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1

Autoreactive antibodies raised by self derived de novo peptides can identify unrelated antigens on protein microarrays. Are autoantibodies really autoantibodies?  

PubMed

The development of arrays of human proteins has been a huge boon to the search for autoantibody diagnostics. Typically, slides with thousands of recombinant human proteins arrayed in an addressable fashion are incubated with sera from diseased or normal people. If an antibody binds a protein more in the diseased than in the normal cohort it is considered an autoantibody response. It is usually presumed that the autoantibody was elicited by the protein bound on the array. However, our studies using human protein and random peptide arrays indicate that antibody specificity may not be as high as commonly thought. Therefore we have tested the assumption of the source of autoantibodies. One test was to generate antibodies to two totally random peptides and bind these antibodies to a human protein array. One of the antibodies generated bound two human proteins. A second test was to generate an antibody to a frameshift peptide occurring in cancers. This antibody also bound several proteins on the array. We conclude that one should be cautious about assuming a particular autoantibody target on an array which elicited the original immune response. PMID:22421442

Kroening, Kaitlin; Johnston, Stephen Albert; Legutki, Joseph Barten

2012-06-01

2

Transfer of the ability of HIV-1 Tat to raise an adjuvant-free humoral immune response to unrelated antigens.  

PubMed

The HIV-1 Tat protein is able to raise a humoral immune response in the absence of adjuvant. Here, we investigated whether this property can be transferred to unrelated antigens. We first observed that Tat self-adjuvanticity is a T cell-dependent phenomenon in which a Th2 profile predominates. Then, we showed that the determinant governing the property is located in the region 1-57 of Tat and that fragment Tat1-57 can make two unrelated model antigens immunogenic in the absence of adjuvant. We found a Th2 pattern of immune response for both antigens, suggesting that Tat1-57 mediates this response. Next, we showed that, although less efficient than Tat1-57, the Tat37-57 fragment suffices to transfer the adjuvant property to other antigens. We also observed that preservation of cysteine 37 is absolutely required for the transfer, suggesting the role of disulphide-mediated dimerization in the transfer of the adjuvant property. Our observations suggest that for various antigens, the use of Tat37-57 or Tat1-57 or Tat22-57C(22-34)A might represent an alternative to adjuvants in humans, thereby opening up new perspectives in vaccination. PMID:22386747

Gadzinski, Adeline; Matz, Delphine; Favre, Emilie; Léonetti, Michel

2012-04-16

3

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

1965-01-01

4

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.

THOMAS, S; THIRUMALAPURA, N; CROSSLEY, E C; ISMAIL, N; WALKER, D H

2009-01-01

5

Antigenic cross reactivity among the venoms and toxins from unrelated diverse sources  

Microsoft Academic Search

Numerous investigators have studied and reported the antigenic reactivity of venoms from the species of snakes belonging to a genus or a family. However, there is very little published data on the inter-family antigenic cross reactivity among the venoms of snakes and absolutely no data on venoms from other sources such as honey bee, scorpion and toad. This report describes

Binie V. Lipps; Abbas A. Khan

2000-01-01

6

Delayed Hypersensitivity to Toxoplasma and Unrelated Antigens in Toxoplasma-Infected Mice: Induction and Elicitation of Delayed-Type Hypersensitivity by Antigen-Pulsed Macrophages  

PubMed Central

Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 106 Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice.

Handman, Emanuela; Chester, Patrice M.; Remington, Jack S.

1980-01-01

7

Antigen Retrieval Causes Protein Unfolding  

PubMed Central

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.

Fowler, Carol B.; Evers, David L.; O'Leary, Timothy J.; Mason, Jeffrey T.

2011-01-01

8

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

PubMed

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

2004-12-01

9

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

2008-01-01

10

ONE ANTIGEN MISMATCHED RELATED VS. HLA-MATCHED UNRELATED DONOR HEMATOPOIETIC TRANSPLANTATION IN ADULTS WITH ACUTE LEUKEMIA: CIBMTR RESULTS IN THE ERA OF MOLECULAR HLA TYPING  

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.

2012-01-01

11

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein  

Microsoft Academic Search

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of

W. H. Gispen; J. Oosterom; K. M. Garner; W. K. den Dekker

2001-01-01

12

Cytotoxic T-lymphocyte antigen-4 single nucleotide polymorphisms are not associated with outcomes after unrelated donor transplantation: a center for international blood and marrow transplant research analysis.  

PubMed

Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays an essential role in T cell homeostasis by restraining immune responses. AG and GG genotypes of donor CTLA-4 SNP rs4553808 in patients after unrelated donor hematopoietic stem cell transplantations (HSCT) have been shown to be an independent predictor of inferior relapse-free survival (RFS) and overall survival (OS) compared with those with the AA genotype, in single-center studies. We tested the hypothesis that SNP rs4553808 is associated with RFS, OS, nonrelapse mortality (NRM) and the cumulative incidence of acute graft-versus-host disease (GVHD) and chronic GVHD in adults with acute myeloid leukemia and advanced myelodysplastic syndrome undergoing a first 8/8 or 7/8 HLA-matched unrelated donor HSCT. Multivariable analysis adjusting for relevant donor and recipient characteristics showed no significant association between SNP rs4553808 and OS, RFS, NRM, and incidence of acute and chronic GVHD. An exploratory analysis of other CTLA-4 SNPs, as well as studying the interaction with antithymocyte globulin, also demonstrated no significant associations. Our results indicate that CTLA-4 SNPs are not associated with HSCT outcomes. PMID:24631737

Sengsayadeth, Salyka; Wang, Tao; Lee, Stephanie J; Haagenson, Michael D; Spellman, Stephen; Fernandez Viña, Marcelo A; Muller, Carlheinz R; Verneris, Michael R; Savani, Bipin N; Jagasia, Madan

2014-06-01

13

THE ANTIGENIC DETERMINANTS OF THE PROTEIN-POLYSACCHARIDES OF CARTILAGE  

PubMed Central

1. Protein-polysaccharides isolated from bovine, human, and porcine cartilage are antigenic in the rabbit. 2. PP-L from each of these species contains a minimum of two antigenic determinants; one is species specific, and one is common to all three species. Human PP-H also contains these two determinants. 3. Both antigenic determinants are in, or closely associated with, the protein moiety of the compound. Chondroitin sulfate and keratan sulfate were not demonstrated to be antigenic in these studies.

Sandson, John; Rosenberg, Lawrence; White, David

1966-01-01

14

Trypanosoma cruzi expresses diverse repetitive protein antigens.  

PubMed Central

We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images

Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

1989-01-01

15

Considering scores between unrelated proteins in the search database improves profile comparison  

PubMed Central

Background Profile-based comparison of multiple sequence alignments is a powerful methodology for the detection remote protein sequence similarity, which is essential for the inference and analysis of protein structure, function, and evolution. Accurate estimation of statistical significance of detected profile similarities is essential for further development of this methodology. Here we analyze a novel approach to estimate the statistical significance of profile similarity: the explicit consideration of background score distributions for each database template (subject). Results Using a simple scheme to combine and analytically approximate query- and subject-based distributions, we show that (i) inclusion of background distributions for the subjects increases the quality of homology detection; (ii) this increase is higher when the distributions are based on the scores to all known non-homologs of the subject rather than a small calibration subset of the database representatives; and (iii) these all known non-homolog distributions of scores for the subject make the dominant contribution to the improved performance: adding the calibration distribution of the query has a negligible additional effect. Conclusion The construction of distributions based on the complete sets of non-homologs for each subject is particularly relevant in the setting of structure prediction where the database consists of proteins with solved 3D structure (PDB, SCOP, CATH, etc.) and therefore structural relationships between proteins are known. These results point to a potential new direction in the development of more powerful methods for remote homology detection.

2009-01-01

16

Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis  

Microsoft Academic Search

Umbilical cord blood (UCB) is being in- creasingly used for hematopoietic stem cell transplantation and has been associ- ated with a reduced incidence of severe graft-versus-host disease (GVHD). To fur- ther investigate the relative merits of unre- lated donor UCB versus bone marrow (BM), a matched-pair analysis comparing the outcomes of recipients of 0 to 3 human leukocyte antigen (HLA)-mis-

Juliet N. Barker; Stella M. Davies; Todd DeFor; Norma K. C. Ramsay; Daniel J. Weisdorf; John E. Wagner

2001-01-01

17

Outcomes of patients with myeloid malignancies treated with allogeneic hematopoietic stem cell transplantation from matched unrelated donors compared with one human leukocyte antigen mismatched related donors using HLA typing at 10 loci.  

PubMed

Most candidates for hematopoietic stem cell transplantation (HSCT) lack a human leukocyte antigen (HLA)-identical sibling donor. Some patients may have a related donor with whom they are mismatched at 1 antigen/allele. It is not known whether such a match is preferable to a matched unrelated donor (MUD). We evaluated the outcomes (survival, relapse, nonrelapse mortality [NRM]) of all 28 patients with a single HLA antigen/allele mismatch identified through high-resolution HLA typing at HLA-A, -B, -C, -DRB1, and -DQB1, and all 318 patients with myeloid malignancies who received transplants from a 10/10 MUD treated during the same period of time at a single institution. Overall, outcomes for patients treated from a 1-antigen/allele mismatch related donor were significantly worse than from a MUD, primarily because of increased NRM. Overall survival (OS) rates at 3 years for 1-antigen/allele mismatched related donor and MUD transplant recipients were 19% and 45% (P = .007), and NRM rates were 40% and 26% (P = .05), respectively. Patients with class I mismatches appeared to have poorer OS than did patients with class II mismatches. A higher incidence of graft rejection was identified in the mismatched related donor group (P = .02). These results indicate that transplant outcomes are better with a MUD than with a 1 antigen/allele-mismatched related donor. PMID:20969970

Ciurea, Stefan O; Saliba, Rima M; Rondon, Gabriela; Patah, Poliana A; Aung, Fleur; Cano, Pedro; Andersson, Borje S; Kebriaei, Partow; Popat, Uday; Fernandez-Vina, Marcelo; Champlin, Richard E; de Lima, Marcos

2011-06-01

18

Outcomes of Patients with Myeloid Malignancies Treated with Allogeneic Hematopoietic Stem Cell Transplantation from Matched Unrelated Donors Compared with One Human Leukocyte Antigen Mismatched Related Donors Using HLA Typing at 10 Loci  

PubMed Central

Most candidates for hematopoietic stem cell transplantation (HSCT) lack a human leukocyte antigen (HLA)-identical sibling donor. Some patients may have a related donor with whom they are mismatched at 1 antigen/allele. It is not known whether such a match is preferable to a matched unrelated donor (MUD). We evaluated the outcomes (survival, relapse, nonrelapse mortality [NRM]) of all 28 patients with a single HLA antigen/allele mismatch identified through high-resolution HLA typing at HLA-A, -B, -C, -DRB1, and -DQB1, and all 318 patients with myeloid malignancies who received transplants from a 10/10 MUD treated during the same period of time at a single institution. Overall, outcomes for patients treated from a 1-antigen/allele mismatch related donor were significantly worse than from a MUD, primarily because of increased NRM. Overall survival (OS) rates at 3 years for 1-antigen/allele mismatched related donor and MUD transplant recipients were 19% and 45% (P =.007), and NRM rates were 40% and 26% (P =.05), respectively. Patients with class I mismatches appeared to have poorer OS than did patients with class II mismatches. A higher incidence of graft rejection was identified in the mismatched related donor group (P =.02). These results indicate that transplant outcomes are better with a MUD than with a 1 antigen/allele-mismatched related donor.

Ciurea, Stefan O.; Saliba, Rima M.; Rondon, Gabriela; Patah, Poliana A.; Aung, Fleur; Cano, Pedro; Andersson, Borje S.; Kebriaei, Partow; Popat, Uday; Fernandez-Vina, Marcelo; Champlin, Richard E.; de Lima, Marcos

2014-01-01

19

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;?http://dx.doi.org/10.1289/ehp.1306607

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

2013-01-01

20

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.

1981-06-01

21

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.  

PubMed Central

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein. Images

Khudyakov, Y E; Favorov, M O; Khudyakova, N S; Cong, M E; Holloway, B P; Padhye, N; Lambert, S B; Jue, D L; Fields, H A

1994-01-01

22

Expression of antigenic crossreactivity to RD114 p 30 protein in a human fibrosarcoma cell line.  

PubMed Central

An antigen crossreacting with the 30,000-molecular-weight protein (p30) of the feline endogenous oncornavirus (RD114) was detected in a well-characterized human fibrosarcoma cell line, HT1080, by indirect immunofluorescence. Three antisera against RD114 p30 gave similar positive results, while two antisera prepared against simian sarcoma virus p30, one antiserum prepared against murine leukemia virus p30, and one antiserum prepared against feline leukemia virus p30 gave no immunofluorescence. The reactivity observed with the antiserum against RD114 p30 was detected in 10-40% of the cells at early passages and was no longer expressed by the forty-first subculture. The reactivity could be removed by adsorption of the antiserum with RD114-infected dog or human cells, but not by uninfected cells or by cells infected with an antigenically unrelated oncornavirus, feline leukemia virus. Neither complete virus particles nor reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity was detected in the culture. These experiments suggest that the fibrosarcoma cell line is expressing an antigen related to the p30 protein of RD114 baboon endogenous virus group of oncornaviruses without producing complete virions. Images

Smith, H S; Riggs, J L; Springer, E L

1977-01-01

23

Common antigens of streptococcal and nonstreptococcal oral bacteria: characterization of wall-associated protein and comparison with extracellular protein antigen.  

PubMed Central

A soluble wall-associated common protein (WAP) has been isolated from cell wall-extracted antigens of exponentially growing cells of Streptococcus sanguis OMZ9, two serotypes (a and e), of Streptococcus mutans and Actinomyces viscosus OMZ104. The WAP antigens from the different strains were obtained by chromatography on an anti-rabbit immunoglobulin column of sodium dodecyl sulfate-dissociated immunoprecipitates. The affinity-isolated WAP antigens from the different oral bacteria were identical, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (showing an apparent molecular weight of 80,000) and amino acid and sugar compositions. This WAP antigen, common to all bacterial strains tested, consisted of 90% protein and 10% sugar. A comparison of the chemical properties of this WAP antigen and of the soluble extracellular common protein antigen showed that, although having similar amino acid and sugar compositions, the two proteins are different. Electron micrographs developed after immunocytological labeling of the WAP antigen on whole cells confirmed that it is located on the cell wall. Images

Scholler, M; Klein, J P; Sommer, P; Frank, R

1983-01-01

24

Changes in structural and antigenic properties of proteins by radiation  

NASA Astrophysics Data System (ADS)

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 N 2. Significant changes in surface hydrophobicity and [ ?] 222 nm of CD were also observed by radiation showing the destruction of secondary structure of proteins. Antigenicity of irradiated OVA measured by the method of immunodiffusion was decreased by radiation, and the reactivity to anti-OVA antibody was almost diminished at 8 kGy in N 2 and 4 kGy in O 2, respectively. The reactivity of BSA was diminished at 4 kGy both in N 2 and O 2. Changes in hydrophobicity of OVA did not correspond to the decrease in antigenicity, whereas the changes in [ ?] 222 nm relatively well corresponded to the antigenicity. The SDS-PAGE and immunoblotting analysis showed that radiation at higher doses induced the production of protein aggregates and degraded fragments with reactivity to the specific antibodies. These results suggest that the main part of conformation-dependent antigenic structure (conformational epitope) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes), remains even at higher dose.

Kume, Tamikazu; Matsuda, Tsukasa

1995-08-01

25

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.

2012-01-01

26

Changes in the clinical impact of high-risk human leukocyte antigen allele mismatch combinations on the outcome of unrelated bone marrow transplantation.  

PubMed

Several high-risk HLA allele mismatch combinations (HR-MMs) for severe acute graft-versus-host disease (GVHD) have been identified by analyzing transplantation outcomes in Japanese unrelated hematopoietic stem cell transplant recipients. In this study, we analyzed the effects of HR-MMs in 3 transplantation time periods. We confirmed that the incidence of grade III to IV acute GVHD in the HR-MM group was significantly higher than that in the low-risk (LR) MM group (hazard ratio [HR], 2.74; P < .0001) in the early time period (1993 to 2001). However, the difference in the incidence of grade III to IV acute GVHD between the HR-MM and LR-MM groups was not statistically significant (HR, 1.06; P = .85 and HR, .40; P = .21, respectively) in the mid (2002 to 2007) and late (2008 to 2011) time periods. Similarly, survival in the HR-MM group was significantly inferior to that in the LR-MM group (HR, 1.46; P = .019) in the early time period, whereas the difference in survival between the 2 groups was not statistically significant in the mid and late time periods (HR, 1.06; P = .75 and HR, .82; P = .58, respectively). In conclusion, the adverse impact of HR-MM has become less significant over time. Unrelated transplantation with a single HR-MM could be a viable option in the absence of a matched unrelated donor or an unrelated donor with a single LR-MM. PMID:24417871

Kanda, Yoshinobu; Kanda, Junya; Atsuta, Yoshiko; Fuji, Shigeo; Maeda, Yoshinobu; Ichinohe, Tastuo; Takanashi, Minoko; Ohashi, Kazuteru; Fukuda, Takahiro; Miyamura, Koichi; Mori, Takehiko; Sao, Hiroshi; Kobayashi, Naoki; Iwato, Koji; Sawada, Akihisa; Mori, Shinichiro

2014-04-01

27

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.

1990-04-01

28

Genetic and Antigenic Diversity of the Surface Protective Antigen Proteins of Erysipelothrix rhusiopathiae  

Microsoft Academic Search

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the

S. Nagai

2007-01-01

29

High-Affinity Reactions Between Antigen-Specific T-Cell Receptors and Peptides Associated with Allogeneic and Syngeneic Major Histocompatibility Complex Class I Proteins  

Microsoft Academic Search

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8^+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (L^d) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL,

Yuri Sykulev; Anders Brunmark; Theodore J. Tsomides; Shigeki Kageyama; Michael Jackson; Per A. Peterson; Herman N. Eisen

1994-01-01

30

Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.  

PubMed

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types ( approximately 60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness. PMID:17475766

To, Ho; Nagai, Shinya

2007-07-01

31

Genetic and Antigenic Diversity of the Surface Protective Antigen Proteins of Erysipelothrix rhusiopathiae?  

PubMed Central

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types (?60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.

To, Ho; Nagai, Shinya

2007-01-01

32

Identification of Antigenic Proteins in Trichomonas vaginalis  

PubMed Central

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ? Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ?-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

Lee, Hye-Yeon; Hyung, Sujin; Lee, Jong Woong; Kim, Juri; Shin, Myeong Heon; Ryu, Jae-Sook

2011-01-01

33

Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest  

Microsoft Academic Search

The INK4a (MTS1, CDKN2) gene encodes an inhibitor (p16INK4a) of the cyclin D-dependent kinases CDK4 and CDK6 that blocks them from phosphorylating the retinoblastoma protein (pRB) and prevents exit from the G1 phase of the cell cycle. Deletions and mutations involving INK4a occur frequently in cancers, implying that p16INK4a, like pRB, suppresses tumor formation. An unrelated protein (p19ARF) arises in

Dawn E. Ouelle; Frédérique Zindy; Richard A. Ashmun; Charles J. Sherr

1995-01-01

34

Virosome-mediated delivery of protein antigens to dendritic cells  

Microsoft Academic Search

Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I

Laura Bungener; Karine Serre; Liesbeth Bijl; Lee Leserman; Jan Wilschut; Toos Daemen; Patrick Machy

2002-01-01

35

Antigenicity and Immunogenicity of Plasmodium vivax Merozoite Surface Protein-3  

PubMed Central

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

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

2013-01-01

36

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.  

PubMed

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner. PMID:23756210

Spierings, Eric; Kim, Yeung-Hyen; Hendriks, Matthijs; Borst, Eric; Sergeant, Ruhena; Canossi, Angelica; Oudshoorn, Machteld; Loiseau, Pascale; Dolstra, Harry; Markiewicz, Miroslaw; Leffell, Mary S; Pereira, Noemi; Kircher, Brigitte; Turpeinen, Hannu; Eliaou, Jean-François; Gervais, Thibaut; Laurin, David; Enczmann, Jürgen; Martinetti, Miryam; Thomson, Jackie; Oguz, Fatma; Santarone, Stella; Partanen, Jukka; Siekiera, Urszula; Alessandrino, Emilio Paolo; Kalayoglu, Sevgi; Brand, Ronald; Goulmy, Els

2013-08-01

37

A metalloantibody that irreversibly binds a protein antigen.  

PubMed

Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics. PMID:17617633

Trisler, Kirk; Looger, Loren L; Sharma, Vikram; Baker, Martin; Benson, David E; Trauger, Sunia; Schultz, Peter G; Smider, Vaughn V

2007-09-01

38

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

PubMed

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

2013-01-01

39

Genomic, protein homogeneity and antigenic variability of Mycoplasma agalactiae  

Microsoft Academic Search

Eleven strains of Mycoplasma agalactiae differing in pathogenicity, animal species origin and geographic localisation, showed similar chromosome restriction profiles with four endonucleases. However the international reference strain PG2 showed a unique profile.The protein and antigenic variabilities of 31 strains of M. agalactiae were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting performed with naturally infected animal sera

Michel Solsona; Maurice Lambert; François Poumarat

1996-01-01

40

Priming of class I-restricted cytotoxic T lymphocytes by vaccination with recombinant protein antigens  

Microsoft Academic Search

We investigated the specific priming of MHC class I-restricted cytotoxic T lymphocytes (CTL) by different protein antigen preparations in mice. The recombinant viral protein antigens tested are of potential relevance for the design of subunit vaccines. They include the hepatitis B virus (HBV) surface antigen (S-antigen), the HIV-1 gp160 envelope protein, and a chimeric HIV-1 Pr55-gag\\/V3-3 retrovirus-like particle. In addition,

Reinhold Schirmbeck; Ludwig Deml; Karl Melber; Hans Wolf; Ralf Wagner; Jörg Reimann

1995-01-01

41

Immunogenicity and antigenicity of Plasmodium vivax merozoite surface protein 10.  

PubMed

Among the proteins involved in the invasion by merozoite, the glycosylphosphatidylinositol-anchored proteins (GPI-APs) are suggested as potential vaccine candidates because of their localization to apical organelles and the surface; these candidates are predicted to play essential roles during invasion. As a GPI-AP, Plasmodium vivax merozoite surface protein 10 (PvMSP-10) induces high antibody titers. However, such high antibody titers have shown no protective efficacy for animals challenged with P. vivax parasites in a previous study. To adequately evaluate the immunogenicity and further characterize PvMSP-10 in order to understand its vaccine potential, we assessed its immunogenicity by immunizing BALB/c mice with cell-free expressed recombinant PvMSP-10 protein. The antigenicity of MSP-10 was analyzed, and we found 42 % sensitivity and 95 % specificity using serum samples from P. vivax-infected Korean patients. The IgG1 and IgG3 were the predominant immunoreactive antibodies against PvMSP-10 in vivax patient sera, and IgG1 and IgG3 and Th1-type cytokines were predominantly secreted in PvMSP-10-immunized mice. We conclude that the immunogenicity and antigenicity of MSP-10 may serve as a potential vaccine against vivax malaria. PMID:24764159

Cheng, Yang; Wang, Bo; Sattabongkot, Jetsumon; Lim, Chae Seung; Tsuboi, Takafumi; Han, Eun-Taek

2014-07-01

42

Stearoyl-Acyl-Carrier-Protein Desaturase from Higher Plants is Structurally Unrelated to the Animal and Fungal Homologs  

Microsoft Academic Search

Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa.

John Shanklin; Chris Somerville

1991-01-01

43

Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry  

PubMed Central

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.

Krummenacher, Claude; Nicola, Anthony V.; Whitbeck, J. Charles; Lou, Huan; Hou, Wangfang; Lambris, John D.; Geraghty, Robert J.; Spear, Patricia G.; Cohen, Gary H.; Eisenberg, Roselyn J.

1998-01-01

44

Sensitization to enhanced green fluorescence protein minor histocompatibility antigen by gene transduction into dendritic cells and peritoneal exudate macrophages  

Microsoft Academic Search

Enhanced green fluorescence protein (EGFP) has been widely applied to gene transduction in cellular and molecular biology as a reporter element. When applied to cell transplantation, it raises fundamental issues concerning cell-associated antigens, in particular, a model of minor histocompatibility antigen(s). Although it is well known that immunological behavior of minor histocompatibility antigens mimic tumor associated antigens (TAA), identified genes

Eigo Satoh; Xiao-Kang Li; Yuhko Hara; Keiichi Ogata; Lei Guo; Yuhsuke Kitazawa; Naoko Funeshima-Fuji; Takashi Satoh; Tohko Miyagi; Wataru Sugiura; Naoki Yamamoto; Kenichi Teramoto; Shigeki Arii; Hiromitsu Kimura

2007-01-01

45

Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen.  

PubMed

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity. PMID:24776487

Temchura, Vladimir V; Kozlova, Diana; Sokolova, Viktoriya; Uberla, Klaus; Epple, Matthias

2014-07-01

46

Investigation of endogenous antigen processing by delivery of an intact protein into cells.  

PubMed

Investigation of MHC class I endogenous antigen processing and presentation is important for understanding non-self immune recognition, vaccine design, immune evasion by pathogens and tumours, and elucidation of the mechanisms of certain auto-immune diseases. To study the MHC class I antigen-processing pathway, the protein antigen of interest has to be expressed inside the cells. To express a desired protein antigen, delivery of protein coding genes into the cell using transfection or infection by a virus carrying the gene of the antigen is a popular method. But there is no satisfactory method using the intact protein as antigen directly for investigation of MHC antigen processing and presentation that can give a quantitative result for the efficiency of antigen processing. Here we describe a novel method to deliver protein antigen directly into the cytosol of cells for quantitative analysis of the efficiency of antigen processing. Adherent mouse fibroblasts, mouse lymphoma cell line RMA, spleen cells and dendritic cells were permeabilized with streptolysin O, and chicken ovalbumin (OVA), a model protein antigen, was successfully introduced into the cells. Mouse fibroblast and RMA cells were able to properly process OVA protein, and OVA-derived peptide OVA(258-265) (or SIINFEKL) was successfully presented via the MHC class I molecule (K(b)) to SIINFEKL-K(b)-specific T cell hybridoma B3Z. As expected, delivery of OVA protein into RMA-S, a cell line with a deficiency in MHC class I antigen processing due to a TAP defect, failed to present the SIINFEKL epitope to B3Z. PMID:18406420

Fu, Hongmei; Ding, Jie; Flutter, Barry; Gao, Bin

2008-06-01

47

Analysis of Antigenic Structure and Human Immune Response to Outer Membrane Protein CD of Moraxella catarrhalis  

Microsoft Academic Search

Moraxella catarrhalis is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Outer membrane protein CD (OMP CD) is a 45-kDa protein which is a potential vaccine antigen to prevent infections caused by M. catarrhalis. Eight monoclonal antibodies were used to study the antigenic structure of the OMP

TIMOTHY F. MURPHY; CHARMAINE KIRKHAM; ERNESTO DENARDIN; SANJAY SETHI

1999-01-01

48

Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.  

PubMed Central

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

Ortiz, B L; Garcia, A M; Restrepo, A; McEwen, J G

1996-01-01

49

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

2010-01-01

50

Consensus classification of human leukocyte antigen class II proteins.  

PubMed

Class II human leukocyte antigens (HLA II) are proteins involved in the human immunological adaptive response by binding and exposing some pre-processed, non-self peptides in the extracellular domain in order to make them recognizable by the CD4+ T lymphocytes. However, the understanding of HLA-peptide binding interaction is a crucial step for designing a peptide-based vaccine because the high rate of polymorphisms in HLA class II molecules creates a big challenge, even though the HLA II proteins can be grouped into supertypes, where members of different class bind a similar pool of peptides. Hence, first we performed the supertype classification of 27 HLA II proteins using their binding affinities and structural-based linear motifs to create a stable group of supertypes. For this purpose, a well-known clustering method was used, and then, a consensus was built to find the stable groups and to show the functional and structural correlation of HLA II proteins. Thus, the overlap of the binding events was measured, confirming a large promiscuity within the HLA II-peptide interactions. Moreover, a very low rate of locus-specific binding events was observed for the HLA-DP genetic locus, suggesting a different binding selectivity of these proteins with respect to HLA-DR and HLA-DQ proteins. Secondly, a predictor based on a support vector machine (SVM) classifier was designed to recognize HLA II-binding peptides. The efficiency of prediction was estimated using precision, recall (sensitivity), specificity, accuracy, F-measure, and area under the ROC curve values of random subsampled dataset in comparison with other supervised classifiers. Also the leave-one-out cross-validation was performed to establish the efficiency of the predictor. The availability of HLA II-peptide interaction dataset, HLA II-binding motifs, high-quality amino acid indices, peptide dataset for SVM training, and MATLAB code of the predictor is available at http://sysbio.icm.edu.pl/HLA . PMID:23229472

Saha, Indrajit; Mazzocco, Giovanni; Plewczynski, Dariusz

2013-02-01

51

Changes in a CSF Antigen Associated with Dementia  

Microsoft Academic Search

An 85-kDa antigen was detected in cerebrospinal fluid (CSF) using a monoclonal antibody, 11.57, raised against a fraction of paired helical filaments extracted from Alzheimer’s disease (AD) brain tissues. The antigen is a thermostable protein that is unrelated either to tau or amyloid ?-protein precursor. Its level was measured in lumbar and post-mortem CSF samples and found to be decreased

M. Teresa Carretero; Charles R. Harrington; Claude M. Wischik

1995-01-01

52

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

2008-01-01

53

Development of an indirect sandwich ELISA for detection of urinary antigen, using Legionella pneumophila PAL protein.  

PubMed

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease. PMID:24293241

Gholipour, Abolfazl; Moosavian, Mojtaba; Makvandi, Manoochehr; Galehdari, Hamid; Alvandi, Amirhooshang; Mard, Seyyed Ali

2014-05-01

54

Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach  

PubMed Central

Background Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens.

2014-01-01

55

A simple technique to enhance the humoral immune response to intracellular protein antigens in genetic immunizations.  

PubMed

A simple technique to enhance the humoral immune response to intracellular protein antigens in genetic immunizations is demonstrated in mice. In this approach, the intracellular protein is intentionally secreted from expressing cells as a chimeric protein, comprising an N-terminal secreted protein fused to the intracellular protein antigen. Using the Leishmania chagasi Ldccys1 cysteine protease (411CP) as an example of an intracellular protein antigen and both human and murine granulocyte colony stimulating factor (GMCSF) as examples of N-terminal secretion competent fusion partners in chimeric proteins, a humoral response in plasmid DNA immunized mice could only be detected by Western blotting when the expressed 411CP was secreted. PMID:22964556

Farrell, Patrick J; Kucknoor, Ashwini S; Iatrou, Kostas; Gedamu, Lashitew

2013-01-31

56

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

SciTech Connect

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

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

2008-01-01

57

A Comparative Study of Cross Reactions of Serum Protein Antigens of Seven Mammals.  

National Technical Information Service (NTIS)

The study on the antigenic cross reactions among the serum proteins of mammals is not only definitely important in bio-taxonomy, embryology, and evolutional theory, but also important for immunobiological and immunochemical researches. If a cross reaction...

Y. Hsieh L. Shih S. Ch'en H. Jao C. Ch'ien

1967-01-01

58

Demonstration of Multiple Antigenic Determinants on 'Mycoplasma pneumoniae' Attachment Protein by Monoclonal Antibodies.  

National Technical Information Service (NTIS)

Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments we...

P. C. Hu C. H. Huang Y. S. Huang A. M. Collier W. A. Clyde

1985-01-01

59

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

PubMed

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

2013-06-01

60

Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells  

Microsoft Academic Search

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription-polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP,

Stephanie K. Dougan; Azucena Salas; Paul Rava; Amma Agyemang; Arthur Kaser; Jamin Morrison; Archana Khurana; Mitchell Kronenberg; Caroline Johnson; Mark Exley; M. Mahmood Hussain; Richard S. Blumberg

61

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.

2010-01-01

62

A study of the antigenicity of Rickettsia helvetica proteins using two-dimensional gel electrophoresis.  

PubMed

Rickettsia helvetica is an obligate intracellular Gram-negative microorganism found in Ixodes ricinus ticks. When R. helvetica was first discovered in 1979, little was known about its physiology and it fell into oblivion until it recently was suspected of being pathogenic to humans. However, all efforts to isolate R. helvetica from patients have been unsuccessful, although serological responses against R. helvetica can be demonstrated. The aim of our study was to investigate the protein profile of R. helvetica and study the antigenicity of its proteins using two-dimensional (2D) immunoblot in order to characterize the immunological response against R. helvetica infection. Our results show that in addition to the known PS120 and OmpB antigenic R. helvetica proteins, three other antigens exist: a 60 kDa GroEL protein, a 10 kDa GroES protein and a hitherto unknown 35 kDa hypothetical protein that has similarities with ORF-RC0799 of Rickettsia conorii. Furthermore, the lipopolysaccharide showed strong antigenicity. In this study, we present the first proteome map and the first 2D immunoblot profile of R. helvetica and finally we present the 35 kDa R. helvetica as an additional antigen to the previously known rickettsial antigens. PMID:19338513

Hajem, Nedaa; Weintraub, Andrej; Nimtz, Manfred; Römling, Ute; Påhlson, Carl

2009-04-01

63

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen.  

PubMed

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis. PMID:1400966

Zygmunt, M S; Gilbert, F B; Dubray, G

1992-10-01

64

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen.  

PubMed Central

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis. Images

Zygmunt, M S; Gilbert, F B; Dubray, G

1992-01-01

65

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

PubMed Central

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

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

1993-01-01

66

Chimeric Influenza Virus Hemagglutinin Proteins Containing Large Domains of the Bacillus anthracis Protective Antigen: Protein Characterization, Incorporation into Infectious Influenza Viruses, and Antigenicity  

Microsoft Academic Search

Large polypeptides of the Bacillus anthracis protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1, the region responsible for binding to the other toxin compo- nents, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD),

Zhu-Nan Li; Scott N. Mueller; Ling Ye; Zhigao Bu; Chinglai Yang; Rafi Ahmed; David A. Steinhauer

2005-01-01

67

Antigenic domains of the open reading frame 2-encoded protein of hepatitis E virus.  

PubMed

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein. PMID:10449466

Khudyakov, Y E; Lopareva, E N; Jue, D L; Crews, T K; Thyagarajan, S P; Fields, H A

1999-09-01

68

Antigenic Domains of the Open Reading Frame 2-Encoded Protein of Hepatitis E Virus  

PubMed Central

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.

Khudyakov, Yury E.; Lopareva, Elena N.; Jue, Danny L.; Crews, Tamara K.; Thyagarajan, S. P.; Fields, Howard A.

1999-01-01

69

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

PubMed

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

2012-01-01

70

A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union.  

PubMed

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the ? and ? light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field. PMID:24503852

Grover, Rajesh K; Zhu, Xueyong; Nieusma, Travis; Jones, Teresa; Boero, Isabel; MacLeod, Amanda S; Mark, Adam; Niessen, Sherry; Kim, Helen J; Kong, Leopold; Assad-Garcia, Nacyra; Kwon, Keehwan; Chesi, Marta; Smider, Vaughn V; Salomon, Daniel R; Jelinek, Diane F; Kyle, Robert A; Pyles, Richard B; Glass, John I; Ward, Andrew B; Wilson, Ian A; Lerner, Richard A

2014-02-01

71

Molecular characterisation of a predominant antigenic region of Giardia lamblia variant surface protein H7.  

PubMed

During infection, the intestinal protozoan parasite Giardia lamblia undergoes continuous antigenic variation which is determined by diversification of the parasite's major surface antigen, named VSP (variant surface protein). One member from this protein family, VSP H7, is expressed by G. lamblia clone GS/M-83-H7. In the present study, we characterised a highly antigenic portion of VSP H7 which is positioned inside a 130 amino acid C-terminal region of the protein. This region overlaps with a cysteine-rich motif that is rather conserved within the VSP family. Detailed molecular dissection of the antigenic portion monitored a 12 amino acid peptidyl structure which constitutes a non-conformational epitope of VSP H7. In the murine host, this epitope is recognised relatively early (before day 10 p.i.) during infection and stimulates a strong intestinal immunoglobulin A response. At late infective stages (after day 10 p.i.) this immune reaction is progressively complemented by reactions against 'late' antigenic epitopes which are also located inside the 130 amino acid antigenic portion but in closer proximity to the C-terminal end of VSP H7 than the 12 amino acid epitope. Both the high antigenicity and the conserved character suggest that the 12 amino acid epitope is a key factor within the immunological interplay between G. lamblia and the experimental murine host. PMID:11403775

Bienz, M; Wittwer, P; Zimmermann, V; Müller, N

2001-06-01

72

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

2000-01-01

73

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development  

PubMed Central

Purpose Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. Materials and Methods Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. Results We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8+T cells and OT-2-specific CD4+T cells. Conclusion Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.

2014-01-01

74

Serum and mucosal antibody responses to pneumococcal protein antigens in children: relationships with carriage status.  

PubMed

Streptococcus pneumoniae causes significant morbidity and mortality especially in children. Some pneumococcal protein antigens can protect mice against infection. Little information is available concerning the nature of naturally acquired protective immunity to pneumococci in humans induced by these antigens. This study investigates the relationships between systemic and local antibody production and carriage in children. Children undergoing adenoidectomy (n=112, ages 2-12 years) were studied. Nasopharyngeal swabs were collected for pneumococcal culture. Serum and saliva were assayed for antibodies to several pneumococcal proteins: choline binding protein A (CbpA), pneumolysin (Ply), pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA). Adenoidal mononuclear cells (MNC) were cultured with pneumococcal culture supernatants or recombinant proteins. Cell culture supernatants were analyzed for antigen-specific antibodies. Carriage rates fell with age and serum levels of anti-CbpA, Ply and PspA rose. Anti-CbpA and -Ply serum and salivary IgG antibody levels were higher in children who were culture negative than those who were colonized. Antigen stimulation increased respective antigen-specific IgG production by adenoidal MNC and these responses were greater in those who were colonized than in culture-negative children. Antibodies to CbpA and Ply may protect children aged 2 years and older against pneumococcal colonization. Adenoids may be important local induction and effector sites for both mucosal and systemic antibody production to pneumococcal proteins in children. PMID:16342325

Zhang, Qibo; Bernatoniene, Jolanta; Bagrade, Linda; Pollard, Andrew J; Mitchell, Timothy J; Paton, James C; Finn, Adam

2006-01-01

75

ANTIGENIC RELATIONSHIPS OF BENCE JONES PROTEINS, MYELOMA GLOBULINS, AND NORMAL HUMAN ?-GLOBULIN  

PubMed Central

By means of immunodiffusion and immunoelectrophoresis study has been made of antigenic relationships of Bence Jones proteins, and the three classes of normal and pathological immunoglobulins, 7S ?, ?2A, and ?2M. All thirty-nine Bence Jones proteins studied could be classified into either one of two distinct antigenic types, A or B. Both types are related to the immunoelectrophoretically slow (S) fragment of a papain digest of normal ?-globulin; B is related more closely than A, but neither has antigenic determinants in common with the fast (F) fragment. The 7S ? myeloma globulins were either immunological type I or II. The papain digests of these proteins produced the S and F precipitin lines in immunoelectrophoresis but multiple bands in starch gel electrophoresis, especially in the F region. The S fraction of type I myeloma globulins is antigenically similar to Bence Jones protein of type B, and the S component of type II myeloma globulins has antigenic determinants in common with type A Bence Jones protein. Correspondingly, myeloma patients with type I globulins and proteinuria usually excrete type B Bence Jones proteins, whereas patients with type II excrete type A proteins. The F fragment is the part common to normal 7S ?-globulin and types I and II myeloma globulins but is absent in ?2A and ?2M pathological globulins and in both types of Bence Jones proteins. Papain digests of ?2A myeloma globulins produced a single precipitin line in immunoelectrophoresis. ?2A myeloma globulins appeared to have two antigenic units, one in common with type B Bence Jones protein and normal ?-globulin, and another specific to ?2A. The ?2A myeloma patients excreted type B Bence Jones protein. The papain digest of a macroglobulin produced two precipitin lines, the faster of which had antigenic determinants in common with type B Bence Jones protein, the slower seemed specific for the macroglobulin. Five serum micromolecular globulins proved to be either type A or B Bence Jones proteins. From the above results, an antigenic map was constructed showing which determinants are shared and which are specific for normal 7S ?-globulin, types I and II myeloma globulins, ?2A myeloma globulins, a macroglobulin, and types A and B Bence Jones proteins.

Migita, Shunsuke; Putnam, Frank W.

1963-01-01

76

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

PubMed Central

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 (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

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

2008-01-01

77

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

78

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

PubMed

Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection. PMID:21927872

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

2012-02-01

79

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens  

PubMed Central

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.

Felgner, Philip L.; Kayala, Matthew A.; Vigil, Adam; Burk, Chad; Nakajima-Sasaki, Rie; Pablo, Jozelyn; Molina, Douglas M.; Hirst, Siddiqua; Chew, Janet S. W.; Wang, Dongling; Tan, Gladys; Duffield, Melanie; Yang, Ron; Neel, Julien; Chantratita, Narisara; Bancroft, Greg; Lertmemongkolchai, Ganjana; Davies, D. Huw; Baldi, Pierre; Peacock, Sharon; Titball, Richard W.

2009-01-01

80

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

2007-01-01

81

Enzymatic Hydrolysis of Heated Whey: Iron-Binding Ability of Peptides and Antigenic Protein Fractions  

Microsoft Academic Search

This study evaluated the influence of various en- zymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydroly- sates. Heated (for 10 min at 100°C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Fla- vourzyme, papain, and trypsin for 30, 60, 90,

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

2007-01-01

82

Heat shock proteins in autoimmune disease. From causative antigen to specific therapy?  

Microsoft Academic Search

Heat shock proteins (hsp) are highly conserved from bacteria to man. Bacterial hsp, with approximate molecular weights of 60 kDa (hsp60), are immunodominant antigens that are immunologically cross-reactive with their mammalian counterparts. Hsp molecules are therefore useful in studies of fundamental questions concerning immune responses to foreign as opposed to self antigens. The finding that immune responses to hsp are

X.-D. Yang; U. Feige

1992-01-01

83

Molecular force probe measurement of antigen I/II-matrix protein interactions.  

PubMed

Viridans streptococci possess a family of immunologically and structurally related cell-surface proteins, termed antigen I/II, which may function as adhesins and enable oral streptococci to adhere to saliva-coated surfaces and matrix proteins. Here we used atomic force microscopy in the molecular force mode to measure the specific interaction forces between antigen I/II and two matrix proteins, collagen and fibronectin. These matrix proteins provide important binding sites for adherence of oral streptococcal in dentinal caries and endocarditis, respectively. Antigen I/II-coated cantilever tips were brought into contact with collagen- or fibronectin-coated silica coverslips. For the protein I/II-fibronectin interaction experiments, the mean strength of the last ruptures was 216 pN, with most of the detachments located around 125 pN. In antigen I/II-collagen interaction experiments, the mean strength of the last rupture forces corresponded to 136 pN, with the most frequent unbinding force around 75 pN. Thus, our findings definitely suggest that, under the present experimental conditions, antigen I/II binds more strongly to fibronectin than to type I collagen. This might be of relevance for the attachment of viridians streptococci to surfaces exposed to strong hydrodynamic shearing forces under in vivo conditions. PMID:21083620

Soell, Martine; Hemmerlé, Joseph; Hannig, Matthias; Haïkel, Youssef; Sano, Hidehiko; Selimovic, Denis

2010-12-01

84

Antigenic epitope characterization of matrix protein of Newcastle disease virus using monoclonal antibody approach: Contrasting variability amongst NDV strains  

Microsoft Academic Search

A panel of 15 monoclonal antibodies (MABs) against matrix (M) protein of Newcastle disease virus (NDV) was obtained and the specificity towards the M protein was proven by radioimmunoprecipitation assay and antigen capture enzyme-linked immunosorbent assay (ELISA). Further studies were directed to antigenic epitope mapping of the M protein by means of this panel. The epitope characterization was performed by

A. Panshin; E. Shihmanter; Y. Weisman; C. Örvell; M. Lipkind

1997-01-01

85

The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein  

PubMed Central

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T-cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freund's adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA-specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat-killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA-specific CD8+ CTL. An OVA-specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re-stimulated in vitro. Adjuvant activity of heat-killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat-shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side-effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial.

Skinner, M A; Prestidge, R; Yuan, S; Strabala, T J; Tan, P L J

2001-01-01

86

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.

2012-01-01

87

Lipid-modified surface protein antigens expressing size variation within the species Mycoplasma hyorhinis.  

PubMed Central

Monoclonal antibodies (MAbs) previously shown to recognize distinct epitopes selectively expressed on the surface of some Mycoplasma hyorhinis strains were used to define two discrete sets of lipid-modified membrane surface proteins showing marked size variation within this species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of Triton X-114 phase-fractionated proteins from six isolates of M. hyorhinis defined a set of amphiphilic integral membrane proteins of 23, 50, and 55 kilodaltons (kDa) recognized on respective isolates by one MAb and a second set of integral proteins of 88, 120, and 100 to 150 kDa recognized by another MAb. The first group of proteins all contained a common, amphiphilic 18-kDa limit tryptic polypeptide bearing the epitope. The size- and strain-variant surface antigens identified by the MAbs were shown to be lipid-modified proteins. Phase fractionation of [3H]palmitate-labeled organisms revealed numerous 3H-labeled proteins in all isolates, which partitioned exclusively into the hydrophobic phase. These proteins generally showed pronounced size variation among isolates and included the antigen variants recognized by the two MAbs, as demonstrated directly by immunoprecipitation of correspondingly sized 3H-labeled proteins from each isolate. A third MAb recognized an invariant, lipid-associated surface protein of 70 kDa on all M. hyorhinis isolates. Covalent modification of lipid-associated proteins was confirmed by identifying 3H-labeled methyl palmitate after acid methanolysis of Triton X-114 phase proteins derived from [3H]palmitate-labeled organisms. However, removal of covalently bound lipid from chloroform-methanol-extracted proteins by alkaline hydroxylamine was selective; complete removal was observed with only a few proteins, possibly including the 120-kDa form of one antigen variant. This suggested potential differences in the nature of covalent linkage among lipid-modified M. hyorhinis surface antigens. Intraspecies antigen variants described here in M. hyorhinis share some characteristics with size-variant antigens reported in phylogenetically related gram-positive eubacteria and may contribute to phenotypic diversification and differences in pathogenicity of mycoplasmas. Images

Boyer, M J; Wise, K S

1989-01-01

88

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

PubMed

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 molecules to CD8+ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4+ and CD8+ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin+ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin+ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4+ and CD8+ T cell responses emphasizes their potential for epicutaneous immunization strategies. PMID:18677477

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

2009-07-01

89

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

1992-01-01

90

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

1997-01-01

91

A plasmid DNA immunogen expressing fifteen protein antigens and complex virus-like particles (VLP +) mimicking naturally occurring HIV  

Microsoft Academic Search

We describe here a single plasmid DNA immunogen representing the broadest antigen repertoire among HIV vaccine candidates. This pDNA was “ANTIGENeered” for the regulated expression of thirteen complete and two non-functional HIV protein antigens. These proteins self assemble into complex virus-like particles (VLP+). Multiple irreversible safety features were introduced by genetic modifications including the complete impairment of integration, reverse transcription,

Eszter Somogyi; Jianqing Xu; Ágnes Gudics; József Tóth; Attila L. Kovács; Franco Lori; Julianna Lisziewicz

2011-01-01

92

Tumor rejection antigen gp96/grp94 is an ATPase: implications for protein folding and antigen presentation.  

PubMed Central

Immunization of mice with gp96/grp94 heat shock proteins (HSPs) elicits tumor-specific cellular immunity to the tumors from which gp96 is isolated. However, the cDNA sequence of gp96 is identical among tumors and normal tissues. This raises the question regarding the structural basis of the specific immunogenicity of gp96. As HSPs bind a wide array of molecules including peptides, we have proposed that gp96 may not be immunogenic per se, but may chaperone antigenic peptides. Furthermore, gp96 is localized predominantly in the lumen of the endoplasmic reticulum (ER) suggesting that it may act as a peptide acceptor and as accessory to peptide loading of MHC class I molecules. We demonstrate here that gp96 molecules contain ATP-binding cassettes, bind ATP and possess an Mg(2+)-dependent ATPase activity. Gp96 preparations are also observed to contain tightly bound peptides, which can be eluted by acid extraction. These properties of gp96 are consistent with its proposed roles in chaperoning antigenic peptides and in facilitating MHC class I--peptide assembly in the ER lumen. We present a model to explain how interaction of gp96 with MHC class I may result in transfer of peptides to the latter. Images

Li, Z; Srivastava, P K

1993-01-01

93

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

2013-01-01

94

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

2004-01-01

95

[Construction and screening of SARS-CoV S protein-specific phage displayed antigen library].  

PubMed

The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics. PMID:23905471

Wu, Rui-Ping; Meng, Jia-Zi; He, Yu-Xian

2013-05-01

96

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

PubMed

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

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

2013-08-01

97

In vivo induction of type 1 and 2 immune responses against protein antigens  

Microsoft Academic Search

Polarization of the immune response towards Th 1o r T h2 profiles is under the control of several, not yet well known, mechanisms. The present study was undertaken to investigate whether immune responses generated against major protein antigens, of parasitic (Schistosoma mansoni) and bacterial (Clostridium tetani) origin, present characteristic Th profiles. Mice were immunized with a single dose of S.

Emmanuel E. Comoy; Georges Thyphronitis

1997-01-01

98

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

1980-01-01

99

Immunogenic CD91 Ligand-Antigenic Molecule Complexes and Fusion Proteins.  

National Technical Information Service (NTIS)

The present invention relates to complexes and fusion proteins comprising a CD91 ligand and an antigenic molecule, for use in the treatment or prevention of a disease. The invention specifically provides complexes comprising a CD91 ligand non-covalently b...

P. K. Srivastava

2004-01-01

100

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

Microsoft Academic Search

The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these anti- gens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5

Armead H. Johnson; Rose G. F. Leke; Nancy R. Mendell; D. Shon; Y. J. Suh; D. Bomba-Nkolo; V. Tchinda; S. Kouontchou; L. W. Thuita; A. M. van der Wel; A. Thomas; A. Stowers; A. Saul; A. Zhou; D. W. Taylor; I. A. Quakyi

2004-01-01

101

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

PubMed

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

2005-01-01

102

The polycomb group proteins, BMI1 and EZH2, are tumour-associated antigens  

Microsoft Academic Search

We used SEREX technology to identify novel tumour-associated antigens in patients with primary hepatocellular carcinoma and found serological responses to the polycomb group (PcG) protein BMI-1, which is overexpressed in a range of different tumour types. Further studies identified T-cell responses to both BMI-1 and another PcG protein, EZH2, in cancer patients and at relatively lower levels in some normal

J C Steele; E E Torr; K L Noakes; E Kalk; P A Moss; G M Reynolds; S G Hubscher; M van Lohuizen; D H Adams; L S Young

2006-01-01

103

Application of an acid proteinase from Monascus purpureus to reduce antigenicity of bovine milk whey protein.  

PubMed

An acid proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637-1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP's comprehensive substrate specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry. MpuAP hydrolyzed primarily five peptide bonds, Gln(4)-His(5), His(10)-Leu(11), Ala(14)-Leu(15), Gly(23)-Phe(24) and Phe(24)-Phe(25) in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30-40°C for 7 days without stabilizers. To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and ?-lactalbumin were efficiently degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, ?-lactalbumin and ?-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP-trypsin combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin-trypsin combination. Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach for making hypoallergic bovine milk whey protein hydrolysates. PMID:21298320

Lakshman, P L Nilantha; Tachibana, Shinjiro; Toyama, Hirohide; Taira, Toki; Suganuma, Toshihiko; Suntornsuk, Worapot; Yasuda, Masaaki

2011-09-01

104

Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity.  

PubMed Central

Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes. Images

Michel, J L; Madoff, L C; Kling, D E; Kasper, D L; Ausubel, F M

1991-01-01

105

Antigenic Changes in Nonhistone Proteins during Azo Dye Hepatocarcinogenesis1  

Microsoft Academic Search

We have investigated the appearance of specific nonhistone proteins during azo dye-induced hepatocarcinogenesis in the rat. Groups of animals fed azo dye-containing diet were sacri ficed at approximately 3-week intervals, portions of their livers were examined histologically, and the remaining material was fractionated into chromatin and cytoplasmic fractions. Livers of the azo dye-fed animals exhibited histological changes that have been

Warren N. Schmidt; Brian J. Gronert; David L. Page; Robert C. Briggs; Lubomir S. Hnilica

1982-01-01

106

Isolation and Characterization of Antibody Fragments Selective for Specific Protein Morphologies from Nanogram Antigen Samples  

PubMed Central

We developed atomic force microscope (AFM) based protocols that enable isolation and characterization of antibody based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single chain antibody fragments (scFvs) that specifically recognize an oligomeric amyloid-beta (A?) species correlated with Alzheimer’s disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We employed several subtractive panning steps to remove all phage binding non-desired antigens and then employed a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain derived oligomeric A?. The protocols described are readily adapted to isolating antibody based reagents against other antigenic targets with limited availability.

Kasturirangan, Srinath; Reasoner, Tim; Schulz, Philip; Boddapati, Shanta; Emadi, Sharareh; Valla, Jon; Sierks, Michael R.

2013-01-01

107

Optimization of fluoroimmunoassay against C-reactive protein exploiting immobilized-antigen glass slide.  

PubMed

An optimization experiment for an indirect-competitive (IC) fluoroimmunoassay (FIA) against C-reactive protein (CRP) was conducted exploiting an immobilized-antigen glass slide and an anti-CRP antibody tagged with fluorescent silica nanoparticles (FSNPs). The optimized conditions for the IC FIA were as follows: time and concentration of treatment with glutaraldehyde, 30 min and 1.5%, respectively; time of reaction with coating antigen and concentration of coating antigen for immobilization, 1 h and 0.1 mg/mL, respectively; concentration of FSNP-anti-CRP antibody conjugate coupled by the biotin-avidin interaction, the bioconjugate, for immune reaction, 0.250 mg/mL; concentration of bovine serum albumin (BSA) for blocking and time of blocking with BSA, 3% and 30 min, respectively. By using the glass slide, a highly sensitive detection against CRP was possible with the limit of detection below 0.1 ng/mL. PMID:23054305

Kim, Namsoo; Cho, Yong-Jin

2013-03-01

108

Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens.  

PubMed

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 125I-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commerically available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas. PMID:316440

Brown, J P; Tamerius, J D; Hellström, I

1979-01-01

109

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response.  

PubMed

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-?. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell-mediated immunity. PMID:20947070

Eisenberg, Galit; Machlenkin, Arthur; Frankenburg, Shoshana; Mansura, Adva; Pitcovski, Jacob; Yefenof, Eitan; Peretz, Tamar; Lotem, Michal

2010-01-01

110

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response  

PubMed Central

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-?. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell mediated immunity.

Eisenberg, Galit; Machlenkin, Arthur; Frankenburg, Shoshana; Mansura, Adva; Pitcovski, Jacob; Yefenof, Eitan; Peretz, Tamar; Lotem, Michal

2010-01-01

111

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

1992-01-01

112

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

1993-01-01

113

Plasmodium vivax Antigen Discovery Based on Alpha-Helical Coiled Coil Protein Motif.  

PubMed

Protein ?-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with ?-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high ?-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the ?-helical coiled coil structures. In addition, ex vivo production of IFN-? by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the ?-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes. PMID:24959747

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

2014-01-01

114

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

PubMed Central

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

Skov S?rensen, U B; Blom, J; Birch-Andersen, A; Henrichsen, J

1988-01-01

115

Plasmodium vivax Antigen Discovery Based on Alpha-Helical Coiled Coil Protein Motif  

PubMed Central

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

Cespedes, Nora; Habel, Catherine; Lopez-Perez, Mary; Castellanos, Angelica; Kajava, Andrey V.; Servis, Catherine; Felger, Ingrid; Moret, Remy; Arevalo-Herrera, Myriam; Corradin, Giampietro; Herrera, Socrates

2014-01-01

116

Successful mimicry of a complex viral antigen by multiple peptide insertions in a carrier protein.  

PubMed

The antigenic properties of a viral peptide from the surface of foot-and-mouth disease virus particles have been successfully mimicked by multiple insertion in solvent-exposed regions of Escherichia coli beta-galactosidase. By increasing the number of viral peptides per enzyme monomer, the average IC(50) of hybrid proteins in a competitive enzyme-linked immunosorbent assay) have decreased to values close to that presented by natural virions. Moreover, the antigenic diversity of these new recombinant enzymes when measured with different anti-virus antibodies has also been largely reduced, indicating a better presentation of the epitopes located in the viral peptide. Although bivalent antibody binding could have been favoured by multiple presentation, conformational modifications of the viral peptide, due to the presence of other insertions or a cooperative antibody binding cannot be excluded. In addition, a multidimensional antigenic analysis have grouped together the multiple-inserted proteins with the native virus, suggesting that increasing the number of insertions could be a good strategy to reproduce the antigenic properties of an immunoreactive peptide in a natural multimeric disposition. PMID:10828457

Feliu, J X; Carbonell, X; Villaverde, A

2000-05-26

117

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

1997-01-01

118

A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.  

PubMed Central

The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever. Images

Anderson, B E; McDonald, G A; Jones, D C; Regnery, R L

1990-01-01

119

MHC Haplotype Matching for Unrelated Hematopoietic Cell Transplantation  

PubMed Central

Background Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined. Methods and Findings We isolated DNA strands extending across 2 million base pairs of the MHC to determine the physical linkage of HLA-A, -B, and -DRB1 alleles in 246 HCT recipients and their HLA-A, -B, -C, -DRB1, -DQB1 allele-matched unrelated donors. MHC haplotype mismatching was associated with a statistically significantly increased risk of severe acute GVHD (odds ratio 4.51; 95% confidence interval [CI], 2.34–8.70, p < 0.0001) and with lower risk of disease recurrence (hazard ratio 0.45; 95% CI, 0.22–0.92, p = 0.03). Conclusions The MHC harbors genes that encode unidentified transplantation antigens. The three-locus HLA-A, -B, -DRB1 haplotype serves as a proxy for GVHD risk among HLA-identical transplant recipients. The phasing method provides an approach for mapping novel MHC-linked transplantation determinants and a means to decrease GVHD-related morbidity after HCT from unrelated donors.

Petersdorf, Effie W; Malkki, Mari; Gooley, Ted A; Martin, Paul J; Guo, Zhen

2007-01-01

120

Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: Effect of charge, fluidity and antigen-to-lipid ratio.  

PubMed

The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens ?-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal adjuvant CAF01 composed of cationic dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6'-dibehenate (TDB) or ii) the neutral adjuvant formulation NAF01, where DDA was replaced with zwitterionic distearoylphosphatidylcholine (DSPC). The effect of liposome charge, bilayer rigidity, isoelectric point and antigen-to-lipid ratio was investigated using dynamic light scattering, transmission electron microscopy, differential scanning calorimetry, intrinsic fluorescence and Langmuir monolayers. The net anionic ?-lactalbumin adsorbed onto the cationic liposomes, while there was no measureable attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of ?-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized the liposomal surface charge, resulting in reduced colloidal stability and liposome aggregation. Langmuir studies revealed that ?-lactalbumin was not squeezed out of DDA monolayers upon compression, which suggests additional hydrophobic interactions. Such interactions are thus likely to affect the way vaccine antigens are presented to antigen-presenting cells, and may play an important role for the efficacy of the vaccine-induced immune response. These studies thus exemplify the importance of characterizing the molecular interactions between the vaccine antigen and adjuvant along with immunogenicity and efficacy studies. PMID:24769435

Hamborg, Mette; Rose, Fabrice; Jorgensen, Lene; Bjorklund, Katrine; Pedersen, Helene B; Christensen, Dennis; Foged, Camilla

2014-08-01

121

The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen  

PubMed Central

Background Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. Methods A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. Results Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. Conclusions S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.

2011-01-01

122

Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry  

Microsoft Academic Search

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus recep- tor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds

CLAUDE KRUMMENACHER; ANTHONY V. NICOLA; J. CHARLES WHITBECK; HUAN LOU; WANGFANG HOU; JOHN D. LAMBRIS; ROBERT J. GERAGHTY; PATRICIA G. SPEAR; GARY H. COHEN; ROSELYN J. EISENBERG

1998-01-01

123

Mapping antibody binding sites on cytochrome c with synthetic peptides: are results representative of the antigenic structure of proteins?  

PubMed Central

Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.

Schwab, C.; Twardek, A.; Lo, T. P.; Brayer, G. D.; Bosshard, H. R.

1993-01-01

124

Identifying large sets of unrelated individuals and unrelated markers  

PubMed Central

Background Genetic Analyses in large sample populations are important for a better understanding of the variation between populations, for designing conservation programs, for detecting rare mutations which may be risk factors for a variety of diseases, among other reasons. However these analyses frequently assume that the participating individuals or animals are mutually unrelated which may not be the case in large samples, leading to erroneous conclusions. In order to retain as much data as possible while minimizing the risk of false positives it is useful to identify a large subset of relatively unrelated individuals in the population. This can be done using a heuristic for finding a large set of independent of nodes in an undirected graph. We describe a fast randomized heuristic for this purpose. The same methodology can also be used for identifying a suitable set of markers for analyzing population stratification, and other instances where a rapid heuristic for maximal independent sets in large graphs is needed. Results We present FastIndep, a fast random heuristic algorithm for finding a maximal independent set of nodes in an arbitrary undirected graph along with an efficient implementation in C++. On a 64 bit Linux or MacOS platform the execution time is a few minutes, even with a graph of several thousand nodes. The algorithm can discover multiple solutions of the same cardinality. FastIndep can be used to discover unlinked markers, and unrelated individuals in populations. Conclusions The methods presented here provide a quick and efficient method for identifying sets of unrelated individuals in large populations and unlinked markers in marker panels. The C++ source code and instructions along with utilities for generating the input files in the appropriate format are available at http://taurus.ansci.iastate.edu/wiki/people/jabr/Joseph_Abraham.html

2014-01-01

125

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

2003-01-01

126

The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of plasmodium falciparum-infected erythrocytes  

Microsoft Academic Search

The human malaria parasite Plasmodium falciparum evades host immunity by varying the antigenic and adhesive character of infected erythrocytes. We describe a large and extremely diverse family of P. falciparum genes (var) that encode 200–350 kDa proteins having the expected properties of antigenically variant adhesion molecules. Predicted amino acid sequences of var genes show a variable extracellular segment with domains

Xin-zhuan Su; Virginia M. Heatwole; Samuel P. Wertheimer; Frangoise Guinet; Jacqueline A. Herrfeldt; David S. Peterson; Jeffrey A. Ravetch; Thomas E. Wellems

1995-01-01

127

Evaluation of dengue nonstructural protein 1 antigen strip for the rapid diagnosis of patients with dengue infection.  

PubMed

Two hundred twenty samples obtained from 104 patients with dengue infection (n = 89) and other febrile illnesses (n = 15) were assayed for dengue nonstructural protein 1 (NS1) antigen by enzyme immunoassay and by an immunochromatography (lateral flow) test strip. The sensitivity and the specificity of dengue NS1 antigen strip were 98.9% and 90.6%, respectively. PMID:19232857

Chaiyaratana, Wathanee; Chuansumrit, Ampaiwan; Pongthanapisith, Viroj; Tangnararatchakit, Kanchana; Lertwongrath, Sarapee; Yoksan, Sutee

2009-05-01

128

Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity.  

PubMed

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding. PMID:21411808

Fowler, Carol B; Evers, David L; O'Leary, Timothy J; Mason, Jeffrey T

2011-04-01

129

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

Microsoft Academic Search

Polysaccharide-protein conjugates as vaccines have proven to be very effective in preventing Haemophilus influenzae type b infections in industrialized countries. However, cost-effective technologies need to be devel- oped for increasing the availability of anti-H. influenzae type b vaccines in countries from the developing world. Consequently, vaccine production with partially synthetic antigens is a desirable goal for many reasons. They may

V. Fernandez-Santana; F. Cardoso; A. Rodriguez; T. Carmenate; L. Pena; Y. Valdes; E. Hardy; F. Mawas; L. Heynngnezz; M. C. Rodriguez; I. Figueroa; J. Chang; M. E. Toledo; A. Musacchio; I. Hernandez; M. Izquierdo; K. Cosme; R. Roy; V. Verez-Bencomo

2004-01-01

130

Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen  

Microsoft Academic Search

Mammalian Heat Shock Proteins (HSP), have potent immune-stimulatory properties due to the natural capability to associate\\u000a with polypeptides and bind receptors on antigen presenting cells. The present study was aimed to explore whether plant HSP,\\u000a and in particular HSP70, share similar properties. We wanted in particular to evaluate if HSP70 extracted in association to\\u000a naturally bound polypeptides from plant tissues

Giampaolo Buriani; Camillo Mancini; Eugenio Benvenuto; Selene Baschieri

2011-01-01

131

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.  

PubMed Central

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.

Neville, J A; Prescott, L E; Bhattacherjee, V; Adams, N; Pike, I; Rodgers, B; El-Zayadi, A; Hamid, S; Dusheiko, G M; Saeed, A A; Haydon, G H; Simmonds, P

1997-01-01

132

Antigenic and immunogenic properties of defined physical forms of tick-borne encephalitis virus structural proteins.  

PubMed Central

Polymeric, delipidated glycoprotein complexes of defined size and composition were prepared from tick-borne encephalitis virus by solubilization with Triton X-100 or cetyltrimethylammonium bromide, followed by centrifugation into detergent-free sucrose density gradients. The antigenic reactivities and immunogenicities of these complexes were compared with those of complete inactivated virus. These glycoprotein preparations induced hemagglutination-inhibiting and neutralizing antibodies which proved to be protective in passive mouse protection tests and monospecifically reacted only with the viral envelope and not with the internal core. In a competitive radioimmunoassay the glycoprotein complexes revealed about 10-fold higher antigenicity than whole virus when tested at equal protein concentrations. The important implications of these results with respect to antigen quantification in vaccines are discussed. As shown in the mouse challenge potency test, glycoprotein complexes prepared after Triton X-100 solubilization actively protected mice almost as well as did complete inactivated virus at the same protein concentration, whereas those prepared after cetyltrimethylammonium bromide solubilization had a somewhat lower protective activity per microgram of protein. Images

Heinz, F X; Tuma, W; Kunz, C

1981-01-01

133

Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity  

PubMed Central

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

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

2014-01-01

134

Mechanisms of protective immunity in experimental cutaneous leishmaniasis of the guinea-pig. III. Inhibition of leishmanial lesion in the guinea-pig by delayed hypersensitivity reaction to unrelated antigens.  

PubMed Central

The purpose of this investigation was to determine whether prior induction of a non-specific delayed reaction at a site of leishmanial infection could modify the course of infection. Groups of animals were made hypersensitive to either DNCB or BCG and a delayed reaction was elicited by corresponding antigen in one or both ears when an infective dose of L. enriettii was inoculated. With various experimental designs the following results were obtained: (a) induction of delayed reaction by DNCB or BCG inhibited the development of leishmanial lesions; (a) the protection was effective only when delayed reaction occurred at the site of infection; (c) to be effective, the reaction had to be continuously present at the site of infection for at least 3--4 weeks; (d) lesions developed normally, in the absence of delayed reaction, in DNCB-tolerant animals treated with DNCB; (E) a protective delayed reaction did not completely eliminate the parasites from the host tissues, since metastatic lesions appeared later at ectopic areas; (f) the suppressed development of a lesion did not confer resistance to reinfection dose of the parasite. It is concluded that cell-mediated immunity plays an important role in healing leishmanial lesions in the guinea-pig and that the final effector mechanism may be sought in the non-specific microbicidal capacity of activated macrophages. The relevance of leishmania-specific delayed reaction in the course of the disease is discussed.

Behin, R; Mauel, J; Rowe, D S

1977-01-01

135

Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins.  

PubMed

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLa-CEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens. PMID:9151893

Chen, T; Grunert, F; Medina-Marino, A; Gotschlich, E C

1997-05-01

136

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

2005-01-01

137

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.

2012-01-01

138

Molecular Basis for the High Degree of Antigenic Cross-Reactivity between Hepatitis B Virus Capsids (HBcAg) and Dimeric Capsid-Related Protein (HBeAg): Insights into the Enigmatic Nature of the e-Antigen  

Microsoft Academic Search

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini.

Norman R. Watts; Joe G. Vethanayagam; R. Bridget Ferns; Richard S. Tedder; Audray Harris; Stephen J. Stahl; Alasdair C. Steven; Paul T. Wingfield

2010-01-01

139

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen.  

PubMed

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group. PMID:3125665

Nakazawa, M; Isayama, Y; Kashiwazaki, M; Yasui, T

1987-10-01

140

Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein  

PubMed Central

SUMMARY Chronic Hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a non-particulate variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. Here, we report the crystal structure of HBeAg, which clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerization relative to HBcAg (~140° rotation), locked into place through f ormation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.

DiMattia, Michael A.; Watts, Norman R.; Stahl, Stephen J.; Grimes, Jonathan M.; Steven, Alasdair C.; Stuart, David I.; Wingfield, Paul T.

2012-01-01

141

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

PubMed

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

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

2014-07-16

142

Protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity?  

PubMed Central

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

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

2014-01-01

143

A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors  

PubMed Central

Background Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. Methods A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium. Results ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining. Conclusions This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.

2013-01-01

144

Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity.  

PubMed

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100 degrees C) WPC (2% protein solution) were incubated at 50 degrees C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The alpha-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and alpha-lactalbumin. The beta-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The beta-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of beta-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey. PMID:17699020

Kim, S B; Ki, K S; Khan, M A; Lee, W S; Lee, H J; Ahn, B S; Kim, H S

2007-09-01

145

Mycoplasma hyorhinis GDL surface protein antigen p120 defined by monoclonal antibody.  

PubMed Central

Four antigens of Mycoplasma hyorhinis GDL were defined by murine monoclonal antibodies. Components of broth-grown mycoplasmas were separated under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent protein blots were stained with individual antibodies. Each antibody reacted with a distinct component with relative molecular weights of 120,000, 73,000, 51,000, and 38,000, respectively (termed p120, p73, p51, and p38). Trypsin treatment of protein blots specifically abrogated binding of antibodies, suggesting that the epitopes recognized were associated with proteins. By using indirect immunofluorescence and immunoferritin techniques, mycoplasmas colonizing the surface of chronically infected BW5147 murine T-lymphoblastoid cells were selectively stained with antibody to p120, indicating the localization of the corresponding epitope at the mycoplasma surface. Protein blots of mycoplasmas derived from BW5147 cell cultures were stained with antibody to p120, revealing a component identical to that observed with broth-grown organisms. These results establish the identity of a surface protein antigen of M. hyorhinis GDL expressed at the surface of organisms during their colonization of host cells. Images

Wise, K S; Watson, R K

1983-01-01

146

A 168-kilodalton protein of Mycoplasma pneumoniae used as antigen in a dot enzyme-linked immunosorbent assay  

Microsoft Academic Search

The attachment protein ofMycoplasma pneumoniae(molecular weight 168 kd) was used as antigen in a special enzyme-linked immunosorbent assay (dot ELISA) and compared with a sonicate of the whole organism. In control sera the intensity of the 168-kd band on immunoblots correlated well with the ELISA IgG values derived from isolated protein. The diagnostic significance of the 168-kd antigen was tested

E. Jacobs; K. Fuchte; W. Bredt

1986-01-01

147

A Single Amino Acid Substitution Changes Antigenicity of ORF2-Encoded Proteins of Hepatitis E Virus  

PubMed Central

Extensive genomic diversity has been observed among hepatitis E virus (HEV) strains. However, the implication of the genetic heterogeneity on HEV antigenic properties is uncertain. In this study, monoclonal antibodies (Mabs) against truncated ORF2-encoded proteins (aa452–617, designated p166 proteins) derived from HEV strains of Burma (genotype 1a, p166Bur), Pakistan (1b, p166Pak) and Morocco (1c, p166Mor) were raised and used for identification of HEV antigenic diversity. Six Mabs reacted to these 3 p166 proteins as well as p166 proteins constructed from strains derived from Mexico (genotype 2), US (genotype 3) and China (genotype 4), indicating the existence of pan-genotypic epitopes. Two Mabs, 1B5 and 6C7, reacted with p166Bur and p166Mor, but not p166Pak or p166s derived from genotypes 2, 3, and 4, indicating that these 2 Mabs recognized strain-specific HEV epitopes. Both the common and specific epitopes could not be mapped by 23 synthetic peptides spanning the p166Bur sequence, suggesting that they are confirmation-dependent. Comparative sequence analysis showed that p166Bur and p166Mor shared an identical aa sequence along their entire lengths, whereas for p166Pak the aas occupying positions 606 and 614 are different from aas at corresponding positions of p166Bur and p166Mor. Reactivity between 1B5 and p166Bur was abrogated with mutation of p166Bur/A606V, whereas p166Pak acquired the reactivity to 1B5 with mutation of p166Pak/V606A. However, mutations of p166Bur/L614M and P166Pak/M614L did not affect the immunoreactivity. Therefore, the aa occupying position 606 plays a critical role in maintaining the antigenicity of the HEV p166 proteins.

Liang, Jiu-Hong; Dai, Xing; Dong, Chen; Meng, Ji-Hong

2010-01-01

148

Linkage of Bacterial Protein Synthesis and Presentation of MHC Class I-Restricted Listeria monocytogenes-Derived Antigenic Peptides  

PubMed Central

The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217–225 and p60 449–457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.

Grauling-Halama, Silke; Schenk, Simone; Bubert, Andreas; Geginat, Gernot

2012-01-01

149

Prostate-specific antigen and gross cystic disease fluid protein-15 are co-expressed in androgen receptor-positive breast tumours.  

PubMed Central

Androgens regulate breast cancer cell proliferation via androgen receptor (AR)-mediated mechanisms. To investigate further the androgen-responsiveness of human breast tumours, we examined the immunohistochemical expression of the AR and two androgen-regulated proteins, prostate-specific antigen (PSA) and gross cystic disease fluid protein-15 (GCDFP-15), in 72 primary breast tumours. AR immunoreactivity was present in the nuclei of breast tumour cells and was correlated with oestrogen receptor (ER; P < 0.05) and progesterone receptor (PR; P < 0.01) status. PSA and GCDFP-15 immunoreactivity was present in the cytoplasm of tumour cells but not the adjacent stromal cells. AR-positive cells were present in 85% (61/72) of breast tumours, and 98% (43/44) of PSA-positive and 92% (44/48) of GCDFP-15-positive tumours were also positive for AR. Positive immunoreactivity for both PSA and GCDFP-15 in breast tumours was highly dependent on AR status (odds ratios of 24.0 and 4.5 respectively), but unrelated to age, ER and PR status and axillary lymph node involvement. PSA immunoreactivity was more frequently observed in moderate and well-differentiated tumours and was significantly (P < 0.001) associated with GCDFP-15 immunoreactivity. In conclusion, PSA and GCDFP-15 immunoreactivity was dependent on the presence of AR, but not ER or PR in primary breast tumours. Images Figure 1

Hall, R. E.; Clements, J. A.; Birrell, S. N.; Tilley, W. D.

1998-01-01

150

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

PubMed

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

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

2004-12-01

151

Engineering the chloroplast genome for hyperexpression of human therapeutic proteins and vaccine antigens.  

PubMed

The chloroplast genome is ideal for engineering because it offers a number of attractive advantages, including high-level gene expression, the feasibility of expressing multiple genes or pathways in a single transformation event, and transgene containment due to lack of pollen transmission. The chloroplast-based expression system is suitable for hyperexpression of foreign proteins, oral delivery of vaccine antigens and therapeutic proteins, via both leaves and fruits. Through the refinement of expression vectors and use of chaperones, chloroplasts produce up to 47% of foreign protein in the total cellular protein in transgenic tissues. This chapter describes various techniques for creating chloroplast transgenic plants and their biochemical and molecular characterization. Suitable examples for application of chloroplast genetic engineering in human medicine are provided. PMID:15269437

Kumar, Shashi; Daniell, Henry

2004-01-01

152

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

PubMed Central

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

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

2014-01-01

153

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

PubMed

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

2012-04-15

154

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  

PubMed Central

The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein Erns and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21ErnsE2, 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) were detected as positive (100%). In contrast, an ELISA based on an Erns fragment (Ernsaa 109-160) or an E2 fragment (E2aa 1-221) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21ErnsE2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi.

Lin, Min; Trottier, Erin; Mallory, Maria

2005-01-01

155

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.  

PubMed

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ?50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ?50 kDa channel protein is a good specific target for typhoid detection test. PMID:21371926

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

156

Changes in lamina propria dendritic cells on the oral administration of exogenous protein antigens during weaning.  

PubMed

Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood. Considering that tolerogenic antigen-presenting cells (APCs) are likely to be a key factor in the acquisition of oral tolerance, in the present study, we examined the changes of dendritic cells (DCs) in the lamina propria (LP) on exposure to food proteins at weaning. C57BL/6 female mice were weaned at the age of 3 weeks and orally administered 10 mg of ovalbumin (OVA) for ten consecutive days after weaning. The administration led to a decrease in the plasma level of immunoglobulin specific for OVA, suggesting the acquisition of oral tolerance. The uptake of fluorescence-labeled OVA was significantly observed for CD11c(+)LPDCs. When we analyzed the changes of two types of LPDCs, PDCA-1(+) MHC II(+) DCs and CD103(+) MHC II(+) DCs, ten consecutive gavages of OVA marginally, but not significantly, augmented only the frequency of PDCA-1(+) MHC II(+) DCs. Considering that the change of APCs likely appears immediately on the response to antigen intake, we found the statistically significant increase in the frequency of PDCA-1(+) DCs, but not in that of CD103(+) DCs, even after two treatments, indicating PDCA-1(+) DCs to be recruited in the LP within 2 days of exposure to food proteins. These results suggest that the behavior of tolerogenic PDCA-1(+) DCs may change at weaning with the removal of the immunoprotective components of maternal milk. PMID:21509613

Ohue, Ryuji; Nakamoto, Masahiro; Kitabatake, Naofumi; Tani, Fumito

2012-05-01

157

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.  

PubMed

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ?90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. J. Med. Virol. 86:1169-1175, 2014. © 2013 Wiley Periodicals, Inc. PMID:24105844

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

158

Cell surface proteins of Helicobacter pylori as antigens in an ELISA and a comparison with three commercial ELISA.  

PubMed

Cell surface proteins of Helicobacter pylori were solubilized by extraction with acidic glycine buffer, N-octyl-glucoside, lithium chloride, and distilled water, and by sonication. The preparations were evaluated as antigens in ELISA to detect serum IgG responses in patients and healthy subjects. SDS-PAGE analyses of the preparations from a type strain (NCTC 11637) and of acidic glycine extracts of 4 clinical isolates showed multiple protein bands. The sera were classified as HP+ve and HP-ve by culture of biopsy and immunoblotting. Sera were considered positive for H. pylori if they detected the specific 120kD antigen or 4-5 other bands. 49 sera were HP+ve; the 51 HP-ve sera did not react in immunoblotting. 35/44 sera (80%) that reacted with the 120kD antigen demonstrated high titers in ELISA with all antigen preparations, and the remaining 9(20%) sera gave discordant results. 4/5 HP+ve sera that did not react with the 120kD antigen, demonstrated high ELISA titers with all 5 antigen preparations. Glycine extracts of 3 isolates did not exhibit the 120kD protein, but were equally sensitive in ELISA. The role of 120kD antigen in our ELISA was not clear. Immunoblotting demonstrated that the 5 antigen preparations share similar antigenic components. All preparations were similarly high in sensitivity and specificity, indicating that surface antigens could be satisfactorily used in our ELISA. Our ELISA using the glycine extract was compared with commercial H. pylori ELISAs developed by Bio-Rad Laboratories, USA (GAP ELISA), Roche, Switzerland (EIA 2G), and Whittaker Bioproducts, USA (Pyloristat).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1411312

Lelwala-Guruge, J; Nilsson, I; Ljungh, A; Wadström, T

1992-01-01

159

Cross-presentation of a human tumor antigen delivered to dendritic cells by HSV VP22-mediated protein translocation.  

PubMed

Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and cross-present them to T cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen-presenting cells. Adenoviral vectors were used to express the tumor-associated-but-self-antigen MART-1 fused to HSV VP22 in MART-1-negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22-MART-1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22-MART-1 fusion by intercellular trafficking efficiently presented the MART-1(27-35) epitope to MART-1(27-35)-specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART-1(27-35)-specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be cross-presented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross-presentation but also for vaccine development. PMID:15368298

Chhabra, Arvind; Mehrotra, Shikhar; Chakraborty, Nitya G; Mukherji, Bijay; Dorsky, David I

2004-10-01

160

Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.  

PubMed Central

The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever. Images

Stover, C K; Marana, D P; Dasch, G A; Oaks, E V

1990-01-01

161

Expression of Recombinant Antigenic Proteins from Angiostrongylus cantonensis: A Brief Report  

PubMed Central

Cerebral angiostrongyliasis is an acute inflammation caused by the infection of the nematode Angiostrongylus cantonensis that results in eosinophilic meningitis. The current immunological assay of choice is an immunoblot that detects antibodies to a 31 kDa protein present in crude extracts of the female worm. Recently we have identified diagnostic targets from excretion and secretion products and determined the composition of the 31 kDa antigen after 2-D gel electrophoresis and mass spectrometry. Here we cloned and expressed five proteins in prokaryotic and eukaryotic systems. Recombinant proteins were purified and analysed by Western blot assays and among them 14-3-3, Lec5 and ES7 were recognized by Angiostrongylus-specific serum, although the signal was weak.

Perelygin, Andrey; Levert, Keith; Lin, Seh-Ching; Lee, Yeuk-Mui; da Silva, Alexandre J; Wilkins, Patricia P; Graeff-Teixeira, Carlos

2013-01-01

162

Salmonella enterica serotype Typhimurium fimbrial proteins serve as antigens during infection of mice.  

PubMed

The Salmonella enterica serotype Typhimurium genome contains 13 operons with homology to fimbrial gene sequences. Here we investigated the role of 11 serotype Typhimurium fimbrial proteins, including FimA, AgfA (CsgA), BcfA, StbA, SthA, LpfA, PefA, StdA, StcA, StiA, and StfA, as antigens during the infection of genetically resistant mice (CBA). Upon the growth of serotype Typhimurium in standard laboratory broth culture, only the expression of FimA could be detected by Western blot analysis. The infection of mice with serotype Typhimurium grown in broth culture, followed by at least one subsequent infection, resulted in seroconversion of animals to FimA, AgfA, BcfA, StbA, SthA, LpfA, PefA, StdA, StcA, StiA, and StfA positivity. Most animals seroconverted to only a subset of these fimbrial antigens. The immunization of mice with glutathione S-transferase (GST)-FimA, GST-AgfA, GST-BcfA, GST-StbA, GST-SthA, GST-LpfA, GST-PefA, GST-StdA, GST-StcA, GST-StiA, and GST-StfA fusion proteins resulted in reduced fecal shedding of serotype Typhimurium during a challenge compared to that by a control group immunized with purified GST protein. Collectively, these data suggest that the expression of serotype Typhimurium fimbrial antigens is induced during the infection of mice. PMID:16113248

Humphries, Andrea; Deridder, Sandra; Bäumler, Andreas J

2005-09-01

163

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor.  

PubMed

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin. PMID:19635491

Ma, Dongying; Gao, Li; An, Shu; Song, Yuzhu; Wu, Jing; Xu, Xueqing; Lai, Ren

2010-01-01

164

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

NASA Astrophysics Data System (ADS)

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

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

2010-08-01

165

Plasmodium falciparum Antigen 332 Is a Resident Peripheral Membrane Protein of Maurer's Clefts  

PubMed Central

During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle.

Nilsson, Sandra; Angeletti, Davide; Wahlgren, Mats; Chen, Qijun; Moll, Kirsten

2012-01-01

166

Characterization of an antigenically conserved heat-modifiable major outer membrane protein of Branhamella catarrhalis.  

PubMed Central

Branhamella catarrhalis is a common cause of otitis media in children and of respiratory infections in adults with chronic bronchitis. Little is known about the antigenic structure of the outer membrane proteins (OMPs). In this study, two murine monoclonal antibodies, 7D6 and 5E8, were developed and used to characterize the major heat-modifiable OMP (OMP C/D) of B. catarrhalis. Immunoblot assays indicated that OMP C/D is heat modifiable, having a molecular mass of 55 kDa at room temperature and a mass of 60 kDa when heated under reducing conditions. Expression of the epitopes is independent of growth phase and growth media. Both epitopes are present in 51 of 51 strains of B. catarrhalis tested and are highly specific for Branhamella strains, being absent from a variety of other gram-negative species. Antibody 5E8 recognizes an epitope which is expressed on the surface of the intact bacterium. We conclude that OMP C/D is a major, heat-modifiable OMP antigen that expresses at least one stable, conserved epitope on the surface of B. catarrhalis. Future studies should focus on the role of OMP C/D in pathogenesis and on its potential role as a vaccine antigen. Images

Sarwar, J; Campagnari, A A; Kirkham, C; Murphy, T F

1992-01-01

167

Association of HLA-DQ7 antigen with cow milk protein allergy in Italian children.  

PubMed

In this study we investigated the HLA association with cow milk allergy. Thirty-seven Italian children with cow milk allergy and 35 randomly selected age-matched healthy children as control group were included in the study. DNA typing was performed by restriction fragment length polymorphism (RFLP) technique. We show the first statistically significant positive association between the expression of the HLA-DQ7 antigen and cow milk allergy. Several immunological tests (skin prick test, RIA, radioallergosorbent test (RAST) and ELISA) were performed to evaluate the humoral immune responses of DQ7 positive and DQ7 negative allergic patients. Our results show that among the DQ7 positive patients the majority presented a high humoral response. Furthermore, the in vitro proliferative response of patients to the beta-lactoglobulin antigen was performed to evaluate their cell-mediated immune response. We observed that the number of the nonresponders was higher in the DQ7 positive patients when compared to the DQ7 negative patients. Our data indicate an association of HLA-DQ7 antigen with cow milk protein allergy and that the DQ7 positive patients had a prevalence of humoral rather than cellular responses. PMID:9617782

Camponeschi, B; Lucarelli, S; Frediani, T; Barbato, M; Quintieri, F

1997-05-01

168

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.

2013-01-01

169

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

PubMed Central

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

Corso, J.; Lugert, R.; Gross, U.

2011-01-01

170

Identification of antigenic proteins of Toxoplasma gondii RH strain recognized by human immunoglobulin G using immunoproteomics.  

PubMed

Toxoplasma gondii, a ubiquitous intracellular protozoan, infects one third of the world human population. It is of great medical significance, especially for pregnant women and immune-compromised patients. Accurate and early detection of T. gondii infection is crucial in the management of this disease. To obtain potential diagnostic markers, immunoproteomics was employed to identify immunodominant proteins separated by 2-D immunobloting and probed with sera collected from Toxoplasma-positive pregnant women. MALDI-TOF MS and MS/MS analyses identified a total of 18 immunoreactive proteins that were recognized by Toxoplasma-positive sera, whereas none was reactive with the negative-control sera from healthy, Toxoplasma-negative volunteers. Pregnant women showed a diverse immunoreactivity pattern with each serum recognizing one to eight identified tachyzoite proteins. The identified proteins were localized in the membrane, cytoplasm and specific organelles of T. gondii, and are involved in host cell invasion, metabolism and cell structure. Among these 18 proteins, actin, catalase, GAPDH, and three hypothetical proteins had a broad reactivity with Toxoplasma-positive sera, indicating their potential as diagnostic markers for toxoplasmosis. Each of several combinations of the identified proteins offered 100% detection of Toxoplasma infections of all 28 Toxoplasma-positive women. The study findings suggest that Toxoplasma tachyzoites are highly immunogenic and highlights the heterogeneity of host responses to Toxoplasma infection and the importance of using combinations of immunogens as diagnostic antigens. The findings have significant implications to the development of diagnostic reagents with high sensitivity and specificity. PMID:23026549

Sun, Xi-Meng; Ji, Yong-Sheng; Elashram, Saeed A; Lu, Zhi-Min; Liu, Xian-Yong; Suo, Xun; Chen, Qi-Jun; Wang, Heng

2012-12-21

171

Functional analysis of the highly antigenic outer capsid protein, Hoc, a virus decoration protein from T4-like bacteriophages.  

PubMed

Bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein, Hoc. The Hoc molecule (40 kDa) is present at the centre of each hexameric capsomer and provides a good platform for surface display of pathogen antigens. Biochemical and modelling 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 beta-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. PMID:20497329

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

2010-07-01

172

Novel proteins associated with MHC class I antigens in cells expressing the adenovirus protein E3/19K.  

PubMed Central

Assembly of histocompatibility class I antigens (MHC) with beta 2-microglobulin (beta 2m) and peptide takes place in the rough endoplasmic reticulum (ER). At present, it is unclear why peptides generated in the cytosol or ER by proteolysis are not further degraded. Also, it is an open question whether assembly and/or peptide binding is self-instructive or is promoted by additional molecules, for example, chaperones. We previously demonstrated that the adenovirus glycoprotein E3/19K binds to human and some mouse MHC molecules in the ER, interfering with their transport to the cell surface. Here we show that immunoprecipitates from human cells that express transfected E3/19K and murine MHC Kd molecules not only contain MHC heavy chain, beta 2m and E3/19K but also two additional proteins with apparent molecular weights of 100 kDa and 110 kDa. Biochemical characterization of these proteins, designated p100 and p110, demonstrates that they are transmembrane glycoproteins with a similar if not identical protein backbone. Consistent with a role as chaperones, we find that glucose starvation induces complex formation between p100/110 and MHC-E3/19K. Most interestingly, p100 and p110 are displaced from the complex by addition of Kd-specific peptides. Therefore, p100 and p110 might be chaperones that promote correct folding of MHC antigens and/or peptide binding to MHC. Images

Feuerbach, D; Burgert, H G

1993-01-01

173

Specific interaction between the p53 cellular tumour antigen and major heat shock proteins  

Microsoft Academic Search

The protein p53 is capable of participating in neoplastic transformation1-3 and can form specific complexes with the large-T antigen of simian virus 40 (SV40)4-6. This interaction probably results in the stabilization of p53 (refs 7, 8) and may contribute to SV40-mediated transformation9,10. Several non-SV40-trans-formed cells also exhibit a stabilized p53 which is present in elevated levels11-13. Recently, this stabilization was

Orit Pinhasi-Kimhi; Dan Michalovitz; Avri Ben-Zeev; Moshe Oren

1986-01-01

174

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

PubMed Central

Objectives We evaluated and compared tumor antigen precursor protein (TAPP) profiles in adult and pediatric brain tumors of 31 genes related to tumor associated antigens (TAA) for possible use in immunotherapy. Antigens were selected based on their potential to stimulate T cell responses against tumors of neuroectodermal origin. Methods Thirty-seven brain tumor specimens from 11 adult and 26 pediatric patients were analyzed by quantitative real-time PCR for the relative expression of 31 TAPP mRNAs. The age range of adults (4F:7M) was 27–77 years (median 51.5 ± 14.5 years) and for pediatrics (12F:14M) was 0.9–19 years (median 8.3 ± 5.5 years). Histological diagnoses consisted of 16 glioblastomas, 4 low grade astrocytomas, 10 juvenile pilocytic astrocytomas, and 7 ependymomas. Results The adult gliomas expressed 94% (29 of 31) of the TAPP mRNAs evaluated compared with pediatric brain tumors that expressed 55–74% of the TAPP mRNAs, dependent on tumor histological subtype. Four types of TAPP expression patterns were observed: (1) equal expression among adult and pediatric cases, (2) greater expression in adult than pediatric cases, (3) expression restricted to adult GBM and (4) a random distribution. The pediatric brain tumors lacked expression of some genes associated with engendering tumor survival, such as hTert and Survivin. Conclusions The potential TAA targets identified from the TAPP profiles of 31 genes associated with adult and pediatric brain tumors may help investigators select specific target antigens for developing dendritic cell- or peptide-based vaccines or T cell-based immunotherapeutic approaches against brain tumors.

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

2014-01-01

175

Large, identical, tandem repeating units in the C protein alpha antigen gene, bca, of group B streptococci.  

PubMed Central

Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C protein alpha antigen of GBS is thought to have a role in both virulence and immunity. We previously cloned the C protein alpha antigen structural gene (named bca for group B, C protein, alpha) into Escherichia coli. Western blots of both the native alpha antigen and the cloned gene product demonstrate a regularly laddered pattern of heterogeneous polypeptides. The nucleotide sequence of the bca locus reveals an open reading frame of 3060 nucleotides encoding a precursor protein of 108,705 Da. Cleavage of a putative signal sequence of 41 amino acids yields a mature protein of 104,106 Da. The 20,417-Da N-terminal region of the alpha antigen shows no homology to previously described protein sequences and is followed by a series of nine tandem repeating units that make up 74% of the mature protein. Each repeating unit is identical and consists of 82 amino acids with a molecular mass of 8665 Da, which is encoded by 246 nucleotides. The size of the repeating units corresponds to the observed size differences in the heterogeneous ladder of alpha C proteins expressed by GBS. The C-terminal region of the alpha antigen contains a membrane anchor domain motif that is shared by a number of Gram-positive surface proteins. The large region of identical repeating units in bca defines protective epitopes and may play a role in generating phenotypic and genotypic diversity of the alpha antigen. Images

Michel, J L; Madoff, L C; Olson, K; Kling, D E; Kasper, D L; Ausubel, F M

1992-01-01

176

Neurofibromatosis Type 2 Tumor Suppressor Protein, NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma  

PubMed Central

Neurofibromatosis type 2 protein (NF2) has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV) tumor antigen (T-antigen) as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.

Beltrami, Sarah; Branchetti, Emanuela; Sariyer, Ilker K.; Otte, Jessica; Weaver, Michael; Gordon, Jennifer

2013-01-01

177

Stage-specific proteins and glycoproteins of plasmodium falciparum: identification of antigens unique to schizonts and merozoites.  

PubMed Central

Establishment of highly synchronized cultures of Plasmodium falciparum enabled identification of stage-specific proteins, glycoproteins, and antigens. Comparison of metabolically labeled constituents of rings, trophozoites, mature schizonts, and merozoites indicated the absence of major proteins or glycorproteins unique to rings or trophozoites. A burst of new synthetic activity occurred during schizogony when several schizont- and merozoite-specific proteins and glycoproteins became apparent. In addition to the knob protein, which was previously shown to be associated with protrusions on the host erythrocyte membrane, a major glycoprotein of parasite origin was identified on the surface membrane of schizonts. Analysis of antigens solubilized from different developmental stages indicated that immune sera, which inhibit growth of parasites in vitro, react mainly with merozoite- and schizont-associated antigens. Images

Kilejian, A

1980-01-01

178

The Two Major Human Metapneumovirus Genetic Lineages Are Highly Related Antigenically, and the Fusion (F) Protein Is a Major Contributor to This Antigenic Relatedness  

Microsoft Academic Search

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (>6.0

Mario H. Skiadopoulos; Stephane Biacchesi; Ursula J. Buchholz; Jeffrey M. Riggs; Sonja R. Surman; Emerito Amaro-Carambot; Josephine M. McAuliffe; William R. Elkins; M. St. Claire; P. L. Collins; B. R. Murphy

2004-01-01

179

Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.  

PubMed Central

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.

Washitani, Y; Kuroda, N; Shiraki, H; Itoyama, Y; Sato, H; Ohshima, K; Kiyokawa, H; Maeda, Y

1992-01-01

180

Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein  

Microsoft Academic Search

Three isolates of feline calicivirus (FCV) designated NADC, KCD and CFI\\/68 were compared for bio- chemical, serological and genetic variation within the capsid protein gene. The M r of the capsid protein from purified virions was approximately 66 000 for the NADC virus isolate, which differed slightly from the relative mobilities of the purified capsid proteins of the KCD and

Bruce S. Seal; Jufia F. Ridpath; William L. Mengeling

1993-01-01

181

Papaya ringspot virus coat protein gene for antigen presentation in Escherichia coli.  

PubMed

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus. PMID:16466633

Chatchen, Supawat; Juricek, Mila; Rueda, Paloma; Kertbundit, Sunee

2006-01-31

182

Immunological Research on Basic Specific and Non-Specific Protein Antigens of the Type Streptococcus Pyogenes (Group a).  

National Technical Information Service (NTIS)

Immunoelectrophoresis and electrophoresis have confirmed that acid-alcohol gamma extracts contained several type specific and non-specific protein antigens which were basic proteins. Their isoelectric point is above 8.5, while that of the non-type specifi...

R. Wahl J. Goichot G. Drach

1966-01-01

183

Delivery of Protein Antigens to the Immune System by Fusion-Active Virosomes: A Comparison with Liposomes and ISCOMs  

Microsoft Academic Search

The induction of effective cellular and humoral immune responses against protein antigens is of major importance in vaccination strategies against infectious diseases and cancer. Immunization with protein alone in general does not result in efficient induction of cytotoxic T lymphocyte (CTL) and antibody responses. Numerous other immunization strategies have been explored. In this review we will discuss a number of

Laura Bungener; Anke Huckriede; Jan Wilschut; Toos Daemen

2002-01-01

184

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

1986-01-01

185

varDB: a pathogen-specific sequence database of protein families involved in antigenic variation  

PubMed Central

Summary: Infectious diseases are a major threat to global public health and prosperity. The causative agents consist of a suite of pathogens, ranging from bacteria to viruses, including fungi, helminthes and protozoa. Although these organisms are extremely varied in their biological structure and interactions with the host, they share similar methods of evading the host immune system. Antigenic variation and drift are mechanisms by which pathogens change their exposed epitopes while maintaining protein function. Accordingly, these traits enable pathogens to establish chronic infections in the host. The varDB database was developed to serve as a central repository of protein and nucleotide sequences as well as associated features (e.g. field isolate data, clinical parameters, etc.) involved in antigenic variation. The data currently contained in varDB were mined from GenBank as well as multiple specialized data repositories (e.g. PlasmoDB, GiardiaDB). Family members and ortholog groups were identified using a hierarchical search strategy, including literature/author-based searches and HMM profiles. Included in the current release are>29 000 sequences from 39 gene families from 25 different pathogens. This resource will enable researchers to compare antigenic variation within and across taxa with the goal of identifying common mechanisms of pathogenicity to assist in the fight against a range of devastating diseases. Availability: varDB is freely accessible at http://www.vardb.org/ Contact: nelson@kuicr.kyoto-u.ac.jp; goto@kuicr.kyoto-u.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online.

Hayes, C. Nelson; Diez, Diego; Joannin, Nicolas; Honda, Wataru; Kanehisa, Minoru; Wahlgren, Mats; Wheelock, Craig E.; Goto, Susumu

2008-01-01

186

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.

1986-08-01

187

Epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in complex biological systems.  

PubMed

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system. PMID:9879358

Yu, L; Gaskell, S J; Brookman, J L

1998-03-01

188

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

1986-01-01

189

Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen  

PubMed Central

Variable lymphocyte receptors (VLRs) are leucine-rich repeat (LRR) proteins that mediate adaptive immunity in jawless vertebrates. VLRs are fundamentally different from the antibodies of jawed vertebrates, which consist of immunoglobulin (Ig) domains. We determined the structure of an anti-hen egg white lysozyme (HEL) VLRB, isolated by yeast display, bound to HEL. The VLR, whose affinity resembles that of IgM antibodies, uses nearly all its concave surface to bind the protein, in addition to a loop that penetrates into the enzyme active site. The VLR-HEL structure, combined with sequence analysis, revealed an almost perfect match between ligand-contacting positions and positions with highest sequence diversity. Thus, we have defined the generalized antigen-binding site of VLRs. We further demonstrated that VLRs can be affinity-matured to affinities as high as those of IgG antibodies, making VLRs potential alternatives to antibodies for biotechnology applications.

Velikovsky, C Alejandro; Deng, Lu; Tasumi, Satoshi; Iyer, Lakshminarayan M; Kerzic, Melissa C; Aravind, L; Pancer, Zeev; Mariuzza, Roy A

2009-01-01

190

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.

2013-01-01

191

A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen.  

PubMed Central

Cell surface-exposed antigenic determinants of several high-molecular-weight outer membrane proteins of Haemophilus influenzae type b (Hib) have been shown to be consistently immunogenic in human infants convalescing from Hib meningitis. A monoclonal antibody (mab), 6G12, directed against one of these cell surface-exposed outer membrane proteins that has an apparent molecular weight of 98,000 (98K) was identified by radioimmunoprecipitation analysis. Of 120 clinical isolates of Hib, 83 were found to possess antigenic determinants which reacted with mab 6G12 in a colony blot-radioimmunoassay procedure, indicating that the antigenic determinant recognized by mab 6G12 is present in the majority of Hib strains. A different radioimmunoassay, which uses whole Hib cells as antigen, confirmed that strains reactive with mab 6G12 in the colony blot-radioimmunoassay procedure possessed a cell surface-exposed and antibody-accessible antigenic determinant recognized by this mab. Hib strains which did not react with mab 6G12 were found to lack a 98K protein. Passive immunization with mab 6G12 reduced the level of bacteremia that developed in infant rats challenged with the homologous Hib strain against which this mab was raised. In contrast, no protection was observed when the challenge strain was one which lacks the antigenic determinant recognized by mab 6G12. Radioimmunoprecipitation analysis of sera from human infants convalescing from Hib meningitis detected an antibody response directed against the 98K protein. The protection against experimental Hib disease provided by antibody to the 98K protein, the immunogenicity of this protein in human infants, and its presence in a majority of Hib strains indicate that the 98K outer membrane protein may have potential for vaccine development. Images

Kimura, A; Gulig, P A; McCracken, G H; Loftus, T A; Hansen, E J

1985-01-01

192

The Two Major Human Metapneumovirus Genetic Lineages Are Highly Related Antigenically, and the Fusion (F) Protein Is a Major Contributor to This Antigenic Relatedness  

PubMed Central

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (?6.0 log10) in the upper respiratory tract of hamsters and to moderate titers (?3.6 log10) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.

Skiadopoulos, Mario H.; Biacchesi, Stephane; Buchholz, Ursula J.; Riggs, Jeffrey M.; Surman, Sonja R.; Amaro-Carambot, Emerito; McAuliffe, Josephine M.; Elkins, William R.; St. Claire, Marisa; Collins, Peter L.; Murphy, Brian R.

2004-01-01

193

The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness.  

PubMed

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (> or =6.0 log(10)) in the upper respiratory tract of hamsters and to moderate titers (> or =3.6 log(10)) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro. PMID:15194769

Skiadopoulos, Mario H; Biacchesi, Stéphane; Buchholz, Ursula J; Riggs, Jeffrey M; Surman, Sonja R; Amaro-Carambot, Emerito; McAuliffe, Josephine M; Elkins, William R; St Claire, Marisa; Collins, Peter L; Murphy, Brian R

2004-07-01

194

Characterization and antigenicity of recombinant Campylobacter jejuni flagellar capping protein FliD.  

PubMed

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry. PMID:24445509

Yeh, Hung-Yueh; Hiett, Kelli L; Line, John E; Seal, Bruce S

2014-04-01

195

B-cell epitopes of antigenic proteins in Leishmania infantum: an in silico analysis.  

PubMed

Serodiagnosis of visceral leishmaniasis is often hindered by cross-reactions to other parasitic diseases. Identifying specific B-cell epitopes in proteins is therefore important for immunodiagnostics, as well as for disease control by vaccines. This study aimed to identify linear and conformational B-cell epitopes and to evaluate the secondary structure of antigen proteins in Leishmania infantum using in silico analysis. Linear epitopes were predicted using the Immune Epitope Database and Analysis Resource (IEDB), BepiPred and BcePred programs. The conformational B-cell epitopes were identified using the CBTOPE server. The combination of the predictions using IEDB, BepiPred and BcePred generated 148 linear epitopes from the calpain-like cysteine peptidase (CP), thiol-dependent reductase 1 (TDR1) and HSP70 proteins. In total, 164 conformational epitopes were predicted, mostly located in the linear epitope region. The predicted epitopes are located in ? helix and random coil regions in the thiol-dependent reductase 1 and HSP70 proteins. New linear and conformational B-cell epitopes of L. infantum proteins were identified in silico, and the prediction using various programs ensures greater accuracy of the results, as suggested by confirmation of previously identified HSP70 epitopes. PMID:24606067

Assis, L M; Sousa, J R; Pinto, N F S; Silva, A A; Vaz, A F M; Andrade, P P; Carvalho, E M; De Melo, M A

2014-07-01

196

A viral vaccine encoding PSA induces antigen spreading to a common set of self proteins in prostate cancer patients  

PubMed Central

Purpose We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding PSA with radiotherapy in patients with localized prostate cancer. Here we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome. Experimental Design Pre- and post-treatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serological screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses. Results Western blotting revealed treatment-associated autoantibody responses in 15/33 (45.5%) patients treated with vaccine + EBRT versus 1/8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that seven of 33 patients (21.2%) treated with vaccine + EBRT demonstrated a vaccine-associated autoantibody response to four ubiquitously expressed self antigens: DIRC2, NDUFS1, MRFAP1 and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend towards decreased biochemical-free survival. Conclusions Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self proteins, which may influence the efficacy of vaccination.

Nesslinger, Nancy J.; Ng, Alvin; Tsang, Kwong-Yok; Ferrara, Theresa; Schlom, Jeff; Gulley, James L.; Nelson, Brad H.

2010-01-01

197

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

2010-01-01

198

Blood Antigen, Serum Protein, and Milk Protein Gene Frequencies and Genetic Interrelationships in Holstein Cattle1  

Microsoft Academic Search

Gene frequencies at ten blood group loci, one serum protein locus, and four milk protein loci were determined for the Holstein-Friesian breed in the United States. The sample consisted of 8630 cows in 51 herds from 10 states. Because of the close linkage among casein subloci and the concomitant rarity of crossover- recombinant groups, casein gene com- bination or haplotype

H. C. Hines; G. F. W. Haenlein; J. P. Zikakis; H. C. Dickey

1977-01-01

199

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.  

PubMed

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza. PMID:15019738

Mena-Rojas, Erika; Vázquez Cruz, Candelario; Vaca Pacheco, Sergio; García González, Octavio; Pérez-Márquez, Víctor M; Pérez-Méndez, Alma; Ibarra-Caballero, Jorge; de la Garza, Mireya; Zenteno, Edgar; Negrete-Abascal, Erasmo

2004-03-12

200

Identification and Characterization of Epitopes on the 120-Kilodalton Surface Protein Antigen ofRickettsia prowazekii with Synthetic Peptides  

Microsoft Academic Search

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were syn- thesizedonderivatizedpolyethylenepins.Amodifiedenzyme-linkedimmunosorbentassaywasusedtoidentify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epi- topes were

W.-M. CHING; H. WANG; B. JAN; A. DASCH

1996-01-01

201

Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus  

Microsoft Academic Search

Summary. This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1–Sp6), one in the S2 (Sp7) and four in the N protein (Np1–Np4) were

J. Ignjatovic; S. Sapats

2005-01-01

202

Identification of outer membrane proteins as target antigens of Pseudomonas aeruginosa Homma serotype M.  

PubMed Central

Pseudomonas aeruginosa is routinely serotyped in Japan by using the Homma scheme. The serotypes (O serotypes) are based on the chemical structure of the O-polysaccharide portion of the lipopolysaccharide (LPS). However, the nature of the Homma serotype M antigen has remained obscure because strains classified as serotype M usually have the rough phenotype. I characterized the target antigen of serotype M. The results of Western blotting (immunoblotting) showed that commercially available typing monoclonal antibody (MAb) against serotype M specifically bound to outer membrane protein (Opr) G and that typing rabbit antiserum specific for serotype M mainly contained antibodies against Oprs F and H2. These Oprs were distributed among all P. aeruginosa strains tested, including the serotype standard, serotype M and nontypeable strains, and a series of LPS-core-defective mutants derived from strain PAC1. However, the rough mutants derived from strain PAC1 agglutinated with the anti-serotype M antibodies, whereas the smooth strains did not. LPS preparations from serotype M strains possessed few or no polysaccharide chains. These strains had higher levels of binding activity with anti-serotype M MAb, as well as with anti-lipid A MAb, which specifically bound to the cell surface of the rough-natured gram-negative bacterial strains with high activity. The anti-serotype M antiserum also contained rough-LPS-specific antibodies, but the epitope was distributed among only a few strains. The results suggested that the Oprs acted as the serotype M antigen and that LPS did not. In conclusion, the rough strains agglutinated with anti-Opr antibodies and were distinguished as serotype M from the smooth strains of other serotypes, because the antibodies were accessible to the cell surface lacking O polysaccharides. I supposed that Homma serotype M is an index of the rough nature of P. aeruginosa strains rather than one of the O serotypes.

Yokota, S

1995-01-01

203

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

SciTech Connect

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

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

1985-02-01

204

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

PubMed

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 identified on the ORF2 protein: one site was not accessible on the surface of infectious virions but the other site was accessible to antibodies and was recognized specifically by antibodies that neutralize the virus. PMID:14615154

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

2003-12-12

205

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

PubMed

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

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

2014-05-01

206

Lineage specific antigenic differences in porcine torovirus hemagglutinin-esterase (PToV-HE) protein  

PubMed Central

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms.

2013-01-01

207

Genetic and antigenic characterization of sigma C protein from avian reovirus.  

PubMed

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine. PMID:20544425

Goldenberg, Dana; Pasmanik-Chor, Metsada; Pirak, Michael; Kass, Nava; Lublin, Avishai; Yeheskel, Adva; Heller, Dan; Pitcovski, Jacob

2010-06-01

208

Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.  

PubMed

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection. PMID:24021883

Zhao, Qin; Sun, Ya-ni; Hu, Shou-bin; Wang, Xin-jie; Xiao, Yi-hong; Hsu, Walter H; Xiao, Shu-qi; Wang, Cheng-bao; Mu, Yang; Hiscox, Julian A; Zhou, En-Min

2013-12-27

209

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

210

Identification of a Nonstructural DNA-Binding Protein (DBP) as an Antigen with Diagnostic Potential for Human Adenovirus  

PubMed Central

Background Human adenoviruses (HAdVs) have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed. Methodology/Principal Findings In this study, a nonstructural antigenic protein, the DNA binding protein (DBP) of human adenovirus 5 and 35 (Ad5, Ad35) - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (?2?=? 44.9, P<0.01) the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive. Conclusions/Significance The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.

Zhou, Hongli; Wu, Chao; Paranhos-Baccala, Glaucia; Vernet, Guy; Jin, Qi; Wang, Jianwei; Hung, Tao

2013-01-01

211

Sperm protein 17: clinical relevance of a cancer/testis antigen, from contraception to cancer immunotherapy, and beyond.  

PubMed

Sperm protein 17 belongs to the cancer/testis antigen family and was found to play a key role in the cell fusion process between the mammalian spermatozoa and egg. Sperm protein 17-specific autoantibodies in vasectomized males suggest its high expression in the testis. Sperm protein 17 expression levels are low or absent in normal tissues, other than the testis. Sperm protein 17 is expressed by tumors, including ovarian cancer, indicating that it is an ideal target for cancer immunotherapy, and plays a role in tumorigenesis and drug resistance. This review recapitulates the milestones of sperm protein 17 research and highlights its potential use in translational medicine. PMID:21557640

Chiriva-Internati, Maurizio

2011-01-01

212

Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications.  

PubMed

Bacterial ghosts (BGs) represent vaccine delivery systems gifted with outstanding natural adjuvant properties. BGs are empty cell envelopes of Gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts. The intact surface make-up of BGs is easily recognized by professional APCs through pattern-recognition receptors, making them ideal for mucosal administration through oral, ocular, intranasal or aerogenic routes, which represent the most desirable methods of application in advanced vaccine use. BGs have been designed to be used as carriers of active substances and foreign antigens (protein and/or DNA) for vaccine development. This review highlights the salient features of the BGs' versatile multipurpose vaccine platform for application in a wide range of human and veterinary medicines. PMID:22149712

Muhammad, Abbas; Champeimont, Jonathan; Mayr, Ulrike Beate; Lubitz, Werner; Kudela, Pavol

2012-01-01

213

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

PubMed

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

2013-07-31

214

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

PubMed

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

2013-01-01

215

THE CATABOLISM OF PROTEIN ANTIGENS IN THE NEWBORN AND MATURING RABBIT  

PubMed Central

The rate of degradation, organ deposition, and blood clearance of SBSA, SRSA, and IRSA has been measured in the newborn, 6-, and 30-day-old rabbits. When the animals were injected with a weight-graded dose of the 3 proteins, differences in their catabolism in the newborns were demonstrable as compared to the 6-, and 30-day-old animals. The capacity to degrade the azo compounds was shown to be incompletely developed at birth. At 6 days of age, however, the rabbits catabolized these proteins much at the same rate as the 30-day-old animals. Addition of the benzenesulfonate moiety determined the rate of degradation organ deposition and excretion rather than the carrier protein when the azo compounds were injected. The biosynthetically labeled S25 rabbit serum albumin (IRSA) was catabolized at a slower rate than the azoproteins in all age groups. Very little difference in the metabolism and organ deposition of the IRSA was shown to exist between the newborn and maturing animals. A dosage schedule, therefore, designed to test the immunological capacity of developing animals may not be valid when calculated upon body weight. The low level of activity of enzyme systems present at birth which degrade anti-genic material may serve as an explanation as to why this period of development is so vulnerable to the induction of tolerance rather than immunity when compared to the adult.

Robbins, John; Eitzman, Donald V.; Smith, Richard T.

1963-01-01

216

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

SciTech Connect

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

Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States)] [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)] [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

2013-01-18

217

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

PubMed Central

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.

Muller, Rebekka; Bachke, Siri; Gilljam, Karin M.; Vatsveen, Thea K.; R?, Torstein B.; Bellacchio, Emanuele; Sundan, Anders; Otterlei, Marit

2013-01-01

218

Modulation of Epstein-Barr Virus Nuclear Antigen 2-Dependent Transcription by Protein Arginine Methyltransferase 5  

PubMed Central

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

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

2013-01-01

219

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

PubMed Central

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

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

2013-01-01

220

Identification of previously unknown antigenic epitopes on the S and N proteins of avian infectious bronchitis virus.  

PubMed

This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection. PMID:15868095

Ignjatovic, J; Sapats, S

2005-09-01

221

Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity  

Microsoft Academic Search

Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can

Laura Bungener; Anke Huckriede; Arjan de Mare; Jacqueline de Vries-Idema; Jan Wilschut; Toos Daemen

2005-01-01

222

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: bio-research@hotmail.co [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, No. 21, Rd 1st Tianbei, 518020 Shenzhen (China)

2009-11-01

223

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.

2000-01-01

224

Detection of polyclonal antibody against any area of the protein-antigen using immobilized protein-antigens: the critical role of the immobilization protocol.  

PubMed

Antigens immobilized on solid supports may be used to detect or purify their corresponding antibodies (Ab) from serum. Direct immobilization of antigens on support surfaces (through short spacer arms) may promote interesting stabilizing effects on the immobilized antigen. However, the proximity of the support may prevent the interaction of some fractions of polyclonal Ab with some regions of the antigen (those placed in close contact with the support surface). Horseradish peroxidase (HRP) was immobilized on agarose by different protocols of multipoint covalent immobilization involving different regions of the antigen surface. Glyoxyl-agarose, BrCN-agarose, and glutaraldehyde-agarose were used as activated supports. Each HRP-immobilized preparation was much more stable than the soluble enzyme, but it was only able to adsorb up to 60-70% of a mixture of polyclonal anti-HRP antibodies. On the other hand, HRP was also immobilized on agarose through a very long, flexible, and hydrophilic spacer arm (dextran). This immobilized HRP was hardly stabilized, but it was able to adsorb 100% of the polyclonal anti-HRP. The absence of steric hindrances seems to play a critical role favoring the complete recognition of all classes of polyclonal Ab. Another solution to achieve a complete adsorption of polyclonal Ab on immobilized-stabilized antigens has been also reached by using a mixture of the differently immobilized and stabilized HRP-agarose preparations. In this case, an improved storage and operational stabilities of the immobilized antigens can be combined with the complete adsorption of any class of antibody. PMID:16471927

Fuentes, Manuel; Mateo, Cesar; Fernández-Lafuente, Roberto; Guisán, J M

2006-02-01

225

Control of protein phosphatase 2A by simian virus 40 small-t antigen.  

PubMed Central

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity. Images

Yang, S I; Lickteig, R L; Estes, R; Rundell, K; Walter, G; Mumby, M C

1991-01-01

226

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

2006-01-01

227

Immunization against Plasmodium chabaudi malaria using combined formulations of apical membrane antigen-1 and merozoite surface protein-1  

Microsoft Academic Search

The control of Plasmodium falciparum malaria by vaccination will require immunization with multiple parasite antigens effectively formulated in combination. In this regard, proteins expressed on the surface of blood-stage merozoites are attractive as vaccine targets given their functional importance in the invasion of erythrocytes and accessibility to serum antibodies. We have utilized a Plasmodium chabaudi vaccine model to begin to

James M Burns; Patrick R Flaherty; Margarita M Romero; William P Weidanz

2003-01-01

228

Protein Array Profiling of Tic Patient Sera Reveals a Broad Range and Enhanced Immune Response against Group A Streptococcus Antigens  

PubMed Central

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.

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

2009-01-01

229

Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing  

PubMed Central

Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients.

Romao, Susana; Gasser, Nathalie; Becker, Andrea C.; Guhl, Bruno; Bajagic, Milica; Vanoaica, Danusia; Ziegler, Urs; Roesler, Joachim; Dengjel, Jorn; Reichenbach, Janine

2013-01-01

230

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

2012-01-01

231

Gene for the major antigenic structural protein (p100) of human herpesvirus 6.  

PubMed

A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions. The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera. The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa. Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus. Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed. The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera. They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus. Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6. PMID:1374813

Neipel, F; Ellinger, K; Fleckenstein, B

1992-06-01

232

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.  

PubMed Central

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus. Images

Gunn, P R; Sato, F; Powell, K F; Bellamy, A R; Napier, J R; Harding, D R; Hancock, W S; Siegman, L J; Both, G W

1985-01-01

233

Monoclonal antibodies against membrane proteins of the rat glomerulus. Immunochemical specificity and immunofluorescence distribution of the antigens.  

PubMed

Monoclonal antibodies (MAbs) were generated to detergent-solubilized glomerular extracts to identify new epithelial and endothelial membrane proteins and to study the possible role of the corresponding antigens in the formation of immune deposits. Triton X-114 extracts of isolated glomeruli were subjected to phase separation, and the resultant detergent and aqueous phases were used to immunize mice. Monoclonal antibodies were prepared by standard techniques, and hybridomas secreting antibodies (IgGs) that recognize glomerular cell surface antigens were selected by enzyme immunoassay (EIA) and indirect immunofluorescence. The IgGs of 13 MAbs selected for study recognized antigens of different molecular weights (45-350 kd) by immunoprecipitation and immunoblotting and had different distributions in the glomerulus and in other renal structures by immunofluorescence. Several proved to recognize known antigens--ie, podocalyxin (MAbs 1A, 5A, 11A, and 20A), gp330 (20B), and dipeptidylpeptidase IV (26C). Others recognized antigens not previously characterized that fell into four groups: 1) those that were detected mainly in glomeruli; 2) those present in both glomeruli and peritubular capillaries; 3) those present in both glomeruli and tubule epithelia; and 4) those detected in all these sites. The pattern of glomerular staining also varied, but most of the antigens appeared to be expressed on either the endothelium or the epithelium, or on both. 27A IgG was specific for podocytes and weakly precipitated a 103-kd protein. 7A and 13A IgG precipitated a 120-kd protein and stained glomeruli as well as the basal aspects of distal tubules. 23A IgG recognized a more-than 350-kd antigen that appeared to be specific for endothelial cells in rat kidney and in all other organs studied. 14A IgG precipitated a 150-kd protein and stained glomeruli, proximal tubule brush borders, and endothelial and epithelial cells in rat kidney and in several other organs. 4B and 9B IgG gave a granular cytoplasmic staining in all cells. When injected intravenously into rats, all of the MAbs except 4B and 9B rapidly bound to glomeruli, demonstrating that the respective antigens are exposed at the cell surface and represent potential targets for antibody-mediated immune injury. It is concluded that selective detergent extraction of glomeruli is a useful approach for generation of antibodies that recognize native, nondenatured membrane components of glomerular endothelial and epithelial cells. PMID:1699424

Miettinen, A; Dekan, G; Farquhar, M G

1990-10-01

234

Epstein-Barr Viral BNLF2a Protein Hijacks the Tail-anchored Protein Insertion Machinery to Block Antigen Processing by the Transport Complex TAP*  

PubMed Central

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.

Wycisk, Agnes I.; Lin, Jiacheng; Loch, Sandra; Hobohm, Kathleen; Funke, Jessica; Wieneke, Ralph; Koch, Joachim; Skach, William R.; Mayerhofer, Peter U.; Tampe, Robert

2011-01-01

235

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.

2011-01-01

236

Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2  

PubMed Central

Background The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection. Results The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%. Conclusions This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera.

2010-01-01

237

Variability of genes encoding surface proteins used as vaccine antigens in meningococcal endemic and epidemic strain panels from Norway.  

PubMed

Surface-expressed protein antigens such as factor H-binding protein (fHbp), Neisserial adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and Porin protein A (PorA); all express sequence variability that can affect their function as protective immunogens when used in meningococcal serogroup B vaccines like the recently-approved 4CMenB (Bexsero(®)). We assessed the sequence variation of genes coding for these proteins and two additional proteins ("fusion partners" to fHbp and NHBA) in pathogenic isolates from a recent low incidence period (endemic situation; 2005-2006) in Norway. Findings among strains from this panel were contrasted to what was found among isolates from a historic outbreak (epidemic situation; 1985-1990). Multilocus sequence typing revealed 14 clonal complexes (cc) among the 66 endemic strains, while cc32 vastly predominated in the 38-strain epidemic panel. Serogroup B isolates accounted for 50/66 among endemic strains and 28/38 among epidemic strains. Potential strain-coverage ("sequence match") for the 4CMenB vaccine was identified among the majority (>70%) of the endemic serogroup B isolates and all of the epidemic serogroup B isolates evaluated. Further information about the degree of expression, surface availability and the true cross-reactivity for the vaccine antigens will be needed to fully characterize the clinical strain-coverage of 4CMenB in various geographic and epidemiological situations. PMID:24631075

Holst, Johan; Comanducci, Maurizio; Bambini, Stefania; Muzzi, Alessandro; Comandi, Sara; Oksnes, Jan; DeTora, Lisa; Pizza, Mariagrazia; Rappuoli, Rino; Caugant, Dominique A

2014-05-13

238

Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes  

PubMed Central

The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.

Hall, Michael; Nylander, ?sa; Jenkinson, Howard F.; Persson, Karina

2014-01-01

239

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody.  

PubMed

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule. PMID:2779474

Yamaguchi, H; Taguchi, H; Katura, T; Kumada, J; Uekusa, T; Ogata, S

1989-01-01

240

Antigenic relationships among the porin proteins of encapsulated Haemophilus influenzae clones.  

PubMed Central

Monoclonal antibodies (Mabs) specific for Haemophilus influenzae were generated to identify antigenic determinants shared among encapsulated H. influenzae clones. Sixteen MAbs reacted by Western immunoblot with a protein of an approximate molecular size of 40 kilodaltons corresponding to the P2 major outer membrane protein (porin). These MAbs also reacted with purified and recombinant H. influenzae porin. Fourteen of the MAbs recognized cell surface-exposed epitopes, and two of the MAbs, P2-16 and P2-17, identified epitopes that are not present or are not accessible on the cell surface. The reactivity spectrum of the MAb panel was studied by dot immunoassay against 32 serologically nontypeable and 119 encapsulated H. influenzae strains recovered worldwide, representing the major serotype a, b, and d clone families. MAbs P2-4 and P2-6 recognized only serotype b clones assigned to primary phylogenetic division I. These clones account for more than 99% of all invasive episodes worldwide. MAbs P2-3, P2-8, and P2-11 reacted with division I serotype b isolates and also identified all genetically allied strains expressing serotype a and d polysaccharide capsules. In contrast, none of the 16 MAbs reacted with genetically divergent serotype a or b clones assigned to primary phylogenetic division II. These results demonstrate that, in general, the patterns of P2 protein surface epitope exposure are cognate with genetic lineages of encapsulated H. influenzae strains and support the hypothesis that the population structure of encapsulated H. influenzae is predominantly clonal. Images

Martin, D; Hamel, J; Brodeur, B R; Musser, J M

1990-01-01

241

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

1999-01-01

242

Folded conformations of antigenic peptides from riboflavin carrier protein in aqueous hexafluoroacetone.  

PubMed Central

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.

Bhattacharjya, S.; Awasthi, S. K.; Adiga, P. R.; Balaram, P.

1998-01-01

243

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

2012-01-01

244

Exemptions from Unrelated Business Tax: Rental Income  

ERIC Educational Resources Information Center

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

Reed, George E.

1975-01-01

245

Analysis of monocyte chemotactic protein-1 production in different major histocompatability complex-restricted antigen presentation systems.  

PubMed

In the present study the production of the CC chemokine monocyte chemotactic protein-1 (MCP-1) in several MHC II-restricted antigen presentation systems was investigated in vitro. To assess which type of antigen-presenting cell (APC) influences MCP-1 production during antigen presentation, cultures enriched for different APC populations were prepared and MCP-1 production was determined. Our results showed that APCs that effectively induce a T cell response also produce elevated amounts of MCP-1. The MCP-1 production is highest in the memory-driven secondary response against a single antigen. Despite a massive T cell proliferation, low MCP-1 concentrations are found in Con A-induced cultures. These results suggest that T cell proliferation alone is not sufficient for MCP-1 production and that stimulation of the APC during the process of antigen presentation results in MCP-1 production. Based on our results and the literature, we propose a model for MCP-1 as an enhancer of the adaptive immune response. PMID:11580229

Tekstra, J; Tjin, E P; Tuk, C W; Broekhuis-Fluitsma, D; Havenith, C E; Beelen, R H

2001-10-01

246

Molecular cloning of the gene for a conserved major immunoreactive 28-kilodalton protein of Ehrlichia canis: a potential serodiagnostic antigen.  

PubMed

A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen. PMID:10225842

McBride, J W; Yu, X j; Walker, D H

1999-05-01

247

Antigenicity of the region encoded by exon8 of the human serine protease, HtrA2\\/Omi, is associated with its protein solubility  

Microsoft Academic Search

HtrA2\\/Omi, a mitochondrial serine protease, is pivotal in regulating apoptotic cell death. To determine the location of antigenic determinants in HtrA2\\/Omi, we expressed a series of the N-terminally truncated HtrA2\\/Omi as GST fusion proteins in E. coli. We assessed protein solubility and antigenic reactivity of various N-terminally truncated HtrA2\\/Omi proteins by binding to glutathione beads and immunoblot analyses, respectively. We identified

Hyo-Jin Park; Ju-Youn Choi; Young-Mo Seong; Chul Han; Sung-Joo Kim Yoon; Seongman Kang; Hyangshuk Rhim

2003-01-01

248

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing  

Microsoft Academic Search

Kaposi's sarcoma-associated herpesvirus (KSHV\\/human herpesvirus 8 (HHV8)) and Epstein-Barr virus (EBV\\/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and

Hyun Jin Kwun; Suzane Ramos da Silva; Ishita M. Shah; Neil Blake; Patrick S. Moore; Yuan Chang

2007-01-01

249

The Wzz (Cld) Protein in Escherichia coli: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity  

PubMed Central

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.

Franco, Agustin V.; Liu, Dan; Reeves, Peter R.

1998-01-01

250

Membrane interactions of simian virus 40 large T-antigen: influence of protein sequences and fatty acid acylation.  

PubMed Central

To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells. Images

Klockmann, U; Staufenbiel, M; Deppert, W

1984-01-01

251

Antigens of Mycobacterium tuberculosis Expressed during Preclinical Tuberculosis: Serological Immunodominance of Proteins with Repetitive Amino Acid Sequences  

PubMed Central

Four antigens of Mycobacterium tuberculosis that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, of M. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB.

Singh, K. K.; Zhang, X.; Patibandla, A. S.; Chien, P.; Laal, S.

2001-01-01

252

Antigenic diversity of the Plasmodium vivax circumsporozoite protein in parasite isolates of Western Colombia.  

PubMed

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

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

2011-02-01

253

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex*  

PubMed Central

The antigen 85 complex (Ag85) consists of three predominantly secreted proteins (Ag85A, Ag85B, and Ag85C), which play a key role in the mycobacterial pathogenesis and also possess enzymatic mycolyltransferase activity involved in cell wall synthesis. Ag85 is not only considered to be a virulence factor because its expression is essential for intracellular survival within macrophages, but also because it contributes to adherence, invasion, and dissemination of mycobacteria in host cells. In this study, we report that the extracellular matrix components, elastin and its precursor (tropoelastin) derived from human aorta, lung, and skin, serve as binding partners of Ag85 from Mycobacterium tuberculosis. The binding affinity of M. tuberculosis Ag85 to human tropoelastin was characterized (KD = 0.13 ± 0.006 ?m), and a novel Ag85-binding motif, AAAKAA(K/Q)(Y/F), on multiple tropoelastin modules was identified. In addition, the negatively charged Glu-258 of Ag85 was demonstrated to participate in an electrostatic interaction with human tropoelastin. Moreover, binding of Ag85 on elastin siRNA-transfected Caco-2 cells was significantly reduced (34.3%), implying that elastin acts as an important ligand contributing to mycobacterial invasion.

Kuo, Chih-Jung; Ptak, Christopher P.; Hsieh, Ching-Lin; Akey, Bruce L.; Chang, Yung-Fu

2013-01-01

254

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))

1991-10-01

255

Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c.  

PubMed Central

The pac gene of the serotype c strain Streptococcus mutans MT8148 encodes a cell surface protein antigen (PAc) of approximate 190 kilodaltons. The serotype c strain S. mutans GS-5 does not produce the 190-kilodalton PAc but produces a lower-molecular-weight protein that reacts with anti-PAc serum. The SphI-BamHI fragment of the pac gene was ligated with the S. mutans-Escherichia coli shuttle vector pSA3. The chimeric shuttle vector was transformed into strain GS-5, and two transformants (TK15 and TK18) were isolated. These transformants produced a large amount of cell-free and cell-bound PAc of 190 kilodaltons. No plasmid was isolated from these transformants, and the EcoRI fragments of their chromosomal DNA hybridized with the erythromycin resistance gene in the shuttle vector DNA, indicating insertion of the chimeric shuttle vector DNA into the chromosomal DNA. The cell hydrophobicity of strains TK15 and TK18 as well as PAc-defective mutants constructed by inserting an erythromycin resistance gene into the pac gene of strain MT8148 was analyzed. Strains MT8148, TK15, and TK18 were hydrophobic. On the other hand, strain GS-5 and PAc-defective MT8148 transformants were hydrophilic. Resting cells of the hydrophobic strains attached in larger numbers to saliva-coated hydroxyapatite than did the hydrophilic strains. Human whole saliva induced the aggregation of cells of the hydrophobic strains but not that of cells of the hydrophilic strains. These results suggest that cell surface PAc of S. mutans serotype c participates in attachment of the streptococcal cell to experimental pellicles. Images

Koga, T; Okahashi, N; Takahashi, I; Kanamoto, T; Asakawa, H; Iwaki, M

1990-01-01

256

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

PubMed

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease. PMID:24905682

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

2014-08-01

257

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.  

PubMed

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease. PMID:14607842

Kraiczy, Peter; Hellwage, Jens; Skerka, Christine; Becker, Heiko; Kirschfink, Michael; Simon, Markus M; Brade, Volker; Zipfel, Peter F; Wallich, Reinhard

2004-01-23

258

Chlamydia pneumoniae Major Outer Membrane Protein Is a Surface-Exposed Antigen That Elicits Antibodies Primarily Directed against Conformation-Dependent Determinants  

Microsoft Academic Search

The major outer membrane protein (MOMP) of Chlamydia trachomatis serovariants is known to be an immunodominant surface antigen. Moreover, it is known that the C. trachomatis MOMP elicits antibodies that recognize both linear and conformational antigenic determinants. In contrast, it has been reported that the MOMP of Chlamydia pneumoniae is not surface exposed and is immunorecessive. We hypothesized that the

KATERINA WOLF; ELIZABETH FISCHER; DAVID MEAD; GUANGMING ZHONG; ROSEANNA PEELING; BILL WHITMIRE; HARLAN D. CALDWELL

2001-01-01

259

Recombinant Exosporium Protein BclA of Bacillus anthracis Is Effective as a Booster for Mice Primed with Suboptimal Amounts of Protective Antigen  

Microsoft Academic Search

Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we

Trupti N. Brahmbhatt; Stephen C. Darnell; Humberto M. Carvalho; Patrick Sanz; Tae J. Kang; Robert L. Bull; Susan B. Rasmussen; Alan S. Cross; Alison D. O'Brien

2007-01-01

260

Simultaneous Purification of Murine Mammary Tumor Virus Structural Proteins: Analysis of Antigenic Reactivities of Native gp34 by Radioimmunocompetition Assays  

PubMed Central

All the structural proteins (gp47, gp34, p27, p23, p16, and p12) of the murine mammary tumor virus (MuMTV) were simultaneously purified utilizing alkylagarose chromatography as the initial fractionation step. Least-hydrophobic MuMTV polypeptides (p23, p16) and the slightly hydrophobic p27 were separated from moderately hydrophobic proteins gp47 and p12 by passage through octylimino (C8)-agarose; the gp47 and p12 could be removed from the matrix by elution with ethylene glycol, whereas the most hydrophobic MuMTV protein, gp34, was eluted using nonionic detergent together with ethylene glycol. Subsequent purification steps involved ion-exchange or gel filtration chromatography. The resulting protein preparations appeared near-homogeneous on analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Recoveries of MuMTV proteins, based on their approximate individual contribution to total virus protein, ranged from about 20% for gp47 to greater than 100% for the minor structural component p23, the major phosphoprotein of MuMTV. Antiserum against purified C3H MuMTV gp34, together with purified, radioiodinated gp34, was used to develop a radioimmunoassay which showed that from 13 to 14% of total MuMTV protein by weight is gp34. Using this assay system, the group-specific antigenic reactivity of gp34 was also demonstrated. When solubilized preparations of C3H, RIII, and GR MuMTV's were used as competing antigens in gp34 radioimmunoassays with anti-C3H MuMTV serum, both group- and type-specific differences in antigenic reactivity were found. Images

Marcus, Stuart L.; Kopelman, Rebecca; Sarkar, Nurul H.

1979-01-01

261

A Cloned MajorSchistosoma mansoniEgg Antigen with Homologies to Small Heat Shock Proteins Elicits Th1 Responsiveness  

Microsoft Academic Search

Inschistosomiasismansoni,solubleeggantigensoftheworminducechronicT-cell-mediatedgranulomatous tissue responses. Since thefirst preparation of crude soluble egg antigen extract, a dearth of highly purified antigenshashamperedtheidentificationofgranulomainducermolecules.Herewereportthatacloned38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a

YINLONG CAI; JANE G. LANGLEY; DAVID I. SMITH; ANDDOV L. BOROS

1996-01-01

262

Development and evaluation of an antigen-capture ELISA for detection of the UL24 antigen of the duck enteritis virus, based on a polyclonal antibody against the UL24 expression protein  

Microsoft Academic Search

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) method was developed for the efficient detection of the UL24 antigen of the duck enteritis virus (DEV) using polyclonal antibodies. Ducks and rabbits were immunized, respectively, with expressed UL24 recombinant protein. The IgG antibodies against UL24 from ducks and rabbits were purified and used as the capture antibodies. The specificity of the optimized AC-ELISA

Renyong Jia; Anchun Cheng; Mingshu Wang; Xuefeng Qi; Dekang Zhu; Han Ge; Qihui Luo; Fei Liu; Yufei Guo; Xiaoyue Chen

2009-01-01

263

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity.  

PubMed

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines. PMID:11994474

Kawa, Diane E; Stephens, Richard S

2002-05-15

264

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

PubMed Central

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

Rockey, D D; Rosquist, J L

1994-01-01

265

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice.  

PubMed

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen. PMID:17972923

Mkrtichyan, M; Ghochikyan, A; Loukinov, D; Davtyan, H; Ichim, T E; Cribbs, D H; Lobanenkov, V V; Agadjanyan, M G

2008-01-01

266

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

PubMed

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

2013-06-01

267

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

2013-01-01

268

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

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. PMID:24093105

Arribillaga, Laura; Durantez, Maika; Lozano, Teresa; Rudilla, Francesc; Rehberger, Federico; Casares, Noelia; Villanueva, Lorea; Martinez, Marta; Gorraiz, Marta; Borrás-Cuesta, Francisco; Sarobe, Pablo; Prieto, Jesús; Lasarte, Juan José

2013-01-01

269

Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage*  

PubMed Central

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.

Balan, Vitaly; Nangia-Makker, Pratima; Kho, Dhong Hyo; Wang, Yi; Raz, Avraham

2012-01-01

270

Peptide Inhibitors of the Malaria Surface Protein, Apical Membrane Antigen 1: Identification of Key Binding Residues  

PubMed Central

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5–10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to > 350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C?H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a non-polar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.

Lee, Erinna F.; Yao, Shenggen; Sabo, Jennifer K.; Fairlie, W. Douglas; Stevenson, Rachel A.; Harris, Karen S.; Anders, Robin F.; Foley, Michael; Norton, Raymond S.

2011-01-01

271

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

1987-01-01

272

Protein kinase Cdelta controls self-antigen-induced B-cell tolerance  

Microsoft Academic Search

Interaction of a B cell expressing self-specific B-cell antigen receptor (BCR) with an auto-antigen results in either clonal deletion or functional inactivation. Both of these processes lead to B-cell tolerance and are essential for the prevention of auto-immune diseases. Whereas clonal deletion results in the death of developing autoreactive B cells, functional inactivation of self-reactive B lymphocytes leads to complex

Ingrid Mecklenbräuker; Kaoru Saijo; Nai-Ying Zheng; Michael Leitges; Alexander Tarakhovsky

2002-01-01

273

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.

2007-01-01

274

Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs.  

PubMed

A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n=5] or IM [n=5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (P<0.05) IgG and IgA responses against all individual antigens present in the chimaera, but it could not confer a significant protection against M. hyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach. PMID:24909331

Marchioro, Silvana B; Sácristan, Rubén Del Pozo; Michiels, Annelies; Haesebrouck, Freddy; Conceição, Fabricio R; Dellagostin, Odir A; Maes, Dominiek

2014-08-01

275

Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56 Knockdown  

Microsoft Academic Search

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein

Carlos S. Moreno; Sumathi Ramachandran; Danita G. Ashby; Noelani Laycock; Courtney A. Plattner; Wen Chen; William C. Hahn; David C. Pallas

2004-01-01

276

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

PubMed

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; El Kirdasy, Ahmed; Salama, Akram; Attia, Mabrouk; Aboulaila, Mahmoud; Zhou, Mo; Kamyingkird, Ketsarin; Moumouni, Paul Franck Adjou; Masatani, Tatsunori; El Aziz, Sami Ahmed Abd; Moussa, Waheed Mohammed; Chahan, Bayin; Fukumoto, Shinya; Nishikawa, Yoshifumi; El Ballal, Salah Sayed; Xuan, Xuenan

2013-10-01

277

[Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].  

PubMed

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein. PMID:18998549

Nazierbieke, Wulumuhan; Yan, Fang; He, Cui; Zhang, Lei; Borrathybay, Entomack

2008-08-01

278

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

2011-01-01

279

Immunization with FSH? fusion protein antigen prevents bone loss in a rat ovariectomy-induced osteoporosis model  

SciTech Connect

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

Geng, Wenxin; Yan, Xingrong; Du, Huicong; Cui, Jihong; Li, Liwen, E-mail: liven@nwu.edu.cn; Chen, Fulin, E-mail: chenfl@nwu.edu.cn

2013-05-03

280

Ultrastructural, protein composition, and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds.  

PubMed

Psittacine beak and feather disease (PBFD) virus, was purified from diseased tissues of a lesser sulphur-crested cockatoo (Cacatua sulphurea), a black palm cockatoo (Probosiger aterrimus), a red-lored Amazon parrot (Amazona autumnalis), and a peach-faced lovebird (Agapornis roseicollis). The histopathology of diseased feathers and follicular epithelium from the different species was compared; basophilic intranuclear inclusion bodies were identified in the follicular epithelium and intracytoplasmic globular inclusions were observed within macrophages located in the feather pulp from the four species. Psittacine beak and feather disease virus antigen was specifically detected by colloidal gold immunoelectron microscopy. The different preparations of purified virions displayed an icosahedral symmetry, were non-enveloped, and had a mean diameter that varied from 12 to 15 nm when negatively stained. Two major viral-associated proteins with approximate molecular weights of 26 and 23 kilodaltons (kd) were consistently demonstrated from the four viral preparations. Purified virions from the four genera were antigenically related. These findings suggest that the PBFD virus purified from numerous genera of diseased birds is similar based on ultrastructural characteristics, protein composition and antigenic reactivity. PMID:2338723

Ritchie, B W; Niagro, F D; Latimer, K S; Lukert, P D; Steffens, W L; Rakich, P M; Pritchard, N

1990-04-01

281

Occult hepatitis B in blood donors in Indonesia: altered antigenicity of the hepatitis B virus surface protein  

PubMed Central

Background and aims Occult hepatitis B virus infection (OBI) poses a challenge to the safety of blood donation. The prevalence of OBI is not well documented in Indonesia, although this information in such an endemic country is needed. This study was aimed to evaluate the prevalence of occult hepatitis B in blood donors from two cities of Indonesia, and to study the genetic variation and its effect on the predicted antigenicity of HBsAg. Methods Serum samples of 309 regular blood donors negative for HBsAg were tested for anti-HBs and anti-HBc. Hepatitis B virus (HBV) DNA isolated from anti-HBc-positive samples were analyzed by polymerase chain reaction, cloned and sequenced. Antigenic properties of identified HBsAg mutants were predicted by calculation of the antigenic index. Results Of the 309 HBsAg-negative samples, anti-HBc was positive in 134 (43.4%) and HBV DNA was detected in 25 (8.1%). Seven of the viremic samples had nucleotide substitutions (A521G, A551T, C582T, and A562G) in the S gene, causing amino acid mutations (T123A, M133L, and T143M) in the ‘a’ determinant of HBsAg that resulted in changes in the predicted antigenicity. Conclusions OBI was detected in blood donors’ samples in Indonesia. Anti-HBc was shown to be a better screening parameter than HBsAg, however, it might result in the loss of donors particularly in endemic countries. HBsAg detection failure in this study might be due to mutations altering the protein antigenicity and/or the low-level carriage of HBV.

Thedja, Meta D.; Roni, Martono; Harahap, Alida R.; Siregar, Nurjati C.; Ie, Susan I.

2010-01-01

282

Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins.  

PubMed

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3-10 and 4-7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions. PMID:17182197

Pinto, Paulo Marcos; Chemale, Gustavo; de Castro, Luiza Amaral; Costa, Ana Paula Metz; Kich, Jalusa Deon; Vainstein, Marilene Henning; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

2007-03-31

283

Unrelated umbilical cord blood transplant for adult acute lymphoblastic leukemia in first and second complete remission: a comparison with allografts from adult unrelated donors.  

PubMed

Allogeneic hematopoietic cell transplantation has an established role in the treatment of adults with acute lymphoblastic leukemia whose survival when recipients of grafts from adult unrelated donors approaches that of recipients of grafts from sibling donors. Our aim was to determine the role of mismatched unrelated cord blood grafts in transplantation for 802 adults with acute lymphoblastic leukemia in first or second complete remission. Using Cox regression we compared outcomes after 116 mismatched single or double cord blood transplants, 546 peripheral blood progenitor cell transplants and 140 bone marrow transplants. The characteristics of the recipients and their diseases were similar except cord blood recipients were younger, more likely to be non-Caucasians and more likely to have a low white blood cell count at diagnosis. There were differences in donor-recipient human leukocyte antigen-match depending on the source of the graft. Most adult donor transplants were matched at the allele-level considering human leukocyte antigens-A, -B, -C and -DRB1. In contrast, most cord blood transplants were mismatched and considered antigen-level matching; 57% were mismatched at two loci and 29% at one locus whereas only 29% of adult donor transplants were mismatched at one locus and none at two loci. There were no differences in the 3-year probabilities of survival between recipients of cord blood (44%), matched adult donor (44%) and mismatched adult donor (43%) transplants. Cord blood transplants engrafted slower and were associated with less grade 2-4 acute but similar chronic graft-versus-host disease, relapse, and transplant-related mortality. The survival of cord blood graft recipients was similar to that of recipients of matched or mismatched unrelated adult donor grafts and so cord blood should be considered a valid alternative source of stem cells for adults with acute lymphoblastic leukemia in the absence of a matched unrelated adult donor. PMID:24056817

Marks, David I; Woo, Kwang Ahn; Zhong, Xiaobo; Appelbaum, Frederick R; Bachanova, Veronika; Barker, Juliet N; Brunstein, Claudio G; Gibson, John; Kebriaei, Partow; Lazarus, Hillard M; Olsson, Richard; Perales, Miguel-Angel; Pidala, Joseph; Savani, Bipin; Rocha, Vanderson; Eapen, Mary

2014-02-01

284

Bayesian Geoadditive Seemingly Unrelated Regression 1  

Microsoft Academic Search

Summary Parametric seemingly unrelated regression (SUR) models are a common tool for multivariate regression analysis when error variables are reason- ably correlated, so that separate univariate analysis may result in inefficient estimates of covariate effects. A weakness of parametric models is that they require strong assumptions on the functional form of possibly nonlinear effects of metrical covariates. In this paper,

Stefan Lang; B. Adebayo; Ludwig Fahrmeir; Winfried J. Steiner

285

Minimizing the makespan for Unrelated Parallel Machines  

Microsoft Academic Search

In this paper, we study the unrelated parallel machine problem for minimizing the makespan, which is NP-hard. We used Simulated Annealing (SA) and Tabu Search (TS) with Neighborhood Search (NS) based on the structure of the problem. We also used a modied SA algorithm, which gives better results than the traditional SA and developed an eectiv e heuristic for the

Yunsong Guo; Andrew Lim; Brian Rodrigues; Liang Yang

2007-01-01

286

RHAMM (CD168) is overexpressed at the protein level and may constitute an immunogenic antigen in advanced prostate cancer disease.  

PubMed

Localized prostate cancer (CaP) can be cured using several strategies. However, the need to identify active substances in advanced tumor stages is tremendous, as the outcome in such cases is still disappointing. One approach is to deliver human tumor antigen-targeted therapy, which is recognized by T cells or antibodies. We used data mining of the Cancer Immunome Database (CID), which comprises potential immunologic targets identified by serological screening of expression libraries. Candidate antigens were screened by DNA microarrays. Genes were then validated at the protein level by tissue microarrays, representing various stages of CaP disease. Of 43 targets identified by CID, 10 showed an overexpression on the complementary DNA array in CaP metastases. The RHAMM (CD168) gene, earlier identified by our group as an immunogenic antigen in acute and chronic leukemia, also showed highly significant overexpression in CaP metastases compared with localized disease and benign prostatic hyperplasia. At the protein level, RHAMM was highest in metastatic tissue samples and significantly higher in neoplastic localized disease compared with benign tissue. High RHAMM expression was associated with clinical parameters known to be linked to better clinical outcome. Patients with high RHAMM expression in the primaries had a significantly lower risk of biochemical failure. The number of viable cells in cell cultures was reduced in blocking experiments using hormone-sensitive and hormone-insensitive metastatic CaP cell lines. Acknowledging the proven immunogenic effects of RHAMM in leukemia, this antigen is intriguing as a therapeutic target in far-advanced CaP. PMID:19724689

Gust, Kilian M; Hofer, Matthias D; Perner, Sven R; Kim, Robert; Chinnaiyan, Arul M; Varambally, Sooryanarayana; Moller, Peter; Rinnab, Ludwig; Rubin, Mark A; Greiner, Jochen; Schmitt, Michael; Kuefer, Rainer; Ringhoffer, Mark

2009-09-01

287

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

2001-01-01

288

Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen.  

PubMed Central

Anti-Mi-2 autoantibody is strongly associated with dermatomyositis and found in sera of 20% of patients. Mi-2 antigen contains at least eight components and previous evidence suggested that the 240-kD protein was the antigenic component for at least some sera. In this study, anti-M-2 patient sera were used to screen human thymocyte and HeLa cell lambda gt11 expression libraries, and two clones from each had plaques specifically reactive with anti-Mi-2 sera. Studies with affinity-purified antibody supported the identification of the clones. All of 44 anti-Mi-2 sera reacted with the plaques, but none of 44 control sera reacted significantly. The cDNAs were identical, and full sequencing of one revealed an open reading frame spanning a 1,054-bp insert. Rescreening the library with the cDNA yielded a 1,589-bp cDNA that continued the open reading frame. The Mi-2 cDNA hybridized to a single 7.5-8.0 kb mRNA of HeLa cells, by Northern blot. Rabbit antiserum directed at a portion of the cDNA product reacted with HeLa 240-kD Mi-2 protein. The sequence was notable for four potential zinc-fingers and several charged regions. The protein encoded by the cDNA produced in vitro reacted with only one of five of the Mi-2 sera. These findings indicate that the Mi-2 240 kD is a novel protein that is antigenic for all Mi-2 sera, and strongly suggests that a major common epitope is conformational in nature. Images

Ge, Q; Nilasena, D S; O'Brien, C A; Frank, M B; Targoff, I N

1995-01-01

289

The Transferrin Binding Protein B of Moraxella catarrhalis Elicits Bactericidal Antibodies and Is a Potential Vaccine Antigen  

PubMed Central

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.

Myers, Lisa E.; Yang, Yan-ping; Du, Run-pan; Wang, Qijun; Harkness, Robin E.; Schryvers, Anthony B.; Klein, Michel H.; Loosmore, Sheena M.

1998-01-01

290

Purification and properties of a 75-kilodalton major protein, an immunodominant surface antigen, from the oral anaerobe Bacteroides gingivalis.  

PubMed Central

A 75-kilodalton major protein (75K protein) was purified to homogeneity from the cell lysate fraction and the envelope of Bacteroides gingivalis 381. The 75K protein was originally present in the outer membrane or the outermost part of this organism as a large, stable complex with an apparent molecular weight of about 2,000,000. Heating at 80 degrees C and at higher temperatures in the presence of sodium dodecyl sulfate was needed to completely dissociate it to monomers. Amino acid analysis revealed that the 75K protein had about 50% nonpolar amino acids. Various strains of B. gingivalis but not other bacteria, including oral Bacteroides species tested, contained serologically related 75K proteins when tested in Western blotting (immunoblotting) analysis. The abundance and localization of the 75K protein in this organism suggest that it has the potential to participate in the host-parasite interaction in infection. The 75K protein was, indeed, strongly recognized in patients with adult periodontal diseases. Immunoblotting with sera from patients and with rabbit antisera generated by intravenous inoculations of whole B. gingivalis cells revealed that the 75K protein was an immunodominant antigen on the surface of B. gingivalis. Images

Yoshimura, F; Watanabe, K; Takasawa, T; Kawanami, M; Kato, H

1989-01-01

291

The Immune Response to a Vesicular Stomatitis Virus Vaccine Vector Is Independent of Particulate Antigen Secretion and Protein Turnover Rate  

PubMed Central

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 (MSC69A) and compared the immunogenicity of this vector to that of the wild-type VSV-MS vector in mice. As expected, the MSC69A 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 MSC69A 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.

Cobleigh, Melissa A.; Bradfield, Clinton; Liu, Yuanjie; Mehta, Anand

2012-01-01

292

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

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. PMID:22069532

Le Moigne, Vincent; Robreau, Georges; Mahana, Wahib

2009-12-01

293

Epstein-Barr viral BNLF2a protein hijacks the tail-anchored protein insertion machinery to block antigen processing by the transport complex TAP.  

PubMed

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors. PMID:21984826

Wycisk, Agnes I; Lin, Jiacheng; Loch, Sandra; Hobohm, Kathleen; Funke, Jessica; Wieneke, Ralph; Koch, Joachim; Skach, William R; Mayerhofer, Peter U; Tampé, Robert

2011-12-01

294

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

2012-01-01

295

Overexpression and purification of PreS region of hepatitis B virus antigenic surface protein adr subtype in Escherichia coli.  

PubMed

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80 degrees. PMID:18047797

Abbas, Naaz; Ahmad, Aftab; Shakoori, Abdul Rauf

2007-11-30

296

Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax.  

PubMed

Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27?kDa, 31?kDa, and 53?kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines. PMID:24757558

Velásquez, Norma P; Camargo, Rocío E; Uzcanga, Graciela L; Bubis, José

2014-01-01

297

Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax  

PubMed Central

Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27?kDa, 31?kDa, and 53?kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines.

Velasquez, Norma P.; Camargo, Rocio E.; Uzcanga, Graciela L.; Bubis, Jose

2014-01-01

298

Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera.  

PubMed

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, ?-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on ?-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines. PMID:24674665

Bazan, Silvia Boschi; Breinig, Tanja; Schmitt, Manfred J; Breinig, Frank

2014-05-01

299

[Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].  

PubMed

Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits. PMID:23012985

Vladyko, A S; Scheslenok, E P; Fomina, E G; Semizhon, P A; Ignat'ev, G M; Shkolina, T V; Kras'ko, A G; Semenov, S F; Vinokurov, N V

2012-01-01

300

Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins.  

PubMed Central

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology. Images

Jenison, S A; Firzlaff, J M; Langenberg, A; Galloway, D A

1988-01-01

301

Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus ?  

PubMed Central

The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA.

Chen, Wuguo; Dittmer, Dirk P.

2011-01-01

302

Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins.  

PubMed Central

Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60 micrograms) from the ear isolate also inhibited bactericidal activity in the respective immune serum. LPSs exhibited minimal inhibition (greater than 110 micrograms). Three human sera (two normal, one immune) were selectively depleted of 80% of antibody activity against OMPs (measured by enzyme-linked immunosorbent assay) by affinity chromatography using OMPs from the pulmonary isolate coupled to a solid phase. These OMP antibody-depleted sera also showed an 88% reduction of bactericidal activity against this strain. Immunopurified antibody against OMPs eluted from the solid phase was bactericidal. Images

Gnehm, H E; Pelton, S I; Gulati, S; Rice, P A

1985-01-01

303

Antibodies to the Plasmodium falciparum Antigens Circumsporozoite Protein, Thrombospondin-Related Adhesive Protein, and Liver-Stage Antigen 1 Vary by Ages of Subjects and by Season in a Highland Area of Kenya  

PubMed Central

Immunoglobulin G (IgG) antibodies to three vaccine candidate preerythrocytic Plasmodium falciparum antigens were evaluated in children and adults in an epidemic-prone highland area of Kenya during rainy (high-transmission) and dry (low-transmission) seasons. The frequencies and median levels of IgG antibodies to circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP) were compared to the frequencies and median levels of IgG antibodies to liver-stage antigen 1 (LSA-1) reported previously. The frequencies and median levels of IgG antibodies to CSP and TRAP were similar in children and adults in the rainy season, but they were lower in children than in adults in the dry season. The frequencies and median levels of antibodies to LSA-1 were lower in children than in adults in both the rainy and dry seasons. Antibodies to CSP and LSA-1 were primarily members of the IgG1 and IgG3 subclasses, while antibodies to TRAP were primarily members of the IgG3 and IgG4 subclasses. In a treatment-reinfection study following dry season testing, antibodies to TRAP were associated with a trend toward protection from infection in children (P = 0.051) but not in adults. Antibodies to LSA-1 and CSP did not correlate with protection in children or adults. In this highland area of Kenya with unstable transmission, IgG antibodies to preerythrocytic P. falciparum antigens vary in subjects by age and season, and the protective effects of these antibodies against infection may be different in adults and children.

John, Chandy C.; Zickafoose, Joseph S.; Sumba, P. Odada; King, Christopher L.; Kazura, James W.

2003-01-01

304

Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis.  

PubMed Central

The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28- and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50- to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band. Images

Zimmer, B L; Pappagianis, D

1986-01-01

305

The effect of serum protein concentrations on the specificity of the radioimmunoassay of carcinoembryonic antigen in malignant neoplasia and non-neoplastic disease  

Microsoft Academic Search

Carcinoembryonic antigen (CEA) has been measured in parallel with seven serum proteins and seromucoids in the sera of patients with malignant neoplasia and non-neoplastic disease. In the total group significant correlations were found between CEA and seromucoids and between CEA and several serum proteins. However, with two exceptions, when the individual disease groups were examined no correlation was seen. It

J. M. Crawley; B. E. Northam; J. P. G. King; J. C. Leonard; S. N. Booth; P. W. Dykes

1974-01-01

306

Recombinant Jembrana disease virus gag proteins identify several different antigenic domains but do not facilitate serological differentiation of JDV and nonpathogenic bovine lentiviruses  

Microsoft Academic Search

In Indonesia, it is suspected that there are two bovine lentiviruses circulating in the cattle population: a pathogenic Jembrana disease virus (JDV), and a nonpathogenic bovine immunodeficiency-like virus (BIV). Both viruses cross-react antigenically and cannot be differentiated by current serological tests using JDV antigens. To identify possible type-specific epitopes, a series of recombinant protein constructs including the matrix, capsid and

Moira Desport; Meredith E. Stewart; Carol A. Sheridan; William G. F. Ditcham; Surachmi Setiyaningsih; W. Masa Tenaya; Nining Hartaningsih; Graham E. Wilcox

2005-01-01

307

Biophysical Analysis of the Endoplasmic Reticulum-Resident Chaperone\\/Heat Shock Protein gp96\\/GRP94 and Its Complex with Peptide Antigen  

Microsoft Academic Search

Animals vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant

Nora A. Linderoth; Martha N. Simon; Natalia A. Rodionova; Martine Cadene; William R. Laws; Brian T. Chait; Srin Sastry

2001-01-01

308

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

2011-01-01

309

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

1999-01-01

310

Human Antibody Responses to VlsE Antigenic Variation Protein of Borrelia burgdorferi  

Microsoft Academic Search

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease,

M. B. LAWRENZ; J. M. HARDHAM; R. T. OWENS; J. NOWAKOWSKI; A. C. STEERE; G. P. WORMSER; S. J. NORRIS

1999-01-01

311

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

2008-01-01

312

Immunization of Aotus monkeys with recombinant proteins of an erythrocyte surface antigen of Plasmodium falciparum  

Microsoft Academic Search

Recent studies have identified and characterized a ring-infected erythrocyte surface antigen (RESA) of the human malaria parasite Plasmodium falciparum with a relative molecular mass (Mr) of ~155,000 (refs 1-7). RESA is localized in the micronemes of merozoites and also the membrane of red cells infected with ring-stage parasites. It is thought to be released through the apical pore from the

William E. Collins; Robin F. Anders; Marguerite Pappaioanou; Gary H. Campbell; Graham V. Brown; David J. Kemp; Ross L. Coppel; Jimmie C. Skinner; Patricia M. Andrysiak; Jenny M. Favaloro; Lynn M. Corcoran; J. Roger Broderson; Graham F. Mitchell; Carlos C. Campbell

1986-01-01

313

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

2010-01-01

314

Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease.  

PubMed

The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. PSMA was captured from serum by anti-PSMA antibody bound to ProteinChip arrays, the captured PSMA detected by surface-enhanced laser desorption/ionization mass spectrometry, and quantitated by comparing the mass signal integrals to a standard curve established using purified recombinant PSMA. The average serum PSMA value for prostate cancer (623.1 ng/ml) was significantly different (P < 0.001) from that for benign prostate hyperplasia (117.1 ng/ml) and the normal groups (age <50, 272.9 ng/ml; age >50, 359.4 ng/ml). These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility. PMID:11507047

Xiao, Z; Adam, B L; Cazares, L H; Clements, M A; Davis, J W; Schellhammer, P F; Dalmasso, E A; Wright, G L

2001-08-15

315

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

2011-01-01

316

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

PubMed Central

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

Seydel, U; Jans, D A

1996-01-01

317

The Major Antigenic Membrane Protein of "Candidatus Phytoplasma asteris" Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors  

PubMed Central

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the ? and ? subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachi, Cristina

2011-01-01

318

Identification and Characterization of a Trypanosoma congolense 46 kDa Protein as a Candidate Serodiagnostic Antigen.  

PubMed

Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T. congolense infection. In this study, we have identified one T. congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T. congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T. congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T. congolense infection. PMID:24492330

Zhou, Mo; Suganuma, Keisuke; Ruttayaporn, Ngasaman; Nguyen, Thu-Thuy; Yamasaki, Shino; Igarashi, Ikuo; Kawazu, Shin-Ichiro; Suzuki, Yasuhiko; Inoue, Noboru

2014-07-01

319

Antigens shared by Leishmania species and Trypanosoma cruzi: immunological comparison of the acidic ribosomal P0 proteins.  

PubMed Central

Patients with visceral leishmaniasis produce high levels of immunoglobulin, but the specificities of antibodies produced are not well characterized. In an effort to identify leishmania antigens that are specific to Leishmania species or are cross-reactive with other parasitic protozoa, we have cloned and characterized full-length genomic and cDNA clones encoding a Leishmania chagasi acidic ribosomal antigen, LcP0, recognized during human infections. The protein is homologous to the Trypanosoma cruzi and human ribosomal proteins TcP0 and HuP0, respectively. Unlike most higher eukaryotes, but similar to TcP0, LcP0 has a C-terminal heptapeptide sequence resembling those of the archaebacterial acidic (P-like) proteins. The highly charged C-terminal acidic domain of LcP0 contains a serine residue typically found in most eukaryotes but lacking in all T. cruzi P proteins we have characterized thus far. L. chagasi-infected individuals as well as those with T. cruzi infections have antibodies cross-reactive with recombinant LcP0 and TcP0 as well as HuP0. However, the properties of anti-P0 antibodies in T. cruzi and L. chagasi infection sera are quite different. Through the use of synthetic peptides, we showed that while T. cruzi infection anti-TcP0 antibodies are exclusively directed against the C-terminal domain of TcP0, L. chagasi infection sera contain antibodies reactive with epitopes other than the C-terminal sequence of LcP0. Thus, anti-LcP0 antibodies in L. chagasi infection sera represent the first characterized deviation from the restricted immunodominant C-terminal epitope involved in the generation of anti-P0 antibodies following infection or autoimmune diseases. Images

Skeiky, Y A; Benson, D R; Elwasila, M; Badaro, R; Burns, J M; Reed, S G

1994-01-01

320

Identification and Characterization of a Trypanosoma congolense 46 kDa Protein as a Candidate Serodiagnostic Antigen  

PubMed Central

ABSTRACT Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T. congolense infection. In this study, we have identified one T. congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T. congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T. congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T. congolense infection.

ZHOU, Mo; SUGANUMA, Keisuke; RUTTAYAPORN, Ngasaman; NGUYEN, Thu-Thuy; YAMASAKI, Shino; IGARASHI, Ikuo; KAWAZU, Shin-ichiro; SUZUKI, Yasuhiko; INOUE, Noboru

2014-01-01

321

IgG response to purified 65- and 70-kDa mycobacterial heat shock proteins and to antigen 85 in leprosy.  

PubMed

IgG antibody response to mycobacterial heat-shock proteins (hsp) (the 70-kDa antigen from Mycobacterium tuberculosis and M. bovis BCG; the 65-kDa antigen from M. leprae and M. bovis BCG) and to the fibronectin-binding antigen 85 from M. bovis BCG was analyzed in a dot-blot assay in plasma from leprosy patients and their contacts. Most plasma--whatever the status of the subjects--reacted to the hsp 70; 8 of 9 (89%) of paucibacillary patients recognized the 65 mycobacterial hsp but only 2 of 9 (22%) recognized the antigen 85. In contrast, 12 of 12 (100%) of multibacillary patients reacted with the antigen 85 and only 4 of 12 (33%) reacted to the hsp 65 from M. leprae. On the one hand, 7 of 25 (28%) of the lepromin-positive contacts and 2 of 9 (22%) of the lepromin-negative contacts recognized the antigen 85. On the other hand, 11 of 25 (44%) of the lepromin-positive contacts but only 1 of 9 (11%) of the lepromin-negative contacts reacted to the hsp65 from M. leprae. Finally, very few (10%) of the lepromin-positive controls showed a positive reaction to the M. leprae 65-kDa antigen, the BCG 65-kDa antigen, and the 85-kDa antigen of BCG. Thus, differences in binding to the hsp65 from M. leprae and to antigen 85 could be helpful in distinguishing different forms of the disease. PMID:8189089

Launois, P; N'Diaye Niang, M; Drowart, A; Van Vooren, J P; Sarthou, J L; Lalu, T; Millan, J; Huygen, K

1994-03-01

322

Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity  

PubMed Central

Background Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel. Results In vitro, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies. Conclusion The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.

Tenbusch, Matthias; Kuate, Seraphin; Tippler, Bettina; Gerlach, Nicole; Schimmer, Simone; Dittmer, Ulf; Uberla, Klaus

2008-01-01

323

A Novel Cancer Testis Antigen, A-Kinase Anchor Protein 4 (AKAP4) Is a Potential Biomarker for Breast Cancer  

PubMed Central

Background Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma. Methodology/Findings We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein. Conclusions Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use.

Saini, Shikha; Jagadish, Nirmala; Gupta, Anju; Bhatnagar, Amar; Suri, Anil

2013-01-01

324

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

PubMed Central

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 chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.

You, Jianxin; Srinivasan, Viswanathan; Denis, Gerald V.; Harrington, William J.; Ballestas, Mary E.; Kaye, Kenneth M.; Howley, Peter M.

2006-01-01

325

Comparative modeling of retinol-binding protein-3 and retinal S-antigen in Eales' disease and prediction of their binding sites using computational methods.  

PubMed

Retinal S-antigen and interphotoreceptor retinoid-binding protein-3 play a significant role in the etiopathogenesis of Eales' disease. Protein 3D structures are functionally very important and play a significant role in progression of the disease, hence these 3D structures are better target for further drug designing and relative studies. We developed 3D model structure of retinol-binding protein-3 and retinal S-antigen protein of human involved in Eales' disease. Functional site prediction is a very important and related step; hence, in the current course of analysis, we predicted putative functional site residues in the target proteins. Molecular models of these proteins of Eales' disease as documented in this study may provide a valuable aid for designing an inhibitor or better ligand against Eales' disease and could play a significant role in drug design. PMID:22833777

Tiwari, Anshul; Trivedi, Ashish Chandra; Srivastava, Prachi; Pant, Aditya Bhusan; Saxena, Sandeep

2010-09-01

326

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

2009-01-01

327

Antigenic properties of a transport-competent influenza HA\\/HIV Env chimeric protein  

Microsoft Academic Search

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

Ling Ye; Yuliang Sun; Jianguo Lin; Zhigao Bu; Qingyang Wu; Shibo Jiang; David A. Steinhauer; Richard W. Compans; Chinglai Yang

2006-01-01

328

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

2003-01-01

329

A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity  

Microsoft Academic Search

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318–510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1

Yuxian He; Jingjing Li; Shibo Jiang

2006-01-01

330

Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells.  

PubMed Central

The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells.

Carrasco-Marin, E; Paz-Miguel, J E; Lopez-Mato, P; Alvarez-Dominguez, C; Leyva-Cobian, F

1998-01-01

331

The ubiquitin-like protein, ISG15, is a novel tumor-associated antigen for cancer immunotherapy.  

PubMed

The recent announcement of the first FDA-approved therapeutic vaccine for prostate cancer, Sipuleucel-T, is a watershed moment for the field of tumor immunotherapy. However, while Sipuleucel-T provides a powerful tool to clinicians for the most prevalent form of cancer in men, there remains an unmet need for a similar therapeutic strategy against breast cancer, the most prevalent cancer in women. While current breast cancer vaccines in development target several antigens, the most prevalent is the tumor-associated antigen, HER2. Initial results with HER2 vaccines appear promising in terms of efficacy; however, the lack of HER2 overexpression by a majority of breast tumors and the safety concerns associated with current HER2-targeted immunotherapy suggest that additional therapeutic strategies would be beneficial. Recently, several studies have identified ISG15 as a molecule highly expressed in numerous malignancies. ISG15 is a small ubiquitin-like protein regulated by type-I interferon and classically associated with viral defense. Elevated ISG15 expression in breast cancer is especially well documented and is independent of HER2, progesterone receptor, and estrogen receptor status. Additionally, high ISG15 expression in breast cancer correlates with an unfavorable prognosis and poor responses to traditional treatment strategies such as chemotherapy and radiation. To overcome these challenges, we employ a novel strategy to specifically target tumor-associated ISG15 expression with immunotherapy. We demonstrate that vaccination against ISG15 results in significant CD8-mediated reductions in both primary and metastatic mammary tumor burden. These results validate ISG15 as a tumor-associated antigen for cancer immunotherapy. PMID:22057675

Wood, Laurence M; Pan, Zhen-Kun; Seavey, Matthew M; Muthukumaran, Geetha; Paterson, Yvonne

2012-05-01

332

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.  

PubMed

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

2013-02-26

333

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

2013-01-01

334

Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.  

PubMed Central

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death. Images

Kosik, K S; Joachim, C L; Selkoe, D J

1986-01-01

335

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: chyang@emory.edu

2006-08-15

336

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5.  

PubMed

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

2013-04-01

337

In Vivo Interaction of the Hepatitis Delta Virus Small Antigen with the ELAV-Like Protein HuR  

PubMed Central

The small and large delta antigens (S-HDAg and L-HDAg, respectively) represent two forms of the only protein encoded by the hepatitis delta virus (HDV) RNA genome. Consequently, HDV relies, at a large extent, on the host cell machinery for replication and transcription. Until now, only a limited number of cellular proteins were identified as S-HDAg or L-HDAg partners being involved in the modulation of the virus life cycle. In an attempt to identify cellular S-HDAg-binding proteins we made use of a yeast two-hybrid approach to screen a human liver cDNA library. We were able to identify HuR, a ubiquitously expressed protein involved in RNA stabilization, as an S-HDAg partner both in vitro and in vivo. HuR was found to be overexpressed and colocalize with HDAg in human hepatoma cells. siRNA knockdown of HuR mRNA resulted in inhibition of S-HDAg and L-HDAg expression.

Casaca, Ana; Fardilha, Margarida; da Cruz e Silva, Edgar; Cunha, Celso

2011-01-01

338

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

2011-01-01

339

A novel DNA vaccine constructed by heat shock protein 70 and melanoma antigen-encoding gene 3 against tumorigenesis.  

PubMed

Melanoma antigen-encoding gene 3 (MAGE-3) is an ideal candidate for a tumor vaccine although its potency need to be increased. Heat shock proteins (HSPs) represents a potential approach for increasing the potency of DNA vaccines. In the present study, a fusion DNA vaccine composed of Mycobacterium tuberculosis HSP70 and MAGE-3 was constructed and used to immunize C57BL/6 mice against B16 or B16-MAGE-3 tumor cells. The results show that the HSP70-MAGE-3 fusion DNA vaccine enhanced the frequency of MAGE-3-specific cytotoxic T-cells as compared to the MAGE-3 DNA vaccine or the HSP70/MAGE-3 cocktail DNA vaccine (P < 0.05). In conclusion, the results indicate that the HSP70-MAGE-3 fusion DNA vaccine can strongly activate MAGE-3 specific cellular immunological reactions and thus significantly inhibit the growth of B16-MAGE-3 tumors, improving the survival of tumor-bearing mice, and the HSP70-MAGE-3 fusion DNA vaccine has a significant therapeutic effect on the tumors that express MAGE-3 antigens. PMID:20795360

Qu, Ping; Ma, Jia-Hai; Zhang, Xiu-Min; Huang, Xiao-Jun; Yang, Xin-Wei; Yan-Fang, Sui

2010-05-01

340

Construction and characterization of Lactobacillus pentosus expressing the D antigenic site of the spike protein of Transmissible gastroenteritis virus.  

PubMed

This study explored the feasibility of Lactobacillus pentosus as a live vehicle to deliver and express antigen. First of all, L. pentosus transformed by electroporation with the plasmids pg611-6D (anchored) and pg612-6D (secretory) based on the xylose operon generated the recombinant strains rLppg611-6D and rLppg612-6D, respectively, expressing the D antigenic site of the spike (S) protein of Transmissible gastroenteritis virus (TGEV), for intragastric administration in mice. Secondly, we collected serum, fecal, nasal, ophthalmic, and vaginal samples from pre-immune mice and after the first immunization (on days 7, 14, 21, 28, 35, and 42) that were used to analyze the levels of immunoglobulins G and A against TGEV by using ELISA. In addition, a plaque reduction assay was performed using sera from groups pg611, pg612-6D, pg11-6D, and phosphate-buffered saline (blank control) to analyze TGEV-neutralizing antibody activity in vitro. A statistically significant difference in serum tests between groups demonstrated that rLppg612-6D induced better immunogenicity than rLppg611-6D, making rLppg612-6D the better candidate for oral vaccine. Taken together, L. pentosus possessed the potential to become a novel vector for mucosal vaccine in the future. PMID:21529121

Di-Qiu, Liu; Xin-Yuan, Qiao; Jun-Wei, Ge; Li-Jie, Tang; Yan-Ping, Jiang; Yi-Jing, Li

2011-05-01

341

Immune Complex Signatures of Patients with Active and Inactive SLE Revealed by Multiplex Protein Binding Analysis on Antigen Microarrays  

PubMed Central

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens. These complement components covalently bind to those microarray features where antibodies and other serum components induce complement activation. Using this technology, we compared functional serum antibody profiles of control subjects (n?=?31) and patients with lupus erythematosus (n?=?61) in the active (n?=?22) and inactive (n?=?39) phase of the disease. Multivariate analysis was applied to identify contributions of binding data on 25 antigens to the discrimination of the study groups. Receiver operating characteristic analysis was used to portray the discriminative property of each measured parameter for each antigen in pairwise group comparisons. Complement C3 and C4 deposition increased on autoantibody targets in spite of the decreased serum complement concentrations, and decreased on other autoantigens, demonstrating the imbalance of complement function in patients with lupus erythematosus. Our observations confirmed previously known markers of disease and showed that C3 and C4 deposition data were at least as powerful as Ig binding data in separating the study groups.

Papp, Krisztian; Vegh, Peter; Hobor, Renata; Szittner, Zoltan; Voko, Zoltan; Podani, Janos; Czirjak, Laszlo; Prechl, Jozsef

2012-01-01

342

Artificial Salmonella vaccines: O-antigenic oligosaccharide-protein conjugates induce protection against infection with Salmonella typhimurium.  

PubMed Central

Outbred mice were vaccinated with various artificial Salmonella vaccines and subsequently challenged intraperitoneally with graded doses of virulent Salmonella typhimurium. The Salmonella vaccines used were: (i) octasaccharide, obtained by hydrolysis of the O-antigenic polysaccharide chain of S. typhimurium strain SH 4809 with phage P22-associated endo-rhamnosidase and covalently linked to either diphtheria toxin or edestine; (ii) purified outer membrane proteins (porins) from S. typhimurium; and (iii) octasaccharide covalently linked to porins. All vaccines induced significant protection against experimental infection of mice with S. typhimurium. However, vaccination with the octasaccharide-porin conjugate resulted in better protection than that obtained by vaccination with octasaccharide or porin vaccines separately. Rabbit antibodies raised against the different vaccines were also passively administered intravenously to mice. Such mice were protected against challenge with virulent S. typhimurium by antibodies specific for the S. typhimurium O-antigen or for the porins. Thus, active immunization with more than one surface component of Salmonella bacteria improved the efficacy of the vaccine. The data from the passive immunization experiments also emphasized the role of humoral immunity for protection against S. typhimurium infection. Images

Svenson, S B; Nurminen, M; Lindberg, A A

1979-01-01

343

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3  

PubMed Central

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.

Martinez-Torrecuadrada, Jorge L.; Lazaro, Beatriz; Rodriguez, Jose F.; Casal, J. Ignacio

2000-01-01

344

B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites  

PubMed Central

Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines.

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F.; Barfod, Lea

2014-01-01

345

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.

1993-01-01

346

The Cancer/Testis Antigen Prostate-associated Gene 4 (PAGE4) Is a Highly Intrinsically Disordered Protein*  

PubMed Central

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.

Zeng, Yu; He, Yanan; Yang, Fan; Mooney, Steven M.; Getzenberg, Robert H.; Orban, John; Kulkarni, Prakash

2011-01-01

347

SEPPA 2.0-more refined server to predict spatial epitope considering species of immune host and subcellular localization of protein antigen.  

PubMed

Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported. PMID:24838566

Qi, Tao; Qiu, Tianyi; Zhang, Qingchen; Tang, Kailin; Fan, Yangyang; Qiu, Jingxuan; Wu, Dingfeng; Zhang, Wei; Chen, Yanan; Gao, Jun; Zhu, Ruixin; Cao, Zhiwei

2014-07-01

348

Requirements for the association of adenovirus type 2 E3/19K wild-type and mutant proteins with HLA antigens.  

PubMed Central

The E3/19K protein of human adenovirus type 2 is a resident of the endoplasmic reticulum (ER). Immediately after synthesis, it associates with major histocompatibility complex class I antigens and prevents their intracellular transport and cell surface expression. We have generated several C-terminal deletion mutants of the E3/19K protein that are preterminated at various positions on both sides of the membrane-spanning segment of the protein. One of these mutants is terminated at the luminal side of the membrane (M310), and two are terminated in the hydrophobic segment (M374 and M392), whereas mutant M621 is terminated on the cytoplasmic side of the ER membrane. The M310, M374, and M392 mutants are soluble proteins. They do not associate with HLA antigens in transfected 293 cells, and they are, to some extent, secreted into the medium. The M621 mutant protein is integrated in the ER membrane, associates immediately after its synthesis with HLA antigens, and exits from the ER. By using either an in vitro translation system supplemented with microsomes or overexpression in insect cells, we showed that M374 and E3/19K are able to associate with HLA antigens. These results indicate that the conformation of the luminal part of the E3/19K protein is not grossly altered by the mutations. Rapid transport of the M374 mutant out of the ER and partial degradation of this protein may prevent the interaction with HLA class I antigens in transfected 293 cells. Images

Gabathuler, R; Levy, F; Kvist, S

1990-01-01

349

Comparison of the Antigenicity and Protein Composition of Hepatitis B Virus e Antigen Subtypes HBeAg\\/1 and HBeAg\\/2  

Microsoft Academic Search

SUMMARY Hepatitis virus B e antigen subtypes HBeAg\\/1 and HBeAg\\/2 can be distinguished by a micro-Ouchterlony immunodiffusion assay and a solid-phase radioimmunoassay using serum IgG-associated HBeAgs and liver- and serum-derived IgG-free HBeAgs. The liver- and serum-derived HBeAg consisted of two different antigenicities, HBeAg\\/I and HBeAg\\/2. Both the liver-derived HBeAg\\/I and HBeAg\\/2 had mol. wt. of 30 000 (monomer) and 90

EI YAMADA; NAKAO ISHIDA; HITOSHI OHORI

1983-01-01

350

Performance of methods for the measurement of natural rubber latex (NRL) proteins, antigens and allergens  

Microsoft Academic Search

RationaleDetermination of natural rubber latex (NRL) allergenic proteins in medical and consumer products promotes rational selection of products to minimize sensitization. The objective of this multi-center study was to assess the variability and relative analytical performance of available laboratory methods to measure NRL proteins.

V. J. Tomazic-Jezic; D. H. Beezhold; H. Hashim; T. Palosuo; M. Raulf-Heimsoth; M. Swanson; R. G. Hamilton

2004-01-01

351

Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins.  

PubMed

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

2013-05-01

352

The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology1  

PubMed Central

Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins).

Wang, Weifang; Hause, Bettina; Peumans, Willy J.; Smagghe, Guy; Mackie, Anne; Fraser, Robin; Van Damme, Els J.M.

2003-01-01

353

Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis.  

PubMed

Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-?g and 50-?g doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis. PMID:24914218

Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M; Murphy, Timothy F

2014-08-01

354

Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein.  

PubMed Central

Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate to homogeneity, obtained the NH2-terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line lambda gt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NH2-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA+ cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types. Images

Shipp, M A; Richardson, N E; Sayre, P H; Brown, N R; Masteller, E L; Clayton, L K; Ritz, J; Reinherz, E L

1988-01-01

355

Babesia microti: molecular and antigenic characterizations of a novel 94-kDa protein (BmP94).  

PubMed

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis. PMID:20599995

Ooka, Hideo; Terkawi, Mohamad Alaa; Goo, Youn-Kyoung; Luo, Yuzi; Li, Yan; Yamagishi, Junya; Nishikawa, Yoshifumi; Igarashi, Ikuo; Xuan, Xuenan

2011-01-01

356

Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.  

PubMed

Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection. PMID:19969431

Mohamad, Khalil Yousef; Rekiki, Abdessalem; Berri, Mustapha; Rodolakis, Annie

2010-07-14

357

Vaccination of mice with an antigenic serine protease-like protein elicits a protective immune response against Trichinella spiralis infection.  

PubMed

Trichinellosis has major economic impacts on animal husbandry and food safety, and the control and elimination of trichinellosis is a major objective of veterinary medicine. A gene encoding serine protease of Trichinella spiralis (Ts-Adsp) was identified by immunoscreening an adult T. spiralis cDNA library. In this study, the recombinant Ts-Adsp protein (rTs-Adsp) was cloned and expressed in a prokaryotic expression system and purified by Ni-affinity chromatography. To determine whether the purified rTs-Adsp is a potential vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with this protein in combination with an alum adjuvant and subsequently challenged with T. spiralis larvae. The results showed that mice vaccinated with rTs-Adsp exhibited an average reduction in the muscle larvae burden of 46.5% relative to the control group. Immunization with the rTs-Adsp antigen induced both humoral and cellular immune responses, which manifested as elevated specific anti-rTs-Adsp IgG and IgE antibodies and a mixed Th1-Th2 response, as determined by Th1 (IFN-? and IL-2) and Th2 (IL-4, IL-10, and IL-13) cytokine profiling, with the Th2 predominant. Thus, purified rTs-Adsp is able to limit the invasion of T. spiralis , and this protein could be an effective vaccine candidate for trichinellosis. PMID:23252743

Feng, Shuang; Wu, Xiuping; Wang, Xuelin; Bai, Xue; Shi, Haining; Tang, Bin; Liu, Xiaolei; Song, Yanxia; Boireau, Pascal; Wang, Feng; Zhao, Ying; Liu, Mingyuan

2013-06-01

358

Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles.  

PubMed

Pneumococcal surface protein A (PspA) is a choline-binding protein which is a virulence factor found on the surface of all Streptococcus pneumoniae strains. Vaccination with PspA has been shown to be protective against a lethal challenge with S. pneumoniae, making it a promising immunogen for use in vaccines. Herein the design of a PspA-based subunit vaccine using polyanhydride nanoparticles as a delivery platform is described. Nanoparticles based on sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), specifically 50:50 CPTEG:CPH and 20:80 CPH:SA, were used to encapsulate and release PspA. The protein released from the nanoparticle formulations retained its primary and secondary structure as well as its antigenicity. The released PspA was also biologically functional based on its ability to bind to apolactoferrin and prevent its bactericidal activity against Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice they elicited a high titer and high avidity anti-PspA antibody response. Together these studies provide a framework for the rational design of a vaccine against S. pneumoniae based on polyanhydride nanoparticles. PMID:23774257

Haughney, Shannon L; Petersen, Latrisha K; Schoofs, Amy D; Ramer-Tait, Amanda E; King, Janice D; Briles, David E; Wannemuehler, Michael J; Narasimhan, Balaji

2013-09-01

359

Identification of two new antigen epitopes on the putative capsid protein encoded by torque teno sus virus type 1 ORF1.  

PubMed

Torque teno sus virus type 1 (TTSuV1) ORF1 is considered to encode the viral capsid (Cap) protein, which is crucial for the induction of TTSuV1-specific antibodies and protective immunity in the host. Eight monoclonal antibodies (mAbs) directed against the Cap protein were generated and biologically characterized. The immunoreactivity of the Cap protein expressed in transfected 293T cells for these mAbs was determined with an immunoperoxidase monolayer assay. The antigen epitopes of the Cap protein were mapped using these mAbs and truncated Cap proteins expressed in Escherichia coli. Fine epitope mapping was then performed with a panel of synthesized polypeptides. All the mAbs reacted with the Cap protein C fragment expressed in E. coli. One antigenic epitope of the Cap protein, which reacted with seven mAbs, had the polypeptide sequence (536)HPKYAGQGGGYTT(548), whereas another epitope recognized by the 1E9 mAb had the polypeptide sequence (549)EIGHQGITAASLR(561). It is interesting that the two new epitopes are adjacent, but mutually independent. This study should facilitate further investigation of the antigenic differences and enable the differential diagnosis of the virus. PMID:24172766

Liu, Jianbo; Zhang, Long; Huang, Liping; Wei, Yanwu; Wu, Hongli; Liu, Changming

2013-12-27

360

Identification of a meiosis-specific protein, MEIOB, as a novel cancer/testis antigen and its augmented expression in demethylated cancer cells.  

PubMed

Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen. Indeed, the MEIOB gene is expressed only in the testis and not in other normal tissues. The mRNA expression of MEIOB was greatly enhanced in several lung cancer cell lines after the treatment with DAC. Furthermore, we identified a variety of helper epitopes of the MEIOB antigen, which were recognized by MEIOB antigen-specific T cells in a HLA-restriction manner. Finally, we demonstrated that IFN-? production of MEIOB peptide-specific helper T cells in response to HLA-matched cancer cells was greatly augmented by treatment with DAC and IFN-?. Taken together, these findings show DAC to be a promising tool for finding novel CT antigens and for developing a future novel combination cancer vaccine chemotherapy. PMID:24440806

Shiohama, Yasuo; Ohtake, Junya; Ohkuri, Takayuki; Noguchi, Daisuke; Togashi, Yuji; Kitamura, Hidemitsu; Nishimura, Takashi

2014-01-01

361

The central nervous sytem-specific myelin oligodendrocytic basic protein (MOBP) is encephalitogenic and a potential target antigen in multiple sclerosis (MS)  

Microsoft Academic Search

Uncovering primary target antigens in multiple sclerosis (MS) is of major significance for understanding the etiology and pathophysiology of the disease, and for designing immunospecific therapy. In this study, a synthetic peptide representing a predicted T cell epitope on myelin oligodendrocytic basic protein (MOBP) was found to be encephalitogenic in C3H.SW mice, inducing experimental autoimmune encephalomyelitis with an abrupt onset.

Joel F Kaye; Nicole Kerlero de Rosbo; Itzhack Mendel; Shlomo Flechter; Michael Hoffman; Israel Yust; Avraham Ben-Nun

2000-01-01

362

Disease-specific analyses of unrelated cord blood transplantation compared with unrelated bone marrow transplantation in adult patients with acute leukemia.  

PubMed

We made a disease-specific comparison of unrelated cord blood (CB) recipients and human leukocyte antigen allele-matched unrelated bone marrow (BM) recipients among 484 patients with acute myeloid leukemia (AML; 173 CB and 311 BM) and 336 patients with acute lymphoblastic leukemia (ALL; 114 CB and 222 BM) who received myeloablative transplantations. In multivariate analyses, among AML cases, lower overall survival (hazard ratio [HR]=1.5; 95% confidence interval [CI], 1.0-2.0, P= .028) and leukemia-free survival (HR=1.5; 95% CI, 1.1-2.0, P= .012) were observed in CB recipients. The relapse rate did not differ between the 2 groups of AML (HR=1.2; 95% CI, 0.8-1.9, P= .38); however, the treatment-related mortality rate showed higher trend in CB recipients (HR=1.5; 95% CI, 1.0-2.3, P= .085). In ALL, there was no significant difference between the groups for relapse (HR=1.4, 95% CI, 0.8-2.4, P= .19) and treatment-related mortality (HR=1.0; 95% CI, 0.6-1.7, P= .98), which contributed to similar overall survival (HR=1.1; 95% CI, 0.7-1.6, P= .78) and leukemia-free survival (HR=1.2; 95% CI, 0.9-1.8, P= .28). Matched or mismatched single-unit CB is a favorable alternative stem cell source for patients without a human leukocyte antigen-matched related or unrelated donor. For patients with AML, decreasing mortality, especially in the early phase of transplantation, is required to improve the outcome for CB recipients. PMID:19104080

Atsuta, Yoshiko; Suzuki, Ritsuro; Nagamura-Inoue, Tokiko; Taniguchi, Shuichi; Takahashi, Satoshi; Kai, Shunro; Sakamaki, Hisashi; Kouzai, Yasushi; Kasai, Masaharu; Fukuda, Takahiro; Azuma, Hiroshi; Takanashi, Minoko; Okamoto, Shinichiro; Tsuchida, Masahiro; Kawa, Keisei; Morishima, Yasuo; Kodera, Yoshihisa; Kato, Shunichi

2009-02-19

363

Targeting and Therapy of Carcinoembryonic Antigen-expressing Tumors in Transgenic Mice with an Antibody-Interleukin 2 Fusion Protein1  

Microsoft Academic Search

The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic antigen (CEA) antibody and an interleukin 2 (IL-2) fusion protein derivative for selective tumor targeting of cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody (single-chain variable fragment joined to the crystallizable fragment, Fc (scFvFc)). The fusion protein (scFvFc.IL-2)

Xiaochuan Xu; Patrick Clarke; Gyorgy Szalai; John E. Shively; Lawrence E. Williams; Yu Shyr; Ergang Shi; F. James

2000-01-01

364

Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8+ T-Cell Epitopes  

Microsoft Academic Search

Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A (LMP2A) is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological

Bing Wang; Kun Yao; Genyan Liu; Fangyi Xie; Feng Zhou; Yun Chen

2009-01-01

365

Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.  

PubMed

Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri. PMID:19756751

Kim, Jong-Hyun; Yang, Ae-Hee; Sohn, Hae-Jin; Kim, Daesik; Song, Kyoung-Ju; Shin, Ho-Joon

2009-11-01

366

Discriminating antigen and non-antigen using proteome dissimilarity: bacterial antigens  

PubMed Central

It has been postulated that immunogenicity results from the overall dissimilarity of pathogenic proteins versus the host proteome. We have sought to use this concept to discriminate between antigens and non-antigens of bacterial origin. Sets of 100 known antigenic and nonantigenic peptide sequences from bacteria were compared to human and mouse proteomes. Both antigenic and non-antigenic sequences lacked human or mouse homologues. Observed distributions were compared using the non-parametric Mann-Whitney test. The statistical null hypothesis was accepted, indicating that antigen and non-antigens did not differ significantly. Likewise, we were unable to determine a threshold able to separate meaningfully antigen from non-antigen. Thus, antigens cannot be predicted from pathogen genomes based solely on their dissimilarity to the human genome.

Ramakrishnan, Kamna; Flower, Darren R

2010-01-01

367

An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses  

PubMed Central

The stable fly, Stomoxys calcitrans, is an economically important pest of livestock. Prior studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27 kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine if this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and if naïve calves can mount an immune response to it. Calves raised in the winter months were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 DPI in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab’)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive, but has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.

AMERI, M.; WANG, X.; WILKERSON, M. J.; KANOST, M. R.; BROCE, A. B.

2008-01-01