Quantity and Quality of Naturally Acquired Antibody Immunity to the Pneumococcal Proteome Throughout Life.

BACKGROUND: Young children and older adults are susceptible for invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Pneumococcal protein-specific antibodies play a protective role against IPD; however, not much is known about the pace of acquisition, maturation, and maintenance of these antibodies throughout life. METHODS: Immunoglobulin G (IgG) and IgA levels, avidity, and/or specificity to the pneumococcal proteome in serum and saliva from healthy young children, adults, and older adults, with known carriage status, were measured by enzyme-linked immunosorbent assay (ELISA) and 2-dimensional western blotting against ΔcpsTIGR4. RESULTS: Eleven-month-old children, the youngest age group tested, had the lowest pneumococcal proteome-specific IgG and IgA levels and avidity in serum and saliva, followed by 24-month-old children and were further elevated in adult groups. Among adult groups, the parents had the highest serum and saliva IgG and IgA antibody levels. In children, antibody levels and avidity correlated with daycare attendance and presence of siblings, posing as proxy for exposure and immunization. Immunodominance patterns slightly varied throughout life. CONCLUSIONS: Humoral immunity against the pneumococcal proteome is acquired through multiple episodes of pneumococcal exposure. Low-level and low-avidity antiproteome antibody profiles in young children may contribute to their IPD susceptibility, while in overall antiproteome antibody-proficient older adults other factors likely play a role.

Nanoflow LC-MS Method Allowing In-Depth Characterization of Natural Heterogeneity of Complex Bacterial Lipopolysaccharides.

The variable modification of the outer membrane lipopolysaccharide (LPS) in Gram-negative bacteria contributes to bacterial pathogenesis through various mechanisms, including the development of antibiotic resistance and evasion of the immune response of the host. Characterizing the natural structural repertoire of LPS is challenging due to the high heterogeneity, branched architecture, and strong amphipathic character of these glycolipids. To address this problem, we have developed a method enabling the separation and structural profiling of complex intact LPS mixtures by using nanoflow reversed-phase high-performance liquid chromatography (nLC) coupled to electrospray ionization Fourier transform mass spectrometry (ESI-FT-MSn). Nanogram quantities of rough-type LPS mixtures from Neisseria meningitidis could be separated and analyzed by nLC-ESI-FT-MS. Furthermore, the method enabled the analysis of highly heterogeneous smooth (S)-type LPS from pathogenic enteric bacteria such as Salmonella enterica serotype Typhimurium and Escherichia coli serotype O111:B4. High-resolution, accurate mass spectra of intact LPS containing various lengths of the O-specific polysaccharide in the range of 3 and 15 kDa were obtained. In addition, MS/MS experiments with collision-induced dissociation of intact LPS provided detailed information on the composition of oligo/polysaccharides and lipid A domains of single S-type LPS species. The structural heterogeneity of S-type LPS was characterized by unprecedented details. Our results demonstrate that nLC-ESI-FT-MSn is an attractive strategy for the structural profiling of small quantities of complex bacterial LPS mixtures in their intact form.

Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.

Systems vaccinology and big data in the vaccine development chain.

Systems vaccinology has proven a fascinating development in the last decade. Where traditionally vaccine development has been dominated by trial and error, systems vaccinology is a tool that provides novel and comprehensive understanding if properly used. Data sets retrieved from systems-based studies endorse rational design and effective development of safe and efficacious vaccines. In this review we first describe different omics-techniques that form the pillars of systems vaccinology. In the second part, the application of systems vaccinology in the different stages of vaccine development is described. Overall, this review shows that systems vaccinology has become an important tool anywhere in the vaccine development chain.

Specific T Cell Responses against Minor Histocompatibility Antigens Cannot Generally Be Explained by Absence of Their Allelic Counterparts on the Cell Surface.

Allogeneic stem cell transplantation has emerged as immunotherapy in the treatment of a variety of hematological malignancies. Its efficacy depends on induction of graft versus leukemia by donor lymphocytes. Both graft versus leukemia and graft versus host disease are induced by T cells reactive against polymorphic peptides, called minor histocompatibility antigens (MiHA), which differ between patient and donor and are presented in the context of self-HLA (where HLA is human leukocyte antigen). The allelic counterpart (AC) of the MiHA is generally considered to be absent at the cell surface, based on the absence of immune responses directed against the AC. To study this in detail, we evaluate the recognition, HLA-binding affinity, and cell surface expression of three selected MiHA. By quantitative MS, we demonstrate the similarly abundant expression of both MiHA and AC at the cell surface. We conclude that the absent recognition of the AC cannot generally be explained by insufficient processing and presentation at the cell surface of the AC.

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.

Proteome Analysis Is a Valuable Tool to Monitor Antigen Expression during Upstream Processing of Whole-Cell Pertussis Vaccines.

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of B. pertussis. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in B. pertussis, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of B. pertussis. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines.

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.

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD).

The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.

Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that (i) B29 administration by itself suppressed disease, (ii) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and (iii) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.

A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products.

Mixed-bed ion exchange chromatography employing a salt-free pH gradient for improved sensitivity and compatibility in MudPIT.

In proteomics, comprehensive analysis of peptides mixtures necessitates multiple dimensions of separation prior to mass spectrometry analysis to reduce sample complexity and increase the dynamic range of analysis. The main goal of this work was to improve the performance of (online) multidimensional protein identification technology (MudPIT) in terms of sensitivity, compatibility and recovery. The method employs weak anion and strong cation mixed-bed ion exchange chromatography (ACE) in the first separation dimension and reversed phase chromatography (RP) in the second separation dimension (Motoyama et.al. Anal. Chem 2007, 79, 3623-34.). We demonstrated that the chromatographic behavior of peptides in ACE chromatography depends on both the WAX/SCX mixing ratio as the ionic strength of the mobile phase system. This property allowed us to replace the conventional salt gradient by a (discontinuous) salt-free, pH gradient. First dimensional separation of peptides was accomplished with mixtures of aqueous formic acid and dimethylsulfoxide with increasing concentrations. The overall performance of this mobile phase system was found comparable to ammonium acetate buffers in application to ACE chromatography, but clearly outperformed strong cation exchange for use in first dimensional peptide separation. The dramatically improved compatibility between (salt-free) ion exchange chromatography and reversed phase chromatography-mass spectrometry allowed us to downscale the dimensions of the RP analytical column down to 25 µm i.d. for an additional 2- to 3-fold improvement in performance compared to current technology. The achieved levels of sensitivity, orthogonality, and compatibility demonstrates the potential of salt-free ACE MudPIT for the ultrasensitive, multidimensional analysis of very modest amounts of sample material.

Mass Spectrometry-Based Quantification of the Antigens in Aluminum Hydroxide-Adjuvanted Diphtheria-Tetanus-Acellular-Pertussis Combination Vaccines.

Vaccines undergo stringent batch-release testing, most often including in-vivo assays for potency. For combination vaccines, such as diphtheria-tetanus-pertussis (DTaP), chemical modification induced by formaldehyde inactivation, as well as adsorption to aluminum-based adjuvants, complicates antigen-specific in-vitro analysis. Here, a mass spectrometric method was developed that allows the identification and quantitation of DTaP antigens in a combination vaccine. Isotopically labeled, antigen-specific internal standard peptides were employed that permitted absolute quantitation of their antigen-derived peptide counterparts and, consequently, the individual antigens. We evaluated the applicability of the method on monovalent non-adjuvanted antigens, on final vaccine lots and on experimental vaccine batches, where certain antigens were omitted from the drug product. Apart from the applicability for final batch release, we demonstrated the suitability of the approach for in-process control monitoring. The peptide quantification method facilitates antigen-specific identification and quantification of combination vaccines in a single assay. This may contribute, as part of the consistency approach, to a reduction in the number of animal tests required for vaccine-batch release.

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells.

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1ß, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.

Aluminum Hydroxide And Aluminum Phosphate Adjuvants Elicit A Different Innate Immune Response.

Aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4) are widely used adjuvants in human vaccines. However, a rationale to choose one or the other is lacking since the differences between molecular mechanisms of action of these adjuvants are unknown. In the current study, we compared the innate immune response induced by both adjuvants in vitro and in vivo. Proteome analysis of human primary monocytes was used to determine the immunological pathways activated by these adjuvants. Subsequently, analysis of immune cells present at the site of injection and proteome analysis of the muscle tissue revealed the differentially regulated processes related to the innate immune response in vivo. Incubation with Al(OH)3 specifically enhanced the activation of antigen processing and presentation pathways in vitro. In vivo experiments showed that only intramuscular (I.M.) immunization with Al(OH)3 attracted neutrophils, while I.M. immunization with AlPO4 attracted monocytes/macrophages to the site of injection. In addition, only I.M. immunization with Al(OH)3 enhanced the process of hemostasis after 96 hours, possibly related to neutrophilic extracellular trap formation. Both adjuvants differentially regulated various immune system-related processes. The results show that Al(OH)3 and AlPO4 act differently on the innate immune system. We speculate that these different regulations affect the interaction with cells, due to the different physicochemical properties of both adjuvants.

Altered linkage of hydroxyacyl chains in lipid A of Campylobacter jejuni reduces TLR4 activation and antimicrobial resistance.

Modification of the lipid A moiety of bacterial lipopolysaccharide influences cell wall properties, endotoxic activity, and bacterial resistance to antimicrobial peptides. Known modifications are variation in the number or length of acyl chains and/or attached phosphoryl groups. Here we identified two genes (gnnA and gnnB) in the major foodborne pathogen Campylobacter jejuni that enable the synthesis of a GlcN3N precursor UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucopyranose (UDP-GlcNAc3N) in the lipid A backbone. Mass spectrometry of purified lipooligosaccharide verified that the gene products facilitate the formation of a 2,3-diamino-2,3-dideoxy-D-glucose (GlcN3N) disaccharide lipid A backbone when compared with the beta-1'-6-linked D-glucosamine (GlcN) disaccharide observed in Escherichia coli lipid A. Functional assays showed that inactivation of the gnnA or gnnB gene enhanced the TLR4-MD2-mediated NF-kappaB activation. The mutants also displayed increased susceptibility to killing by the antimicrobial peptides polymyxin B, colistin and the chicken cathelicidin-1. The gnnA and gnnB genes are organized in one operon with hemH, encoding a ferrochelatase catalyzing the last step in heme biosynthesis. These results indicate that lipid A modification resulting in amide-linked acyl chains in the lipid A is an effective mechanism to evade activation of the innate host defense and killing by antimicrobial peptides.

Novel mumps virus epitopes reveal robust cytotoxic T cell responses after natural infection but not after vaccination.

Mumps is nowadays re-emerging despite vaccination. The contribution of T cell immunity to protection against mumps has not been clearly defined. Previously, we described a set of 41 peptides that were eluted from human leukocyte antigen (HLA) class I molecules of mumps virus (MuV)-infected cells. Here, we confirmed immunogenicity of five novel HLA-B*07:02- and HLA-A*01:01-restricted MuV T cell epitopes from this set of peptides. High frequencies of T cells against these five MuV epitopes could be detected ex vivo in all tested mumps patients. Moreover, these epitope-specific T cells derived from mumps patients displayed strong cytotoxic activity. In contrast, only marginal T cell responses against these novel MuV epitopes could be detected in recently vaccinated persons, corroborating earlier findings. Identifying which MuV epitopes are dominantly targeted in the mumps-specific CD8+ T- response is an important step towards better understanding in the discrepancies between natural infection or vaccination-induced cell-mediated immune protection.

Genetic Analysis Reveals Differences in CD8+ T Cell Epitope Regions That May Impact Cross-Reactivity of Vaccine-Induced T Cells against Wild-Type Mumps Viruses.

Nowadays, mumps is re-emerging in highly vaccinated populations. Waning of vaccine-induced immunity plays a role, but antigenic differences between vaccine and mumps outbreak strains could also contribute to reduced vaccine effectiveness. CD8+ T cells play a critical role in immunity to viruses. However, limited data are available about sequence variability in CD8+ T cell epitope regions of mumps virus (MuV) proteins. Recently, the first set of naturally presented human leukocyte antigen Class I (HLA-I) epitopes of MuV was identified by us. In the present study, sequences of 40 CD8+ T cell epitope candidates, including previously and newly identified, obtained from Jeryl-Lynn mumps vaccine strains were compared with genomes from 462 circulating MuV strains. In 31 epitope candidates (78%) amino acid differences were detected, and in 17 (43%) of the epitope candidates the corresponding sequences in wild-type strains had reduced predicted HLA-I-binding compared to the vaccine strains. These findings suggest that vaccinated persons may have reduced T cell immunity to circulating mumps viruses due to antigenic differences.