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Chronic bystander infections and immunity to unrelated antigens  

PubMed Central

Chronic infections with persistent pathogens such as helminths, mycobacteria, Plasmodium and hepatitis viruses affect more than a third of the human population and are associated with increased susceptibility to other pathogens as well as reduced vaccine efficacy. Although these observations suggest an impact of chronic infections in modulating immunity to unrelated antigens, little is known regarding the underlying mechanisms. Here, we summarize evidence of the most prevalent infections affecting immunity to unrelated pathogens and vaccines, and discuss potential mechanisms of how different bystander chronic infections might impact immune responses. We suggest that bystander chronic infections affect different stages of host responses and may impact transmission of other pathogens, recognition and innate immune responses, priming and differentiation of adaptive effector responses, as well as the development and maintenance of immunological memory. Further understanding of the immunological effects of co-infection should provide opportunities to enhance vaccine efficacy and control infectious diseases.

Stelekati, Erietta; Wherry, E. John



Anti-tumoral effect of active immunotherapy in C57BL\\/6 mice using a recombinant human VEGF protein as antigen and three chemically unrelated adjuvants  

Microsoft Academic Search

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that affects the interaction between vascular\\u000a endothelial growth factor (VEGF) and its receptors, blocking tumor-induced angiogenesis has become one of the most important\\u000a targets for the development of new cancer therapeutic drugs and procedures. Among the latter, therapeutic vaccination using\\u000a VEGF as antigen presents itself as very attractive, with the

Yanelys Morera; Mónica Bequet-Romero; Marta Ayala; Humberto Lamdán; Else-Marie Agger; Peter Andersen; Jorge V. Gavilondo



A common antigenic determinant found in two functionally unrelated toxins  

PubMed Central

The heat-stable enterotoxin ST Ib produced by enterotoxigenic E. coli strains shares a sequence homology with the sea snail neurotoxin, conotoxin GI. Rabbit antisera were raised against synthetic analogs of these toxins and to a six-residue peptide representing the region common to both toxins. Results from enzyme-linked immunosorbent assays indicate that the homologous region of both toxins represents part of their antigenic site. The lack of cross-reactivity exhibited by the six- residue common domain with serum directed against either toxin suggests that this region probably retains a similar conformation in the intact toxins but not in the isolated fragment.



Actinomycin D: Inhibition of Protein Synthesis Unrelated to Effect on Template RNA Synthesis  

Microsoft Academic Search

Incubation of sarcoma-37 ascites cells in vitro with actinomycin D resulted in inhibition of synthesis of nuclear and cytoplasmic proteins. The overall inhibition could be prevented or relieved by glucose; it is thus unrelated to breakdown of template RNA.

George R. Honig; Marco Rabinovitz



Antigenic protein modifications in Ehrlichia  

PubMed Central

To develop effective vaccination strategies againstEhrlichia, we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed withE. muris were resistant to lethal infection withIxodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming withE. muris and later injected with IOE-detected antigenic proteins inE. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both theE. muris and IOE cell lysates with IOE andE. muris-specific polyclonal antibody. Analysis of the total proteins ofE. muris and IOE by two dimensional electrophoresis showed that bothE. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed thatE. muris proteins are more lipoylated and glycosylated than those of IOE.




Quantitating T Cell Cross-Reactivity for Unrelated Peptide Antigens1  

PubMed Central

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.

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.



Insulin Resistance Is Unrelated to Circulating Retinol Binding Protein and Protein C Inhibitor  

Microsoft Academic Search

Context:Recentdatasuggestthatcirculatingretinol-bindingprotein (RBP) might be involved in the pathogenesis of insulin resistance. Moreover, protein C inhibitor (PCI), which specifically binds retinoic acid,wasfoundtobeincreasedinmyocardialinfarctionsurvivorswho are also insulin resistant. Objective: The objective of this study was to investigate the asso- ciation of insulin resistance with RBP factors and PCI active antigen. Design and Setting: This was a clinical study. Patients: Nondiabetic humans with high

Miriam Promintzer; Michael Krebs; Jelena Todoric; Anton Luger; Martin Georg Bischof; Peter Nowotny; Oswald Wagner; Harald Esterbauer; Christian Anderwald


Efficient Mapping of Protein Antigenic Determinants  

Microsoft Academic Search

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic

Vijay Mehra; Douglas Sweetser; Richard A. Young



Immune recognition of protein antigens  

SciTech Connect

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.

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



Protein folding: Independent unrelated pathways or predetermined pathway with optional errors  

PubMed Central

The observation of heterogeneous protein folding kinetics has been widely interpreted in terms of multiple independent unrelated pathways (IUP model), both experimentally and in theoretical calculations. However, direct structural information on folding intermediates and their properties now indicates that all of a protein population folds through essentially the same stepwise pathway, determined by cooperative native-like foldon units and the way that the foldons fit together in the native protein. It is essential to decide between these fundamentally different folding mechanisms. This article shows, contrary to previous supposition, that the heterogeneous folding kinetics observed for the staphylococcal nuclease protein (SNase) does not require alternative parallel pathways. SNase folding kinetics can be fit equally well by a single predetermined pathway that allows for optional misfolding errors, which are known to occur ubiquitously in protein folding. Structural, kinetic, and thermodynamic information for the folding intermediates and pathways of many proteins is consistent with the predetermined pathway–optional error (PPOE) model but contrary to the properties implied in IUP models.

Bedard, Sabrina; Krishna, Mallela M. G.; Mayne, Leland; Englander, S. Walter



Cyclorraphan yolk proteins and lepidopteran minor yolk proteins originate from two unrelated lipase families.  


Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins. PMID:15606810

Hens, K; Lemey, P; Macours, N; Francis, C; Huybrechts, R



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

PubMed Central

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.

Rodriguez-Almazan, Claudia; Torner, Francisco J.; Costas, Miguel; Perez-Montfort, Ruy; de Gomez-Puyou, Marieta Tuena; Puyou, Armando Gomez



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

PubMed Central

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.

Tribouillard-Tanvier, Deborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cecile; Beringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stephane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chedin, Stephane; Vilette, Didier; Galons, Herve; Sanyal, Suparna; Blondel, Marc



Antigenic Structure of Flavivirus Proteins  

Microsoft Academic Search

The increased activity of Dengue virus in the tropical regions of the world and the recent movement of West Nile virus from the eastern to the western hemisphere emphasize the fact that vector-borne flaviviruses are medically important emerging infectious diseases. These facts warrant continued efforts to decode all facets of flavivirus immunology. This chapter reviews current understanding of the antigenic

John T. Roehrig



Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins.  

PubMed Central

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution.

Manning, Charlene M; Mathews, Wendy R; Fico, Leah P; Thackeray, Justin R




PubMed Central

Purpose Approximately 13% of patients lacking an HLA-identical sibling have a 1-antigen-mismatched related donor (MMRD). Historically, outcomes using a 1-antigen MMRD were considered equivalent to a matched unrelated donor (UD). Recent improvements in unrelated donor (UD) stem cell transplantation (SCT) due to better molecular HLA-matching justifies investigating if UD should be preferred to MMRD in adult patients with acute leukemia. Patients and Methods The outcomes of MMRD (n=89) and HLA-A, B, C, DRB1 allele matched UD (n=700) SCT reported to the CIBMTR between 1995 and 2005 were compared. Patients were transplanted for acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) in first or second complete remission. Results Donor type was not associated with hematological recovery. Univariate and multivariate comparisons of MMRD vs. HLA-matched UD transplants showed no statistically significant differences in overall survival, disease free survival, transplant related mortality, relapse, and 100-day grade III–IV acute graft-versus-host disease (GVHD). MMRD SCT was associated with a lower rate of chronic GVHD at 1-year, 35% vs 47% p=0.03, which was confirmed in multivariate analysis (RR 0.58, 95% CI 0.39-0.85, p<0.01). Conclusion HLA-matched UD and MMRD SCT are associated with comparable survival. Since less chronic GVHD was observed in MMRD, this option when available remains the first choice in acute leukemia patients without an HLA-identical sibling in need of allogeneic transplantation.

Valcarcel, David; Sierra, Jorge; Wang, Tao; Kan, Fangyu; Gupta, Vikas; Hale, Gregory A.; Marks, David I.; McCarthy, Philip L; Oudshoorn, Machteld; Petersdorf, Effie W; Ringden, Olle; Setterholm, Michelle; Spellman, Stephen R; Waller, Edmund K.; Gajewski, James L; Marino, Susana R.; Senitzer, David; Lee, Stephanie J.



Sperm fibrous sheath proteins: a potential new class of target antigens for use in human therapeutic cancer vaccines  

PubMed Central

Cancer vaccines have been demonstrated to be a promising strategy for treating human neoplastic disease, but one of the limitations of these vaccines remains the paucity of target antigens to which to direct an effective immune response. We hypothesize that sperm fibrous sheath proteins may be a new class of useful antigens for developing successful cancer vaccines. This hypothesis is supported by the expression of two sperm fibrous sheath proteins, called sperm protein 17 and calcium-binding tyrosine-phosphorylation regulated protein, in tumors of unrelated histological origin and their capability to induce T cell-based immune responses.

Chiriva-Internati, Maurizio; Cobos, Everardo; Da Silva, Diane M.



Structural features of the reactions between antibodies and protein antigens  

Microsoft Academic Search

Antibodies bind protein antigens over large sterically and electrostatically complementary sur- faces. Van der Waals forces, hydrogen bonds, and occa- sionally ion pairs provide stability to antibody-antigen complexes. In addition, water molecules contribute hydrogen bonds linking antigen and antibody, and in- crease the complementarity of antigen-antibody inter- faces. In qualification to a strict 'lock and key' mechan- ism, evidence of



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...


Estimation and efficient computation of the true probability of recurrence of short linear protein sequence motifs in unrelated proteins  

PubMed Central

Background Large datasets of protein interactions provide a rich resource for the discovery of Short Linear Motifs (SLiMs) that recur in unrelated proteins. However, existing methods for estimating the probability of motif recurrence may be biased by the size and composition of the search dataset, such that p-value estimates from different datasets, or from motifs containing different numbers of non-wildcard positions, are not strictly comparable. Here, we develop more exact methods and explore the potential biases of computationally efficient approximations. Results A widely used heuristic for the calculation of motif over-representation approximates motif probability by assuming that all proteins have the same length and composition. We introduce pv, which calculates the probability exactly. Secondly, the recently introduced SLiMFinder statistic Sig, accounts for multiple testing (across all possible motifs) in motif discovery. However, it approximates the probability of all other possible motifs, occurring with a score of p or less, as being equal to p. Here, we show that the exhaustive calculation of the probability of all possible motif occurrences that are as rare or rarer than the motif of interest, Sig', may be carried out efficiently by grouping motifs of a common probability (i.e. those which have permuted orders of the same residues). Sig'v, which corrects both approximations, is shown to be uniformly distributed in a random dataset when searching for non-ambiguous motifs, indicating that it is a robust significance measure. Conclusions A method is presented to compute exactly the true probability of a non-ambiguous short protein sequence motif, and the utility of an approximate approach for novel motif discovery across a large number of datasets is demonstrated.



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

Microsoft Academic Search

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

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



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  

Microsoft Academic Search

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

Karl-Otto Fresen; Harald Zur Hausen



Identification of antigenic proteins of setaria cervi by immunoblotting technique.  


Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites. PMID:2442100

Kaushal, N A; Kaushal, D C; Ghatak, S



Antigen-specific suppressor factors of noncytotoxic CD8+ suppressor T cells downregulate antibody responses also to unrelated antigens when the latter are presented as covalently linked adducts with the specific antigen.  


We had previously shown (i) that conjugates of a given antigen A (AgA) and monomethoxypolyethylene glycol (mPEG) induced AgA-specific tolerance in mice which was mediated by polyclonal CD8+ suppressor T (Ts) cells, as well as by soluble factor(s) of these cells (TsF), and (ii) that clones of nonhybridized CD8+ Ts cells could be derived from the above single cells, and monoclonal AgA-specific TsF could be released from these cloned cells. In the present study, we demonstrate that mice pretolerized by injection of AgA(mPEG)n are also unresponsive to an unrelated antigen B (AgB), or to its haptenated derivative AgB-Hpn, when AgB or AgB-Hpn is injected in the form of a covalent adduct with AgA, i.e., AgA-AgB or AgA-AgB-Hpn, but not when it is injected as a mixture with AgA; in this study human (myeloma) IgG (HIgG) served as AgA, and ovalbumin (OVA) or OVA-DNP3 served as AgB or AgB-Hpn. Moreover, this phenomenon was reproduced in vitro; i.e., Ts cells of mice tolerized with HIgG(mPEG)30, or the soluble monoclonal TsF of cloned Ts cells, exerted their associative suppressive effector function--in the obligatory presence of CD8+ T cells of syngeneic naive mice (Tn cells)--on antibody (Ab) formation to an Hp (DNP), when the Hp was present as a covalent adduct linked either directly to HIgG (e.g., HIgG-DNP7) or indirectly via OVA (as in HIgG-OVA-DNP3); however, no suppression of the anti-DNP Ab response was observed when OVA-DNP3 was present as a mixture with HIgG. Furthermore, it was established that the accessory cells involved in processing the specific Ag in the presence of the Ts cells were also downregulated, as reflected by their reduced capacity for presentation of the Ag to HIgG-specific helper T (Th) cells in proliferation assays. All these results demonstrate that (i) the phenomenon of linked immunological suppression may involve the downregulation of Th cells which recognize, concomitantly with the Ts cell, the appropriate epitopes of AgA and AgB on the same Ac cell, (ii) the downregulation of these Th cells may be a consequence of the downregulation of Ac cells by Ts cells interacting with the appropriate epitope(s) present on the Ac cells, and (iii) most remarkably the CD8+ Ts cells could be substituted by Tn cells "armed" with the specific monoclonal TsF. PMID:8343965

Bitoh, S; Takata, M; Maiti, P K; Holford-Strevens, V; Kierek-Jaszczuk, D; Sehon, A H



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

PubMed Central

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.

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



Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors  

PubMed Central

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110–130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent ?-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-? fold with eight predicted ?-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin–cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.

Rigden, Daniel J.; Mosolov, Vladimir V.; Galperin, Michael Y.



Protein and antigenic heterogeneity among isolates of Bacillus piliformis.  

PubMed Central

Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels and Western blot (immunoblot) analyses, respectively. Results showed differences in protein and antigen banding patterns, indicating diversity among isolates. Western blots probed with serum preabsorbed with a heterologous bacterial extract revealed that numerous antigens have different electrophoretic mobilities among isolates but apparently share common epitopes. Immunodominant cross-reactive antigens may be candidate proteins useful for development of improved serologic diagnostic tests, allowing identification of animals infected with a wide range of B. piliformis isolates. Images

Riley, L K; Besch-Williford, C; Waggie, K S



Reagentless fluorescent biosensors from artificial families of antigen binding proteins.  


Antibodies and artificial families of antigen binding proteins (AgBP) are constituted by a connected set of hypervariable (or randomized) residue positions, supported by a constant polypeptide backbone. The residues that form the binding site for a given antigen, are selected among the hypervariable residues. We showed that it is possible to transform any AgBP of these families into a reagentless fluorescent biosensor, specific of the target antigen, simply by coupling a solvatochromic fluorophore to one of the hypervariable residues that have little or no importance for the interaction with the antigen, after changing this residue into cysteine by mutagenesis. We validated this approach with a DARPin (Designed Ankyrin Repeat Protein) and a Nanofitin (also known as Affitin) with high success rates. Reagentless fluorescent biosensors recognize their antigen in an immediate, quantitative, selective and specific way, without any manipulation of the sample to analyze or addition of reagent. PMID:21565483

Miranda, Frederico F; Brient-Litzler, Elodie; Zidane, Nora; Pecorari, Frédéric; Bedouelle, Hugues



Associations of Urinary Cadmium with Age and Urinary Proteins: Further Evidence of Physiological Variations Unrelated to Metal Accumulation and Toxicity  

PubMed Central

Background: The current risk assessment for environmental cadmium (Cd) largely relies on the assumption that urinary Cd (U-Cd) is a reliable biomarker of the Cd body burden. Recent studies have questioned the validity of this assumption. Objectives: We studied the lifetime trend of U-Cd as a function of diuresis, gender, smoking status, and protein tubular reabsorption. We also analyzed the associations between U-Cd and urinary proteins. Methods: Cd, retinol-binding protein, and albumin were measured in the urine of six cohorts of the general population of Belgium, with a mean age ranging from 5.7 to 88.1 years (n = 1,567). Variations of U-Cd with age were modeled using natural cubic splines. Results: In both genders, U-Cd decreased to a minimum (~ 0.20 ?g/L) at the end of adolescence, then increased until 60–70 years of age (~ 0.60 ?g/L in never-smokers) before leveling off or decreasing. When U-Cd was expressed in micrograms per gram of creatinine, these variations were amplified (minimum, 0.15 µg/g creatinine; maximum, 0.70 µg/g creatinine) and much higher U-Cd values were observed in women. We observed no difference in U-Cd levels between never-smokers and former smokers, and the difference with current smokers did not increase over time. Lifetime curves of U-Cd were higher with increasing urinary retinol-binding protein or albumin, a consequence of the coexcretion of Cd with proteins. Conclusions: At low Cd exposure levels, U-Cd and age are associated through nonlinear and nonmonotonic relationships that appear to be driven mainly by recent Cd intake and physiological variations in the excretion of creatinine and proteins. Citation: Chaumont A, Voisin C, Deumer G, Haufroid V, Annesi-Maesano I, Roels H, Thijs L, Staessen J, Bernard A. 2013. Associations of urinary cadmium with age and urinary proteins: further evidence of physiological variations unrelated to metal accumulation and toxicity. Environ Health Perspect 121:1047–1053;?

Chaumont, Agnes; Voisin, Catherine; Deumer, Gladys; Haufroid, Vincent; Annesi-Maesano, Isabella; Roels, Harry; Thijs, Lutgarde; Staessen, Jan



Prediction of protein antigenic determinants from amino acid sequences  

SciTech Connect

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinis, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.

Hopp, T.P.; Woods, K.R.



Protein polymorphism of a human plasma apolipoprotein D antigenic epitope.  


Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein. PMID:2477480

Camato, R; Marcel, Y L; Milne, R W; Lussier-Cacan, S; Weech, P K



The Role of Heat Shock Proteins in Antigen Cross Presentation  

PubMed Central

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.

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



The role of heat shock proteins in antigen cross presentation.  


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

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



Antibody Profiling with Protein Antigen Microarrays in Early Stage Cancer  

PubMed Central

Introduction Proteins not present in normal cells, i.e., cancer cells, may elicit a host immune response that leads to the generation of antibodies that might react with these tumor-associated proteins. In recent years, a growing number of reports have showed that autoantibody profiling may provide an alternative approach for the detection of cancer. However, most studies of antigen-autoantibody reactivity have relied on recombinant proteins. Recombinant proteins lack the proper post-translational modifications present in native proteins. Because of this limitation, native or natural protein antigen microarrays are gaining popularity for profiling antibody responses. Areas covered 1) to illustrate some examples of autoantibodies as signatures for early stage cancer; 2) to briefly outline the various protein antigen microarray platforms; 3) to illustrate the use of native or natural protein microarrays in the discovery of potential biomarkers; and, 4) to discuss the advantages of native protein antigen microarrays over other approaches. Expert opinion The nature of protein microarray platforms is conducive to multiplexing, which amplifies the potential for uncovering effective biomarkers for many significant diseases. However, the major challenge will be in integrating microarray platforms into multiplexed clinical diagnostic tools, as the main drawback is the reproducibility and coefficient of variation of the results from array to array, and the transportability of the array platform to a more automatable platform.

Liu, Brian C.-S.; DiJohnson, Daniel A.; O'Rourke, Dennis J.



Molecular cloning of genes encoding oncosphere proteins reveals conservation of modular protein structure in cestode antigens  

Microsoft Academic Search

Recombinant oncosphere antigens have been found to be remarkably effective when used as vaccines against cysticercosis and hydatid disease. Comparison of the structural features of these proteins and their associated genes suggest common features between antigens. Here molecular cloning is used to complete comparisons of Taenia solium, Taenia saginata, Taenia ovis, Echinococcus granulosus and Echinococcus multilocularis oncosphere antigens and genes.

Charles Gauci; Marshall W. Lightowlers



Extracellular Release of Antigenic Proteins by Helicobacter pylori  

PubMed Central

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation.

Cao, Ping; McClain, Mark S.; Forsyth, Mark H.; Cover, Timothy L.



Immunological Properties of Hepatitis B Core Antigen Fusion Proteins  

NASA Astrophysics Data System (ADS)

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.

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



Hyaluronan conjugation of antigenic protein to modify immunogenic information  

Microsoft Academic Search

Hyaluronan is an anionic polysaccharide and a major component of extracellular matrices. The major physiological roll of hyaluronan (hyaluronic acid, HA) is thought to be a space filling material and HA is an attractive biomaterial due to its biocompatibility and biodegradability. We chemically modified ovalbumin (OVA), a well defined protein antigen, with HA to reduce its immunogenicity. HA was enzymatically

S. Mochizuki; A. Kano; A. Yamayoshi; A. Maruyama



Comparative Analyses of the Proteins and Antigens of Five Herpesviruses  

Microsoft Academic Search

SUMMARY The proteins of five herpesviruses - herpes simplex types I and 2 (HSV-I and HSV-2), bovine mammillitis virus (BMV), pseudorabies virus (PRV) and equine abortion virus (EAV) - have been compared by polyacrylamide gel electrophoresis of purified virus and by antigenic analysis using virus neutralization and agar-gel immunodiffusion tests. Although there were general similarities in the number, size range

R. A. Killington; JANE YEO; R. W. Honess; D. H. Watson; BARBARA E. DUNCAN; I. W. Halliburton; JENNIFER MUMFORD



The Effect of Alternatives to Formocresol on Antigenicity of Proteins  

Microsoft Academic Search

As part of an overall evaluation of possible substitutes for the pulpotomy agent formocresol, this study was initiated to compare the antigenicity of the reaction products of protein with formaldehyde, glutaraldehyde, or dimethylsuberimidate (DMS).Rabbits were injected with rabbit serum albumin (RSA) which had been treated with one of the following solutions: phosphate-buffered saline, 2% glutaraldehyde, 4% formaldehyde, or 2% DMS.

D. M. Ranly; D. Horn; T. Zislis



Chloroplast-derived vaccine antigens and other therapeutic proteins  

Microsoft Academic Search

The chloroplast genetic engineering offers a number of unique advantages including high level of transgene expression, multi-gene expression in single transformation event and transgene containment due to maternal inheritance. Hyper-expression of vaccine antigens or therapeutic proteins in transgenic chloroplasts (leaves) or chromoplasts (fruits\\/roots) facilitates efficient oral delivery. Ability of chloroplasts to correctly fold human blood proteins with proper disulfide bridges

Henry Daniell; Seethamahalakshmi Chebolu; Shashi Kumar; Michael Singleton; Regina Falconer



Antigenicity, storage, and aging: physiologic autoantibodies to cell membrane and serum proteins and the senescent cell antigen  

Microsoft Academic Search

Normal human serum contains autoantibodies to a wide range of cellular and serum proteins. IgG autoantibodies to cell membrane proteins spectrin, syndein (Band 2.1), Band 3 degradation products, and the senescent cell antigen are among them. Physiologic autoantibodies to the senescent cell antigen, a ~62 000 dalton glycopeptide derived from Band 3, initiate removal of senescent, damaged, and stored cells

M. M. B. Kay; K. Sorensen; P. Wong; P. Bolton



Peripheral blood stem cell transplantation from human leukocyte antigen-matched sibling donors and unrelated donors in acute myeloid leukemia patients.  


There have been rare comparative studies of hematopoietic stem cell transplantation from matched sibling donors (MSDs) and unrelated donors (URDs) with regard to peripheral blood stem cell transplantation (PBSCT). We performed a retrospective study of 104 consecutive acute myeloid leukemia (AML) patients who had received an allogeneic PBSCT from an MSD or a URD in order to compare transplant outcomes and posttransplant complications between the 2 groups of patients. The cumulative incidence of grade 2-4 acute graft-versus-host disease (aGVHD) at 100 days (22.6% with MSD vs. 35.3% with URD; p = 0.107) and that of chronic GVHD (cGVHD) at 2 years (72.9% with MSD vs. 56.1% with URD; p = 0.153) was not significantly different between the 2 groups. Multivariate analysis also indicated that a URD was not an independent predictor of grade 2-4 aGVHD or cGVHD. No statistically significant differences were observed in terms of relapse incidence (p = 0.371), nonrelapse mortality (p = 0.473), disease-free survival (p = 0.925) or overall survival (p = 0.534) at 2 years. URDs are comparable with MSDs as a donor type for PBSCT in AML patients if risk-stratified GVHD prophylaxis is adopted. PMID:23816761

Kim, Hee-Je; Kim, Sung-Yong; Lee, Mark Hong; Min, Woo-Sung



Changes in structural and antigenic properties of proteins by radiation  

Microsoft Academic Search

Radiation effect on structural and antigenic properties of proteins (0.2% in 0.01 M phosphate buffer, pH 7.4) were investigated using ovalbumin (OVA) and bovine serum albumin (BSA). Aggregation of OVA and BSA was induced by radiation and the molecular mass increased significantly in N2. Significant changes in surface hydrophobicity and [?]222 nm of CD were also observed by radiation showing

Tamikazu Kume; Tsukasa Matsuda



Antibodies to Citrullinated Protein Antigens (ACPAs): Clinical and Pathophysiologic Significance  

Microsoft Academic Search

Antibodies to citrullinated protein antigens (ACPAs) are highly specific for rheumatoid arthritis (RA) and are useful in the\\u000a diagnosis of RA as well as the prediction of the course and outcomes of disease. Multiple methodologies exist for measuring\\u000a ACPAs, including the widely available tests for anticyclic citrullinated peptide antibodies and for antibodies to mutated\\/modified\\u000a citrullinated vimentin. These methodologies overall have

M. Kristen Demoruelle; Kevin Deane


Antigenic differences among Campylobacter fetus S-layer proteins.  

PubMed Central

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein. Images

Dubreuil, J D; Kostrzynska, M; Austin, J W; Trust, T J



Isolation of Antigens of Pasteurella Pestis. I. Lipopolysaccharide-Protein Complex and R and S Antigens.  

National Technical Information Service (NTIS)

Pasteurella pestis contains at least 18 different antigens, 2 of which will protect experimental animals from challenge infection. A specific polysaccharide isolated and described as a hapten was isolated as a complete antigen. Two additional antigens wer...

A. R. Larrabee J. D. Marshall D. Crozier



Quantitating protein synthesis, degradation, and endogenous antigen processing.  


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

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



Identification of major antigenic proteins of Pasteurella piscicida.  


Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicida rabbit serum from a genomic DNA library of P. piscicida strain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins in Escherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins of P. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein of P. piscicida with previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci in E. coli for the degQ and degS serine protease genes. A sequence in the 3' non-coding region of Vibrio hollisae thermostable hemolysin gene that is highly homologous with a similar located sequence in the Pseudomonas putida p-cresol methylhydroxylase gene is also found in the 3' non-coding region of the degS homolog gene of the PPA2. PMID:9441863

Hirono, I; Kato, M; Aoki, T



A novel Lawsonia intracellularis autotransporter protein is a prominent antigen.  


Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ? 72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent. PMID:21697340

Watson, Eleanor; Clark, Ewan M; Alberdi, M Pilar; Inglis, Neil F; Porter, Megan; Imrie, Lisa; McLean, Kevin; Manson, Erin; Lainson, Alex; Smith, David G E



A Novel Lawsonia intracellularis Autotransporter Protein Is a Prominent Antigen?  

PubMed Central

Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ?72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.

Watson, Eleanor; Clark, Ewan M.; Alberdi, M. Pilar; Inglis, Neil F.; Porter, Megan; Imrie, Lisa; Mclean, Kevin; Manson, Erin; Lainson, Alex; Smith, David G. E.



Identification of antigenic epitopes in a surface protein antigen of Streptococcus mutans in humans.  

PubMed Central

The reactivities of antibodies in human serum and saliva to a cell surface protein antigen (PAc) of Streptococcus mutans and synthetic peptides covering the PAc molecule were examined. Both an enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting) showed that all the serum samples from five adult subjects harboring serotype c S. mutans in their oral cavity reacted with recombinant PAc (rPAc). On the other hand, the serum from a 4-month-old infant did not react with rPAc in ELISA. The immunoglobulin A (IgA) antibodies in saliva samples from the five adult subjects reacted with rPAc. However, in saliva samples from these subjects, the titers of IgA antibody to rPAc did not correlate with the titers of serum antibody to the antigen. To map continuous antigenic epitopes in the PAc molecule, we synthesized 153 decapeptides covering the entire mature PAc molecule, 121 overlapping decapeptides covering the alanine-rich repeating region (A-region) of the PAc molecule, and 21 overlapping decapeptides covering the middle region (residues 824 to 853) according to multiple pin-coupled peptide synthesis technology. Of 153 decapeptides covering the mature PAc, 27 decapeptides showed a strong reaction with the antibodies in serum from the adult subjects. The epitope-scanning patterns in the serum samples from these subjects were also very similar to each other. The antigenic epitope patterns in the saliva resembled those in the serum. However, the ELISA titers of salivary IgA antibodies to these decapeptides differed from the titers of the serum antibody. Of the 121 overlapping decapeptides covering the A-region, 27 decapeptides showed a positive reaction with the antibodies in serum from the adult subjects. All of these 27 decapeptides had either one or two of the five common sequences YQAXL, NADAKA, VQKAN, NNAKNA, and IKKRNA. Six decapeptides of the 21 overlapping decapeptides covering the middle region reacted strongly with the serum antibodies from a high PAc responder, and each of the six decapeptides had one of the two common sequences KVTKEKP and VKPTAPTK. These epitopes might therefore be relevant to the humoral responses against the PAc protein during natural infection with S. mutans in humans. Images

Matsushita, K; Nisizawa, T; Nagaoka, S; Kawagoe, M; Koga, T



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

SciTech Connect

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.

Shanklin, J.; Somerville, C. (Michigan State Univ., East Lansing (United States))



Stress proteins and the immune response to mycobacteria — Antigens as virulence factors?  

Microsoft Academic Search

The immune response to mycobacterial infection includes pathogenic as well as protective activities. It is possible that different types of immune responses are associated with recognition of different antigenic determinants. Amongst the antigens which are prominent in antibody and T cell recognition of mycobacteria, we have identified members of highly conserved stress protein families. Mapping of antigenic determinants on stress

D B Young; A Mehlert; V Bal; P Mendez-Samperio; J Ivanyi; J R Lamb



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


Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 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. PMID:2006187

Shanklin, J; Somerville, C



Hallmarks of homology recognition by RecA-like recombinases are exhibited by the unrelated Escherichia coli RecT protein  

PubMed Central

Homologous recombination is a fundamental process for genome maintenance and evolution. Various proteins capable of performing homology recognition and pairing of DNA strands have been isolated from many organisms. The RecA family of proteins exhibits a number of biochemical properties that are considered hallmarks of homology recognition. Here, we investigated whether the unrelated Escherichia coli RecT protein, which mediates homologous pairing and strand exchange, also exhibits such properties. We found that, like RecA and known RecA homologs: (i) RecT promotes the co-aggregation of ssDNA with duplex DNA, which is known to facilitate homologous contacts; (ii) RecT binding to ssDNA mediates unstacking of the bases, a key step in homology recognition; (iii) RecT mediates the formation of a three-strand synaptic intermediate where pairing is facilitated by local helix destabilization, and the preferential switching of A:T base pairs mediates recognition of homology; and (iv) RecT-mediated pairing occurs from both 3?- and 5?-single-stranded ends. Taken together, our results show that RecT shares fundamental homology-recognition properties with the RecA homologs, and provide new insights on an underlying universal mechanism of homologous recognition.

Noirot, Philippe; Gupta, Ravindra C.; Radding, Charles M.; Kolodner, Richard D.



Comparison of Different Live Vaccine Strategies In Vivo for Delivery of Protein Antigen or Antigen-Encoding DNA and mRNA by Virulence-Attenuated Listeria monocytogenes  

Microsoft Academic Search

Listeria monocytogenes can be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and CD4 T cells when the antigen is secreted by L. monocy- togenes or when antigen-encoding plasmid DNA

Daniela I. M. Loeffler; Christoph U. Schoen; Werner Goebel; Sabine Pilgrim



Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins  

Microsoft Academic Search

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different

Rosário Gonçalves; José Regalla; Jacques Nicolet; Joachim Frey; Robin Nicholas; John Bashiruddin; Paola de Santis; Aires Penha Gonçalves



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

Microsoft Academic Search

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

Yen-Ling Yu; Kai-Wun Yeh; C.-P. Lin



Towards Preserving the Immunogenicity of Protein Antigens Carried by Nanoparticles While Avoiding the Cold Chain  

PubMed Central

Nanoparticles are an attractive vaccine carrier with potent adjuvant activity. Data from our previous studies showed that immunization of mice with lecithin/glyceryl monostearate-based nanoparticles with protein antigens conjugated onto their surface induced a strong, quick, and long-lasting antigen-specific immune response. In the present study, we evaluated the feasibility of preserving the immunogenicity of protein antigens carried by nanoparticles without refrigeration using these antigen-conjugated nanoparticles as a model. The nanoparticles were lyophilized, and the immunogenicity of the antigens was evaluated in a mouse model using bovine serum albumin or the Bacillus anthracis protective antigen protein as model antigens. With proper excipients, the nanoparticles can be lyophilized while maintaining the immunogenicity of the antigens. Moreover, the immunogenicity of the model antigen conjugated onto the nanoparticles was undamaged after a relatively extended period of storage at room temperature or under accelerated conditions (37°C) when the nanoparticles were lyophilized with 5% mannitol plus 1% polyvinylpyrrolidone. To our knowledge, the present study represents an early attempt to preserve the immunogenicity of the protein antigens carried by nanoparticles without refrigeration.

Sloat, Brian R.; Sandoval, Michael A.; Cui, Zhengrong



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

PubMed Central

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.

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



Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis  

Microsoft Academic Search

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was

Young-Bae Chung; Mejeong Lee; Hyun-Jong Yang; Byung-Suk Chung; Shun-Yu Lee; Min-Ho Choi; Sung-Tae Hong



Novel related cDNAs (C184L, C184M, and C184S) from developing mouse brain encoding two apparently unrelated proteins.  


Three related cDNAs (C184L, C184M, and C184S) were isolated from a developing mouse brain cDNA library. C184S is the 5'-end portion and C184M is the 3'-end portion, respectively, of C184L. C184S and C184M have open reading frames of 199 amino acids (ORF1) and 189 amino acids (ORF2), respectively; C184L has both ORF1 and ORF2 (dicistronic structure), but seems to translate only ORF1. Southern blot analysis suggests that all of the three related mRNAs are transcribed from the same single gene. The intervening region of C184L cDNA between ORF1 and ORF2 contained a promoter sequence for C184M mRNA, which is transcribed from the corresponding genomic sequence. Very recently, a cDNA encoding human homologue of ORF1 (human autoantigen p27) and a cDNA encoding a different mouse isoform of ORF2 (mammary tumor virus receptor) were reported. Our results indicate that the mRNAs encoding these apparently unrelated proteins are transcribed within an adjacent or overlapping area on the genome, suggesting the same origin of the two transcription units. PMID:10512749

Sakuma-Takagi, M; Tohyama, Y; Kasama-Yoshida, H; Sakagami, H; Kondo, H; Kurihara, T



Characterization of proteins Msp22 and Msp75 as vaccine antigens of Moraxella catarrhalis.  


Moraxella catarrhalis is a respiratory tract pathogen causing otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. This study examined two newly identified proteins as potential vaccine antigens. Antisera raised to recombinant purified proteins Msp22 and Msp75 recognized corresponding native proteins in multiple strains of M. catarrhalis. Vaccine formulations individually administered subcutaneously and intranasally showed enhanced clearance of M. catarrhalis in a mouse pulmonary clearance model by both routes of administration. Msp22 and Msp75 are antigenically conserved proteins that induce potentially protective immune responses and should be examined further as vaccine antigens for M. catarrhalis. PMID:19786139

Ruckdeschel, Elizabeth A; Brauer, Aimee L; Johnson, Antoinette; Murphy, Timothy F



Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin.  


The trophozoites of Giardia lamblia possess several protein antigens, predominant among them a protein of approximately 32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety of G. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to alpha-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is alpha-1-giardin. PMID:8278341

Wenman, W M; Meuser, R U; Nyugen, Q; Kilani, R T; el-Shewy, K; Sherburne, R



Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool  

Microsoft Academic Search

The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by




Tumor antigen precursor protein profiles of adult and pediatric brain tumors identify potential targets for immunotherapy  

Microsoft Academic Search

Objectives We evaluated and compared tumor antigen precursor protein (TAPP) profiles in adult and pediatric brain tumors of 31 genes\\u000a related to tumor associated antigens (TAA) for possible use in immunotherapy. Antigens were selected based on their potential\\u000a to stimulate T cell responses against tumors of neuroectodermal origin. Methods Thirty-seven brain tumor specimens from 11 adult and 26 pediatric patients

Jian Gang Zhang; Carol A. Kruse; Lara Driggers; Neil Hoa; Jeffrey Wisoff; Jeffrey C. Allen; David Zagzag; Elizabeth W. Newcomb; Martin R. Jadus



Importance of the Crystalline Surface Layer Protein Antigens of Rickettsiae in T-cell Immunity.  

National Technical Information Service (NTIS)

Studies in animal models have demonstrated that solid immunity to typhus rickettsiae is dependent on immune T cells. In addition, the surface protein antigen (SPA) of typhus rickettsiae has been shown to be an effective immunogen, protecting vaccinated an...

M. Carl G. A. Dasch



Inhibition of MHC class I antigen presentation by viral proteins  

Microsoft Academic Search

An essential part of the immune response to viral infections is the recognition and elimination of infected cells by cytotoxic\\u000a T lymphocytes. For this purpose a display mechanism has evolved which is present in almost all nucleated cells: the major\\u000a histocompatibility complex class I antigen processing pathway. Both self and foreign antigens are degraded in the cytosol\\u000a to peptides which

K. Früh; K. Ahn; P. A. Peterson



Analysis of transcutaneous antigenic protein delivery by a hydrogel patch formulation.  


We have developed a hydrogel patch, which could promote antigen penetration through stratum corneum (SC), and have demonstrated its safety and efficacy in animals and humans. For the availability improvement of our system, it is important to develop a device, which enhances antigen penetration through SC more efficiently. In this study, we have tried to collect the basic information involved in transcutaneous antigen delivery by investigating the immune event induced by our system and examining the effect of physical property of antigens or patch component on antigen penetration. A hydrogel patch delivered antigens through SC into skin, and some of Langerhans cells captured antigens, activated, and migrated to regional lymph nodes. We also showed that protein distribution into SC was regulated by various complexly-intertwined factors of proteins but not one particular parameter. Additionally, glycerin as the patch component contributed to the formation of SC hydration by patch application, which might be one of the factors of acceleration of protein penetration. On the basis of the present information, we are planning to modify the patch composition and establish the antigen modification technology for improvement in the efficacy of transcutaneous immunization. PMID:23585300

Matsuo, Kazuhiko; Ishii, Yumiko; Kawai, Yasuaki; Saiba, Yuki; Quan, Ying-Shu; Kamiyama, Fumio; Hirobe, Sachiko; Okada, Naoki; Nakagawa, Shinsaku



A model for the hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins.  

PubMed Central

The sequences of the core proteins from several serotypes of human hepatitis B virus and related mammalian and avian hepadnaviruses are aligned with the vp3 capsid protein of mengo virus, a picornavirus. The homology indicates an eight-stranded antiparallel beta-barrel fold for the hepatitis protein, as observed in the tertiary structure of the picornavirus protein. The locations of known antigenic sites and other modifications are consistent with this structure for the core protein. The predicted folding suggests additional exposed antigenic sites and supports an evolutionary relationship between this family of enveloped DNA viruses and enveloped and non-enveloped RNA viruses.

Argos, P; Fuller, S D



Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments  

PubMed Central

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive. ImagesFig. 2.

Parish, C. R.; Wistar, R.; Ada, G. L.



Responses of Bovine WC1+ ?? T Cells to Protein and Nonprotein Antigens of Mycobacterium bovis  

PubMed Central

WC1+ ?? T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, ?? T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1+ ?? T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1+ ?? T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1+ ?? T cells in comparison with CD4+ ?? T cells. Both cell types proliferated strongly in response to MBSE, with CD4+ T cells being the major producers of gamma interferon (IFN-?). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4+ T-cell responses, whereas some WC1+ ?? T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-? secretion in WC1+ ?? T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1+ ?? T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1+ ?? T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1+ ?? T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1+ ?? T-cell proliferation and IFN-? secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.

Welsh, Michael D.; Kennedy, Hilary E.; Smyth, Allister J.; Girvin, R. Martyn; Andersen, Peter; Pollock, John M.



Responses of Bovine WC1+   T Cells to Protein and Nonprotein Antigens of Mycobacterium bovis  

Microsoft Academic Search

WC1 T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1 T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought

Michael D. Welsh; Hilary E. Kennedy; Allister J. Smyth; R. Martyn Girvin; Peter Andersen; John M. Pollock



HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen  

PubMed Central

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule High mobility group box (HMGB1) protein potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-?, TNF-?, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines.

Saenz, R.; da Silva Souza, C.; Huang, C-T; Larsson, M.; Esener, S.; Messmer, D.



Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and Neisserial heparin binding antigen  

Microsoft Academic Search

A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies

David M. Vu; Tracy T. Wong; Dan M. Granoff



Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.  

PubMed Central

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation. Images

Wang, E; Garcia, M M; Blake, M S; Pei, Z; Blaser, M J



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

SciTech Connect

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.

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



Protein and antigenic composition of adult and microfilarial stages of Setaria cervi.  


The protein and antigenic pattern of adult (female/male) and microfilarial stages of Setaria cervi, a bovine filarial parasite, was studied using certain immunochemical techniques. SDS-polyacrylamide gel electrophoretic analysis showed the presence of 35-40 protein bands in adults and 25-29 protein bands in microfilariae in molecular weight range of 10,000-200,000. Immunoelectrophoresis revealed the presence of 9-10 precipitin lines in adult and only 4 precipitin lines in microfilarial antigenic preparations. Crossed immunoelectrophoresis resolved these antigenic preparations further, and revealed the presence of 22-24 antigens in adults and 12-14 in microfilariae. These results demonstrate complex nature of somatic extracts of adult stage as compared to microfilariae and also reveal some qualitative and quantitative differences between these stages. PMID:2432003

Malhotra, A; Kaushal, N A; Ghatak, S



The identification of antigenic proteins: 14-3-3 protein and propionyl-CoA carboxylase in Clonorchis sinensis.  


Clonorchis sinensis, the causative agent of clonorchiasis, is widespread in East and Southeast Asia, including China, Vietnam and the Republic of Korea. We identified antigenic proteins from adult C. sinensis liver flukes using immunoproteomic analysis. In this study, we found 23 candidate antigenic proteins with a pI in the range of 5.4-6.2 in total lysates of C. sinensis. The antigenic protein spots reacted against sera from clonorchiasis patients and were identified as cysteine proteases, glutathione transferases, gelsolin, propionyl-CoA carboxylase (PCC), prohibitin and 14-3-3 protein (14-3-3) using LC-coupled ESI-MS/MS and an EST database for C. sinensis. PCC and 14-3-3 were identified for the first time as serological antigens for the diagnosis of C. sinensis. To validate the antigenicity of PCC and 14-3-3, recombinant proteins were immunoblotted with sera from clonorchiasis patients. The structural, functional and immunological characteristics of the putative amino acid sequence were predicted by bioinformatics analysis. Our novel finding will contribute to the development of diagnostics for clonorchiasis. These results suggest that immunoproteomic approaches are valuable tools to identify antigens that could be used as targets for effective parasitic infection control strategies. PMID:22119288

Lee, Myoung-Ro; Kim, Yu-Jung; Kim, Dae-Won; Yoo, Won Gi; Cho, Shin-Hyeong; Hwang, Kwang Yeon; Ju, Jung-Won; Lee, Won-Ja



Characterization of a protective protein antigen of Erysipelothrix rhusiopathiae.  

PubMed Central

Although vaccination is widely practiced against infection by Erysipelothrix rhusiopathiae in pigs and turkeys, the protective antigen(s) involved have not been fully characterized or purified to homogeneity. Antigens of E. rhusiopathiae strain T28, serotype 2b, and of FRANKFURT XI, serotype N, in culture supernatant and in extracts made with hot acid, 10 mM NaOH, ultrasound or EDTA were compared by SDS-PAGE and immunoblotting and in a mouse protection test. EDTA and 10 mM NaOH yielded highly protective extracts; culture supernatant was less protective and ultrasonic or hot acid extracts stimulated little or no protection in mice. Protective antisera from swine, horses and mice recognized prominent bands of molecular mass (m.m.) of 66-64 and 40-39 kDa in EDTA and 10 mM NaOH extracts. Mice immunized with preparations of the 66-64 kDa band purified by preparative electrophoresis were protected. Both antigens were trypsin sensitive, contained no detectable polysaccharide, and showed a marked tendency to aggregate in the absence of SDS. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Groschup, M. H.; Cussler, K.; Weiss, R.; Timoney, J. F.



Mycobacterium tuberculosis-Secreted Protein Antigens: Immunogenicity in Baboons  

Microsoft Academic Search

The effective control of tuberculosis (TB) requires the development of improved vaccines. It is now well established that Mycobacterium tuberculosis-secreted antigens represent promising candidates to be included in subunit vaccine preparations. It also is accepted that studies in nonhuman primate models will be required to further develop these vaccine preparations. As a necessary step in this direction, we have assessed

Karen Pehler; Kathleen M. Brasky; Thomas M. Butler; Roberta Attanasio



Antigenic determinants of the chlamydial major outer membrane protein resolved at a single amino acid level.  

PubMed Central

Antigenic determinants were identified from seven chlamydial major outer membrane proteins by using overlapping hexapeptides and polyclonal antisera. Sixty-one determinants were detected, and 30 were surface exposed on the native organisms. The two negatively charged residues, aspartic acid and glutamic acid, were found most often in determinants. Thirteen antigenic sites were further characterized by alanine substitution. Differences in fine specificities of these linear determinants were observed in alanine substitution profiles. Five determinants had adjacent critical residues, while eight had critical residues alternated with noncritical residues. Complete replacement analysis of two antigenic determinants provided more detailed information for elucidating the structural basis of the specificity of antigen-antibody interaction and suggested a correlation between sequence conservation and tolerance to amino acid substitution for antigenic sites subject to intense immune selection pressure.

Zhong, G M; Brunham, R C



Expression of Bcl-2 protein and Fas antigen in non-Hodgkin's lymphomas.  

PubMed Central

Expression of Bcl-2 protein and Fas antigens was analyzed in 12 cases of follicular lymphoma and 32 cases of diffuse lymphoma, including 22 B-cell and 10 T-cell lymphomas. It was shown that 75% of follicular lymphomas had clear expression of both Bcl-2 protein and Fas antigen. Thus, follicular lymphomas may have a growth advantage due to their high expression of Bcl-2 protein, which tended to impede apoptosis mediated by Fas antigen. On the other hand, diffuse lymphomas showed various patterns; 28% were double positive, 16% were only Bcl-2 protein-positive, 28% were only Fas antigen-positive, and 28% were double negative or equivocal. Cytocidal assay of seven leukemia/lymphoma cell lines using anti-human Fas monoclonal antibody revealed that overexpression of Bcl-2 protein tended to impede apoptosis mediated by Fas antigen. However, this inhibitory effect of Bcl-2 protein was incomplete and its effect might be dependent upon cell type. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

Kondo, E.; Yoshino, T.; Yamadori, I.; Matsuo, Y.; Kawasaki, N.; Minowada, J.; Akagi, T.



Receptor-mediated Uptake of Antigen\\/Heat Shock Protein Complexes Results in Major Histocompatibility Complex Class I Antigen Presentation via Two Distinct Processing Pathways  

Microsoft Academic Search

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen- specific CD8 1 T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we

Flora Castellino; Philip E. Boucher; Katrin Eichelberg; Mark Mayhew; James E. Rothman; Alan N. Houghton; Ronald N. Germain


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


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. PMID:752255

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


Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells  

Microsoft Academic Search

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a

Michael Majewski; Tina O. Bose; Fenna C. M. Sille; Annette M. Pollington; Edda Fiebiger; Marianne Boes



Structural elements of a protein antigen determine immunogenicity of the embedded MHC class I-restricted T cell epitope.  


Substantial effort has been invested into optimization of vector structure, DNA formulation, or delivery methods to increase the effectiveness of DNA vaccines. In contrast, it has been only insufficiently explored how the higher order structure of an antigenic protein influences immunogenicity of embedded epitopes in vivo. Potent CD8+ T cell responses specific for a single immunogenic epitope are induced upon electrovaccination with plasmid DNA encoding the full-length heavy chain of the human HLA-Cw3 molecule. Contrary to expectations, a minimal construct, which provoked a substantial release of IFN-gamma from specific CTLs in vitro, did not induce a significant response in vivo. Systematically altered variants of the Cw3 molecule were thus tested both in vivo and in vitro to determine which structural parts are responsible for this discrepancy. In complementation experiments the participation of trans-acting helper epitopes was ruled out. Successive C-terminal truncations, human/mouse domain swap variants, and subdomain modifications defined the alpha3 region of the HLA heavy chain and membrane anchoring as critical elements. Based on these data, refined minimal constructs were engineered that triggered very high in vivo responses. The most advanced variant consisted only of an adenoviral leader, antigenic epitope, alpha3 domain, and 16 aa of the transmembrane domain. When a tumor Ag epitope was incorporated into one of these high performer minimal constructs, protection against melanoma metastases was attained upon vaccination. Thus, structural elements of the Ag can dominantly influence immunogenicity in vivo. These elements can also markedly improve the immunogenicity of unrelated Ags and may form the basis of a new generation of DNA vaccines. PMID:12218107

Paster, Wolfgang; Kalat, Milena; Zehetner, Margit; Schweighoffer, Tamás



Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague  

Microsoft Academic Search

The pathogenicYersiniaeproduce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection withY. pestisin murine models, and is considered to be a protective antigen. In this

Sophie E. C. Leary; Kate F. Griffin; Edouard E. Galyov; Jason Hewer; E. Diane Williamson; Anna Holmström; Åke Forsberg; Richard W. Titball



Epicutaneous Application of Protein Antigens Incorporated into Cosmetic Cream Induces Antigen-Nonspecific Unresponsiveness in Mice and Affects the Cell-Mediated Immune Response  

Microsoft Academic Search

Background: Protein antigens applied epicutaneously by the patch method induce allergic dermatitis mediated by IgE antibodies in mice and simultaneously significant suppression of Th1-mediated delayed-type hypersensitivity (DTH) reactions. Methods: We developed a method in which protein antigens (calf collagen, elastin, keratin, TNP-substituted mouse Ig) were homogenized with neutral cream. Animals treated epicutaneously with such preparations were tested for contact sensitivity

Marian Szczepanik; Krzysztof Bryniarski; Monika Tutaj; Maria Ptak



Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses.  


Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress. PMID:11855626

Jiang, X; Zhong, W M; Farkas, T; Huang, P W; Wilton, N; Barrett, E; Fulton, D; Morrow, R; Matson, D O



Enhancing immunoassay detection of antigens with multimeric protein Gs  

Microsoft Academic Search

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)3 linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO2-MNPs, Fe3O4@SiO2) were prepared and coupled to the protein

Jin Hyung Lee; Hong Kyung Choi; Soo Youn Lee; Myung-Woon Lim; Jeong Ho Chang



HAGE, a cancer/testis antigen expressed at the protein level in a variety of cancers  

PubMed Central

The search for novel tumour antigens that are either uniquely expressed or over-expressed in a wide variety of tumours is still ongoing. Because of their expression in a broad spectrum of cancers and limited expression in normal tissues, cancer/testis antigens are considered to be potentially reliable targets for immunotherapy of cancer in general. The helicase antigen HAGE has been identified as a cancer/testis antigen. However, little is known about its expression in normal and cancer tissues. Using a newly developed antibody against HAGE, specific staining of its expression by immunohistochemistry was validated and optimised on murine tumours transfected to express the HAGE protein. The antibody was subsequently used to determine HAGE expression in normal human and cancer tissue microarrays. HAGE protein expression was confirmed in 75% (12/16) of carcinomas as compared to normal tissues, which either did not express HAGE at all or expressed HAGE at very low levels with the exception of testis. Interestingly, discrepancies were also found between mRNA analysis by real time quantitative PCR (RT-qPCR) and protein analysis by immunohistochemistry, emphasising the need to validate the expression of cancer/testis antigens at the protein level prior to the development of new vaccine strategies. HAGE is therefore proposed to be a valid candidate for designing a broad spectrum vaccine against cancer.

Mathieu, Morgan G.; Linley, Adam J.; Reeder, Stephen P.; Badoual, Cecile; Tartour, Eric; Rees, Robert C.



Antigen vehiculization particles based on the Z protein of Junin virus  

PubMed Central

Background Arenavirus matrix protein Z plays an important role in virus budding and is able to generate enveloped virus-like-particles (VLPs) in absence of any other viral proteins. In these VLPs, Z protein is associated to the plasma membrane inner surface by its myristoyl residue. Budding induction and vesicle formation properties can be exploited to generate enveloped VLPs platform. These structures can be designed to carry specific antigen in the inner side or on the surface of VLPs. Vaccines based on VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles in absence of viral nucleic acid, being noninfectious. In this work we assayed the capacity of Junin Z protein to produce VLPs carrying the green fluorescent protein (eGFP), as a model antigen. Results In this report the Junin Z protein ability to produce VLPs from 293T cells and its capacity to deliver a specific antigen (eGFP) fused to Z was evaluated. Confocal microscopy showed a particular membrane bending in cells expressing Z and a spot welded distribution in the cytoplasm. VLPs were detected by TEM (transmission electron microscopy) and were purified from cell supernatant. The proteinase protection assay demonstrated the VLPs integrity and the absence of degradation of the fused antigen, thus indicating its internal localization. Finally, immunization of mice with purified VLPs produced high titres of anti-eGFP antibodies compared to the controls. Conclusions It was proved that VLPs can be generated from cells transfected with a fusion Junin virus Z-eGFP protein in absence of any other viral protein, and the capacity of Z protein to support fusions at the C-terminal, without impairing its budding activity, allowing vehiculization of specific antigens into VLPs.



Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins  

Microsoft Academic Search

Summary.  ?A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of\\u000a the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54\\/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia\\u000a ni larvae, retained antigenic determinants present in both proteins and reacted in

M. G. Barderas; F. Rodríguez; P. Gómez-Puertas; M. Avilés; F. Beitia; C. Alonso; J. M. Escribano



The scaffold protein prohibitin is required for antigen-stimulated signaling in mast cells.  


The protein prohibitin (PHB) is implicated in diverse cellular processes, including cell signaling, transcriptional control, and mitochondrial function. We found that PHB was abundant in the intracellular granules of mast cells, which are critical for allergic responses to antigens. Thus, we investigated whether PHB played a role in signaling mediated by the high-affinity receptor for antigen-bound immunoglobulin E (IgE), Fc?RI. PHB-specific small interfering RNAs (siRNAs) inhibited antigen-mediated signaling, degranulation, and cytokine secretion by mast cells in vitro. Knockdown of PHB inhibited the antigen-dependent association of the tyrosine kinase Syk with Fc?RI and inhibited the activation of Syk. Fractionation studies revealed that PHB translocated from intracellular granules to plasma membrane lipid rafts in response to antigen, and knockdown of PHB suppressed the movement of Fc?RI? and Syk into lipid rafts. Tyrosine phosphorylation of PHB by Lyn was observed early after exposure to antigen, and point mutations in PHB indicated that Tyr(114) and Tyr(259) were required for the recruitment of Syk to Fc?RI? and mast cell activation. In mice, PHB-specific siRNAs inhibited antigen-initiated mast cell degranulation, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. Together, these results suggest that PHB is essential for Fc?RI-mediated mast cell activation and allergic responses in vivo, raising the possibility that PHB might serve as a therapeutic target for the treatment of allergic diseases. PMID:24023254

Kim, Do Kyun; Kim, Hyuk Soon; Kim, A-Ram; Jang, Geun Hyo; Kim, Hyun Woo; Park, Young Hwan; Kim, Bokyung; Park, Yeong Min; Beaven, Michael A; Kim, Young Mi; Choi, Wahn Soo



Modified anthrax fusion proteins deliver HIV antigens through MHC Class I and II pathways.  


T cell-based HIV vaccine candidates have focused on eliciting both CD4- and CD8-mediated responses. One challenge in vaccine development is the successful introduction and presentation of exogenous antigen to elicit an immune response. Modified bacterial toxins have been studied extensively as intracellular delivery agents because of their unique capability to translocate antigen across the cell membrane without affecting cell viability. Modified anthrax toxin lethal factor (LFn) fusion protein is able to effectively induce anti-HIV cytotoxic T lymphocytes in the absence of protective antigen (PA) and is being evaluated as a vaccine candidate. Here we describe, for the first time, the processing and presentation of LFn fusion proteins by the MHC Class II pathway. The ability of LFn--HIV to induce both CD8- and CD4-mediated responses may have relevance in current approaches to vaccine design. Furthermore, the translocation and presentation of antigens occurs in the absence of PA, which proposes a modified molecular mechanism of antigen presentation by the anthrax toxin model. Additionally, we found that LFn--HIV is specific and sensitive in detecting HIV-specific CD4(+) and CD8(+) T cell responses in T cell assays, further broadening the value of this antigen delivery system as a useful immunologic tool. PMID:15964481

McEvers, K; Elrefaei, M; Norris, P; Deeks, S; Martin, J; Lu, Y; Cao, H



Responses of bovine WC1(+) gammadelta T cells to protein and nonprotein antigens of Mycobacterium bovis.  


WC1(+) gammadelta T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, gammadelta T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1(+) gammadelta T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1(+) gammadelta T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1(+) gammadelta T cells in comparison with CD4(+) alphabeta T cells. Both cell types proliferated strongly in response to MBSE, with CD4(+) T cells being the major producers of gamma interferon (IFN-gamma). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4(+) T-cell responses, whereas some WC1(+) gammadelta T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-gamma secretion in WC1(+) gammadelta T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1(+) gammadelta T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1(+) gammadelta T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1(+) gammadelta T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1(+) gammadelta T-cell proliferation and IFN-gamma secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development. PMID:12379688

Welsh, Michael D; Kennedy, Hilary E; Smyth, Allister J; Girvin, R Martyn; Andersen, Peter; Pollock, John M



Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis  

PubMed Central

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.

Chung, Young-Bae; Lee, Mejeong; Yang, Hyun-Jong; Chung, Byung-Suk; Lee, Shun-Yu; Hong, Sung-Tae



Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis.  


The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins. PMID:12073733

Chung, Young-Bae; Lee, Mejeong; Yang, Hyun-Jong; Chung, Byung-Suk; Lee, Shun-Yu; Choi, Min-Ho; Hong, Sung-Tae



Antigenic characterization of ehrlichiae: protein immunoblotting of Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii.  

PubMed Central

In recent years a febrile illness apparently associated with tick bite in patients in the United States has been attributed to infection by an Ehrlichia species. This implication is based on serologic responses to E. canis, morphologic demonstration of ehrlichiae in clinical materials, and a single isolate distinct from E. canis which was obtained from a human patient by the Centers for Disease Control. Little is known about the antigens of the ehrlichiae. This report expands the breadth of available knowledge concerning the antigenic components and serologic responses to component antigens of E. canis, E. sennetsu, and E. risticii. Protein immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using density gradient-purified ehrlichiae and homologous antisera demonstrated reproducible and characteristic antigens within each species (for E. sennetsu, 91, 64, 54, 44, 36, 34, 28, 25, and 24 kDa; for E. risticii, 70, 52, 48, 44, 35, 28, 24, 23, and 20 kDa; for E. canis, 110, 64, 52, 42, 33, 28, 24, 23, and 20 kDa). When antisera were reacted with heterologous antigens, cross-reactivity among these species was virtually restricted to the 70-kDa antigen. Furthermore, when serum samples obtained from 10 patients who were convalescing from ehrlichiosis were tested against each antigen, only three serum samples had any reactivities, and these serum samples reacted with only a few of the antigenic bands. These results documented the molecular sizes of electrophoretically separated antigens of the three Ehrlichia species, confirm their serologic relationships, and support the novel nature of the agent(s) of human ehrlichiosis in the United States. Images

Brouqui, P; Dumler, J S; Raoult, D; Walker, D H



Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.  

PubMed Central

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Seong, S Y; Park, S G; Huh, M S; Jang, W J; Kim, H R; Han, T H; Choi, M S; Chang, W H; Kim, I S



Occurrence of antibodies to pneumococcal protein antigens in experimental acute otitis media.  


To study serological antibody response to pneumococcal protein antigens, experimental pneumococcal (type 3) acute otitis media (AOM) was induced in 6 rats, sera being analysed with the Western blot technique at different intervals after bacterial challenge. The most striking finding was a distinct antibody response to a protein of about 35 kDa visible in 5 of the rat sera within 14 days, and persisting throughout the remainder of the study (i.e., 56 days in all). Moreover, appear to be a phenomenon restricted to type 3 pneumococci. Both the pathogenetic importance of this protein and the immunological response it evokes are still unclear. However, as antibodies to protein antigens may contribute to inflammatory reactions in the middle ear (e.g., otitis media with effusion), this 35-kDa protein might be important for the development of sequelae to AOM. PMID:1441994

Svinhufvud, M; Akkoyunlu, M; Renneberg, J; Christensen, P; Prellner, K



Baculovirus expression and antigenic characterization of classical swine fever virus E2 proteins.  


Genes encoding a major structural glycoprotein, E2, of classical swine fever viruses (CSFV) Brescia (subgroup 1.2), Paderborn (subgroup 2.1) and Kanagawa (subgroup 3.4) were constructed by removing the transmembrane domain and adding a C-terminal 6 histidine (His) tag. All the E2 constructs were efficiently expressed in a baculovirus system as 53-kDa glycosylated proteins that were identified in Western blots by their reaction with anti-His and CSFV-specific antibodies. These proteins were used as ELISA antigens to confirm the existence of an antigenic relationship between the viruses using group-specific polyclonal antisera. Antigenic differences were identified by Western blot and ELISA reactivity of the E2 proteins with a panel of monoclonal antibodies. Specifically, one monoclonal antibody (WH303) reacted with all three proteins, two monoclonal antibodies (M1660 and M1665) reacted with only the Brescia E2 protein, and three monoclonal antibodies (M1654, M1664 and M1669) reacted equally well with only Brescia and Kanagawa E2 proteins. Therefore, antibody reactivity profiles, established using recombinant E2 proteins, could be used to quickly identify novel CSFV strains as illustrated in this report with only a limited number of monoclonal antibodies. These proteins could also have added utility in the production of monoclonal antibodies and as critical reagents in diagnostic assays. PMID:22510427

Luo, L; Nishi, K; Macleod, E; Sabara, M I; Lin, M; Handel, K; Pasick, J



IFN-? enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation  

PubMed Central

We compared monocyte-derived dendritic cells and transforming growth factor-?1-induced Langerhans-like cells (LCs) for their capacity to cross-present exogenous NY-ESO-1 protein/antibody immune complexes to an NY-ESO-1-specific CD8+ T cell clone. In contrast to dendritic cells, LCs were not able to cross-present NY-ESO-1 to the T cell clone constitutively but did so after treatment with IFN-?. Remarkably, this IFN-?-inducible characteristic was due neither to enhanced antigen uptake nor to facilitated antigen processing in LCs. Rather, IFN-? acted at least in part by potentiating the maturation of otherwise refractory LCs, enabling in turn exogenous antigen to reach the processing machinery. This model of conditional cross-presentation establishes an original level of action for IFN-? as an effective immune modulator and supports the use of IFN-? in protein vaccination strategies targeting LCs.

Matsuo, Mitsutoshi; Nagata, Yasuhiro; Sato, Eiichi; Atanackovic, Djordje; Valmori, Danila; Chen, Yao-Tseng; Ritter, Gerd; Mellman, Ira; Old, Lloyd J.; Gnjatic, Sacha



Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses II. Mammalian Leukemia-Sarcoma Viruses  

PubMed Central

Proteins of leukemia and sarcoma viruses from the chicken, mouse, hamster, and cat were analyzed by gel filtration in guanidine hydrochloride. The mammalian viruses were found to contain six major proteins, whereas the avian viruses contained seven proteins. Proteins of viruses from different mammalian species had the same molecular weights which also closely resembled the molecular weights of the six equivalent avian viral proteins. These results defined a basic similarity in protein composition for the C-type viruses of avian and mammalian origin. The two glyco-proteins of murine leukemia virus were identified immunologically as constituents of the viral membrane. Antisera prepared against other proteins distinguished individual internal viral antigens in immunodiffusion tests and also reacted in immunofluorescence with cytoplasmic components of infected cells. Antisera which reacted with the major internal viral proteins did not contain antibodies inhibitory to the viral reverse transcriptase. Images

Nowinski, Robert C.; Fleissner, Erwin; Sarkar, Nurul H.; Aoki, Tadao



Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite.  


Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite. PMID:2516802

Souto-Padrón, T; Reyes, M B; Leguizamon, S; Campetella, O E; Frasch, A C; de Souza, W



Fusion Protein Vaccines Targeting Two Tumor Antigens Generate Synergistic Anti-Tumor Effects  

PubMed Central

Introduction Human papillomavirus (HPV) has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(?III)/E6 and PE(?III)/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. Materials and Methods In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. Results PE(?III)/E6+PE(?III)/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(?III)/E6 group compared to 100% in the PE(?III)/E7 and PE(?III)/E6+PE(?III)/E7 groups. Mice vaccinated with the PE(?III)/E6+PE(?III)/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(?III)/E6 or PE(?III)/E7 fusion proteins alone. Conclusion Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies.

Cheng, Wen-Fang; Chang, Ming-Cheng; Sun, Wei-Zen; Jen, Yu-Wei; Liao, Chao-Wei; Chen, Yun-Yuan; Chen, Chi-An



Molecular Analysis of Group B Protective Surface Protein, a New Cell Surface Protective Antigen of Group B Streptococci  

Microsoft Academic Search

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary

Sezgin Erdogan; Peter K. Fagan; Susanne R. Talay; Manfred Rohde; Patricia Ferrieri; Aurea E. Flores; Carlos A. Guzmán; Mark J. Walker; Gursharan S. Chhatwal



Heat Stress Increases Protein Antigen Transport Across the Intestinal Epithelium Via a Mechanism of Impairing Proteolytic Enzymatic Activity  

Microsoft Academic Search

It has not been fully understood how intact protein antigens escape digestion in the course of absorption. The present study was designed to investigate the mechanism that heat stress induced an increase in intact protein antigen absorption. Human colonic cell line Caco-2 cells were treated with high temperature (37 to 43°C) for 60 min. Epithelial permeability was evaluated by horseradish peroxidase

P.-C. Yang; C.-S. Wang



[Antigenic relationship between nucleocapsid proteins of phyto- and zoorhabdoviruses].  


The methods of electrophoresis in PAAG and immunological method were used for comparative analysis of structural proteins of phytorhabdovirus of potato curly dwarf (PCDV) and zoorhabdoviruses-vesicular stomatitis virus (VSV) and fixed rabies Virus (RV). Molecular weight of viral proteins was determined by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The proteins with molecular weight 45-51 kD, are probably, the major component of the viral nucleocapsid. Nucleocapsid protein 45 kD RV virus was isolated by the method of preparative electrophoresis and then the monospecific serum was obtained. The Ouchterlony and immunoblotting method were used to show, that nucleocapsid proteins with molecular weights 51 and 45 kD both of phytorhabdovirus PCDV and zoorhabdoviruses VSV and RV are serologically related. The obtained data may be used in biotechnology as the basis for creation of a new class of diagnostic preparations with the purpose to detect RV virus using proteins of curly potato dwarf virus and may be also used in serological tests to reveal viruses of Rhabdoviridae family in various eukaryotic objects. PMID:16018215

Maksymenko, L O; Parkhomenko, N I; Didenko, L F; Diachenko, N S; Olevyns'ka, Z M


Enhancement of DNA vaccine potency by antigen linkage to IFN-?-inducible protein-10.  


DNA vaccines have emerged as an attractive approach to generate antigen-specific T-cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1-polarizing chemokine, IP-10, to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, enhanced DNA vaccine potency. IP-10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP-10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP-10/E7 DNA exhibited a dramatic increase in the number of E7-specific CD4(+) Th1 T-cells and CD8(+) T-cells and, consequently, were strongly resistant over the long term to E7-expressing tumors compared to mice vaccinated with wild-type E7 DNA. Thus, because of the increase in tumor antigen-specific T-cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP-10 linked to a tumor antigen holds great promise for treating tumors. PMID:20473881

Kang, Tae Heung; Kim, Keon Woo; Bae, Hyun Cheol; Seong, Seung-Yong; Kim, Tae Woo



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

PubMed Central

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.

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



Protection against Mycobacterium avium by DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein  

PubMed Central

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.

Velaz-Faircloth, Maria; Cobb, Alison J.; Horstman, Amanda L.; Henry, Stanley C.; Frothingham, Richard



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

Microsoft Academic Search

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.

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



Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains  

Microsoft Academic Search

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates ob- tained from three continents over 27 years. Anti- genic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the pre- viously established subgroups A and AB. A third pattern was produced

M. Elvander; C. Baule; A. Ballagi-Porda; S. Bela


Display of the antigenic region of Nipah virus nucleocapsid protein on hepatitis B virus capsid.  


The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP???????) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP??????? increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera. PMID:22024533

Yap, Wei Boon; Tey, Beng Ti; Alitheen, Noorjahan Banu Mohamed; Tan, Wen Siang



Association of B lymphocyte antigen receptor polypeptides with multiple chaperone proteins  

Microsoft Academic Search

The B cell antigen receptor (BCR) is comprised of four different polypeptides, immunoglobulin (Ig) heavy chain, Ig light chain, and the two signaling subunits of this receptor, Ig-? and Ig-?. These four chains must assemble correctly in the endoplasmic reticulum (ER) before the BCR can be transported to the cell surface. The roles of the different chaperone proteins in mediating

Shaun P Foy; Linda Matsuuchi



Development of a Fish Stress Protein Antibody/Antigen-Based Approach for Biomonitoring of Water Quality.  

National Technical Information Service (NTIS)

The purpose of this research was to extend the qualitative and quantitative approach to the application of an antibody/antigen-based method which assess actual levels of stress proteins in fish and other aquatic organisms exposed to both acute and chronic...

E. G. Zimmerman K. L. Dickson M. J. Donahue



Structural Characterizations of Protein Antigens for The Next- Generation Vaccines Against Anthrax and Plague.  

National Technical Information Service (NTIS)

Two recombinant protein antigens called rPA and F1-V are recommended by the U.S. Army as active pharmaceutical ingredients (API) for the next- generation vaccines to protect the warfighter against aerosolized anthrax and plague. Separate candidate vaccine...

B. Powell W. Ribot J. Adamovicz G. Andrews J. Enama



Human Hepatocellular Carcinoma Cell Lines Secrete the Major Plasma Proteins and Hepatitis B Surface Antigen  

Microsoft Academic Search

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B is injected into athymic mice, metastatic hepatocellular carcinomas

Barbara B. Knowles; Chin C. Howe; David P. Aden



Identification of Novel Mycobacterium bovis Antigens by Dissection of Crude Protein Fractions? †  

PubMed Central

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-?) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-? release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.

Meikle, V.; Alito, A.; Llera, A. S.; Gioffre, A.; Peralta, A.; Buddle, B. M.; Cataldi, A.



Plasmodium falciparum Merozoite Surface Protein 6 Is a Dimorphic Antigen  

PubMed Central

Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition.

Pearce, J. Andrew; Triglia, Tony; Hodder, Anthony N.; Jackson, David C.; Cowman, Alan F.; Anders, Robin F.



Antigenic analysis of the major outer membrane protein of Haemophilus somnus with monoclonal antibodies.  

PubMed Central

The major outer membrane protein of Haemophilus somnus possesses at least five distinct epitopes. Three surface-exposed epitopes on the major outer membrane protein include a conserved epitope with potential for development of a vaccine and a diagnostic test and two variable epitopes responsible for antigenic differences among strains; the remaining two epitopes are well preserved among strains but not exposed on the cell surface. Images

Tagawa, Y; Haritani, M; Ishikawa, H; Yuasa, N



Mapping of antigenic determinants of hepatitis C virus proteins using phage display  

Microsoft Academic Search

Analysis of the properties for individual hepatitis C virus (HCV) proteins makes it possible to establish their molecular\\u000a structure and conformation, to localize antigenic and immunogenic determinants, to identify protective epitopes, and to solve\\u000a applied problems (e.g., design of diagnostic tests, vaccines, and drugs). Linear and conformational epitopes of HCV proteins\\u000a were localized using the phage display technique, and the

E. A. Rechkina; G. F. Denisova; O. V. Masalova; L. F. Lideman; D. A. Denisov; E. I. Lesnova; R. I. Ataullakhanov; S. V. Gurianova; A. A. Kushch



Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.  

PubMed Central

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells. Images

Taniuchi, I; Kitamura, D; Maekawa, Y; Fukuda, T; Kishi, H; Watanabe, T



Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)  

PubMed Central

Background Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.



Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).  

PubMed Central

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K). Images

Ito, Y; Hamagishi, Y; Segawa, K; Dalianis, T; Appella, E; Willingham, M



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

PubMed Central

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 mass spectrometry. 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. 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 mass spectrometry and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

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



Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins  

PubMed Central

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.



Immunoassays with protein misfolding cycle amplification: a platform for ultrasensitive detection of antigen.  


Protein misfolding cycle amplification (PMCA), a novel technology on amplifying cyclically misfolded proteins in vitro, is conceptually analogous to DNA amplification by polymerase chain reaction (PCR) and has tremendous implications for the researches and diagnosis. Here we first introduce the protein amplification technology into the classic immunoassay and develop a PMCA-based immunoassay (immuno-PMCA) for highly sensitive detection of antigen. This method takes advantage of sandwich binding of two affinity aptamers for increased specificity, magnetic nanoparticles for fast magnetic separation, PMCA for signal amplification, and conjugated polyelectrolytes for visual detection, allowing the detection limit of antigen by colorimetry down to femtomolar level with a wide linear range from 10 to 10(4) fM. More importantly, no specialized facilities or enzymes are needed either in the amplification reaction or the evaluation of results, which indicates its great potential application in immunological research and clinical diagnostics. PMID:22888831

Zhou, Peng; Luo, Qingying; Lin, Yi; Chen, Lu; Li, Sha; Zhou, Guohua; Ji, Xinghu; He, Zhike



Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.  

PubMed Central

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D. Images

Abou-Zeid, C; Harboe, M; Rook, G A



Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis  

PubMed Central

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.

Kim, Tae Yun; Kang, Shin-Yong; Ahn, Il-Young; Cho, Seung-Yull



Application of a Novel Radioimmunoassay to Identify Baculovirus Structural Proteins That Share Interspecies Antigenic Determinants  

PubMed Central

Immunological comparisons were made of baculovirus structural proteins by using a modification of the radioimmunological techniques described by Renart et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 3116-3120, 1979) and Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354, 1979). Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five baculoviruses: Autographa californica, Porthetria dispar, Trichoplusia ni, and Heliothis zea nuclear polyhedrosis viruses (NPVs) and T. ni granulosis virus (GV). These antisera were tested against the virion structural polypeptides of 17 different species of baculoviruses. Specific multiple-nucleocapsid NPV (MNPV), single-nucleocapsid NPV (SNPV), and GV virion polypeptides were shown to have similar antigenic determinants and thus be immunologically related. The molecular weights of the virion polypeptides with cross-reacting antigenic determinants were identified. Antisera prepared to purified A. californica and H. zea MNPV polyhedrin (the occlusion body protein from NPVs) recognized antigenic determinants on all the polyhedrins and granulins (occlusion body protein from GVs) that were tested. No immunological relationship was detected between A. californica MNPV polyhedrin and any of the A. californica MNPV virion structural polypeptides present on either the virus isolated from occlusion bodies or A. californica MNPV extracellular virus from infected-cell cultures. Images

Smith, Gale E.; Summers, Max D.



Incorporation of disulfide containing protein modules into multivalent antigenic conjugates: generation of antibodies against the thrombin-sensitive region of murine protein S.  


Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S. PMID:23066897

Van de Vijver, Pieter; Schmitt, Martin; Suylen, Dennis; Scheer, Liesbeth; Thomassen, M Christella L G D; Schurgers, Leon J; Griffin, John H; Koenen, Rory R; Hackeng, Tilman M



Echinococcus antigens  

PubMed Central

Much of the work on immunology of hydatidosis has so far been devoted to the development of suitable methods for serological diagnosis. The precise nature of hydatid antigens and their chemical characterization has still not been worked out, largely because of the complex life-history of the parasite and the difficulties of in vitro cultivation. The most widely used antigen for routine serological testing in hydatidosis caused by Echinococcus granulosus is fluid taken from the cyst. This fluid is, however, a complex mixture of substances and contains several protein and carbohydrate fractions as well as end-products of carbohydrate and protein metabolism. The cyst fluid from different sources is variable in its antigenic properties, and the fluid from sterile cysts is especially lacking in antigenic activity. Antigens from tissue extracts of hydatid cysts appear to have greater specificity. Cyst extracts of E. multilocularis, the cysts of which contain relatively little fluid, have also been used but are poor antigens, and contain measurable amounts of host protein. Antigens prepared from other cestodes and metabolic antigens are also reviewed. Biochemical analysis of Echinococcus antigens covering polysaccharides, proteins, lipids, and blood-group substances is considered, together with the characterization of antigens by electrophoresis, column chromatography and gel-diffusion methods. The problems associated with the standardization of antigens are discussed. Recent data on the character and reactivity of antigens employed in Echinococcus studies are summarized.

Kagan, Irving G.; Agosin, Moises



Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.  

PubMed Central

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src. Images

Luo, K; Sefton, B M



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


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. PMID:23982308

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



Receptor-Mediated Uptake of Antigen/Heat Shock Protein Complexes Results in Major Histocompatibility Complex Class I Antigen Presentation via Two Distinct Processing Pathways  

PubMed Central

Heat shock proteins (HSPs) derived from tumors or virally infected cells can stimulate antigen-specific CD8+ T cell responses in vitro and in vivo. Although this antigenicity is known to arise from HSP-associated peptides presented to the immune system by major histocompatibility complex (MHC) class I molecules, the cell biology underlying this presentation process remains poorly understood. Here we show that HSP 70 binds to the surface of antigen presenting cells by a mechanism with the characteristics of a saturable receptor system. After this membrane interaction, processing and MHC class I presentation of the HSP-associated antigen can occur via either a cytosolic (transporter associated with antigen processing [TAP] and proteasome–dependent) or an endosomal (TAP and proteasome–independent) route, with the preferred pathway determined by the sequence context of the optimal antigenic peptide within the HSP-associated material. These findings not only characterize two highly efficient, specific pathways leading to the conversion of HSP-associated antigens into ligands for CD8+ T cells, they also imply the existence of a mechanism for receptor-facilitated transmembrane transport of HSP or HSP-associated ligands from the plasma membrane or lumen of endosomes into the cytosol.

Castellino, Flora; Boucher, Philip E.; Eichelberg, Katrin; Mayhew, Mark; Rothman, James E.; Houghton, Alan N.; Germain, Ronald N.



Proteins antigenically related to methyl-accepting chemotaxis proteins of Escherichia coli detected in a wide range of bacterial species.  

PubMed Central

The four methyl-accepting chemotaxis proteins of Escherichia coli, often called transducers, are transmembrane receptor proteins that exhibit substantial identity among the sequences of their cytoplasmic domains. Thus, antiserum raised to one of these proteins recognizes the others and might be expected to recognize related proteins in other bacteria. We used antiserum raised to the transducer Trg in immunoblot experiments to probe a wide range of bacterial species for the presence of antigenically related proteins. Such proteins were detected in over 20 different species, representing 6 of the 11 eubacterial phyla defined by analysis of rRNA sequences as well as one archaebacterial group. Species containing proteins antigenically related to the transducers of E. coli included members of all four subdivisions of the phylum in which E. coli is placed, members of four of the six subdivisions of spirochetes, and two gliding bacteria. These observations provide substantial support for the notion that methyl-accepting taxis proteins are widely distributed among the diversity of bacterial species. Images

Morgan, D G; Baumgartner, J W; Hazelbauer, G L



Practical considerations for analyzing antigene RNAs (agRNAs): RNA immunoprecipitation of argonaute protein.  


Target validation for small RNAs in cells can be a confusing task wrought with pitfalls and false leads. One technique for validating in vivo targets of small RNAs is immunoprecipitation of target RNAs using antibodies again the RNAi machinery. Antigene RNAs (agRNAs) regulate transcription in human cells using machinery from the RNAi regulatory pathway - namely argonaute proteins. Here we describe a technique for validating targets of agRNAs using RNA immunoprecipitation with antibodies against human argonaute proteins. This technique can be used to detect interactions of argonaute proteins in the cell nucleus with their targets, lowly expressed noncoding RNA transcripts. PMID:21748649

Schwartz, Jacob C; Corey, David R



Determinants of antigenicity and specificity in immune response for protein sequences  

PubMed Central

Background Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore, it is important to understand the properties of protein sequences that are important for antigenicity and to identify small peptide epitopes and large regions in the linear sequence of the proteins whose utilization result in specific antibodies. Results Our analysis using protein properties suggested that sequence composition combined with evolutionary information and predicted secondary structure, as well as solvent accessibility is sufficient to predict successful peptide epitopes. The antigenicity and the specificity in immune response were also found to depend on the epitope length. We trained the B-Cell Epitope Oracle (BEOracle), a support vector machine (SVM) classifier, for the identification of continuous B-Cell epitopes with these protein properties as learning features. The BEOracle achieved an F1-measure of 81.37% on a large validation set. The BEOracle classifier outperformed the classical methods based on propensity and sophisticated methods like BCPred and Bepipred for B-Cell epitope prediction. The BEOracle classifier also identified peptides for the ChIP-grade antibodies from the modENCODE/ENCODE projects with 96.88% accuracy. High BEOracle score for peptides showed some correlation with the antibody intensity on Immunofluorescence studies done on fly embryos. Finally, a second SVM classifier, the B-Cell Region Oracle (BROracle) was trained with the BEOracle scores as features to predict the performance of antibodies generated with large protein regions with high accuracy. The BROracle classifier achieved accuracies of 75.26-63.88% on a validation set with immunofluorescence, immunohistochemistry, protein arrays and western blot results from Protein Atlas database. Conclusions Together our results suggest that antigenicity is a local property of the protein sequences and that protein sequence properties of composition, secondary structure, solvent accessibility and evolutionary conservation are the determinants of antigenicity and specificity in immune response. Moreover, specificity in immune response could also be accurately predicted for large protein regions without the knowledge of the protein tertiary structure or the presence of discontinuous epitopes. The dataset prepared in this work and the classifier models are available for download at



Identification of two antigenic epitopes on SARS-CoV spike protein  

Microsoft Academic Search

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1–S2–S3–S4–S5–S6

Ronghong Hua; Yanjun Zhou; Yunfeng Wang; Yuzhuo Hua; Guangzhi Tong



Expression of a Gene Encoding the Major Antigenic Protein 2 Homolog of Ehrlichia chaffeensis and Potential Application for Serodiagnosis  

Microsoft Academic Search

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified




Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1Based Vaccines  

Microsoft Academic Search

BackgroundTwo current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP119) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the

Shigeto Yoshida; Hiroshi Nagumo; Takashi Yokomine; Hitomi Araki; Ayaka Suzuki; Hiroyuki Matsuoka; Laurent Rénia



Mutated mitogen-activated protein kinase: A tumor rejection antigen of mouse sarcoma  

PubMed Central

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.

Ikeda, Hiroaki; Ohta, Nobuyoshi; Furukawa, Keiko; Miyazaki, Hiroshi; Wang, Lijie; Furukawa, Koichi; Kuribayashi, Kagemasa; Old, Lloyd J.; Shiku, Hiroshi



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

SciTech Connect

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.

Lee, C.Y.G. (Univ. of British Columbia, Vancouver, Canada); Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.



Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer  

PubMed Central

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.

Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua



Influence of cephalosporins and iron on surface protein antigens of Klebsiella pneumoniae in vivo.  

PubMed Central

The outer membrane protein (OMP) profiles of Klebsiella pneumoniae grown in a rabbit peritonitis model in the presence or absence of cephalosporins were investigated. Six high-molecular-weight OMPs (Mr 69,000 to 83,000) were induced under iron-depleted conditions in vitro. Three of these proteins (the 69,000-Mr protein [69K protein] and the 70K and 78K proteins) and trace amounts of the 73K and 75K proteins were induced in the OM of bacteria infecting the peritoneal cavity of rabbits. Addition of iron either to the growth medium in vitro or to the peritoneum in vivo repressed the expression of these proteins. Cephaloridine had no significant effect on the OMP profiles. An additional 56,000-Mr protein was observed in the OM of bacteria cultivated in vivo in the presence of CGP 17520 and also to a lesser extent in vivo under conditions of iron excess. A difference in recognition of OM antigens between cells grown in vitro and in vivo was observed by immunoblotting techniques. The 26K, 27.5K, and 28.5K antigens present in the OM of cells grown in vitro (but not in vivo) were recognized by antibodies raised against bacteria cultivated in vitro under conditions of iron depletion, but were not recognized by antisera raised against bacteria harvested directly from infections. Antisera raised against a nonencapsulated K. pneumoniae strain caused no agglutination of encapsulated K. pneumoniae grown in vivo in the absence of cephalosporins. Rapid agglutination was observed with this antiserum when the same encapsulated strain was grown in vivo in the presence of either cephalosporin, indicating less occlusion of critical antigens by the capsule. Images

Kadurugamuwa, J L; Anwar, H; Brown, M R; Hengstler, B; Kunz, S; Zak, O



Haemophilus somnus complex: antigenicity and specificity of fractions of Haemophilus somnus.  


Five Haemophilus somnus type 8025 preparations (whole cell, sonicate, crude polysaccharide, purified polysaccharide, and protein) were produced for studies of their antigenicity in rabbits. Bacterial agglutination and passive hemagglutination tests were used to assess the level of antibody produced in rabbits inoculated with the different antigenic preparations. Cross-reactions were seen between the antiserums against the H sumnus 8025 antigens and a variety of related and unrelated bovine pathogens. The strongest cross-reaction occurred between antiserums against H somnus 8025 whole cell and crude polysaccharide antigens and Haemophilus agni and Actinobacillus lignieresii cell suspensions. PMID:50755

Miller, R J; Renshaw, H W; Evans, J A



Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine  

Microsoft Academic Search

In order to obtain monoclonal antibodies that might have prophylactic applications and to understand better the immune response to hepatitis E virus (HEV), we used phage display to isolate chimpanzee antibodies to HEV. The panning antigen was an two open reading frame (ORF2) recombinant protein that elicits a broadly protective immune response in vaccinated monkeys. Two major antigenic sites were

Darren J Schofield; Robert H Purcell; Hanh T Nguyen; Suzanne U Emerson



?-NAP, a cerebellar degeneration antigen, is a neuron-specific vesicle coat protein  

Microsoft Academic Search

We have identified a target antigen in autoimmune cerebellar degeneration, ?-NAP, that is closely related to the ?-adaptin and ?-COP coat proteins. ?-NAP is a non-clathrin-associated phosphoprotein expressed exclusively in neurons, from E12 through adulthood. ?-NAP is present in the neuronal soma and nerve terminal as soluble and membrane-bound pools and is associated with a discrete set of nerve-terminal vesicles.

Lori S Newman; Matthew O McKeever; Hirotaka J Okano; Robert B Darnell



Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein  

Microsoft Academic Search

Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length

Zeliang Chen; Decui Pei; Lingxiao Jiang; Jin Wang; Hongxia Wang; Dongsheng Zhou; Junhui Zhai; Maofeng Qiu; Yanping Han; Zhaobiao Guo; Ruifu Yang



Mapping Antigenic Domains Expressed by Chlamydia trachomatis Major Outer Membrane Protein Genes  

Microsoft Academic Search

Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells. Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants. To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs).

Wolfgang Baehr; You-Xun Zhang; Theresa Joseph; Hua Su; Francis E. Nano; Karin D. E. Everett; Harlan D. Caldwell



Role of the mitogen-activated protein kinase signaling pathway in the regulation of human melanocytic antigen expression.  


Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy. PMID:17050671

Kono, Michihiro; Dunn, Ian S; Durda, Paul J; Butera, David; Rose, Lenora B; Haggerty, Timothy J; Benson, Elizabeth M; Kurnick, James T



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


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. PMID:10347754

Langella, P; Le Loir, Y



Antigen Cross-Priming of Cell-Associated Proteins is Enhanced by Macroautophagy within the Antigen Donor Cell  

PubMed Central

Phagocytosis of dying cells constitutes an important mechanism of antigen capture for the cross-priming of CD8+ T cells. This process has been shown to be critical for achieving tumor and viral immunity. While most studies have focused on the mechanisms inherent in the dendritic cell that account for exogenous antigen accessing MHC I, several recent reports have highlighted the important contribution made by the antigen donor cell. Specifically, the cell stress and cell death pathways that precede antigen transfer are now known to impact cross-presentation and cross-priming. Herein, we review the current literature regarding a role for macroautophagy within the antigen donor cell. Further examination of this point of immune regulation is warranted and may contribute to a better understanding of how to optimize immunotherapy for treatment of cancer and chronic infectious disease.

Joubert, Pierre-Emmanuel; Albert, Matthew L.



Fournier's gangrene after unrelated cord blood stem cell transplantation  

Microsoft Academic Search

A 16-year-old boy with refractory acute myelogenous leukemia developed Fournier's gangrene as an early complication after two-antigen HLA-mismatched unrelated cord blood stem cell transplantation. On day 25 after the transplantation, he noted abrupt onset of penile swelling with miction pain. The penile inflammation rapidly extended posteriorly to involve the scrotum and perianal tissues, inferiorly to involve the thighs, and superiorly

C. Yoshida; K. Kojima; K. Shinagawa; D. Hashimoto; S. Asakura; S. Takata; M. Tanimoto



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

Microsoft Academic Search

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

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



Definition of purified enzyme-linked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis.  


Enzyme-linked immunosorbent assay (ELISA) has been developed as the ancillary diagnosis of bovine tuberculosis at ante-mortem to overcome the disadvantages of intradermal skin test. In this study, the antigenic proteins were purified, applied to bTB ELISA, and identified through proteomic analysis. Culture filtrate protein of Mycobacterium bovis was fractionated by MonoQ column chromatography, and examined the antigenicity by immunoblotting. The antigenic 20 kDa protein was in-gel digested and identified the antigenome by LTQ mass spectrometer and peptide match fingerprinting, which were MPB64, MPB70, MPB83, Fas, Smc, Nrp, RpoC, Transposase, LeuA, and MtbE. The 20 kDa protein exhibited the highest antigenicity to bTB positive cattle in ELISA and would be useful for bTB serological diagnosis. PMID:19591042

Cho, Yun Sang; Lee, Sang-Eun; Ko, Young Joon; Cho, Donghee; Lee, Hyang Shim; Hwang, Inyeong; Nam, Hyangmi; Heo, Eunjung; Kim, Jong Man; Jung, Sukchan



Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA.  


Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. A number of serological assays have been developed for diagnosis of M. synoviae infection; however, they lack sensitivity and/or are prone to false-positive reactions. Using a combination of PCR and expression cloning, four overlapping regions (regions 1-4) of the surface antigen MSPB of M. synoviae WVU-1853 were expressed in a bacterial expression system. Immunostaining of the resultant polypeptides with chicken sera raised against different M. synoviae strains, or Mycoplasma gallisepticum S6, suggested that region 4 contained a highly antigenic and species-specific domain (amino acids 212-317) [corrected] of MSPB. A fusion protein of region 4 was expressed in the pMAL expression system and purified from cold-osmotic-shock fluids of Escherichia coli cells for use in an indirect ELISA. The potential of the purified antigen for detection of M. synoviae antibodies was assessed with sera obtained from chickens experimentally infected with different strains of M. synoviae or M. gallisepticum, or from uninoculated chickens. All the sera from M. synoviae-inoculated chickens provided higher absorbance values than those from M. gallisepticum-inoculated or uninoculated chickens. Chickens inoculated with M. synoviae 86079/7NS had detectable increases of serum anti-MSPB immunoglobulins at day 7 after inoculation. These studies have identified the most antigenic region of one of the major species-specific surface proteins of M. synoviae, and shown the potential of this protein as a serodiagnostic reagent. PMID:10463175

Noormohammadi, A H; Markham, P F; Markham, J F; Whithear, K G; Browning, G F



Antibody affinity maturation using yeast display with detergent-solubilized membrane proteins as antigen sources.  


Antigen preparations in the form of detergent-solubilized cell lysates could, in principle, render membrane proteins (MPs) compatible with in vitro antibody engineering technologies. To this end, detergent-solubilized cell lysates were coupled with the yeast surface display platform to affinity mature an anti-transferrin receptor (TfR) single-chain antibody (scFv). Lysates were generated from TfR-expressing HEK293 cells by solubilization with detergent-containing buffer after undergoing plasma membrane-restricted biotinylation. Lysate-resident TfR was then combined with a mutagenic anti-TfR scFv library in a competitive, dissociation rate screen, and scFvs were identified with up to 4-fold improved dissociation rates on the surface of yeast. Importantly, although the lysates contained a complex mixture of biotinylated proteins, the engineered scFvs retained their TfR binding specificity. When secreted by yeast as soluble proteins, mutant scFvs bound to cell surface TfR with 3-7-fold improvements in equilibrium binding affinity. Although a known MP antigen was targeted for purposes of this study, employing biotin tagging as a means of antigen detection makes the lysate-based approach particularly flexible. We have previously shown that yeast display can be used to identify lead antibodies using cell lysate-resident MP antigens, and combined with this work showing that antibodies can also be quantitatively engineered using cell lysates, these approaches may provide a high-throughput platform for generation and optimization of antibodies against MPs. PMID:23109730

Tillotson, Benjamin J; de Larrinoa, Iñigo F; Skinner, Colin A; Klavas, Derek M; Shusta, Eric V



Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A  

Microsoft Academic Search

Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase a-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to




Structural, antigenic, and evolutionary characterizations of the envelope protein of newly emerging duck tembusu virus.  


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

Yu, Kexiang; Sheng, Zhi-Zhang; Huang, Bing; Ma, Xiuli; Li, Yufeng; Yuan, Xiaoyuan; Qin, Zhuoming; Wang, Dan; Chakravarty, Suvobrata; Li, Feng; Song, Minxun; Sun, Huaichang



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

PubMed Central

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.

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



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

PubMed Central

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.

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



Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology.  


We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15 nM to 250 nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation. PMID:20129075

Ohiro, Yoshiyuki; Ueda, Hiroshi; Shibata, Norio; Nagamune, Teruyuki



Definition of Purified Enzyme-Linked Immunosorbent Assay Antigens from the Culture Filtrate Protein of Mycobacterium bovis by Proteomic Analysis  

Microsoft Academic Search

Enzyme-linked immunosorbent assay (ELISA) has been developed as the ancillary diagnosis of bovine tuberculosis at ante-mortem to overcome the disadvantages of intradermal skin test. In this study, the antigenic proteins were purified, applied to bTB ELISA, and identified through proteomic analysis. Culture filtrate protein of Mycobacterium bovis was fractionated by MonoQ® column chromatography, and examined the antigenicity by immunoblotting. The

Yun Sang Cho; Sang-Eun Lee; Young Joon Ko; Donghee Cho; Hyang Shim Lee; Inyeong Hwang; Hyangmi Nam; Eunjung Heo; Jong Man Kim; Sukchan Jung



Innate immunity mediates follicular transport of particulate but not soluble protein antigen.  


Ag retention on follicular dendritic cells (FDCs) is essential for B cell activation and clonal selection within germinal centers. Protein Ag is deposited on FDCs after formation of immune complexes with specific Abs. In this study, by comparing the same antigenic determinant either as soluble protein or virus-like particle (VLP), we demonstrate that VLPs are transported efficiently to murine splenic FDCs in vivo in the absence of prior immunity. Natural IgM Abs and complement were required and sufficient to mediate capture and transport of VLPs by noncognate B cells. In contrast, soluble protein was only deposited on FDCs in the presence of specifically induced IgM or IgG Abs. Unexpectedly, IgG Abs had the opposite effect on viral particles and inhibited FDC deposition. These findings identify size and repetitive structure as critical factors for efficient Ag presentation to B cells and highlight important differences between soluble proteins and viral particles. PMID:22427639

Link, Alexander; Zabel, Franziska; Schnetzler, Yvonne; Titz, Alexander; Brombacher, Frank; Bachmann, Martin F



Assessing the Serodiagnostic Potential of 35 Mycobacterium tuberculosis Proteins and Identification of Four Novel Serological Antigens  

PubMed Central

Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods. The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB). We cloned and expressed 35 M. tuberculosis proteins as recombinant proteins in Escherichia coli and analyzed their serodiagnostic potentials. By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before. The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97%. The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients. Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect M. tuberculosis-specific antibodies.

Weldingh, Karin; Rosenkrands, Ida; Okkels, Limei Meng; Doherty, T. Mark; Andersen, Peter



Human HER-2/neu protein immunization circumvents tolerance to rat neu: a vaccine strategy for 'self' tumour antigens.  

PubMed Central

Many newly defined tumour antigens are 'self' proteins. Immunizing cancer patients against these antigens may be difficult due to tolerance. The HER-2/neu oncogenic protein is such a 'self' tumour antigen. Rat neu is homologous with human HER-2/neu and provides a model system for studying vaccination strategies. Rats are tolerant to rat neu. Vaccination with this 'self' protein elicits no detectable immune response. The current studies evaluated whether tolerance to rat neu can be circumvented by immunizing with the highly homologous foreign human HER-2/neu protein. Rats were immunized with human HER-2/neu intracellular domain (hICD) protein that is 92% homologous to rat neu ICD. Animals immunized with hICD developed significant antibody and T-cell responses that were specific for both human HER-2/neu and rat neu. Neu-specific antibodies were present in titres of greater than 1:200,000. Analysis of the specificity of the antibody response using synthetic peptides demonstrated substantial reactivity to an epitope with 100% homology between rat and human protein. Significant T-cell responses (stimulation index > 10) to hICD and rat neu protein (stimulation index > 4) were detected. The T cells also responded to both human and rat ICD. The results imply that immunization with foreign proteins, which are highly homologous to 'self' tumour antigens, may be an effective vaccine strategy for 'self' tumour antigens. Images Figure 2 Figure 3

Disis, M L; Shiota, F M; Cheever, M A



Identification of 62-kDa protein as an immunogenic antigen of Vibrio vulnificus for humans.  


Vibrio vulnificus infection can cause necrotizing fasciitis and sepsis and can develop within a few days despite intensive care. The mortality rate is up to 60% in vulnerable people. Most patients infected with this microbe have chronic liver disease, especially liver cirrhosis or cancer, as an underlying disease. V. vulnificus infection is opportunistic, and there is an urgent need to develop an anti- V. vulnificus vaccine. Thus, it is important to identify immunogenic antigens. We collected human sera from three subject groups: patients with V. vulnificus infection, patients with chronic liver disease but without V. vulnificus infection, and healthy volunteers with normal liver function. Immunoblots of cytosolic and membrane proteins of seven strains of V. vulnificus and one of V. parahaemolyticus were performed with sera from these groups. Although we could not demonstrate differences in antibody response between the groups, all sera showed a strong antibody response to a 62-kDa protein that was common to all strains examined. Immunoblots of Escherichia coli and Klebsiella pneumoniae also showed strong antibody response to this 62-kDa protein, and the possibility of cross-reaction cannot be denied. We identified this 62-kDa protein as an immunogenic antigen of V. vulnificus for humans. PMID:24040694

Tomita, Yukiko; Higashibata, Akira; Oishi, Hirotaka; Hara, Hiromitsu; Sakagucmhi, Yoshiro



Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.  


Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods. PMID:16478273

Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K



Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil.  


New Mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (MDT) monitoring. This study describes seroreactivity to M. leprae proteins among participants from three highly endemic leprosy areas in Brazil: central-western Goiânia/Goiás (GO) (n = 225), Rondonópolis/Mato Grosso (MT) (n = 764) and northern Prata Village/Pará (PA) (n = 93). ELISA was performed to detect IgG to proteins (92f, 46f, leprosy IDRI diagnostic-1, ML0405, ML1213) and IgM to phenolic glycolipid-I (PGL-I). Multibacillary (MB) leprosy had positive rates for PGL-I that were similar to those for proteins; however, some anti-PGL-I-negative subjects were positive for proteins, suggesting that adding protein antigen to PGL-I can enhance the sensitivity of MB leprosy detection. In MT, different degrees of seroreactivity were observed and ranked for MB, former patients after MDT, paucibacillary (PB) leprosy, household contact (HHC) and endemic control (EC) groups. The seroreactivity of PB patients was low in GO and MT. HHCs from different endemic sites had similar IgG antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ECs or HHCs within GO, an area with high BCG vaccination coverage. Low positivity in EC and HHC was observed in PA and MT. Our results provide evidence for the development of an improved serologic test that could be widely applicable for MB leprosy testing in Brazil. PMID:23283461

Hungria, Emerith Mayra; de Oliveira, Regiane Morillas; de Souza, Ana Lúcia Osório Maroclo; Costa, Maurício Barcelos; de Souza, Vânia Nieto Brito; Silva, Eliane Aparecida; Moreno, Fátima Regina Vilani; Nogueira, Maria Esther Salles; Costa, Maria Renata Sales Nogueira; Silva, Sônia Maria Usó Ruiz; Bührer-Sékula, Samira; Reed, Steven G; Duthie, Malcolm S; Stefani, Mariane Martins de Araújo



Label-Free Electrochemical Diagnosis of Viral Antigens with Genetically Engineered Fusion Protein  

PubMed Central

We have developed a simple electrochemical biosensing strategy for the label-free diagnosis of hepatitis B virus (HBV) on a gold electrode surface. Gold-binding polypeptide (GBP) fused with single-chain antibody (ScFv) against HBV surface antigen (HBsAg), in forms of genetically engineered protein, was utilized. This GBP-ScFv fusion protein can directly bind onto the gold substrate with the strong binding affinity between the GBP and the gold surface, while the recognition site orients toward the sample for target binding at the same time. Furthermore, this one-step immobilization strategy greatly simplifies a fabrication process without any chemical modification as well as maintaining activity of biological recognition elements. This system allows specific immobilization of proteins and sensitive detection of targets, which were verified by surface plasmon resonance analysis and successfully applied to electrochemical cyclic voltammetry and impedance spectroscopy upto 0.14 ng/mL HBsAg.

Heo, Nam Su; Zheng, Shun; Yang, MinHo; Lee, Seok Jae; Lee, Sang Yup; Kim, Hwa-Jung; Park, Jung Youn; Lee, Chang-Soo; Park, Tae Jung



Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer.  


Serological analysis of expression cDNA libraries (SEREX) derived from two small cell lung cancer (SCLC) cell lines using pooled sera of SCLC patients led to the isolation of 14 genes, including 4 SOX group B genes (SOX1, SOX2, SOX3, and SOX21) and ZIC2. SOX group B genes and ZIC2 encode DNA-binding proteins; SOX group B proteins regulate transcription of target genes in the presence of cofactors, whereas ZIC2 is also suspected to be a transcriptional regulator. These genes are expressed at early developmental stages in the embryonic nervous system, but are down-regulated in the adult. Although SOX2 mRNA can be detected in some adult tissues, ZIC2 is expressed only in brain and testis, and SOX1, SOX3, and SOX21 transcripts are not detectable in normal adult tissues. Of SCLC cell lines tested, 80% expressed ZIC2 mRNA, and SOX1, SOX2, and SOX3 expression was detected in 40%, 50%, and 10%, respectively. SOX group B and ZIC2 antigens elicited serological responses in 30-40% of SCLC patients in this series, at titers up to 1:10(6). In sera from 23 normal adults, no antibody was detected against SOX group B or ZIC2 proteins except for one individual with low-titer anti-SOX2 antibody. Seroreactivity against SOX1 and 2 was consistently higher titered than SOX3 and 21 reactivity, suggesting SOX1 and/or SOX2 as the main antigens eliciting anti-SOX responses. Although paraneoplastic neurological syndromes have been associated with several SCLC antigens, neurological symptoms have not been observed in patients with anti-SOX or anti-ZIC2 antibodies. PMID:10760287

Güre, A O; Stockert, E; Scanlan, M J; Keresztes, R S; Jäger, D; Altorki, N K; Old, L J; Chen, Y T



Bovine Rotavirus Nonstructural Protein 4 Produced by Lactococcus lactis Is Antigenic and Immunogenic  

PubMed Central

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.

Enouf, Vincent; Langella, Philippe; Commissaire, Jacqueline; Cohen, Jean; Corthier, Gerard



Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes?  

PubMed Central

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A.; Lacroix-Desmazes, Sebastien; Stadler, Beda M.; Horn, Michael P.



Cellular proteins which can specifically associate with simian virus 40 small t antigen.  

PubMed Central

When crude, radiolabeled extracts of various cells were applied to homogeneous simian virus 40 small t antigen-Sepharose adsorbents, three cell proteins (57, 32, and 20 kilodaltons [kDa]) bound specifically. Each also bound to an insoluble, truncated t derivative composed of the COOH-terminal 123 residues of the protein. The binding of these proteins was greatly inhibited after reduction and alkylation of the t ligand. Therefore, some element of native conformation, but not all of the primary structure of t, is necessary for this binding property, which may constitute a discrete, in vitro biochemical function of this protein. Results of cell fractionation experiments suggested that the 57- and 32-kDa proteins are nonnuclear cell constituents, whereas the 20-kDa protein was closely associated with a detergent-washed nuclear fraction. Specific immunoblotting and comparative partial proteolytic digestion analyses indicated that the 57-kDa protein is tubulin, a major component of the cytoskeleton. In this regard, t and tubulin were observed to coimmunoprecipitate from crude cell extracts after incubation with monospecific anti-t antibody. Therefore, it is possible that t and tubulin interact in vivo. Images

Murphy, C I; Bikel, I; Livingston, D M



Functional Analysis of the Highly Antigenic Outer Capsid Protein, Hoc, a Virus Decoration Protein from T4-like Bacteriophages  

PubMed Central

Summary Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the center of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modeling studies show that Hoc consists of a string of four domains, three immunoglobulin (Ig)-like and one non-Ig domain at the C-terminus. Biochemical data suggest that the Hoc protein has two functional modules, a capsid binding module containing domains 1 and 4 and a solvent-exposed module containing domains 2 and 3. This model is consistent with the dumbbell-shaped cryo-EM density of Hoc observed in the reconstruction of the T4 capsid. Mutagenesis localized the capsid binding site to the C-terminal 25 amino acids, which are predicted to form two ?-strands flanking a capsid binding loop. Mutations in the loop residues, ESRNG, abolished capsid binding, suggesting that these residues might interact with the major capsid protein, gp23*. With the conserved capsid binding module forming a foothold on the virus and the solvent-exposed module able to adapt to bind to a variety of surfaces, Hoc probably provides survival advantages to the phage, such as increasing the virus concentration near the host, efficient dispersion of the virus, and exposing the tail for more efficient contact with the host cell surface prior to infection.

Sathaliyawala, Taheri; Islam, Mohammad Z.; Li, Qin; Fokine, Andrei; Rossmann, Michael G.; Rao, Venigalla B.



Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV) serotype Asia 1  

PubMed Central

Foot and mouth disease virus (FMDV), with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.

Alam, SM Sabbir; Amin, Ruhul; Rahman, Mohammed Ziaur; Hossain, M Anwar; Sultana, Munawar



Heat shock protein 70: role in antigen presentation and immune stimulation.  


Heat shock proteins (HSP) when released into the extracellular milieu can act simultaneously as a source of antigen due to their ability to chaperone peptides and as a maturation signal for dendritic cells, thereby inducing DCs to cross-present antigens to CD8+ T-cells. HSP can also act independently from associated peptides, stimulating the innate immune system. Previous results regarding the activation of NK cells by HSP70 cell surface expression on tumour cells and soluble HSP70 will be further covered elsewhere within this issue. For cross-presentation, HSP70-peptide complexes (HSP70-PC) were used from two human melanoma cell lines that differ in the expression of the tumour-associated antigen tyrosinase. Purified HSP70-PC consists of both the constitutively expressed HSC70 and the inducible HSP70. HSP70-peptide complexes purified from tyrosinase positive (HSP70-PC/tyr+) human melanoma cells, incubated with immature DCs, results in the activation of HLA-*A0201-restricted tyrosinase peptide-specific T-cells. Receptor-mediated uptake of HSP70-PC by DCs and intracellular transport are required for efficient MHC class I restricted cross-presentation of chaperoned peptides. Demonstration of HSP70-PC mediated cross-presentation of such non-mutated naturally expressed tumour antigens is of special clinical interest with regard to hyperthermia. Tumour regression and improved local control have been shown within clinical phase II/III trials integrating regional hyperthermia combined with radiation and/or chemotherapy in multimodal treatment strategies. According to the proposed concept, local necrosis induced by hyperthermic treatment induces the release of HSPs, followed by uptake, processing and presentation of associated peptides by DCs. By acting as chaperone and a signal for DC maturation, HSP70-PC might efficiently prime circulating T-cells. Therefore, upregulating HSP70 and causing local necrosis in tumour tissue by hyperthermia offers great potential as a new approach to directly activate the immune system. PMID:12537755

Milani, V; Noessner, E; Ghose, S; Kuppner, M; Ahrens, B; Scharner, A; Gastpar, R; Issels, R D


[Prokaryotic expression and detective application of the main antigenic region of VP2 protein of Aleutian mink disease parvovirus].  


To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD. PMID:18271270

Zeng, Xiang-Wei; Hua, Yu-Ping; Liang, Dong-Ying



Enzyme-Linked Immunosorbent Assay Based on a Chimeric Antigen Bearing Antigenic Regions of Structural Proteins Erns and E2 for Serodiagnosis of Classical Swine Fever Virus Infection  

Microsoft Academic Search

protein, C21E rns E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210)

Min Lin; Erin Trottier; Maria Mallory



Value of measuring serum procalcitonin, C-reactive protein, and mannan antigens to distinguish fungal from bacterial infections  

Microsoft Academic Search

The study presented here was conducted to determine the diagnostic value of measuring procalcitonin, C-reactive protein, and mannan antigens to distinguish fungal from bacterial infections. The sensitivity and specificity of these measurements ranged from 35% to 97%. On days 1 and 3 following the onset of fever, both serum procalcitonin and C-reactive protein levels were lower in patients with fungal

G. L. Petrikkos; S. A. Christofilopoulou; N. K. Tentolouris; E. A. Charvalos; C. J. Kosmidis; G. L. Daikos



Kinetic analysis of a protein antigen-antibody interaction limited by mass transport on an optical biosensor  

Microsoft Academic Search

Using BIAcore™ technology, we determined the rate constants for a protein antigen-antibody interaction that was mass transport limited on the optical biosensor. The antigen consisted of a soluble form of the human T-cell receptor CD4 (two amino terminal domains, D1D2) and the antibody was an anti-CD4 monoclonal from monkey engineered with the constant domains from human IgG1. High quality response

David G. Myszka; Thomas A. Morton; Michael L. Doyle; Irwin M. Chaiken



Experimental bullous pemphigoid generated in mice with an antigenic epitope of the human hemidesmosomal protein BP230  

Microsoft Academic Search

Bullous pemphigoid (BP) is an IgG-mediated autoimmune blistering disease that targets the hemidesmosomal proteins BP230 and BP180. To investigate the pathogenic role of anti-BP230 antibodies, rabbit polyclonal antibodies were generated against an antigenic sequence of the human BP230 antigen (BPAG 1, 2479–2499), which shows 67% homology in the human and the mouse BP230. Purified IgG from the rabbit anti-serum was

Mária Kiss; Sándor Husz; Tamás Jánossy; Ilona Marczinovits; János Molnár; Irma Korom; Attila Dobozy



D6STNFa Microsatellite Locus Correlates with CTLp Frequency in Unrelated Bone Marrow Donor-Recipient Pairs  

Microsoft Academic Search

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings, primarily due to an increased rate of graft-versus-host-disease. HLA matching for donors and recipients is the most important factor influencing the outcome of BMT. However, unrelated donor selection generally relies on matching only for HLA antigens without

Payman Hanifi Moghaddam; Ailko Zwinderman; Marzieh Kazemi; Minke van der Voort Maarschalk; Marieke Ruigrok; Albert Naipal; Arno van der Slik; Machteld Oudshoorn; Marius J Giphart



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

PubMed Central

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 ability to complex with a 68-kilodalton cellular protein in monkey cells. Images

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



Analysis of Immune Responses Directed toward a Recombinant Early Secretory Antigenic Target Six-Kilodalton Protein-Culture Filtrate Protein 10 Fusion Protein in Mycobacterium bovis-Infected Cattle  

Microsoft Academic Search

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To

Alexander C. Maue; W. Ray Waters; William C. Davis; Mitchell V. Palmer; F. Chris Minion; D. Mark Estes




PubMed Central

A new technique for the enumeration of antigen-reactive cells (ARC) responsive to the polymer antigen of S. adelaide flagellin (POL) is described for two strains of mice. Foci have been shown to be antibody dependent, may be mimicked by IgM as well as IgG antibodies, and contain specific antibody-forming cells (AFC). The use of POL offers a system unencumbered by relatively high numbers of background foci which, when present, appear to be basically different from those found using the SRBC antigen. The response of 1 antigen-reactive cell (ARC) focus/1 x 106 CBAT6T6 mouse spleen cells is linearly related to the injected number between 1 x 106–3 x 106 donor spleen cells and since 5% of injected cells remain in the spleen, there are an estimated 2400 ARC/spleen. The number of ARC foci does not increase significantly after the 5th postantigen day, and by the 8th day the AFC progeny of ARC have reached the maximum mean of 280 AFC/ARC focus. In response to increasing antigen concentrations, an initial rise in the number of AFC as well as ARC is observed, resulting in a relatively constant AFC/ARC ratio. This suggests that the number of ARC stimulated determines the total number of AFC produced under these conditions rather than a variable mitotic rate of the ARC offspring. The main significance of this technique is that it will allow a study of the kinetics of the ARC in the primary and secondary immune response as well as in immunological tolerance.

Armstrong, W. D.; Diener, E.



Transfer of protein antigens into milk after intravenous injection into lactating mice  

SciTech Connect

We investigated the transfer of bovine serum /sup 125/I-albumin (/sup 125/I-BSA), bovine /sup 125/I-gamma-globulin (/sup 125/I-BGG), /sup 125/I-ovalbumin (/sup 125/I-OVA), and /sup 125/I-beta-lactoglobulin (/sup 125/I-BLG) from the blood into the milk of lactating mice. Equal amounts (by weight) of the radiolabeled proteins were injected intravenously into mice 1 wk postpartum. Total radioactivity, trichloroacetic acid-precipitable radioactivity, and specifically immunoprecipitable radioactivity were measured in serum, mammary gland homogenate, and milk. Clearance of immunoreactive OVA (iOVA) and iBLG from the circulation was more rapid than iBSA and iBGG. The radioactivity in mammary tissue associated with BSA and BGG was greater than 70% immunoprecipitable throughout the 4-h test interval; /sup 125/I-OVA and /sup 125/I-BLG were less than 12% precipitable 1 and 4 h after injection. In milk obtained at 4 h, there was an approximately 10-fold greater accumulation of iBSA or iBGG than of iOVA or iBLG. These experiments demonstrate that protein antigens differ in their ability to transfer from maternal circulation into milk. The transfer into milk appeared to be in proportion to persistence of the antigens in the maternal circulation.

Harmatz, P.R.; Hanson, D.G.; Walsh, M.K.; Kleinman, R.E.; Bloch, K.J.; Walker, W.A.



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


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. PMID:12438590

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



Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins.  

PubMed Central

Rabies virus isolates from terrestrial animals in six areas of the United States were examined with a panel of monoclonal antibodies to nucleocapsid proteins. Characteristic differences in immunofluorescence reactions permitted the formation of four antigenically distinct reaction groups from the 231 isolates tested. The geographic distribution of these groups corresponded well with separate rabies enzootic areas recognized by surveillance of sylvatic rabies in the United States. Distinctive reaction patterns were also identified for viral proteins from four infected bat species, and identical patterns were found in eight isolated cases of rabies in terrestrial animals. These findings suggest that monoclonal antibodies can be used to study the prevalence, distribution, and transmission of rabies among wildlife species.

Smith, J S; Reid-Sanden, F L; Roumillat, L F; Trimarchi, C; Clark, K; Baer, G M; Winkler, W G



Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen  

PubMed Central

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented.

Pandey, Binod; Demchenko, Alexei V.; Stine, Keith J.



Effects of pH, temperature and enzyme-to-substrate ratio on the antigenicity of whey protein hydrolysates prepared by Alcalase  

Microsoft Academic Search

Response surface methodology was used to study the effects of pH (7.0–11.0), temperature (30–60°C), and enzyme-to-substrate ratio (4000–8000unitsg?1protein) on the residual antigenicity of whey protein concentrate (WPC, 77.5% protein) hydrolysates obtained with Alcalase. It was shown that enzymatic hydrolysis with Alcalase could reduce the antigenicity of WPC for ?-lactalbumin (?-LA) and ?-lactoglobulin (?-LG) effectively and the reduction of antigenicity could

Hai Zheng; Xiaoqin Shen; Guanhao Bu; Yongkang Luo



Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle.  


Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future. PMID:21123520

Terkawi, Mohamad Alaa; Huyen, Nguyen Xuan; Wibowo, Putut Eko; Seuseu, Faasoa Junior; Aboulaila, Mahmoud; Ueno, Akio; Goo, Youn-Kyoung; Yokoyama, Naoaki; Xuan, Xuenan; Igarashi, Ikuo



Expression of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus in soybean seed yields an immunogenic antigenic protein.  


Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a serious disease of swine and contributes to severe worldwide economic losses in swine production. Current vaccines against PRRS rely on the use of an attenuated-live virus; however, these are unreliable. Thus, alternative effective vaccines against PRRS are needed. Plant-based subunit vaccines offer viable, safe, and environmentally friendly alternatives to conventional vaccines. In this study, efforts have been undertaken to develop a soybean-based vaccine against PRRSV. A construct carrying a synthesized PRRSV-ORF7 antigen, nucleocapsid N protein of PRRSV, has been introduced into soybean, Glycine max (L.) Merrill. cvs. Jack and Kunitz, using Agrobacterium-mediated transformation. Transgenic plants carrying the sORF7 transgene have been successfully generated. Molecular analyses of T(0) plants confirmed integration of the transgene and transcription of the PRRSV-ORF7. Presence of a 15-kDa protein in seeds of T(1) transgenic lines was confirmed by Western blot analysis using PRRSV-ORF7 antisera. The amount of the antigenic protein accumulating in seeds of these transgenic lines was up to 0.65% of the total soluble protein (TSP). A significant induction of a specific immune response, both humoral and mucosal, against PRRSV-ORF7 was observed following intragastric immunization of BALB/c female mice with transgenic soybean seeds. These findings provide a 'proof of concept', and serve as a critical step in the development of a subunit plant-based vaccine against PRRS. PMID:21971995

Vimolmangkang, Sornkanok; Gasic, Ksenija; Soria-Guerra, Ruth; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; Korban, Schuyler S



Protein antigens of encapsulated Klebsiella pneumoniae surface exposed after growth in the presence of subinhibitory concentrations of cephalosporins.  

PubMed Central

It recently has been reported by us that cephalosporins, at a concentration below that influencing growth rate, reduced the production of enterochelin and capsule formation of iron-depleted Klebsiella pneumoniae. We now report on the antigenicity of the outer membrane components and surface-exposed protein antigens of iron-depleted cells grown in the presence or absence of cephalosporins. All major outer membrane proteins, including iron-regulated membrane proteins, were immunogenic. Encapsulated K. pneumoniae grown in antibiotic-free media had three protein antigens (60, 35.5, and 32.5 kilodaltons) exposed on the surface that were accessible to antibodies. Growth of the same cultures in the presence of subinhibitory concentrations of cephalosporins resulted in the exposure of a greater number of protein antigenic determinants, including iron-regulated membrane proteins, which become readily accessible to antibodies. It was also found that immunoblotting was generally more sensitive than conventional staining of the acrylamide gel with Coomassie blue in the detection of proteins. Images

Kadurugamuwa, J L; Anwar, H; Brown, M R; Zak, O



Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis  

PubMed Central

Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

Esteban, L. E.; Temprana, C. F.; Arguelles, M. H.; Glikmann, G.; Castello, A. A.



Genetic diversity of polymorphic vaccine candidate antigens (apical membrane antigen-1, merozoite surface protein-3, and erythrocyte binding antigen-175) in Plasmodium falciparum isolates from western and central Africa.  


The malaria vaccine candidate antigens erythrocyte binding antigen 175 (EBA-175), merozoite surface protein 3 (MSP-3), and apical membrane antigen (AMA-1) from Plasmodium falciparum isolates from countries in central and west Africa were assessed for allelic diversity. Samples were collected on filter paper from 600 P. falciparum-infected symptomatic patients in Cameroon, Republic of Congo, Burkina Faso, Ghana, and Senegal and screened for class-specific amplification fragments. Genetic diversity, assessed by mean heterozygosity, was comparable among countries. We detected a clinical increase in eba 175 F-allele frequency from west to east across the study region. No statistical difference in msp-3 allele distribution between countries was observed. The ama-1 3D7 alleles were present at a lower frequency in central Africa than in West Africa. We also detected little to no genetic differentiation among sampling locations. This finding indicates that, at least at the level of resolution offered by restriction fragment length polymorphism analysis, these antigens showed remarkable genetic homogeneity throughout the region sampled, perhaps caused by balancing selection to maintain a diverse array of antigen haplotyes. PMID:21292899

Soulama, Issiaka; Bigoga, Jude D; Ndiaye, Magatte; Bougouma, Edith C; Quagraine, Josephine; Casimiro, Prisca N; Stedman, Timothy T; Sirima, Sodiomon B



Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines  

PubMed Central

A unique multicomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ?80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if one antigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria.

Donnelly, John; Medini, Duccio; Boccadifuoco, Giuseppe; Biolchi, Alessia; Ward, Joel; Frasch, Carl; Moxon, E. Richard; Stella, Maria; Comanducci, Maurizio; Bambini, Stefania; Muzzi, Alessandro; Andrews, William; Chen, Jie; Santos, George; Santini, Laura; Boucher, Philip; Serruto, Davide; Pizza, Mariagrazia; Rappuoli, Rino; Giuliani, Marzia Monica



Cytotoxic T Cell Specificity for Respiratory Syncytial Virus Proteins: Fusion Protein Is an Important Target Antigen  

Microsoft Academic Search

SUMMARY We examined the specificity of BALB\\/c cytotoxic T (To) cells for respiratory syncytial virus (RSV) components, using recombinant vaccinia viruses (VV) coding for several individual RSV proteins. We found that immunization with the different VVs yielded the following T~ memory cell populations: high levels of RSV-specific To cells were induced with the fusion protein VV, but low levels were

R. M. Pemberton; M. J. Cannon; P. J. M. Openshaw; L. A. Ball; G. W. Wertz; B. A. Askonas



Adsorption of multimeric T cell antigens on carbon nanotubes: effect on protein structure and antigen-specific T cell stimulation.  


Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin. PMID:23090793

Fadel, Tarek R; Li, Nan; Shah, Smith; Look, Michael; Pfefferle, Lisa D; Haller, Gary L; Justesen, Sune; Wilson, Corey J; Fahmy, Tarek M



Antigenic reactivity and electrophoretic migrational heterogeneity of the three polymerase proteins of type A human and animal influenza viruses  

Microsoft Academic Search

Summary Antigenic reactivity of the three polymerase proteins PB1, PB2, and PA of type A influenza viruses of animal and human origin were analysed by radioimmunoprecipitation using monospecific antisera. Each of the polymerase monospecific antisera made against the polymerase proteins of the human A\\/WSN\\/33 (H1N1) influenza virus reacted efficiently with the homologous proteins of all the known thirteen HA subtype

R. K. Akkina; Accepted December



Common distribution of antigenic determinants and complementation activity on matrix proteins of two vesicular stomatitis virus serotypes  

Microsoft Academic Search

To compare the antigenic and functional domains of the matrix (M) proteins of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ), deletion mutants and chimeras were cloned in pBSM13 and expressed as in-frame lacZ fusion proteins in Escheriehia eoli. Non-cross-reactive monoclonal anti- bodies directed to the two antigenieally distinct M proteins were tested by Western blot analysis

Wei Sun; Liping Huang; Robert R. Wagner



SH3P7 Is a Cytoskeleton Adapter Protein and Is Coupled to Signal Transduction from Lymphocyte Antigen Receptors  

Microsoft Academic Search

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple




Molecular analyses of the possible RNA-binding protein gene located in the human leukocyte antigen (HLA) — DR subregion  

Microsoft Academic Search

We have previously found the sequence having potential for encoding a new protein in the human leukocyte antigen (HLA) class II region. The predicted amino acid sequence showed a significant sequence homology to the Xenopus double-stranded RNA-binding protein (Xlrbp) and the human cellular protein bound to the transactivation response (TAR) of human immunodeficiency virus type-1 (HIV-1) RNA (TRBP). Reverse transcription-polymerase

Shohei Chida; Hirohiko Hohjoh; Katsushi Tokunaga



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

PubMed Central

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 potential vaccine candidates or virulence markers.



Use of Pre-S Protein-Containing Hepatitis B Virus Surface Antigens and a Powerful Adjuvant To Develop an Immune Therapy for Chronic Hepatitis B Virus Infection  

PubMed Central

A hepatitis B virus (HBV) vaccine has been developed using a new adjuvant and HBV surface antigens produced from a CHO cell line. The purified HBV surface antigens are composed of L protein, M protein, and S protein in a mixture of 20- and 40-nm-diameter particles and filamentous forms. This HBV surface antigen, formulated with L-pampo, a proprietary adjuvant, induced 10 times more antibody than the same antigen with alum and was capable of inducing strong immune responses in three different HBV transgenic mice. In spite of the presence of a large amount of HBV antigen in the blood, no antibody against HBV surface antigen was normally detected in these transgenic mice. After immunization, the HBV antigen was also cleared from the blood.

Yum, Jung Sun; Ahn, Byung Cheol; Jo, Hyun Jin; Kim, Dong Yeon; Kim, Ki Hyun; Kim, Hyo Sun; Sung, Young Chul; Yoon, Jaeseung; Morrey, John



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

PubMed Central

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.

Jaoude, Georges Abou; Sureau, Camille



Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of hepatitis delta virus.  


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

Jaoudé, Georges Abou; Sureau, Camille



Detection of antigenic determinants in the Treponema pallidum membrane protein TmpA using overlapping synthetic peptides.  


The antigenic structure of the 42 kDa membrane protein of Treponema Pallidum, TmpA, was studied using synthetic peptides. Ten overlapping peptides, 35-40 residues each, were synthesized in order to cover the entire sequence of the molecule. The antigenic activity of the fragments was examined by enzyme-linked immunosorbent assay (ELISA). In this way it was possible to demonstrate a significant antigenic activity of four peptides which were reactive with syphilitic sera. The N-terminal fragment TmpA1, 38 residues long, proved to be the most reactive. Its antigenic structure was therefore studied in more detail, by examining shorter fragments. The N-terminal portion of TmpA1, consisting of 19 residues, (ASGAKEEAEKKAAEQRALL) represents an important fragment of the molecule, and was specifically interactive with most of the syphilitic sera examined. PMID:8576575

Antoni, G; dal Maso, G; Berti, B; Soldatini, C; Cocola, F



Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes).  


The ability of non-ionic surfactant vesicles to induce systemic immune responses in mice following oral immunisation was studied using a standard antigen (bovine serum albumin), a synthetic measles peptide and an influenza sub-unit vaccine. The effectiveness of this formulation was significantly increased by incorporating bile salts (in particular deoxycholate) into the formulation. We have named the resulting vesicles bilosomes. We found that the most effective immunisation protocol was to give two doses of vaccine three days apart and then repeat this protocol two weeks later. Following this method, preparation of measles peptide in bilosomes produced a specific cell mediated response, as measured by splenocyte proliferation and IL-2 production. Of particular significance, these studies demonstrate that oral administration of bilosomes incorporating the influenza sub-unit vaccine could induce as potent an antibody response as the parenterally administered vaccine containing the same quantity of antigen. In addition, the Th1/Th2 balance, as measured by antibody subclasses, was similar whether animals were immunised by the oral or the parenteral vaccine route. As bilosomes are prepared from naturally occurring lipids and have no apparent toxicity associated with their use, they represent a useful modification of conventional lipid vesicle based systems for the oral delivery of proteins and peptides. PMID:11282208

Conacher, M; Alexander, J; Brewer, J M



Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen  

PubMed Central

Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans.

Ono, Toshiro; Kurashige, Takushi; Harada, Naoki; Noguchi, Yuji; Saika, Takashi; Niikawa, Norio; Aoe, Motoi; Nakamura, Shinichiro; Higashi, Toshihiro; Hiraki, Akio; Wada, Hisashi; Kumon, Hiromi; Old, Lloyd J.; Nakayama, Eiichi



Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen.  


Serological expression cloning of antigens eliciting a humoral immune response to a syngeneic mouse sarcoma identified pem (mouse placenta and embryonic expression gene) as a new member of the cancer/testis family. To identify the human homologue of pem, mouse pem sequences and pem-related expressed sequence tags from human testis were used as PCR primers for amplification using human testis cDNA. However, rather than pem, another gene, designated OY-TES-1, was isolated and found to be the human homologue of proacrosin binding protein sp32 precursor originally identified in mouse, guinea pig, and pig. OY-TES-1 maps to chromosome 12p12-p13 and contains 10 exons. Southern blot analysis suggests the presence of two OY-TES-1-related genes in the human genome. In normal tissues, OY-TES-1 mRNA was expressed only in testis, whereas in malignant tissues, a variable proportion of a wide array of cancers, including bladder, breast, lung, liver, and colon cancers, expressed OY-TES-1. Serological survey of 362 cancer patients with a range of different cancers showed antibody to OY-TES-1 in 25 patients. No OY-TES-1 sera reactivity was found in 20 normal individuals. These findings indicate that OY-TES-1 is an additional member of the cancer/testis family of antigens and that OY-TES-1 is immunogenic in humans. PMID:11248070

Ono, T; Kurashige, T; Harada, N; Noguchi, Y; Saika, T; Niikawa, N; Aoe, M; Nakamura, S; Higashi, T; Hiraki, A; Wada, H; Kumon, H; Old, L J; Nakayama, E



Repeated amphetamine administration induces a prolonged augmentation of phosphorylated cyclase response element-binding protein and Fos-related antigen immunoreactivity in rat striatum  

Microsoft Academic Search

Semi-quantitative immunocytochemistry was used to investigate the levels of cyclase response element-binding protein, phosphorylated cyclase response element-binding protein, Fos and Fos-related antigen immunoreactivity in the striatum of rats after acute or repeated amphetamine administration. Rats were perfused 20 min (phosphorylated cyclase response element-binding protein) or 2 h (cyclase response element-binding protein, phosphorylated cyclase response element-binding protein, Fos, Fos-related antigen) after

J. N. Simpson; J. Q. Wang; J. F. McGinty



Successful Unrelated Cord Blood Transplantation For Homozygous ?-Thalassemia.  


A now 10-year-old Laotian female was delivered at 30-week gestation by cesarean section because of severe hydrops. Fetal blood sampling revealed homozygous ?-thalassemia. After immediate resuscitation, the infant was supported with frequent red cell transfusions. At 44 months of age, she received a 5 of 6 human leukocyte antigen-matched unrelated cord blood transplantation. She was treated with phlebotomy and chelation therapy with Deferasirox for correction of hemosiderosis and has been transfusion-independent since 41 days after transplant. She is currently 6 years after transplantation with stable, 100% donor engraftment, resolved iron overload, and normal growth and development. PMID:23337553

Gumuscu, Burak; Thompson, Elizabeth I; Grovas, Alfred C; Zach, Terrence L; Warkentin, Phyllis I; Coccia, Peter F



Dendritic Cells Pulsed with Protein Antigens In Vitro Can Prime Antigen-specific, MHC-restricted T Cells In Situ  

Microsoft Academic Search

Summary T cellsrecognizepeptidesthatarebound toMHC moleculeson thesurfaceofdifferent types ofantigen-presenting cells(APC).Antigenpresentation most oftenisstudiedusingT cellsthat haveundergonepriminginsitu,orcelllinesthathavebeenchronically stimulatedinvitro.The useofprimedcellsprovidessufficient numbersofantigen-reactive lymphocytesforexperimental study.A more completeunderstandingofimmunogenicity,however,requiresthatone develop systemsforstudyingtheonsetofa T cellresponsefrom unprimed lymphocytes,especially in situ.Here itisshown thatmouse T cellscanbe reliably primed insituusingdendriticcells asAPC .The dendritic cellswereisolated from spleen,pulsedwithproteinantigens,andthen administered tonaivemice.Antigen-responsive T cellsdevelopedinthedraininglymphoidtissue, andtheseT cellsonlyrecognizedproteinwhen presentedon cells bearingthesameMHC products astheoriginal primingdendritic cells .Incontrast, little ornoprimingwas seenifantigen-pulsed spleencellsor peritoneal

Kayo Inaba; Joshua P. Metlay; Mary T. Crowley; Ralph M. Steinman


Identification of a protein isolated from senescent human cells that binds to hepatitis B virus X antigen.  


Hepatitis B virus-encoded X antigen contributes to the development of hepatocellular carcinoma. Given that X antigen functions by binding to other proteins, additional X-binding proteins were sought from an adult human liver cDNA library in a yeast two-hybrid system. The results yielded a clone encoding a 55-kd protein that is associated with replicative senescence (p55sen). Binding of p55sen to X antigen was confirmed in vitro by immunoprecipitation and affinity chromatography. The expression of endogenous p55sen inversely correlated with cell growth. Transient transfection of X antigen or p55sen into HepG2 cells stimulated DNA synthesis by twofold to threefold, whereas cotransfection did not, suggesting that these molecules functionally interact. The detection of p55sen in embryonic mouse liver, its absence in adult mouse and human livers, and its reappearance in livers from carriers with chronic liver disease, suggest that it may play important roles in the regulation of liver cell growth. The similarity between p55sen and a notch ligand, which is involved in cell fate determinations during embryogenesis, implies that the binding of p55sen by X antigen may also contribute to an alteration in cell fate, which is characteristic of carcinogenesis. PMID:9425942

Sun, B S; Zhu, X; Clayton, M M; Pan, J; Feitelson, M A



Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines.  


Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases. PMID:21899371

Xu, Peng; Alam, Mohammad Murshid; Kalsy, Anuj; Charles, Richelle C; Calderwood, Stephen B; Qadri, Firdausi; Ryan, Edward T; Ková?, Pavol



Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.  

PubMed Central

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events. Images

Collins, K L; Kelly, T J



Identification, cloning and characterization of BmP41, a common antigenic protein of Babesia microti.  


Babesia microti is a rodent tick-borne blood parasite and the major causative agent of emerging human babesiosis. Here, we identified a candidate of common antigenic protein BmP41 of B. microti by serological screening of cDNA library of human-pathogenic Gray strain with antisera against rodent Munich strain. Immunofluorescent antibody test using mouse anti-recombinant BmP41 (rBmP41) serum revealed that native BmP41 was expressed in each of the developmental stages of B. microti merozoites. An enzyme-linked immunosorbent assay (ELISA) using rBmP41 detected specific antibodies in sera from hamsters infected with B. microti Gray strain and mice infected with B. microti Munich strain. Taken together, BmP41 could be a promising universal serological marker for diagnosis of human babesiosis. PMID:23428774

Masatani, Tatsunori; Ooka, Hideo; Terkawi, Mohamad A; Cao, Shinuo; Luo, Yuzi; Asada, Masahito; Hayashi, Kei; Nishikawa, Yoshifumi; Xuan, Xuenan



Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry  

PubMed Central

Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies. Conclusion Our data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.



Challenges for the evaluation of Staphylococcus aureus protein based vaccines: monitoring antigenic diversity.  


Clumping factors A (ClfA) and B (ClfB) are Staphylococcus aureus virulence proteins that are displayed on the cell surface of the organism and have potential as vaccine antigens for the prevention of S. aureus disease. Here we evaluate the phylogeny of S. aureus in the context of antigenic variation of these two surface proteins. ClfA and ClfB gene sequences, along with epidemiological markers (MLST, spa and capsule genotype) were obtained for 224 S. aureus isolates including both historical strains and a collection representative of current MRSA isolates from the United States. Variation within ClfA and ClfB was consistent with the established population biology of S. aureus, namely, that S. aureus strains belong to a relatively small number of clonal lineages, with evolution proceeding mainly by mutation and with little to no recombination between clades. Thus most variation in ClfA and ClfB occurs between but not within lineages, and particular groups of ClfA and ClfB variants are closely linked. This has important implications for vaccine development and assessment as it suggests that a relatively small survey of strains will be representative of the total population variation, whereas for species that evolve mainly by recombination, such as Neisseria meningitidis, analysis of a much larger number of strains is needed to accomplish the same purpose. Our study also revealed evidence for the de-evolution of ClfB and therefore its reduced suitability as a target for vaccine development compared to ClfA. PMID:21245656

Murphy, Ellen; Lin, Shuo L; Nunez, Lorna; Andrew, Lubomira; Fink, Pamela S; Dilts, Deborah A; Hoiseth, Susan K; Jansen, Kathrin U; Anderson, Annaliesa S



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

NASA Astrophysics Data System (ADS)

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.

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



Isolation of Mycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography  

Microsoft Academic Search

Proteins secreted into the culture medium byMycobacterium tuberculosis (M. tb) are shown to be source of antigens of immunodiagnostic interest. Anin vitro released 31 kDa antigen ESAS-7F isolated fromM.tb H37Ra culture filtrate by salt precipitation, SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown\\u000a earlier to be a diagnostically important antigen fraction. In this report, we describe the

E. Raji Nair; Swati Banerjee; Satish Kumar; M. V. R. Reddy; B. C. Harinath



Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.  


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

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



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

PubMed Central

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.

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



Human H-Y: A Male-Specific Histocompatibility Antigen Derived from the SMCY Protein  

Microsoft Academic Search

H-Y is a transplantation antigen that can lead to rejection of male organ and bone marrow grafts by female recipients, even if the donor and recipient match at the major histocompatibility locus of humans, the HLA (human leukocyte antigen) locus. However, the origin and function of H-Y antigens has eluded researchers for 40 years. One human H-Y antigen presented by

Wei Wang; Leslie R. Meadows; Joke M. M. den Haan; Nicholas E. Sherman; Ye Chen; Els Blokland; Jeffrey Shabanowitz; Alexander I. Agulnik; Ronald C. Hendrickson; Colin E. Bishop; Donald F. Hunt; Els Goulmy; Victor H. Engelhard



Molecular and antigenic characteristics and synthesis of rubella virus structural proteins.  


The molecular and antigenic properties and synthesis of the structural proteins as well as the virus-specific RNAs of rubella virus were analyzed. Virions contain three major polypeptides--E1 (relative molecular weight [Mr] 58,000), E2 (Mr 42,000-47,000), and C (Mr 33,000). E1 and E2 are glycosylated and located externally on the viral membrane. C is associated with the genomic RNA to form the nucleocapsid. E2 occurs in two forms, E2a and E2b; the protein moieties of the two are indistinguishable. E1 is the viral hemagglutinin. IgG antibodies react with all the structural proteins, whereas IgM and IgA antibodies react predominantly with E1 and C proteins, respectively. After rubella vaccination, the reactivity of IgG antibodies matured slowly and reactivity of IgM and IgA antibodies remained at a low level. Rubella virus contains a 40S (Mr approximately 3.8 X 10(6)) single-stranded RNA with a 5' cap structure and a 3' poly(A) tract. In infected cells a 24S (Mr approximately 1.2 X 10(6)) subgenomic, polyadenylated mRNA is synthesized; it codes for a precursor (Mr 110,000 [p110]) to the structural proteins. The gene order in the 24S RNA is NH2-C-E2-E1-COOH. The overall structure and strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses of the Togaviridae family. PMID:4001721

Pettersson, R F; Oker-Blom, C; Kalkkinen, N; Kallio, A; Ulmanen, I; Kääriäinen, L; Partanen, P; Vaheri, A


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

SciTech Connect

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.

Liu Jianjun; Xing Xiumei; Huang Haiyan; Jiang Yingzhi; He Haowei; Xu Xinyun; Yuan Jianhui; Zhou Li; Yang Linqing [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 21, Rd 1st Tianbei, 518020 Shenzhen (China); Zhuang Zhixiong, E-mail: [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 21, Rd 1st Tianbei, 518020 Shenzhen (China)



The POU domain protein Tst-1 and papovaviral large tumor antigen function synergistically to stimulate glia-specific gene expression of JC virus.  

PubMed Central

Synergism between transcriptional activators is a powerful way of potentiating their function. Here we show that the glial POU domain protein Tst-1 (also known as Oct-6 and SCIP) and large tumor antigen (T antigen) synergistically increased transcription from both the early and the late promoters of papovavirus JC in glial cells. Synergism between both proteins did not require T-antigen-mediated DNA replication or direct binding of T antigen to the promoter. The ability of T antigen to functionally cooperate with Tst-1 was contained within its N-terminal region, shown by the fact that small tumor antigen (t antigen) could substitute for T antigen in transfection experiments. In addition to this functional synergism, a direct interaction between Tst-1 and T antigen was observed in vitro. Using deletion mutants of Tst-1 and T antigen, the POU domain of Tst-1 and the N-terminal region of T antigen were found to participate in this interaction. Because of the low levels of Tst-1 present in oligodendrocytes, synergism between Tst-1 and T antigen could be an important factor in establishing the lytic infection of oligodendrocytes by JC virus during the course of the fatal demyelinating disease progressive multifocal leukoencephalopathy. Images

Renner, K; Leger, H; Wegner, M



Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin.  


Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites. PMID:12189150

Osaki, Tsukasa; Okino, Nozomu; Tokunaga, Fuminori; Iwanaga, Sadaaki; Kawabata, Shun-Ichiro



Antigenic Equivalence of Human T-Cell Responses to Mycobacterium tuberculosis-Specific RD1-Encoded Protein Antigens ESAT-6 and Culture Filtrate Protein 10 and to Mixtures of Synthetic Peptides  

PubMed Central

The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96, P < 0.0001 for ESAT-6; r = 0.98, P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6; r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.

Arend, Sandra M.; Geluk, Annemieke; van Meijgaarden, Krista E.; van Dissel, Jaap T.; Theisen, Michael; Andersen, Peter; Ottenhoff, Tom H. M.



Unrelated parallel machine scheduling with job splitting  

Microsoft Academic Search

Scheduling jobs on unrelated parallel machines is an activity that is very much a part of industrial scheduling. We report a methodology for minimizing the total weighted tardiness of all jobs intended to be processed on unrelated parallel machines in the presence of dynamic job releases and dynamic machine availability. More importantly, the mixed (binary) integer linear programming model formulated




Tumor antigen delivered by Salmonella III secretion protein fused with heat shock protein 70 induces protection and eradication against murine melanoma.  


Attenuated Salmonella typhimurium possess the ability to stimulate innate immune responses and preferentially allocate within the solid tumor. These two main characteristics make attenuated Salmonella one of the most attractive vehicles for development of vaccine and also targeted cancer therapies. However, location of Salmonella prevents the process of antigen presentation. Salmonella Type III secretion system can be utilized to circumvent this problem because this system secretes the protein it encoded outside the cells. Heat shock protein 70 (Hsp70) is referred to as an "immunochaperone" for its capacity to elicit tumor-specific adaptive immune responses in the form of Hsp70-TAA (tumor associated antigen) complex. Hsp70 facilitates the cross-presentation of exogenous antigens through its receptor on antigen-presenting cells and therefore activates an antigen-specific cytotoxic T lymphocyte (CTL) response, which can directly contribute to potent anti-tumor immunity. Here, we designed a novel therapeutic vaccine utilizing the type III secretion system and Hsp70 to deliver and present the tumor-specific antigen. This live recombinant bacteria vaccine, when administrated orally, successfully broke the immune tolerance, induced a specific CTL response against tumor cells, and therefore revealed protective and therapeutic effects against generation and growth of B16F10 melanoma in C57BL/6J mice. PMID:20880334

Zhu, Xiangying; Zhou, Ping; Cai, Jianguo; Yang, Guimei; Liang, Shenghua; Ren, Daming



Human immunodeficiency virus (HIV) antigens: Structure and serology of multivalent human mucin MUC1-HIV V3 chimeric proteins  

SciTech Connect

Molecular modeling and two-dimensional NMR techniques enable us to identify structural features in the third variable region (V3) loop of the human immunodeficiency virus (HIV) surface glycoprotein gp120, in particular the principal neutralizing determinant (PND), that remain conserved despite the sequence variation. The conserved structure of the PND is a solvent-accessible protruding motif or a knob, structurally isomorphous with the immunodominant knobs in the tandem repeat protein of human mucin 1 (MUC1) (a tumor antigen for breast, pancreatic, and ovarian cancer). We have replaced the mucin antigenic knobs by the PND knobs of the HIV MN isolate in a set of chimeric human MUC1/HIV V3 antigens. This produced multivalent HIV antigens in which PNDs are located at regular intervals and separated by extended mucin spacers. In this article we show by two-dimensional NMR spectroscopy that the multivalent antigens preserve the PNDs in their native structure. We also demonstrate by ELISA that the antigens correctly present the PNDs for binding to monoclonal antibodies or polyclonal antisera from HIV-infected patients. 38 refs., 3 figs., 1 tab.

Fontenot, J.D.; Gatewood, J.M.; Mariappan, S.V.S. [Los Alamos National Laboratory, NM (United States)] [and others



Sequence Diversity of the Bacillus thuringiensis and B. cereus Sensu Lato Flagellin (H Antigen) Protein: Comparison with H Serotype Diversity  

Microsoft Academic Search

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen (Hag)) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin (hag) alleles from 80 Bacillus thurin- giensis strains and 16 strains from the B. cereus sensu lato

Dong Xu; Jean-Charles Cote ´



Prevention of antigen-induced microtubule organizing center reorientation in cytotoxic T cells by modulation of protein kinase C activity  

Microsoft Academic Search

Lysis of target cells (TC) by cytotoxic T lymphocytes (CTL) is achieved by directional exocytosis of cytolytic molecules—perforin and granzymes. They are stored within lytic granules which can be readily released following antigenic stimulation. Secretion of lytic molecules appears to be controlled by protein kinase C (PKC) activity, since specific modulators of PKC activity abolish the lysis of TC. We

Dragana Nesic; Scott Henderson; Michael Heidelberger



In Vivo Selection for Neisseria gonorrhoeae Opacity Protein Expression in the Absence of Human Carcinoembryonic Antigen Cell Adhesion Molecules  

Microsoft Academic Search

phenicol-resistant (Cmr) strain and following Cmr Opa populations mixed with a higher percentage of Opa variants of the wild-type (Cms) strain. Reciprocal experiments (Opa Cmr gonococci spiked with Opa Cms bacteria) were consistent with selection of Opa variants. Based on the absence in mice of human carcino- embryonic antigen cell adhesion molecules, the major class of Opa protein adherence receptors,

Amy N. Simms; Ann E. Jerse



Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.  


The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders. PMID:19623252

Bombaci, Mauro; Grifantini, Renata; Mora, Marirosa; Reguzzi, Valerio; Petracca, Roberto; Meoni, Eva; Balloni, Sergio; Zingaretti, Chiara; Falugi, Fabiana; Manetti, Andrea G O; Margarit, Immaculada; Musser, James M; Cardona, Francesco; Orefici, Graziella; Grandi, Guido; Bensi, Giuliano



Chlamydial Serology: Comparative Diagnostic Value of Immunoblotting, Microimmunofluorescence Test, and Immunoassays Using Different Recombinant Proteins as Antigens  

Microsoft Academic Search

To improve the reliability of the serodiagnosis of Chlamydia trachomatis infections, an immunoblot analysis, a microimmunofluorescence titration, and different immunoassays using synthetic peptides derived from spe- cies-specific epitopes in variable domain IV of the major outer membrane protein or recombinant antigens (heat shock protein 70 (hsp70), hsp60, hsp10, polypeptide encoded by open reading frame 3 of the plasmid (pgp3), macrophage

S. Bas; P. Muzzin; B. Ninet; J. E. Bornand; C. Scieux; T. L. Vischer



A synthetic parvovirus B19 capsid protein can replace viral antigen in antibody-capture enzyme immunoassays  

Microsoft Academic Search

To establish a renewable source of parvovirus B19 antigens for diagnostic tests, gene sequences for the viral capsid proteins, VP1 and VP2, were cloned into baculovirus expression vectors and the recombinant viruses used to infect Sf9 insect cells. Cell lysates examined by immunoblotting demonstrated reactive proteins corresponding to the expected sizes of native VP1 (83 kDa) and VP2 (58 kDa).

William C. Koch



GILT Modulates CD4+ T-Cell Tolerance to the Melanocyte Differentiation Antigen Tyrosinase-Related Protein 1  

Microsoft Academic Search

Gamma-IFN-inducible lysosomal thiol reductase (GILT) facilitates major histocompatibility complex class II-restricted processing through endocytic reduction of protein disulfide bonds and is necessary for efficient class II-restricted processing of melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). Using class II-restricted, TRP1-specific T-cell receptor transgenic mice, we identify a role, to our knowledge, previously unreported, for GILT in the maintenance of tolerance to

Matthew P Rausch; K Taraszka Hastings



Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Bromodomain Protein Brd4 on Host Mitotic Chromosomes  

Microsoft Academic Search

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA\\/viral episomes to the host mitotic chromosomes, and LANA chromosome- binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic

Jianxin You; Viswanathan Srinivasan; Gerald V. Denis; William J. Harrington; M. E. Ballestas; K. M. Kaye; P. M. Howley



Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens.  

PubMed Central

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-dalton protein is a major surface antigen, and like the fimbrial subunit, exhibited variation in electrophoretic mobility between serotypically different isolates. Images

Mattick, J S; Anderson, B J; Mott, M R; Egerton, J R



Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis.  


Bovine tuberculosis is an important infectious disease caused by Mycobacterium bovis, which is responsible for considerable economic losses. This disease constitutes a serious public health problem. Control programs in most countries, including Brazil, are based on the identification and slaughter of infected animals, as defined by the skin tuberculin test, which has its constraints. In the present study, the recombinant proteins CFP-10, ESAT-6, Mb0143, MPB83, PE5, PE13, TB10.4, TB15.3 and a chimera of ESAT-6/MPB70/MPB83 (fusion protein) were tested as ELISA antigens for the diagnosis of bovine tuberculosis. The proteins were produced in Escherichia coli, purified and tested in ELISAs with sera from 126 cattle having tested negative in the comparative intradermal tuberculin test (CITT) and 107 sera from cattle having tested positive in the CITT. Also, 236 sera from two BTB-free beef cattle herds were tested. Among the proteins tested, only the ESAT-6/MPB70/MPB83 chimera demonstrated satisfactory agreement with the CITT (kappa index: 0.688), reflecting in 83.2% sensitivity and 86.5% specificity. The ELISA absorbances of the cattle sera from BTB-free herds showed similar levels to those of CITT positive cattle, probably as the result of successive skin tuberculinizations to define the BTB-free status of the herds. However, the ELISA with the ESAT-6/MPB70/MPB83 chimera was useful to discriminate BTB positive and negative cattle in herds prior to the tuberculin skin test. PMID:23419946

Souza, Ingrid If; Melo, Elaine Sp; Ramos, Carlos An; Farias, Thaís A; Osório, Ana Luiza Ar; Jorge, Klaudia Sg; Vidal, Carlos Es; Silva, Altino S; Silva, Márcio R; Pellegrin, Aiesca O; Araújo, Flábio R



Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1  

PubMed Central

Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to therapeutic development through the use of anti-sense strategies or small molecules targeting EBNA1 mRNA structure.

Tellam, Judy T.; Lekieffre, Lea; Zhong, Jie; Lynn, David J.; Khanna, Rajiv



Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death  

PubMed Central

In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-? response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro.

Hartmann, Wiebke; Brenz, Yannick; Kingsley, Manchang Tanyi; Ajonina-Ekoti, Irene; Brattig, Norbert W.; Liebau, Eva; Breloer, Minka



Association of protein kinase C-delta with the B cell antigen receptor complex.  


Protein kinase C (PKC)-delta is a diacylglycerol-dependent, calcium-independent novel PKC isoform and has been demonstrated to exert negative regulatory functions in B lymphocytes as well as in mast cells. Whereas in mast cells PKC-delta functionally interacts with the high-affinity receptor for IgE, FcepsilonR1, no such association has been described for the B cell antigen receptor (BCR). In this report, for the first time, we demonstrate the interaction of PKC-delta with different classes of BCR by means of affinity purification and native protein complex analysis. Using a C-terminally truncated Ig-alpha as well as non-phosphorylated and phosphorylated peptides representing C-terminal regions of Ig-alpha, the dependence of this BCR/PKC-delta interaction on tyrosine-phosphorylated Ig-alpha is shown. Finally, splenocytes from PKC-delta-deficient mice are found to exert reduced phosphorylation of PKD (a.k.a. PKC-mu) in response to BCR engagement, suggesting the early, membrane-proximal activation of an attenuating kinase complex including PKC-delta and PKD. PMID:17098397

Pracht, Catrin; Minguet, Susana; Leitges, Michael; Reth, Michael; Huber, Michael



Formation of potential titanium antigens based on protein binding to titanium dioxide nanoparticles  

PubMed Central

Degradation products of titanium implants include free ions, organo-metallic complexes, and particles, ranging from nano to macro sizes. The biological effects, especially of nanoparticles, is yet unknown. The main objective of this study was to develop Ti-protein antigens in physiological solutions that can be used in testing of cellular responses. For this purpose, 0.1% TiO2 nanoparticles less than 100 nm were mixed with human serum albumin (HSA), 0.1% and 1%, in cell culture medium (DMEM, pH 7.2). The Ti concentrations in the resulting solutions were analyzed by inductively coupled plasma mass spectrometry. The stability of the nanoparticles in suspension was analyzed by UV-vis spectrophotometer and Dynamic Light Scattering. The concentration of Ti in suspension was dependent on the presence and concentration of HSA. Albumin prevented high aggregation rate of TiO2 nanoparticles in cell culture medium. It is shown that nano TiO2-protein stable aggregates can be produced under physiological conditions at high concentrations, and are candidates for use in cellular tests.

Vamanu, Carmen Irina; H?l, Paul Johan; Allouni, Zouhir Ekeland; Elsayed, Said; Gjerdet, Nils Roar



The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells5  

PubMed Central

The hematopoietic actin regulatory protein HS1 is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in antigen presentation has not been addressed. We show that dendritic cells (DCs) from HS1?/? mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1?/? DCs present OVA peptide efficiently to CD4+ T cells. However, presentation of ovalbumin protein is defective. Similarly, MHC Class I-dependent presentation of VSV8 peptide to CD8+ T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of antigen uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1?/? DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient antigen presentation of exogenous antigen by DCs.

Huang, Yanping; Biswas, Chhanda; Dehring, Deborah A. Klos; Sriram, Uma; Williamson, Edward K.; Li, Shuixing; Clarke, Fiona; Gallucci, Stefania; Argon, Yair; Burkhardt, Janis K.



A plant signal peptide-hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells  

PubMed Central

The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage protein vspA (VSP?S) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSP?S-HBsAg fusion accumulation. This product was more stable and presented the protective “a” antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSP?S-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSP?S-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.

Sojikul, Punchapat; Buehner, Norene; Mason, Hugh S.



Presentation of Antigen in Immune Complexes Is Boosted by Soluble Bacterial Immunoglobulin Binding Proteins  

PubMed Central

Using a snake toxin as a proteic antigen (Ag), two murine toxin–specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag–specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20–100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP–Ab–Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS® analyses showed that an Ag–Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag–Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor–containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.

Leonetti, Michel; Galon, Jerome; Thai, Robert; Sautes-Fridman, Catherine; Moine, Gervaise; Menez, Andre



Enzymatic hydrolysis of heated whey: iron-binding ability of peptides and antigenic protein fractions.  


This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100 degrees C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50 degrees C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (beta-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of beta-lactoglobulin, alpha-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4 x 7 H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC. PMID:17699019

Kim, S B; Seo, I S; Khan, M A; Ki, K S; Lee, W S; Lee, H J; Shin, H S; Kim, H S



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

PubMed Central

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-?, 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.

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




PubMed Central

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.

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



A 230-kD Basic Protein Is the Major Bullous Pemphigoid Antigen  

Microsoft Academic Search

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of EP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP

Stephan Mueller; Vera Klaus-Kovtun; John R. Stanley



Marrow Transplantation from Unrelated Volunteer Donors  

Microsoft Academic Search

less than 30% of the patients in North America have an HLA-matched sibling and 3-5% have a one HLA-locus mismatched relative, for most patients in need of an allogeneic marrow transplant the only chance of finding a suitable donor is through the identification of an HLA-compatible unrelated volunteer. Three accomplishments have allowed unrelated donor transplants to become feasible and successful:

Claudio Anasetti


Fasciola hepatica: An Antigen Fraction Derived from Newly Excysted Juveniles, Containing an Immunoreactive 32-kDa Protein, Induces Strong Protective Immunity in Rats  

Microsoft Academic Search

van Milligen, F. J., Cornelissen, J. B. W. J., and Bokhout, B. A. 2000. Fasciola hepatica: An antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats. Experimental Parasitology94, 163–171. Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested

Florine J. van Milligen; Jan B. W. J. Cornelissen; Ben A. Bokhout



Binding kinetics of an antibody against HIV p24 core protein measured with real-time biomolecular interaction analysis suggest a slow conformational change in antigen p24  

Microsoft Academic Search

The interaction between HIV core protein p24 and the murine monoclonal antibody CB-4\\/1 or its Fab fragment showed unusual kinetics. Recombinant p24 was immobilised in a hydrophilic carboxymethyldextran matrix. At high concentration of CB-4\\/1 Fab the association of the antigen-antibody complex proceeds in two phases, while dissociation is mono-exponential. The antigen has a ‘memory’ i.e. shortly after dissociation of Fab-antigen

Ralf W. Glaser; Gert Hausdorf



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

PubMed Central

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.

Hernandez-Martinez, Miguel Angel; Escalante, Ananias A.; Arevalo-Herrera, Myriam; Herrera, Socrates



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

SciTech Connect

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.

Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio (Aichi Cancer Center Research Inst., Nagoya (Japan))



Antigenic relationships among penicillin-binding proteins 1 from members of the families Pasteurellaceae and Enterobacteriaceae.  

PubMed Central

Penicillin-binding proteins (PBPs) from Haemophilus influenzae RD purified by a combination of affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electroelution were used to immunize rabbits to obtain specific antisera. Antisera directed against PBP 1 (1b) of H. influenzae cross-reacted with representative organisms of the family Pasteurellaceae and with many members of the family Enterobacteriaceae but not with other gram-negative organisms. Immunization with purified PBP 3 of H. influenzae produced antisera that reacted with PBP 1 (1b) of H. influenzae and showed the same cross-reactive pattern with other species as the anti-PBP 1 antiserum. A 24,000-molecular-weight polypeptide of H. influenzae, not radiolabeled by [35S]penicillin, reacted with antisera against purified PBPs 1 (1a, 1b), 2, and 3. The results suggest that antigenic epitopes are shared among similar PBPs from related species and even among different PBPs within the same species. Images

Schryvers, A B; Wong, S S; Bryan, L E



Lack of antigenic diversification of major outer membrane proteins during clonal waves of Neisseria meningitidis serogroup A colonization and disease.  


In particular in the 'meningitis belt' of sub-Saharan Africa, epidemic meningococcal meningitis is a severe public health problem. In the past decades, serogroup A lineages have been the dominant etiologic agents, but also other serogroups have caused outbreaks. A comprehensive vaccine based on subcapsular outer membrane proteins (OMPs) is not available. Here, we have investigated whether meningococcal populations overcome herd immunity by changing antigenic properties of their OMPs. Meningococcal isolates were collected in the context of longitudinal studies in Ghana between 2002 and 2008 and in Burkina Faso between 2006 and 2007. Serogroup A strains isolated during two clonal waves of colonization and disease showed no diversification in the genes encoding their PorA, PorB, and FetA proteins. However, we detected occasional allelic exchange of opa genes, as well as wide variation in the number of intragenic tandem repeats, showing that phase variation of Opa protein expression is a frequent event. Altogether we observed a remarkable antigenic stability of the PorA, PorB and FetA proteins over years. Our results indicate that while herd immunity may be responsible for the disappearance of meningococcal clones over time, it is not a strong driving force for antigenic diversification of the major OMPs analyzed here. PMID:23620114

Huber, Charlotte A; Pflüger, Valentin; Hamid, Abdul-Wahab M; Forgor, Abudulai A; Hodgson, Abraham; Sié, Ali; Junghanss, Thomas; Pluschke, Gerd



Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum  

PubMed Central

Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO2-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expression is very rare calling for further investigations in this highly promising field. In this study, we present data on the expression of a monoclonal human IgG antibody against the Hepatitis B surface protein and the respective antigen in the diatom Phaeodactylum tricornutum. Antibodies are fully-assembled and functional and accumulate to 8.7% of total soluble protein, which complies with 21 mg antibody per gram algal dry weight. The Hepatitis B surface protein is functional as well and is recognized by algae-produced and commercial antibodies.

Hempel, Franziska; Lau, Julia; Klingl, Andreas; Maier, Uwe G.



Glucose Transport Protein is Structurally and Immunologically Related to Band 3 and Senescent Cell Antigen  

Microsoft Academic Search

Senescent cell antigen, a polypeptide that appears on the surface of senescent and damaged cells, has been shown to be derived from band 3. In the present study, the relationship between the as yet unidentified glucose transporter and senescent cell antigen is examined. Since cytochalasin B is a specific and potent competitive inhibitor of glucose transport in human erythrocytes, the

Marguerite M. B. Kay



Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray  

Microsoft Academic Search

Received 18 August 2008\\/Returned for modification 10 September 2008\\/Accepted 19 September 2008 Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include

Paul A. Beare; Chen Chen; Timo Bouman; Jozelyn Pablo; Berkay Unal; Diane C. Cockrell; Wendy C. Brown; Kent D. Barbian; Stephen F. Porcella; James E. Samuel; Philip L. Felgner; Robert A. Heinzen



Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.  

PubMed Central

Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas. Images

Thompson, A R



[Prokaryotic expression for fusion protein of human metapneumovirus and its preliminary application as an antigen for antibody detection].  


To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old. PMID:21528539

Zhu, Ru-nan; Qian, Yuan; Zhao, Lin-qing; Sun, Yu; Deng, Jie; Wang, Fang



Tissue-Specific Expression of the Gene Encoding a Mouse RNA Binding Protein Homologous to Human HuD Antigen  

Microsoft Academic Search

Abstract We have cloned and sequenced cDNAs encoding amouse ,RNA-binding protein that is homologous to human,HuD antigen. The amino acid sequence deduced from the nucleotide sequence has revealed that the mouse Hu is identical to the human ,counterpart except for two amino-acid substitutions outside the three RNA recognition motifs (RRMs) and,a difference in theN-terminus. The mouse ,HuD gene produces two

Ryoichi Abe; Yutaka Uyeno; Koichi Yamamoto; Hiroshi Sakamoto



Cellular Protein TIA-1 Regulates the Expression of HBV Surface Antigen by Binding the HBV Posttranscriptional Regulatory Element  

Microsoft Academic Search

Objective: To identify T-cell intracellular antigen 1 (TIA-1) interacting with the posttranscriptional regulatory element (PRE) of hepatitis B virus (HBV), and reveal TIA-1 function on HBV gene expression. Methods: In order to demonstrate TIA-1 binding to PRE, electrophoretic mobility shift and RNA and protein pull-down assays were used. The pDM138-PRE reporter system, HepG2.2.15 cell line and HBs-PRE transient expression cells

Hua Tang; Ying Huang; Jing Chen; Chao Yu; Ai-long Huang



Predicted and observed alleles of Plasmodium falciparum merozoite surface protein-1 (MSP-1), a potential malaria vaccine antigen  

Microsoft Academic Search

The 19-kDa antigenic domain of Plasmodium falciparum merozoite surface protein (MSP)-1 is a potential malaria vaccine candidate. Based on the amino acid substitution, four known alleles, E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type) of this domain have been identified. Using single or double crossover recombinational events, we predicted the existence of additional alleles of

Shoukat H Qari; Ya-Ping Shi; Ira F Goldman; Bernard L Nahlen; Michel Tibayrenc; Altaf A Lal



Enhanced Immunogenicity of a Genetic Chimeric Protein Consisting of Two Virulence Antigens of Streptococcus mutans and Protection against Infection  

Microsoft Academic Search

The saliva-binding region (SBR) of the cell surface antigen I\\/II (AgI\\/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of

Ping Zhang; Christina Jespersgaard; Leticia Lamberty-Mallory; Jannet Katz; Yan Huang; George Hajishengallis; Suzanne M. Michalek



Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients  

Microsoft Academic Search

Summary  Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen\\/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions,

Rishab K. Gupta; A. Marilyn Leitch; Donald L. Morton



Virus-like particles with host protein-like antigenic determinants protect an insect parasitoid from encapsulation  

Microsoft Academic Search

Summary Virus-like particles from several species of parasitoid wasps are known to interfere with encapsulation. We raised vertebrate antibodies against the virus-like particles from the waspVenturia canescens and used them to show that antigenic determinants of the particles display similarity to a protein component of the host. When particles on the egg surface are in turn covered with particle-specific antibodies,

I. Feddersen; K. Sander; O. Schmidt



The Babesia bovis Merozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins  

Microsoft Academic Search

Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus.

Monica Florin-Christensen; Carlos E. Suarez; Stephen A. Hines; Guy H. Palmer; Wendy C. Brown; Terry F. McElwain



Heterogeneous antigen recognition behavior of induced polyspecific antibodies.  


Polyspecific antibodies represent a significant fraction of the antibody repertoires in healthy animals and humans. Interestingly, certain antibodies only acquire a polyspecific antigen-binding behavior after exposure to protein-modifying conditions, such as those found at inflammation sites, or used in small- and large-scale immunoglobulin purification. This phenomenon is referred to as "criptic polyspecificity". In the present study, we compare the potential of different chemical agents to induce IgG polyspecificity. Depending on the treatment used, quantitative and qualitative differences in the recognition of individual antigens from a standard panel were observed. Antibodies with cryptic polyspecificity utilized common mechanisms for the recognition of structurally unrelated antigens when exposed to a particular inductor of polyspecificity. Our study contributes to the understanding of the mechanisms underlying the cryptic polyspecificity. PMID:20599726

Dimitrov, Jordan D; Planchais, Cyril; Kang, Jonghoon; Pashov, Anastas; Vassilev, Tchavdar L; Kaveri, Srinivas V; Lacroix-Desmazes, Sebastien



Cloning and expression of recombinant MPB70 protein antigen from Mycobacterium bovis BCG for diagnosis of tuberculosis.  


In a search for developing new skin test reagents, MPB70 protein antigen was a candidate antigen for the Diagnosis of bovine tuberculosis. First M. bovis BCG genomic DNA was extracted purified and the mpb70 gene was amplified by PCR. The gene was then ligated to an expression vector, PQE. After transformation of the expression E. coil M15 host strain with the PQE plasmid, the expression was induced using 10 mM of IPTG. Two bands were seen in the SDS-PAGE analysis the 44 and 50 KDa represents the dimmers of the nonglycosylated and glycosylated form of the reMPB70 antigen. The His-tagged reMPB70 antigen was then purified by metal affinity chromatography using Ni-NTA agarose. Protein refolding was done by the use of the co solvent Polyethylene glycol MW 3000. The diagnostic potential of the re-MPB70 was evaluated using sera from experimentally sensitized guinea pigs with different strains of mycobacteria (M. bovis BCG, M. tuberculosis, M. kansasii and M. intracellular) using ELISA test. The results indicated the efficiency of MPB70 but not bovine PPD to discriminate between M. bovis sensitized guinea pigs and those sensitized with other mycobacterial strains at serum dilution of 1150. In a field trials to using reMPB70 antigen for the serodiagnosis of bovine tuberculosis using ELISA test. Fifty serum samples from tuberculin +ve and 6 from tuberculin -ve cattle were used as well as 10 tuberculin +ve buffaloes. All +ve animals were confirmed to be M. bovis infected by P/M analysis, bacteriological examination. ELISA results revealed that reMPB70 could recognize the tuberculin +ve infected animals at serum dilution of 1/50 and that it could diagnose tuberculosis in cattle as well as buffaloes. PMID:16734114

Soliman, Y A; Ghonim, M; Nasr, E A; Mousse, I; Ebiary, E A; Selim, S A



The Major Cicatricial Pemphigoid Antigen Is a 180-kD Protein that Shows Immunologic Cross-Reactivities with the Bullous Pemphigoid Antigen  

Microsoft Academic Search

Recent studies have shown that sera from patients with cicatricial pemphigoid (CP) contained autoantibodies against epidermal antigens of molecular weight 230 kD and\\/or 180 kD by immunoblotting, similar to those recognized by bullous pemphigoid (BP) sera. Previous immunoprecipitation studies have shown that BP sera only precipitated the 230-kD antigen. To characterize the CP antigen(s) we tested 10 CP sera, 10

Philippe Bernard; Catherine Prost; Nathalie Durepaire; Nicole Basset-Seguin; Liliane Didierjean; Jean-Hilaire Saurat



Priming protective CD8 T cell immunity by DNA vaccines encoding chimeric, stress protein-capturing tumor-associated antigen.  


DNA vaccines encoding heat shock protein (hsp)-capturing, chimeric peptides containing antigenic determinants of the tumor-associated Ag (TAA) gp70 (an envelope protein of endogenous retrovirus) primed stable, specific, and tumor-protective CD8 T cell immunity. Expression of gp70 transcripts was detectable in most normal tissues but was particularly striking in some (but not all) tumor cell lines tested (including the adenocarcinoma cell line CT26). An approximately 200 residue gp70 fragment or its L(d)-binding antigenic AH1 peptide cloned in-frame behind an hsp-capturing (cT(272)) or noncapturing (T(60)) N-terminal large SV40 tumor Ag sequence was expressed as either hsp-binding or -nonbinding chimeric Ags. Only hsp-capturing, chimeric fusion proteins were expressed efficiently in transfected cell lines and primed TAA-specific CD8 T cell immunity. This immunity mediated protection in the CT26 and mKSA models. A vaccination strategy based on delivering antigenic, hsp-associated TAA fragments can thus prime protective CD8 T cell immunity even if these TAA are of low intrinsic immunogenicity. PMID:16849460

Schirmbeck, Reinhold; Riedl, Petra; Kupferschmitt, Mark; Wegenka, Ursula; Hauser, Hansjörg; Rice, Jason; Kröger, Andrea; Reimann, Jörg



Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles.  


Using poly(propylene sulfide) (PPS) and poly(ethylene glycol) (PEG) as components of a nanocarrier platform, we sought to compare immune responses induced by PPS-bl-PEG polymersomes (PSs; watery-core structures, with antigen incorporated within the PSs) and PEG-stabilized PPS nanoparticles (NPs; solid-core structures, with antigen conjugated upon the NP surface). We have previously shown strong CD8(+) T cell responses to antigen conjugated to NPs via a disulfide link, and here we investigated the extent to which antigen incorporated within oxidatively-sensitive PSs could induce CD4(+) or CD8(+) T cell responses. C57BL/6 mice were subcutaneously immunized with free ovalbumin (OVA) as a model antigen, or equivalent doses of OVA-loaded into PSs, conjugated onto NPs, or given as a mixture of the two. Free CpG was used as an adjuvant. Antigen-loaded PSs induced enhanced frequencies of antigen-specific CD4(+) T cells in the spleen, lymph nodes and lungs as compared to the NP formulation, whereas antigen-conjugated NPs induced stronger CD8(+) T cell responses. Co-administration of both PSs and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time, i.e. both strong CD4(+) and CD8(+) T cell responses. These results have important implications for particulate-based vaccine design and highlight the potential of using different antigen-delivery systems for the induction of both T helper and cytotoxic T lymphocyte immune responses. PMID:23478034

Stano, Armando; Scott, Evan A; Dane, Karen Y; Swartz, Melody A; Hubbell, Jeffrey A



Diversity of Ehrlichia ruminantium Major Antigenic Protein 1-2 in Field Isolates and Infected Sheep?  

PubMed Central

Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.

Barbet, Anthony F.; Byrom, Barbara; Mahan, Suman M.



A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo  

PubMed Central

The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10?14?mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-?? by TLR4-expressing cells, as well as the production of TNF-? by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

Durantez, Maika; Lozano, Teresa; Rudilla, Francesc; Rehberger, Federico; Casares, Noelia; Villanueva, Lorea; Martinez, Marta; Gorraiz, Marta; Borras-Cuesta, Francisco; Sarobe, Pablo; Prieto, Jesus; Lasarte, Juan Jose



A quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.  


A mass spectrometry-based antibody selection procedure was developed to evaluate optimal 'capture' monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K(d)) were determined for 'bound mAb-antigen' vs. 'unbound antigen' using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal 'capture' mAbs were determined through evaluation of relative K(d) constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K(d) constants as determined using NIST Standard Reference Material (SRM) 2921 - Human Cardiac Troponin Complex. This ID LC-MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification. PMID:21856254

Lowenthal, Mark S; Gasca-Aragon, Hugo; Schiel, John E; Dodder, Nathan G; Bunk, David M



pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor  

Microsoft Academic Search

It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.

Vladimir P. Zav'yalov; Vyacheslav M. Abramov; Peter G. Cherepanov; Galina V. Spirina; Tatiana V. Chernovskaya; Anatolii M. Vasiliev; Galina A. Zav'yalova



Antigenic complementarity between HIV and other AIDS-associated infections results in idiotype-antiidiotype antibody complexes that cross react with lymphocyte proteins.  


HIV proteins mimic HLA proteins, and the proteins of cofactor infections (cytomegalovirus, hepatitis viruses, mycobacteria, mycoplasmas) mimic CD4 proteins, making some HIV antigens molecularly complementary to cofactor antigens. Antibodies to HIV and its cofactors should therefore act like idiotype-antiidiotype pairs. Over 2000 combinations of antibodies were tested for such complementarity using modified forms of Ouchterlony immunodiffusion and ELISA. HIV antibodies do precipitate some antibodies against cofactor infections including CMV, HBV, mycobacteria and mycoplasmas. These antibodies also mimic antibodies against HLA and CD4 proteins. Thus, some combinations of HIV with cofactor infections may induce lymphocytotoxic antibodies (LCTA), a risk that must be addressed in vaccine development. PMID:15755587

Root-Bernstein, Robert



Immunological Unresponsiveness to Protein Antigens in Rabbits Exposed to X-Irradiation or 6-Mercaptopurine Treatment  

PubMed Central

Immune unresponsiveness to HSA and BSA was induced in adult rabbits exposed to 550 R. total-body irradiation. Immunogenic doses of antigen, 20–75 mg., were found to be tolerogenic in this system. The degree of unresponsiveness was found to be a function of the time interval between X-irradiation and the beginning of antigen administration. The unresponsive animals retained the circulating antigen in quantities detectable by the gel-diffusion technique. These results were compared with the state of unresponsiveness induced by means of 6-mercaptopurine, and similar parameters were found to be operative. Some aspects of the significance of the ratio of antigen to competent cells, in the induction of immune unresponsiveness, are discussed. ImagesFIG. 2FIG. 3FIG. 4FIG. 5FIG. 7

Nachtigal, D.; Feldman, M.



Modified anthrax fusion proteins deliver HIV antigens through MHC Class I and II pathways  

Microsoft Academic Search

T cell-based HIV vaccine candidates have focused on eliciting both CD4- and CD8-mediated responses. One challenge in vaccine development is the successful introduction and presentation of exogenous antigen to elicit an immune response. Modified bacterial toxins have been studied extensively as intracellular delivery agents because of their unique capability to translocate antigen across the cell membrane without affecting cell viability.

K. McEvers; M. Elrefaei; P. Norris; S. Deeks; J. Martin; Y. Lu; H. Cao



Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy  

Microsoft Academic Search

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent\\u000a functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility\\u000a complexes (MHC)-class II molecules to CD4+ T cells, but also on MHC-class I molecules to CD8+ T cells. Immune responses against topically applied antigen

Vincent Flacher; Florian Sparber; Christoph H. Tripp; Nikolaus Romani; Patrizia Stoitzner



Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein  

Microsoft Academic Search

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication1. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication2, so the majority of replication enzymes must be cellular. Indeed, a number of

Gregory Prelich; Cheng-Keat Tan; Matthew Kostura; Michael B. Mathews; Antero G. So; Kathleen M. Downey; Bruce Stillman



Genetic, Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species? †  

PubMed Central

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.

B?rud, Bente; Aas, Finn Erik; Vik, Ashild; Winther-Larsen, Hanne C.; Egge-Jacobsen, Wolfgang; Koomey, Michael



Proteomic and Immunoblot Analyses of Bartonella quintana Total Membrane Proteins Identify Antigens Recognized by Sera from Infected Patients?  

PubMed Central

Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.

Boonjakuakul, Jenni K.; Gerns, Helen L.; Chen, Yu-Ting; Hicks, Linda D.; Minnick, Michael F.; Dixon, Scott E.; Hall, Steven C.; Koehler, Jane E.



Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).  


Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs. PMID:23968686

Mousa, Ahmed Abdelmoniem; Cao, Shinuo; Aboge, Gabriel Oluga; Terkawi, Mohamad Alaa; Kirdasy, Ahmed El; Salama, Akram; Attia, Mabrouk; Aboulaila, Mahmoud; Zhou, Mo; Kamyingkird, Ketsarin; Moumouni, Paul Franck Adjou; Masatani, Tatsunori; Aziz, Sami Ahmed Abd El; Moussa, Waheed Mohammed; Chahan, Bayin; Fukumoto, Shinya; Nishikawa, Yoshifumi; Ballal, Salah Sayed El; Xuan, Xuenan



Incidental CD8 T cell reactivity against caspase-cleaved apoptotic self-antigens from ubiquitously expressed proteins in islets from prediabetic human leucocyte antigen-A2 transgenic non-obese diabetic mice  

PubMed Central

Apoptosis is known as a major mechanism which contributes to beta cell decay in type 1 diabetes. Commitment to this pathway generally involves caspase-mediated protein cleavage and was found to induce cross-presentation of a specific antigen repertoire under certain inflammatory conditions. We aimed to assess the significance of the CD8 T cell population reactive against such caspase-cleaved apoptotic self-antigens in pancreatic islets of prediabetic human leucocyte antigen (HLA)-A2 transgenic non-obese diabetic chimeric monochain transgene construct (NOD.HHD) mice. We have reproduced a unique peptide library consisting of human CD8 T cell-derived apoptosis-specific antigens, all of which belong to structural proteins expressed ubiquitously in human islets. Pancreatic islets from prediabetic NOD.HHD mice, harbouring humanized major histocompatibilty complex (MHC) class I, were isolated and handpicked at various ages, and islet-infiltrating CD8 T cells were expanded in vitro and used as responders in an interferon (IFN)-? enzyme-linked immunospot (ELISPOT) assay. Human T2 cells were used as antigen-presenting cells (APC) to avoid endogenous antigen presentation. Analogous to the interindividual variability found with peptides from known islet autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) and insulin, some mice showed variable, low-degree CD8 T cell reactivity against caspase-cleaved self-antigens. Because reactivity was predominantly minor and often undetectable, we conclude that beta cell apoptosis does not routinely provoke the development of dominant cytotoxic T lymphocyte (CTL) reactive against caspase-cleaved self-antigens in the NOD.HHD model.

Coppieters, K T; Amirian, N; von Herrath, M G



Treatment of relapsed leukemia after unrelated donor marrow transplantation with unrelated donor leukocyte infusions  

Microsoft Academic Search

The efficacy and toxicity of donor leuko- cyte infusions (DLI) after unrelated donor bone marrow transplantation (BMT) is largely unknown. We identified 58 recipi- ents of unrelated DLI (UDLI) for the treatment of relapsed disease from the National Marrow Donor Program data- base. A retrospective analysis was per- formed to determine response, toxicity, and survival after UDLI and to identify

David L. Porter; Robert H. Collins; Christine Hardy; Nancy A. Kernan; William R. Drobyski; Sergio Giralt; Mary E. D. Flowers; James Casper; Pablo Parker; Rosemarie Mick; Bev Bate-Boyle; Joseph H. Antin



Neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid beta-protein of Alzheimer's disease.  

PubMed Central

Formalin-fixed, paraffin-embedded temporal lobe sections from eight former boxers' brains were examined using an immunohistochemical method with antibodies to amyloid beta protein. In accord with recent observations in Alzheimer's disease, significant numbers of beta-protein immunoreactive neurofibrillary tangles (NFT) were observed in three cases. Most of these immunoreactive NFTs appeared to be tombstone tangles, although not all such tangles were stained. This immunoreaction was completely abolished by preincubation of antibodies with synthetic beta-protein peptides, and the identity of the immunostained NFTs was confirmed by polarization microscopy of sections counterstained with Congo red. However, it is not yet clear if the beta-protein antigens are, in fact, an integral part of paired helical filaments. These observations, together with our recent finding of beta-immunoreactive plaque-like lesions in dementia pugilistica, also emphasize the many similarities in pathology between this condition and Alzheimer's disease. Images Figure 1

Allsop, D.; Haga, S.; Bruton, C.; Ishii, T.; Roberts, G. W.



Human Antibody Response to Thioredoxin Peroxidase-1 and Tandem Repeat Proteins as Immunodiagnostic Antigen Candidates for Schistosoma japonicum Infection  

PubMed Central

Schistosomiasis continues to be a public health problem in many tropical and subtropical countries. Improving the diagnostic tools for surveillance and monitoring in areas that have reached elimination level will help hasten the possible elimination of this disease. This study therefore aims to develop enzyme-linked immunosorbent assay through the use of recombinant proteins such as thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, and Sj7TR). Cutoff values were calculated using 38 serum samples from healthy Japanese volunteers. Sera from 35 schistosomiasis-confirmed patients, four cured from the disease by chemotherapy, and 15 endemic negative controls were used to assess these antigens. SjTPx-1 and Sj7TR both had 85.71% sensitivity. Furthermore, these antigens were also tested against human sera positive for other parasitic infections and showed no or very minimal cross-reaction. These results suggest the potential defined antigens for development of an accurate diagnostic test for schistosomiasis.

Angeles, Jose Ma.; Goto, Yasuyuki; Kirinoki, Masashi; Leonardo, Lydia; Tongol-Rivera, Pilarita; Villacorte, Elena; Inoue, Noboru; Chigusa, Yuichi; Kawazu, Shin-ichiro



Transfer of Proteins from Gels to Diazobenzyloxymethyl-Paper and Detection with Antisera: A Method for Studying Antibody Specificity and Antigen Structure  

Microsoft Academic Search

We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide\\/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected

Jaime Renart; Jakob Reiser; George R. Stark



Mapping of Monoclonal Antibody Binding Sites on CNBr Fragments of the S-Layer Protein Antigens of Rickettsia Typhi and Rickettsia Prowazekii. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsia...

W. M. Ching M. Carl G. A. Dasch



Presence of common antigens, including major surface protein epitopes, between the cattle (intraerythrocytic) and tick stages of Anaplasma marginale.  


Epitopes of major surface proteins of the intraerythrocytic cattle stage of Anaplasma marginale were demonstrated in the midgut stage of the organism within the infective tick host Dermacentor andersoni. These proteins were common to all A. marginale isolates tested and at all stages of parasitemia. Sera from cattle immunized with the tick midgut stage of A. marginale immunoprecipitated multiple-erythrocyte-stage proteins, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major proteins recognized (primarily greater than 14 and less than 200 kilodaltons [kDa]) included two major-erythrocyte-stage surface proteins of 36 and 105 kDa molecular size. To confirm the presence of common tick and erythrocyte A. marginale antigens with the immunized cattle sera, we purified the 36-kDa erythrocyte-stage protein by monoclonal immunoaffinity chromatography and developed an enzyme-linked immunosorbent assay based on the purified protein. All sera from cattle immunized with tick-stage A. marginale and cattle infected with various isolates of A. marginale developed antibodies to the 36-kDa protein. The potential immunoprophylactic, diagnostic, and epidemiologic value of the major epitopes common to both the invertebrate and mammalian stages of A. marginale, especially the 36-kDa protein, is discussed. PMID:2415457

Palmer, G H; Kocan, K M; Barron, S J; Hair, J A; Barbet, A F; Davis, W C; McGuire, T C



Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.  


Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines. PMID:22221920

Buriani, Giampaolo; Mancini, Camillo; Benvenuto, Eugenio; Baschieri, Selene



Characteristics of carbohydrate antigen binding to the presentation protein HLA-DR  

PubMed Central

Zwitterionic polysaccharide antigens (ZPSs) were recently shown to activate T cells in a class II major histocompatibility complex (MHCII)-dependent fashion requiring antigen presenting cell (APC)-mediated oxidative processing although little is known about the mechanism or affinity of carbohydrate presentation (Cobb BA, Wang Q, Tzianabos AO, Kasper DL. 2004. Polysaccharide processing and presentation by the MHCII pathway. Cell. 117:677–687). A recent study showed that the helical conformation of ZPSs (Wang Y, Kalka-Moll WM, Roehrl MH, Kasper DL. 2000. Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis. Proc Natl Acad Sci USA. 97:13478–13483; Choi YH, Roehrl MH, Kasper DL, Wang JY. 2002. A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides. Biochemistry. 41:15144–15151) is closely linked with immunogenic activity (Tzianabos AO, Onderdonk AB, Rosner B, Cisneros RL, Kasper DL. 1993. Structural features of polysaccharides that induce intra-abdominal abscesses. Science. 262:416–419) and is stabilized by a zwitterionic charge motif (Kreisman LS, Friedman JH, Neaga A, Cobb BA. 2007. Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism. Glycobiology. 17:46–55), suggesting a strong carbohydrate structure–function relationship. In this study, we show that PSA, the ZPS from Bacteroides fragilis, associates with MHCII at high affinity and 1:1 stoichiometry through a mechanism mirroring peptide presentation. Interestingly, PSA binding was mutually exclusive with common MHCII antigens and showed significant allelic differences in binding affinity. The antigen exchange factor HLA-DM that catalyzes peptide antigen association with MHCII also increased the rate of ZPS association and was required for APC presentation and ZPS-mediated T cell activation. Finally, the zwitterionic nature of these antigens was required only for MHCII binding, and not endocytosis, processing, or vesicular trafficking to MHCII-containing vesicles. This report is the first quantitative analysis of the binding mechanism of carbohydrate antigens with MHCII and leads to a novel model for nontraditional MHCII antigen presentation during bacterial infections.

Cobb, Brian A; Kasper, Dennis L



Outer membrane proteins of bovine strains of Pasteurella multocida type A and their doubtful role as protective antigens.  


Outer membranes were prepared by the Sarkosyl method from 30 strains of Pasteurella multocida and the closely related Taxon 13, which had been isolated from cattle. The patterns of the outer membrane proteins (OMPs) on SDS-PAGE were generally similar to one another, though the four major proteins (a-d) varied somewhat in molecular mass; these patterns allowed the strains to be arranged into 12 groups. Taxon 13 strains and typical P. multocida strains were indistinguishable, both types being found within the same group. Mice were vaccinated with heat-killed bacteria of three strains and challenged with 10 LD50 of homologous and heterologous live bacteria, representing groups based on OMP patterns; the best protection was afforded by strain W674, which protected against nine of the 17 challenge strains; but there was no correlation between protection and PAGE pattern. Pre-vaccination and pre-challenge sera were used in immunoblotting to probe OMPs from protective and non-protective strains. All three vaccines produced antibody to proteins a and d; these proteins appeared to be common to all strains, varying in molecular mass but not in overall antigenic expression. The antibody response to the other two major OMPs appeared to be PAGE-group specific. There was no correlation between protection and the antigen pattern seen by immunoblotting. PMID:2342087

Abdullahi, M Z; Gilmour, N J; Poxton, I R



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

PubMed Central

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

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



Determination of antigenic domain in GST fused major surface protein (Nc-p43) of Neospora caninum  

PubMed Central

The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins. The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T; OFR without signal sequence and C-terminal hydrophobic sequence, S; N-terminal 2/3 parts of S, A; C-terminal 2/3 parts, P; N-terminal 1/3 part, X; middle 1/3 part, Y; and C-terminal 1/3 part, Z, respectively. The DNA fragments were cloned into pGEX-4T vector. Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T, 66 kDa for S, 52 kDa for A, 53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE. The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot. These findings suggest that the antigenic domain of Nc-p43 of N. caninum may be localized in the C-terminal 2/3 parts. Together with A19 clone in SAG1 of Toxoplasma gondii (Nam et al., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species.

Son, Eui-Sun; Ahn, Hye-Jin; Kim, Jae-Hoon; Kim, Dae-Yong



Dynamics of the antigen-binding grooves in CD1 proteins: reversible hydrophobic collapse in the lipid-free state.  


CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a meta-analysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two ?-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these ?-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal ?-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization. PMID:23677998

Garzón, Diana; Anselmi, Claudio; Bond, Peter J; Faraldo-Gómez, José D



New strategies for selection of unrelated bone marrow donors.  


An important problem in the selection of unrelated donors for bone marrow transplantation (UD-BMT), is HLA matching, between selected donor and recipient. Serological screening, mixed lymphocyte culture (MLC), and sequence specific oligonucleotide genotyping (PCR-SSO) are the methods commonly used for typing of HLA-genes. These ways to select donor candidates are time-expensive. We set up new applications of the "fingerprinting-PCR" technique, to analyse the polymorphic second exon of DRB, DQB, DQA, DPB of HLA Class II and second exon A, B, C HLA-Class I genes, and to search for identity between patient and serologically selected unrelated donors. In an assessment of the technique, 50 normal samples, and 4 unrelated HLA-A and HLA-B serological matched patient-donor pairs were analysed for HLA polymorphic regions. In 3 of the 4 cases (UD-BMT) at least HLA-DRB mismatched different donor-transplanted patterns were identified. In all cases PCR-SSO analysis was performed as control. Based on our data, we suggest that identification of UD for allogeneic BMT should follow these steps: 1) serological HLA-Class I and II genes screening; 2) HLA-Class II DRB gene PCR fingerprinting; 3) confirmation by SSO analysis in case of fingerprinting identity. 4) HLA-Class II DQA, DQB, DPB PCR fingerprinting. Moreover, confirmation by PCR fingerprinting or protein isoelectrofocusing of HLA-Class I identity is recommended. This "strategy" may contribute to rapid and specific selection of unrelated marrow donors. PMID:8448542

Martinelli, G; Buzzi, M; Farabegoli, P; Testoni, N; Mantovani, V; Calori, E; Rosti, G; Bandini, G; Bragliani, M; Panzica, G



Bootstrap Bartlett Adjustment in Seemingly Unrelated Regression  

Microsoft Academic Search

Seemingly unrelated regression (SUR) is a method introduced by Zellner (1962) for estimating several regression equations simultaneously, a common and important problem in econometrics. This article considers hypothesis testing in SUR using the log-likelihood ratio (LLR) test. Although the asymptotic distribution of this statistic is well known, substantial departures occur in samples of the size commonly employed by economists. Significance

David M. Rocke



The human non-lineage antigen CD46 (HuLy-m5) and primate retroviral gp70 molecules share protein-defined antigenic determinants  

Microsoft Academic Search

The CD46 lymphocyte surface antigen of man (until recently called HuLy-m5), and defined by the E4-3 monoclonal antibody (MoAb), shares cross-reactive antigenic epitopes with the envelope gp70 glycoproteins of gibbon ape leukaemia virus (GaLV) and Mason Pfizer monkey virus (MPMV) primate retroviruses. It is now shown that the cross-reactive antigenic epitope shared by these three molecules is determined solely by

DFJ Purcell; NJ Deacon; IFC McKenzie



Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen ?  

PubMed Central

Anaplasma platys infects peripheral blood platelets and causes infectious cyclic thrombocytopenia in canines. The genes, proteins, and antigens of A. platys are largely unknown, and an antigen for serodiagnosis of A. platys has not yet been identified. In this study, we cloned the A. platys major outer membrane protein cluster, including the P44/Msp2 expression locus (p44ES/msp2ES) and outer membrane protein (OMP), using DNA isolated from the blood of four naturally infected dogs from Venezuela and Taiwan, Republic of China. A. platys p44ES is located within a 4-kb genomic region downstream from a putative transcriptional regulator, tr1, and a homolog of the Anaplasma phagocytophilum, identified here as A. platys omp-1X. The predicted molecular masses of the four mature A. platys P44ES proteins ranged from 43.3 to 43.5 kDa. Comparative analyses of the deduced amino acid sequences of Tr1, OMP-1X, and P44/Msp2 proteins from A. platys with those from A. phagocytophilum showed sequence identities of 86.4% for Tr1, 45.9% to 46.3% for OMP-1X, and 55.0% to 56.9% for P44/Msp2. Comparison between A. platys and Anaplasma marginale proteins showed sequence identities of 73.1% for Tr1/Tr, 39.8% for OMP-1X/OMP1, and 41.5% to 42.1% for P44/Msp2. A synthetic OMP-1X peptide was shown to react with A. platys-positive sera but not with A. platys-negative sera or A. phagocytophilum-positive sera. Together, determination of the genomic locus of A. platys outer membrane proteins not only contributes to the fundamental understanding of this enigmatic pathogen but also helps in developing A. platys-specific PCR and serodiagnosis.

Lai, Tzung-Huei; Orellana, Nelson G.; Yuasa, Yumi; Rikihisa, Yasuko



A single Cys sup 706 to Phe substitution in the retinoblastoma protein causes the loss of binding to SV40 T antigen  

SciTech Connect

Most naturally occurring mutants of the retinoblastoma (RB) protein contain large deletions or truncations. The small cell lung carcinoma cell line H209 contains a normal-sized but unphosphorylated RB protein, which fails to form a complex with SV40 T antigen, suggesting that the RB gene of H209 may contain a subtle mutation. To define this mutation, the RB complementary DNA and genomic DNA were sequenced, revealing a point mutation in exon 21 that changed a G to a T. This results in an amino acid substitution of a Phe for Cys{sup 706}. The mutant RB complementary DNA was used as a template for in vitro transcription and translation to synthesize the mutated protein. The resulting protein failed to bind to SV40 T antigen, demonstrating that a single missense mutation of the RB gene led to the complete inactivation of the ability of the RB protein to bind T antigen.

Bignon, Y.J.; Jinyuh Shew; Lee, E.Y.H.P.; Schnier, J.; Wenhwa Lee (Univ. of California, San Diego, La Jolla (United States)); Rappolee, D. (Univ. of California, San Francisco (United States)); Naylor, S.L. (Univ. of Texas Health Science Center, San Antonio (United States))



Characterization of the membrane attack complex inhibitory protein CD59 antigen on human amniotic cells and in amniotic fluid.  

PubMed Central

A functional complement system and the potential for its activation are present in human amniotic fluid. We have recently demonstrated that CD59 antigen is present and functionally active on human amniotic epithelial cells (HAEC). We have now further examined the role of this protein on HAEC and have also demonstrated its presence in amniotic fluid (AF). CD59 Ag on HAEC is similar in size to the erythrocyte protein and is anchored via glycosyl phosphatidylinositol. The AF protein retained the capacity to incorporate into target cells and protect against lysis by complement. These data suggest that HAEC secrete into AF a form of CD59 Ag which retains inhibitory activity and which may be important in protection of the foetus from maternal complement in utero. Images Figure 1 Figure 3 Figure 4

Rooney, I A; Morgan, B P



The immune response to a vesicular stomatitis virus vaccine vector is independent of particulate antigen secretion and protein turnover rate.  


Vesicular stomatitis virus (VSV) is a highly cytopathic virus being developed as a vaccine vector due to its ability to induce strong protective T cell and antibody responses after a single dose. However, little is known regarding the mechanisms underlying the potent immune responses elicited by VSV. We previously generated a VSV vector expressing the hepatitis B virus middle envelope surface glycoprotein (MS) that induces strong MS-specific T cell and antibody responses in mice. After synthesis in the cytoplasm, the MS protein translocates to the endoplasmic reticulum, where it forms subviral particles that are secreted from the cell. To better understand the contributions of secreted and intracellular protein to the VSV-induced immune response, we produced a vector expressing a secretion-deficient MS mutant (MS(C69A)) and compared the immunogenicity of this vector to that of the wild-type VSV-MS vector in mice. As expected, the MS(C69A) protein was not secreted from VSV-infected cells and displayed enhanced proteasome-mediated degradation. Surprisingly, despite these differences in intracellular protein processing, the T cell and antibody responses generated to MS(C69A) were comparable to those elicited by virus expressing wild-type MS protein. Therefore, when it is expressed from VSV, the immune responses to MS are independent of particulate antigen secretion and the turnover rate of cytoplasmic protein. These results are consistent with a model in which the immune responses to VSV are strongly influenced by the replication cycle of the vector and demonstrate that characteristics of the vector have the capacity to affect vaccine efficacy more than do the properties of the antigen itself. PMID:22345454

Cobleigh, Melissa A; Bradfield, Clinton; Liu, Yuanjie; Mehta, Anand; Robek, Michael D



Use of Recombinant Flagellin Protein as a Tracer Antigen in a Fluorescence Polarization Assay for Diagnosis of Leptospirosis  

PubMed Central

The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of Leptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against L. interrogans serovar pomona and L. bergpetersenii serovars hardjo and sejroe to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83.7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-Leptospira antibodies.

Bughio, Nasreen I.; Lin, Min; Surujballi, Om P.



Helper T-Cell Antigenic Site Identification in the Acquired Immunodeficiency Syndrome Virus gp120 Envelope Protein and Induction of Immunity in Mice to the Native Protein Using a 16Residue Synthetic Peptide  

Microsoft Academic Search

Much effort has been devoted to the analysis of antibodies to acquired immunodeficiency syndrome virus antigens, but no studies, to our knowledge, have defined antigenic sites of this virus that elicit T-cell immunity, even though such immunity is important in protection against many other viruses. T cells tend to recognize only a limited number of discrete sites on a protein

Kemp B. Cease; Hanah Margalit; James L. Cornette; Scott D. Putney; W. Gerard Robey; Cecilia Ouyang; Howard Z. Streicher; Peter J. Fischinger; Robert C. Gallo; Charles Delisi; Jay A. Berzofsky



Identification of L. infantum chagasi proteins in VL patients' urine: a promising antigen discovery approach of vaccine candidates  

PubMed Central

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 E. 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 + BpMPLASE 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 kala-azar and opens novel possibilities for vaccine development to other serious infectious diseases.

Kashino, Suely S.; Abeijon, Claudia; Qin, Lizeng; Kanunfre, Kelly A.; Kubrusly, Flavia S.; Silva, Fernando O.; Costa, Dorcas L.; Campos, Dioclecio; Costa, Carlos H.N.; Raw, Isaias; Campos-Neto, Antonio



Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection  

Microsoft Academic Search

Within the multiform liver\\/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis

M Lenzi; P Manotti; L Muratori; M Cataleta; G Ballardini; F Cassani; F B Bianchi



Nucleotide sequence of the gene coding for the major protein of hepatitis B virus surface antigen  

Microsoft Academic Search

DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the

Pablo Valenzuela; Patrick Gray; Margarita Quiroga; Josefina Zaldivar; Howard M. Goodman; William J. Rutter



Enhancing Effect of Dietary Oil Emulsions on Immune Responses to Protein Antigens Fed to Mice  

Microsoft Academic Search

Repeated intragastric administration of ?-lactoglobulin (?-Lg) with emulsified soybean oil elicited an antigen-specific, systemic humoral immune response in different strains of mice. The antibody response was enhanced as the dose of oil was increased and the particle size of emulsions was decreased. Feeding of aqueous ?-Lg could induce the antibody response only when emulsified oil was fed almost simultaneously. However,

Tetsuo Kaneko; Yuka Terasawa; Yukiko Senoo; Masashi Nagata; Tamotsu Kuwata



Invariant Chain Modulates HLA Class II Protein Recycling and Peptide Presentation in Nonprofessional Antigen Presenting Cells  

PubMed Central

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.

Haque, Azizul; Hajiaghamohseni, Laela M.; Li, Ping; Toomy, Katherine; Blum, Janice S.



Identification of RD5-encoded Mycobacterium tuberculosis proteins as B-cell antigens used for serodiagnosis of tuberculosis.  


Comparative genomic studies have identified several Mycobacterium tuberculosis-specific genomic regions of difference (RDs) which are absent in the vaccine strains of Mycobacterium bovis BCG and which may be useful in the specific diagnosis of tuberculosis (TB). In this study, all encoded proteins from DNA segment RD5 of Mycobacterium tuberculosis, that is, Rv3117-Rv3121, were recombined and evaluated by enzyme-linked immunosorbent assays for antibody reactivity with sera from HIV-negative pulmonary TB patients (n = 60) and healthy controls (n = 32). The results identified two immunodominant antigens, that is, Rv3117 and Rv3120, both of which revealed a statistically significant antigenic distinction between healthy controls and TB patients (P < 0.05). In comparison with the well-known early-secreted antigen target 6?kDa (ESAT-6) (sensitivity 21.7%, specificity 90.6%), the higher detection sensitivity and higher specificity were achieved (Rv3117: sensitivity 25%, specificity 96.9%; Rv3120: sensitivity 31.7%, specificity 96.9%). Thus, the results highlight the immunosensitive and immunospecific nature of Rv3117 and Rv3120 and indicate promise for their use in the serodiagnosis of TB. PMID:22701501

Zhang, Miao-Miao; Zhao, Jun-Wei; Sun, Zhan-Qiang; Liu, Jun; Guo, Xiao-Kui; Liu, Wen-Di; Zhang, Shu-Lin



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


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 PKC?, we demonstrate an essential role of PKC? in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKC? 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 PKC?-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKC? in the metabolic switch to glycolysis following BCR engagement of naïve B-cells. PMID:22994860

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



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

PubMed Central

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.

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



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


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 ?X174 gene A* protein and Z(mab25) (A*-Zmab). The ?X174 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 ?1 (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-? (IFN-?) 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. PMID:22571843

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



Immune Response to Chlamydophila abortus POMP91B Protein in the Context of Different Pathogen Associated Molecular Patterns (PAMP); Role of Antigen in the Orientation of Immune Response  

PubMed Central

In a previous study, we used bacterial flagellin to deliver antigens such as p27 of Mycobacterium tuberculosis to a host immune system and obtained a potent Th1 response compared to those obtained with Freund’s adjuvant and DNA immunization. In the current study, using a POMP91B antigen of Chlamydophila abortus, a human and animal pathogen, as a model, we found that this antigen is unable to promote Th1 response. However, this antigen, unlike others, was able to induce a good Th2 response and IL-4 production after immunization by recombinant protein in Freund’s adjuvant or in phosphate buffered saline. Our results suggest that immune response is not only dependent on the immunization adjuvant, but also dependent on the nature of antigen used.

Moigne, Vincent Le; Robreau, Georges; Mahana, Wahib



Antibody Responses to Recombinant Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients: Complementary Test of ZEBRA Protein and Early Antigens p54 and p138  

Microsoft Academic Search

Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with

R. Dardari; W. Hinderer; D. Lang; A. Benider; B. El Gueddari; I. Joab; A. Benslimane; M. Khyatti



Delivery of heterologous protein antigens via hemolysin or autotransporter systems by an attenuated ler mutant of rabbit enteropathogenic Escherichia coli.  


In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8 passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30-40 ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p<0.008) when compared to rabbits inoculated with the parent ler mutant. Our results suggest that attenuated rEPEC are capable of serving as vaccine vectors to express heterologous protein antigens from different cellular locations and deliver these antigens to the intestinal mucosa. With this system, secreted proteins may be more effective than cell-associated antigens in generating protection. PMID:16098637

Zhu, Chengru; Ruiz-Perez, Fernando; Yang, Zhuolu; Mao, Ying; Hackethal, Veronica L; Greco, Karla M; Choy, Wendy; Davis, Katherine; Butterton, Joan R; Boedeker, Edgar C



Tacrolimus for prevention of graft-versus-host disease after mismatched unrelated donor cord blood transplantation  

Microsoft Academic Search

Ten children with hematologic malignancies or a storage disease underwent transplantation using cord blood cells from an unrelated donor mismatched for 1 (n = 7) or 2 (n = 3) HLA antigens. The median total nucleated cell dose was 4.0 (range, 2.2–7.1) × 107\\/kg. GVHD prophylaxis consisted of tacrolimus dose-adjusted to maintain a whole blood level of 5–15 ng\\/ml with

D Przepiorka; D Petropoulos; CA Mullen; M Danielson; V Mattewada; K-W Chan



Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays  

Microsoft Academic Search

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body




Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.  

PubMed Central

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities. Images

Van Roy, F; Fransen, L; Fiers, W



Cysticercosis Immunodiagnosis Using 18- and 14-Kilodalton Proteins from Taenia crassiceps Cysticercus Antigens Obtained by Immunoaffinity Chromatography  

PubMed Central

Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18- and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.

Espindola, Noeli Maria; Iha, Alberto Hiroshi; Fernandes, Irene; Takayanagui, Osvaldo Massaiti; Machado, Luis dos Ramos; Livramento, Jose Antonio; Mendes Maia, Antonio Augusto; Peralta, Jose Mauro; Vaz, Adelaide Jose



CD4+ T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens.  


We characterized cytokine profiles of CD4(+) T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4(+) T cells producing IL-2, (p=0.004). Vaccine antigen-specific CD4(+) T-cell populations in adults were largely of effector (TEM) and/or central memory (TCM) phenotypes as defined by CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) respectively; however among young children antigen-specific IL-2 producing CD4(+) T cells demonstrated CD45RA(+) expression (non-memory cells). We conclude that adults have circulating memory CD4(+) T cells (CD45RA(-)) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response. PMID:23632305

Sharma, Sharad K; Roumanes, David; Almudevar, Anthony; Mosmann, Tim R; Pichichero, Michael E



Aggregations of unrelated Apis florea colonies  

Microsoft Academic Search

Intensive surveys of an area of woodland in Phitsanulok province, Thailand, revealed 15 colonies of Apis florea. The colonies had a highly aggregated spatial distribution (Standardized Morisita’s Index of Dispersion = 0.59). Microsatellite\\u000a analysis based on 5 loci showed that no colonies were related as mother-daughter, suggesting that unrelated colonies tend\\u000a to nest near existing colonies.

Wandee Wattanachaiyingcharoen; Siriwat Wongsiri; Benjamin P. Oldroyd



Localization and expression profiling of a 31 kDa antigenic repetitive protein Sjp_0110390 in Schistosoma japonicum life stages.  


Sj7TR is a 13 kDa repetitive region of a 31 kDa protein in Schistosoma japonicum known as Sjp_0110390 that showed high sensitivity and specificity in antibody detection against schistosomiasis patients. However, the current database for S. japonicum genes characterized it only as an expressed protein. A more thorough understanding of this antigenic protein is therefore necessary to possibly give more information about the nature of this protein and its role in the parasite. In this study, immunolocalization and expression profiling were done for Sjp_0110390 on the different stages of the parasite. Immunofluorescent assay showed that Sjp_0110390 was expressed in the young stages of the parasites including the schistosomula, eggs, aquatic and intra-molluscan stages. This was supported by the reverse-transcriptase PCR which confirmed the stage-specific expression of Sjp_0110390 and Western blot test which detected the protein in the extracted eggs proteins, but not in the adults. Furthermore, it was also highly expressed in infected Oncomelania hupensis nosophora snails suggesting that Sjp_0110390 might have a role in the development of the parasite inside the intermediate host. This result also suggests that Sj7TR might be used not only for human diagnosis but to detect snail infection as well. PMID:23254201

Angeles, Jose Ma M; Kirinoki, Masashi; Goto, Yasuyuki; Asada, Masahito; Hakimi, Hassan; Leonardo, Lydia R; Tongol-Rivera, Pilarita; Villacorte, Elena A; Inoue, Noboru; Chigusa, Yuichi; Kawazu, Shin-ichiro



Influence of the primary emulsification procedure on the characteristics of small protein-loaded PLGA microparticles for antigen delivery.  


Microparticles prepared from poly(lactic-co-glycolic acid) (PLGA) using a W1/O/W2 double emulsion solvent evaporation method are suitable vehicles for the delivery of proteins to antigen presenting cells, e.g. dendritic cells. In this study, the influence of different techniques for the preparation of the primary W1/O emulsion was investigated with respect to the protein localization within the microparticles, morphological characteristics of these particles, protein burst release and the native state of the released protein. Bovine serum albumin bearing fluorescein isothiocyanate (FITC-BSA) was used as model protein. A static micromixer was applied for the preparation of the W1/O/W2 double emulsion. Employing a rotor-stator homogenizer (Ultra-Turrax) for primary emulsification, microcapsules with a high burst release were produced, because nearly all FITC-BSA was attached to the outside of the particle wall. Using a high pressure homogenizer or an ultrasonic procedure resulted in the formation of microspheres with homogeneous protein distribution and a reduced burst release. PMID:16854818

Wischke, C; Borchert, H-H



Method to Map Antigenic Determinants Recognized by Monoclonal Antibodies: Localization of a Determinant of Virus Neutralization on the Feline Leukemia Virus Envelope Protein gp70  

Microsoft Academic Search

A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta -galactosidase gene of phage lambda Charon 16 so as to obtain expression of

J. H. Nunberg; G. Rodgers; J. H. Gilbert; R. M. Snead



Does binding of complement factor H to the meningococcal vaccine antigen, factor H binding protein, decrease protective serum antibody responses?  


Factor H binding protein (fHbp) is a principal antigen in a multicomponent meningococcal vaccine recently licensed in Europe for prevention of serogroup B diseases. The protein recruits the complement downregulator, factor H (fH), to the bacterial surface, which enables the organism to resist complement-mediated bacteriolysis. Binding is specific for human fH. In preclinical studies, mice and rabbits immunized with fHbp vaccines developed serum bactericidal antibody responses, which in humans predict protection against developing meningococcal disease. These studies, however, were in animals whose fH did not bind to the vaccine antigen. Here we review the immunogenicity of fHbp vaccines in human fH transgenic mice. The data suggest that animals with high serum human fH concentrations have impaired protective antibody responses. Further, mutant fHbp vaccines with single amino acid substitutions that decrease fH binding are superior immunogens, possibly by unmasking epitopes in the fH binding site that are important for eliciting serum bactericidal antibody responses. Humans immunized with fHbp vaccines develop serum bactericidal antibody, but achieving broad coverage in infants required incorporation of additional antigens, including outer membrane vesicles, which increased rates of fever and local reactions at the injection site. The experimental results in transgenic mice predict that fHbp immunogenicity can be improved in humans by using mutant fHbp vaccines with decreased fH binding. These results have important public health implications for developing improved fHbp vaccines for control of serogroup B meningococcal disease and for development of vaccines against other microbes that bind host molecules. PMID:23740919

Granoff, Dan M; Ram, Sanjay; Beernink, Peter T



[Serum antibodies to brain protein antigens in cerebral palsy in children].  


Using special brain antigen test-system ELITEST-24, ELISA assays of the serum of children with cerebral palsy were conducted. The data obtained were compared to relevant characteristics of the sera from neurologically and somatically healthy persons and patients with diagnoses of multiple sclerosis, schizophrenia, epilepsy and hepato-cerebral dystrophy according to special PC program VIZUAL and DIAGNOST. High specificity of the cerebral palsy anti-brain antigens reactivity was revealed by ELITEST technique. Moreover, the comparison of the mother-child pair immunoreactivity has been conducted. Evidence for hypothesis of epigenetically performing of children antibodies repertoires by mother-during-pregnancy immune status were obtained. Possible immunopathologic mechanisms of the disease are discussed. PMID:1391899

Rubtsova, M Iu; Lobanov, D S; Kloss, T Iu; Semenov, A S; Poletaev, A B



The 14-3-3 Protein Detectable in the Cerebrospinal Fluid of Patients with Prion-Unrelated Neurological Diseases Is Expressed Constitutively in Neurons and Glial Cells in Culture  

Microsoft Academic Search

The 14-3-3 protein belongs to a family of 30-kD proteins originally identified by two-dimensional analysis of brain protein extracts. Recently, the detection of the 14-3-3 protein in the cerebrospinal fluid (CSF) is utilized as a highly reliable test for the premortem diagnosis of prion diseases such as Creutzfeldt-Jakob disease. For the initial step, to clarify the biological implication of the

Jun-ichi Satoh; Kazuhiro Kurohara; Motohiro Yukitake; Yasuo Kuroda



Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.  

PubMed Central

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases. Images

Jindal, S; Dudani, A K; Singh, B; Harley, C B; Gupta, R S



Polyelectrolyte LbL microcapsules versus PLGA microparticles for immunization with a protein antigen  

Microsoft Academic Search

The transition from organism-based traditional vaccines to the use of safer subunit vaccines has implemented the use of adjuvants to enhance immunogenicity. This study compares the potential of two types of polymeric microparticles as delivery systems for the model antigen ovalbumin. The delivery systems encompassed polyelectrolyte microcapsules, assembled via Layer-by-Layer technology, and PLGA microparticles fabricated by spray-drying. Mice were immunized

Marie-Luce De Temmerman; Joanna Rejman; Roosmarijn E. Vandenbroucke; Stefaan De Koker; Claude Libert; Johan Grooten; Jo Demeester; Bruno Gander; Stefaan C. De Smedt


The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene  

Microsoft Academic Search

To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific\\u000a antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter.\\u000a In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility\\u000a for PSA promoter. The in vitro DNA

Weiwen Chen; Jianye Zhang; Charles Y. F. Young; Lianying Zhang; Liucun Chen; Jian Zhao



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

Microsoft Academic Search

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

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



Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy  

Microsoft Academic Search

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4+ T cells, but also on MHC-class I mole- cules to CD8+ T cells. Immune responses against topically applied

Vincent Flacher; Florian Sparber; Christoph H. Tripp; Nikolaus Romani; Patrizia Stoitzner



Antigens in Penicillin Allergy  

Microsoft Academic Search

The present communication reveals a relationship between the epitope density of penicilloylated protein antigens and their antigenic activities in a radioimmunoassay (RIA), in passive cutaneous anaphylaxis (PCA) and in inducing antibody formation in mice. In the RIA and PCA a critical number of 2–4 penicilloyl residues per protein molecule was noted. At this level small changes in the number of

A. Kristofferson; S. Ahlstedt; P. O. Svärd



GILT modulates CD4+ T-cell tolerance to the melanocyte differentiation antigen tyrosinase-related protein 1.  


Gamma-IFN-inducible lysosomal thiol reductase (GILT) facilitates major histocompatibility complex class II-restricted processing through endocytic reduction of protein disulfide bonds and is necessary for efficient class II-restricted processing of melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). Using class II-restricted, TRP1-specific T-cell receptor transgenic mice, we identify a role, to our knowledge, previously unreported, for GILT in the maintenance of tolerance to TRP1. TRP1-specific thymocytes are centrally deleted in the presence of GILT and TRP1. In contrast, CD4 single-positive thymocytes and peripheral T cells develop in the absence of GILT or TRP1, demonstrating that GILT is required for negative selection of TRP1-specific thymocytes. Although TRP1-specific T cells escape thymic deletion in the absence of GILT, they are tolerant to TRP1 and do not induce vilitigo. TRP1-specific T cells that develop in the absence of GILT have diminished IL-2 and IFN-? production. Furthermore, GILT-deficient mice have a 4-fold increase in the percentage of TRP1-specific regulatory T (Treg) cells compared with TRP1-deficient mice, and depletion of Treg cells partially restores the ability of GILT-deficient TRP1-specific CD4(+) T cells to induce vitiligo. Thus, GILT has a critical role in regulating CD4(+) T-cell tolerance to an endogenous skin-restricted antigen relevant to controlling autoimmunity and generating effective immunotherapy for melanoma. PMID:21833020

Rausch, Matthew P; Hastings, K Taraszka



GILT modulates CD4+ T cell tolerance to the melanocyte differentiation antigen tyrosinase-related protein 1  

PubMed Central

Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates MHC class II-restricted processing though endocytic reduction of protein disulfide bonds and is necessary for efficient class II-restricted processing of melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). Using class II-restricted, TRP1-specific T cell repector transgenic mice, we identify a novel role for GILT in the maintenance of tolerance to TRP1. TRP1-specific thymocytes are centrally deleted in the presence of GILT and TRP1. In contrast, CD4 single positive thymocytes and peripheral T cells develop in the absence of GILT or TRP1, demonstrating that GILT is required for negative selection of TRP1-specific thymocytes. Although TRP1-specific T cells escape thymic deletion in the absence of GILT, they are tolerant to TRP1 and do not induce vilitigo. TRP1-specific T cells that develop in the absence of GILT have diminished IL-2 and IFN-? production. Furthermore, GILT-deficient mice have a four-fold increase in the percentage of TRP1-specific regulatory T cells compared to TRP1-deficient mice, and depletion of regulatory T cells partially restores the ability of GILT-deficient TRP1-specific CD4+ T cells to induce vitiligo. Thus, GILT plays a critical role in regulating CD4+ T cell tolerance to an endogenous skin-restricted antigen relevant to controlling autoimmunity and generating effective immunotherapy for melanoma.

Rausch, Matthew P.; Hastings, Karen Taraszka



Tubular antigen-binding proteins repress transcription of type IV collagen in the autoimmune target epithelium of experimental interstitial nephritis.  

PubMed Central

We have been studying immune interactions with somatic cells using a tubular antigen-binding protein (ThF) secreted by helper T lymphocytes harvested from mice that have an autoimmune form of interstitial nephritis called anti-tubular basement membrane disease. This ThF, although characterized originally because of its ability to induce effector T cells, additionally recognizes the nephritogenic 3M-1 antigen expressed by its target renal tubular epithelium. We believe these proteins, in general, may modulate directly some homeostatic functions in organ-derived cells, and now report that our ThF represses specifically the cellular transcription and secretion of basement membrane type IV collagen in tubular epithelium. These in vitro findings of reduced levels of mRNA encoding type IV collagen correlate well with in situ hybridization studies performed on kidneys expressing early autoimmune lesions, and predict a progressive drop in the expression of type IV collagen in the interstitium. Such a novel and unexpected repression of transcription of type IV collagen might easily impart or facilitate permanent change in the infrastructure of kidney architecture during autoimmune injury and, perhaps, contributes to the process of tubular atrophy attendant to prolonged renal inflammation. Images

Haverty, T P; Kelly, C J; Hoyer, J R; Alvarez, R; Neilson, E G



The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin  

PubMed Central

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein ?-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of ?-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same ?-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its ?-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro



Antibodies to neuron-specific antigens in children with autism: possible cross-reaction with encephalitogenic proteins from milk, Chlamydia pneumoniae and Streptococcus group A.  


We measured autoantibodies against nine different neuron-specific antigens and three cross-reactive peptides in the sera of autistic subjects and healthy controls by means of enzyme-linked immunosorbent assay (ELISA) testing. The antigens were myelin basic protein (MBP), myelin-associated glycoprotein (MAG), ganglioside (GM1), sulfatide (SULF), chondroitin sulfate (CONSO4), myelin oligodendrocyte glycoprotein (MOG), alpha,beta-crystallin (alpha,beta-CRYS), neurofilament proteins (NAFP), tubulin and three cross-reactive peptides, Chlamydia pneumoniae (CPP), streptococcal M protein (STM6P) and milk butyrophilin (BTN). Autistic children showed the highest levels of IgG, IgM and IgA antibodies against all neurologic antigens as well as the three cross-reactive peptides. These antibodies are specific because immune absorption demonstrated that only neuron-specific antigens or their cross-reactive epitopes could significantly reduce antibody levels. These antibodies may have been synthesized as a result of an alteration in the blood-brain barrier. This barrier promotes access of preexisting T-cells and central nervous system antigens to immunocompetent cells, which may start a vicious cycle. These results suggest a mechanism by which bacterial infections and milk antigens may modulate autoimmune responses in autism. PMID:12161033

Vojdani, A; Campbell, A W; Anyanwu, E; Kashanian, A; Bock, K; Vojdani, E



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

SciTech Connect

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.

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



Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa.  


During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination. PMID:22177563

Giovannini, Donatella; Späth, Stephan; Lacroix, Céline; Perazzi, Audrey; Bargieri, Daniel; Lagal, Vanessa; Lebugle, Camille; Combe, Audrey; Thiberge, Sabine; Baldacci, Patricia; Tardieux, Isabelle; Ménard, Robert



Problems and possible solutions in finding an unrelated bone marrow donor. Results of consecutive searches for 240 Dutch patients  

Microsoft Academic Search

To evaluate the efficiency of our protocol for finding an HLA matched unrelated bone marrow donor, search results obtained between 1990 and 1995 for 240 Dutch patients were analyzed. The percentage of patients for whom, according to information given by the registries, a fully split-HLA antigen matched donor is available, increased from 24% in 1990 to over 70% in 1995.

M Oudshoorn; JJ Cornelissen; WE Fibbe; ER de Graeff-Meeder; JLWT Lie; GMTh Schreuder; K Sintnicolaas; R Willemze; JMJJ Vossen; JJ van Rood



High Affinity Antigen Recognition of the Dual Specific Variants of Herceptin Is Entropy-Driven in Spite of Structural Plasticity  

PubMed Central

The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2) antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF) to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called “structural plasticity”. Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

Bostrom, Jenny; Haber, Lauric; Koenig, Patrick; Kelley, Robert F.; Fuh, Germaine



The human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX44 antigens  

Microsoft Academic Search

CD53 is an N-glycosylated pan-leucocyte antigen of 35–42 000 Mr. The sequence of the CD53 polypeptide deduced from a cDNA clone is 219 amino acids in length. It appears to lack a conventional leader sequence because the deduced NH2-terminal amino acid sequence is very similar to the rat MRC OX-44 and human CD37 antigens. The CD53 molecule is likely to

Pavla Angelisová; ?estmír Vl?ek; Irena Štefanová; Marie Lipoldová; Václav Ho?ejší



Malaria Sporozoite Surface Antigen Distinct from the Circumsporozoite Protein. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

The sporozoite of Plasmodium is uniformly covered by a proteinaceous membrane coat which is thought to be composed entirely of the circumsporozoite (CS) protein. Being the first parasite molecule encountered by the host, the CS protein has been intensivel...

J. R. Campbell M. Carter M. L. Leef R. C. Hedstrom Y. Charoenvit



Application of a Novel Radioimmunoassay to Identify Baculovirus Structural Proteins that Share Interspecies Antigenic Determinants.  

National Technical Information Service (NTIS)

Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125 I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intac...

G. E. Smith M. D. Summers



Structural and Antigenic Properties of Merozoite Surface Protein 4 of Plasmodium falciparum  

Microsoft Academic Search

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Esche- richia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific




Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay? †  

PubMed Central

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10?6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

Neiman, Maja; Hamsten, Carl; Schwenk, Jochen M.; Bolske, Goran; Persson, Anja



Screening of 71 P. multocida Proteins for Protective Efficacy in a Fowl Cholera Infection Model and Characterization of the Protective Antigen PlpE  

Microsoft Academic Search

BackgroundThere is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens.Principal FindingsA high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested,

Tamás Hatfaludi; Keith Al-Hasani; Lan Gong; John D. Boyce; Mark Ford; Ian W. Wilkie; Noelene Quinsey; Michelle A. Dunstone; David E. Hoke; Ben Adler



A major T-cell-inducing cytosolic 23 kDa protein antigen of the vaccine candidate Mycobacterium habana is superoxide dismutase  

Microsoft Academic Search

This study describes the purification and immunochemical characterization of a major 23 kDa cytosolic protein antigen of the vaccine candidate Mycobacterium habana (TMC 5135). The 23 kDa protein alone was salted out from the cytosol at an ammonium sulfate saturation of 8&95%. It represented about 1*5% of the total cytosolic protein, appeared glycosylated by staining with periodic acid\\/Schiff's reagent, and

Deepa Bisht; J. Mehrotra; M. S. Dhindsa; N. B. Singh; S. Sinha



An Enterococcus faecium Secreted Antigen, SagA, Exhibits Broad-Spectrum Binding to Extracellular Matrix Proteins and Appears Essential for E. faecium Growth  

Microsoft Academic Search

A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recom- binant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of

Fang Teng; Magdalena Kawalec; George M. Weinstock; Waleria Hryniewicz; Barbara E. Murray



Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays  

PubMed Central

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

Ching, W.-M.; Wang, H.; Eamsila, C.; Kelly, D. J.; Dasch, G. A.



Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design  

SciTech Connect

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.

Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M. (Notre)



Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein  

SciTech Connect

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.

Ye Ling [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Sun Yuliang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Lin Jianguo [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Bu Zhigao [Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Nangang District, Harbin 150001 (China); Wu Qingyang [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Jiang, Shibo [New York Blood Center, 310 E. 67 Street, New York, NY 10021 (United States); Steinhauer, David A. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Compans, Richard W. [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States); Yang Chinglai [Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3086 Rollins Research Center, Atlanta, GA 30322 (United States)]. E-mail:



Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen  

PubMed Central

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.

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, Sebastien; 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



Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.  


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. PMID:23396847

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



The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication  

PubMed Central

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase ?-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

Weisshart, Klaus; Taneja, Poonam; Fanning, Ellen



Transcriptional Synergy between Melanoma Antigen Gene Protein-A11 (MAGE-11) and p300 in Androgen Receptor Signaling*  

PubMed Central

Androgen receptor (AR)-mediated gene regulation involves interactions with coregulatory proteins that include the melanoma antigen gene protein-A11 (MAGE-11). To understand the functional significance of sequence similarity between MAGE-11 and the adenovirus early protein E1A, we determined whether MAGE-11 contributes to AR transcriptional activity through an interaction with p300, a potent and ubiquitous transcriptional regulator. Here, we report that MAGE-11 interacts with the NH2-terminal region of p300 through the MAGE-11 MXXIF motif 185MXXIF189, with transcriptional activity depending on the MAGE-11 F-box and MAPK phosphorylation. The MAGE-11- and p300-dependent increase in AR transactivation required the NH2-terminal regions of AR and p300, p300 acetyltransferase activity, and the AR FXXLF motif 23FQNLF27 interaction with MAGE-11. MAGE-11 linked AR to p300 and the p160 coactivator, transcriptional intermediary protein 2 (TIF2). The p300 NH2-terminal FXXLF motif 33FGSLF37 was required for transcriptional activation by TIF2. Increased expression of p300 decreased the ubiquitinylation of MAGE-11 and transiently increased endogenous MAGE-11 levels. Autoacetylation of p300 and decreased acetylation of TIF2 were evident in the MAGE-11, p300, and TIF2 complex. The studies suggest that MAGE-11 links NH2-terminal domains of AR and p300 to promote transcriptional synergy through a cadre of FXXLF-related interacting motifs.

Askew, Emily B.; Bai, Suxia; Blackwelder, Amanda J.; Wilson, Elizabeth M.



Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5.  


Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5. PMID:23350689

Van Vaerenbergh, M; Cardoen, D; Formesyn, E M; Brunain, M; Van Driessche, G; Blank, S; Spillner, E; Verleyen, P; Wenseleers, T; Schoofs, L; Devreese, B; de Graaf, D C



Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation.  


The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4(+) and CD8(+) T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8(+) T-cell activation. PMID:23836147

Rosalia, Rodney A; Quakkelaar, Esther D; Redeker, Anke; Khan, Selina; Camps, Marcel; Drijfhout, Jan W; Silva, Ana Luisa; Jiskoot, Wim; van Hall, Thorbald; van Veelen, Peter A; Janssen, George; Franken, Kees; Cruz, Luis J; Tromp, Angelino; Oostendorp, Jaap; van der Burg, Sjoerd H; Ossendorp, Ferry; Melief, Cornelis J M



Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein  

PubMed Central

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

O'Connor, Kevin C; McLaughlin, Katherine A; De Jager, Philip L; Chitnis, Tanuja; Bettelli, Estelle; Xu, Chenqi; Robinson, William H; Cherry, Sunil V; Bar-Or, Amit; Banwell, Brenda; Fukaura, Hikoaki; Fukazawa, Toshiyuki; Tenembaum, Silvia; Wong, Susan J; Tavakoli, Norma P; Idrissova, Zhannat; Viglietta, Vissia; Rostasy, Kevin; Pohl, Daniela; Dale, Russell C; Freedman, Mark; Steinman, Lawrence; Buckle, Guy J; Kuchroo, Vijay K; Hafler, David A; Wucherpfennig, Kai W



Permeation of antigen protein-conjugated nanoparticles and live bacteria through microneedle-treated mouse skin  

PubMed Central

Background: The present study was designed to evaluate the extent to which pretreatment with microneedles can enhance skin permeation of nanoparticles in vitro and in vivo. Permeation of live bacteria, which are physically nanoparticles or microparticles, through mouse skin pretreated with microneedles was also studied to evaluate the potential risk of microbial infection. Methods and results: It was found that pretreatment of mouse skin with microneedles allowed permeation of solid lipid nanoparticles, size 230 nm, with ovalbumin conjugated on their surface. Transcutaneous immunization in a mouse skin area pretreated with microneedles with ovalbumin nanoparticles induced a stronger antiovalbumin antibody response than using ovalbumin alone. The dose of ovalbumin antigen determined whether microneedle-mediated transcutaneous immunization with ovalbumin nanoparticles induced a stronger immune response than subcutaneous injection of the same ovalbumin nanoparticles. Microneedle treatment permitted skin permeation of live Escherichia coli, but the extent of the permeation was not greater than that enabled by hypodermic injection. Conclusion: Transcutaneous immunization on a microneedle-treated skin area with antigens carried by nanoparticles can potentially induce a strong immune response, and the risk of bacterial infection associated with microneedle treatment is no greater than that with a hypodermic injection.

Kumar, Amit; Li, Xinran; Sandoval, Michael A; Rodriguez, B Leticia; Sloat, Brian R; Cui, Zhengrong



The Babesia bovis Merozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins  

PubMed Central

Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus. The four genes, designated msa-2a1 (which corresponds to the originally described msa-2 gene), msa-2a2, msa-2b, and msa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a1) amino acids. MSA-2a1 and -2a2 are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a2 and -2b. In contrast, msa-2c is most closely related to the previously described babr 0.8 gene in Australia strains of B. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain of B. bovis with the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a1, -2a2, -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a1, -2b, and -2c. The results support the evolution of the msa-2 locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

Florin-Christensen, Monica; Suarez, Carlos E.; Hines, Stephen A.; Palmer, Guy H.; Brown, Wendy C.; McElwain, Terry F.



Unrelated donor umbilical cord blood transplantation in adults  

Microsoft Academic Search

Umbilical cord blood (UCB) has emerged as an appealing alternative source of hematopoietic stem cells for unrelated donor transplantation. Shorter time to transplant and an improved chance of finding a suitable graft are evident advantages over bone marrow transplantation from unrelated donors. The majority of UCB transplants from unrelated donors have been performed in children, but the number in adults

MA Sanz; GF Sanz



Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy.  


Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C'-PSA) and ?-fetoprotein (rHSP70C'-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C'-PSA or rHsp70C'-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use. PMID:23112956

Wang, Yu-Shan; Liu, Shih-Jen; Huang, Su-Chen; Chang, Chao-Chun; Huang, Yi-Chun; Fong, Weng-Lam; Chi, Mau-Shin; Chi, Kwan-Hwa



Surfactant proteins A and D protect mice against pulmonary hypersensitivity induced by Aspergillus fumigatus antigens and allergens  

PubMed Central

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic disorder caused by an opportunistic fungal pathogen, Aspergillus fumigatus (Afu). Lung surfactant proteins SP-A and SP-D can interact with the glycosylated antigens and allergens of Afu, inhibit specific IgE binding to these allergens, and block histamine release from sensitized basophils. We have now examined the therapeutic effect of exogenous administration of human SP-A, SP-D, and a recombinant fragment of SP-D (rSP-D), in a murine model of pulmonary hypersensitivity induced by Afu antigens and allergens, which resembles human ABPA immunologically. The ABPA mice exhibited high levels of Afu-specific IgG and IgE, blood eosinophilia, extensive infiltration of lymphocytes and eosinophils in the lung sections, and a Th2 cytokine response. Treatment with SP-A, SP-D, and rSP-D lowered blood eosinophilia, pulmonary infiltration, and specific Ab levels considerably, which persisted up to 4 days in the SP-A–treated ABPA mice, and up to 16 days in the SP-D– or rSP-D–treated ABPA mice. The levels of IL-2, IL-4, and IL-5 were decreased, while the level of IFN-? was raised in the splenic supernatants of the treated mice, indicating a marked shift from Th2 to Th1 response. These results clearly implicate pulmonary SP-A and SP-D in the modulation of allergic reactions.

Madan, Taruna; Kishore, Uday; Singh, Mamta; Strong, Peter; Clark, Howard; Hussain, Ejaj M.; Reid, Kenneth B.M.; Sarma, P. Usha



Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy  

PubMed Central

Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C?-PSA) and ?-fetoprotein (rHSP70C?-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C?-PSA or rHsp70C?-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

Wang, Yu-Shan; Liu, Shih-Jen; Huang, Su-Chen; Chang, Chao-Chun; Huang, Yi-Chun; Fong, Weng-Lam; Chi, Mau-Shin; Chi, Kwan-Hwa



New PHA products using unrelated carbon sources  

PubMed Central

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.

Matias, Fernanda; de Andrade Rodrigues, Maria Filomena



New PHA products using unrelated carbon sources.  


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

Matias, Fernanda; de Andrade Rodrigues, Maria Filomena



Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.  

PubMed Central

Anti-PM-Scl antibodies are associated with polymyositis-scleroderma overlap or either disease alone. Among sera from 39 patients with anti-PM-Scl, 23 recognized the 100-kD band in immunoblot against HeLa cell extract, 16 of which also stained the 70-kD band. A human thymocyte lambda gt11 cDNA expression library was screened with anti-PM-Scl serum, and two clones were identified whose products reacted with 33 and 37 of 39 anti-PM-Scl sera, respectively, but none of 26 negative control sera. Affinity-purified antibody reacting specifically with plaques of the clone stained the 100-kD band on immunoblot, reacted with nucleoli of HEp-2 cells, and immunoprecipitated the PM-Scl protein complex. Partial sequences of both inserts were identical. One insert was fully sequenced, and additional 5' and 3' sequence was obtained using a gene-specific primer to form a cDNA with HeLa cell RNA as template followed by PCR. The complete nucleotide sequence included 2,739-bp coding for a predicted full-length protein of 98,088 D. There was no homology with the PM-Scl 75-kD protein and no significant homology with other proteins. A mixed-charge cluster was identified, with 22 charged amino acids of 37. In conclusion, the full-length cDNA sequence was determined coding for the PM-Scl 100-kD protein, the most commonly antigenic protein of the PM-Scl complex. Images

Ge, Q; Frank, M B; O'Brien, C; Targoff, I N



Transactivation of both early and late simian virus 40 promoters by large tumor antigen does not require nuclear localization of the protein.  

PubMed Central

The early gene product of simian virus 40, large tumor antigen (T antigen), is required for the onset of viral replication. This protein has also been reported to transactivate viral late gene expression, independently of replication. In this study I have used a vector that permits simultaneously a precise quantitation of simian virus 40 early and late promoter activity with a single nuclease S1 mapping probe. The results show that T antigen can activate the early promoter as well as the late promoter and that only on replicating templates does a shift occur in the ratio of late-to-early transcription. This simultaneous transactivation of early and late promoters occurs in human (HeLa) and monkey (CV-1) cells but does not occur in mouse embryonal carcinoma cells. It is seen with either wild-type T antigen or with a T antigen protein that carries a mutation in the nuclear localization signal. The mutant protein cannot bring about an early-to-late shift, consistent with its inability to support viral replication. Images

Wildeman, A G



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

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.



Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein  

Microsoft Academic Search

The haemagglutinin (H) protein is the dominant en- velope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide

Aizhong Hu; Erling Norrby



Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb1  

Microsoft Academic Search

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the

Stephen A. Hines; Guy H. Palmer; Wendy C. Brown; Terry F. McElwain; Carlos E. Suarez; Odillon Vidotto; Allison C. Rice-Ficht



A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes  

Microsoft Academic Search

Summary Herpes simplex virus (HSV) infection of human fibro- blasts rapidly renders the cells resistant to lysis by HSV-specific CDS+ cytotoxic T lymphocytes (CTLs), which normally recognize cell surface major histocom- patibillty complex (MHC) class I proteins presenting viral peptides. Within 3 hr of infection with HSV, MHC class I protein complexes are retained in the endoplas- mic reticulum (ER)\\/cis

Ian A. York; Cindy Roop; David W. Andrews; Stanley R. Riddell; Frank L. Graham; David C. Johnson



Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins  

PubMed Central

Introduction Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results Circulating IC in the serum of RA patients and healthy controls contain fibrinogen? and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogen? and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogen? were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions Citrullinated fibrinogen? and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.



Alternative endogenous protein processing via an autophagy-dependent pathway compensates for Yersinia-mediated inhibition of endosomal major histocompatibility complex class II antigen presentation.  


Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen. PMID:20876292

Rüssmann, Holger; Panthel, Klaus; Köhn, Brigitte; Jellbauer, Stefan; Winter, Sebastian E; Garbom, Sara; Wolf-Watz, Hans; Hoffmann, Sigrid; Grauling-Halama, Silke; Geginat, Gernot



The mechanism of breakdown of immune tolerance to a protein antigen in rabbits  

PubMed Central

Rabbits made tolerant to human serum albumin (HSA) were hyperimmunized with either HSA or bovine serum albumin (BSA) incorporated in Freund's complete adjuvant. Eventually, anti-HSA antibodies could be demonstrated which represented most likely a boosted response to minor determinants to which the animals had not been made tolerant. This response was suppressed by a series of intravenous antigen injections and the animals relapsed into a state of unresponsiveness. Renewal of hyperimmunization with BSA in adjuvant did not terminate tolerance a second time, while identical treatment with suphanilated HSA did induce an anti-HSA response again. The theoretical aspects of tolerance breakdown are discussed in the light of these findings. ImagesFIG. 2

Nachtigal, David



Cloning of the structural genes of three H8 antigens and of protein III of Neisseria gonorrhoeae  

PubMed Central

A bank of gonococcal DNA was constructed in the lambda gt11 expression vector. immunological screening of the bank resulted in the isolation of a clone that contains the structural gene of protein III. In addition, several clones reactive with mAbs specific for the H8 antigen were isolated. DNA hybridization studies revealed that these H8- reactive clones were derived from three different gonococcal genes. When the products produced by these clones were used to absorb antibodies from a rabbit antiserum, and the eluted antibodies were used in immunological studies, it could be shown that the parent gonococcus expressed the product of two of these H8 genes, and in strain R10, these had Mr of approximately 19,700 21,200 respectively. The larger form has not been recognized hitherto because the epitope reactive with the H8 mAb may be masked in this product.



An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.  


The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 ?g/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies will determine if autoclaving treatments affect the allergenicity of the 24-kDa buckwheat protein. PMID:22691491

Tomotake, Hiroyuki; Yamazaki, Rikio; Yamato, Masayuki



Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins.  


Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni. PMID:23312848

Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E; Oakley, Brian B; Seal, Bruce S



Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.  

PubMed Central

The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteri