Human-Fungal Pathogen Interactions from the Perspective of Immunoproteomics Analyses.

Antibody immunity is now known to play a critical role in combating mycotic infections. The identification of molecules that can elicit an antibody response against fungal pathogens is the first step in developing antibody-based therapeutic strategies. Antigenic proteins are molecules recognized by the immune system that can stimulate antibody production and, therefore, can be a direct target for studying human-fungal pathogen interactions. Advances in recent immunoproteomic approaches have substantially aided in determining the key antigenic proteins on a large scale. In this review, we present a collection of antigenic proteins identified in yeast, dimorphic, and filamentous fungal pathogens to date. The general features of antigenic proteins are summarized and reveal that the proteins could commonly function in antistress responses, protein synthesis, and metabolism. The antigenic proteins listed here could serve as starting materials for developing species-specific or broad-spectrum diagnostic tests, therapeutic antibodies, and even vaccines against fungal infections.

Antigen Discovery for Next-Generation Pertussis Vaccines Using Immunoproteomics and Transposon-Directed Insertion Sequencing.

BACKGROUND: Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. METHODS: Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. RESULTS: In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized: BP0840 (OmpP), Pal, OmpA2, BP1485, BamA, Pcp, MlaA, YfgL, BP2197, BP1569, MlaD, ComL, and BP0183. CONCLUSIONS: The B. pertussis antigens identified as immunogenic, essential for persistence in the airway, and membrane-localized warrant further investigation for inclusion in vaccines designed to reduce or prevent carriage of bacteria in the airway of vaccinated individuals.

EgSeverin and Eg14-3-3zeta from Echinococcus granulosus are potential antigens for serological diagnosis of echinococcosis in dogs and sheep.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode larva of Echinococcus granulosus. In this study, two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis revealed that E. granulosus severin and 14-3-3zeta proteins (named EgSeverin and Eg14-3-3zeta, respectively) might be two potential biomarkers for serological diagnosis of echinococcosis. The recombinant EgSeverin (rEgSeverin, 45 kDa) and Eg14-3-3zeta (rEg14-3-3zeta, 35 kDa) were administered subcutaneously to BALB/c mice to obtain polyclonal antibodies for immunofluorescence analyses (IFAs). And IFAs showed that both proteins were located on the surface of protoscoleces (PSCs). Western blotting showed that both proteins could react with sera from E. granulosus-infected sheep, dog, and mice. Indirect ELISAs (rEgSeverin- and rEg14-3-3zeta-iELISA) were developed, respectively, with sensitivities and specificities ranging from 83.33% to 100% and a coefficient of variation (CV %) of less than 10%. The rEgSeverin-iELISA showed cross-reaction with both E. granulosus and E. multilocularis, while the rEg14-3-3zeta-iELISA showed no cross-reaction with other sera except for the E. granulosus-infected ones. The field sheep sera from Xinjiang and Qinghai were analyzed using rEgSeverin-iELISA, rEg14-3-3zeta-iELISA, and a commercial kit respectively, and no significant differences were found among the three methods (p > 0.05). However, the CE positive rates in sheep sera from Qinghai were significantly higher than those from Xinjiang (p < 0.01). Overall, the results suggest that EgSeverin and Eg14-3-3zeta could be promising diagnostic antigens for E. granulosus infection.

HLA Class II Presentation Is Specifically Altered at Elevated Temperatures in the B-Lymphoblastic Cell Line JY.

Human leukocyte antigen (HLA) molecules play critical roles in our adaptive immune system by signaling a cell's health status to the immune system, through presentation of small peptides. Understanding HLA biology is important because of its prominent role in autoimmune diseases and cancer immunotherapy. Although both the HLA class I and class II antigen processing and presentation pathways have been studied extensively, the fundamental rules in HLA class II antigen presentation still remain less understood. To clarify the mechanistic and adaptive differences between the HLA systems, we challenged a B lymphoblastic cell line (JY), widely used as model system in studying antigen presentation, with a high temperature treatment to mimic a "fever-like state", representing one of the most common physiological responses to infection. In the absence of real invading pathogenic peptides to present, we could focus on delineating the intrinsic HLA pathway adaptations in response to high temperature in this particular cell line. Following a three-pronged approach, we performed quantitative analyses of the proteome, the HLA class I ligandome, as well as the HLA class II ligandome. The data reveals that elevated temperature may already prepare these cells for an immune-like response through increased HLA class II presentation capacity and specific release of, from the invariant chain originating, CLIP peptides. Interestingly, at high temperature, prominent changes in the composition of the CLIP repertoire were observed, with enrichment of peptides containing C-terminal extensions beyond the CLIP-core region. Collectively, these illustrate intriguing temperature sensitive adaptations in this B cell line.

Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15.

Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 min, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-peptides were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 pSer5, STMN1 pSer25, CHEK1 pSer286, STIM1 pSer608, and VDAC1 pSer104. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

Proteomic and immunoproteomic insights into the exoproteome of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae affects pig health status and the swine industry worldwide. Despite the extensive number of studies focused on A. pleuropneumoniae infection and vaccine development, a thorough analysis of the A. pleuropneumoniae exoproteome is still missing. Using a complementary approach of quantitative proteomics and immunoproteomics we gained an in-depth insight into the A. pleuropneumoniae serotype 2 exoproteome, which provides the basis for future functional studies. Label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 593 exoproteins, of which 104 were predicted to be virulence factors. The RTX toxins ApxIIA and ApxIIIA -were found to be the most abundant proteins in the A. pleuropneumoniae serotype 2 exoproteome. Furthermore, the ApxIVA toxin was one of the proteins showing the highest abundance, although ApxIVA is commonly assumed to be expressed exclusively in vivo. Our study revealed several antigens, including proteins with moonlight functions, such as the elongation factor (EF)-Tu, and proteins linked to specific metabolic traits, such as the maltodextrin-binding protein MalE, that warrant future functional characterization and might present potential targets for novel therapeutics and vaccines. Our Ig-classes specific serological proteome analysis (SERPA) approach allowed us to explore the development of the host humoral immune response over the course of the infection. These SERPAs pinpointed proteins that might play a key role in virulence and persistence and showed that the immune response to the different Apx toxins is distinct. For instance, our results indicate that the ApxIIIA toxin has properties of a thymus-independent antigen, which should be studied in more detail.

Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

The role of proteomics in defining autoimmunity.

INTRODUCTION: Proteomics, i.e. the study of the set of proteins produced in a cell, tissue, organism, or biological entity, has made possible analyses and contextual comparisons of proteomes/proteins and biological functions among the most disparate entities, from viruses to the human being. In this way, proteomic scrutiny of tumor-associated proteins, autoantigens, and pathogen antigens offers the tools for fighting cancer, autoimmunity, and infections. AREAS COVERED: Comparative proteomics and immunoproteomics, the new scientific disciplines generated by proteomics, are the main themes of the present review that describes how comparative analyses of pathogen and human proteomes led to re-modulate the molecular mimicry concept of the pre-proteomic era. I.e. before proteomics, molecular mimicry - the sharing of peptide sequences between two biological entities - was considered as intrinsically endowed with immunologic properties and was related to cross-reactivity. Proteomics allowed to redefine such an assumption using physicochemical parameters according to which frequency and hydrophobicity preferentially confer an immunologic potential to shared peptide sequences. EXPERT OPINION: Proteomics is outlining peptide platforms to be used for the diagnostics and management of human diseases. A Molecular Medicine targeted to obtain healing without paying the price for adverse events is on the horizon. The next step is to take up the challenge and operate the paradigm shift that the current proteomic era requires.

Immunoproteomic analysis of fish ectoparasite, Argulus siamensis antigens.

AIM: An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. MATERIALS AND RESULTS: The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4-7 and SDS-PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti-A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver-stained gel and subjected to mass spectrometry in MALDI-TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against A siamensis is discussed in the paper. CONCLUSION: Three of the identified proteins namely, bromodomain-containing protein, anaphase-promoting complex subunit 5 and elongation factor-2 could possibly serve as vaccine candidates against argulosis in carps.

Analysis of the Repertoires of Circulating Autoantibodies' Specificities as a Tool for Identification of the Tumor-Associated Antigens: Current Problems and Solutions.

Circulating autoantibodies against tumor-associated autoantigens (TAA) may serve as valuable biomarkers for a wide range of diagnostic purposes. Modern immunology offers a large variety of methods for in-depth comparative analysis of the repertoires of circulating antibodies' antigenic specificities in health and disease. Nevertheless, this research field so far has met somewhat limited clinical success, while numerous data on the repertoires of circulating autoantibodies' specificities in cancer patients are poorly integrated into the contemporary picture of the immunological and molecular landscapes of human tumors. This review is an attempt to identify and systematize the key and essentially universal conceptual and methodological limitations of analyses of the repertoires of circulating antibodies' antigenic specificities in cancer (expression bias, redundancy of TAA repertoires, identification of natural IgG, the absence of the pathogenetically relevant context in the experimental systems used to detect TAA), as well as to discuss potential and already known methodological improvements that may significantly increase the detectability of the pathogenetically relevant and diagnostically significant bona fide TAA.

Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism.

OBJECTIVES: To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. RESULTS: In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. CONCLUSIONS: Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry.

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

Immunoproteomics approach revealed elevated autoantibody levels against ANXA1 in early stage gallbladder carcinoma.

BACKGROUND: Early diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of 'tumor-associated antigens (TAAs) that elicit humoral response' in early stage GBC patients. METHODS: Total protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins. RESULTS: Image analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed 'high' expression levels in 72% of GBC cases whereas all the controls showed 'low' expression levels. CONCLUSIONS: The study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

An immunoproteomics approach to identify Leishmania infantum proteins to be applied for the diagnosis of visceral leishmaniasis and human immunodeficiency virus co-infection.

The co-infection between visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) has increased in several countries in the world. The current serological tests are not suitable since they present low sensitivity to detect the most of VL/HIV cases, and a more precise diagnosis should be performed. In this context, in the present study, an immunoproteomics approach was performed using Leishmania infantum antigenic extracts and VL, HIV and VL/HIV patients sera, besides healthy subjects samples; aiming to identify antigenic markers for these clinical conditions. Results showed that 43 spots were recognized by antibodies in VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. Between them, ß-tubulin was expressed, purified and tested in ELISA experiments as a proof of concept for validation of our immunoproteomics findings and results showed high sensitivity and specificity values to detect VL and VL/HIV patients. In conclusion, the identified proteins in the present work could be considered as candidates for future studies aiming to improvement of the diagnosis of VL and VL/HIV co-infection.

Comparison of antibody repertories against Staphylococcus aureus in healthy and infected dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine.

Staphylococcus aureus is one of the pathogens most frequently isolated from cases of mastitis worldwide. To decrease the effect of S. aureus mastitis in dairy farming, alternative strategies for controlling mastitis are needed that depend on a better knowledge of cow-to-cow variations in S. aureus antibody production. The present study sought to explore the diversity of S. aureus antibodies produced by dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine. We obtained protein extracts from S. aureus isolates derived from persistent subclinical mastitis. Proteins were fractionated using 2-dimensional gel electrophoresis and Western blotting. Then, Western blotting membranes were exposed to sera from 24 dairy cows that had been divided into the following groups: vaccinated dairy cows that were infected with S. aureus, further subdivided according to whether they (a) remained infected by S. aureus or (b) recovered from the intramammary infection; unvaccinated dairy cows infected with S. aureus; and vaccinated healthy dairy cows with no history of S. aureus mastitis. Proteins found to be reactive by Western blot were identified by mass spectrometry (MALDI/TOF-TOF). Our most important finding was that F0F1 ATP synthase subunit α, succinyl-diaminopimelate desuccinylase, and cysteinyl-tRNA synthetase were potential candidate proteins for the prevention of S. aureus mastitis. This study strengthens the notion that variations among animals should not be ignored and shows that the heterogeneity of antibody production against anti-staphylococcal antigens in animals may enable the identification of new immunotherapy targets.

Proteomics Analysis Reveals That Structural Proteins of the Virion Core and Involved in Gene Expression Are the Main Source for HLA Class II Ligands in Vaccinia Virus-Infected Cells.

Protective cellular and humoral immune responses require previous recognition of viral antigenic peptides complexed with human leukocyte antigen (HLA) class II molecules on the surface of the antigen presenting cells. The HLA class II-restricted immune response is important for the control and the clearance of poxvirus infection including vaccinia virus (VACV), the vaccine used in the worldwide eradication of smallpox. In this study, a mass spectrometry analysis was used to identify VACV ligands bound to HLA-DR and -DP class II molecules present on the surface of VACV-infected cells. Twenty-six naturally processed viral ligands among the tens of thousands of cell peptides bound to HLA class II proteins were identified. These viral ligands arose from 19 parental VACV proteins: A4, A5, A18, A35, A38, B5, B13, D1, D5, D7, D12, D13, E3, E8, H5, I2, I3, J2, and K2. The majority of these VACV proteins yielded one HLA ligand and were generated mainly, but not exclusively, by the classical HLA class II antigen processing pathway. Medium-sized and abundant proteins from the virion core and/or involved in the viral gene expression were the major source of VACV ligands bound to HLA-DR and -DP class II molecules. These findings will help to understand the effectiveness of current poxvirus-based vaccines and will be important in the design of new ones.

An immunoproteomic approach to identify antigenic proteins in Nocardia farcinica IFM 10152.

Nocardia farcinica is the etiological agent of nocardiosis, leading to serious pulmonary or systemic infections. To uncover virulence factors and early diagnostic markers, secreted proteins of N. farcinica IFM 10152 were analyzed using an immunoproteome-based approach. A total of 5 proteins were identified by matrix-assisted laser desorption (MALDI-TOF-MS). Bioinformatic analyses showed that the identified proteins were involved in defense against the host innate immune system and required for pathogenesis. All proteins were expressed in E. coli and antigenicity was analyzed with Western blot. To our knowledge, these proteins with antigenicity were identified for the first time in N. farcinica and they may help elucidate the pathogenesis underlying Nocardia and provide potential future diagnostic markers.

Identification of common antigens of three pathogenic Nocardia species and development of DNA vaccine against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease and three pathogens have been reported so far, including Nocardia asteroids, N. seriolae and N. salmonicida. However, the absence of antigen markers is a bottleneck for developing effective vaccines against fish nocardiosis. In this study, the antigenicity of whole-cell protein of these three pathogenic Nocardia species were profiled by immunoproteomic analysis and 7 common immunogenic proteins were identified as follows: molecular chaperone DnaK (DnaK), molecular chaperone GroEL (GroEL), 30 S ribosomal protein S1 (RpsA), TerD family protein (TerD), FHA domain-containing protein (FHA), 50 S ribosomal protein L7/L12 (RplL) and PspA/IM30 family protein (PspA). Furthermore, the DNA vaccine encoding FHA gene against fish nocardiosis was developed and its efficacy was investigated in hybrid snakehead. The results suggested that it needed at least 7 d to transport pcDNA-FHA DNA vaccine from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection. In addition, non-specific immunity paraments (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune response induced in pcDNA-FHA vaccinated hybrid snakehead, it proved that all these mentioned immune activities were significantly enhanced after immunization. The results also showed hybrid snakehead vaccinated with pcDNA-FHA had higher survival rate (79.33%) compared with the controls after challenge with N. seriolae, indicating that the pcDNA-FHA DNA vaccine can supply immune protection against N. seriolae infection. Taken together, this study may warrant further development of these common immunogenic proteins as the antigens for vaccine or diagnosis and facilitate the prevention and treatment of fish nocardiosis.

Screening diagnostic candidates from Leishmania infantum proteins for human visceral leishmaniasis using an immunoproteomics approach.

There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.

Immunome and immune complex-forming components of Brugia malayi identified by microfilaremic human sera.

Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents.