Competitive ELISA for the identification of 35-55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan.

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG35-55 ) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG35-55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG35-55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG35-55 peptide may undergo when it is bound to mannan during production and stability studies.

Integrating genomics and proteomics permits identification of immunodominant antigens associated with drug resistance in human visceral leishmaniasis in India.

Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.

Desmoglein 3 and p40 immunoreactivity in neoplastic and nonneoplastic thymus: a potential adjunct to help resolve selected diagnostic and staging problems.

The potential usefulness of the squamous markers p40 and desmoglein 3 (DSG-3) for the diagnosis and staging of selected thymic lesions is uncertain. We investigated their expression and distribution pattern in 66 thymomas, 12 thymic squamous carcinomas, 6 undifferentiated thymic carcinomas, 5 hyperplastic thymi, and 5 normal thymi. p40 nuclear and DSG-3 cytoplasmic/membranous immunoreactivity in greater than or equal to 10% of thymic epithelial cells was interpreted as positive, and DSG-3 distribution pattern was classified as organotypic and nonorganotypic. All nonneoplastic thymic tissues, 100% of thymic squamous carcinomas, 97% of thymomas, and 50% of undifferentiated thymic carcinomas were positive for p40. Expression of p40 in almost all thymomas and in 50% of undifferentiated carcinomas that lacked squamous features suggests that p40 is not a good marker for the diagnosis of thymic squamous carcinoma. All normal and hyperplastic thymi, 51.5% of thymomas, and 0% of thymic squamous carcinomas expressed DSG-3 in an organotypic pattern, and 13.6% of thymomas and 83% of thymic squamous carcinomas were DSG-3 positive in a nonorganotypic pattern. Findings suggest that nonorganotypic DSG-3 expression favors the diagnosis of squamous cell carcinoma over thymoma. In 26 (60.5%) of the 43 cases where neoplastic and nonneoplastic thymus were present on the same slide, the presence/absence or distribution pattern of DSG-3 immunoreactivity was different in the 2 components, suggesting that this marker can be helpful in staging thymomas with incomplete encapsulation. The presence of DSG-3-positive and DSG-3-negative thymomas raises the possibility that these tumors may originate from 2 different types of thymic epithelial cells.

T cell epitope mapping of JC polyoma virus-encoded proteome reveals reduced T cell responses in HLA-DRB1*04:01+ donors.

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

Crystallization and preliminary X-ray crystallographic analysis of the rhesus macaque MHC class I molecule Mamu-B*17 complexed with an immunodominant SIVmac239 Env epitope.

Long-term nonprogression during simian immunodeficiency virus (SIV) infection has been strongly associated with the major histocompatibility complex (MHC) class I allele Mamu-B*17. Here, a complex of rhesus macaque Mamu-B*17 with rhesus macaque ß2-microglobulin (ß2m) and an immunodominant peptide (SIVmac239 Env241-251; LRCNDTNYSGF; Env LF11) derived from the SIV Env protein was crystallized by the hanging-drop method using PEG 3350 as a precipitating agent. The crystals belonged to the primitive monoclinic space group P2, with unit-cell parameters a = 68.3, b = 45.0, c = 81.5 Å, ß = 96.5°. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.96 Å(3) Da(-1) and 58.5%, respectively.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies.

Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.

Identification and validation of novel serum markers for early diagnosis of endometriosis.

BACKGROUND: Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS: A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS: The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS: Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

Identification of carbonic anhydrase I immunodominant epitopes recognized by specific autoantibodies which indicate an improved prognosis in patients with malignancy after autologous stem cell transplantation.

This work employs an epitope mapping of carbonic anhydrase (CA), isoform I (CA I), for detection of the main immunodominant epitopes. Our interest has arisen from an observed spontaneous tumor regression in patients who developed an aplastic anemia type syndrome after a high-dose therapy with autologous stem cell transplantation and whose sera contained high titer of anti carbonic anhydrase (anti-CA) autoantibodies. There are many indications that the presence of these autoantibodies may provide significant survival benefit for the patients. Western blot analysis confirmed strong immunoreactivity of the patients' sera with several CA isoforms and the CA I has been selected for our study as a highly abundant and widely distributed isoform. The applied analytical approach consists of specific fragmentation of CA I protein followed by immunospecific isolation of peptides reacting with polyclonal anti-CA I autoantibodies of patients in spontaneous remission. We improved the standard epitope mapping schema by incorporating the benefits of magnetic carriers and biomagnetic separation techniques. Mass spectrometry has been applied for detection and identification of epitopes and the acquired results were verified by bioinformatic tools. The candidate epitopes of CA I (NVGHS, DGLAV, SSEQL, and SLKPI) are discussed herein as potential therapeutic targets. This work highlights the usefulness of the epitope mapping technique based on magnetic microspheres for effective and rapid determination of immunodominant epitopes of the target protein.

QUASI analysis of host immune responses to Gag polyproteins of human immunodeficiency virus type 1 by a systematic bioinformatics approach.

There is a consensus that Gag-specific cytotoxic T lymphocyte (CTL) response plays a key role in the immune control of human immunodeficiency virus type 1 (HIV-1) infection. In this study, we analyzed all currently available gag sequences in the Los Alamos HIV sequence database and identified positive selection (PS) sites likely restricted by the host immune responses. We found that between 23.4% and 47.4% of PS sites were shared by clades A, B, and C of Gag, indicating similar positive selection pressure on Gag in different subtypes of HIV-1. Furthermore, a significant correlation was observed between the combined CTL and antibody responses and PS sites. The Gag regions of free from PS contained 9 CTL epitopes restricted by 11 HLA class I alleles associated with disease progression to acquired immune deficiency syndrome (AIDS). These analyses provide information important for the identification of cross-clade epitopes and development of a global HIV-1 vaccine.

Discordance Between the Predicted Versus the Actually Recognized CD8+ T Cell Epitopes of HCMV pp65 Antigen and Aleatory Epitope Dominance.

CD8+ T cell immune monitoring aims at measuring the size and functions of antigen-specific CD8+ T cell populations, thereby providing insights into cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical because within a complex antigen exists a multitude of potential epitopes that can be presented by HLA class I molecules. Further complicating this task, there is HLA class I polygenism and polymorphism which predisposes CD8+ T cell responses towards individualized epitope recognition profiles. In this study, we compare the actual CD8+ T cell recognition of a well-characterized model antigen, human cytomegalovirus (HCMV) pp65 protein, with its anticipated epitope coverage. Due to the abundance of experimentally defined HLA-A*02:01-restricted pp65 epitopes, and because in silico epitope predictions are most advanced for HLA-A*02:01, we elected to focus on subjects expressing this allele. In each test subject, every possible CD8+ T cell epitope was systematically covered testing 553 individual peptides that walk the sequence of pp65 in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. No correlation was found between epitopes' ranking on the prediction scale and their actual immune dominance. Collectively, these data suggest that accurate CD8+ T cell immune monitoring may necessitate reliance on agnostic mega peptide pools, or brute force mapping, rather than electing individual peptides as representative epitopes for tetramer and other multimer labeling of surface antigen receptors.

Atomic-level mapping of antibody epitopes on a GPCR.

Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines.

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most-if not the most-frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.

Identification of a nephritogenic immunodominant B and T cell epitope in experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis (EAG) can be induced in Wistar Kyoto (WKY) rats by immunization with the non-collagenous domain (NC1) of the alpha 3 chain of type IV collagen, alpha3(IV)NC1. In patients with Goodpasture's disease, the major B cell epitope is located at the N-terminus of alpha3(IV)NC1. In order to investigate whether B and T cell responses in EAG are directed towards immunodominant peptides within the same region of rat alpha3(IV)NC1, we immunized WKY rats with recombinant rat alpha3(IV)NC1 (positive control) and five 15-mer overlapping synthetic peptides from the N-terminus of rat alpha3(IV)NC1: pCol(17-31), pCol(24-38), pCol(31-45), pCol(38-52) and pCol(45-59). Positive control animals immunized with alpha3(IV)NC1 produced an antibody response directed towards alpha3(IV)NC1 and pCol(24-38). Splenic T cells from these animals proliferated in response to alpha3(IV)NC1 and pCol(24-38). No significant antibody or T cell responses were observed to the other peptides examined. Animals immunized with pCol(24-38) developed linear deposits of immunoglobulin G on the glomerular basement membrane, albuminuria and focal necrotizing glomerulonephritis with crescent formation by week 6 after immunization. Circulating antibodies from these animals recognized pCol(24-38) and alpha3(IV)NC1, and their T cells proliferated in response to pCol(24-38) and alpha3(IV)NC1. Animals immunized with the other peptides developed no significant immune response to alpha3(IV)NC1 and no disease. In conclusion, these results demonstrate that a 15-mer peptide from the N-terminus of alpha3(IV)NC1 [pCol(24-38)] is recognized by B and T cells from rats immunized with recombinant alpha3(IV)NC1, and that the same peptide is capable of inducing crescentic glomerulonephritis. Identification of this immunodominant peptide will be of value in designing new therapeutic strategies for inducing mucosal tolerance in EAG, which may be applicable to patients with glomerulonephritis.

Correlations between Terasaki's HLA class II epitopes and HLAMatchmaker-defined eplets on HLA-DR and -DQ antigens.

Human leukocyte antigen (HLA) class II-specific antibodies increase the risk of transplant failure, and their characterization must consider epitopes rather than antigens. There are two strategies to determine HLA epitope structure. Terasaki's group has analyzed antibody reactivity patterns with single antigen panels with a computer program based on shared amino acid residues of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. This report describes how 49 of 53 Terasaki's HLA-DR epitopes correspond to HLAMatchmaker-defined eplets. Most of them are equivalent to single eplets (n = 33) or two or more possible eplets (n = 10), but six had corresponding eplet pairs. There were 10 cases whereby eplets have permissible residue combinations, and in 5 cases, we found that eplet specificity might be influenced by nearby hidden residues. We could assign corresponding eplets to 17 of 18 Terasaki's HLA-DQ epitopes. This study demonstrates how the HLAMatchmaker interpretation of amino acid residues shared between antibody-reactive antigens can increase our understanding of the structural basis of HLA epitopes.

Identification of novel immunodominant epididymal sperm proteins using combinatorial approach.

Functionally immature spermatozoa leave the testis mature during epididymal transit. This process of maturation involves either addition of new proteins or modification of existing proteins onto the sperm domains that are responsible for domain-specific functions. Epididymal proteins are preferred targets for immunocontraception. In an attempt to identify epididymis-specific sperm proteins, we used a novel combinatorial approach comprising subtractive immunization (SI) followed by proteomics. Following SI, sera of mice were used for immunoproteomics, which led to the identification of 30 proteins, of which four proteins namely sperm head protein 1, sperm flagella protein 2 (SFP2), SFP3, and SFP4 are being reported for the first time on sperm. Another group of four proteins namely collagen alpha-2 (I) chain precursor, homeodomain-interacting protein kinase 1, GTP-binding protein Rab1, and ubiquinol cytochrome c reductase core protein II although reported earlier in testis are being reported for the first time in epididymal sperm. Furthermore, seven out of these eight novel proteins could be validated using peptide ELISA. These data are a useful repository, which could be exploited to develop targets for post-testicular immunocontraception or biomarkers for infertility diagnosis and management.

Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors.

Varicella zoster viru (VZV)-specific T cell responses are believed to be vital in recovery from primary VZV infection and also in the prevention of viral reactivation. While glycoprotein E (gE) is the most abundant and one of the most immunogenic proteins of the virus, there are no data addressing potential T cell epitopes within gE, nor the phenotype of specific T cells. Using interferon gamma enzyme-linked immunospot assays and intracellular cytokine assays, we identified gE-specific immune responses in 20 adult healthy immune donors which were found to be dominated by the CD4+ subset of T cells. We characterized three immune dominant epitopes within gE restricted through DRB1*1501, DRB1*07 and DRB4*01, and used DRB1*1501 class II tetrameric complexes to determine the ex vivo frequency and phenotype of specific T cells. In healthy immune donors, the cells were largely positive for CCR7, CD28 and CD27, but expressed variable CD62L and low levels of cutaneous lymphocyte associated antigen with evidence of recent activation. In summary, we show that circulating gE-specific CD4+ T cells are detected at a relatively high frequency in healthy immune donors and show evidence of recent activation and mixed central and effector memory phenotype. These data would be compatible with frequent exposure to replicative cycle antigens in healthy donors and are consistent with a role for gE-specific CD4+ T cells in the control of viral replication.

Lyme disease: laboratory issues.

This article describes the laboratory modalities available to confirm the diagnosis of Lyme borreliosis. Use and limitations of these methods are discussed. Current guidelines for the use of recommended serologic methods and discussion of newer methods also are provided.

An epitope-specific PCR test for diagnosis of Leishmania donovani infections.

The carboxy-terminal region of recombinant heat shock protein 70 of Leishmania donovani has been shown to have an immunodominant epitope. We designed a PCR assay using a new set of primers encompassing the gene sequence from 457bp to 927bp, which amplified a segment of the L. donovani genome in a species-specific manner. The assay was sensitive enough to detect 0.5pg of parasite DNA, which increased 10-fold (0.05pg) when an internal probe (583-609bp) was used in the Southern blot. The assay was able to detect parasite DNA from the liver and spleen of L. donovani-infected hamsters as well as lesion aspirates from parasitologically confirmed post-kala-azar dermal leishmaniasis (PKDL) and bone marrow aspirates from visceral leishmaniasis (VL) patients. No amplification was seen with axenically cultured promastigotes of L. infantum, L. tropica, L. major, L. mexicana, L. aethiopica or other intracellular organisms such as Entamoeba histolytica, Mycobacterium tuberculosis, Plasmodium vivax or human peripheral blood mononuclear cells. This PCR provides a specific tool for the diagnosis of VL and PKDL with simultaneous species identification.