characterization of B. pertussis (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). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4+ T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8+ T cells were also analyzed. Results: We demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4+ T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ+ CD8+ T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses. Discussion: Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines.

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.

Uncovering Distinct Primary Vaccination-Dependent Profiles in Human Bordetella Pertussis 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 Bordetella pertussis (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 in vitro Bp antigen stimulation of diluted whole blood. We show for the first time that CD4+ T-cell memory of Th1, Th2, and Th17 lineages can be identified simultaneously in whole blood. Participants ranging from 7 to 70 years of age with different priming backgrounds of whole-cell pertussis (wP) and acellular pertussis (aP) vaccination were analyzed around an acellular booster vaccination. The assay allowed detection of low frequent antigen-specific CD4+ T-cells and revealed significantly elevated numbers of activated and cytokine-producing CD4+ T-cells, with a significant tendency to segregate recall responses based on primary vaccination background. A stronger Th2 response hallmarked an aP primed cohort compared to a wP primed cohort. In conclusion, analysis of Bp specific CD4+ T-cell responses in whole blood showed separation based on vaccination background and provides a promising tool to assess the quantity and quality of CD4+ T-cell responses induced by vaccine candidates.

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). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.

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. Unraveling the MuV epitope repertoire can provide insight in the specificity, functionality, and breadth of the T-cell response against MuV. METHODS: Peptides were eluted from human leukocyte antigen (HLA) class I molecules of MuV-infected cells and characterized by advanced mass spectrometry. Selected identified MuV peptides were tested for in vitro and ex vivo immunogenicity. RESULTS: In this study, we identified a broad landscape of 83 CD8+ T-cell epitopes of MuV, 41 of which were confirmed based on synthetic peptide standards. For 6 epitopes, we showed induction of an HLA-A*02-restriced CD8+ T-cell response. Moreover, robust T-cell responses against 5 selected MuV epitopes could be detected in all tested mumps patients using peptide/HLA-A*02:01 dextramers. CONCLUSIONS: The identified CD8+ T-cell epitopes will help to further characterize MuV-specific T-cell immunity after natural MuV infection or vaccination. These MuV epitopes may provide clues for a better understanding of, and possibly for preventing, mumps vaccine failure.We identified for the first time 41 mumps virus (MuV)-specific HLA-A*02 epitopes. For 6 epitopes, CD8+ T-cell responses were confirmed in T cells derived from several mumps cases, and MuV-specific CD8+ T cells could be identified by peptide/dextramer staining.

Impact of age and vaccination history on long-term serological responses after symptomatic B. pertussis infection, a high dimensional data analysis.

Capturing the complexity and waning patterns of co-occurring immunoglobulin (Ig) responses after clinical B. pertussis infection may help understand how the human host gradually loses protection against whooping cough. We applied bi-exponential modelling to characterise and compare B. pertussis specific serological dynamics in a comprehensive database of IgG, IgG subclass and IgA responses to Ptx, FHA, Prn, Fim2/3 and OMV antigens of (ex-) symptomatic pertussis cases across all age groups. The decay model revealed that antigen type and age group were major factors determining differences in levels and kinetics of Ig (sub) classes. IgG-Ptx waned fastest in all age groups, while IgA to Ptx, FHA, Prn and Fim2/3 decreased fast in the younger but remained high in older (ex-) cases, indicating an age-effect. While IgG1 was the main IgG subclass in response to most antigens, IgG2 and IgG3 dominated the anti-OMV response. Moreover, vaccination history plays an important role in post-infection Ig responses, demonstrated by low responsiveness to Fim2/3 in unvaccinated elderly and by elevated IgG4 responses to multiple antigens only in children primed with acellular pertussis vaccine (aP). This work highlights the complexity of the immune response to this re-emerging pathogen and factors determining its Ig quantity and quality.

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. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy.

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. Two methods based on IgG-Ptx plasma levels alone were used to evaluate the proportion of renewed seroconversions in the past year in a cohort of retrospective pertussis cases ≥ 24 months after a proven earlier symptomatic infection. A Dutch population database was used as a baseline. Applying a classical 62.5 IU/ml IgG-Ptx cut-off, we calculated a seroprevalence of 15% in retrospective cases, higher than the 10% observed in the population baseline. However, this method could not discriminate between renewed seroconversion and waning of previously infection-enhanced IgG-Ptx levels. Two-component cluster analysis of the IgG-Ptx datasets of both pertussis cases and the general population revealed a continuum of intermediate IgG-Ptx levels, preventing the establishment of a positive population and the comparison of prevalence by this alternative method. Next, we investigated the complementary serodiagnostic value of IgA-Ptx levels. When modelling datasets including both convalescent and retrospective cases we obtained new cut-offs for both IgG-Ptx and IgA-Ptx that were optimized to evaluate renewed seroconversions in the ex-cases target population. Combining these cut-offs two-dimensionally, we calculated 8.0% reinfections in retrospective cases, being below the baseline seroprevalence. Our study for the first time revealed the shortcomings of using only IgG-Ptx data in conventional serodiagnostic methods to determine pertussis reinfections. Improved results can be obtained with two-dimensional serodiagnostic profiling. The proportion of reinfections thus established suggests a relatively increased period of protection to renewed infection after clinical pertussis.

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.e.the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4(+)T cell epitopes relevant in health and disease.

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. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC-MS/MS. -Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV.

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. Altogether, 7897 unique self-peptides, derived of 4344 proteins, were eluted. After viral infection, the number of unique self-peptides eluted significantly decreased compared to uninfected cells, paralleled by a decrease in the number of source proteins. In the overall dataset, the total number of unique self-peptides eluted from HLA-B molecules was larger than from HLA-A molecules, and they were derived from a larger number of source proteins. These results in B cells suggest that HLA-B molecules possibly present a more diverse repertoire compared to their HLA-A counterparts, which may contribute to their immunodominance. This study provides a unique data set giving new insights into the complex system of antigen presentation for a broad panel of HLA molecules, many of which were never studied this extensively before.

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. We also report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may thus be important targets for immune surveillance.

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. Blood was collected before the 11-month booster or 7-9 days afterward. Serotype-specific immunoglobulin G (IgG) levels were determined in serum samples by multiplex immunoassay. Circulating specific PCs and Bmems against shared serotypes 1, 6B, 7F, and 19F and against PCV13 serotypes 6A and 19A were measured in peripheral blood mononuclear cells by enzyme-linked immunospot assay. RESULTS: No major differences in IgG levels and PC frequencies between groups were found for the 4 shared serotypes. Notably, PCV13 vaccination resulted in higher frequencies of Bmems than PCV10 vaccination, both before and after the booster dose, for all 4 shared serotypes except for serotype 1 postbooster. For PCV13-specific serotypes 6A and 19A, the IgG levels and frequencies of PCs and Bmems were higher in the PCV13 group, pre- and postbooster, except for PC frequencies prebooster. CONCLUSIONS: Both PCVs are immunogenic and induce measurable IgG, PC, and Bmem booster responses at 11 months. Compared to PCV10, vaccination with PCV13 was associated with overall similar IgG levels and PC frequencies but with higher Bmem frequencies before and after the 11-month booster. The clinical implications of these results need further follow-up. CLINICAL TRIALS REGISTRATION: NTR3069.

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. The well-known PTMs in Mtb are glycosylation, lipidation, or phosphorylation, known regulators of protein function or compartmentalization. Other PTMs include methylation, acetylation, and pupylation, involved in protein stability. While all PTMs add variability to the Mtb proteome, relatively little is understood about their role in the anti-Mtb immune responses. Here, we review Mtb protein PTMs and methods to assess their role in protective immunity against 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. Pertussis epitopes identified in the context of HLA-DR molecules were derived from two envelope proteins, i.e., putative periplasmic protein (PPP) and putative peptidoglycan-associated lipoprotein (PAL), and from two cytosolic proteins, i.e., 10-kDa chaperonin groES protein (groES) and adenylosuccinate synthetase (ASS). No epitopes were detectable from known virulence factors. CD4(+) T cell responsiveness in healthy adults against peptide pools representing epitope regions or full proteins confirmed the immunogenicity of PAL, PPP, groES, and ASS. Elevated lymphoproliferative activity to PPP, groES, and ASS in subjects within a year after the diagnosis of symptomatic pertussis suggested immunogenic exposure to these proteins during clinical infection. The PAL-, PPP-, groES-, and ASS-specific responses were associated with secretion of functional Th1 (tumor necrosis factor alpha [TNF-α] and gamma interferon [IFN-γ]) and Th2 (interleukin 5 [IL-5] and IL-13) cytokines. Relative paucity in the natural B. pertussis epitope display of MDDC, not dominated by epitopes from known protective antigens, can interfere with the effectiveness of immune recognition of B. pertussis. A more complete understanding of hallmarks in B. pertussis-specific immunity may advance the design of novel immunological assays and prevention strategies.

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. Memory B (Bmem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and Bmem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of Bmem cell populations; higher levels of Bmem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects' levels of specific Bmem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific Bmem cell and plasma IgG levels.

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 (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.

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. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.

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. While MV-M(211-219), MV-F(438-446) and MV-H(30-38) epitopes were already presented by HLA class I molecules early in infection, expression of MV-C(84-92) was restricted to the later phases of infection. These dynamics in epitope densities correlated with features of MV protein expression. Synthesis of C protein mainly focused towards the final stages of infection, while the other MV proteins were more readily synthesised from earlier time points on, in line with the emergence of their respective epitopes. Furthermore, the most abundant MV epitope was derived from the most unstable viral protein and vice versa, suggesting that the stability of viral proteins may be an indicator for the final abundance of their epitopes. Thus, even though many other factors may influence the generation of peptide-MHC class I complexes, we here report that the regulation of viral protein expression seems closely linked to the viral MHC class I epitope display. Finally, the observed dynamics in viral epitope hierarchy may have important implications for the induction of antiviral T cell immunity.