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Sample records for unrelated protein antigens

  1. Globally, unrelated protein sequences appear random

    PubMed Central

    Lavelle, Daniel T.; Pearson, William R.

    2010-01-01

    Motivation: To test whether protein folding constraints and secondary structure sequence preferences significantly reduce the space of amino acid words in proteins, we compared the frequencies of four- and five-amino acid word clumps (independent words) in proteins to the frequencies predicted by four random sequence models. Results: While the human proteome has many overrepresented word clumps, these words come from large protein families with biased compositions (e.g. Zn-fingers). In contrast, in a non-redundant sample of Pfam-AB, only 1% of four-amino acid word clumps (4.7% of 5mer words) are 2-fold overrepresented compared with our simplest random model [MC(0)], and 0.1% (4mers) to 0.5% (5mers) are 2-fold overrepresented compared with a window-shuffled random model. Using a false discovery rate q-value analysis, the number of exceptional four- or five-letter words in real proteins is similar to the number found when comparing words from one random model to another. Consensus overrepresented words are not enriched in conserved regions of proteins, but four-letter words are enriched 1.18- to 1.56-fold in α-helical secondary structures (but not β-strands). Five-residue consensus exceptional words are enriched for α-helix 1.43- to 1.61-fold. Protein word preferences in regular secondary structure do not appear to significantly restrict the use of sequence words in unrelated proteins, although the consensus exceptional words have a secondary structure bias for α-helix. Globally, words in protein sequences appear to be under very few constraints; for the most part, they appear to be random. Contact: wrp@virginia.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19948773

  2. The Recognition of Identical Ligands by Unrelated Proteins.

    PubMed

    Barelier, Sarah; Sterling, Teague; O'Meara, Matthew J; Shoichet, Brian K

    2015-12-18

    The binding of drugs and reagents to off-targets is well-known. Whereas many off-targets are related to the primary target by sequence and fold, many ligands bind to unrelated pairs of proteins, and these are harder to anticipate. If the binding site in the off-target can be related to that of the primary target, this challenge resolves into aligning the two pockets. However, other cases are possible: the ligand might interact with entirely different residues and environments in the off-target, or wholly different ligand atoms may be implicated in the two complexes. To investigate these scenarios at atomic resolution, the structures of 59 ligands in 116 complexes (62 pairs in total), where the protein pairs were unrelated by fold but bound an identical ligand, were examined. In almost half of the pairs, the ligand interacted with unrelated residues in the two proteins (29 pairs), and in 14 of the pairs wholly different ligand moieties were implicated in each complex. Even in those 19 pairs of complexes that presented similar environments to the ligand, ligand superposition rarely resulted in the overlap of related residues. There appears to be no single pattern-matching "code" for identifying binding sites in unrelated proteins that bind identical ligands, though modeling suggests that there might be a limited number of different patterns that suffice to recognize different ligand functional groups.

  3. Monoclonal antibodies against an identical short peptide sequence shared by two unrelated proteins.

    PubMed

    Schulze-Gahmen, U; Wilson, I A

    1989-01-01

    Antipeptide antibodies provide the opportunity to explore the molecular basis for antigen-antibody recognition and to test theories of immune recognition. We investigated the possibility of raising monoclonal antipeptide antibodies against a specific epitope consisting of six amino acid residues, which is common to two unrelated proteins. The goal of this investigation was to analyze the reactivity of these epitope specific antibodies towards the same sequence in these two different proteins. A correlation between antibody reactivity and secondary structures of the same peptide sequence in different proteins could help to understand the ability of antipeptide antibodies to react with their cognate sequence in intact folded proteins. Monoclonal antibodies were raised against one hexamer sequence, PGTAPK, that is present in both thioredoxin and Fab New lambda-light chain. The antipeptide antibodies reacted only with thioredoxin but not with Fab New in ELISA's, immune precipitation and Western blots. Determination of the antibody specificity through binding tests with peptide analogs revealed the influence of the residue N-terminal from the hexamer epitope on antibody binding. Because of the observed influence of the N-1 adjacent residue in peptide analogs, the discrimination between the protein antigens could not be interpreted clearly as the result of the different hexamer conformations present in the native structures of the two proteins. However, analysis of the antibody reactivity with peptide analogs with varying "frame residues" surrounding the hexamer epitope indicates the possible discrimination of different peptide conformations by the antibody.

  4. Meningococcal protein antigens and vaccines.

    PubMed

    Feavers, Ian M; Pizza, Mariagrazia

    2009-06-24

    The development of a comprehensive vaccine against meningococcal disease has been challenging. Recent developments in molecular genetics have provided both explanations for these challenges and possible solutions. Since genome sequence data became available there has been a marked increase in number of protein antigens that have been suggested as prospective vaccine components. This review catalogues the proposed vaccine candidates and examines the evidence for their inclusion in potential protein vaccine formulations.

  5. Quantitating T Cell Cross-Reactivity for Unrelated Peptide Antigens1

    PubMed Central

    Ishizuka, Jeffrey; Grebe, Kristie; Shenderov, Eugene; Peters, Bjoern; Chen, Qiongyu; Peng, YanChun; Wang, Lili; Dong, Tao; Pasquetto, Valerie; Osroff, Carla; Sidney, John; Hickman, Heather; Cerundolo, Vincenzo; Sette, Alessandro; Bennink, Jack R.; McMchael, Andrew; Yewdell, Jonathan W.

    2009-01-01

    Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides (∼1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance. PMID:19734234

  6. Antigenic specificities of delayed hypersensitivity in mice to dinitrophenylated proteins

    PubMed Central

    Yonemasu, K.; Crowle, A. J.

    1973-01-01

    Humoral antibodies capable of suppressing induction of delayed hypersensitivity to dinitrophenylated proteins in mice were tested for specific absorbability onto chemically insolubilized antigen and for the antigenic determinant specificities of their immunosuppressive (i.e. contrasensitizing) effects. The activity of an antiserum could be completely removed by absorption with homologous antigen, and it could be recovered by dissociating the absorbed antibodies at low pH and high salt concentration. The immunosuppressive antibodies therefore are specific for determinants on the native antigen, and non-antibody serum constituents are non-essential. By selective immunoabsorptions and elutions, antibodies specific for carrier protein, for dinitrophenyl hapten, and for new determinants unique to the hapten—protein complexes were prepared and were compared with unfractionated antiserum for contrasensitizing activity. Nearly all activity could be accounted for by the anti-hapten antibodies, although anti-carrier antibodies also had some. Despite this evidence that immunosuppressiveness was hapten-specific, the delayed hypersensitivity being suppressed by these antibodies was shown to be directed against hapten—carrier complexes or against carrier but not against hapten alone. Hence, humoral antibodies against a portion of an antigen molecule can suppress induction of delayed hypersensitivity specific for other sometimes unrelated parts of this same molecule, and it is possible to induce antibody-mediated tolerance to a multi-determinant antigen with an antibody response against just a part of the antigen. The theoretical and practical implications of these findings are discussed. Notable theoretically is the observation that although determinant specificities of humoral antibody and delayed hypersensitivity responses to one antigen usually differ, the former can regulate the latter for a given species of antigen molecule. Most important practically is the implication

  7. Immune recognition of protein antigens

    SciTech Connect

    Laver, W.G.; Air, G.M.

    1985-01-01

    This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

  8. Staphylococcal Enterotoxin B (SEB) Induces Memory CD4 T Cell Anergy in vivo and Impairs Recall Immunity to Unrelated Antigens

    PubMed Central

    Janik, David K; Lee, William T

    2015-01-01

    Introduction Naïve and memory T cells can utilize unique regulatory pathways to promote protection but prevent self-reactivity. A bacterial superantigen SEB exploits unique TCR proximal signaling processes in memory CD4 T cells to induce clonal anergy. The aim of this study was to determine if SEB could antagonize memory CD4 T cells in vivo and whether there would be consequences on recall immune responses. We evaluated Ab responses to a T-dependent antigen as a measurement of memory T cell helper function. Method BALB/c mice were primed with TNP-RGG to elicit memory B cells and also immunized with an ovalbumin peptide to elicit memory helper T cells. Another group of TNP-RGG immunized mice were used as adoptive transfer recipients of exogenous DO11.10 memory T cells. Mice were challenged with TNP-OVA with or without prior administration of SEB. B cells secreting IgM or IgG TNP-specific Ab were enumerated by ELISPOT as indicators of primary versus secondary humoral immunity. Results Comparing the SEB and non-SEB-treated groups, the SEB-treated group failed to produce TNP-specific IgG in response to challenge with TNP-OVA, even if they were previously immunized with OVA. All groups produced IgM, indicating that the primary Ab responses and naïve helper T cells were not impacted by SEB. SEB had no negative impact when DO11.10 × Fyn−/− memory T cells were used as donor cells. Conclusion The present study indicated that SEB selectively targeted memory CD4 T cells in vivo and prevented helper function. Consequently, recall humoral immunity was lost. The data are most consistent with in vivo T cell anergy as opposed to indirect suppression as elimination of Fyn kinase restored helper function. These data suggest that bacterial superantigens can impair post-vaccination memory cell responses to unrelated antigens via their ability to target Vb families and antagonize memory cell activation. PMID:26807307

  9. Antigenic Properties of N Protein of Hantavirus

    PubMed Central

    Yoshimatsu, Kumiko; Arikawa, Jiro

    2014-01-01

    Hantavirus causes two important rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in North and South America. Twenty-four species that represent sero- and genotypes have been registered within the genus Hantavirus by the International Committee on Taxonomy of Viruses (ICTV). Among the viral proteins, nucleocapsid (N) protein possesses an immunodominant antigen. The antigenicitiy of N protein is conserved compared with that of envelope glycoproteins. Therefore, N protein has been used for serological diagnoses and seroepidemiological studies. An understanding of the antigenic properties of N protein is important for the interpretation of results from serological tests using N antigen. N protein consists of about 430 amino acids and possesses various epitopes. The N-terminal quarter of N protein bears linear and immunodominant epitopes. However, a serotype-specific and multimerization-dependent antigenic site was found in the C-terminal half of N protein. In this paper, the structure, function, and antigenicity of N protein are reviewed. PMID:25123683

  10. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins.

    PubMed

    Teytelman, Leonid; Thurtle, Deborah M; Rine, Jasper; van Oudenaarden, Alexander

    2013-11-12

    Chromatin immunoprecipitation (ChIP) is the gold-standard technique for localizing nuclear proteins in the genome. We used ChIP, in combination with deep sequencing (Seq), to study the genome-wide distribution of the Silent information regulator (Sir) complex in Saccharomyces cerevisiae. We analyzed ChIP-Seq peaks of the Sir2, Sir3, and Sir4 silencing proteins and discovered 238 unexpected euchromatic loci that exhibited enrichment of all three. Surprisingly, published ChIP-Seq datasets for the Ste12 transcription factor and the centromeric Cse4 protein indicated that these proteins were also enriched in the same euchromatic regions with the high Sir protein levels. The 238 loci, termed "hyper-ChIPable", were in highly expressed regions with strong polymerase II and polymerase III enrichment signals, and the correlation between transcription level and ChIP enrichment was not limited to these 238 loci but extended genome-wide. The apparent enrichment of various proteins at hyper-ChIPable loci was not a consequence of artifacts associated with deep sequencing methods, as confirmed by ChIP-quantitative PCR. The localization of unrelated proteins, including the entire silencing complex, to the most highly transcribed genes was highly suggestive of a technical issue with the immunoprecipitations. ChIP-Seq on chromatin immunoprecipitated with a nuclear-localized GFP reproduced the above enrichment in an expression-dependent manner: induction of the GAL genes resulted in an increased ChIP signal of the GFP protein at these loci, with presumably no biological relevance. Whereas ChIP is a broadly valuable technique, some published conclusions based upon ChIP procedures may merit reevaluation in light of these findings.

  11. Marrow grafts between phenotypically DLA-identical and haploidentical unrelated dogs: additional antigens controlling engraftment are not detected by cell-mediated lympholysis

    SciTech Connect

    Deeg, H.J.; Storb, R.; Raff, R.F.; Weiden, P.L.; DeRose, S.; Thomas, E.D.

    1982-01-01

    Bone marrow transplants with low marrow cell doses (less than or equal to4 X 10/sup 8/ cells/kg) from unrelated donors were carried out in 16 dogs conditioned with 9 Gy (900 rad) of total body irradiation. No immunosuppression was given after grafting. Eleven donor-recipient pairs were phenotypically identical (group 1) for the known antigens of the canine major histocompatibility complex (DLA) and in five the donor was homozygous and the recipient heterozygous for DLA (group 2), as determined by serological histocompatibility typing and mixed leukocyte cultures including homozygous cell typing. In addition, lymphocytes from donors and recipients in group 1 were mutually nonreactive in cell-mediated lympholysis; lymphocytes from recipients in group 2 were not cytotoxic against donor cells. Eight dogs rejected their grafts and eight showed sustained engraftment; of these, four died from graft-versus-host disease. The incidence of rejection was higher than in DLA-identical littermates but lower than in DLA-nonidentical unrelated or littermate dogs. These results indicate that antigens different from the recognized alleles at DLA are involved in the control of engraftment. These antigens most likely represent the expression of unrecognized differences within DLA or are coded for by a locus different from but linked to DLA-A, B, C or D; they are not recognized in the cell-mediated lympholysis assay.

  12. The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins

    PubMed Central

    Rodríguez-Almazán, Claudia; Torner, Francisco J.; Costas, Miguel; Pérez-Montfort, Ruy; de Gómez-Puyou, Marieta Tuena; Puyou, Armando Gómez

    2007-01-01

    The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins. PMID:17551578

  13. Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

    PubMed Central

    Tribouillard-Tanvier, Déborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cécile; Béringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stéphane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chédin, Stéphane; Vilette, Didier; Galons, Hervé; Sanyal, Suparna; Blondel, Marc

    2008-01-01

    Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity. PMID:18478094

  14. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    PubMed Central

    Esparo, Nicolle M.; Child, Stephanie J.; Geballe, Adam P.

    2016-01-01

    During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α. PMID:27780231

  15. Transient induction of a nuclear antigen unrelated to Epstein-Barr nuclear antigen in cells of two human B-lymphoma lines converted by Epstein-Barr virus.

    PubMed

    Fresen, K O; zur Hausen, H

    1977-01-01

    Infection of cells of the Epstein-Barr virus (EBV)-negative human B-lymphoma lines BJAB and Ramos with EBV preparations from P3HR-1 or B 95-8 cells converted these cells to EBV genome carriers expressing Epstein-Barr nuclear antigen (EBNA) in almost 100% of these cells. Induction of these cells as well as of clones from P3HR-1 EBV-converted BJAB cells with iododeoxyuridine, aminopterin, and hypoxanthine resulted in the appearance of a nuclear antigen in about 1-6% of the cells 1-4 days after induction. The antigen is different from known EBV-induced antigens like EBNA, viral capsid antigen (VCA) or the D- and R-subspecificities of the early antigen (EA) complex. It is demonstrated by indirect immunofluorescence and inactivated after acetone fixation. The antigen was not detectable after induction of uninfected BJAB and Ramos cells nor has it been found in noninduced or induced P3HR-1 and Raji cells. Thus, it appears that EBV-infection mediates the expression of this antigen, for which the name TINA (transiently induced nuclear antigen) is suggested. Sera reacting against TINA generally contained high antibody titers against EBV-induced EA. Only a limited number of highly EA-reactive sera, however, were also positive for TINA. Among 200 sera tested thus far, TINA reactivity was most frequently observed in sera of patients with nasopharyngeal carcinoma (7 out of 28), in sera of the only two patients with immunoblastoma tested and occasionally in sera from patients with Hodgkin's disease and chronic lymphatic leukemia. Among 70 sera from nontumor patients, TINA reactivity was observed three times: two patients suffered from "chronic" infectious mononucleosis, the other revealed persistent splenomegaly. PMID:189313

  16. Protein microarrays for parasite antigen discovery.

    PubMed

    Driguez, Patrick; Doolan, Denise L; Molina, Douglas M; Loukas, Alex; Trieu, Angela; Felgner, Phil L; McManus, Donald P

    2015-01-01

    The host serological profile to a parasitic infection, such as schistosomiasis, can be used to define potential vaccine and diagnostic targets. Determining the host antibody response using traditional approaches is hindered by the large number of putative antigens in any parasite proteome. Parasite protein microarrays offer the potential for a high-throughput host antibody screen to simplify this task. In order to construct the array, parasite proteins are selected from available genomic sequence and protein databases using bioinformatic tools. Selected open reading frames are PCR amplified, incorporated into a vector for cell-free protein expression, and printed robotically onto glass slides. The protein microarrays can be probed with antisera from infected/immune animals or humans and the antibody reactivity measured with fluorophore labeled antibodies on a confocal laser microarray scanner to identify potential targets for diagnosis or therapeutic or prophylactic intervention. PMID:25388117

  17. The human leucocyte antigen-G 14-basepair polymorphism correlates with graft-versus-host disease in unrelated bone marrow transplantation for thalassaemia.

    PubMed

    La Nasa, Giorgio; Littera, Roberto; Locatelli, Franco; Lai, Sara; Alba, Francesco; Caocci, Giovanni; Lisini, Daniela; Nesci, Sonia; Vacca, Adriana; Piras, Eugenia; Bernardo, Maria Ester; Di Cesare-Merlone, Alessandra; Orrù, Sandro; Carcassi, Carlo

    2007-10-01

    The presence of the 14-bp insertion polymorphism of the human leucocyte antigen (HLA)-G gene (HLA-G) promotes immune tolerance through increased synthesis of HLA-G molecules. We investigated this polymorphism in a large cohort of 53 thalassaemia patients transplanted from an unrelated donor. Sixteen patients (30.2%) homozygous for the 14-bp deletion had a higher risk of developing acute graft-versus-host disease (aGvHD) than patients homozygous for the 14-bp insertion (-14-bp/-14-bp vs +14-bp/+14-bp: Relative Risk = 15.0; 95% confidence interval 1.59-141.24; P = 0.008). Therefore, the 14-bp polymorphism could be an important predictive factor for aGvHD following bone marrow transplantation. PMID:17897304

  18. Protection of chickens to antigenically variant avian influenza virus challenge after immunization with two antigenically unrelated strains of the same subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antigenic diversity of avian influenza virus (AIV) within a subtype has been well established and is believed to be driven by the selection of immunologic escape mutants. In regions where vaccination against AIV has been implemented for prolonged periods (e.g. Vietnam and Egypt), vaccines which...

  19. Probability of Finding Marrow Unrelated Donor (MUD) for an Indian patient in a Multi-national Human Leukocyte Antigen (HLA) Registry.

    PubMed

    Tiwari, Aseem K; Bhati-Kushwaha, Himakshi; Kukreja, Pooja; Mishra, Vikash C; Tyagi, Neetu; Sharma, Ashish; Raina, Vimarsh

    2015-06-01

    With an increase in the number of transplants happening globally, hematopoietic stem cells (HSC) transplantation from matched unrelated donor (MUD) has begun. The increasing trend of MUD transplants across countries has been largely facilitated with the conspicuous growth of volunteer HSC donor noted in the last decade i.e. 8 million HSC donors in 2002 to more than 22 million in 2013 registered in 71 member registries of the Bone Marrow Donor Worldwide (BMDW). Some populations of the world are still very poorly represented in these registries. Since, the chances of successful engraftment and disease free survival are directly proportional to the HLA compatibility between the recipient and the prospective donor, the diversity of the HLA system at the antigenic and allelic level and the heterogeneity of HLA data of the registered donors has a bearing on the probability of finding a volunteer unrelated HSC donor for patients from such populations. In the present study 126 patients were identified suffering from hematological diseases requiring MUD transplant. Their HLA typing was performed and search was done using BMDW database. The search results for these Indian patients in the multinational registry as well as in the Indian Registries were analyzed using mean, range, standard deviation and finally evaluated in terms of probability for finding matched donor (MUD). Total Asian population is only 11 % in the BMDW making it difficult to find a MUD for an Asian patient. The current study supports this, experimentally; revealing that the probability of finding an allele match for an Indian patient in the multinational Human Leukocyte Antigen (HLA) registries is 16 % and a dismal 0.008 % in the Indian registries (donors in Indian registries is just 33,678 as compared to 22.5 million in BMDW). This greatly, emphasizes on enhancing the number of Indian donors in Indian and multi-national registries. PMID:25825557

  20. Protein antigen delivery by gene gun-mediated epidermal antigen incorporation (EAI).

    PubMed

    Scheiblhofer, Sandra; Ritter, Uwe; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

  1. Tresyl-based conjugation of protein antigen to lipid nanoparticles increases antigen immunogenicity.

    PubMed

    Jain, Anekant; Yan, Weili; Miller, Keith R; O'Carra, Ronan; Woodward, Jerold G; Mumper, Russell J

    2010-11-30

    The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system. PMID:20837122

  2. Tresyl-Based Conjugation of Protein Antigen to Lipid Nanoparticles Increases Antigen Immunogencity

    PubMed Central

    Jain, Anekant; Yan, Weili; Miller, Keith R.; O'Carra, Ronan; Woodward, Jerold G.; Mumper, Russell J.

    2010-01-01

    The present studies were aimed at investigating the engineering of NPs with protein-conjugated-surfactant at their surface. In order to increase the immunogenicity of a protein antigen, Brij 78 was functionalized by tresyl chloride and then further reacted with the primary amine of the model proteins ovalbumin (OVA) or horseradish peroxide (HRP). The reaction yielded Brij 78-OVA and Brij 78-HRP conjugates which were then used directly to form NP-OVA or NP-HRP using a one-step warm oil-in-water microemulsion precursor method with emulsifying wax as the oil phase, and Brij 78 and the Brij 78-OVA or Brij 78-HRP conjugate as surfactants. Similarly, Brij 700 was conjugated to HIV p24 antigen to yield Brij 700-p24 conjugate. The utility of these NPs for enhancing the immune responses to protein-based vaccines was evaluated in vivo using ovalbumin (OVA) as model protein and p24 as a relevant HIV antigen. In separate in vivo studies, female BALB/c mice were immunized by subcutaneous (s.c.) injection with NP-OVA and NP-p24 formulations along with several control formulations. These results suggested that with multiple antigens, covalent attachment of the antigen to the NP significantly enhanced antigen-specific immune responses. This facile covalent conjugation and incorporation method may be utilized to further incorporate other protein antigens, even multiple antigens, into an enhanced vaccine delivery system. PMID:20837122

  3. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    NASA Astrophysics Data System (ADS)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  4. Finding local structural similarities among families of unrelated protein structures: a generic non-linear alignment algorithm.

    PubMed

    Lehtonen, J V; Denessiouk, K; May, A C; Johnson, M S

    1999-02-15

    We have developed a generic tool for the automatic identification of regions of local structural similarity in unrelated proteins having different folds, as well as for defining more global similarities that result from homologous protein structures. The computer program GENFIT has evolved from the genetic algorithm-based three-dimensional protein structure comparison program GA_FIT. GENFIT, however, can locate and superimpose regions of local structural homology regardless of their position in a pair of structures, the fold topology, or the chain direction. Furthermore, it is possible to restrict the search to a volume centered about a region of interest (e.g., catalytic site, ligand-binding site) in two protein structures. We present a number of examples to illustrate the function of the program, which is a parallel processing implementation designed for distribution to multiple machines over a local network or to run on a single multiprocessor computer.

  5. Heat shock protein derivatives for delivery of antigens to antigen presenting cells.

    PubMed

    Nishikawa, Makiya; Takemoto, Seiji; Takakura, Yoshinobu

    2008-04-16

    Delivery of antigens to antigen presenting cells (APCs) is a key issue for developing effective cancer vaccines. Controlling the tissue distribution of antigens can increase antigen-specific immune responses, including the induction of cytotoxic T lymphocytes (CTL). Heat shock protein 70 (Hsp70) forms complexes with a variety of tumor-related antigens via its polypeptide-binding domain. Because Hsp70 is taken up by APCs through recognition by Hsp receptors, such as CD91 and LOX-1, its application to antigen delivery systems has been examined both in experimental and clinical settings. A tissue distribution study revealed that Hsp70 is mainly taken up by the liver, especially by hepatocytes, after intravenous injection in mice. A significant amount of Hsp70 was also delivered to regional lymph nodes when it was injected subcutaneously, supporting the hypothesis that Hsp70 is a natural targeting system for APCs. Model antigens were complexed with or conjugated to Hsp70, resulting in greater antigen-specific immune responses. Cytoplasmic delivery of Hsp70-antigen further increased the efficacy of the Hsp70-based vaccines. These findings indicate that effective cancer therapy can be achieved by developing Hsp70-based anticancer vaccines when their tissue and intracellular distribution is properly controlled. PMID:17980980

  6. Clinical and genetic features of protein C deficiency in 23 unrelated Chinese patients.

    PubMed

    Ding, Qiulan; Shen, Wei; Ye, Xu; Wu, Yingting; Wang, Xuefeng; Wang, Hongli

    2013-01-01

    In this study, we investigated the clinical and genetic features of protein C deficiency in the Chinese population. A total of 23 symptomatic patients with protein C deficiency were identified by thrombophilic assays. Detailed clinical data about the patients with respect to their personal and family history of venous thromboembolism (VTE) were collected. Mutational analysis was then performed by direct sequencing of the protein C gene (PROC) in the patients and their family members. Of the 23 patients, 30.4% (7/23) had additional risk factors, 51.2% (12/23) suffered from recurrent thrombotic episodes, and 50.0% (6/12) of the patients with recurrent thrombosis had more than one heterozygous mutation in PROC itself or combined with protein S gene (PROS). The sex distribution of male:female was 19:4 in the 23 symptomatic patients and 10:2 in the 12 recurrent patients. Almost all patients (22/23) had lower extremity deep vein thrombosis (DVT) and one had pulmonary embolism (PE) only. A total of 15 different causative mutations were identified from the 23 subjects with 6 (40.0%) of the mutations being novel. Among the mutations identified, the Arg147Trp substitution was hotspot mutation in the Chinese population with a high frequency of 43.5%. Our finding suggests that complex genotypes of PROC or combined with protein S deficiency are primarily responsible for an increased risk of recurrent VTE. Our data further provides a framework for correlating the clinical pathogenesis of protein C deficiency to ethnic backgrounds in the Chinese population.

  7. The Role of Heat Shock Proteins in Antigen Cross Presentation

    PubMed Central

    Murshid, Ayesha; Gong, Jianlin; Calderwood, Stuart K.

    2012-01-01

    Heat shock proteins (HSPs) are molecular chaperones that bind tumor antigens and mediate their uptake into antigen presenting cells. HSP–antigen complexes are then directed toward either the MHC class I pathway through antigen cross presentation or the conventional class II pathway, leading to activation of T cell subsets. Uptake of HSP-chaperoned polypeptides can involve both receptor-mediated and receptor-independent routes, and mechanisms of antigen sorting between the Class I and II pathways after uptake are currently under investigation. The processes involved in internalization of HSP–antigen complexes differ somewhat from the mechanisms previously determined for (unchaperoned) particulate and free soluble antigens. A number of studies show that HSP-facilitated antigen cross presentation requires uptake of the complexes by scavenger receptors (SR) followed by processing in the proteasome, and loading onto MHC class I molecules. In this review we have examined the roles of HSPs and SR in antigen uptake, sorting, processing, cell signaling, and activation of innate and adaptive immunity. PMID:22566944

  8. Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

    PubMed Central

    Andrew, Dean; Krishnarjuna, Bankala; Nováček, Jiří; Žídek, Lukáš; Sklenář, Vladimír; Richards, Jack S.; Beeson, James G.; Anders, Robin F.; Norton, Raymond S.

    2015-01-01

    Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design. PMID:25742002

  9. Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

    PubMed Central

    Hoppe, Sebastian; Bier, Frank F.; von Nickisch-Rosenegk, Markus

    2014-01-01

    The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL). After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens and their linear

  10. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

  11. Protein stains to detect antigen on membranes.

    PubMed

    D'souza, Anil; Scofield, R Hal

    2009-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:19378080

  12. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

    1990-04-01

    The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

  13. Antigenic properties of avian hepatitis E virus capsid protein.

    PubMed

    Zhao, Qin; Syed, Shahid Faraz; Zhou, En-Min

    2015-10-22

    Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail. PMID:26340899

  14. Autophagy proteins in antigen processing for presentation on MHC molecules.

    PubMed

    Münz, Christian

    2016-07-01

    Autophagy describes catabolic pathways that deliver cytoplasmic constituents for lysosomal degradation. Since major histocompatibility complex (MHC) molecules sample protein degradation products and present them to T cells for adaptive immunity, it is maybe not too surprising that autophagy contributes to this protein antigen processing for MHC presentation. However, the recently recognized breath of pathways, by which autophagy contributes to MHC antigen processing, is exciting. Macroautophagy does not only seem to deliver intracellular but facilitates also extracellular antigen processing by lysosomal hydrolysis for MHC class II presentation. Moreover, even MHC class I molecules that usually display proteasomal products are regulated by macroautophagy, probably using a pool of these molecules outside the endoplasmic reticulum, where MHC class I molecules are loaded with peptide during canonical MHC class I antigen processing. This review aims to summarize these recent developments and point out gaps of knowledge, which should be filled by further investigation, in order to harness the different antigen-processing pathways via autophagy for vaccine improvement. PMID:27319339

  15. Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

    PubMed

    Bitencourt, Amanda R; Vicentin, Elaine C; Jimenez, Maria C; Ricci, Ricardo; Leite, Juliana A; Costa, Fabio T; Ferreira, Luis C; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R; Barnwell, John W; Rodrigues, Mauricio M; Soares, Irene S

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

  16. Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3

    PubMed Central

    Bitencourt, Amanda R.; Vicentin, Elaine C.; Jimenez, Maria C.; Ricci, Ricardo; Leite, Juliana A.; Costa, Fabio T.; Ferreira, Luis C.; Russell, Bruce; Nosten, François; Rénia, Laurent; Galinski, Mary R.; Barnwell, John W.; Rodrigues, Mauricio M.; Soares, Irene S.

    2013-01-01

    A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential. PMID:23457498

  17. Antigenic proteins of Helicobacter pylori of potential diagnostic value.

    PubMed

    Khalilpour, Akbar; Santhanam, Amutha; Wei, Lee Chun; Saadatnia, Geita; Velusamy, Nagarajan; Osman, Sabariah; Mohamad, Ahmad Munir; Noordin, Rahmah

    2013-01-01

    Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (≥ 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by mass spectrometry. The relative molecular weights and the identities of the proteins, based on MALDI TOF/ TOF, were as follows: CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline- 5-carboxylate dehydrogenase (118 KDa). These identified proteins, singly and/or in combinations, may be useful for diagnosis of H. pylori infection in patients.

  18. The antigenicity and allergenicity of microparticulated proteins: Simplesse.

    PubMed

    Sampson, H A; Cooke, S

    1992-10-01

    New technologies are allowing the food industry to develop products from standard foods which may not be recognized in its modified form by food allergic patients. One such product, Simplesse, has been formulated by microparticulation of egg white and/or cows' milk proteins and is used as a fat substitute in many fat-laden foods. The purpose of this study was to determine whether the process of microparticulation altered the allergenicity/antigenicity of egg white and cows' milk proteins compared to the starting materials. Soluble protein fractions of Simplesse and its respective starting materials were compared to egg white, cows' milk protein, an ultra-filtered egg white/condensed milk mixture, and/or a whey concentrate by SDS-polyacrylamide gel electrophoresis. In addition, sera from 16 patients with documented egg and/or cows' milk hypersensitivity and two controls who were not allergic to egg or milk were used to assess potential allergenicity/antigenicity of these products by immunoblot (Western blot) analysis. There were heterogeneous IgE and IgG binding patterns to the food fractions among these food allergic patients suggesting differing sensitivity patterns among the individuals tested. However, utilizing both SDS-PAGE and immunoblot analyses, the major allergens in the microparticulated products were the same as those found in the starting materials, egg and cows' milk. In addition, there was no evidence of 'novel' protein fractions in the Simplesse test materials compared to the starting materials. PMID:1464052

  19. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  20. Factor H-binding protein, a unique meningococcal vaccine antigen.

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

    GNA1870, also named factor H-binding protein (fHbp) or rLP-2086, is a genome-derived antigen and one of the components of a rationally designed vaccine against Neisseria meningitidis serogroup B, which has entered phase III clinical trials. It has been classified into three main non-cross-protective variant groups. GNA1870 has also been termed fHbp because of its ability to bind factor H, a key regulatory component of the alternative complement pathway. fHbp is important for survival in human blood, human sera, and in presence of antimicrobial peptides, independently of its expression level. All these properties make fHbp a unique vaccine antigen.

  1. A newly identified immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic variation.

    PubMed

    Behrens, A; Poumarat, F; Le Grand, D; Heller, M; Rosengarten, R

    1996-09-01

    Mycoplasma bovis is a bovine pathogen able to cause systemic disease. It possesses a series of prominent, structurally related yet clearly distinguishable membrane lipoproteins on the cell surface. These variable surface proteins (Vsps) undergo highly dynamic and spontaneous changes in size and expression and are key immunogenic components. They may play a critical role as mediators of adherence to host cells and in escaping immune destruction. In this report, we define a novel, Vsp-unrelated membrane protein also associated with M. bovis surface antigenic variation. This protein has an apparent molecular mass of 67,000 Da in the type strain PG45 and was designated pMB67. Immunological and biochemical characterization of pMB67 demonstrated that it: (i) contains a specific epitope, (ii) is not modified by lipid but does contain cysteine, (iii) does not contain a Vsp-like repetitive periodic protein structure, (iv) is a predominant antigen recognized during M. bovis infections, (v) undergoes a high rate of phase variation in vitro and (vi) is size-variable. These results showed that M. bovis employs two types of specialized membrane proteins for surface diversification. The pMB67 protein may be useful in diagnostic assays and as a vaccine component.

  2. Goodpasture Antigen-binding Protein (GPBP) Directs Myofibril Formation

    PubMed Central

    Revert-Ros, Francisco; López-Pascual, Ernesto; Granero-Moltó, Froilán; Macías, Jesús; Breyer, Richard; Zent, Roy; Hudson, Billy G.; Saadeddin, Anas; Revert, Fernando; Blasco, Raül; Navarro, Carmen; Burks, Deborah; Saus, Juan

    2011-01-01

    Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment. PMID:21832087

  3. Antigenic characterization of dimorphic surface protein in Mycobacterium tuberculosis.

    PubMed

    Matsuba, Takashi; Siddiqi, Umme Ruman; Hattori, Toshio; Nakajima, Chie; Fujii, Jun; Suzuki, Yasuhiko

    2016-05-01

    The Mycobacterium tuberculosis Rv0679c protein is a surface protein that contributes to host cell invasion. We previously showed that a single nucleotide transition of the Rv0679c gene leads to a single amino acid substitution from asparagine to lysine at codon 142 in the Beijing genotype family. In this study, we examined the immunological effect of this substitution. Several recombinant proteins were expressed in Escherichia coli and Mycobacterium smegmatis and characterized with antisera and two monoclonal antibodies named 5D4-C2 and 8G10-H2. A significant reduction of antibody binding was detected by enzyme-linked immunosorbent assay (ELISA) and western blot analysis in the Lys142-type protein. This reduction of 8G10-H2 binding was more significant, with the disappearance of a signal in the proteins expressed by recombinant mycobacteria in western blot analysis. In addition, epitope mapping analysis of the recombinant proteins showed a linear epitope by 5D4-C2 and a discontinuous epitope by 8G10-H2. The antibody recognizing the conformational epitope detected only mycobacterial Asn142-type recombinant protein. Our results suggest that a single amino acid substitution of Rv0679c has potency for antigenic change in Beijing genotype strains. PMID:27190237

  4. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

    PubMed Central

    Ducken, Deirdre R.; Brown, Wendy C.; Alperin, Debra C.; Brayton, Kelly A.; Reif, Kathryn E.; Turse, Joshua E.; Palmer, Guy H.; Noh, Susan M.

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  5. The generation of a 'tolerogen' after the ingestion of ovalbumin is time-dependent and unrelated to serum levels of immunoreactive antigen.

    PubMed Central

    Peng, H J; Turner, M W; Strobel, S

    1990-01-01

    In an attempt to investigate the molecular basis of the mechanisms underlying oral tolerance, we have evaluated the molecular and biological features of ovalbumin subjected to intestinal processing. Immunoreactive ovalbumin absorbed by the gut was measured by a sandwich ELISA at different times after feeding 25 mg ovalbumin to adult mice. Ovalbumin was detected as early as 5 min after the feed (36.7 +/- 16 ng/ml; mean +/- 1 s.d.) and reached maximal levels at 1 h (73.3 +/- 20 ng/ml). Pooled mouse serum, collected 5 min or 1 h after the feed, was transferred intraperitoneally into the naive recipients. Suppression of systemic delayed-type hypersensitivity (DTH) was found in mice receiving 0.8 ml of serum obtained 1 h after ovalbumin feeding but not when using serum obtained 5 min after feeding. In order to transfer samples containing similar levels of ovalbumin, an increased amount (1.3 ml) of serum collected 5 min post-feed was used in further experiments but again failed to induce DTH tolerance. Serum samples obtained 5 and 60 min after ovalbumin feeding were analysed by fast-protein liquid chromatography (FPLC) fractionation followed by ELISA. Both the charge characteristics and molecular weight of intestinally absorbed ovalbumin were indistinguishable from native ovalbumin. Although intact native ovalbumin is the only molecular species detected by ELISA, the results suggest that this has no role in the suppression of DTH responses. PMID:2397616

  6. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

    PubMed

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  7. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

    PubMed Central

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  8. Surface antigens of brain synapses: identification of minor proteins using polyclonal antisera

    PubMed Central

    1984-01-01

    Antigenic proteins of brain synaptic plasma membranes (SPM) and postsynaptic densities (PSD) were characterized using antisera raised against SPM. Immunostaining of brain sections showed that the antigens were restricted to synapses, and electron microscopy revealed staining at both presynaptic terminals and PSDs. In primary brain cell cultures the antisera were also neuron-specific but the antigens were distributed throughout the entire neuronal plasma membrane, suggesting that some restrictive influence present in whole tissue is absent when neurons are grown dispersed. The antigenic proteins with which these antisera react were identified using SDS gel immunoblots. SPM and PSD differed from one another in their characteristic antigenic proteins. Comparison with amido-black stained gel blots showed that in both cases most of these did not correspond to known abundant proteins of SPM or PSDs revealed by conventional biochemical techniques. None of the antigens revealed by the polyclonal antisera were detected by any of a large series of monoclonal antibodies against SPM. PMID:6368568

  9. Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection

    PubMed Central

    Kim, Tae Yun; Song, Kye-Yong; Sohn, Woon-Mok; Kang, Shin-Yong

    2001-01-01

    In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection. PMID:11775331

  10. Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

    PubMed Central

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

  11. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs

    SciTech Connect

    Shanklin, J.; Somerville, C. )

    1991-03-15

    Stearoyl-acyl-carrier-protein (ACP) desaturase was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the {Delta}{sup 9} desaturase is developmentally regulated.

  12. Human leukocyte antigen supertype matching after myeloablative hematopoietic cell transplantation with 7/8 matched unrelated donor allografts: a report from the Center for International Blood and Marrow Transplant Research

    PubMed Central

    Lazaryan, Aleksandr; Wang, Tao; Spellman, Stephen R.; Wang, Hai-Lin; Pidala, Joseph; Nishihori, Taiga; Askar, Medhat; Olsson, Richard; Oudshoorn, Machteld; Abdel-Azim, Hisham; Yong, Agnes; Gandhi, Manish; Dandoy, Christopher; Savani, Bipin; Hale, Gregory; Page, Kristin; Bitan, Menachem; Reshef, Ran; Drobyski, William; Marsh, Steven GE; Schultz, Kirk; Müller, Carlheinz R.; Fernandez-Viña, Marcelo A.; Verneris, Michael R.; Horowitz, Mary M.; Arora, Mukta; Weisdorf, Daniel J.; Lee, Stephanie J.

    2016-01-01

    The diversity of the human leukocyte antigen (HLA) class I and II alleles can be simplified by consolidating them into fewer supertypes based on functional or predicted structural similarities in epitope-binding grooves of HLA molecules. We studied the impact of matched and mismatched HLA-A (265 versus 429), -B (230 versus 92), -C (365 versus 349), and -DRB1 (153 versus 51) supertypes on clinical outcomes of 1934 patients with acute leukemias or myelodysplasia/myeloproliferative disorders. All patients were reported to the Center for International Blood and Marrow Transplant Research following single-allele mismatched unrelated donor myeloablative conditioning hematopoietic cell transplantation. Single mismatched alleles were categorized into six HLA-A (A01, A01A03, A01A24, A02, A03, A24), six HLA-B (B07, B08, B27, B44, B58, B62), two HLA-C (C1, C2), and five HLA-DRB1 (DR1, DR3, DR4, DR5, DR9) supertypes. Supertype B mismatch was associated with increased risk of grade II–IV acute graft-versus-host disease (hazard ratio =1.78, P=0.0025) compared to supertype B match. Supertype B07-B44 mismatch was associated with a higher incidence of both grade II–IV (hazard ratio=3.11, P=0.002) and III–IV (hazard ratio=3.15, P=0.01) acute graft-versus-host disease. No significant associations were detected between supertype-matched versus -mismatched groups at other HLA loci. These data suggest that avoiding HLA-B supertype mismatches can mitigate the risk of grade II–IV acute graft-versus-host disease in 7/8-mismatched unrelated donor hematopoietic cell transplantation when multiple HLA-B supertype-matched donors are available. Future studies are needed to define the mechanisms by which supertype mismatching affects outcomes after alternative donor hematopoietic cell transplantation. PMID:27247320

  13. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs.

    PubMed

    Shanklin, J; Somerville, C

    1991-03-15

    Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently. However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated.

  14. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs.

    PubMed Central

    Shanklin, J; Somerville, C

    1991-01-01

    Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently. However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated. Images PMID:2006187

  15. Molecular analysis of three Ljungan virus isolates reveals a new, close-to-root lineage of the Picornaviridae with a cluster of two unrelated 2A proteins.

    PubMed

    Johansson, Susanne; Niklasson, Bo; Maizel, Jacob; Gorbalenya, Alexander E; Lindberg, A Michael

    2002-09-01

    Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles (Clethrionomys glareolus). In the present study, it is revealed through comparative sequence analysis that three newly determined Swedish LV genomes are closely related and possess a deviant picornavirus-like organization: 5' untranslated region-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' untranslated region. The LV genomes and the polyproteins encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho-, and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses, and avian encephalomyelitis virus. The unprecedented association of two structurally different 2A proteins is a feature never previously observed among picornaviruses and implies that their functions are not mutually exclusive. Secondary polyprotein processing of the LV polyprotein is mediated by proteinase 3C (3C(pro)) possessing canonical affinity to Glu and Gln at the P1 position and small amino acid residues at the P1' position. In addition, LV 3C(pro) appears to have unique substrate specificity to Asn, Gln, and Asp and to bulky hydrophobic residues at the P2 and P4 positions, respectively. Phylogenetic analysis suggests that LVs form a separate division, which, together with the Parechovirus genus, has branched off the picornavirus tree most closely to its root. The presence of two 2A proteins indicates that some contemporary picornaviruses with a single 2A may have evolved from the ancestral multi-2A picornavirus.

  16. Identification of Antigenic Proteins from Lichtheimia corymbifera for Farmer’s Lung Disease Diagnosis

    PubMed Central

    Rognon, Bénédicte; Barrera, Coralie; Monod, Michel; Valot, Benoit; Roussel, Sandrine; Quadroni, Manfredo; Jouneau, Stephane; Court-Fortune, Isabelle; Caillaud, Denis; Fellrath, Jean-Marc; Dalphin, Jean-Charles; Reboux, Gabriel; Millon, Laurence

    2016-01-01

    The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer’s lung disease (FLD). The aim of this study was to complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis. PMID:27490813

  17. The in vitro antibacterial activity of ceftriaxone against Streptococcus pyogenes is unrelated to penicillin-binding protein 4.

    PubMed

    Yan, S; Mendelman, P M; Stevens, D L

    1993-07-01

    The in vitro activities of penicillin and ceftriaxone were compared against 29 strains of Streptococcus pyogenes with the result that ceftriaxone showed greater activity than penicillin. The morphological changes induced by 1/2 and 1x MIC concentrations of penicillin and ceftriaxone, respectively, were very similar using scanning electron microscopy. Competitive binding studies using 'cold' penicillin or ceftriaxone as inhibitors of radiolabeled penicillin binding demonstrated that ceftriaxone had a very low affinity for penicillin binding protein (PBP) 4 compared to that of penicillin. Since ceftriaxone had greater antibacterial activity, this suggests that PBP 4 may not be important to the in vitro activity of ceftriaxone. In contrast, the IC50 for ceftriaxone was much lower (> 200 fold) for PBPs 2 and 3 compared to PBP 4, suggesting greater avidity of these high molecular mass PBPs for ceftriaxone. These data may at least in part explain the superior in vitro activity of ceftriaxone compared to penicillin against S. pyogenes. These data, together with the observation that PBP 1 was saturated at a lower concentration of penicillin than any of the other PBPs, suggest that the inhibition of PBPs 1, 2, and 3 mediates the bactericidal activity of beta-lactam antibiotics against group A streptococci. PMID:8354465

  18. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    PubMed

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

  19. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    PubMed

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis. PMID:27379025

  20. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis

    PubMed Central

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis. PMID:27379025

  1. Identification of a peptide binding protein that plays a role in antigen presentation

    SciTech Connect

    Lakey, E.K.; Margoliash, E.; Pierce, S.K.

    1987-03-01

    The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from (/sup 35/S)methionine-labeled cells, appears as two discrete bands of approx. =72 and 74 kDa after NaDodSO/sub 4//PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation.

  2. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    SciTech Connect

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  3. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein.

    PubMed Central

    Buchanan, T M; Hildebrandt, J F

    1981-01-01

    Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2. Images PMID:6166568

  4. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    PubMed

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  5. Haploidentical T Cell-Replete Transplantation with Post-Transplantation Cyclophosphamide for Patients in or above the Sixth Decade of Age Compared with Allogeneic Hematopoietic Stem Cell Transplantation from an Human Leukocyte Antigen-Matched Related or Unrelated Donor.

    PubMed

    Blaise, Didier; Fürst, Sabine; Crocchiolo, Roberto; El-Cheikh, Jean; Granata, Angela; Harbi, Samia; Bouabdallah, Reda; Devillier, Raynier; Bramanti, Stephania; Lemarie, Claude; Picard, Christophe; Chabannon, Christian; Weiller, Pierre-Jean; Faucher, Catherine; Mohty, Bilal; Vey, Norbert; Castagna, Luca

    2016-01-01

    It has recently been shown that a T cell-replete allogeneic (allo) hematopoietic stem cell transplantation (HSCT) from a haploidentical donor (haplo-ID) could be a valid treatment for hematological malignancies. However, little data exist concerning older populations. We provided transplantation to 31 patients over the age of 55 years from a haplo-ID and compared their outcomes with patients of the same ages who underwent transplantation from a matched related (MRD) or an unrelated donor (UD). All 3 groups were comparable, except for their conditioning. Patients in haplo-ID group received 2 days of post-transplantation high-dose cyclophosphamide followed by cyclosporine A and mycophenolate mofetil, whereas patients in other groups received pretransplantation antithymocyte globulin, cyclosporine A, and additional mycophenolate mofetil in case of 1-antigen mismatch. All patients but 1 in the haplo-ID group engrafted. The incidence of grades 2 to 4 acute graft-versus-host disease (GVHD) was not statistically different between recipients from haplo-ID (cumulative incidence, 23%) and MRD (cumulative incidence, 21%) transplantations but it was lower than after UD HSCT (cumulative incidence, 44%). No patient in the haplo-ID group developed severe chronic GVHD, compared with cumulative incidences of 16% and 14% after MRD (P = .02) and UD (P = .03) grafts, respectively. The cumulative incidences of relapse were similar in the 3 groups, whereas nonrelapse mortality after UD HSCT was 3-fold higher than after haplo-ID or MRD HSCT. Overall, 2-year overall survival (70%), progression-free survival (67%), and progression and severe chronic GVHD-free survival (67%) probabilities after haplo-ID did not statistically differ from MRD transplantation (78%, 64%, and 51%, respectively), although they were higher than after UD transplantation (51% [P = .08], 38% [P = .02], and 31% [P = .007]). We conclude that T cell-replete haplo-ID HSCT followed by post-transplantation high

  6. Expression of S-100 protein, epithelial membrane antigen, carcinoembryonic antigen and alpha fetoprotein in normal salivary glands and primary salivary gland tumors.

    PubMed

    Günhan, O; Evren, G; Demiriz, M; Can, C; Celasun, B; Finci, R

    1992-12-01

    The distribution of S-100 protein, epithelial membrane antigen, carcinoembryonic antigen and alpha fetoprotein was studied in 38 primary salivary gland tumors. S-100 protein, a useful marker of myoepithelial cells, was demonstrated in some benign tumors. Carcinoembryonic antigen expression was consistently positive in adenoid cystic carcinoma. Demonstration of epithelial membrane antigen helped to confirm the epithelial nature of some neoplastic cells. Alpha fetoprotein was not expressed in any of the cases examined. No correlation was found between immunopositivity and tumor behavior in the present series.

  7. Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens.

    PubMed Central

    Sandstrom, E G; Knapp, J S; Buchanan, T B

    1982-01-01

    A total of 224 strains were serogrouped by coagglutination (COA) and serotyped by protein I enzyme-linked immunosorbent assay (ELISA). Of these strains, 61 were from patients with disseminated gonococcal infection, 21 were from patients with pelvic inflammatory disease, and 115 were from patients with uncomplicated gonococcal infection in Singapore, the Philippines, and Denmark. Twenty-seven were laboratory reference strains. Of the patient strains, 102 belonged to COA serogroup WI, and all of the 100 strains that typed with protein I serotypes 1, 2, or 3 were in this group. Most of the strains of gonococci from the 61 patients with disseminated gonococcal infection were within this group (COA WI, 53 or 87%; protein I serotypes 1, 2, or 3, 51 or 84%). All 46 strains that were protein I serotypes 4 through 7 were also COA serogroup WII. Protein I serotypes 8 and 9 accounted for 49 (25%) of the 197 patient strains. Twenty-eight of these strains typed as COA serogroup WII, 20 typed as serogroup WIII, and 1 typed as serogroups WII and WIII. COA W serogrouping and protein I ELISA both appeared to detect antigens on the protein I molecule of the outer membrane of Neisseria gonorrhoeae. Protein I serotyping, which uses unboiled organisms, may generally recognize more variable and surface-exposed antigenic determinants. In contrast, COA W serogrouping, which uses boiled organisms, may recognize less exposed shared antigenic determinants in addition to variable protein I antigenic determinants. Both methods may prove useful for further studies of the epidemiology and pathogenesis of gonorrhea. Images PMID:6172380

  8. Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

    PubMed Central

    Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

    1993-01-01

    A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

  9. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  10. 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A

    SciTech Connect

    Samelson, L.E.; Harford, J.; Schwartz, R.H.; Klausner, R.D.

    1985-04-01

    Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell receptor.

  11. The I-antigens of Ichthyophthirius multifiliis are GPI-anchored proteins.

    PubMed

    Clark, T G; Gao, Y; Gaertig, J; Wang, X; Cheng, G

    2001-01-01

    The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.

  12. Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

    PubMed

    Navarrete-Perea, José; Orozco-Ramírez, Rodrigo; Moguel, Bárbara; Sciutto, Edda; Bobes, Raúl J; Laclette, Juan P

    2015-07-01

    Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures.

  13. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  14. Mature proteins derived from Epstein-Barr virus fail to feed into the MHC class I antigenic pool.

    PubMed

    Fiebiger, Benjamin M; Moosmann, Andreas; Behrends, Uta; Mautner, Josef

    2012-12-01

    The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein-Barr virus (EBV) antigens was induced were rapidly recognized by antigen-specific CD8(+) T cells in a time- and dosage-dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half-lives and are hence overexpressed in tumors.

  15. [Incomplete antigens derived from milk proteins in the serum of infants allergic to milk].

    PubMed

    Vanella, L M; de González Lascano, A M; Miguez, V M

    1978-01-01

    1. Sera of 22 children with cow's milk clinical hypersensitivity were studied to demonstrate the presence of substances immunologically related with milk. They were compared with 23 controls. The infants of both groups were feed with bovine milk. The immunogenic capacity of cow's milk and their major proteins were experimentally investigated. 2. Specific rabbit antisera were obtained by injection of antigens with incomplete Freund adjuvant. Double difussion gel, passive hemagglutination and ultramicromethod for the determination of antigen antibody precipitated were performed. 3. Immunogenicity was proved by precipitation and hemagglutination methods. by precipitation cow's milk antigens were present in 5 of 22 sera of antigenic patients, in 3 of them ALA antigens were present and in only 1 of them, caseina were present. By hemagglutination, 12 of 22 allergic infants showed ALA and BLG and 11 caseine (C). In 2 of 7 controls, beta lactoglobuline (BLG) was present and in an other one C. It was possible to detect incomplete antigens related with ALA, BLG, and C in allergic infants as well as controls. A significative difference was found for BLG (P less than 0.01) and it was highest (P less than 0.003) in infants with protein calorie malnutrition. 4. It is concluded that sensitization depends not only on stimulation of incomplete or complete antigens, as were observed in this study but on the host's capacity to form citrotropic antibody in humoral hypersensitivity or to stimulate lymphocytes in cellular immunity field.

  16. Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection.

    PubMed

    Thuy, Nguyen Thu; Goto, Yasuyuki; Lun, Zhao-Rong; Kawazu, Shin-Ichiro; Inoue, Noboru

    2012-02-01

    Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.

  17. Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies

    SciTech Connect

    van Wyke, K.L.; Yewdell, J.W.; Reck, L.J.; Murphy, B.R.

    1984-01-01

    Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. The authors found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of three epitopes are located in nonoverlapping domains on the matrix protein molecule.

  18. Computational antigenic epitope prediction by calculating electrostatic desolvation penalties of protein surfaces.

    PubMed

    Fiorucci, Sébastien; Zacharias, Martin

    2014-01-01

    The prediction of antigenic epitopes on the surface of proteins is of great importance for vaccine development and to specifically design recombinant antibodies. Computational methods based on the three-dimensional structure of the protein allow for the detection of noncontinuous epitopes in contrast to methods based on the primary amino-acid sequence only. A method recently developed to predict protein-protein binding sites is presented, and the application to predict putative antigenic epitopes is described in detail. The prediction approach is based on the local perturbation of the electrostatic field at the surface of a protein due to a neutral probe of low dielectric constant that represents an approaching binding partner. The calculated change in electrostatic energy corresponds to an energy penalty of desolvating a protein surface region, and antigenic epitope surface regions tend to be associated with a lower penalty compared to the average protein surface. The protocol to perform the calculations is described and illustrated on an example antigen, the outer surface protein A of Borrelia burgdorferi, a pathogenic organism causing lyme disease.

  19. Evaluation of Salmonella enterica type III secretion system effector proteins as carriers for heterologous vaccine antigens.

    PubMed

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid; Hensel, Michael

    2012-03-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines.

  20. Overexpression and Enzymatic Assessment of Antigenic Fragments of Hyaluronidase Recombinant Protein From Streptococcus pyogenes

    PubMed Central

    Sadoogh Abbasian, Shabnam; Ghaznavi Rad, Ehsanollah; Akbari, Neda; Zolfaghari, Mohammad Reza; pakzad, Iraj; Abtahi, Hamid

    2014-01-01

    Background: Hyaluronidase catalyzes the hydrolysis of hyaluronan polymers to N-acetyl-D-glucosamine and D-glucuronic acid. This enzyme is a dimer of identical subunits. Hyaluronidase has different pharmaceutical and medical applications. Previously, we produced a recombinant hyaluronidase antigenic fragment of Streptococcus pyogenes. Objectives: This study aimed to improve the protein production and purity of hyaluronidase recombinant protein from S. pyogenes. In addition, the enzymatic activity of this protein was investigated. Materials and Methods: The expression of hyaluronidase antigenic fragments was optimized using IPTG concentration, time of induction, temperature, culture, and absorbance of 0.6-0.8-1 at 600 nm. Afterwards, the expressed proteins were purified and the enzymatic activity was assessed by turbid metric method. Results: Data indicated that maximum protein is produced in OD = 0.8, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), 37ºC, NB 1.5x, without glucose, incubated for overnight. The enzymatic activity of the recombinant protein was similar to the commercial form of hyaluronidase. Conclusions: The results showed that an antigenic fragment of the recombinant hyaluronidase protein from S. pyogenes has a considerable enzymatic activity. It can be suggested to use it for medical purposes. In addition, applications of bioinformatics software would facilitate the production of a smaller protein with same antigenic properties and enzymatic activity. PMID:25789122

  1. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  2. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens

    PubMed Central

    Yanasarn, Nijaporn; Sloat, Brian R.; Cui, Zhengrong

    2011-01-01

    Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome’s ability (i) to up-regulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. PMID:21615153

  3. A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic

    PubMed Central

    Moudallal, Z. Al.; Briand, J. P.; Regenmortel, M. H. V. Van

    1985-01-01

    Eighteen synthetic peptides representing virtually the entire length of the polypeptide chain of tobacco mosaic virus coat protein (TMVP) have been analyzed for their ability to bind in an enzyme immunoassay to 30 monoclonal antibodies raised against the dissociated viral subunits. Only five of the monoclonal antibodies were able to bind a number of peptides while the other 25 antibodies recognized only the complete molecule and seemed to be specific for conformational features that are absent in the peptide fragments. The 18 peptides were also tested for their ability to bind to several antisera to TMVP. Virtually the entire sequence of TMVP possessed antigenic activity. Four new epitopes were identified in the vicinity of residues 19–32, 90–95, 115–134 and 134–146. These results bring to 11 the number of continuous epitopes that have been identified in the TMVP molecule and show that the entire surface of the molecule is antigenic. When peptides of TMVP of a length of 6–8 residues were tested for antigenic activity previously a correlation was found between the location of short continuous epitopes and mobile segments of the protein. In the present study, in which longer peptides as well as monoclonal antibodies were used to probe the antigenicity of TMVP, additional conformation-dependent epitopes were shown to be present. Our results illustrate the operational nature of any definition of antigenicity and caution against the use of any single criterion for distinguishing between antigenic and non-antigenic regions of a protein. PMID:16453613

  4. Analysis of Known Bacterial Protein Vaccine Antigens Reveals Biased Physical Properties and Amino Acid Composition

    PubMed Central

    Mayers, Carl; Rowe, Sonya; Miller, Julie; Lingard, Bryan; Hayward, Sarah; Titball, Richard W.

    2003-01-01

    Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location. PMID:18629010

  5. [Hyperprolactinemia unrelated to prolactinoma].

    PubMed

    Krysiak, Robert; Okopień, Bogusław

    2014-01-01

    Hyperprolactinemia, defined as prolactin levels above the upper limit of normal range, is the most frequent hypothalamus-pituitary dysfunction. Clinical symptoms of hyperprolactinemia in women include oligomenorrhea, infertility, and galactorrhea, while in men the condition may lead to hypogonadism, decreased libido, erectile dysfunction, infertility, gynecomastia, and, in rare instances, galactorrhea. In many patients, hyperprolactinemia results from the presence of prolactinoma, which is considered as the most common hormone-secreting pituitary tumors. However, transient or long-term hyperprolactinemia may also develop during different physiological situations or due to several diseases. It is also a frequent but often neglected side effect of many drugs, particularly of antipsychotics. Finally, hyperprolactinemia may be secondary to the predominance of high molecular mass circulating prolactin forms that have been postulated to represent complexes of prolactin and anti-prolactin immunoglobulins (macroprolactinemia). The cause of hyperprolactinemia determines its treatment. In this paper, we review the causes of hyperprolactinemia unrelated to prolactinoma, providing a differential diagnosis of this condition.

  6. Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins.

    PubMed

    Venkatachalam, M; Teuber, S S; Roux, K H; Sathe, S K

    2002-06-01

    Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart. PMID:12033826

  7. Comprehensive Antigen Screening Identifies Moraxella catarrhalis Proteins That Induce Protection in a Mouse Pulmonary Clearance Model

    PubMed Central

    Verhaegh, Suzanne J. C.; Niebisch, Axel; Hanner, Markus; Selak, Sanja; Schüler, Wolfgang; Morfeldt, Eva; Hellberg, Christel; Nagy, Eszter; Lundberg, Urban; Hays, John P.; Meinke, Andreas; Henriques-Normark, Birgitta

    2013-01-01

    Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis. PMID:23671716

  8. Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein

    PubMed Central

    Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W.; Garcia, Valentino; Kappe, Stefan H. I.; Krzych, Urszula; Duffy, Patrick E.

    2016-01-01

    Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP. PMID:27434123

  9. Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

    PubMed

    Speake, Cate; Pichugin, Alexander; Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W; Garcia, Valentino; Kappe, Stefan H I; Krzych, Urszula; Duffy, Patrick E

    2016-01-01

    Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP. PMID:27434123

  10. An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom.

    PubMed

    Yu, Y L; Yeh, K W; Lin, C P

    1998-05-01

    A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.

  11. Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

    PubMed

    Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

    2013-08-01

    Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band. PMID:23680184

  12. Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

    PubMed

    Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

    2013-08-01

    Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

  13. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    PubMed Central

    Permyakova, Natalia V.; Zagorskaya, Alla A.; Belavin, Pavel A.; Uvarova, Elena A.; Nosareva, Olesya V.; Nesterov, Andrey E.; Novikovskaya, Anna A.; Zav'yalov, Evgeniy L.; Moshkin, Mikhail P.; Deineko, Elena V.

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells. PMID:25949997

  14. A protein chip approach for high-throughput antigen identification and characterization.

    PubMed

    Hu, Shaohui; Li, Yu; Liu, Guozhen; Song, Qifeng; Wang, Li; Han, Yuning; Zhang, Yang; Song, Yali; Yao, Xiying; Tao, Yong; Zeng, Haipan; Yang, Huanming; Wang, Jian; Zhu, Heng; Chen, Zhi-Nan; Wu, Lin

    2007-06-01

    Proteomics research in humans and other eukaryotes demands a large number of high-quality mAbs. Here, we report a new approach to produce high-quality mAbs against human liver proteins using a combined force of high-throughput mAb production and protein microarrays. After immunizing mice with live cells from human livers, we isolated 54 hybridomas with binding activities to human cells and identified the corresponding antigens for five mAbs via screening on a protein microarray of 1058 unique human liver proteins. Finally, we demonstrated that using the five mAbs we could characterize the expression profiles of their corresponding antigens by using tissue microarrays. Among them, we discovered that eIF1A expressed only in normal liver tissues, not in hepatocellular carcinoma in humans.

  15. In Silico Design of a Chimeric Protein Containing Antigenic Fragments of Helicobacter pylori; A Bioinformatic Approach

    PubMed Central

    Mohammad, Nazanin; Karsabet, Mehrnaz Taghipour; Amani, Jafar; Ardjmand, Abolfazl; Zadeh, Mohsen Razavi; Gholi, Mohammad Khalifeh; Saffari, Mahmood; Ghasemi, Amir

    2016-01-01

    Helicobacter pylori is a global health problem which has encouraged scientists to find new ways to diagnose, immunize and eradicate the H. pylori infection. In silico studies are a promising approach to design new chimeric antigen having the immunogenic potential of several antigens. In order to obtain such benefit in H. pylori vaccine study, a chimeric gene containing four fragments of FliD sequence (1-600 bp), UreB (327-334 bp),VacA (744-805 bp) and CagL(51-100 bp) which have a high density of B- and T-cell epitopes was designed. The secondary and tertiary structures of the chimeric protein and other properties such as stability, solubility and antigenicity were analyzed. The in silico results showed that after optimizing for the purpose of expression in Escherichia coli BL21, the solubility and antigenicity of the construct fragments were highly retained. Most regions of the chimeric protein were found to have a high antigenic propensity and surface accessibility. These results would be useful in animal model application and accounted for the development of an epitope-based vaccine against the H. pylori. PMID:27335622

  16. Uncoupling protein 2 regulates metabolic reprogramming and fate of antigen-stimulated CD8+ T cells.

    PubMed

    Chaudhuri, Leena; Srivastava, Rupesh K; Kos, Ferdynand; Shrikant, Protul A

    2016-07-01

    Adoptive cell therapy (ACT) employing ex vivo-generated tumor antigen-specific CD8+ T cells shows tumor efficacy when the transferred cells possess both effector and memory functions. New strategies based on understanding of mechanisms that balance CD8+ T cell differentiation toward effector and memory responses are highly desirable. Emerging information confirms a central role for antigen-induced metabolic reprogramming in CD8+ T cell differentiation and clonal expansion. The mitochondrial protein uncoupling protein 2 (UCP2) is induced by antigen stimulation of CD8+ T cells; however, its role in metabolic reprogramming underlying differentiation and clonal expansion has not been reported. Employing genetic (siRNA) and pharmacologic (Genipin) approaches, we note that antigen-induced UCP2 expression reduces glycolysis, fatty acid synthesis and production of reactive oxygen species to balance differentiation with survival of effector CD8+ T cells. Inhibition of UCP2 promotes CD8+ T cell terminal differentiation into short-lived effector cells (CD62L(lo)KLRG1(Hi)IFNγ(Hi)) that undergo clonal contraction. These findings are the first to reveal a role for antigen-induced UCP2 expression in balancing CD8+ T cell differentiation and survival. Targeting UCP2 to regulate metabolic reprogramming of CD8+ T cells is an attractive new approach to augment efficacy of tumor therapy by ACT. PMID:27271549

  17. Neisseria meningitidis antigen NMB0088: sequence variability, protein topology and vaccine potential.

    PubMed

    Sardiñas, Gretel; Yero, Daniel; Climent, Yanet; Caballero, Evelin; Cobas, Karem; Niebla, Olivia

    2009-02-01

    The significance of Neisseria meningitidis serogroup B membrane proteins as vaccine candidates is continually growing. Here, we studied different aspects of antigen NMB0088, a protein that is abundant in outer-membrane vesicle preparations and is thought to be a surface protein. The gene encoding protein NMB0088 was sequenced in a panel of 34 different meningococcal strains with clinical and epidemiological relevance. After this analysis, four variants of NMB0088 were identified; the variability was confined to three specific segments, designated VR1, VR2 and VR3. Secondary structure predictions, refined with alignment analysis and homology modelling using FadL of Escherichia coli, revealed that almost all the variable regions were located in extracellular loop domains. In addition, the NMB0088 antigen was expressed in E. coli and a procedure for obtaining purified recombinant NMB0088 is described. The humoral immune response elicited in BALB/c mice was measured by ELISA and Western blotting, while the functional activity of these antibodies was determined in a serum bactericidal assay and an animal protection model. After immunization in mice, the recombinant protein was capable of inducing a protective response when it was administered inserted into liposomes. According to our results, the recombinant NMB0088 protein may represent a novel antigen for a vaccine against meningococcal disease. However, results from the variability study should be considered for designing a cross-protective formulation in future studies.

  18. Occurrence of protein a in staphylococcal strains: quantitative aspects and correlation to antigenic and bacteriophage types.

    PubMed

    Kronvall, G; Dossett, J H; Quie, P G; Williams, R C

    1971-01-01

    Protein A of Staphylococcus aureus can be detected on cell walls of intact bacteria by use of radioactively labeled myeloma globulin. Of 156 strains of S. aureus, 141 (90%) contained protein A. None of 47 S. epidermidis strains was positive for protein A. The production of protein A was influenced by incubation temperature but not by differences in incubation time or inoculum size. A medium containing a high concentration of NaCl suppressed the production of protein A by 90%. Formalin treatment of protein A-containing strains caused a decrease in the amount detected, but no further decrease was detected after storage at 4 C. No correlation was found between absence or presence of protein A and phage type or phage group. Sixteen S. aureus strains were studied extensively. There was no correlation between protein A and any of the 26 antigenic characteristics which have been previously described in these strains.

  19. Occurrence of protein a in staphylococcal strains: quantitative aspects and correlation to antigenic and bacteriophage types.

    PubMed

    Kronvall, G; Dossett, J H; Quie, P G; Williams, R C

    1971-01-01

    Protein A of Staphylococcus aureus can be detected on cell walls of intact bacteria by use of radioactively labeled myeloma globulin. Of 156 strains of S. aureus, 141 (90%) contained protein A. None of 47 S. epidermidis strains was positive for protein A. The production of protein A was influenced by incubation temperature but not by differences in incubation time or inoculum size. A medium containing a high concentration of NaCl suppressed the production of protein A by 90%. Formalin treatment of protein A-containing strains caused a decrease in the amount detected, but no further decrease was detected after storage at 4 C. No correlation was found between absence or presence of protein A and phage type or phage group. Sixteen S. aureus strains were studied extensively. There was no correlation between protein A and any of the 26 antigenic characteristics which have been previously described in these strains. PMID:16557923

  20. Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases.

    PubMed

    Yue, Tingting; Partyka, Katie; Maupin, Kevin A; Hurley, Mary; Andrews, Philip; Kaul, Karen; Moser, A James; Zeh, Herbert; Brand, Randall E; Haab, Brian B

    2011-09-01

    The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

  1. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens?

    PubMed

    Banu, Sayera; Honoré, Nadine; Saint-Joanis, Brigitte; Philpott, Dana; Prévost, Marie-Christine; Cole, Stewart T

    2002-04-01

    Mycobacterium tuberculosis H37Rv contains 67 PE-PGRS genes, with multiple tandem repetitive sequences, encoding closely related proteins that are exceptionally rich in glycine and alanine. As no functional information was available, 10 of these genes were selected and shown to be expressed in vitro by reverse transcription-polymerase chain reaction (RT-PCR). Antibodies against five PE-PGRS proteins, raised in mice by DNA vaccination, detected single proteins when the same plasmid constructs used for immunization were expressed in epithelial cells or in reticulocyte extracts, confirming that the PE-PGRS proteins are antigenic. As expected from the conserved repetitive structure, the antibodies cross-reacted with more than one PE-PGRS protein, suggesting that different proteins share common epitopes. PE-PGRS proteins were detected by West-ern blotting in five different mycobacterial species (M. tuberculosis, M. bovis BCG, M. smegmatis, M. marinum and M. gordonae) and 11 clinical isolates of M. tuberculosis. Whole-genome comparisons of M. tuberculosis predicted allelic diversity in the PE-PGRS family, and this was confirmed by immunoblot studies as size variants were detected in clinical strains. Subcellular fractionation studies and immunoelectron microscopy localized many PE-PGRS proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE-PGRS proteins are variable surface antigens.

  2. A mutant chaperone converts a wild-type protein into a tumor-specific antigen.

    PubMed

    Schietinger, Andrea; Philip, Mary; Yoshida, Barbara A; Azadi, Parastoo; Liu, Hui; Meredith, Stephen C; Schreiber, Hans

    2006-10-13

    Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.

  3. The Fimbrial Protein is a Virulence Factor and Potential Vaccine Antigen of Avibacterium paragallinarum.

    PubMed

    Liu, C-C; Ou, S-C; Tan, D-H; Hsieh, M-K; Shien, J-H; Chang, P-C

    2016-09-01

    Fimbriae are recognized as virulence factors and potential vaccine antigens of several pathogenic bacteria, but the function of the fimbriae from Avibacterium paragallinarum is not well known. In this study, a gene encoding the fimbrial protein FlfA was identified in A. paragallinarum . Sequencing analysis of the putative promoter region of flfA suggests that flfA expression in A. paragallinarum might be controlled by phase variation. The flfA gene from A. paragallinarum was expressed as a recombinant protein (r-FlfA) in Escherichia coli . Immunization with r-FlfA conferred chickens protection against challenge infection with A. paragallinarum . Virulence assays showed that the flfA-deficient mutants of A. paragallinarum were less virulent than their parental wild-type strains. These results indicated that the fimbrial protein FlfA is a virulence factor and potential vaccine antigen from A. paragallinarum . PMID:27610725

  4. Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation.

    PubMed

    Matheoud, Diana; Sugiura, Ayumu; Bellemare-Pelletier, Angélique; Laplante, Annie; Rondeau, Christiane; Chemali, Magali; Fazel, Ali; Bergeron, John J; Trudeau, Louis-Eric; Burelle, Yan; Gagnon, Etienne; McBride, Heidi M; Desjardins, Michel

    2016-07-14

    Antigen presentation is essential for establishing immune tolerance and for immune responses against infectious disease and cancer. Although antigen presentation can be mediated by autophagy, here we demonstrate a pathway for mitochondrial antigen presentation (MitAP) that relies on the generation and trafficking of mitochondrial-derived vesicles (MDVs) rather than on autophagy/mitophagy. We find that PINK1 and Parkin, two mitochondrial proteins linked to Parkinson's disease (PD), actively inhibit MDV formation and MitAP. In absence of PINK1 or Parkin, inflammatory conditions trigger MitAP in immune cells, both in vitro and in vivo. MitAP and the formation of MDVs require Rab9 and Sorting nexin 9, whose recruitment to mitochondria is inhibited by Parkin. The identification of PINK1 and Parkin as suppressors of an immune-response-eliciting pathway provoked by inflammation suggests new insights into PD pathology. PMID:27345367

  5. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity

    PubMed Central

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP+ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  6. CHARACTERIZATION OF THE CARBOHYDRATE COMPONENTS OF Taenia solium ONCOSPHERE PROTEINS AND THEIR ROLE IN THE ANTIGENICITY

    PubMed Central

    Arana, Yanina; Verastegui, Manuela; Tuero, Iskra; Grandjean, Louis; Garcia, Hector H.; Gilman, Robert H.

    2015-01-01

    This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that post-translational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells. PMID:23982308

  7. Characterization of the carbohydrate components of Taenia solium oncosphere proteins and their role in the antigenicity.

    PubMed

    Arana, Yanina; Verastegui, Manuela; Tuero, Iskra; Grandjean, Louis; Garcia, Hector H; Gilman, Robert H

    2013-10-01

    This study examines the carbohydrate composition of Taenia solium whole oncosphere antigens (WOAs), in order to improve the understanding of the antigenicity of the T. solium. Better knowledge of oncosphere antigens is crucial to accurately diagnose previous exposure to T. solium eggs and thus predict the development of neurocysticercosis. A set of seven lectins conjugates with wide carbohydrate specificity were used on parasite fixations and somatic extracts. Lectin fluorescence revealed that D-mannose, D-glucose, D-galactose and N-acetyl-D-galactosamine residues were the most abundant constituents of carbohydrate chains on the surface of T. solium oncosphere. Lectin blotting showed that posttranslational modification with N-glycosylation was abundant while little evidence of O-linked carbohydrates was observed. Chemical oxidation and enzymatic deglycosylation in situ were performed to investigate the immunoreactivity of the carbohydrate moieties. Linearizing or removing the carbohydrate moieties from the protein backbones did not diminish the immunoreactivity of these antigens, suggesting that a substantial part of the host immune response against T. solium oncosphere is directed against the peptide epitopes on the parasite antigens. Finally, using carbohydrate probes, we demonstrated for the first time that the presence of several lectins on the surface of the oncosphere was specific to carbohydrates found in intestinal mucus, suggesting a possible role in initial attachment of the parasite to host cells.

  8. Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

    PubMed Central

    2014-01-01

    While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

  9. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  10. Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

    PubMed Central

    Skov Sørensen, U B; Blom, J; Birch-Andersen, A; Henrichsen, J

    1988-01-01

    The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed. Images PMID:3397179

  11. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  12. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    SciTech Connect

    Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-06-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.

  13. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

    PubMed Central

    Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  14. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

    PubMed Central

    Kushner, Nicholas; Zhang, Dong; Touzjian, Neal; Essex, Max; Lieberman, Judy; Lu, Yichen

    2003-01-01

    Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin. PMID:12740437

  15. Evaluation of Mdh1 protein as an antigenic candidate for a vaccine against candidiasis.

    PubMed

    Shibasaki, Seiji; Aoki, Wataru; Nomura, Takashi; Karasaki, Miki; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

    2014-01-01

    Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

  16. Antigenic determinants of a plant proteoglycan, the Gladiolus style arabinogalactan-protein.

    PubMed

    Gleeson, P A; Clarke, A E

    1980-11-01

    Antiserum has been raised to the arabinogalactan-protein of Gladiolus style mucilage. This macromolecule has been characterized and has a structure consistent with a 1 leads to 3-linked beta-galactan backbone with side branches of 1 leads to 6-linked beta-galactosyl residues, some of which carry terminal alpha-L-arabinofuranoside residues [Gleeson & Clarke (1979) Biochem. J. 181, 607-621]. The specificity of the antiserum has been investigated by immunoprecipitation with [3H]arabinogalactan-protein. THe 3H label was introduced into the arabinogalactan-protein by oxidation of the terminal galactose residues with galactose oxidase, followed by reduction with NaB3H4. The antigenic specificity of the antiserum was shown to be directed towards the carbohydrate component of the arabinogalactan-protein. D-galactose and L-arabinose were the most effective hapten inhibitors of the antiserum; other monosaccharides, N-acetyl-D-galactono-1,4-lactone, D-glucose, D-mannose, L-rhamnose. L-fucose and D-xylose, were all poor inhibitors. The antiserum showed preference for beta-galactosides over alpha-galactosides. Of the haptens examined, the disaccharide 6-O-beta-D-galactopyranosyl-D-galactopyranose was the most potent inhibitor. The antigenic features of the arabinogalactan-protein were investigated by examining the interaction of the antiserum with chemically and enzymically modified arabinogalactan-protein. Also, the cross-reactivity of structurally related polysaccharides and glycoproteins with the specific antiserum was assessed by a haemagglutination assay using erythrocytes coupled with specific antiserum. The results indicate that the dominant antigenic determinants of the arabinogalactan-protein are probably the side branches of 1 leads to 6 -linked beta-galactose residues bearing the terminal alpha-L-arabinose residues.

  17. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system.

    PubMed

    Langella, P; Le Loir, Y

    1999-02-01

    Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitopeprotein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

  18. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    PubMed

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  19. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. PMID:26297626

  20. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

  1. Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis.

    PubMed

    Lokanathan, Y; Mohd-Adnan, A; Kua, B-C; Nathan, S

    2016-09-01

    Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens. PMID:27086498

  2. Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity

    PubMed Central

    Sung, Dong-Eun; Lee, Jeongok; Han, Youngshin; Shon, Dong-Hwa; Ahn, Kangmo

    2014-01-01

    BACKGROUND/OBJECTIVES Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis. PMID:24944772

  3. Chitosan based nanoparticles as protein carriers for efficient oral antigen delivery.

    PubMed

    Gao, Ping; Xia, Guixue; Bao, Zixian; Feng, Chao; Cheng, Xiaojie; Kong, Ming; Liu, Ya; Chen, Xiguang

    2016-10-01

    This study aimed to investigate the efficacy of nanoparticles based on chitosan as a vehicle for oral antigen delivery in fish vaccination. Carboxymethyl chitosan/chitosan nanoparticles (CMCS/CS-NPs) loaded extracellular products (ECPs) of Vibrio anguillarum were successfully developed by ionic gelation method. The prepared ECPs-loaded CMCS/CS-NPs were characterized for various parameters including morphology, particle size (312±7.18nm), zeta potential (+17.4±0.38mV), loading efficiency (57.8±2.54%) and stability under the simulated gastrointestinal (GI) tract conditions in turbot. The in vitro profile showed that the cumulative release of ECPs from nanoparticles was higher in pH 7.4 (58%) than in pH 2.0 (37%) and pH 4.5 (29%) after 48h. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was used as model protein antigen and encapsulated in CMCS/CS-NPs for investigating the biodistribution of antigen after oral delivery to turbot in 24h. Oral immunization of ECPs-loaded CMCS/CS-NPs group in turbot showed elevated specific antibody and higher concentrations of lysozyme activity and complement activity in fish serum than ECPs solution. CMCS/CS-NPs loaded with ECPs could enhance both adaptive and innate immune responses than the group treated with ECPs solution and suggested to be a potential antigen delivery system. PMID:27287772

  4. Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans.

    PubMed

    Okahashi, N; Koga, T; Hamada, S

    1986-01-01

    A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests.

  5. Antigenic composition and immunoreactivity differences between HEV recombinant capsid proteins generated from different genotypes.

    PubMed

    Behloul, Nouredine; Wen, Jiyue; Dai, Xing; Dong, Chen; Meng, Jihong

    2015-08-01

    Appreciable variability has been observed in hepatitis E virus (HEV) serological diagnostics. Four recombinant proteins (p166s) were generated from position 452 to 617 aa of ORF2 of different HEV genotypes and used in an indirect ELISA to detect anti-HEV IgMs and IgGs in serially diluted sera of patients infected with different HEV genotypes (genotype 1, n=15; genotype 3, n=12; genotype 4, n=17). To evaluate the differences at a conformational level, 3D-structure models of p166s were predicted, and different bioinformatics tools were used to analyze the antigenic composition. With both anti-HEV IgMs and IgGs antibodies, there was a considerable variability between the four antigens immunoreactivities. In silico results revealed the region 483-533 aa with the highest antigenic potential and contains six key aa at positions 488, 489, 512, 533, 483 and 530. This immunoreactivity variation could affect diagnosis results and seroprevalence estimations and the identification in silico of a region highly antigenic would guide the development of efficient serological assays and epitope-based vaccines. PMID:26122075

  6. Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity☆

    PubMed Central

    Jones, Sarah; Asokanathan, Catpagavalli; Kmiec, Dorota; Irvine, June; Fleck, Roland; Xing, Dorothy; Moore, Barry; Parton, Roger; Coote, John

    2014-01-01

    Protein-coated microcrystals (PCMCs) were investigated as potential vaccine formulations for a range of model antigens. Presentation of antigens as PCMCs increased the antigen-specific IgG responses for all antigens tested, compared to soluble antigens. When compared to conventional aluminium-adjuvanted formulations, PCMCs modified with calcium phosphate (CaP) showed enhanced antigen-specific IgG responses and a decreased antigen-specific IgG1:IgG2a ratio, indicating the induction of a more balanced Th1/Th2 response. The rate of antigen release from CaP PCMCs, in vitro, decreased strongly with increasing CaP loading but their immunogenicity in vivo was not significantly different, suggesting the adjuvanticity was not due to a depot effect. Notably, it was found that CaP modification enhanced the phagocytosis of fluorescent antigen-PCMC particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen or soluble PCMCs. Thus, CaP PCMCs may provide an alternative to conventional aluminium-based acellular vaccines to provide a more balanced Th1/Th2 immune response. PMID:24120484

  7. Structural, Antigenic, and Evolutionary Characterizations of the Envelope Protein of Newly Emerging Duck Tembusu Virus

    PubMed Central

    Huang, Bing; Ma, Xiuli; Li, Yufeng; Yuan, Xiaoyuan; Qin, Zhuoming; Wang, Dan; Chakravarty, Suvobrata; Li, Feng; Song, Minxun; Sun, Huaichang

    2013-01-01

    Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010–2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed. PMID:23990944

  8. A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection.

    PubMed

    Olaya-Abril, Alfonso; Jiménez-Munguía, Irene; Gómez-Gascón, Lidia; Obando, Ignacio; Rodríguez-Ortega, Manuel J

    2015-10-01

    Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of "shaving" the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics.

  9. A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection*

    PubMed Central

    Olaya-Abril, Alfonso; Jiménez-Munguía, Irene; Gómez-Gascón, Lidia; Obando, Ignacio; Rodríguez-Ortega, Manuel J.

    2015-01-01

    Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of “shaving” the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics. PMID:26183717

  10. Relationship between protein C antigen and anticoagulant activity during oral anticoagulation and in selected disease states.

    PubMed Central

    Vigano D'Angelo, S; Comp, P C; Esmon, C T; D'Angelo, A

    1986-01-01

    Protein C is a natural vitamin K-dependent plasma anticoagulant, deficiencies of which have been found in patients with recurrent thrombosis and warfarin-induced skin necrosis. To appreciate more fully the role of protein C in disease states and during oral anticoagulation, a new functional assay for protein C involving adsorption of plasma protein C on a Ca+2-dependent monoclonal antibody, elution, quantitative activation, and assessment of plasma anticoagulant activity, has been developed. When oral anticoagulation is initiated, the anticoagulant activity of protein C decreases to a greater extent than either the amidolytic or immunologic levels. During stabilized warfarin treatment, there is no correlation between either amidolytic or antigenic levels and the functional protein C activity, suggesting that measurement of protein C anticoagulant activity may be necessary to reflect adequately the anticoagulant protection afforded by this protein. In contrast, there was a strong correlation between anticoagulant and amidolytic and immunologic levels in liver failure and disseminated intravascular coagulation. Two patients with thromboembolic disease have been identified who exhibit a marked decrease in anticoagulant activity, but who have normal immunologic and amidolytic levels. Thus, this assay permits assessment of protein C in individuals who have received anticoagulant treatment and identification of a new class of protein C-deficient individuals. PMID:3511097

  11. Potential of Translationally Controlled Tumor Protein-Derived Protein Transduction Domains as Antigen Carriers for Nasal Vaccine Delivery.

    PubMed

    Bae, Hae-Duck; Lee, Joohyun; Jin, Xing-Hai; Lee, Kyunglim

    2016-09-01

    Nasal vaccination offers a promising alternative to intramuscular (i.m.) vaccination because it can induce both mucosal and systemic immunity. However, its major drawback is poor absorption of large antigens in the nasal epithelium. Protein transduction domains (PTDs), also called cell-penetrating peptides, have been proposed as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated the potential of a mutant PTD derived from translationally controlled tumor protein (designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared the l- and d-forms of TCTP-PTD 13 isomers (l- or d-TCTP-PTD 13) as antigen carriers. Studies in mice demonstrated that nasally administered mixtures of the model antigen ovalbumin (OVA) and d-TCTP-PTD 13 induced higher plasma IgG titers and secretory IgA levels in nasal washes than nasally administered OVA alone, OVA/l-TCTP-PTD 13, or i.m.-injected OVA. Plasma IgG subclass responses (IgG1 and IgG2a) of mice nasally administered OVA/d-TCTP-PTD 13 showed that the predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing adjuvant. Nasally administered CpG plus OVA/d-TCTP-PTD 13 was superior in eliciting systemic and mucosal immune responses compared to those induced by nasally administered OVA/d-TCTP-PTD 13. Furthermore, the OVA/CpG/d-TCTP-PTD 13 combination skewed IgG1 and IgG2a profiles of humoral immune responses toward a Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to efficiently carry antigens and to induce more powerful antigen-specific immune responses and a more balanced Th1/Th2 response when combined with a DNA adjuvant. PMID:27454469

  12. Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

    PubMed Central

    Bonino, F; Heermann, K H; Rizzetto, M; Gerlich, W H

    1986-01-01

    Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d. Images PMID:3701932

  13. Antigenicity and immunogenicity of a synthetic oligosaccharide-protein conjugate vaccine against Haemophilus influenzae type b.

    PubMed

    Fernández-Santana, V; Cardoso, Félix; Rodriguez, Arlene; Carmenate, Tania; Peña, Luis; Valdés, Yuri; Hardy, Eugenio; Mawas, Fatme; Heynngnezz, Lazaro; Rodríguez, Maria C; Figueroa, Ignacio; Chang, Janoi; Toledo, Maria E; Musacchio, Alexis; Hernández, Ibis; Izquierdo, Mabel; Cosme, Karelia; Roy, Rene; Verez-Bencomo, V

    2004-12-01

    Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal.

  14. Antigenicity and Immunogenicity of a Synthetic Oligosaccharide-Protein Conjugate Vaccine against Haemophilus influenzae Type b

    PubMed Central

    Fernández-Santana, V.; Cardoso, Félix; Rodriguez, Arlene; Carmenate, Tania; Peña, Luis; Valdés, Yuri; Hardy, Eugenio; Mawas, Fatme; Heynngnezz, Lazaro; Rodríguez, Maria C.; Figueroa, Ignacio; Chang, Janoi; Toledo, Maria E.; Musacchio, Alexis; Hernández, Ibis; Izquierdo, Mabel; Cosme, Karelia; Roy, Rene; Verez-Bencomo, V.

    2004-01-01

    Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be developed for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may be rigidly controlled for purity and effectiveness while at the same time being cheap enough that they may be made universally available. We describe here the antigenicity and immunogenicity of several H. influenzae type b synthetic oligosaccharide-protein conjugates in laboratory animals. The serum of H. influenzae type b-immunized animals recognized our synthetic H. influenzae type b antigens to the same extent as the native bacterial capsular polysaccharide. Compared to the anti-H. influenzae type b vaccine employed, these synthetic versions induced similar antibody response patterns in terms of titer, specificity, and functional capacity. The further development of synthetic vaccines will meet urgent needs in the less prosperous parts of the world and remains our major goal. PMID:15557635

  15. Isolation of a peptide binding protein and its role in antigen presentation

    SciTech Connect

    Lakey, E.; Pierce, S.K.; Margoliash, E.

    1986-03-05

    A mouse T cell hybrid, TPc9.1, recognizes pigeon cytochrome c (Pc) as processed and presented by histocompatible antigen presenting cells (APC). When paraformaldehyde fixed APC are employed, only a peptide fragment of Pc, Pc 81-104, and not the native Pc, is capable of stimulating TPc9.1 cells. Pc 81-104 appears to associate tightly with the APC surface since paraformaldehyde fixed APC which have been incubated with Pc 81-104 remain stimulatory following extensive washing. When APC are surface labeled with /sup 125/I, solubilized and affinity purified on Pc 81-104-Sepharose 4B columns, two predominant polypeptides of approximately 72 and 74 kd are isolated. Little or no immunoglobulin, Class I or Class II proteins are obtained under these conditions. Antisera from rabbits immunized with the affinity purified material, but not preimmune sera, block the activation of TPc 9.1 cells by Pc as well as Pc 81-104 when presented by live APC. Furthermore, these antisera are even more effective in blocking the activation of TPc9.1 cells by either APC which had been pulsed with Pc and then paraformaldehyde fixed, or by Pc 81-104 when added to paraformaldehyde fixed APC, suggesting that these antisera were not affecting antigen processing. Thus, these peptide binding proteins may play a role in antigen presentation, and they are being further characterized.

  16. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    PubMed Central

    Kempsell, Karen E.; Kidd, Stephen P.; Lewandowski, Kuiama; Elmore, Michael J.; Charlton, Sue; Yeates, Annemarie; Cuthbertson, Hannah; Hallis, Bassam; Altmann, Daniel M.; Rogers, Mitch; Wattiau, Pierre; Ingram, Rebecca J.; Brooks, Tim; Vipond, Richard

    2015-01-01

    A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis “infectome.” These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from

  17. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis.

    PubMed

    Kempsell, Karen E; Kidd, Stephen P; Lewandowski, Kuiama; Elmore, Michael J; Charlton, Sue; Yeates, Annemarie; Cuthbertson, Hannah; Hallis, Bassam; Altmann, Daniel M; Rogers, Mitch; Wattiau, Pierre; Ingram, Rebecca J; Brooks, Tim; Vipond, Richard

    2015-01-01

    A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis "infectome." These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  18. Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry.

    PubMed

    Tengattini, Sara; Domínguez-Vega, Elena; Temporini, Caterina; Terreni, Marco; Somsen, Govert W

    2016-09-01

    A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical Abstract Flowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass

  19. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

    PubMed

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary; Castellanos, Angélica; Kajava, Andrey V; Servis, Catherine; Felger, Ingrid; Moret, Remy; Arévalo-Herrera, Myriam; Corradin, Giampietro; Herrera, Sócrates

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  20. LAPTM4B-35 protein is a weak tumor-associated antigen candidate

    PubMed Central

    SHI, GUILAN; ZHOU, CHUNXIA; WANG, DONGMEI; MA, WENBO; ZHANG, SHUREN

    2014-01-01

    Lysosome-associated protein transmembrane 4β (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation. PMID:24396432

  1. Fluorescence Evaluation of Antigen-Antibody Reactivity on Surface of Proteinaceous Occlusion Body: Toward Application in Reusable Protein Chip

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Hiroshi Y.; Hosokawa, Yoichiroh; Suzuki, Kenji; Ikeda, Keiko; Mori, Hajime; Masuhara, Hiroshi

    2006-01-01

    A proteinaceous occlusion body, which is produced by insect viruses and consists of polyhedrin protein, has attracted much attention as a capsule for occluding an antigen protein. The occlusion body is called polyhedron. Its shape is cubic and its size is a few μm. Because several antigen proteins will be on the surface of polyhedra and several chemically active sites will be exposed, the polyhedra can be used as elements of protein chips to monitor antigen-antibody reactions. This idea is demonstrated by fixing a single polyhedron on a glass substrate and inducing an antigen-antibody reaction individually, and confirmed using relevant fluorescence microscopic images. Furthermore, a technique of cleaning the reacted surface is developed on the basis of the solubility of the polyhedrin matrix in an alkaline solution. The antigen-antibody complex on the surface can be removed by washing with the alkaline solution, and the antigen inside the polyhedron is exposed to the surface. On the basis of these results, the possibility of developing a “reusable protein chip” using the polyhedron is proposed.

  2. Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

    PubMed Central

    Rosengarten, R; Behrens, A; Stetefeld, A; Heller, M; Ahrens, M; Sachse, K; Yogev, D; Kirchhoff, H

    1994-01-01

    The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated

  3. An analysis of beta-lactam-derived antigens on spleen cell and serum proteins by ELISA and Western blotting.

    PubMed

    Warbrick, E V; Thomas, A L; Stejskal, V; Coleman, J W

    1995-11-01

    Penicillins and related beta-lactam antibiotics are known to conjugate to proteins to generate potentially antigenic (haptenic) determinants. In the present study, we used a rabbit polyclonal antibody raised against benzylpenicillin (BP) to investigate the capacity of six penicillins and one cephalosporin to generate haptenic groups in vitro on cultured mouse spleen cells and on serum proteins in the culture medium. All of the drugs tested, namely, BP, amoxicillin (AMX), ampicillin (AMP), cephalothin (CEP), cloxacillin (CLX), flucloxacillin (FLX), and phenoxymethylpenicillin (PMP) generated antigens in a concentration-dependent manner on cell and serum proteins, which could be detected by ELISA, although antigens generated by BP, CEP, FLX, or PMP in either cell- or serum-conjugated form were more readily detected than those generated by AMX, AMP, or CLX. Western blot analysis revealed that BP-derived antigens were generated relatively slowly on cell proteins (maximum binding was not yet reached after 8 h), compared to serum proteins (maximum binding within 1 h). BP, CEP, and PMP all generated similar distinctive patterns of immunostaining of electrophoresed cell or serum proteins which did not reflect the relative abundance of different proteins as revealed by Coomassie brilliant blue staining. FLX, CLX, AMP, and AMX did not generate antigens that could be detected on Western blots. In conclusion, we have shown that various beta-lactam antibiotics generate antigens on cell and serum proteins that can be detected and characterized immunochemically with polyclonal antiserum. Further application of these methods may offer potential for further identification of immunologically relevant cellular and serum antigens generated by these drugs.

  4. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR)

    PubMed Central

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J.; Laclette, Juan P.; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-01-01

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest. PMID:25989346

  5. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    PubMed

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  6. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    PubMed

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. PMID:26777581

  7. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    PubMed

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.

  8. Preparation of dichlorvos-protein complete antigen by Mannich-type reaction

    NASA Astrophysics Data System (ADS)

    Feng, Qianqian; Xu, Ying; Zhou, Youxiang; Lu, Liang; Chen, Fusheng; Wang, Xiaohong

    2010-08-01

    Dichlorvos (DDVP) residues have been linked to substantial adverse health effects on several organ systems. To ensure food safety, rapid and low-cost immunological methods must be applied to detect DDVP residues in food. In immunological methods, a key step is coupling DDVP to carrier proteins to obtain a complete antigen due to DDVP being hapten. In the current research, DDVP was coupled with cationized bovine serum albumin (cBSA) using a method based on Mannich-type reaction. A DDVP-cBSA conjugate, with a molar ratio of 40:1 DDVP to cBSA was synthesized. The cationized proteins and their conjugates were identified by UV-Vis and FT-IR spectra, which showed the characteristic bands of the ethylenediamine group and DDVP, respectively. BALB/c mice were immunized with DDVP-cBSA. One hybridoma cell line secreted anti-DDVP monoclonal antibody (Mab) that had high sensitivity and specificity for DDVP. Competitive ELISA identified an IC50 of 600 ng/mL and a limit of detection of 1 ng/mL in aqueous solution. The Mab had some cross-reactivity with phosmet, but no cross-reactivity with chlorothalonil and procymidone. We also detected a trace of DDVP in waste water. In conclusion the Mannich-type reaction couples DDVP to protein, yielding an antigen for the production of Mab to detect residual DDVP in the environment.

  9. A Review of Pneumococcal Vaccines: Current Polysaccharide Vaccine Recommendations and Future Protein Antigens

    PubMed Central

    Daniels, Calvin C.; Rogers, P. David

    2016-01-01

    This review describes development of currently available pneumococcal vaccines, provides summary tables of current pneumococcal vaccine recommendations in children and adults, and describes new potential vaccine antigens in the pipeline. Streptococcus pneumoniae, the bacteria responsible for pneumonia, otitis media, meningitis and bacteremia, remains a cause of morbidity and mortality in both children and adults. Introductions of unconjugated and conjugated pneumococcal polysaccharide vaccines have each reduced the rate of pneumococcal infections caused by the organism S. pneumoniae. The first vaccine developed, the 23-valent pneumococcal polysaccharide vaccine (PPSV23), protected adults and children older than 2 years of age against invasive disease caused by the 23 capsular serotypes contained in the vaccine. Because PPSV23 did not elicit a protective immune response in children younger than 2 years of age, the 7-valent pneumococcal conjugate vaccine (PCV7) containing seven of the most common serotypes from PPSV23 in pediatric invasive disease was developed for use in children younger than 2 years of age. The last vaccine to be developed, the 13-valent pneumococcal conjugate vaccine (PCV13), contains the seven serotypes in PCV7, five additional serotypes from PPSV23, and a new serotype not contained in PPSV23 or PCV7. Serotype replacement with virulent strains that are not contained in the polysaccharide vaccines has been observed after vaccine implementation and stresses the need for continued research into novel vaccine antigens. We describe eight potential protein antigens that are in the pipeline for new pneumococcal vaccines. PMID:26997927

  10. Is the Campylobacter jejuni secretory protein Cj0069 a suitable antigen for serodiagnostics?

    PubMed

    Corso, J; Lugert, R; Groß, U; Zautner, A E

    2011-03-01

    Campylobacter spp. is the most common bacterial pathogen of gastroenteritis worldwide. Poultry is the main reservoir and consequently the main origin of infections for humans. As a consequence of a primary Campylobacter infection which typically manifests as diarrhea, there is an increased risk to suffer from post-infectious complications such as reactive arthritis, neuropathia, myositis or a Guillain-Barré Syndrome. Usually the verification of acute campylobacteriosis is made by stool culture. In contrast, post-infectious complications can be diagnosed by serological assays. Since most of them are based on whole cell lysates, an insufficient specificity results from cross-reactions between related species. Therefore, the use of recombinant antigens becomes more and more favorable. Campylobacter is able to secrete a number of proteins, which are amongst others necessary for cell invasion and therefore play a crucial role for virulence. One of these, Cj0069, has a similar specificity and sensitivity in the detection of anti-Campylobacter jejuni IgG compared to the well-established antigens OMP18 and P39. This makes it a suitable antigen for diagnosing C. jejuni post-infectious complications.

  11. Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

    PubMed Central

    Ryu, Jae Kyu; Petersen, Mark A.; Murray, Sara G.; Baeten, Kim M.; Meyer-Franke, Anke; Chan, Justin P.; Vagena, Eirini; Bedard, Catherine; Machado, Michael R.; Coronado, Pamela E. Rios; Prod'homme, Thomas; Charo, Israel F.; Lassmann, Hans; Degen, Jay L.; Zamvil, Scott S.; Akassoglou, Katerina

    2015-01-01

    Autoimmunity and macrophage recruitment into the central nervous system (CNS) are critical determinants of neuroinflammatory diseases. However, the mechanisms that drive immunological responses targeted to the CNS remain largely unknown. Here we show that fibrinogen, a central blood coagulation protein deposited in the CNS after blood–brain barrier disruption, induces encephalitogenic adaptive immune responses and peripheral macrophage recruitment into the CNS leading to demyelination. Fibrinogen stimulates a unique transcriptional signature in CD11b+ antigen-presenting cells inducing the recruitment and local CNS activation of myelin antigen-specific Th1 cells. Fibrinogen depletion reduces Th1 cells in the multiple sclerosis model, experimental autoimmune encephalomyelitis. Major histocompatibility complex (MHC) II-dependent antigen presentation, CXCL10- and CCL2-mediated recruitment of T cells and macrophages, respectively, are required for fibrinogen-induced encephalomyelitis. Inhibition of the fibrinogen receptor CD11b/CD18 protects from all immune and neuropathologic effects. Our results show that the final product of the coagulation cascade is a key determinant of CNS autoimmunity. PMID:26353940

  12. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein

    PubMed Central

    1989-01-01

    Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis. The pathogenic process(es) that cause these sequelae are thought to be immunological. A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection. This protein is thought to mediate the immunopathology that follows chlamydial infection. To more thoroughly characterize this chlamydial component, we cloned its gene from a C. psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide. The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae. Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed. These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins. Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins. Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms. PMID:2571668

  13. Hypothyroidism attenuates protein tyrosine nitration, oxidative stress and renal damage induced by ischemia and reperfusion: effect unrelated to antioxidant enzymes activities

    PubMed Central

    Tenorio-Velázquez, Verónica M; Barrera, Diana; Franco, Martha; Tapia, Edilia; Hernández-Pando, Rogelio; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

    2005-01-01

    Background It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia. Methods Male Wistar rats weighing 380 ± 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test. Results The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4

  14. Identification and Characterization of an Antigen I/II Family Protein Produced by Group A Streptococcus

    PubMed Central

    Zhang, Shizhen; Green, Nicole M.; Sitkiewicz, Izabela; LeFebvre, Rance B.; Musser, James M.

    2006-01-01

    Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci. PMID:16790795

  15. Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis

    PubMed Central

    Khalid, Ruqyya; Afzal, Madeeha; Khurshid, Sana; Paracha, Rehan Zafar; Khan, Imran H.

    2016-01-01

    Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli. PMID:27654048

  16. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders.

    PubMed

    Mimori, T; Hardin, J A; Steitz, J A

    1986-02-15

    We have characterized the biochemical nature of the Ku protein, the antigen recognized by autoantibodies from certain patients with scleroderma-polymyositis overlap syndrome. From extracts of HeLa cells labeled with [32P]orthophosphate, anti-Ku antibodies precipitated a high molecular weight nucleic acid identified as DNA because of sensitivity to DNase I and resistance to RNase. From extracts of cells labeled with [35S] methionine, these antibodies precipitated two polypeptides of 70,000 and 80,000 Da. These proteins were purified using immunoaffinity column chromatography. In immunoblots most sera containing anti-Ku antibodies recognized both Ku proteins but one serum bound only to the 70,000-Da subunit. When nucleosomal segments of chromatin were used as antigen, anti-Ku antibodies precipitated dinucleosomes and larger forms of chromatin but not mononucleosomes. Thus, the Ku antigen is a novel DNA-binding protein that is at least partially exposed on nucleosomal segments of chromatin.

  17. Detection of antibody-antigen reaction by silicon nitride slot-ring biosensors using protein G

    NASA Astrophysics Data System (ADS)

    Taniguchi, Tomoya; Hirowatari, Anna; Ikeda, Takeshi; Fukuyama, Masataka; Amemiya, Yoshiteru; Kuroda, Akio; Yokoyama, Shin

    2016-04-01

    Biosensors using ring resonators with silicon nitride (SiN) slot waveguides have been fabricated. The temperature coefficient of the resonance wavelength of the SiN resonator is 0.006 nm/°C, which is one order of magnitude smaller than that of Si. The sensitivity of the biosensor has been improved by using slot waveguide together with Si-binding protein (designated as Si-tag), which bonds to SiN or SiO2 surface, as an anchoring molecule to immobilize bioreceptors on the SiN rings in an oriented manner. Furthermore, the protein G, which strongly bonds to many kinds of mammalian antibodies only by mixing the antibody solution, is used to efficiently immobilize the antigen on the sensor surface. By means of these devises the sensitivity of the biosensor has been improved by factor of 10-100 compared with that of normal Si ring resonator sensors without slot. Then the detection of prostate specific antigen (PSA) with the sensitivity of ~1×10-8 g/ml, which is the concentration of strongly suspicious for the prostate cancer, has been achieved.

  18. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

  19. Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation.

    PubMed

    Fernández-Fernández, M Rosario; Martínez-Torrecuadrada, Jorge L; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

    2002-12-01

    PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-gamma, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.

  20. [VLP vaccines and effects of HIV-1 Env protein modifications on their antigenic properties].

    PubMed

    Vzorov, A N; Compans, R W

    2016-01-01

    An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV. PMID:27414779

  1. Echinococcus granulosus antigen B: a Hydrophobic Ligand Binding Protein at the host-parasite interface.

    PubMed

    Silva-Álvarez, Valeria; Folle, Ana Maite; Ramos, Ana Lía; Zamarreño, Fernando; Costabel, Marcelo D; García-Zepeda, Eduardo; Salinas, Gustavo; Córsico, Betina; Ferreira, Ana María

    2015-02-01

    Lipids are mainly solubilized by various families of lipid binding proteins which participate in their transport between tissues as well as cell compartments. Among these families, Hydrophobic Ligand Binding Proteins (HLBPs) deserve special consideration since they comprise intracellular and extracellular members, are able to bind a variety of fatty acids, retinoids and some sterols, and are present exclusively in cestodes. Since these parasites have lost catabolic and biosynthetic pathways for fatty acids and cholesterol, HLBPs are likely relevant for lipid uptake and transportation between parasite and host cells. Echinococcus granulosus antigen B (EgAgB) is a lipoprotein belonging to the HLBP family, which is very abundant in the larval stage of this parasite. Herein, we review the literature on EgAgB composition, structural organization and biological properties, and propose an integrated scenario in which this parasite HLBP contributes to adaptation to mammalian hosts by meeting both metabolic and immunomodulatory parasite demands.

  2. Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis.

    PubMed

    Rojas, Agustina; García-Lugo, Pablo; Crisante, Gladys; Añez-Rojas, Néstor; Añez, Néstor

    2008-02-01

    GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.

  3. Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

    PubMed Central

    Tan, T H; Wallis, J; Levine, A J

    1986-01-01

    An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced

  4. [Comparative study of the antigenic and immunogenic properties of native and recombinant Marburg virus VP35 proteins].

    PubMed

    Sorokin, A V; Kazachinskaia, E I; Kachko, A V; Ivanova, A V; Bukreev, A A; Razumov, I A

    1999-01-01

    Antigenic structure of Marburg virus (MBG) VP35 was compared with that of the recombinant VP35 (f35) expressed in a prokaryotic system. For this purpose, a gene encoding the full-length VP35 was cloned in vector pQE31(QIAGEN) and expressed at about 70 mg/liter culture fluid in Escherichia coli JM103 as a recombinant fusion protein f35. BALB/c mice were immunized with soluble f35 or purified inactivated virions of MBG. Antibodies cross-reacting with VP35 and f35 antigens were detected by ELISA and Western Blot analysis in immune sera. Serum from a convalescent after Marburg disease and polyclonal antibodies from animals immunized with MBG recognized f35 and the MBG VP35. VP35 and its recombinant analog induced the production of specific antiviral antibodies in mice, which cross-reacted with the studied antigens. Competitive EIA showed that VP35 and f35 cross-inhibit the antigenic reactivity with polyclonal antibodies of immune sera. Antigenic structure of f35 protein corresponded to antigenic structure of MBG VP35 protein.

  5. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.

  6. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever. PMID:27317266

  7. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  8. Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

    NASA Technical Reports Server (NTRS)

    Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

    2004-01-01

    Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

  9. Human Leukocyte Antigen Class II Alleles Influence Levels of Antibodies to the Plasmodium falciparum Asexual-Stage Apical Membrane Antigen 1 but Not to Merozoite Surface Antigen 2 and Merozoite Surface Protein 1

    PubMed Central

    Johnson, Armead H.; Leke, Rose G. F.; Mendell, Nancy R.; Shon, Dewon; Suh, Young Ju; Bomba-Nkolo, Dennis; Tchinda, Viviane; Kouontchou, Samuel; Thuita, Lucy W.; van der Wel, Anne Marie; Thomas, Alan; Stowers, Anthony; Saul, Allan; Zhou, Ainong; Taylor, Diane W.; Quakyi, Isabella A.

    2004-01-01

    The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP119) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP119 variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP119 but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP119 variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated. PMID:15102786

  10. Effect of ultraviolet B radiation on S-100 protein antigen in epidermal Langerhans cells

    SciTech Connect

    Schneider, S.A.; Fukuyama, K.; Maceira, J.; Epstein, W.L.

    1985-02-01

    Ultraviolet B (UVB) radiation has been shown to induce significant alterations in both function and surface antigen expression of epidermal Langerhans cells (ELC). In this study the effect of UVB radiation on ELC marker S-100 protein antigen (S-100 Ag) which is present in the nucleus and cytoplasm of human ELC was investigated. A total of 34 sites on 31 volunteers were exposed to 3 MED (minimal erythema dose) of UVB and biopsied at various times up to 7 days after irradiation. Skin from 9 noninjured and 7 slice-wounded subjects served as controls. The avidin-biotin-peroxidase staining technique was used to identify S-100 Ag in sections of formalin-fixed, paraffin-embedded tissue, and the numbers of stained suprabasal dendritic cells were then counted over a 200 basal cell length of interfollicular epidermis. Noninjured skin had 3.56 +/- 3.01 cells, whereas slice-wounded skin had elevated numbers (greater than 10.0 cells) at 1, 24, and 48 h after injury. Following UVB irradiation, a significant (p less than 0.001) increase in antigen-positive cells (14 +/- 3.46) was found at 1 h; this number declined to just below normal at 12 h, but by 48 h returned to and remained at preinjury levels. In contrast to previous observations of the depletion of ELC surface markers by UVB radiation, the authors demonstrate here that the numbers of S-100 Ag-positive ELC actually increase following comparable doses of radiation. Since this increase occurs so rapidly following both UVB irradiation and slice injury, S-100 Ag may be synthesized or unmasked within the ELC as a response to wounding of the epidermis.

  11. Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.

    PubMed

    Lawrenz, M B; Hardham, J M; Owens, R T; Nowakowski, J; Steere, A C; Wormser, G P; Norris, S J

    1999-12-01

    VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

  12. Human Antibody Responses to VlsE Antigenic Variation Protein of Borrelia burgdorferi

    PubMed Central

    Lawrenz, M. B.; Hardham, J. M.; Owens, R. T.; Nowakowski, J.; Steere, A. C.; Wormser, G. P.; Norris, S. J.

    1999-01-01

    VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the “whole-cell” ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen. PMID:10565921

  13. Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

    PubMed Central

    2009-01-01

    Background Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, in vivo-induced antigen technology (IVIAT), an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated in vivo during SS2 infection. Results Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against in vitro antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The in vivo-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT)-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the in vivo condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins. Conclusion Collectively, our results suggest that these in vivo-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified using IVIAT may be useful

  14. Incorporating structure context of HA protein to improve antigenicity calculation for influenza virus A/H3N2.

    PubMed

    Qiu, Jingxuan; Qiu, Tianyi; Yang, Yiyan; Wu, Dingfeng; Cao, Zhiwei

    2016-01-01

    The rapid and consistent mutation of influenza requires frequent evaluation of antigenicity variation among newly emerged strains, during which several in-silico methods have been reported to facilitate the assays. In this paper, we designed a structure-based antigenicity scoring model instead of those sequence-based previously published. Protein structural context was adopted to derive the antigenicity-dominant positions, as well as the physic-chemical change of local micro-environment in correlation with antigenicity change. Then a position specific scoring matrix (PSSM) profile and local environmental change over above positions were integrated to predict the antigenicity variance. Independent testing showed a high accuracy of 0.875, and sensitivity of 0.986, with a significant ability to discover antigenic-escaping strains. When applying this model to the historical data, global and regional antigenic drift events can be successfully detected. Furthermore, two well-known vaccine failure events were clearly suggested. Therefore, this structure-context model may be particularly useful to identify those to-be-failed vaccine strains, in addition to suggest potential new vaccine strains. PMID:27498613

  15. Incorporating structure context of HA protein to improve antigenicity calculation for influenza virus A/H3N2

    PubMed Central

    Qiu, Jingxuan; Qiu, Tianyi; Yang, Yiyan; Wu, Dingfeng; Cao, Zhiwei

    2016-01-01

    The rapid and consistent mutation of influenza requires frequent evaluation of antigenicity variation among newly emerged strains, during which several in-silico methods have been reported to facilitate the assays. In this paper, we designed a structure-based antigenicity scoring model instead of those sequence-based previously published. Protein structural context was adopted to derive the antigenicity-dominant positions, as well as the physic-chemical change of local micro-environment in correlation with antigenicity change. Then a position specific scoring matrix (PSSM) profile and local environmental change over above positions were integrated to predict the antigenicity variance. Independent testing showed a high accuracy of 0.875, and sensitivity of 0.986, with a significant ability to discover antigenic-escaping strains. When applying this model to the historical data, global and regional antigenic drift events can be successfully detected. Furthermore, two well-known vaccine failure events were clearly suggested. Therefore, this structure-context model may be particularly useful to identify those to-be-failed vaccine strains, in addition to suggest potential new vaccine strains. PMID:27498613

  16. A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein

    PubMed Central

    Li, Zhuo; Huang, Richard Y.-C.; Yopp, Daniel C.; Hileman, Travis H.; Santangelo, Thomas J.; Hurwitz, Jerard; Hudgens, Jeffrey W.; Kelman, Zvi

    2014-01-01

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA. PMID:24728986

  17. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

    PubMed

    Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

    2016-03-18

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

  18. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

    PubMed Central

    Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

    2016-01-01

    Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Methodology/Principal Findings Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Conclusions/Significance Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody

  19. Role of the Antigenic Loop of the Hepatitis B Virus Envelope Proteins in Infectivity of Hepatitis Delta Virus

    PubMed Central

    Jaoudé, Georges Abou; Sureau, Camille

    2005-01-01

    The infectious particles of hepatitis B virus (HBV) and hepatitis delta virus (HDV) are coated with the large, middle, and small envelope proteins encoded by HBV. While it is clear that the N-terminal pre-S1 domain of the large protein, which is exposed at the virion surface, is implicated in binding to a cellular receptor at viral entry, the role in infectivity of the envelope protein antigenic loop, also exposed to the virion surface and accessible to neutralizing antibodies, remains to be established. In the present study, mutations were created in the antigenic loop of the three envelope proteins, and the resulting mutants were evaluated for their capacity to assist in the maturation and infectivity of HDV. We observed that short internal combined deletions and insertions, affecting residues 109 to 133 in the antigenic loop, were tolerated for secretion of both subviral HBV particles and HDV virions. However, when assayed for infectivity on primary cultures of human hepatocytes or on the recently described HepaRG cell line, virions carrying deletions between residues 118 and 129 were defective. Single amino acid substitutions in this region revealed that Gly-119, Pro-120, Cys-121, Arg-122, and Cys-124 were instrumental in viral entry. These results demonstrate that in addition to a receptor-binding site previously identified in the pre-S1 domain of the L protein, a determinant of infectivity resides in the antigenic loop of HBV envelope proteins. PMID:16051838

  20. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines.

    PubMed

    Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

    2010-07-01

    Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination. PMID:20562880

  1. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines

    NASA Astrophysics Data System (ADS)

    Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-Ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

    2010-07-01

    Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel (`nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

  2. Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells.

    PubMed

    Lu, Xiao-Ling; Liang, Zhi-Hui; Zhang, Cai-E; Lu, Sheng-Jun; Weng, Xiu-Fang; Wu, Xiong-Wen

    2006-03-01

    Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies, such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy. PMID:16518539

  3. Interphotoreceptor retinoid binding protein is a potent tolerogen in Lewis rat: suppression of experimental autoimmune uveoretinitis is retinal antigen specific

    PubMed Central

    Laliotou, B.; Liversidge, J.; Forrester, J.; Dick, A.

    1997-01-01

    AIMS—Administration of unfractionated retinal antigen(s) (retinal extract, RE) suppresses RE induced experimental autoimmune uveoretinitis (EAU) and offers a potential therapeutic alternative to non-specific immunosuppressive therapies for posterior uveitis and autoimmune diseases. S-Ag and interphotoreceptor retinoid binding protein (IRBP) are two major autoantigens within soluble RE. It was aimed to assess, firstly, as has previously been shown with S-Ag, if IRBP can induce intranasal tolerance and, secondly, the contribution of both these major autoantigens to tolerance induction by whole RE.
METHODS—Animals were tolerised by intranasal administration with S-Ag or IRBP, either alone or in combination, or RE before immunisation with either IRBP or RE. Control animals were administered nasally either PBS or MBP. Daily clinical responses were recorded biomicroscopically and histological grades were obtained using a semiquantitative scoring system. Weekly serum antibody levels to retinal antigens were measured by ELISA and delayed hypersensitivity responses (DTH) were assessed by skin reactivity to intradermal inoculation with retinal or non-specific antigens.
RESULTS—Microgram doses of IRBP successfully suppressed both clinically and histologically IRBP induced EAU. This suppression was accompanied by reduced antigen specific DTH reactivity but maintained T cell dependent (IgG2a) antibody responses. Furthermore, combined S-Ag and IRBP administration afforded equal suppression of RE induced EAU when compared with RE therapy alone. Suppression of RE induced EAU was not achieved with administration of a non-retinal specific autoantigen, MBP. Although individually, both S-Ag and IRBP suppressed RE induced EAU, whole RE was unable to protect against IRBP induced disease.
CONCLUSIONS—Intranasal administration of IRBP suppressed IRBP induced EAU in the Lewis rat. S-Ag and IRBP are the major contributors to the tolerogenicity within RE, despite the known

  4. Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike protein

    PubMed Central

    Reguera, Juan; Ordoño, Desiderio; Santiago, César; Enjuanes, Luis

    2011-01-01

    The N-terminal S1 region of the transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein contains four antigenic sites (C, B, D and A, from the N- to the C-terminal end) and is engaged in host-cell receptor recognition. The most N-terminal portion of the S1 region, which comprises antigenic sites C and B, is needed for the enteric tropism of TGEV, whereas the major antigenic site A at the C-terminal moiety is required for both respiratory and enteric cell tropism, and is engaged in recognition of the aminopeptidase N (APN) receptor. This study determined the kinetics for binding of a soluble S1 protein to the APN protein. Moreover, the S1 region of the TGEV S protein was dissected, with the aim of identifying discrete modules displaying unique antigenic sites and receptor-binding functions. Following protease treatments and mammalian cell expression methods, four modules or domains (D1–D4) were defined at the S1 region. Papain treatment identified an N-terminal domain (D1) resistant to proteolysis, whereas receptor binding defined a soluble and functional APN receptor-binding domain (D3). This domain was recognized by neutralizing antibodies belonging to the antigenic site A and therefore could be used as an immunogen for the prevention of viral infection. The organization of the four modules in the S1 region of the TGEV S glycoprotein is discussed. PMID:21228126

  5. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    NASA Astrophysics Data System (ADS)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  6. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    PubMed

    Catherine, Christy; Lee, Seung-Won; Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  7. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

    PubMed Central

    Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  8. Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein.

    PubMed Central

    Walter, G; Ferre, F; Espiritu, O; Carbone-Wiley, A

    1989-01-01

    Polyoma virus medium tumor antigen forms specific complexes with several cellular proteins; among these is a protein of approximately 61 kDa. With antibodies directed against medium tumor antigen, the 61-kDa protein was purified from human 293 cells that were infected with a hybrid adenovirus and overexpressed medium tumor antigen. The purified 61-kDa protein was partially digested with protease V8, and one of the protease V8 fragments was isolated and partially sequenced. The amino acid sequence information was used to design mixed oligonucleotide probes for screening a cDNA library from human placenta. A clone was isolated that hybridized with two separate probes; the clone contained an insert with an open reading frame for 589 amino acids. By in vitro translation of the transcript from this insert, a protein was generated that had the same size and yielded the same pattern of protease V8 fragments as the original 61-kDa protein. Its amino acid sequence reveals 15 repeats, the majority of which are 39 amino acids long. This protein bears no resemblance to proteins in the data bank that was searched. Images PMID:2554323

  9. Antigenic Characterization of Recombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes

    PubMed Central

    Mueller, Matthias; Renzullo, Sandra; Brooks, Roxann; Ruggli, Nicolas; Hofmann, Martin A.

    2010-01-01

    Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA. PMID:20140098

  10. Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells

    PubMed Central

    Müller, Rebekka; Bachke, Siri; Gilljam, Karin M.; Våtsveen, Thea K.; Rø, Torstein B.; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit

    2013-01-01

    Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment. PMID:23936203

  11. Tartrate/tripolyphosphate as co-crosslinker for water soluble chitosan used in protein antigens encapsulation.

    PubMed

    Srivastava, Gopal; Walke, Shilratna; Dhavale, Dilip; Gade, Wasudeo; Doshi, Jignesh; Kumar, Rakesh; Ravetkar, Satish; Doshi, Pooja

    2016-10-01

    In drug delivery research, several toxic chemical crosslinkers and non-toxic ionic crosslinkers have been exploited for the synthesis of microparticles from acetic acid soluble chitosan. This paper hypothesized the implementation of sodium potassium tartrate (SPT) as an alternative crosslinker for sodium tripolyphosphate (TPP) and SPT/TPP co-crosslinkers for synthesis of the microparticles using water soluble chitosan (WSC) for encapsulation of Bovine serum albumin (BSA) as a model protein, and Tetanus toxoid (TT) as a model vaccine. The crosslinking was confirmed by FT-IR, SEM with EDS. The XRD entailed molecular dispersion of proteins and thermal analysis confirmed the higher stability of STP/TPP co-crosslinked formulations. The resultant microparticles were exhibiting crosslinking degree (52-67%), entrapment efficiency (72-80%), particle size (0.3-1.7μm), zeta potential (+24 to 46mV) and mucoadhesion (41-68%). The superiority of SPT over TPP was confirmed by higher crosslinking degree and entrapment efficiency. However, co-crosslinking were advantageous in higher regression values for Langmuir adsorption isotherm, slower swelling tendency and extended 30days controlled in-vitro release study. TT release obeyed the Quasi-Fickian diffusion mechanism for single and cocrosslinked formulations. Overall, in crosslinking of chitosan as biological macromolecules, STP/TPP may be alternative for single ionic crosslinked formulations for protein antigen delivery. PMID:27246374

  12. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  13. Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

    PubMed Central

    Griffiths, David A.; Abdul-Sada, Hussein; Knight, Laura M.; Jackson, Brian R.; Richards, Kathryn; Prescott, Emma L.; Peach, A. Howard S.; Blair, G. Eric

    2013-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell. PMID:24109239

  14. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

    PubMed

    Cahill, Bethaney K; Seeley, Kent W; Gutel, Dedra; Ellis, Terri N

    2015-11-01

    Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

  15. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    NASA Astrophysics Data System (ADS)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  16. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    PubMed Central

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-01-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8–9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies. PMID:10884430

  17. "Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

    PubMed

    Lavergne, S N; Wang, H; Callan, H E; Park, B K; Naisbitt, D J

    2009-11-01

    Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant

  18. Identification of antigenic proteins associated with trichloroethylene-induced autoimmune disease by serological proteome analysis

    SciTech Connect

    Liu Jianjun; Xing Xiumei; Huang Haiyan; Jiang Yingzhi; He Haowei; Xu Xinyun; Yuan Jianhui; Zhou Li; Yang Linqing; Zhuang Zhixiong

    2009-11-01

    Although many studies indicated that trichloroethylene (TCE) could induce autoimmune diseases and some protein adducts were detected, the proteins were not identified and mechanisms remain unknown. To screen and identify autoantigens which might be involved in TCE-induced autoimmune diseases, three groups of sera were collected from healthy donors (I), patients suffering from TCE-induced exfoliative dermatitis (ED) (II), and the healed ones (III). Serological proteome analysis (SERPA) was performed with total proteins of TCE-treated L-02 liver cells as antigen sources and immunoglobins of the above sera as probes. Highly immunogenic spots (2-fold or above increase compared with group I) in group II and III were submitted to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing. Western blot analysis was followed using commercial antibodies and individual serum. Six proteins were identified. Among them, Enoyl Coenzyme A hydratase peroxisoma 1 and lactate dehydrogenase B only showed stronger immunogenicity for group II sera, while Purine nucleoside phosphorylase, ribosomal protein P0 and proteasome activator subunit1 isoform1 also showed stronger immunogenicity for group III sera. Noteworthy, NM23 reacted only with group II sera. Western blot analysis of NM23 expression indicated that all of the individual serum of group II showed immune activity, which confirmed the validity of SERPA result. These findings revealed that there exist autoantibodies in group II and III sera. Besides, autoantibodies of the two stages of disease course were different. These autoantigens might serve as biomarkers to elucidate mechanisms underlying TCE toxicity and are helpful for diagnosis, therapy and prognosis of TCE-induced autoimmune diseases.

  19. Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

    PubMed Central

    McAuley, Alexander J.; Torres, Maricela; Plante, Jessica A.; Huang, Claire Y.-H.; Bente, Dennis A.

    2016-01-01

    ABSTRACT Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, with in vivo pathogenesis often not being correlated with in vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. IMPORTANCE The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric

  20. The prostate cancer immunome: In silico functional analysis of antigenic proteins from microarray profiling with IgG.

    PubMed

    Luna-Coronell, Johana A; Vierlinger, Klemens; Gamperl, Magdalena; Hofbauer, Johann; Berger, Ingrid; Weinhäusel, Andreas

    2016-04-01

    The study of the immunome of prostate cancer (PCa) and characterization of autoantibody signature from differentially reactive antigens can uncover disease stage proteins, reveal enriched networks and even expose aberrant cellular mechanisms during the disease process. By conducting plasma IgG profiling on protein microarrays presenting 5449 unique human proteins expressed in 15 417 E. coli human cDNA expression clones, we elucidated 471 (21 higher reactive in PCa) differentially reactive antigens in 50 PCa versus 49 patients with benign prostate hyperplasia (BPH) at initial diagnosis. Functional analyzes show that the immune-profile of PCa compared to BPH control samples is significantly enriched in features targeting Cellular assembly, Cell death and pathways involved in Cell cycle, translation, and assembly of proteins as EIF2 signaling, PCa related genes as AXIN1 and TP53, and ribosomal proteins (e.g. RPS10). An overlap of 61 (out of 471) DIRAGs with the published 1545 antigens from the SEREX database has been found, however those were higher reactive in BPH. Clinical relevance is shown when antibody-reactivities against eight proteins were significantly (p < 0.001) correlated with Gleason-score. Herewith we provide a biological and pathophysiological characterization of the immunological layer of cancerous (PCa) versus benign (BPH) disease, derived from antibody profiling on protein microarrays. PMID:27089054

  1. Molecular characterization of a 35-kilodalton protein of Borrelia burgdorferi, an antigen of diagnostic importance in early Lyme disease.

    PubMed Central

    Gilmore, R D; Kappel, K J; Johnson, B J

    1997-01-01

    Antibodies against a 35-kDa antigen of Borrelia burgdorferi are detectable in the serum of about half of patients with early Lyme disease. The gene encoding this antigen was isolated from a genomic library of B. burgdorferi B31 (low passage), and full-length expression of the recombinant gene product was achieved in Escherichia coli. Antiserum raised against the recombinant protein was reactive with a B. burgdorferi protein of the same molecular size as the diagnostic 35-kDa antigen cited in an earlier study of criteria for the sero-diagnosis of early Lyme disease. Also, the recombinant protein was reactive with serum from patients with early Lyme disease who were seropositive for the 35-kDa antigen. DNA sequence analysis of the gene indicated an open reading frame of 909 bp encoding a protein with a calculated molecular mass of 34.3 kDa. This gene did not possess the usual initiation codon ATG but rather probably used a TTG codon. The deduced amino acid sequence of the N terminus exhibited a motif similar to that for signal peptides of lipoproteins. Southern blotting revealed a chromosomal location for this gene; and it was specific for B. burgdorferi, B. afzellii, and B. garinii but not for B. hermsii, B. coriaciae, or B. turicatae. PMID:8968885

  2. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  3. Discovery of GAMA, a Plasmodium falciparum Merozoite Micronemal Protein, as a Novel Blood-Stage Vaccine Candidate Antigen ▿ ‡

    PubMed Central

    Arumugam, Thangavelu U.; Takeo, Satoru; Yamasaki, Tsutomu; Thonkukiatkul, Amporn; Miura, Kazutoyo; Otsuki, Hitoshi; Zhou, Hong; Long, Carole A.; Sattabongkot, Jetsumon; Thompson, Jennifer; Wilson, Danny W.; Beeson, James G.; Healer, Julie; Crabb, Brendan S.; Cowman, Alan F.; Torii, Motomi; Tsuboi, Takafumi

    2011-01-01

    One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen. PMID:21896773

  4. Conservation of antigen components from two recombinant hybrid proteins protective against malaria.

    PubMed Central

    Knapp, B; Nau, U; Hundt, E

    1993-01-01

    Recently, we have shown that two hybrid proteins carrying partial sequences of the blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum confer protective immunity on Aotus monkeys against an experimental parasite infection (B. Knapp, E. Hundt, B. Enders, and H. A. Küpper, Infect. Immun. 60:2397-2401, 1992). The malarial components of the hybrid proteins consist of amino acid residues 630 to 892 of SERP, amino acid residues 146 to 260 of MSAI, and the 189 C-terminal residues of HRPII. We have studied the diversity of these protein regions in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from two different areas where malaria is endemic. The gene regions of SERP and MSAI coding for the corresponding sequences of the protective hybrid proteins and the exon II region of the HRPII gene were amplified by polymerase chain reaction and sequenced. All three regions were found to be highly conserved. In the 262-amino-acid fragment of SERP, one single conservative amino acid substitution was found. The exon II region of HRPII showed only a slight variability in number and arrangement of the repeat units. The 115-amino-acid fragment of MSAI which is located within an N-terminal region known to be conserved among different parasite strains was shown to be the most variable among the vaccine components: amino acid substitutions were found in 14 different positions of this MSAI region when both laboratory strains and field isolates were compared. PMID:8432609

  5. Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis▿

    PubMed Central

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S.; Pujato, Nazarena; Ribone, María E.; Lagier, Claudia M.; Marcipar, Iván S.

    2009-01-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp. PMID:19339486

  6. Murine carcinoma expressing carcinoembryonic antigen-like protein is restricted by antibody against neem leaf glycoprotein.

    PubMed

    Das, Arnab; Barik, Subhasis; Bose, Anamika; Roy, Soumyabrata; Biswas, Jaydip; Baral, Rathindranath; Pal, Smarajit

    2014-11-01

    We have generated a polyclonal antibody against a novel immunomodulator, neem leaf glycoprotein (NLGP) that can react to a specific 47 kDa subunit of NLGP. Generated anti-NLGP antibody (primarily IgG2a) was tested for its anti-tumor activity in murine carcinoma (EC, CT-26), sarcoma (S180) and melanoma (B16Mel) tumor models. Surprisingly, tumor growth restriction was only observed in CT-26 carcinoma models, without any alteration in other tumor systems. Comparative examination of antigenicity between four different tumor models revealed high expression of CEA-like protein on the surface of CT-26 tumors. Subsequent examination of the cross-reactivity of anti-NLGP antibody with purified or cell bound CEA revealed prominent recognition of CEA by anti-NLGP antibody, as detected by ELISA, Western Blotting and immunohistochemistry. This recognition seems to be responsible for anti-tumor function of anti-NLGP antibody only on CEA-like protein expressing CT-26 tumor models, as confirmed by ADCC reaction in CEA(+) tumor systems where dependency to anti-NLGP antibody is equivalent to anti-CEA antibody. Obtained result with enormous therapeutic potential for CEA(+) tumors may be explained in view of the epitope spreading concept, however, further investigation is crucial.

  7. Development of antigen capture ELISA for the quantification of EIAV p26 protein.

    PubMed

    Hu, Zhe; Chang, Hao; Ge, Man; Lin, Yuezhi; Wang, Xuefeng; Guo, Wei; Wang, Xiaojun

    2014-11-01

    An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

  8. Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

    PubMed

    Holland, S J; Liao, X C; Mendenhall, M K; Zhou, X; Pardo, J; Chu, P; Spencer, C; Fu, A; Sheng, N; Yu, P; Pali, E; Nagin, A; Shen, M; Yu, S; Chan, E; Wu, X; Li, C; Woisetschlager, M; Aversa, G; Kolbinger, F; Bennett, M K; Molineaux, S; Luo, Y; Payan, D G; Mancebo, H S; Wu, J

    2001-11-01

    In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

  9. Functional Cloning of Src-like Adapter Protein-2 (SLAP-2), a Novel Inhibitor of Antigen Receptor Signaling

    PubMed Central

    Holland, Sacha J.; Liao, X. Charlene; Mendenhall, Marcy K.; Zhou, Xiulan; Pardo, Jorge; Chu, Peter; Spencer, Collin; Fu, Alan; Sheng, Ning; Yu, Peiwen; Pali, Erlina; Nagin, Anup; Shen, Mary; Yu, Simon; Chan, Eva; Wu, Xian; Li, Connie; Woisetschlager, Max; Aversa, Gregorio; Kolbinger, Frank; Bennett, Mark K.; Molineaux, Susan; Luo, Ying; Payan, Donald G.; Mancebo, Helena S.Y.; Wu, Jun

    2001-01-01

    In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor–stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems. PMID:11696592

  10. Intracellular proteins of feline immunodeficiency virus and their antigenic relationship with equine infectious anaemia virus proteins.

    PubMed

    Egberink, H F; Ederveen, J; Montelaro, R C; Pedersen, N C; Horzinek, M C; Koolen, M J

    1990-03-01

    Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. Pulse-chase experiments suggested that the smaller polypeptides p24 and p15 are cleavage products of both p160 and p50. Western blot analysis using a rabbit serum directed against p26 of equine infectious anaemia virus (EIAV) and an anti-EIAV horse serum from a field case of infection revealed a cross-reactivity with p24 of FIV. Cat sera collected late after experimental FIV infection recognized p26 of EIAV, indicating a reciprocal cross-reactivity. PMID:1690264

  11. Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses

    PubMed Central

    Johansson, Tomas; Nilsson, Anki; Chatzissavidou, Nathalie; Sjöblom, Magnus; Rova, Ulrika; Holgersson, Jan

    2012-01-01

    Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented. OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays. Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect. Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses. PMID:23071675

  12. Specific radioimmunoprecipitation of histone H2A antigens by protein A conjugated sepharose.

    PubMed

    Ruder, F J; Frasch, M; Büsen, W

    1988-04-15

    A modified radioimmunoprecipitation technique is described which allows the specific detection of histone H2A antigens. The technique circumvents unspecific binding of histones to the bacterial adsorbent.

  13. Polymeric hepatitis C virus non-structural protein 5A nanocapsules induce intrahepatic antigen-specific immune responses.

    PubMed

    Fichter, Michael; Piradashvili, Keti; Pietrzak-Nguyen, Anette; Pretsch, Leah; Kuhn, Gabor; Strand, Susanne; Knuf, Markus; Zepp, Fred; Wurm, Frederik R; Mailänder, Volker; Landfester, Katharina; Gehring, Stephan

    2016-11-01

    Targeting antigen combined with adjuvants to hepatic antigen-presenting cells (APCs) is essential for the induction of intrahepatic T cellular immunity controlling and resolving viral infections of the liver. Intravenous injection of antigen-loaded nanoparticles is a promising approach for the delivery of antigens to liver APCs. Accordingly, polymeric nanocapsules (NCs) synthesized exclusively of hepatitis C virus non-structural protein 5A (NS5A) and the adjuvant monophosphoryl lipid A (MPLA) adsorbed to the nanocapsule surface were developed. Aim of the present study was the evaluation of the in vitro and in vivo behavior of MPLA-functionalized NS5A-NCs regarding the interaction with liver dendritic cells (DCs) and the potential to induce intrahepatic immune responses in a mouse model. Maturation of DCs was significantly increased by application of NS5A+MPLA-NCs compared to non-functionalized NS5A-NCs promoting a vigorous expression of CD40, CD80, CD86 and a strong secretion of the Th1-related cytokine IL-12. NS5A-NCs were preferentially deposited in DCs and Kupffer cells residing in the liver after intravenous administration. Immunization with NS5A-NCs induced intrahepatic antigen-specific CD4(+) T cellular immune responses determined by the secretion of IFNγ and IL-2. Furthermore, supplementation with MPLA induced significant levels of NS5A-specific antibodies. The application of polymeric nanocapsules synthesized exclusively out of antigen avoids the risk of unintended side effects caused by additional carrier substances. Functionalization with adjuvants like MPLA and the efficient targeting to liver-resident APCs inherits the potential for application of antigen nanocapsules in further vaccination approaches against pathogens affecting the liver. PMID:27614817

  14. Identification of the major T-cell antigens present in the Brucella melitensis B115 protein preparation, Brucellergene OCB.

    PubMed

    Denoel, P A; Vo, T K; Weynants, V E; Tibor, A; Gilson, D; Zygmunt, M S; Limet, J N; Letesson, J J

    1997-09-01

    Brucellergene is a commercial allergen prepared from Brucella melitensis strain B115 and containing at least 20 cytoplasmic proteins. These proteins were separated by SDS-PAGE. The unstained gel was divided into 18 fractions and proteins were eluted from the gel fractions. The capacity of the separated proteins to elicit delayed-type hypersensitivity (DTH) in infected guinea-pigs or to induce the production of interferon-gamma (IFN-gamma) by blood cells from infected cattle was evaluated. The biological activity of the corresponding protein fractions blotted on to nitrocellulose was measured in a lymphocyte blastogenesis assay. Among the 18 fractions tested, two-spanning the mol. wt ranges 17-22 (fraction 8) and 35-42-kDa (fraction 17)-showed the maximum biological activity in the three tests. These fractions contain two antigens, the Brucella bacterioferritin (BFR) and P39 proteins. Both proteins are good candidates for the detection of cellular immunity to Brucella. PMID:9291893

  15. Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia

    PubMed Central

    Hernández-Martínez, Miguel Ángel; Escalante, Ananías A.; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2011-01-01

    Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly → Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4+, and T-CD8+ cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions. PMID:21292878

  16. CD301b+ dendritic cells suppress T follicular helper cells and antibody responses to protein antigens

    PubMed Central

    Kumamoto, Yosuke; Hirai, Toshiro; Wong, Patrick W; Kaplan, Daniel H; Iwasaki, Akiko

    2016-01-01

    Strong antibody response is considered a hallmark of a successful vaccine. While dendritic cells (DCs) are important for T follicular helper (Tfh) cell priming, how this process is regulated in vivo is unclear. We show here that the depletion of CD301b+ DCs specifically enhanced the development of Tfh cells, germinal center B cells and antibody responses against protein antigens. Exaggerated antibody responses in mice depleted of CD301b+ DCs occurred in the absence of any adjuvants, and resulting antibodies had broader specificity and higher affinity to the immunogen. CD301b+ DCs express high levels of PD-1 ligands, PD-L1 and PD-L2. Blocking PD-1 or PD-L1 during priming in wild-type mice partially mimicked the phenotype of CD301b+ DC-depleted animals, suggesting their role in Tfh suppression. Transient depletion of CD301b+ DC results in the generation of autoreactive IgG responses. These results revealed a novel regulatory mechanism and a key role of CD301b+ DCs in blocking autoantibody generation. DOI: http://dx.doi.org/10.7554/eLife.17979.001 PMID:27657168

  17. Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

    PubMed Central

    Shen, Chih-Lung; Liu, Cheng-Der; You, Ren-In; Ching, Yung-Hao; Liang, Jun; Ke, Liangru; Chen, Ya-Lin; Chen, Hong-Chi; Hsu, Hao-Jen; Liou, Je-Wen; Kieff, Elliott; Peng, Chih-Wen

    2016-01-01

    Epstein–Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. PMID:26858444

  18. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  19. Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

    PubMed Central

    Fukuma, Aiko; Fukushi, Shuetsu; Yoshikawa, Tomoki; Tani, Hideki; Taniguchi, Satoshi; Kurosu, Takeshi; Egawa, Kazutaka; Suda, Yuto; Singh, Harpal; Nomachi, Taro; Gokuden, Mutsuyo; Ando, Katsuyuki; Kida, Kouji; Kan, Miki; Kato, Nobuyuki; Yoshikawa, Akira; Kitamoto, Hiroaki; Sato, Yuko; Suzuki, Tadaki; Hasegawa, Hideki; Morikawa, Shigeru; Shimojima, Masayuki; Saijo, Masayuki

    2016-01-01

    Background Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. Methodology/Principal Findings We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. Conclusions The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. PMID:27045364

  20. Identification and determination of antigenic proteins of Korean ranavirus-1 (KRV-1) using MALDI-TOF/TOF MS analysis.

    PubMed

    Kim, Young Rim; Hikima, Jun-Ichi; Jang, Ho Bin; Nho, Seong Won; Park, Seong Bin; Cha, In Seok; Ohtani, Maki; Eom, Ahn Heume; Aoki, Takashi; Jung, Tae Sung

    2011-05-01

    Ranaviruses are serious pathogens of fish, amphibians, and reptiles, and pose a major threat to global biodiversity. A ranavirus isolated from tissues of diseased tadpoles and frogs in Gangwon province, Korea, in 2006 and 2007, was designated Korean ranavirus-1 (KRV-1) and was infectious in a variety of fish cell lines with highest titers (10(10)TCID(50)/ml) in Epithelioma papulosum cyprini cells (EPCs) and baby hamster kidney-21 (BHK-21) cells. Bullfrog (Rana catesbeiana) tadpoles challenged by immersion in 10(5)TCID(50)/ml of KRV-1 showed 60% mortality within 10 days. SDS-PAGE of frog virus 3 (FV3) and KRV-1 proteins yielded several bands 35-49kDa in size, which were identified as major capsid proteins (MCPs) by MALDI-TOF MS. Immunoblotting of FV3 proteins showed antigenic bands 34kDa and 93kDa in size which were identified by MALDI-TOF/TOF MS as MCP and neurofilament triplet H1-like protein (NF-H1), respectively. In KRV-1, antigenic bands at 32kDa, 69kDa, and 72kDa were identified as MCP, Hypothetical protein, and NF-H1, respectively. The genes encoding these KRV-1 proteins were sequenced. KRV-1 appeared to be closely related to the soft-shelled turtle iridovirus (STIV), based on alignments of amino acid sequences from various ranaviruses. Variability in ranavirus antigenic proteins was apparent in an earlier study. It is expected that use of the methods employed here, together with the results of the present work, will contribute to an understanding of the pathogenesis of ranaviruses, and will further the development of DNA- or protein-based bait vaccines for conservation of natural habitats.

  1. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

    PubMed

    Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

    2014-08-01

    The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

  2. Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

    PubMed

    Altman, Meghan O; Bennink, Jack R; Yewdell, Jonathan W; Herrin, Brantley R

    2015-01-01

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens. PMID:26252514

  3. Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity.

    PubMed

    Altman, Meghan O; Bennink, Jack R; Yewdell, Jonathan W; Herrin, Brantley R

    2015-08-07

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.

  4. Human CD8+ herpes simplex virus-specific cytotoxic T-lymphocyte clones recognize diverse virion protein antigens.

    PubMed Central

    Tigges, M A; Koelle, D; Hartog, K; Sekulovich, R E; Corey, L; Burke, R L

    1992-01-01

    The role of the HLA class I-restricted, CD8+, herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL) in the control of human HSV infections is controversial because previous reports suggest that a substantial portion of the antigen-specific lytic response is mediated by CD4+ cells. To address this question directly, we isolated HSV-specific CD8+ CTL clones from a patient with recurrent genital herpes. These CTL were cloned by coculturing responder peripheral blood mononuclear cells (PBMC) with phytohemagglutinin-stimulated PBMC that had been infected with live HSV-2 and then irradiated prior to the addition of responder cells. After 1 week, CTL were cloned by limiting dilution using phytohemagglutinin stimulation and allogeneic feeder PBMC. Seven clones were isolated; all seven clones were CD8+ CD4- CD3+ DRbright, six lysed only HSV-2-infected targets, and one lysed both HSV-1- and HSV-2-infected targets. Antigen presentation was restricted by two to three different HLA class I loci. To determine the antigens recognized by these HSV-specific CTL, target cells were infected with HSV in the presence of acyclovir, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, or cycloheximide in a series of drug block/release protocols to limit the repertoire of viral gene expression to select transcriptional classes. Five of the clones exhibited a different pattern of cytotoxicity, suggesting that each recognized a distinct HSV antigen. One of the clones appears to be directed against an immediate-early antigen; six of the clones recognize virion proteins. Five of these clones recognized internal virion proteins that could be introduced into target cells by HSV infection in the absence of virus gene expression. Antigen specificity was further tested by using vaccinia virus vectors that express glycoproteins gD2 and gB2 or the tegument protein VP16. One clone lysed vaccinia virus/gD2-infected target cells; the remaining clones did not recognize any of these gene

  5. Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.

    PubMed

    Kay, M M; Marchalonis, J J; Hughes, J; Watanabe, K; Schluter, S F

    1990-08-01

    Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration. PMID:1696010

  6. Sugar–Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines

    PubMed Central

    Stefanetti, Giuseppe; Hu, Qi-Ying; Usera, Aimee; Robinson, Zack; Allan, Martin; Singh, Alok; Imase, Hidetomo; Cobb, Jennifer; Zhai, Huili; Quinn, Douglas; Lei, Ming; Saul, Allan; Adamo, Roberto; MacLennan, Calman A; Micoli, Francesca

    2015-01-01

    A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies. PMID:26350581

  7. Functional insights from a comparative study on the dynamics of Antigen85 proteins and MPT51 from Mycobacterium tuberculosis.

    PubMed

    Sundar, Shobana; Annaraj, David; Selvan, Anitha; Biswas, Pallavi Guha; Vijayakumaran, Reshma; Anishetty, Sharmila

    2015-12-01

    Antigen85 (Ag85) proteins of Mycobacterium tuberculosis are mycolyl transferases that aid in cell wall biosynthesis. MPT51 (Ag85D) is closely related to Ag85 proteins. We have performed a comparative molecular dynamics (MD) simulation study of Ag85 proteins (Ag85A, Ag85B, and Ag85C) and MPT51. We observe that helix α5, β7-α9 loop, and N-terminal region of helix α9 of Ag85 proteins are mobile, suggestive of lid like movement over the active site. Further, in Ag85B, we observe the proposed scooting mode of the hydrophobic gating residue Phe232. Our simulations also show a similar scooting mode for Phe232 of Ag85A and Trp158 of Ag85C. We also found aromatic residue clusters at the ends of the hydrophobic channel of Ag85 proteins, which may have functional significance. Although MPT51 lacks the tunnel, it has the aromatic clusters. The aromatic cluster region has the ability to bind trehalose. From an immunoinformatics study, a promiscuous linear epitope was identified in MPT51 which could be useful in subunit vaccine studies. Recent studies have shown that a mycobacterial protein HupB, interacts with Ag85 proteins and has a regulatory role in cell wall biogenesis, with implications in growth rate and latency. We performed molecular docking studies of HupB protein with Ag85 proteins and predicted potential sites of interaction in Ag85 proteins. The insights gained through the current study can potentially pave way for newer therapeutic interventions. Graphical Abstract Dynamics of antigen85 proteins and MPT51 from Mycobacterium tuberculosis. PMID:26564147

  8. Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

    PubMed

    Ding, Meng-Die; Wang, Hong-Ning; Cao, Hai-Peng; Fan, Wen-Qiao; Ma, Bing-Cun; Xu, Peng-Wei; Zhang, An-Yun; Yang, Xin

    2015-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.

  9. Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

    PubMed

    Rockey, D D; Rosquist, J L

    1994-01-01

    Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.

  10. BINDING OF ANTIGEN BY IMMUNOCYTES

    PubMed Central

    Bystryn, Jean-Claude; Siskind, Gregory W.; Uhr, Jonathan W.

    1973-01-01

    The binding of antigen to cells with antibody on their surface has been studied in a model system consisting of murine myeloma cells (MOPC 315) and DNP conjugates. Specific binding occurred between the DNP groups of DNP conjugates and cell surface immunoglobulin. Using this model, the binding affinities of multivalent and univalent DNP conjugates were measured directly by equilibrium-binding techniques and indirectly by displacement of bound conjugate with univalent hapten. With both approaches the multivalent conjugate was shown to bind to cells with an avidity 100–300 fold greater than the univalent hapten. Nonspecific binding of unrelated protein and repeated washing of cells was found to markedly dedecrease the specific binding of univalent conjugates, presumably because the relatively weak bonds dissociate readily. PMID:4734402

  11. Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

    PubMed

    Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

    2015-05-01

    Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. PMID:25792295

  12. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  13. Evaluation of the cross‐reactivity of antigens in Glupearl 19S and other hydrolysed wheat proteins in cosmetics

    PubMed Central

    Nakamura, Masashi; Yagami, Akiko; Hara, Kazuhiro; Sano‐Nagai, Akiyo; Kobayashi, Tsukane

    2016-01-01

    Summary Background In Japan, over 2000 users of a facial soap containing Glupearl 19S (GP19S), a hydrolysed wheat protein (HWP), developed immediate‐type systemic wheat allergy (HWP‐IWA), and ∼70% of them developed associated contact urticaria. Objectives We investigated whether HWP‐IWA patients cross‐react with other HWPs, and analysed HWP antigenic characteristics. Methods We used 10 types of HWP that are commercially available as cosmetic ingredients, and 16 subjects with HWP‐IWA. We performed an enzyme‐linked immunosorbent assay (ELISA) to evaluate the reactivity to each HWP, and western blotting to evaluate the characteristics of the antigens by using HWP‐IWA patients' serum IgE antibodies. We also performed prick tests with the HWPs. Results The patients reacted to four other HWPs in addition to GP19S, according to ELISA, and this was confirmed by strong reactions in the prick tests to the same four types of HWP. Smears of antigens with molecular weights ranging from the high range to the low range were seen on western blotting with the four HWPs that showed strong reactions in the ELISA and prick tests. Conclusions HWP‐IWA patients cross‐react with other HWPs. The antigens that they cross‐reacted to had a molecular weight distribution similar to that of GP19S present in the HWPs. PMID:27027256

  14. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    PubMed Central

    2015-01-01

    Background Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased the number of potential vaccine targets compared to the number of targets discovered using the traditional approach where low-frequency peptides are excluded. Conclusions We developed a webserver with an intuitive visualization scheme for summarizing the T cell-based antigenic potential of any given protein or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/. PMID:26679766

  15. T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

    PubMed Central

    Held, W A; Mullins, J J; Kuhn, N J; Gallagher, J F; Gu, G D; Gross, K W

    1989-01-01

    A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2714250

  16. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  17. Prokaryotic expression, purification and antigenicity analysis of African swine fever virus pK205R protein.

    PubMed

    Wu, Xulong; Xiao, Lu; Peng, Bin; Wang, Yin; Yang, Zexiao; Yao, Xueping; Hu, Ling; Lin, Xingyu

    2016-01-01

    African swine fever is an acute, febrile and highly virulent porcine disease causing serious economic losses worldwide. The pK205R protein of the African swine fever virus (ASFV) is largely expressed in the early stages of infection, which has given the K205R gene extensive attention. In this study, the ASFV K205R was cloned and expressed in Escherichia coli BL21 (DE3). Expression of histidine-tagged pK205R with a molecular mass of 44 kDa was determined by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Optimisation of culture conditions allowed induction of the recombinant protein with 0.4 mM Isopropyl β-D-thiogalactoside (IPTG) at 37°C for 2 h. The protein existed in cellular supernatant and was purified using a Ni-NTA resin column. The purified protein was used to immunize rabbits four times to enable the production of polyclonal antibodies, and the antiserum titre was detected by ELISA. The results showed that the purified pK205R can react with ASFV positive serum specifically by Western blotting. The pK205R had high antigenicity, which indicated that pK205R could be used as an antigen for detection of ASFV-specific antibodies in ELISA testing, and the recombinant protein could contribute to further research of the action and structure of pK205R. PMID:27096786

  18. Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

    PubMed

    Børud, Bente; Aas, Finn Erik; Vik, Ashild; Winther-Larsen, Hanne C; Egge-Jacobsen, Wolfgang; Koomey, Michael

    2010-06-01

    Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

  19. Effect of prior dietary exposure to cows' milk protein on antigen-specific and nonspecific cellular proliferation in mice.

    PubMed

    Brix, Susanne; Magyar, Orit H; Barkholt, Vibeke; Frøkiaer, Hanne

    2005-05-01

    The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies, which are mainly based on experiments in vitro, it is important to be able to differentiate nonspecific activation of immune cells induced by dietary components from ex vivo restimulation of antigen-specific cells that might be present in cell cultures owing to prior dietary exposure to the antigens in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by beta-casein peptides was further assessed on cells from mice bred on a cows'-milk-free diet. Regarding the dietary effect, we found that prior oral intake of cows' milk proteins affected cell proliferation induced by culturing with cows' milk proteins in vitro, as spleen cells from mice fed a milk-containing diet showed a significantly greater proliferative response than did cells from mice bred on a cows'-milk-free diet. Studies of immune enhancing potentials of beta-casein peptides showed that some peptides stimulate proliferation of immune cells nonspecifically. In conclusion, these findings stress the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some beta-casein peptides possess the potential to induce proliferation in immune cells in a nonspecific manner. PMID:15909688

  20. Identification of an iron-responsive protein that is antigenic in patients with Chlamydia trachomatis genital infections.

    PubMed

    Raulston, Jane E; Miller, Jeffrey D; Davis, Caroyn H; Schell, Maria; Baldwin, Amy; Ferguson, Kaethe; Lane, Heather

    2007-12-01

    Chlamydia trachomatis is an important cause of immune-mediated damage to the reproductive tract of infected patients. Certain chlamydial antigens and host genetic factors have been identified as contributing to immunopathological events, but a comprehensive understanding of specific components involved in destructive vs. protective immune responses to chlamydial infections is far from clear. In this study, it is shown that C. trachomatis-infected patients generate antibodies against an iron-responsive chlamydial protein, YtgA. The identity of YtgA was confirmed by mass spectrometry following two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. This finding underscores a necessity to examine patient sera samples to identify chlamydial antigens that are likely encountered and important to the immune response during human infections.

  1. A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats.

    PubMed Central

    Beall, E L; Admon, A; Rio, D C

    1994-01-01

    P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. This mode of transposition requires repair of both a double-strand break at the donor DNA site and gapped DNA at the target site. Biochemical studies have identified a cellular non-P element-encoded DNA binding protein, termed the inverted repeat binding protein (IRBP), that specifically interacts with the outer half of the 31-bp terminal inverted repeats. Protein sequence information was used to isolate cDNA clones encoding IRBP. Sequence analysis shows that IRBP is related to the 70-kDa subunit of the human Ku autoimmune antigen. The mammalian Ku antigen binds free DNA termini and has been implicated in immunoglobulin VDJ recombination, DNA repair, and transcription. In addition, Ku is the DNA binding subunit of the double-strand DNA-dependent protein kinase. Cytogenetic mapping indicates that the IRBP gene maps to chromosomal position 86E on the right arm of the third chromosome. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7809101

  2. Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii.

    PubMed

    Irrgang, Alexandra; Murugaiyan, Jayaseelan; Weise, Christoph; Azab, Walid; Roesler, Uwe

    2015-01-01

    Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae. PMID:26484314

  3. Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

    PubMed Central

    Irrgang, Alexandra; Murugaiyan, Jayaseelan; Weise, Christoph; Azab, Walid; Roesler, Uwe

    2015-01-01

    Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae. PMID:26484314

  4. An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection

    PubMed Central

    França, Camila T.; Hostetler, Jessica B.; Sharma, Sumana; White, Michael T.; Lin, Enmoore; Kiniboro, Benson; Waltmann, Andreea; Darcy, Andrew W.; Li Wai Suen, Connie S. N.; Siba, Peter; King, Christopher L.; Rayner, Julian C.; Fairhurst, Rick M.; Mueller, Ivo

    2016-01-01

    Background Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. Methodology/Principal Findings In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001–0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46–0.74; P<0.001–0.041). Conclusion/Significance These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both. PMID:27182597

  5. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

    PubMed Central

    Weeratna, R; Stamler, D A; Edelstein, P H; Ripley, M; Marrie, T; Hoskin, D; Hoffman, P S

    1994-01-01

    We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in

  6. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    PubMed

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  7. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  8. A new Toxoplasma gondii chimeric antigen containing fragments of SAG2, GRA1, and ROP1 proteins-impact of immunodominant sequences size on its diagnostic usefulness.

    PubMed

    Ferra, Bartłomiej; Holec-Gąsior, Lucyna; Kur, Józef

    2015-09-01

    This study presents the first evaluation of new Toxoplasma gondii recombinant chimeric antigens containing three immunodominant regions of SAG2, GRA1, and one of two ROP1 fragments differing in length for the serodiagnosis of human toxoplasmosis. The recombinant chimeric antigens SAG2-GRA1-ROP1L (with large fragment of ROP1, 85-396 amino acid residues) and SAG2-GRA1-ROP1S (with a small fragment of ROP1, 85-250 amino acid residues) were obtained as fusion proteins containing His6-tags at both ends using an Escherichia coli expression system. The diagnostic utility of these chimeric antigens was determined using the enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-T. gondii immunoglobulin G (IgG). The IgG ELISA results obtained for the chimeric antigens were compared to those obtained for the use of Toxoplasma lysate antigen (TLA) and for a mixture of recombinant antigens containing rSAG2, rGRA1, and rROP1. The sensitivity of the IgG ELISA was similar for the SAG2-GRA1-ROP1L chimeric antigen (100 %), the mixture of three proteins (99.4 %) and the TLA (97.1 %), whereas the sensitivity of IgG ELISA with the SAG2-GRA1-ROP1S chimeric antigen was definitely lower, reaching 88.4 %. In conclusion, this study shows that SAG2-GRA1-ROP1L chimeric antigen can be useful for serodiagnosis of human toxoplasmosis with the use of the IgG ELISA assay. Therefore, the importance of proper selection of protein fragments for the construction of chimeric antigen with the highest reactivity in ELISA test is demonstrated.

  9. African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever

    Technology Transfer Automated Retrieval System (TEKTRAN)

    African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of African swine fever virus (ASFV) strain diversity and the viral antigens conferring type specific protective im...

  10. Antigen-antibody binding in reverse micelles: interaction of monoclonal antibodies with a myelin basic protein peptide.

    PubMed

    Groome, N P; Vacher, M; Nicot, C; Waks, M

    1990-01-01

    Reverse micelles can be used to mimic biological processes occurring at interfaces. To investigate antigen-antibody binding in a membrane-like environment, we first obtained Fab fragments from monoclonal antibodies against bovine myelin basic protein (MBP), an encephalitogenic protein. The binding of the fragments to a dansylated synthetic human MBP peptide gly(119)-gly(131), presenting sequence homologies with a viral protein, was measured in buffer and for the first time in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate, in isooctane. Analysis of the fluorescence polarisation titration curves discloses that the Fab fragments in reverse micelles have retained the high affinity for the peptide found in buffer, and similar to that for intact MBP.

  11. Exemptions from Unrelated Business Tax: Rental Income

    ERIC Educational Resources Information Center

    Reed, George E.

    1975-01-01

    Section 512(b) of the Internal Revenue Code contains several categorical exemptions from the unrelated business tax including rental income. The article covers various problems faced by nonprofit organizations such as parochial schools in leasing or selling property. (LBH)

  12. [A comparative analysis of various antigenic proteins found in Haemonchus contortus--a review].

    PubMed

    Tak, I R; Dar, J S; Dar, S A; Ganai, B A; Chishti, M Z; Ahmad, F

    2015-01-01

    Many innovative researches on the development and introduction of recombinant vaccines against many economically important parasites were carried out in the 20th century. Research continues to hold promise with the development of immunological and molecular approaches for control of these parasites and in this regard it has already been seen that blood-sucking parasites such as Haemonchus contortus and Ostertagia ostertagi are susceptible to control by vaccines containing "novel" or "concealed" antigens. Haemonchus contortus is primarily pathogenic to sheep and its blood-feeding behaviour causes effects ranging from mild anaemia to mortality in young animals. Current means of control which are dependent on repeated treatment with anthelmintics are responsible for the increasing drug resistance of this parasite. Together with the growing concern of residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with an aim to develop alternate means of control, including the development of molecular vaccines. More problematic and also important is the formulation and delivery strategy to induce expulsion of this parasite, using vaccines containing recombinant "conventional" antigens. Tremendous progress has been made in the last decade in identifying several antigens from Haemonchus contortus which in their native form stimulate useful levels of protective immunity. Vaccines have been developed against H. contortus using 'novel' gut antigens from the parasite, but variable responsiveness of the host sheep has resulted in varying degrees of protection which are stimulated by these vaccines. Computer models have also been used to simulate vaccine efficacy in worm control and have yielded good results. This review will try to summarise the protective efficacy and also the molecular properties of principal candidate antigens which are expressed by this parasite. The review will try to cover the aspirations

  13. [A comparative analysis of various antigenic proteins found in Haemonchus contortus--a review].

    PubMed

    Tak, I R; Dar, J S; Dar, S A; Ganai, B A; Chishti, M Z; Ahmad, F

    2015-01-01

    Many innovative researches on the development and introduction of recombinant vaccines against many economically important parasites were carried out in the 20th century. Research continues to hold promise with the development of immunological and molecular approaches for control of these parasites and in this regard it has already been seen that blood-sucking parasites such as Haemonchus contortus and Ostertagia ostertagi are susceptible to control by vaccines containing "novel" or "concealed" antigens. Haemonchus contortus is primarily pathogenic to sheep and its blood-feeding behaviour causes effects ranging from mild anaemia to mortality in young animals. Current means of control which are dependent on repeated treatment with anthelmintics are responsible for the increasing drug resistance of this parasite. Together with the growing concern of residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with an aim to develop alternate means of control, including the development of molecular vaccines. More problematic and also important is the formulation and delivery strategy to induce expulsion of this parasite, using vaccines containing recombinant "conventional" antigens. Tremendous progress has been made in the last decade in identifying several antigens from Haemonchus contortus which in their native form stimulate useful levels of protective immunity. Vaccines have been developed against H. contortus using 'novel' gut antigens from the parasite, but variable responsiveness of the host sheep has resulted in varying degrees of protection which are stimulated by these vaccines. Computer models have also been used to simulate vaccine efficacy in worm control and have yielded good results. This review will try to summarise the protective efficacy and also the molecular properties of principal candidate antigens which are expressed by this parasite. The review will try to cover the aspirations

  14. Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.

    PubMed

    Royuela, Enrique; Sánchez-Fauquier, Alicia

    2010-01-01

    The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.

  15. Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.

    PubMed

    García-Lunar, P; Regidor-Cerrillo, J; Ortega-Mora, L M; Gutiérrez-Expósito, D; Alvarez-García, G

    2014-10-15

    Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of

  16. Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit.

    PubMed

    Satoh, M; Ajmani, A K; Stojanov, L; Langdon, J J; Ogasawara, T; Wang, J; Dooley, M A; Richards, H B; Winfield, J B; Carter, T H; Reeves, W H

    1996-09-01

    DNA-dependent protein kinase (DNA-PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA-PKcs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA-PK were identified. One type of autoantibody stabilized the interaction of DNA-PKcs with Ku and recognized the DNA-PKcs -Ku complex, but not bio-chemically purified DNA-PKcs. Another type recognized purified DNA-PKcs. Autoantibodies to Ku (p70/p80 heterodimer), 'stabilizing' antibodies, and antibodies to DNA-PKcs comprise a linked autoantibody set, since antibodies recognizing purified DNA-PKcs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti-Ku. This hierarchical pattern of autoantibodies specific for components of DNA-PK (anti-Ku > stabilizing antibodies > anti-DNA-PKcs) may have implications for the pathogenesis of autoimmunity to DNA-PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA-PKcs-Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA-PKcs.

  17. Protein and Antigen Diversity in the Vesicular Fluid of Taenia Solium Cysticerci Dissected from Naturally Infected Pigs

    PubMed Central

    Esquivel-Velázquez, Marcela; Larralde, Carlos; Morales, Julio; Ostoa-Saloma, Pedro

    2011-01-01

    Cysticercosis caused by Taenia solium is a health threat for humans and pigs living in developing countries, for which there is neither a flawless immunodiagnostic test nor a totally effective vaccine. Suspecting of individual diversity of hosts and parasites as possible sources of the variations of the parasite loads among cysticercotic animals and of the limited success of such immunological applications as well as, we explored and measured both in nine cases of naturally acquired porcine cysticercosis. For this purpose, 2-Dimensional IgG immunoblots were performed by reacting the sera of each cysticercotic pig with the antigens contained in the vesicular fluid (VF) of their own cysticerci. We found an unexpectedly large diversity among the proteins and antigens contained in each of the nine VFs. Also diverse were the serum IgG antibody responses of the nine pigs, as none of their 2D- immunoblot images exhibited the same number of spots and resembled each other in only 6.3% to 65.3% of their features. So large an individual immunological diversity of the cysticercal antigens and of the infected pigs´ IgG antibody response should be taken into account in the design of immunological tools for diagnosis and prevention of cysticercosis and should also be considered as a possibly significant source of diversity in Taenia solium´s infectiveness and pathogenicity. PMID:22110381

  18. Protein and antigen diversity in the vesicular fluid of Taenia solium cysticerci dissected from naturally infected pigs.

    PubMed

    Esquivel-Velázquez, Marcela; Larralde, Carlos; Morales, Julio; Ostoa-Saloma, Pedro

    2011-01-01

    Cysticercosis caused by Taenia solium is a health threat for humans and pigs living in developing countries, for which there is neither a flawless immunodiagnostic test nor a totally effective vaccine. Suspecting of individual diversity of hosts and parasites as possible sources of the variations of the parasite loads among cysticercotic animals and of the limited success of such immunological applications as well as, we explored and measured both in nine cases of naturally acquired porcine cysticercosis. For this purpose, 2-Dimensional IgG immunoblots were performed by reacting the sera of each cysticercotic pig with the antigens contained in the vesicular fluid (VF) of their own cysticerci. We found an unexpectedly large diversity among the proteins and antigens contained in each of the nine VFs. Also diverse were the serum IgG antibody responses of the nine pigs, as none of their 2D- immunoblot images exhibited the same number of spots and resembled each other in only 6.3% to 65.3% of their features. So large an individual immunological diversity of the cysticercal antigens and of the infected pigs´ IgG antibody response should be taken into account in the design of immunological tools for diagnosis and prevention of cysticercosis and should also be considered as a possibly significant source of diversity in Taenia solium´s infectiveness and pathogenicity.

  19. Protein kinase Cβ is critical for the metabolic switch to glycolysis following B-cell antigen receptor engagement.

    PubMed

    Blair, Derek; Dufort, Fay J; Chiles, Thomas C

    2012-11-15

    Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

  20. Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*.

    PubMed

    Akter, Farhima; Mie, Masayasu; Grimm, Sebastian; Nygren, Per-Åke; Kobatake, Eiry

    2012-06-01

    The chemical reactions used to make antibody-DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin γ1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.

  1. Identification of ribosomal protein L19 as a novel tumor antigen recognized by autologous cytotoxic T lymphocytes in lung adenocarcinoma.

    PubMed

    Kuroda, Koji; Takenoyama, Mitsuhiro; Baba, Tetsuro; Shigematsu, Yoshiki; Shiota, Hironobu; Ichiki, Yoshinobu; Yasuda, Manabu; Uramoto, Hidetaka; Hanagiri, Takeshi; Yasumoto, Kosei

    2010-01-01

    The purpose of the present study was to identify a novel tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. The co-culture of regional lymph node lymphocytes and the CD80-transfected autologous lung adenocarcinoma cell line H1224L resulted in a successful induction of bulk cytotoxic T lymphocytes (CTL). CTL clone L7/8 was established by the limiting dilution method from these bulk CTLs and lysed H1224L but not autologous Epstein-Barr virus-transformed B cells or K562. The CTL clone also recognized allogeneic lung cancer cell lines in an HLA-A*31012-restricted manner. Using the CTL clone, an antigen-coding gene was identified using the cDNA expression cloning technique, which encodes ribosomal protein L19 (RPL19). Finally, a 9 mer antigenic peptide was identified by means of construction of mini-genes. RPL19 was overexpressed in the lung cancer tissue from patient H1224. All of the normal tissues examined expressed lower levels of RPL19 mRNA than that of the lung cancer tissue. RPL19 was also found to be overexpressed in 12 of 30 (40%) non-small-cell lung cancer tissues by immunohistochemical staining. The expression level of RPL19 in tumor cell lines correlated positively with the production of interferon (IFN)-gammaby CTL clone L7/8 in response to such cell lines. In addition, the suppression of RPL19 expression by transfection with small interfering RNA resulted in the suppression of cyclinD1, D3 synthesis, and the growth inhibition of lung cancer cell lines overexpressing RPL19. Therefore, this growth suppression could be ascribed to the inhibition of the cell cycle. These results may indicate that RPL19 is a novel overexpressed antigen which may therefore be a useful candidate as a target for specific immunotherapy.

  2. The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses

    PubMed Central

    Mokhtar, Helen; Pedrera, Miriam; Frossard, Jean-Pierre; Biffar, Lucia; Hammer, Sabine E.; Kvisgaard, Lise K.; Larsen, Lars E.; Stewart, Graham R.; Somavarapu, Satyanarayana; Steinbach, Falko; Graham, Simon P.

    2016-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-α and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development. PMID:26909080

  3. Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

    PubMed

    Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

    2016-07-27

    Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. PMID:27045683

  4. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

  5. Chemically modified inulin microparticles serving dual function as a protein antigen delivery vehicle and immunostimulatory adjuvant.

    PubMed

    Gallovic, Matthew D; Montjoy, Douglas G; Collier, Michael A; Do, Clement; Wyslouzil, Barbara E; Bachelder, Eric M; Ainslie, Kristy M

    2016-03-01

    To develop a new subunit vaccine adjuvant, we chemically modified a naturally-occurring, immunostimulatory inulin polysaccharide to produce an acid-sensitive biopolymer (acetalated inulin, Ace-IN). Various hydrophobic Ace-IN polymers were formed into microparticles (MPs) by oil-in-water emulsions followed by solvent evaporation These Ace-IN MPs possessed tunable degradation characteristics that, unlike polyesters used in FDA-approved microparticulate formulations, had only pH-neutral hydrolytic byproducts. Macrophages were passively targeted with cytocompatible Ace-IN MPs. TNF-α production by macrophages treated with Ace-IN MPs could be altered by adjusting the polymers' chemistry. Mice immunized with Ace-IN MPs encapsulating a model ovalbumin (OVA) antigen showed higher production of anti-OVA IgG antibody levels relative to soluble antigen. The antibody titers were also comparable to an alum-based formulation. This proof-of-concept establishes the potential for chemically-modified inulin MPs to simultaneously enable dual functionality as a stimuli-controlled antigen delivery vehicle and immunostimulatory adjuvant.

  6. A method to identify protein antigens of Dermanyssus gallinae for the protection of birds from poultry mites.

    PubMed

    Makert, Gustavo R; Vorbrüggen, Susanne; Krautwald-Junghanns, Maria-Elisabeth; Voss, Matthias; Sohn, Kai; Buschmann, Tilo; Ulbert, Sebastian

    2016-07-01

    The poultry red mite (PRM) Dermanyssus gallinae causes high economic losses and is among the most important parasites in poultry farming worldwide. Different chemical, physical, and biological strategies try to control the expansion of PRM. However, effective solutions to this problem still have to be found. Here, we present a method for the development of an immunological control strategy, based on the identification of mite protein antigens which elicit antibodies with anti-mite activity in the immunized chicken. Hens were immunized with different PRM protein extracts formulated with two different adjuvants, and IgY-antibodies were isolated from the eggs. A PRM in vitro feeding assay which used chicken blood spiked with these IgY-preparations was used to detect antibodies which caused PRM mortality. In vitro feeding of mites with IgY isolated from hens immunized with PRM extract formulated with one of the adjuvants showed a statistically significant increase in the mortality as compared to control mites. After the separation of total PRM extracts in two-dimensional gels, several protein spots were recognized by such IgY preparations. Ten protein spots were subjected to mass spectrometry (MS/MS) for the identification of the corresponding proteins. Complete protein sequences were deduced from genomic and transcriptomic assemblies derived from high throughput sequencing of total PRM DNA and RNA. The results may contribute to the development of an immunological control strategy of D. gallinae. PMID:27026505

  7. A method to identify protein antigens of Dermanyssus gallinae for the protection of birds from poultry mites.

    PubMed

    Makert, Gustavo R; Vorbrüggen, Susanne; Krautwald-Junghanns, Maria-Elisabeth; Voss, Matthias; Sohn, Kai; Buschmann, Tilo; Ulbert, Sebastian

    2016-07-01

    The poultry red mite (PRM) Dermanyssus gallinae causes high economic losses and is among the most important parasites in poultry farming worldwide. Different chemical, physical, and biological strategies try to control the expansion of PRM. However, effective solutions to this problem still have to be found. Here, we present a method for the development of an immunological control strategy, based on the identification of mite protein antigens which elicit antibodies with anti-mite activity in the immunized chicken. Hens were immunized with different PRM protein extracts formulated with two different adjuvants, and IgY-antibodies were isolated from the eggs. A PRM in vitro feeding assay which used chicken blood spiked with these IgY-preparations was used to detect antibodies which caused PRM mortality. In vitro feeding of mites with IgY isolated from hens immunized with PRM extract formulated with one of the adjuvants showed a statistically significant increase in the mortality as compared to control mites. After the separation of total PRM extracts in two-dimensional gels, several protein spots were recognized by such IgY preparations. Ten protein spots were subjected to mass spectrometry (MS/MS) for the identification of the corresponding proteins. Complete protein sequences were deduced from genomic and transcriptomic assemblies derived from high throughput sequencing of total PRM DNA and RNA. The results may contribute to the development of an immunological control strategy of D. gallinae.

  8. Immunization against HTLV-I with chitosan and tri-methylchitosan nanoparticles loaded with recombinant env23 and env13 antigens of envelope protein gp46.

    PubMed

    Amirnasr, Maryam; Fallah Tafti, Tannan; Sankian, Mojtaba; Rezaei, Abdorrahim; Tafaghodi, Mohsen

    2016-08-01

    To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P < 0.001). After nasal administration, for env23, IgG2a levels and IgG2a/IgG1 ratio was significantly higher than groups with subcutaneous administration (P < 0.001). Both nanoparticles showed good immunoadjuvant potential. Env23 antigen was a better candidate for vaccination against HTLV-I, as it induced higher cellular immune responses, compared with env13. PMID:27235335

  9. Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment*

    PubMed Central

    Geng, Jie; Sivaramakrishnan, Sivaraj; Raghavan, Malini

    2013-01-01

    The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function. PMID:24196954

  10. Do antibodies to myelin basic protein isolated from multiple sclerosis cross-react with measles and other common virus antigens?

    PubMed Central

    Bernard, C C; Townsend, E; Randell, V B; Williamson, H G

    1983-01-01

    Immunological activity to various antigens, including brain components, measles and other viruses, has been associated with IgG in multiple sclerosis (MS). One possible explanation for the presence of anti-viral antibodies and antibody to myelin basic protein (MBP) in MS patients is that there are antigenic determinants common to certain viruses and MBP. To assess this possibility, IgG from individual brains and sera from patients with MS, subacute sclerosing panencephalitis (SSPE) and controls was isolated by protein A and MBP-Sepharose affinity chromatography. Antibody to MBP was measured with a solid phase radioimmunoassay and antibody to measles and other viruses by immunofluorescence and/or complement fixation. Anti-MBP activity was detected in brain extracts and sera of all MS patients tested. In contrast to the low levels of antibody to MBP in control brains, high levels of anti-MBP antibodies were found in most of the normal sera. There was no correlation between the presence and levels of serum anti-measles antibodies and the anti-MBP activity. None of the anti-MBP antibodies affinity purified from brain and serum of MS patients reacted with any of the viruses tested, including measles. IgG purified from SSPE patients or from a rabbit hyperimmunized with measles antigen had no reactivity to MBP, despite high levels of anti-measles antibody. It is concluded that there is not direct link between the presence of antibody to MBP and antibody to measles and other viruses in MS patients. PMID:6190599

  11. Immunization with FSHβ fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model

    SciTech Connect

    Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen Chen, Fulin

    2013-05-03

    Highlights: •A GST-FSH fusion protein was successfully expressed in E. coli. •Immunization with GST-FSH antigen can raise high-titer anti-FSH polyclonal sera. •Anti-FSH polyclonal sera can neutralize osteoclastogenic effect of FSH in vitro. •FSH immunization can prevent bone loss in a rat osteoporosis model. -- Abstract: Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally. Hormone replacement therapy (HTR), especially estrogen replacement therapy (ERT), is used widely in the clinic because it has been generally accepted that postmenopausal osteoporosis is caused by estrogen deficiency. However, hypogonadal α and β estrogen receptor null mice were only mildly osteopenic, and mice with either receptor deleted had normal bone mass, indicating that estrogen may not be the only mediator that induces osteoporosis. Recently, follicle-stimulating hormone (FSH), the serum concentration of which increases from the very beginning of menopause, has been found to play a key role in postmenopausal osteoporosis by promoting osteoclastogenesis. In this article, we confirmed that exogenous FSH can enhance osteoclast differentiation in vitro and that this effect can be neutralized by either an anti-FSH monoclonal antibody or anti-FSH polyclonal sera raised by immunizing animals with a recombinant GST-FSHβ fusion protein antigen. Moreover, immunizing ovariectomized rats with the GST-FSHβ antigen does significantly prevent trabecular bone loss and thereby enhance the bone strength, indicating that a FSH-based vaccine may be a promising therapeutic strategy to slow down bone loss in postmenopausal women.

  12. Inferring epitopes of a polymorphic antigen amidst broadly cross-reactive antibodies using protein microarrays: a study of OspC proteins of Borrelia burgdorferi.

    PubMed

    Baum, Elisabeth; Randall, Arlo Z; Zeller, Michael; Barbour, Alan G

    2013-01-01

    Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming 'wet bench' techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation

  13. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system. PMID:26481477

  14. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus.

    PubMed

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-05-01

    We developed a rapid dot-enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available.

  15. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

    PubMed Central

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-01-01

    We developed a rapid dot–enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available. PMID:25802430

  16. Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.

    PubMed Central

    Seydel, U; Jans, D A

    1996-01-01

    Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import. PMID:8670127

  17. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis.

    PubMed

    Agallou, Maria; Athanasiou, Evita; Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  18. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

  19. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis

    PubMed Central

    Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  20. Detergent pretreatment of solid phase globular proteins in ELISA`s. Enhanced antigenicity and subsequent sensitivity. Final report, September 1989-September 1991

    SciTech Connect

    Blanchard, G.C.; Bouhmadouche, M.; Williamson, M.L.

    1994-10-01

    Methods for pretreatment and rejuvenation of preimmobilized globular proteins used in immunodiagnostics were investigated using reagents routinely used in ELISA`s. Rabbit and goat gamma globulins, functioning as antigens, and antibodies on non-covalent, and covalent solid surfaces, were monitored for detergent mediated desorption, denaturation, non-specific binding and altered antigenicity. The results from fourteen commercially supplied polyvinyl- and polystyrene-derivatized microtiter plates coated with antibody or antigenic lgG were compared with commercial microtiter diagnostic plates with preimmobilized lgG. Wash solutions had no effect on immobilized gamma globulins when the solid phase protein functioned as an antibody on covalent or noncovalent surfaces. In addition to tween 20 removing up to 50% of noncovalently bound protein additional binding sites are apparently exposed on solid phase antigens, evident by an increase in signal, which cannot be explained by nonspecific binding. However, no increase in signal was evident when antigen was preimmobilized covalently. The role of between 20 and other reagent components in ELISA-based assays are explored. The screening of noncovalent preimmobilized antigen coated surfaces prior to use for deteraent mediated enhancement is suggested.

  1. Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein.

    PubMed Central

    Luka, J; Miller, G; Jörnvall, H; Pearson, G R

    1986-01-01

    Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines. Images PMID:2422401

  2. Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins

    PubMed Central

    1990-01-01

    Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands. PMID:2277087

  3. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    PubMed

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

  4. Expression, characterisation and antigenicity of a truncated Hendra virus attachment protein expressed in the protozoan host Leishmania tarentolae.

    PubMed

    Fischer, Kerstin; dos Reis, Vinicius Pinho; Finke, Stefan; Sauerhering, Lucie; Stroh, Eileen; Karger, Axel; Maisner, Andrea; Groschup, Martin H; Diederich, Sandra; Balkema-Buschmann, Anne

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein.

  5. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    PubMed

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  6. Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins.

    PubMed

    Futami, Junichiro; Nonomura, Hidenori; Kido, Momoko; Niidoi, Naomi; Fujieda, Nao; Hosoi, Akihiro; Fujita, Kana; Mandai, Komako; Atago, Yuki; Kinoshita, Rie; Honjo, Tomoko; Matsushita, Hirokazu; Uenaka, Akiko; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-10-21

    Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy. PMID:26355635

  7. Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

    PubMed Central

    Sampson, J S; Plikaytis, B B; Wilkinson, H W

    1986-01-01

    Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis. Images PMID:3517046

  8. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  9. Pharmaceutical proteins in plants. A strategic genetic engineering approach for the production of tuberculosis antigens.

    PubMed

    Frutos, Roger; Denise, Hubert; Vivares, Christian; Neuhaus, Jean-Marc; Vitale, Sandro; Pedrazzini, Emmanuela; Ma, Julian; Dix, Phil; Gray, John; Pezzotti, Mario; Conrad, Udo; Robinson, David

    2008-12-01

    Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy.

  10. Evaluation of the antigenicity of hydrolyzed cow's milk protein formulas using the mouse basophil activation test.

    PubMed

    Iwamoto, Hiroshi; Matsubara, Takeshi; Nakazato, Yuki; Namba, Kazuyoshi; Takeda, Yasuhiro

    2016-02-01

    Hypoallergenic infant formulas are widely used for infants with cow's milk allergy. The aim of this study was to assess the utility of the mouse basophil activation test (BAT) in the evaluation of residual antigenicity in these formulas. Whole blood samples derived from β-lactoglobulin- or casein-immunized mice were incubated with one of the following formulas: conventional, partially hydrolyzed, or extensively hydrolyzed. Basophilic activation was analyzed by flow cytometry using an IgE-dependent activation marker CD200R1 and an IgG-dependent activation marker CD200R3. Systemic anaphylaxis was induced by i.v. injection of milk formula and results were compared. Conventional formula induced pronounced changes in CD200R1 and CD200R3 expression on basophils, whereas extensively hydrolyzed formulas did not elicit any changes in these markers. Similarly, challenge with conventional formula induced anaphylaxis, whereas extensively hydrolyzed formulas did not induce anaphylaxis. Although the partially hydrolyzed formula also induced basophilic activation and systemic anaphylaxis, the magnitude of these effects was smaller than that observed with the conventional formula. Compared to CD200R1, the observed trend in CD200R3 expression resembled the results obtained from systemic anaphylaxis test more closely. These findings show that mouse BAT, in particular using CD200R3, is highly useful for the evaluation of antigenicity of milk formulas. PMID:26626100

  11. Pharmaceutical proteins in plants. A strategic genetic engineering approach for the production of tuberculosis antigens.

    PubMed

    Frutos, Roger; Denise, Hubert; Vivares, Christian; Neuhaus, Jean-Marc; Vitale, Sandro; Pedrazzini, Emmanuela; Ma, Julian; Dix, Phil; Gray, John; Pezzotti, Mario; Conrad, Udo; Robinson, David

    2008-12-01

    Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy. PMID:19120228

  12. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    PubMed

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination. PMID:24384300

  13. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    PubMed

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination.

  14. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  15. Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

    PubMed

    Malito, Enrico; Faleri, Agnese; Lo Surdo, Paola; Veggi, Daniele; Maruggi, Giulietta; Grassi, Eva; Cartocci, Elena; Bertoldi, Isabella; Genovese, Alessia; Santini, Laura; Romagnoli, Giacomo; Borgogni, Erica; Brier, Sébastien; Lo Passo, Carla; Domina, Maria; Castellino, Flora; Felici, Franco; van der Veen, Stijn; Johnson, Steven; Lea, Susan M; Tang, Christoph M; Pizza, Mariagrazia; Savino, Silvana; Norais, Nathalie; Rappuoli, Rino; Bottomley, Matthew J; Masignani, Vega

    2013-02-26

    Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

  16. Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.

    PubMed Central

    Kosik, K S; Joachim, C L; Selkoe, D J

    1986-01-01

    The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death. Images PMID:2424016

  17. Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

    SciTech Connect

    Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M.

    2012-05-08

    Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

  18. Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.

    PubMed

    Raynaud, Cécile; Sozzani, Rosangela; Glab, Nathalie; Domenichini, Séverine; Perennes, Claudette; Cella, Rino; Kondorosi, Eva; Bergounioux, Catherine

    2006-08-01

    The proliferating cell nuclear antigen (PCNA) functions as a sliding clamp for DNA polymerase, and is thus a key actor in DNA replication. It is also involved in DNA repair, maintenance of heterochromatic regions throughout replication, cell cycle regulation and programmed cell death. Identification of PCNA partners is therefore necessary for understanding these processes. Here we identify two Arabidopsis SET-domain proteins that interact with PCNA: ATXR5 and ATXR6. A truncated ATXR5Deltaex2, incapable of interacting with PCNA, also occurs in planta. ATXR6, upregulated during the S phase, is upregulated by AtE2F transcription factors, suggesting that it is required for S-phase progression. The two proteins differ in their subcellular localization: ATXR5 has a dual localization in plastids and in the nucleus, whereas ATXR6 is solely nuclear. This indicates that the two proteins may play different roles in plant cells. However, overexpression of either ATXR5 or ATXR6 causes male sterility because of the degeneration of defined cell types. Taken together, our results suggest that both proteins may play a role in the cell cycle or DNA replication, and that the activity of ATXR5 may be regulated via its subcellular localization.

  19. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

    PubMed

    Kraushar, Matthew L; Thompson, Kevin; Wijeratne, H R Sagara; Viljetic, Barbara; Sakers, Kristina; Marson, Justin W; Kontoyiannis, Dimitris L; Buyske, Steven; Hart, Ronald P; Rasin, Mladen-Roko

    2014-09-01

    Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

  20. Antigenic cross-reactivity among avian pneumoviruses of subgroups A, B, and C at the matrix but not nucleocapsid proteins.

    PubMed

    Lwamba, Humphrey C M; Halvorson, David A; Nagaraja, Kakambi V; Turpin, Elizabeth A; Swayne, David; Seal, Bruce S; Njenga, M Kariuki

    2002-01-01

    Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein-based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.

  1. Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

    PubMed

    Babij, Konrad; Bajzert, Joanna; Dąbrowska, Anna; Szołtysik, Marek; Zambrowicz, Aleksandra; Lubec, Gert; Stefaniak, Tadeusz; Willak-Janc, Ewa; Chrzanowska, Józefa

    2015-11-01

    In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

  2. Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells.

    PubMed Central

    Carrasco-Marín, E; Paz-Miguel, J E; López-Mato, P; Alvarez-Domínguez, C; Leyva-Cobián, F

    1998-01-01

    The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells. PMID:9824492

  3. Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane-associated antigen critical to niche homing

    PubMed Central

    Dubovsky, Jason A.; Chappell, Danielle L.; Harrington, Bonnie K.; Agrawal, Kitty; Andritsos, Leslie A.; Flynn, Joseph M.; Jones, Jeffrey A.; Paulaitis, Michael E.; Bolon, Brad; Johnson, Amy J.

    2013-01-01

    Membrane antigens are critical to the pathogenesis of chronic lymphocytic leukemia (CLL) as they facilitate microenvironment homing, proliferation, and survival. Targeting the CLL membrane and associated signaling patterns is a current focus of therapeutic development. Many tumor membrane targets are simultaneously targeted by humoral immunity, thus forming recognizable immunoglobulin responses. We sought to use this immune response to identify novel membrane-associated targets for CLL. Using a novel strategy, we interrogated CLL membrane-specific autologous immunoglobulin G reactivity. Our analysis unveiled lymphocyte cytosolic protein 1 (LCP1), a lymphocyte-specific target that is highly expressed in CLL. LCP1 plays a critical role in B-cell biology by crosslinking F-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for critical signaling pathways. Small interfering RNA knockdown of LCP1 blocked migration toward CXCL12 in transwell assays and to bone marrow in an in vivo xenotransplant model, confirming a role for LCP1 in leukemia migration. Furthermore, we demonstrate that the Bruton’s tyrosine kinase inhibitor ibrutinib or the PI3K inhibitor idelalisib block B-cell receptor induced activation of LCP1. Our data demonstrate a novel strategy to identify cancer membrane target antigens using humoral anti-tumor immunity. In addition, we identify LCP1 as a membrane-associated target in CLL with confirmed pathogenic significance. This clinical trial was registered at clinicaltrials.gov; study ID number: OSU-0025 OSU-0156. PMID:24009233

  4. Human MHC class I antigens are associated with a 90-kDa cell surface protein.

    PubMed

    Ferm, M T; Grönberg, A

    1991-08-01

    Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non-covalently associated with beta 2-microglobulin (12 kDa). In this report we show that MHC class I (MHC-I) proteins can also be associated with a 90-kDa protein in the cell membrane. Surface-radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS-PAGE and autoradiography. Immunoprecipitation of human MHC-I proteins co-precipitated another protein of about 90 kDa in molecular weight-p90. p90 was coprecipitated from all the MHC-I expressing cells tested: U937, Raji, Molt-4 and IFN-gamma treated K562, but not from untreated, MHC-I negative K562. A 90-kDa protein was also co-precipitated with MHC-I from fresh peripheral blood mononuclear cells (PBMC). Furthermore, p90 was coprecipitated by different MoAbs to the MHC-I heavy chain or beta 2-microglobulin, but not by control antibodies. Two additional co-precipitating proteins at 34 kDa and 28 kDa were seen in MHC-I precipitates from Raji cells. Our results suggest that MHC-I proteins and the 90-kDa protein are associated in the cell membrane, probably by a close but weak, non-covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC-I associated on Raji Burkitt's lymphoma cells.

  5. Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response.

    PubMed

    Rodák, L

    1975-10-01

    An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.

  6. Production of antigens in Chlamydomonas reinhardtii: green microalgae as a novel source of recombinant proteins.

    PubMed

    Fuhrmann, Markus

    2004-01-01

    Recombinant small-scale proteins are produced in a number of systems, from bacteria like Escherichia coli, through lower eukaryotes like baker's yeast, up to mammalian cell cultures. However, the need for safe and cheap sources of large amounts of recombinant proteins for different purposes, including material sciences, diagnostics, and, of course, medical therapy, has forced the development of alternative production systems. Green microalgae are cheap and easily grown and offer a high protein content, which would seem to make them ideal hosts for the large-scale sustainable production of recombinant proteins in the future. In selected species, recombinant DNA can be introduced into the genomes of the nucleus, the chloroplast, and even the mitochondria, and thus the system offers both prokaryotic (chloroplast, mitochondria) and eukaryotic translation systems for a tailored expression of virtually any protein.

  7. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  8. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-02-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  9. Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

    PubMed Central

    Biedroń, Rafał; Konopiński, Maciej K.; Marcinkiewicz, Janusz; Józefowski, Szczepan

    2015-01-01

    The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect

  10. Cultured human Langerhans' cells are superior to fresh cells at presenting native HIV-1 protein antigens to specific CD4+ T-cell lines.

    PubMed Central

    Girolomoni, G; Valle, M T; Zacchi, V; Costa, M G; Giannetti, A; Manca, F

    1996-01-01

    Cultured Langerhans' cells (CLC) exhibit enhanced antigen-presenting function compared to freshly isolated LC (FLC), but they are commonly believed to be inefficient at processing intact proteins. In this study, FLC and CLC from normal, human immunodeficiency virus (HIV) seronegative volunteers were compared for their ability to present the HIV-1 envelope glycoprotein gp120 or reverse transcriptase (p66) antigens to autologous, specific CD4+ T cell lines. Epidermal cell suspensions enriched for LC were prepared from suction blister roofs. FLC stimulated T cells at lower antigen concentrations compared to unfractionated peripheral blood mononuclear cells (PBMC). CLC were more potent on a per cell basis than FLC, PBMC or adherent monocytes at presenting native gp120, native p66 or immunogenic peptides. CLC were also more efficient than FLC or PBMC in terms of the amount of antigen required for T-cell activation. Chloroquine and leupeptin inhibited presentation of intact p66, but not of an immunodominant peptide, by FLC or CLC, thus indicating that both cells utilize antigen-processing mechanisms that are based on intracellular acidification and protease activity. Incubation of CLC with monoclonal antibodies against HLA-DR, CD11b, CD18, CD50, CD54, CD58 or CD80, but not anti-major histocompatibility complex class I (MHC-I), inhibited antigen-specific T-cell proliferation to varying degrees. We conclude that human CLC retain the ability to process and present protein antigens potently to CD4+ T cells. Thus, CLC have the capacity to participate actively in the generation and maintenance of T-helper cell immunity to viral antigens during HIV-1 infection. PMID:8698396

  11. Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

    NASA Astrophysics Data System (ADS)

    Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

    2016-03-01

    We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

  12. Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

    PubMed Central

    Roy, S; Das, A B; Ghosh, A N; Biswas, T

    1994-01-01

    The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study. Images PMID:7927692

  13. Cattle response to foot-and-mouth disease virus nonstructural proteins as antigens within vaccines produced using different concentrations.

    PubMed

    Lubroth, J; López, A; Ramalho, A K; Meyer, R F; Brown, F; Darsie, G C

    1998-05-01

    Abstract Four groups of ten nine-month-old Nelore heifers were used for this study. Each group received one of four foot-and-mouth disease (FMD) trivalent vaccines for the duration of the experiment. The four vaccine formulations (Normal, 2X, 4X and 8X) differed in 140S content to determine the serological reactivities to FMD virus (FMDV) nonstructural proteins 2C, 3ABC and 3D. Vaccination was by the intramuscular administration of vaccine on day 0, 180 and 360. Bleedings were done at 30 days post vaccination (dpv), 90 dpv, 30 days post revaccination (dpr), 90 dpr, and 30 days post third administration (dprr). There was a general tendency to have higher mean 3D responses with increased vaccine application but not with increased concentration of antigen. With 2C and 3ABC this tendency was not seen, neither with repeated application of vaccine nor with increased antigen concentration. All individual animal observations to 2C and 3ABC remained within three standard deviations of the average observed for naive bovids. Percent of positive (PP) reactions was determined using an ELISA for nonstructural proteins 2C, 3ABC and 3D expressed in baculovirus as previously described. A value of >25 PP to 2C or 3ABC could be considered as an indication of previous infection or of the presence of viral activity. PP results between 18 and 25 PP suggest viral activity and animals should be retested. Those responses below 15 PP are suggestive of vaccination or naive status. As diagnosis in the laboratory is not divorced from the field epidemiological scene, the intermediate zone between 10 and 20 PP should be considered and acted upon according to the overall zoosanitary situation of that country or region and the purposes of the ongoing FMD control efforts. PMID:22077290

  14. SEPPA 2.0--more refined server to predict spatial epitope considering species of immune host and subcellular localization of protein antigen.

    PubMed

    Qi, Tao; Qiu, Tianyi; Zhang, Qingchen; Tang, Kailin; Fan, Yangyang; Qiu, Jingxuan; Wu, Dingfeng; Zhang, Wei; Chen, Yanan; Gao, Jun; Zhu, Ruixin; Cao, Zhiwei

    2014-07-01

    Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported.

  15. Vesicular Stomatitis Virus Matrix Protein Impairs CD1d-Mediated Antigen Presentation through Activation of the p38 MAPK Pathway▿

    PubMed Central

    Renukaradhya, Gourapura J.; Khan, Masood A.; Shaji, Daniel; Brutkiewicz, Randy R.

    2008-01-01

    Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d+ cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways. PMID:18815300

  16. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins

    PubMed Central

    1993-01-01

    The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins. PMID:8227122

  17. The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen

    PubMed Central

    Hofbauer, Anna; Melnik, Stanislav; Tschofen, Marc; Arcalis, Elsa; Phan, Hoang T.; Gresch, Ulrike; Lampel, Johannes; Conrad, Udo; Stoger, Eva

    2016-01-01

    Zein is a water-insoluble polymer from maize seeds that has been widely used to produce carrier particles for the delivery of therapeutic molecules. We encapsulated a recombinant model vaccine antigen in newly formed zein bodies in planta by generating a fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γ-zein. The chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs) were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control, and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5-zein bodies did not enhance the immune response any further, suggesting that the zein portion itself mediates an adjuvant effect. While the zein portion used to induce protein body formation was only weakly immunogenic, our results indicate that zein-induced PBs are promising production and delivery vehicles for subunit vaccines. PMID:26909090

  18. Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M.

    2014-01-01

    Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-μg and 50-μg doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis. PMID:24914218

  19. Immunogenicity and antigenicity of a recombinant chimeric protein containing epitopes of poliovirus type 1.

    PubMed

    Pan, X-X; Wang, J; Xia, W-Y; Li, X-F; Yang, L-J; Huang, C; Chen, Y-D

    2016-01-01

    To design a vaccine that simultaneously prevents both rotavirus (RV) and poliovirus (PV), a PV type 1 (PV1) chimeric protein using RV VP6 as a vector (VP6F) was constructed, expressed in Escherichia coli expression system and characterized by SDS-PAGE, Western blot, immunofluorescence assay and neutralization test. The results showed that the chimeric protein reacted with anti-VP6F and anti-PV1 antibodies and elicited production of serum antibodies against the chimeric protein in guinea pigs. Antibodies against the chimeric protein neutralized RV Wa and PV1 infection in vitro. The results provided a relevant possibility of developing novel approaches in the rational design of vaccines effective against both RV and PV. PMID:27640433

  20. Adult Schistosoma mansoni worms positively modulate soluble egg antigen-induced inflammatory hepatic granuloma formation in vivo. Stereological analysis and immunophenotyping of extracellular matrix proteins, adhesion molecules, and chemokines.

    PubMed Central

    Jacobs, W.; Bogers, J.; Deelder, A.; Wéry, M.; Van Marck, E.

    1997-01-01

    Synchronized liver granulomas were induced by injecting Sepharose beads to which SEA soluble egg antigen (SEA) or the concanavalin A binding fraction of SEA had been coupled into a mesenteric vein in naive, single-sex (35 days) and bisexually (28 days) Schistosoma mansoni-infected and Plasmodium berghei-immunized mice. Stereological analysis revealed that peak granuloma formation was already reached 8 days after injection in single-sex infected mice compared with 16 days in naive animals. No difference in granuloma formation between naive and P. berghei-immunized animals and between unisexually and bisexually S. mansoni-infected mice was observed. This suggests that the positive immunomodulatory effect on the granulomogenesis is worm specific and not likely to be due to arousal of the immune system by unrelated factors, nor is it influenced by the gender or degree of maturation of female worms. At all stages in time, the concanavalin A binding-fraction-induced granulomas reached only 65 to 70% of the volume of SEA-induced granulomas. Immunophenotyping of extracellular matrix proteins around deposited heads revealed that fibronectin was the dominant extracellular matrix protein and that also type I and IV collagen and laminin were deposited. Temporal analysis of the expression of the adhesion molecules ICAM-1, LFA-1, VLA-4, and VLA-6 was performed. Morphological evidence is presented for the role of adhesion molecules in the initiation and maintenance of hepatic granuloma formation. The chemokine monocyte chemoattractant protein-1 was expressed in the granuloma and in hepatic artery branches. From these data, it is concluded that adult S. mansoni worms positively modulate schistosomal hepatic granuloma formation in vivo. Adhesion molecules and chemokines play important roles in schistosomal granuloma formation. Images Figure 1 Figure 2 Figure 3 PMID:9176396

  1. Scrapie and Creutzfeldt-Jakob disease prion proteins share physical properties and antigenic determinants.

    PubMed Central

    Bendheim, P E; Bockman, J M; McKinley, M P; Kingsbury, D T; Prusiner, S B

    1985-01-01

    Scrapie of sheep and goats as well as Creutzfeldt-Jakob disease (CJD) of humans are neurologic disorders caused by slow infectious pathogens. The novel molecular properties of the pathogen causing scrapie have prompted introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by nucleic acid-modifying procedures. Antiserum to the major hamster scrapie prion protein (PrP 27-30) was found to cross-react with murine CJD proteins. The CJD proteins had molecular weights similar to those observed for scrapie prion proteins as determined by NaDodSO4 gel electrophoresis. In addition, the CJD proteins were resistant to digestion by proteinase K and appear to polymerize into rod-shaped particles. The purification procedure developed for scrapie prions was found to be useful in purifying the CJD agent. Purification of the two infectious pathogens by virtually identical procedures provided further evidence for similarities in their molecular structures. We conclude that the molecular and biologic properties of the CJD agent are sufficiently similar to those of the scrapie prion protein that CJD should be classified as a prion disease. Images PMID:2579394

  2. Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

    PubMed Central

    Mungre, S; Enderle, K; Turk, B; Porrás, A; Wu, Y Q; Mumby, M C; Rundell, K

    1994-01-01

    Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45. Images PMID:8107228

  3. Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

    PubMed

    Kim, Jong-Hyun; Yang, Ae-Hee; Sohn, Hae-Jin; Kim, Daesik; Song, Kyoung-Ju; Shin, Ho-Joon

    2009-11-01

    Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

  4. Synthesis of human parainfluenza virus 2 nucleocapsid protein in yeast as nucleocapsid-like particles and investigation of its antigenic structure.

    PubMed

    Bulavaitė, Aistė; Lasickienė, Rita; Vaitiekaitė, Aušra; Sasnauskas, Kęstutis; Žvirblienė, Aurelija

    2016-05-01

    The aim of this study was to investigate the suitability of yeast Saccharomyces cerevisiae expression system for the production of human parainfluenza virus type 2 (HPIV2) nucleocapsid (N) protein in the form of nucleocapsid-like particles (NLPs) and to characterize its antigenic structure. The gene encoding HPIV2 N amino acid (aa) sequence RefSeq NP_598401.1 was cloned into the galactose-inducible S. cerevisiae expression vector and its high-level expression was achieved. However, this recombinant HPIV2 N protein did not form NLPs. The PCR mutagenesis was carried out to change the encoded aa residues to the ones conserved across HPIV2 isolates. Synthesis of the modified proteins in yeast demonstrated that the single aa substitution NP_598401.1:p.D331V was sufficient for the self-assembly of NLPs. The significance of certain aa residues in this position was confirmed by analysing HPIV2 N protein structure models. To characterize the antigenic structure of NLP-forming HPIV2 N protein, a panel of monoclonal antibodies (MAbs) was generated. The majority of the MAbs raised against the recombinant NLPs recognized HPIV2-infected cells suggesting the antigenic similarity between the recombinant and virus-derived HPIV2 N protein. Fine epitope mapping revealed the C-terminal part (aa 386-504) as the main antigenic region of the HPIV2 N protein. In conclusion, the current study provides new data on the impact of HPIV2 N protein sequence variants on the NLP self-assembly and demonstrates an efficient production of recombinant HPIV2 N protein in the form of NLPs. PMID:26821928

  5. Analysis of automatically generated peptide mass fingerprints of cellular proteins and antigens from Helicobacter pylori 26695 separated by two-dimensional electrophoresis.

    PubMed

    Krah, Alexander; Schmidt, Frank; Becher, Dörte; Schmid, Monika; Albrecht, Dirk; Rack, Axel; Büttner, Knut; Jungblut, Peter R

    2003-12-01

    Helicobacter pylori is a causative agent of severe diseases of the gastric tract ranging from chronic gastritis to gastric cancer. Cellular proteins of H. pylori were separated by high resolution two-dimensional gel electrophoresis. A dataset of 384 spots was automatically picked, digested, spotted, and analyzed by matrix-assisted laser desorption ionization mass spectrometry peptide mass fingerprint in triple replicates. This procedure resulted in 960 evaluable mass spectra. Using a new version of our data analysis software MS-Screener we improved identification and tested reliability of automatically generated data by comparing with manually produced data. Antigenic proteins from H. pylori are candidates for vaccines and diagnostic tests. Previous immunoproteomics studies of our group revealed antigen candidates, and 24 of them were now closely analyzed using the MS-Screener software. Only in three spots minor components were found that may have influenced their antigenicities. These findings affirm the value of immunoproteomics as a hypothesis-free approach. Additionally, the protein species distribution of the known antigen GroEL was investigated, dimers of the protein alkyl hydroperoxide reductase were found, and the fragmentation of gamma-glutamyltranspeptidase was demonstrated.

  6. The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...

  7. Inverse relation between prostate-specific antigen and insulin-like growth factor-binding protein 3 in bone metastases and serum of patients with prostate cancer.

    PubMed

    Smith, G L; Doherty, A P; Mitchell, H; Hanham, I W; Christmas, T J; Epstein, R J

    1999-12-11

    The usual osteoblastic phenotype of metastatic prostate cancer is unexplained. Here we show that tissue and serum concentrations of prostate-specific antigen (PSA)-vary inversely with a substrate protein that binds a growth factor known to activate osteoblasts. These findings suggest that PSA may contribute to the osteoblastic phenotype, and could thus represent a new drug target devoid of antiandrogenic toxicity.

  8. Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

    PubMed Central

    Evans, C M; Fryer, A D; Jacoby, D B; Gleich, G J; Costello, R W

    1997-01-01

    In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs. PMID:9410903

  9. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

    PubMed

    Chang, Hsin-Hou; Shyu, Huey-Fen; Wang, Yo-Ming; Sun, Der-Shan; Shyu, Rong-Hwa; Tang, Shiao-Shek; Huang, Yao-Shine

    2002-09-15

    Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

  10. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

    PubMed Central

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  11. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    PubMed

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-11-13

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

  12. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    PubMed

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  13. Identification of Leishmania proteins preferentially released in infected cells using change mediated antigen technology (CMAT).

    PubMed

    Kima, Peter E; Bonilla, J Alfredo; Cho, Eumin; Ndjamen, Blaise; Canton, Johnathan; Leal, Nicole; Handfield, Martin

    2010-01-01

    Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.

  14. Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

    PubMed Central

    Kima, Peter E.; Bonilla, J. Alfredo; Cho, Eumin; Ndjamen, Blaise; Canton, Johnathan; Leal, Nicole; Handfield, Martin

    2010-01-01

    Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus. PMID:20957202

  15. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  16. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  17. Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Celiac disease is an immune-mediated enteropathy that is generally understood to be triggered by the ingestion of gluten proteins of wheat and related cereals. The skin manifestation of the condition is known as dermatitis herpetiformis. Antibody response to native and deamidated seque...

  18. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of...

  19. An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses

    PubMed Central

    AMERI, M.; WANG, X.; WILKERSON, M. J.; KANOST, M. R.; BROCE, A. B.

    2008-01-01

    The stable fly, Stomoxys calcitrans, is an economically important pest of livestock. Prior studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27 kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine if this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and if naïve calves can mount an immune response to it. Calves raised in the winter months were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 DPI in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab’)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive, but has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves. PMID:18283948

  20. Interspecies antigenic determinants of structural proteins of mammalian type-C viruses as detected by a competitive sepharose bead immunofluorescence assay.

    PubMed

    Micheel, B; Fiebach, H; Wunderlich, V

    1979-01-01

    The present paper describes a competitive immunoassay using antiserum to FeLV p 27 and SSV-conjugated Sepharose beads. The assay is applied to compare the interspecies-specific antigenic determinants of the major structural proteins of type-C viruses of different mammalian species. The test proves to be highly sensitive and specific and may be used for the demonstration of viral proteins in crude cellular extracts.

  1. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  2. Azurin-like protein blocks invasion of Toxoplasma gondii through potential interactions with parasite surface antigen SAG1.

    PubMed

    Naguleswaran, Arunasalam; Fialho, Arsenio M; Chaudhari, Anita; Hong, Chang Soo; Chakrabarty, Ananda M; Sullivan, William J

    2008-02-01

    Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents. PMID:18070964

  3. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

    PubMed Central

    Chu, D H; Spits, H; Peyron, J F; Rowley, R B; Bolen, J B; Weiss, A

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling. Images PMID:8947048

  4. The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

    PubMed

    Floridia-Yapur, N; Monje Rumi, M; Ragone, P; Lauthier, J J; Tomasini, N; Alberti D'Amato, A; Diosque, P; Cimino, R; Marco, J D; Barroso, P; Sanchez, D O; Nasser, J R; Tekiel, V

    2016-09-01

    In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans. PMID:27173912

  5. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    NASA Astrophysics Data System (ADS)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  6. The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

    PubMed

    Floridia-Yapur, N; Monje Rumi, M; Ragone, P; Lauthier, J J; Tomasini, N; Alberti D'Amato, A; Diosque, P; Cimino, R; Marco, J D; Barroso, P; Sanchez, D O; Nasser, J R; Tekiel, V

    2016-09-01

    In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

  7. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.

    PubMed

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway. PMID:26752992

  8. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens

    PubMed Central

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway. PMID:26752992

  9. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

    PubMed Central

    Barber, E K; Dasgupta, J D; Schlossman, S F; Trevillyan, J M; Rudd, C E

    1989-01-01

    Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex. Images PMID:2470098

  10. Discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens

    PubMed Central

    Ramakrishnan, Kamna; Flower, Darren R

    2010-01-01

    It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome. PMID:20975907

  11. Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages

    PubMed Central

    Hua, Chun-Zhen; Hu, Wei-Lin; Li, Jian-Ping; Hong, Li-Quan

    2015-01-01

    Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae. PMID:26677200

  12. Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2.

    PubMed

    Mason, Lyndel; Tribolet, Leon; Simon, Anne; von Gnielinski, Natascha; Nienaber, Lisa; Taylor, Paul; Willis, Charlene; Jones, Malcolm K; Sternberg, Paul W; Gasser, Robin B; Loukas, Alex; Hofmann, Andreas

    2014-05-01

    Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.

  13. [Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

    PubMed

    Liang, Cheng-Zhu; Cao, Rui-Bing; Wei, Jian-Chao; Zhu, Lai-Hua; Chen, Pu-Yan

    2006-06-01

    According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

  14. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  15. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-09-27

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. PMID:7524079

  16. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed Central

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-01-01

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. Images PMID:7524079

  17. A Rapid Diagnostic Test for Toxoplasmosis using Recombinant Antigenic N-terminal Half of SAG1 Linked with Intrinsically Unstructured Domain of GRA2 Protein

    PubMed Central

    Song, Kyoung Ju; Yang, Zhaoshou; Chong, Chom-Kyu; Kim, Jin-Soo; Lee, Kyung Chan; Kim, Tong-Soo

    2013-01-01

    Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis. PMID:24327774

  18. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli.

    PubMed Central

    Spindler, K R; Rosser, D S; Berk, A J

    1984-01-01

    Plasmid vectors were constructed which expressed three adenovirus tumor antigens fused to a portion of the trpE protein of Escherichia coli. Insertion of adenovirus type 2 DNA from early region 1A (E1A) into such a plasmid led to a fusion protein which contained the C-terminal 266 amino acids of the 289-amino acid protein encoded by the viral 13S mRNA. Similarly, insertion of adenovirus type 5 DNA corresponding to the E1B 55- and 21-kilodalton proteins led to production of fusion proteins containing amino acid sequences from these proteins. After induction with indoleacrylic acid, fusion proteins accumulated stably in the E. coli cells. By using a simple extraction of insoluble protein, 1 to 10 mg of fusion protein per liter of culture was obtained. The fusion proteins were purified on preparative polyacrylamide gels and used to immunize rabbits. Specific antisera for the E1A 289- and closely related 243-amino acid proteins and the E1B 55- and 21-kilodalton proteins were obtained. These sera were used to immunoprecipitate the tumor antigens in cells infected with wild-type and various mutants of adenovirus or to analyze them by an immunoblotting procedure. Mutant E1A proteins in which the C-terminal 70 amino acids are deleted were phosphorylated to much lower extents than the wild-type E1A proteins. This indicates that the deleted region is important for the process of phosphorylation. The E1A proteins were extracted, sedimented in glycerol gradients, analyzed by immunoprecipitation, and found to sediment primarily as monomers. Images PMID:6361277

  19. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  20. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  1. Serological diagnosis of pneumococcal infection in children with pneumonia using protein antigens: A study of cut-offs with positive and negative controls.

    PubMed

    Andrade, Dafne Carvalho; Borges, Igor Carmo; Ivaska, Lauri; Peltola, Ville; Meinke, Andreas; Barral, Aldina; Käyhty, Helena; Ruuskanen, Olli; Nascimento-Carvalho, Cristiana Maria

    2016-06-01

    The etiological diagnosis of infection by Streptococcus pneumoniae in children is difficult, and the use of indirect techniques is frequently warranted. We aimed to study the use of pneumococcal proteins for the serological diagnosis of pneumococcal infection in children with pneumonia. We analyzed paired serum samples from 13 Brazilian children with invasive pneumococcal pneumonia (positive control group) and 23 Finnish children with viral pharyngitis (negative control group), all aged <5years-old. Children with pharyngitis were evaluated for oropharyngeal colonization, and none of them carried S. pneumoniae. We used a multiplex bead-based assay with eight proteins: Ply, CbpA, PspA1 and 2, PcpA, PhtD, StkP and PcsB. The optimal cut-off for increase in antibody level for the diagnosis of pneumococcal infection was determined for each antigen by ROC curve analysis. The positive control group had a significantly higher rate of ≥2-fold rise in antibody levels against all pneumococcal proteins, except Ply, compared to the negative controls. The cut-off of ≥2-fold increase in antibody levels was accurate for pneumococcal infection diagnosis for all investigated antigens. However, there was a substantial increase in the accuracy of the test with a cut-off of ≥1.52-fold rise in antibody levels for PcpA. When using the investigated protein antigens for the diagnosis of pneumococcal infection, the detection of response against at least one antigen was highly sensitive (92.31%) and specific (91.30%). The use of serology with pneumococcal proteins is a promising method for the diagnosis of pneumococcal infection in children with pneumonia. The use of a ≥2-fold increase cut-off is adequate for most pneumococcal proteins. PMID:26928648

  2. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  3. A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

    PubMed Central

    Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

    2014-01-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

  4. Utilization of protein-A in immuno-histochemical techniques for detection of Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats.

    PubMed

    Abu Elzein, E M E; Al-Naeem, A

    2009-01-01

    This paper constitutes the first record of utilizing the S. aureus protein-A (PA), conjugated to peroxidase enzyme, for the detection of the Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats. The goats were experimentally infected with a virulent PPR virus, which was previously isolated from a severe natural disease outbreak in gazelles, during 2002 in Saudi Arabia. The technique is rapid, and has the superiority over the peroxidase -anti-peroxidase (PAP) test in that, inactivation of the indigenous peroxidase in the tissues is not required and that it can be used against a wide range of animal species. An advantage over the other immunolabelled conjugates is that PA attaches specifically to the crystalizable fraction (Fc) of the IgG molecule, thus allowing the antigen binding fraction (Fab) of the molecule, free to interact specifically with the antigen. So, it doesn't actually compete with the antigen for the Fab portion of the IgG molecule. In the present study, PA conjugate detected the PPR virus antigens in various tissues of the experimentally infected goats. PMID:19052895

  5. Utilization of protein-A in immuno-histochemical techniques for detection of Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats.

    PubMed

    Abu Elzein, E M E; Al-Naeem, A

    2009-01-01

    This paper constitutes the first record of utilizing the S. aureus protein-A (PA), conjugated to peroxidase enzyme, for the detection of the Peste des Petits Ruminants (PPR) virus antigens in tissues of experimentally infected goats. The goats were experimentally infected with a virulent PPR virus, which was previously isolated from a severe natural disease outbreak in gazelles, during 2002 in Saudi Arabia. The technique is rapid, and has the superiority over the peroxidase -anti-peroxidase (PAP) test in that, inactivation of the indigenous peroxidase in the tissues is not required and that it can be used against a wide range of animal species. An advantage over the other immunolabelled conjugates is that PA attaches specifically to the crystalizable fraction (Fc) of the IgG molecule, thus allowing the antigen binding fraction (Fab) of the molecule, free to interact specifically with the antigen. So, it doesn't actually compete with the antigen for the Fab portion of the IgG molecule. In the present study, PA conjugate detected the PPR virus antigens in various tissues of the experimentally infected goats.

  6. Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

    PubMed Central

    Boire, G; Gendron, M; Monast, N; Bastin, B; Ménard, H A

    1995-01-01

    Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7774061

  7. Surface-exposed antigenic cleavage fragments of Neisseria gonorrhoeae proteins 1A and IB.

    PubMed Central

    Schmitt, S; Layh, G; Buchanan, T B

    1986-01-01

    Whole bacteria, isolated outer membranes, and purified protein I (PI) from one transparent (O-) and two different opaque (O+) phenotype gonococcal strains (serogroups I, II, and III; PI serotypes 1, 5, and 9b) were each treated with tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin, alpha-chymotrypsin, and proteinase K. Protein IA (PIA) of strain 7122 (O-, serotype 1, serogroup I) was resistant to proteolysis by tolysulfonyl phenylalanyl chloromethyl ketone-trypsin and alpha-chymotrypsin and only slightly affected by proteinase K, as long as it was associated with intact bacteria or isolated outer membranes. Purified PIA however was cleaved by these enzymes, resulting in two to five fragments. In contrast, all preparations of strains 5766 opaque phenotype (O+, serotype 7, serogroup II) and 1955 (O+, serotype 9b, serogroup III) were accessible to proteolysis, resulting in cleavage fragments of PIB compatible to those described previously by O. Barrera and J. Swanson (Infect. Immun. 44:565-568, 1984), M. S. Blake et al. (Infect. Immun. 33:212-222, 1981), and Blake (in G. K. Schoolnik, ed., The Pathogenic Neisseriae, 1985). Our data indicated that the purified PIB fraction was more accessible to proteases than the PIBs of whole bacteria or outer membranes. The fragmentation pattern of PIA cleavage products were quite different from PIB fragments, consistent with the different structure of these two groups of PI molecules. Time-dependent cleavage experiments with proteases, i.e., alpha-chymotrypsin, indicated that PIA was subsequently cleaved into smaller fragments. Highly reactive monoclonal antibodies, each specific for a surface-exposed epitope of PIA of strain 7122 or PIB of strains 5766 and 1955, as assessed by coagglutination, Western blot, and immunofluorescence, were reacted with PIA and PIB cleavage fragments in Western blot experiments. All cleavage fragments of the purified PIA and PIB preparations with molecular weights of greater than or equal to 14

  8. Structural and Biochemical Insights into the Regulation of Protein Phosphatase 2A by Small t Antigen of SV40

    SciTech Connect

    Chen,Y.; Xu, Y.; Bao, Q.; Xing, Y.; Li, Z.; Lin, Z.; Stock, J.; Jeffrey, P.; Shi, Y.

    2007-01-01

    The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.

  9. Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators

    PubMed Central

    Schnabel, Renate B.; Baumert, Jens; Barbalic, Maja; Dupuis, Josée; Ellinor, Patrick T.; Durda, Peter; Dehghan, Abbas; Bis, Joshua C.; Illig, Thomas; Morrison, Alanna C.; Jenny, Nancy S.; Keaney, John F.; Gieger, Christian; Tilley, Cathy; Yamamoto, Jennifer F.; Khuseyinova, Natalie; Heiss, Gerardo; Doyle, Margaret; Blankenberg, Stefan; Herder, Christian; Walston, Jeremy D.; Zhu, Yanyan; Vasan, Ramachandran S.; Klopp, Norman; Boerwinkle, Eric; Larson, Martin G.; Psaty, Bruce M.; Peters, Annette; Ballantyne, Christie M.; Witteman, Jacqueline C. M.

    2010-01-01

    To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10−323). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10−13). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications. PMID:20040767

  10. Melanoma antigen-D2: A nucleolar protein undergoing delocalization during cell cycle and after cellular stress.

    PubMed

    Pirlot, Céline; Thiry, Marc; Trussart, Charlotte; Di Valentin, Emmanuel; Piette, Jacques; Habraken, Yvette

    2016-04-01

    Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203-254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248-254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation. PMID:26705694

  11. New PHA products using unrelated carbon sources

    PubMed Central

    Matias, Fernanda; de Andrade Rodrigues, Maria Filomena

    2011-01-01

    Polyhydroxyalkanoates (PHA) are natural polyesters stored by a wide range of bacteria as carbon source reserve. Due to its chemical characteristics and biodegradability PHA can be used in chemical, medical and pharmaceutical industry for many human purposes. Over the past years, few Burkholderia species have become known for production of PHA. Aside from that, these bacteria seem to be interesting for discovering new PHA compositions which is important to different industrial applications. In this paper, we introduce two new strains which belong either to Burkholderia cepacia complex (Bcc) or genomovar-type, Burkholderia cepacia SA3J and Burkholderia contaminans I29B, both PHA producers from unrelated carbon sources. The classification was based on 16S rDNA and recA partial sequence genes and cell wall fatty acids composition. These two strains were capable to produce different types of PHA monomers or precursors. Unrelated carbon sources were used for growth and PHA accumulation. The amount of carbon source evaluated, or mixtures of them, was increased with every new experiment until it reaches eighteen carbon sources. As first bioprospection experiments staining methods were used with colony fluorescent dye Nile Red and the cell fluorescent dye Nile Blue A. Gas chromatography analysis coupled to mass spectrometry was used to evaluate the PHA composition on each strain cultivated on different carbon sources. The synthesized polymers were composed by short chain length-PHA (scl-PHA), especially polyhydroxybutyrate, and medium chain length-PHA (mcl-PHA) depending on the carbon source used. PMID:24031764

  12. Antigenic Analysis of Monoclonal Antibodies against Different Epitopes of σB Protein of Avian Reovirus

    PubMed Central

    Yin, Chun-hong; Qin, Li-ting; Sun, Mei-yu; Gao, Yu-long; Qi, Xiao-le; Gao, Hong-lei; Wang, Yong-qiang; Wang, Xiao-mei

    2013-01-01

    Background Avian reovirus (ARV) causes arthritis, tenosynovitis, runting-stunting syndrome (RSS), malabsorption syndrome (MAS) and immunosuppression in chickens. σB is one of the major structural proteins of ARV, which is able to induce group-specific antibodies against the virus. Methods and Results The present study described the identification of two linear B-cell epitopes in ARV σB through expressing a set of partially overlapping and consecutive truncated peptides spanning σB screened with two monoclonal antibodies (mAbs) 1F4 and 1H3-1.The data indicated that 21KTPACW26 (epitope A) and 32WDTVTFH38 (epitope B) were minimal determinants of the linear B cell epitopes. Antibodies present in the serum of ARV-positive chickens recognized the minimal linear epitopes in Western blot analyses. By sequence alignment analysis, we determined that the epitopes A and B were not conserved among ARV, duck reovirus (DRV) and turkey reovirus (TRV) strains. Western blot assays, confirmed that epitopes A and B were ARV-specific epitopes, and they could not react with the corresponding peptides of DRV and TRV. Conclusions and Significance We identified 21KTPACW26 and 32WDTVTFH38 as σB -specific epitopes recognized by mAbs 1F4 and 1H3-1, respectively. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against ARV groups. PMID:24312314

  13. Evaluation of different heterologous prime-boost immunization strategies against Babesia bovis using viral vectored and protein-adjuvant vaccines based on a chimeric multi-antigen.

    PubMed

    Jaramillo Ortiz, José Manuel; Molinari, María Paula; Gravisaco, María José; Paoletta, Martina Soledad; Montenegro, Valeria Noely; Wilkowsky, Silvina Elizabeth

    2016-07-19

    Protection against the intraerythrocytic bovine parasite Babesia bovis requires both humoral and cellular immune responses. Therefore, tailored combinations of immunogens targeted at both arms of the immune system are strategies of choice to pursue sterilizing immunity. In this study, different heterologous prime-boost vaccination schemes were evaluated in mice to compare the immunogenicity induced by a recombinant adenovirus, a modified vaccinia Ankara vector or a subunit vaccine all expressing a chimeric multi-antigen. This multi-antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: Merozoite Surface Antigen - 2c (MSA-2c), Rhoptry Associated Protein - 1 (RAP-1) and Heat Shock Protein 20 (HSP20). Both priming with the adenovirus or recombinant multi-antigen and boosting with the modified vaccinia Ankara vector achieved a high degree of activation of TNFα and IFNγ-secreting CD4(+) and CD8(+) specific T cells 60days after the first immunization. High titers of specific IgG antibodies were also detected at the same time point and lasted up to day 120 of the first immunization. Only the adenovirus - MVA combination triggered a marked isotype skew for the IgG2a antibody subclass meanwhile for the other immune traits analyzed here, both vaccination schemes showed similar performances. The immunological characterization in the murine model of these rationally designed immunogens led us to propose that adenoviruses as well as the bacterially expressed multi-antigen are highly reliable primer candidates to be considered in future experiments in cattle to test protection against bovine babesiosis. PMID:27269058

  14. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System.

    PubMed

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo.

  15. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System.

    PubMed

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

  16. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System

    PubMed Central

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

  17. The preparation of specific immune sera against type 3 poliovirus D-antigen and C-antigen and the demonstration of two C-antigenic components in vaccine strain populations.

    PubMed

    Minor, P D; Schild, G C; Wood, J M; Dandawate, C N

    1980-11-01

    Animals were immunized with purified D-antigen or C-antigen of type 3 poliovirus to produce specific antisera which were used to analyse the antigenic characteristics of the progeny virus in harvests from poliovirus type 3-infected cells. An examination of the virus progeny present at 24 h p.i. of cells with Sabin type 3 vaccine strain virus revealed a large population of particles sedimenting at a slightly lower rate (130S) than infectious virus (155S) in addition to slowly sedimenting (80S) empty capsids. Such 130S particles were not detected in the progeny from cells infected with strains genetically unrelated to the Sabin vaccine strains. They were non-infectious, contained RNA in an RNase-resistant form unless heated, and lacked the virion protein VP4. They expressed C-antigen rather than the D-antigen of infectious virus, and, therefore, had the properties previously described for poliovirus particles eluted from cells. The amount of incorporation of radio-isotope into the proteins or nucleic acids of such particles varied from 15 to 20% to 300% of the amount incorporated into infectious virus depending on the cells and virus strains studied. Virus strains genetically related to Sabin type 3 vaccine virus which were isolated from cases of paralytic poliomyelitis produced the particles in either low or undetectable quantities.

  18. Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid.

    PubMed

    Di Genova, Gianfranco; Savelyeva, Natalia; Suchacki, Amy; Thirdborough, Stephen M; Stevenson, Freda K

    2010-04-01

    Antigen-specific CD4(+) T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4(+) T cells against TT in vivo would influence injected CD4(+) TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4(+) memory T-cell response and CD4(+) T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.

  19. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine.

    PubMed

    Nawaz-Tipu, Hamid

    2015-10-01

    The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1) protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA) class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine's candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Three alleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens more prevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of 50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  20. [Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

    PubMed

    Yu, Xing-Long; Tu, Chang-Chun; Xu, Xing-Ran; Zhang, Mao-Lin; Chen, Yi-Xiang; Liu, Bo-Hua

    2003-07-01

    Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The

  1. Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments.

    PubMed Central

    Karvelas, M; Nossal, G J

    1992-01-01

    rendered tolerant by this manipulation. The results argue for a major T-cell component in the process whereby soluble protein antigens ablate affinity maturation and memory cell generation. PMID:1348366

  2. Peripheral benzodiazepine receptor (PBR) ligand cytotoxicity unrelated to PBR expression.

    PubMed

    Hans, Gregory; Wislet-Gendebien, Sabine; Lallemend, François; Robe, Pierre; Rogister, Bernard; Belachew, Shibeshih; Nguyen, Laurent; Malgrange, Brigitte; Moonen, Gustave; Rigo, Jean-Michel

    2005-03-01

    Some synthetic ligands of the peripheral-type benzodiazepine receptor (PBR), an 18 kDa protein of the outer mitochondrial membrane, are cytotoxic for several tumor cell lines and arise as promising chemotherapeutic candidates. However, conflicting results were reported regarding the actual effect of these drugs on cellular survival ranging from protection to toxicity. Moreover, the concentrations needed to observe such a toxicity were usually high, far above the affinity range for their receptor, hence questioning its specificity. In the present study, we have shown that micromolar concentrations of FGIN-1-27 and Ro 5-4864, two chemically unrelated PBR ligands are toxic for both PBR-expressing SK-N-BE neuroblastoma cells and PBR-deficient Jurkat lymphoma cells. We have thereby demonstrated that the cytotoxicity of these drugs is unrelated to their PBR-binding activity. Moreover, Ro 5-4864-induced cell death differed strikingly between both cell types, being apoptotic in Jurkat cells while necrotic in SK-N-BE cells. Again, this did not seem to be related to PBR expression since Ro 5-4864-induced death of PBR-transfected Jurkat cells remained apoptotic. Taken together, our results show that PBR is unlikely to mediate all the effects of these PBR ligands. They however confirm that some of these ligands are very effective cytotoxic drugs towards various cancer cells, even for reputed chemoresistant tumors such as neuroblastoma, and, surprisingly, also for PBR-lacking tumor cells.

  3. Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection

    PubMed Central

    2014-01-01

    Background Ticks represent a significant health risk to animals and humans due to the variety of pathogens they can transmit during feeding. The traditional use of chemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and environmental contamination with chemical residues. Vaccination with the tick midgut antigen BM86 was shown to be a good alternative for cattle tick control. However, results vary considerably between tick species and geographic location. Therefore, new antigens are required for the development of vaccines controlling both tick infestations and pathogen infection/transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these vaccines, but appropriate screening procedures are needed to select the best candidates. Methods In this study, we selected proteins involved in tick-Anaplasma (Subolesin and SILK) and tick-Babesia (TROSPA) interactions and used in vitro capillary feeding to characterize their potential as antigens for the control of cattle tick infestations and infection with Anaplasma marginale and Babesia bigemina. Purified rabbit polyclonal antibodies were generated against recombinant SUB, SILK and TROSPA and added to uninfected or infected bovine blood to capillary-feed female Rhipicephalus (Boophilus) microplus ticks. Tick weight, oviposition and pathogen DNA levels were determined in treated and control ticks. Results The specificity of purified rabbit polyclonal antibodies against tick recombinant proteins was confirmed by Western blot and against native proteins in tick cell lines and tick tissues using immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect of these antibodies on tick weight and oviposition was shown. However, no effect was observed on pathogen DNA levels. Conclusions These results highlighted the advantages and some of the disadvantages of in vitro tick capillary

  4. Protease-like sequence in hepatitis B virus core antigen is not involved in the cleavage processes of core protein in Escherichia coli.

    PubMed

    Hwang, L H; Lin, Y J; Lai, W C; Lo, M S

    1991-02-01

    A DNA fragment, coding for hepatitis core antigen (HBcAg), was amplified by polymerase chain reaction and inserted into a lambda PL promoter-derived expression vector. The recombinant plasmid was transformed into Escherichia coli and proteins produced after heat induction were analyzed. In addition to the 21 kDa HBcAg protein, several smaller related polypeptides, particularly one of 17 kDa in size, were also detected with rabbit anti-HBcAg antiserum. Whether the protease-like sequence of core protein involved in the self-cleavage process to form the 17 kDa polypeptide was investigated by a deletion experiment. Our results with a mutant in which 7 amino acids of the conserved protease-like region in the core protein have been deleted suggest that the cleavage does not depend on the presence of these protease-like sequence. In addition, the core protein synthesized from in vitro translation reaction was not cleaved. Core particles from E. coli lysate were purified by sucrose and cesium chloride density gradient centrifugations and subsequently treated with 0.2% of SDS and 0.2% of beta-mecaptoethanol. Immunoblotting analysis, however, did not reveal any conversion of the 21 kDa protein to smaller ones. In conclusion, our results suggest that the protease-like domain at the N-terminus of the core protein does not contain intrinsic autocleavage activity, nor could the HBcAg be converted to smaller antigens by detergent treatment. PMID:1935370

  5. [Target antigens for therapeutic antibodies in oncology: many candidates, few successes].

    PubMed

    Ceze, Nicolas; Probst, Alicia; Lecomte, Thierry; Ohresser, Marc; Paintaud, Gilles; Watier, Hervé

    2007-01-01

    Targeted therapies, especially monoclonal antibodies, have reached an increasing importance in oncology. High-throughput techniques have allowed the identification of numerous transcripts, proteins, or non-protein antigens, which have generated the concept of immunome. This epitope library constitutes a huge reservoir of candidate antigens susceptible to become some say the target of an antibody for passive immunotherapy. However, the conception and development of a therapeutic antibody represent a very important investment, both in terms of human power and finance, such that there is a requirement for an early identification of the best candidates among the potential target antigens. Among multiple criteria, the function of the antigen is crucial when it has been identified. A receptor antigen can be targeted by an agonistic or an antagonistic antibody, according to what is sought. When the antigen function is unknown, a therapeutic antibody can be useful, for instance through induction of apoptosis or through accrual of immuno-competent cells, via its Fc portion (complement-dependent cytotoxicity or antibody-dependent cytotoxicity). Other antibody features, unrelated to its function, can also be exploited, such as its internalisation or its translocation in membrane lipid rafts. The expression of the target antigen may also be crucial, in terms of localisation and levels, as is its tumour specificity, which can influence the efficacy and toxicity of the targeting antibody. The multiplicity of the factors to be taken into account and the complexity of the mechanism of action of therapeutic antibodies renders the choice of a target antigen a hazardous bet. Very often, this is only when the clinical efficacy of a targeting antibody is demonstrated that the antigen can be considered as a good target. PMID:17964990

  6. Unrelated business income tax: an update.

    PubMed

    Fama, A J

    1984-02-01

    To meet spiraling costs, tax-exempt hospitals increasingly are operating businesses unrelated to direct patient care. Knowing which activities may be open to challenge by the Internal Revenue Service (IRS) is essential to avoid the unrelated business income (UBI) tax. Three criteria must be met for an activity to be taxable as UBI: It must constitute a trade or business; It must be regularly carried on; and It must be unrelated to the organization's exempt purpose. The Internal Revenue Code and IRS rulings clearly exclude the following areas from UBI taxation: Activities performed by unpaid volunteers (e.g., hospital auxiliaries' fund-raising dinners and bazaars and the operation of thrift stores); Operations conducted for the convenience of the organization's members, students, patients, or employees (e.g., gift shops, cafeterias, coffee shops, parking lots, lounges, vending machines, pharmaceutical sales to inpatients and emergency room outpatients, and research activities for students' benefit; The sale of merchandise that has been received by gift (e.g., flea markets, baked goods sales, book sales, and rummage sales); Investment income such as dividends, interest, annuities, royalties, certain rents, and capital gains from the sale of investment assets; Gifts or contributions made directly to the facility; and Bingo games that are conducted commercially. Areas which may be subject to UBI taxation, or in which there have been controversial or contradictory court rulings, include: Pharmaceutical sales to the public or private physicians' patients; and Laboratory services provided to private physicians for treating their patients. IRS private letter rulings, though not precedential, have excluded from UBI taxation the x-ray income from a hospital's branch facility and rental income from property leased for use as a clinic or medical office building that is substantially related to the hospital's exempt functions. Private letter rulings have subjected to UBI

  7. Antibody production in vitamin A-depleted rats is impaired after immunization with bacterial polysaccharide or protein antigens.

    PubMed

    Pasatiempo, A M; Kinoshita, M; Taylor, C E; Ross, A C

    1990-05-01

    Vitamin A nutritional status has been implicated as important in maintaining the integrity of immune functions. We have determined the effect of vitamin A (retinol) depletion on the ability of young animals to produce antibodies after challenge with various bacterial antigens. Male Lewis rats raised on vitamin A-free or adequate diets were immunized either near 40 days of age, before signs of vitamin A deficiency were apparent, or near 47 days of age when symptoms of deficiency were beginning to be manifest. For rats immunized with polysaccharide antigens from Streptococcus pneumoniae or Neisseria meningitidis, antibody production did not exceed 0-19% of the response of control rats. Vitamin A depletion also severely compromised the response to two T cell-dependent antigens, tetanus toxoid and sheep red blood cells. In striking contrast, retinol-depleted rats immunized with lipopolysaccharides from Pseudomonas aeruginosa and Serratia marcesens produced an antibody response indistinguishable from retinol-sufficient animals. These lipopolysaccharides could elicit antibodies in rat pups, whereas the capsular polysaccharide antigens could not. This is consistent with the characteristics of type 1 and type 2 antigens, respectively. These studies indicate that retinol status is an important determinant of the humoral immune response to certain types of antigen and suggest that antibody production to capsular polysaccharides and T cell-dependent antigens is particularly dependent on adequate retinol status.

  8. Expression, immunolocalization, and serological reactivity of a novel sphingomyelin phosphodiesterase-like protein, an excretory/secretory antigen from Clonorchis sinensis.

    PubMed

    Huang, Yanwei; Zheng, Youwei; Li, Yuzhe; Yang, Mei; Li, Ting; Zeng, Suxiang; Yu, Xinbing; Huang, Huaiqiu; Hu, Xuchu

    2013-06-01

    Clonorchiasis, caused by Clonorchis sinensis infection, is a zoonotic parasitic disease of hepatobiliary system in which the proteins released by adult are major pathogenetic factors. In this study, we first characterized a putative sphingomyelin phosphodiesterase (CsSMPase) A-like secretory protein, which was highly expressed in the adult worm. The full-length gene was cloned. The putative protein is of relatively low homology comparing with SMPase from other species, and of rich T cell and B cell epitopes, suggesting that it is an antigen of strong antigenicity. The complete coding sequence of the gene was expressed in the Escherichia coli. The recombinant CsSMPase (rCsSMPase) can be recognized by C. sinensis-infected serum, and the protein immunoserum can recognize a specific band in excretory/secretory products (ESPs) of C. sinensis adult by western blotting. Immunolocalization revealed that CsSMPase was not only localized on tegument, ventral sucker of metacercaria, and the intestine of adult but also on the nearby epithelium of bile duct of the infected Sprague-Dawley rats, implying that CsSMPase was mainly secreted and excreted through adult intestine and directly interacted with bile duct epithelium. Although immunized rats evoked high level antibody response, the antigen level was low in clonorchiasis patients. And the sensitivity and specificity of rCsSMPase were 50.0 % (12/24) and 88.4 % (61/69), in sera IgG-ELISA, respectively. It is likely due to the fact that CsSMPase binding to the plasma membrane of biliary epithelium decreases the antigen immune stimulation.

  9. Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

    PubMed

    Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

    2014-04-01

    The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. PMID:24593905

  10. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  11. Effect of Chemical Modification and Fragmentation on Antigenic Determinants of Internal Protein p30 and Surface Glycoprotein gp70 of Type C Retroviruses

    PubMed Central

    Versteegen, Rosemary J.; Oroszlan, Stephen

    1980-01-01

    The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of α and ε amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH2-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27. Images PMID:6154154

  12. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis

    PubMed Central

    Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B.; Volkin, David B.; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

    2013-01-01

    A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink™, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0–8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine. PMID:23880663

  13. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

    PubMed Central

    Vodkin, M H; Williams, J C

    1988-01-01

    A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen. Images PMID:3343219

  14. Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen

    PubMed Central

    Malito, Enrico; Faleri, Agnese; Lo Surdo, Paola; Veggi, Daniele; Maruggi, Giulietta; Grassi, Eva; Cartocci, Elena; Bertoldi, Isabella; Genovese, Alessia; Santini, Laura; Romagnoli, Giacomo; Borgogni, Erica; Brier, Sébastien; Lo Passo, Carla; Domina, Maria; Castellino, Flora; Felici, Franco; van der Veen, Stijn; Johnson, Steven; Lea, Susan M.; Tang, Christoph M.; Pizza, Mariagrazia; Savino, Silvana; Norais, Nathalie; Rappuoli, Rino; Bottomley, Matthew J.; Masignani, Vega

    2013-01-01

    Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen–antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å2 on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen–antibody interfaces are required to understand and design effective immunogens. PMID:23396847

  15. Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen

    PubMed Central

    Hu, Liya; Crawford, Sue E.; Czako, Rita; Cortes-Penfield, Nicolas W; Smith, David F.; Le Pendu, Jacques; Estes, Mary K.; Venkataram Prasad, B. V.

    2012-01-01

    As with many other viruses, the initial cell attachment of rotaviruses, major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans1–4. The distally located VP8* domain of the rotavirus spike protein VP45 mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus (HR) strains bind to glycans with internal Sia such as GM13. Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies1,3,6,7, it is not yet known how VP8* of HRs interacts with Sia and whether their cell attachment necessarily involves sialoglycans. We found that VP8* of a HR strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-Atype antibodies as well as significantly enhanced in CHO cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of HR. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelial and on red blood cells8, and are recognized as susceptibility and cell attachment factors for gastric pathogens like H. pylori9 and noroviruses10. Our crystallographic studies show that the A-type HBGA binds to the HR VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific HR strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population. PMID:22504179

  16. Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance

    PubMed Central

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C.; Steinman, Ralph M.

    2002-01-01

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation. PMID:12486105

  17. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.

    PubMed

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C; Steinman, Ralph M

    2002-12-16

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

  18. Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

    PubMed

    Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

    2014-09-01

    The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients.

  19. Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many gram-negative bacteria.

    PubMed

    Menozzi, F D; Menozzi-Dejaiffe, C; Nano, F E

    1989-03-01

    In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.

  20. Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III.

    PubMed

    Fan, Yi-Chin; Chiu, Hsien-Chung; Chen, Li-Kuang; Chang, Gwong-Jen J; Chiou, Shyan-Song

    2015-10-01

    Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines. PMID:26495991

  1. Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

    PubMed Central

    Fan, Yi-Chin; Chiu, Hsien-Chung; Chen, Li-Kuang; Chang, Gwong-Jen J.; Chiou, Shyan-Song

    2015-01-01

    Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines. PMID:26495991

  2. Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma.

    PubMed

    Hwang, Guang-Yuh; Lin, Chuang-Yu; Huang, Li-Mei; Wang, Yan-Hsiung; Wang, Jing-Chyi; Hsu, Ching-Ting; Yang, Sheng-Shun; Wu, Cheng-Chung

    2003-12-01

    Hepatitis B virus X protein (HBx) expressed in Escherichia coli DH5alpha by recombinant DNA technology was purified to homogeneity by use of glutathione-Sepharose beads. Immunological characterization of the recombinant HBx protein was performed. Specific binding between the anti-HBx monoclonal antibody and HBx protein showed the specificity of the recombinant HBx protein. The intact HBx protein of the factor Xa-digested glutathione S-transferase-HBx fusion protein was further purified and was used as an antigen for screening the titers of anti-HBx antibodies in sera. Titers of anti-HBx in sera from 20 patients with hepatocellular carcinoma (HCC), 20 patients with chronic hepatitis (CH), and 20 healthy individuals were evaluated by Western blotting and a quantitative enzyme-linked immunosorbent assay. The results indicated that 70% of sera from HCC patients and 5% of sera from CH patients contained antibodies with significant binding to the HBx protein. Western blotting of HBx protein in liver extracts from 20 HCC patients was also performed by using the anti-HBx monoclonal antibody. Results showed that 85% of HCC patients' liver tissues contained a specific HBx protein with the same molecular size as the purified intact HBx. Full correlation was found between anti-HBx antibody positivity in serum and HBx protein positivity in HCC tissues. The data demonstrated that the etiology of HCC is involved with hepatitis B virus (HBV) infection and that HBx in particular plays a role in the development of HBV-related HCC.

  3. Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

    PubMed Central

    Wang, Anqi; Welch, Rene; Zhao, Bo; Ta, Tram; Keleş, Sündüz

    2015-01-01

    ABSTRACT Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for

  4. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

    NASA Astrophysics Data System (ADS)

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

    2016-06-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

  5. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins

    PubMed Central

    1996-01-01

    T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum. PMID:8627164

  6. Soluble antigen derived from IV larva of Angiostrongylus cantonensis promotes chitinase-like protein 3 (Chil3) expression induced by interleukin-13.

    PubMed

    Wu, Feng; Wei, Jie; Liu, Zhen; Zeng, Xin; Yu, Zilong; Lv, Zhiyue; Sun, Xi; Wu, Zhongdao

    2016-10-01

    Angiostrongyliasis caused by Angiostrongylus cantonensis (A. cantonensis) is an emerging food-borne parasitic disease, which refers basically to eosinophilic meningitis. Chitinase-like protein 3 (Chil3), a member of chitinase-like protein family which has chemotactic activity for eosinophils, is reported to be highly upregulated in brain of mouse infected with A. cantonensis. The mechanisms of high expression of Chil3 and the association between A. cantonensis and Chil3 are rarely reported. In order to understand the mechanism of high expression of Chil3 in A. cantonensis-infected mouse, we measured the level of Chil3 in RAW 264.7 and BV2 cell lines stimulated with soluble antigen of A. cantonensis by qPCR and ELISA. To explore the role of Chil3 in inflammation caused by A. cantonensis, we extracted and cultured brain mononuclear cells (BMNCs) and detected the eosinophil chemotactic activity of Chil3 using transwell assay and flow cytometer. Furthermore, we treated the infected mice by injection with rmChil3 and then counted the number of larvae in brains of infected mice and treated mice to examine the association between the worm and Chil3. Our results showed the soluble antigen from A. cantonensis could promote the Chil3 expression in macrophage and microglial cell lines induced by interleukin-13. In conclusion, we supposed that high expression of Chil3 enhanced by soluble antigens from A. cantonensis might be the reason of serious eosinophil infiltration in mouse brain after A. cantonensis infection.

  7. Past and future of providing matched, unrelated donors for marrow transplantation.

    PubMed

    Roeckel, I E; Baker, J

    1997-01-01

    Prior to 1979, bone marrow transplants were only performed with histocompatible sibling donors. Once it was established that histocompatible, unrelated donors could donate marrow for transplantation, the recruitment of such donors needed to be standardized. Blood donor centers had already identified the histocompatibility locus antigen (HLA) typing for donors who could be recruited to donate bone marrow. Recruiting a large number of donors required systematic evaluation and testing according to defined standards which were published in 1988 by the National Marrow Donor Program (NMDP). Peripheral stem cell collection (PBS) has been added as a transplant source. It promises additional therapeutic modalities, such as gene splicing to address other than cancer therapy.

  8. Predicting promiscuous antigenic T cell epitopes of Mycobacterium tuberculosis mymA operon proteins binding to MHC Class I and Class II molecules.

    PubMed

    Saraav, Iti; Pandey, Kirti; Sharma, Monika; Singh, Swati; Dutta, Prasun; Bhardwaj, Anshu; Sharma, Sadhna

    2016-10-01

    Limited efficacy of Bacillus Calmette-Guérin vaccine has raised the need to explore other immunogenic candidates to develop an effective vaccine against Mycobacterium tuberculosis (Mtb). Both CD4+ and CD8+ T cells play a critical role in host immunity to Mtb. Infection of macrophages with Mtb results in upregulation of mymA operon genes thereby suggesting their importance as immune targets. In the present study, after exclusion of self-peptides mymA operon proteins of Mtb were analyzed in silico for the presence of Human Leukocyte Antigen (HLA) Class I and Class II binding peptides using Bioinformatics and molecular analysis section, NetMHC 3.4, ProPred and Immune epitope database software. Out of 56 promiscuous epitopes obtained, 41 epitopes were predicted to be antigenic for MHC Class I. In MHC Class II, out of 336 promiscuous epitopes obtained, 142 epitopes were predicted to be antigenic. The comparative bioinformatics analysis of mymA operon proteins found Rv3083 to be the best vaccine candidate. Molecular docking was performed with the most antigenic peptides of Rv3083 (LASGAASVV with alleles HLA-B51:01, HAATSGTLI with HLA-A02, IVTATGLNI and EKIHYGLKVNTA with HLA-DRB1_01:01) to study the structural basis for recognition of peptides by various HLA molecules. The software binding prediction was validated by the obtained molecular docking score of peptide-HLA complex. These peptides can be further investigated for their immunological relevance in patients of tuberculosis using major histocompatibility complex tetramer approach. PMID:27389362

  9. An adhesin-like protein, Lam29, from Lactobacillus mucosae ME-340 binds to histone H3 and blood group antigens in human colonic mucus.

    PubMed

    Watanabe, Masamichi; Kinoshita, Hideki; Huang, I-Nung; Eguchi, Kei; Tsurumi, Takuya; Kawai, Yasushi; Kitazawa, Haruki; Kimura, Katsunori; Taketomo, Naoki; Kikuchi, Daisuke; Sase, Tomohiko; Miura, Koh; Ogawa, Hitoshi; Shibata, Chikashi; Horii, Akira; Saito, Tadao

    2012-01-01

    A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

  10. [Eukaryotic expression of NS1 major antigen region of PPV and development of an indirect ELISA based on the expressed protein].

    PubMed

    Ma, Hui; Zhao, Xu-Yong; Bian, Chuan-Zhou

    2012-11-01

    To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revealed 94.2% and 92.1% agreement respectively. The assay demonstrates good specificity and sensitivity, and can be applied in the detection of porcine parvovirus.

  11. Characterization of a 60-kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2015-08-01

    A sandwich enzyme-linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60-kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60-kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma-derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60-kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2-dimensional gel electrophoresis (2-D GE) and amino acid sequencing revealed the 60-kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons. PMID:26172875

  12. Comparative studies of strains Ictero No. I and RGA as the type strain of Leptospira interrogans: agglutinin absorption test, protein and antigen profiles, and enzyme activities.

    PubMed

    Hata, K; Ono, E; Yanagawa, R

    1988-01-01

    Strain Ictero No. I, the first isolate of Leptospira, isolated by Inada and Ido in 1914, was found to be sufficiently qualified to be the type strain of Leptospira interrogans rather than strain RGA. In an agglutinin absorption test, anti-Ictero No. I serum was not absorbed completely with strain RGA, and 25% of the homologous titer remained unabsorbed, while anti-RGA serum was completely absorbed with strain Ictero No. I. Thus, strain Ictero No. I was not serologically identical with strain RGA, and the two strains were considered to be different serovars. A protein band with a molecular weight of approximately 33,000 daltons was detected in strain Ictero No. I but not in strain RGA by SDS-PAGE. By Western blotting, this protein band was detectable with anti-Ictero No. I serum but not with anti-RGA serum. The presence of the 33K protein in strain Ictero No. I, but not in strain RGA, was confirmed by radioimmunoprecipitation using [125I]-labeled antigens, indicating that the protein antigen was surface-exposed. Only 8 of the 89 enzymes activities were different between strains Ictero No. I and RGA (line Sapporo). From the above results, we propose that strain Ictero No. I should be designated as the type strain of L. interrogans instead of strain RGA.

  13. Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

    PubMed Central

    Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  14. Characterization of a 60-kDa Thermally Stable Antigenic Protein as a Marker for the Immunodetection of Bovine Plasma-Derived Food Ingredients.

    PubMed

    Ofori, Jack A; Hsieh, Yun-Hwa P

    2015-08-01

    A sandwich enzyme-linked immunosorbent assay (sELISA) based on 2 monoclonal antibodies (Bb3D6 and Bb6G12) that recognize a 60-kDa antigenic protein in bovine blood was previously developed for detecting bovine blood in animal feed for the prevention of mad cow disease. This study sought to establish the identity of this 60-kDa antigenic protein and consequently determine the suitability of the sELISA for detecting bovine plasma-derived food ingredients (BPFIs), which are widely used in dietary products without explicit labeling. Results from western blot confirmed the 60-kDa protein to be present in the plasma fraction of bovine blood. Further proteomic analyses involving 2-dimensional gel electrophoresis (2-D GE) and amino acid sequencing revealed the 60-kDa protein to be bovine serum albumin (BSA). The sELISA proved capable of detecting BPFIs in all the commercial dietary supplements tested, including those that were formulated with hydrolyzed BPFIs. The assay could also detect 0.01% and 0.5% of different BPFIs in spiked raw and cooked ground beef, respectively. This assay based on the detection of BSA therefore has the potential to become a valuable analytical tool to protect consumers who avoid consuming BPFIs for religious, health, or ethical reasons.

  15. Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

    PubMed

    Josepriya, T A; Chien, Kuo-Hsuan; Lin, Hsin-Yun; Huang, Han-Ning; Wu, Chang-Jer; Song, Yen-Ling

    2015-08-01

    The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered. PMID

  16. Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen

    PubMed Central

    Beachey, EH; Stollerman, GH; Chiang, EY; Chiang, TM; Seyer, JM; Kang, AH

    1977-01-01

    M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24. PMID:325168

  17. Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection.

    PubMed

    Bai, Xiaofei; Shaozhou, Wulin; Zhang, Qingshan; Li, Chenxi; Qiu, Na; Meng, Runzhe; Liu, Ming; Zhang, Yun

    2015-03-01

    The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds. PMID:25588821

  18. Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity

    PubMed Central

    Altman, Meghan O; Bennink, Jack R; Yewdell, Jonathan W; Herrin, Brantley R

    2015-01-01

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ∼80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens. DOI: http://dx.doi.org/10.7554/eLife.07467.001 PMID:26252514

  19. Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

    PubMed

    Maddaluno, Marcella; MacRitchie, Neil; Grassia, Gianluca; Ialenti, Armando; Butcher, John P; Garside, Paul; Brewer, James M; Maffia, Pasquale

    2014-01-01

    In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

  20. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    PubMed

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  1. Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability

    PubMed Central

    2012-01-01

    Background The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins. Results Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20–40 nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability. Conclusion We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages. PMID:23110350

  2. The HtrA-Like Serine Protease PepD Interacts with and Modulates the Mycobacterium tuberculosis 35-kDa Antigen Outer Envelope Protein

    PubMed Central

    White, Mark J.; Savaryn, John P.; Bretl, Daniel J.; He, Hongjun; Penoske, Renee M.; Terhune, Scott S.; Zahrt, Thomas C.

    2011-01-01

    Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress. PMID:21445360

  3. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    PubMed

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  4. Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial

    PubMed Central

    Hodgson, Susanne H; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Rampling, Thomas W; Biswas, Sumi; Poulton, Ian D; Miura, Kazutoyo; Douglas, Alexander D; Alanine, Daniel GW; Illingworth, Joseph J; de Cassan, Simone C; Zhu, Daming; Nicosia, Alfredo; Long, Carole A; Moyle, Sarah; Berrie, Eleanor; Lawrie, Alison M; Wu, Yimin; Ellis, Ruth D; Hill, Adrian V S; Draper, Simon J

    2014-01-01

    The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines—chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising “mixed-modality” regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible. PMID:25156127

  5. Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.

    PubMed

    Hodgson, Susanne H; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Rampling, Thomas W; Biswas, Sumi; Poulton, Ian D; Miura, Kazutoyo; Douglas, Alexander D; Alanine, Daniel Gw; Illingworth, Joseph J; de Cassan, Simone C; Zhu, Daming; Nicosia, Alfredo; Long, Carole A; Moyle, Sarah; Berrie, Eleanor; Lawrie, Alison M; Wu, Yimin; Ellis, Ruth D; Hill, Adrian V S; Draper, Simon J

    2014-12-01

    The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines--chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising "mixed-modality" regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible. PMID:25156127

  6. Targeting of Nasal Mucosa-Associated Antigen-Presenting Cells In Vivo with an Outer Membrane Protein A Derived from Klebsiella pneumoniae

    PubMed Central

    Goetsch, Liliane; Gonzalez, Alexandra; Plotnicky-Gilquin, Hélène; Haeuw, Jean François; Aubry, Jean Pierre; Beck, Alain; Bonnefoy, Jean Yves; Corvaïa, Nathalie

    2001-01-01

    Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection. PMID:11553588

  7. Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

    PubMed

    Kashino, S S; Abeijon, C; Qin, L; Kanunfre, K A; Kubrusly, F S; Silva, F O; Costa, D L; Campos, D; Costa, C H N; Raw, I; Campos-Neto, A

    2012-07-01

    Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

  8. Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

    PubMed

    Moriguchi, K; Mitamura, Y; Iwami, J; Hasegawa, Y; Higuchi, N; Murakami, Y; Maeda, H; Yoshimura, F; Nakamura, H; Ohno, N

    2012-11-01

    Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

  9. SLA/LP/tRNP((Ser)Sec) antigen in autoimmune hepatitis: identification of the native protein in human hepatic cell extract.

    PubMed

    Volkmann, Martin; Luithle, Daniel; Zentgraf, Hanswalter; Schnölzer, Martina; Fiedler, Sabine; Heid, Hans; Schulze-Bergkamen, Andrea; Strassburg, Christian P; Gehrke, Sven G; Manns, Michael P

    2010-02-01

    A diagnostic subgroup of AIH type 1 is characterized by specific serum antibodies against soluble liver protein. The respective autoantigen was named SLA/LP/tRNP((Ser)Sec), after three homologous recombinant polypeptides were isolated from expression gene libraries. We analyzed human cultured liver cells for the human homologue of recombinant SLA/LP/tRNP((Ser)Sec) by antigen purification. In addition, a monoclonal antibody was generated against recombinant SLA-p35, a truncated recombinant SLA-reactive polypeptide. With a positive patient serum, immune affinity chromatography was performed on the 52 kD-SLA main antigenic determinant pre-enriched by ion exchange chromatography. By mass spectrometry, the 52 kD-SLA/LP/tRNP ((Ser)Sec) autoantigen was unambiguously identified in the purification product. The identity of the recombinant SLA-p35 and its human homologue was further confirmed by a specific signal of the anti SLA-p35 monoclonal antibody with purified human SLA/LP/tRNP((Ser)Sec). The 48 kD-SLA species frequently comigrating in SLA-immunoblotting however was not identified by either approach. We conclude that the native counterpart of recombinant tRNP((Ser)(Sec)) indeed is detectable with a molecular weight of 52 kD in soluble liver extract of human cells as the major antigenic component of SLA/LP/tRNP((Ser)Sec).

  10. Epitope analysis of immunoglobulins against gp20, a GPI-anchored protein of the human sperm surface homologous to leukocyte antigen CD52.

    PubMed

    Flori, F; Giovampaola, C Della; Focarelli, R; Secciani, F; La Sala, G B; Nicoli, A; Hale, G; Rosati, F

    2005-09-01

    Gp20 is a sialylglycoprotein of the human sperm surface related to maturation and capacitation and is homologous to CD52, a glycosyl- phosphatidyl-inositol (GPI)-anchored protein highly expressed in lymphocytes, monocytes, eosinophils, and epididymal cells, described by the monoclonal antibody family CAMPATH. The CAMPATH antigen is characterized by a very short peptide (12 amino acids) and an N-linked oligosaccharide chain bound to the asparagine located in the third position and a GPI anchor bound to the C-terminal serine. The CAMPATH epitope includes three amino acids at the C-terminus and part of the GPI anchor. It has been suggested that anti-gp20 interacts with the same peptide recognized by CAMPATH antibodies but with a different epitope, since it describes the corresponding antigen in a different way. For example, it localizes the corresponding antigen in the equatorial region of the sperm head when sperm are capacitated, whereas CAMPATH antibodies bind all over the sperm surface. Our results indicate that the anti-gp20 epitope does not include the peptide backbone, the GPI anchor, or the N-glycans but consists of O-linked oligosaccharide chains bound to a unique CD52 glycoform present both in sperm and leukocytes. This is suggested by results obtained using many different approaches, such as immunoblot analysis of gp20 after removal of N- and O-glycans and after jacalin (Artocarpus integrifolia agglutinin)-affinity chromatography.

  11. Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease.

    PubMed

    Rasiah, C; Rauer, S; Gassmann, G S; Vogt, A

    1994-04-01

    A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples, negative in routine B. burgdorferi serology, were tested as controls. The hybrid protein, made up of specific epitopes of an early (p18) and late (p83) antigen, is recognized by almost the same number of patient serum samples as the individual antigens.

  12. Consequences of point mutations in melanoma-associated antigen 4 (MAGE-A4) protein: Insights from structural and biophysical studies

    PubMed Central

    Hagiwara, Yoshio; Sieverling, Lina; Hanif , Farina; Anton, Jensy; Dickinson, Eleanor R.; Bui, Tam T. T.; Andreeva, Antonina; Barran, Perdita E.; Cota, Ernesto; Nikolova, Penka V.

    2016-01-01

    The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers. PMID:27121989

  13. Potential Impact of Seasonal Malaria Chemoprevention on the Acquisition of Antibodies against Glutamate-Rich Protein and Apical Membrane Antigen 1 in Children Living in Southern Senegal

    PubMed Central

    Ndiaye, Magatte; Sylla, Khadime; Sow, Doudou; Tine, Roger; Faye, Babacar; Ndiaye, Jean Louis; Dieng, Yemou; Lo, Aminata Collé; Abiola, Annie; Cisse, Badara; Ndiaye, Daouda; Theisen, Michael; Gaye, Oumar; Alifrangis, Michael

    2015-01-01

    Seasonal malaria chemoprevention (SMC) is defined as the intermittent administration of full treatment courses of an antimalarial drug to children during the peak of malaria transmission season with the aim of preventing malaria-associated mortality and morbidity. SMC using sulfadoxine–pyrimethamine (SP) combined with amodiaquine (AQ) is a promising strategy to control malaria morbidity in areas of highly seasonal malaria transmission. However, a concern is whether SMC can delay the natural acquisition of immunity toward malaria parasites in areas with intense SMC delivery. To investigate this, total IgG antibody (Ab) responses to Plasmodium falciparum antigens glutamate-rich protein R0 (GLURP-R0) and apical membrane antigen 1 (AMA-1) were measured by enzyme-linked immunosorbent assay in Senegalese children under the age of 10 years in 2010 living in Saraya and Velingara districts (with SMC using SP+AQ [SMC+] since 2007) and Tambacounda district (without SMC (SMC−)). For both P. falciparum antigens, total IgG response were significantly higher in the SMC− compared with the SMC+ group (for GLURP-R0, P < 0.001 and for AMA-1, P = 0.001). There was as well a nonsignificant tendency for higher percentage of positive responders in the SMC− compared with the SMC+ group (for GLURP-R0: 22.2% versus 14.4%, respectively [P = 0.06]; for AMA-1: 45.6% versus 40.0%, respectively [P = 0.24]). Results suggest that long-term malaria chemoprevention by SMC/SP+AQ have limited impact on the development of acquired immunity, as tested using the P. falciparum antigens GLURP-R0 and AMA-1. However, other factors, not measured in this study, may interfere as well. PMID:26283746

  14. Variations in the Electrostatic Landscape of Class II Human Leukocyte Antigen Molecule Induced by Modifications in the Myelin Basic Protein Peptide: A Theoretical Approach

    PubMed Central

    Agudelo, William A.; Galindo, Johan F.; Ortiz, Marysol; Villaveces, José L.; Daza, Edgar E.; Patarroyo, Manuel E.

    2009-01-01

    The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA) - Myelin Basic Protein (MBP) peptide complex (HLA-DRβ1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen - peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets' amino acids: “anchoring” located in Pocket 1 and 4, and “recognition” located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9≫Pocket 4≈Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions β13R, β26F, β28D, β9W, β74A, β47F and β57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC–antigenic peptide complex, which could have potential implications for synthetic vaccine design. PMID:19132105

  15. Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis.

    PubMed

    Martins, Vivian Tamietti; Chávez-Fumagalli, Miguel Angel; Lage, Daniela Pagliara; Duarte, Mariana Costa; Garde, Esther; Costa, Lourena Emanuele; da Silva, Viviane Grazielle; Silva, Viviane Gomes da; Oliveira, Jamil Silvano; Magalhães-Soares, Danielle Ferreira de; Teixeira, Santuza Maria Ribeiro; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2015-01-01

    In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite

  16. Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

    PubMed Central

    Martins, Vivian Tamietti; Chávez-Fumagalli, Miguel Angel; Lage, Daniela Pagliara; Duarte, Mariana Costa; Garde, Esther; Costa, Lourena Emanuele; da Silva, Viviane Gomes; Oliveira, Jamil Silvano; de Magalhães-Soares, Danielle Ferreira; Teixeira, Santuza Maria Ribeiro; Fernandes, Ana Paula; Soto, Manuel; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2015-01-01

    In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite

  17. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  18. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian . E-mail: yhe@nybloodcenter.org; Li Jingjing; Jiang Shibo

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  19. A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection.

    PubMed Central

    Santori F, R; Paranhos-Bacalla, G S; Franco DA Silveira, J; Yamauchi, L M; Araya, J E; Yoshida, N

    1996-01-01

    To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection. PMID:8606064

  20. Association of Streptococcus pneumoniae common protein antigen (CPA) antibodies and pneumococcal nasopharyngeal colonization in HIV-infected and HIV-uninfected African children.

    PubMed

    Ditse, Z; Adrian, P V; Kuwanda, L; Madhi, S A

    2013-09-13

    Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4-7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against "cell-wall associated or surface-exposed" proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), "membrane-associated" proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n=212) and HIV-infected (n=74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease. PMID:23845819

  1. Effect of cholinomimetics and adrenomimetics on proliferation of mouse B lymphocytes during primary immune response to protein antigen

    SciTech Connect

    Ado, A.D.; Dontsov, V.I.; Gol'dshtein, M.M.

    1985-12-01

    The aim of this investigation was to study the effect of neurotransmitters on proliferation of B lymphocytes induced by specific antigen. Experiments were carried out on female mice. To estimate proliferative activity, lymphocytes enriched with B cells were incubated in medium 199 for 2 h at 37 degrees C in a dose of 2.10/sup 6/-5.10/sup 6/ cells with 2 microCi of /sup 3/H-(methyl)-thymidine. The effect of acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of antigen during culture is shown. Discordance of effects of adrenalin and acetylcholine on incorporation of /sup 3/H-thymidine into B lymphocytes of mice immunized with different doses of ovalbumin is also shown.

  2. Nonmyeloablative Unrelated Donor Hematopoietic Cell Transplantation for the Treatment of Patients with Poor-Risk, Relapsed or Refractory Multiple Myeloma

    PubMed Central

    Georges, George E.; Maris, Michael B.; Maloney, David G.; Sandmaier, Brenda M.; Sorror, Mohamed L.; Shizuru, Judith A.; Niederwieser, Dietger W.; Agura, Edward D.; Bruno, Benedetto; McSweeney, Peter A.; Pulsipher, Michael A.; Chauncey, Thomas R.; Mielcarek, Marco; Storer, Barry E.; Storb, Rainer

    2007-01-01

    The purpose of this study was to determine long-term outcome of unrelated donor nonmyeloablative hematopoietic cell transplantation in patients with poor-risk multiple myeloma. Twenty-four patients were enrolled. Seventeen patients (71%) had chemotherapy-refractory disease and 14 patients (58%) had disease relapse or progression after prior autologous transp