Immunoproteomic Approach Research Articles

Immunoproteomic Identification of Graft-Versus-Leukemia Targets in Patients with Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is susceptible to immune-mediated destruction, as demonstrated by the potentially curative graft-versus-leukemia (GvL) effects of allogeneic hematopoietic stem cell transplant and donor lymphocyte infusion (DLI). Identification of the target antigens of GvL may lead to the development of novel reagents to monitor, diagnose or vaccinate against CLL. Our previous studies suggest that B cell responses are critical elements of successful GvL following DLI. In the current study, we used plasma from 2 patients who achieved durable complete remission after DLI for relapsed CLL as a source of immunoglobulin to probe high-density protein microarrays that express ∼5000 opening reading frames. As neither patient developed graft-versus-host disease (GvHD) after DLI, we hypothesized that immunologic dissection of responses in these patients would lead to the identification of CLL-associated antigens. Our studies identified 16 and 21 antigens with significantly higher reactivity post- versus pre-DLI for patients A and B, respectively. Specificity of candidate antigen-antibody interactions was validated by immunoprecipitation of individual recombinant proteins with patient plasma. Of 29 candidate antigen interactions tested, 21 (72%) were reactive, whereas all 5 control antigen interactions were unreactive. While most of the candidate antigens represent intracellular proteins of known function, nine are unknown. Of the known antigens, 8 bind nucleic acid and 2 are tumor suppressors, while others play roles in cellular functions such as ubiquitination, spermatogenesis, cell cycle, and apoptosis. Three proteins (C6orf130, MDS032, ZFYVE19) were recognized by both patients and therefore represent possible shared targets of anti-CLL immunity. By quantitative RT-PCR, all 3 shared antigens were more significantly expressed in malignant B cells than peripheral blood mononuclear cells, while one candidate, OGFOD1, was more significantly expressed in CLL than B cells, suggesting that immunogenicity may result from overexpression. Serologic characterization of reactivity patterns showed ZFYVE19 and DAPK3, another candidate, to be commonly immunogenic, as they elicited reactivity in 3 of 12 CLL patients in remission without GvHD post-transplant, in 0 of 12 normal volunteers and in only 1 of 12 untreated CLL patients. Confirming the close association of humoral immunity to GvL effects, histopathologic tumor regression was temporally related to presence of antibody reactivity against ZFYVE19 in both patients, to DAPK3 in patient A, and to OGFOD1 in patient B. Our results demonstrate that clinically evident anti-tumor immune responses in CLL are associated with the development of polyclonal humoral immunity, and that an immunoproteomic approach can rapidly identify CLL-associated antigens that are possible informative biomarkers of effective anti-CLL immunity. Ongoing studies will define the extent to which these targets elicit coordinated T cell mediated tumor rejection, and will elucidate their potential as novel immunogens for defined-antigen vaccines for CLL.

Immunoproteomics approach for identification of novel allergens of aspergillus fumigatus

Immunoproteomics approach for identification of novel allergens of aspergillus fumigatus

Chlamydia trachomatis Species-Specific Induction of Ezrin Tyrosine Phosphorylation Functions in Pathogen Entry

Chlamydia trachomatis is an obligate intracellular pathogen of humans that exhibits species-specific biological characteristics in its early interactions with host cells that are likely important to pathogenesis. One such characteristic is the tyrosine phosphorylation (Tyr-P) of an approximately 70-kDa polypeptide that occurs only after infection of mammalian cells by human strains. We sought to identify this protein because of its potential significance to the pathogenesis of human chlamydial infections. Using an immunoproteomic approach we identified the host protein ezrin, a member of the ezrin-radixin-moesin (ERM) protein family that serves as a physical link between host cell receptors and the actin cytoskeleton. Confocal microscopy studies showed colocalization of ezrin and actin at the tips and crypts of microvilli, the site of chlamydial attachment and entry, respectively. To demonstrate a functional role for ezrin we infected cells with a dominant-negative (DN) ezrin phenotype or treated cells with ezrin-specific small interfering RNA (siRNA). We found that both DN and siRNA-treated cells were significantly less susceptible to infection by human chlamydial strains. Moreover, we demonstrated that inhibition of infection in ezrin DN cells occurred at the stage of chlamydial entry. We hypothesize that the C. trachomatis-specific Tyr-P of ezrin might relate to an undefined species-specific mechanism of pathogen entry that involves chlamydial specific ligand(s) and host cell coreceptor usage.

Characterization of discriminant human brain antigenic targets in neuropsychiatric systemic lupus erythematosus using an immunoproteomic approach

To characterize discriminant human brain antigenic targets in patients with neuropsychiatric systemic lupus erythematosus (NPSLE), using a standardized immunoproteomic approach. Self-IgG reactivity against normal and injured human brain tissues was studied by Western blotting of sera from 169 subjects, 16 patients with NPSLE, 12 patients with SLE without neuropsychiatric manifestations (non-NPSLE), 32 patients with Sjögren's syndrome with or without central nervous involvement, 82 patients with multiple sclerosis, and 27 healthy subjects. A proteomic approach was then applied to characterize discriminant antigens identified after comparisons of all patterns. The serum self-IgG reactivity patterns against human brain tissue differed significantly between patients with NPSLE and the control groups. Four normal brain antigenic bands were specifically or preferentially recognized by sera from NPSLE patients (p240, p90, p77, and p24). Protein band p240 was characterized as microtubule-associated protein 2B (MAP-2B), p77 as Hsp70-71, and p24 as triosephosphate isomerase. Protein band p90 was not characterized. In contrast, 1 other protein band (p56, characterized as septin 7) was never recognized by sera from NPSLE patients but was recognized by a majority of sera from non-NPSLE patients. Our findings show that the immunoproteomic approach is a reliable method for assessing serum self-IgG reactivities against human brain tissue in NPSLE. Our characterization of some of the identified discriminant antigens, such as MAP-2B, triosephosphate isomerase, and septin 7, suggests that the stability of neuronal microtubules might be involved in the pathophysiology of NPSLE.

Immunoproteomic Assay of Membrane‐associated Proteins ofStreptococcus suisType 2 China Vaccine Strain HA9801

Immunoproteomic approaches were undertaken to study the immunogenicity of the membrane-associated proteins of the Streptococcus suis type 2 (SS2) China vaccine strain HA9801. The membrane-associated proteins were enriched using the Triton X-114 extraction protocol and were analysed by two-dimensional gel electrophoresis (2-DE) and subsequent immunoblotting using the hyperimmune serum of SS2-HA9801-immunized specific pathogen free (SPF) minipigs. A total of 11 proteins were recognized, and the corresponding spots on a duplicate gel were excised and identified by MALDI-TOF MS.

Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

Approximately 20% of the world's asthmatics are suffering from Aspergillus fumigatus (Afu)-induced allergies. The characterization of specific IgE-inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy. Immunoproteomics combined with mass spectrometric analysis was used to identify proteins of third-week culture filtrate (3wcf) potentially responsible for Afu-specific IgE immunoreactivity, using pooled sera from Afu-sensitized asthmatics. Their allergenic potential was also tested against patients with allergic bronchopulmonary aspergillosis (ABPA), by two-dimensional (2-D) gel electrophoresis immunoblotting of 3wcf proteins with individual sera from such patients. This helped us to establish a set of candidate allergens, which could be explored further for diagnostic application in allergic aspergillosis asthmatics including ABPA. Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or de novo sequencing by MS/MS analysis of the protein spots from 2-D gels led to the identification of a total of 16 allergens of Afu. Eleven of them are being reported as allergens for the first time and five had been reported earlier. Putative isoforms of the proteins Asp f 13 and chitosanase have been observed for the first time. When studied for reactivity of these proteins among patients with ABPA using their individual sera, these patients exhibited sensitization although the pattern was varying. Taken together, these proteins could thus be considered as potential allergens even among patients with ABPA. Three of these proteins viz. the hypothetical protein (# spot no. 5), extracellular arabinase (# spot no. 6) and chitosanase (# spot no. 11) could be major allergens with specific IgE immunoreactivity with six out of eight patients' sera. The immunoproteomic approach applied to the analysis of culture filtrate proteins resulted in the identification of several candidate allergens, many of them novel, contributing to the catalogue of Afu allergenic proteins, which would facilitate improved serodiagnosis for allergic aspergillosis. In addition, the immunoreactivity of these proteins observed among the patients with ABPA may be potentially useful for its serodiagnosis and opens up further opportunities for the development of personalized immunotherapeutics for patients with ABPA.

Uropathogenic Escherichia coli Outer Membrane Antigens Expressed during Urinary Tract Infection

Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) represents a prevalent and potentially severe infectious disease. In this study, we describe the application of an immunoproteomics approach to vaccine development that has been used successfully to identify vaccine targets in other pathogenic bacteria. Outer membranes were isolated from pyelonephritis strain E. coli CFT073 cultured under conditions that mimic the urinary tract environment, including iron limitation, osmotic stress, human urine, and exposure to uroepithelial cells. To identify antigens that elicit a humoral response during experimental UTI, outer membrane proteins were separated by two-dimensional gel electrophoresis and probed using pooled antisera from 20 CBA/J mice chronically infected with E. coli CFT073. In total, 23 outer membrane antigens, including a novel iron compound receptor, reacted with the antisera and were identified by mass spectrometry. These antigens also included proteins with known roles in UPEC pathogenesis, such as ChuA, IroN, IreA, Iha, IutA, and FliC. These data demonstrate that an antibody response is directed against these virulence-associated factors during UTI. We also show that the genes encoding ChuA, IroN, hypothetical protein c2482, and IutA are significantly more prevalent (P < 0.01) among UPEC strains than among fecal-commensal E. coli isolates. Thus, we suggest that the conserved outer membrane antigens identified in this study could be rational candidates for a UTI vaccine designed to elicit protective immunity against UPEC infection.

Proteomic analysis of antibody response in a case of laboratory-acquired infection withFrancisella tularensis subsp.tularensis

Immunoproteomic analysis was applied to study the immunoreactivity of serum samples collected at different time points from a laboratory assistant accidentally infected with highly virulent strain of Francisella tularensis subsp. tularensis. Immunoblotting showed that the spectrum of F. tularensis antigens recognized specifically by immune sera remained with the exception for 1 antigen stable for up to 16 years after infection. Using immunoproteomics approach 10 immunoreactive antigens were successfully identified. Several new immunogenic F. tularensis proteins were described for the first time.

Immunization of Pigs To Prevent Disease in Humans: Construction and Protective Efficacy of a Salmonella enterica Serovar Typhimurium Live Negative-Marker Vaccine

Zoonotic infections caused by Salmonella enterica serovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcDeltaompD, were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcDeltaompD and challenge with a multiresistant S. enterica serovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcDeltaompD in pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F(100) to Y(108)) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.

Reactivity of an Anti NS3-HCV-Induced Autoantibody in HCV-Positive Gastric Tissue by 2D Gel Immunoblot.

In a previous study we demonstrated an IgM cross reactivity against Fc region of human IgG and HCV-NS3 antigen in HCV-induced type II mixed cryoglobulinemia (MC). We identified NS3 and Fc epitopes recognized by these IgM by epitope excision approach. HLA determination confirms their ability to present these epitopes. The same cross reactivity has been observed with a monoclonal antibody, (mAb B3-18), obtained after immunization of a mouse with one of the identified NS3 epitopes. Previous, observations indicate that HCV may localize in several tissues besides the liver (such as gastric mucosa) possibly inducing some of the extra hepatic manifestations of chronic HCV infection. The aim of this study is to characterize the mAb B3-18 reactivity by an immuno-proteomic approach. Such novel information may be of interest to identify new potential antigens implicated in HCV pathogenesis useful to design new molecules for diagnostic or therapeutic approaches. Proteins were extracted from 12 gastric biopsies of patients with gastric disturb and HCV-infection (one of them with a concomitant malt lymphoma of the stomach) and from 3 patients with gastric disturb but without HCV infection as control. Proteins were analysed by 2D gel electrophoresis and mAb B3-18 immunoblot. Proteins recognized by mAb B3-18 were identified by MALDI-TOF mass spectrometry. Differential protein expression levels between patients and controls were detected by 2D-DIGE. Samples were also tested by immunohistochemistry with the mAb B3-18, by VDJ-BCR and VDJ-TCR genscan analysis and by KIR/ligands haplotypes. MAb B3-18 is functional both in 2D gel electrophoresis and in immunohistochemistry. Two specific proteins were identified by B3-18 immunoblot in HCV-infected patients: 18 kDa antrum mucosa protein (AMP 18) and TFIZ1. Both proteins function as growth factors at least partly responsible for maintaining a functional gastric epithelium by seeking evidence of epithelial cell mitogenic activity. They are expressed and secreted in normal gastric mucosa but down-regulated in gastric cancer. These proteins, as well as some others, also showed an up-regulation in gastric mucosa from HCV-infected patients. Mab B3–18 reactivity in immunohistochemistry was restricted to gastric tissue surrounding the NHL lesion. An oligo/monoclonal B cell pattern was found in 3 patients. The characterization of KIR/ligand is in course. The use of a monoclonal antibody miming IgM autoantibodies from HCV+ patients with type II MC and a concomitant immunocytoma, is useful to identify autoantigens that could sustain HCV-related B cell proliferations and HCV infection-associated antigens.

Advances in immunoproteomics for serological characterization of microbial antigens

We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.

Decoding Serological Response to Candida Cell Wall Immunome into Novel Diagnostic, Prognostic, and Therapeutic Candidates for Systemic Candidiasis by Proteomic and Bioinformatic Analyses

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach coupled with bioinformatic analysis was used to decode the serological response to Candida wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in 45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way hierarchical clustering and principal component analysis of their serum anti-Candida wall antibody expression patterns discriminated SC patients from controls and highlighted the heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) and the anti-wall phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC. Receiver operating characteristic curve analysis revealed no difference between their combined evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody levels or a positive anti-wall enolase antibody status, which correlated with each other, had a reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that these mediated the protective effect. No association between serum anti-cytoplasmic enolase antibody levels and outcomes was established, suggesting a specific mechanism of enolase processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and wall-associated enolase could be valuable candidates for future vaccine development.

An Immunoproteomic Approach for Identification of Clinical Biomarkers for Monitoring Disease: Application to Cystic Fibrosis

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia

Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.