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Sample records for unrelated protein antigens

  1. Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins.

    PubMed

    Ramos, Gustavo C; Rodrigues, Claudiney M; Azevedo, Geraldo M; Pinho, Vanessa; Carvalho, Cláudia R; Vaz, Nelson M

    2009-03-01

    Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

  2. The Recognition of Identical Ligands by Unrelated Proteins

    PubMed Central

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

    2015-01-01

    The binding of drugs and reagents to off-targets is well-known. Whereas many off-targets are related to the primary target by sequence and fold, many ligands bind to unrelated pairs of proteins, and these are harder to anticipate. If the binding site in the off-target can be related to that of the primary target, this challenge resolves into aligning the two pockets. However, other cases are possible: the ligand might interact with entirely different residues and environments in the off-target, or wholly different ligand atoms may be implicated in the two complexes. To investigate these scenarios at atomic resolution, the structures of 59 ligands in 116 complexes (62 pairs in total), where the protein pairs were unrelated by fold but bound an identical ligand, were examined. In almost half of the pairs, the ligand interacted with unrelated residues in the two proteins (29 pairs), and in 14 of the pairs wholly different ligand moieties were implicated in each complex. Even in those 19 pairs of complexes that presented similar environments to the ligand, ligand superposition rarely resulted in the overlap of related residues. There appears to be no single pattern-matching “code” for identifying binding sites in unrelated proteins that bind identical ligands, though modeling suggests that there might be a limited number of different patterns that suffice to recognize different ligand functional groups. PMID:26421501

  3. Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil

    PubMed Central

    Bub, Carolina Bonet; Torres, Margareth Afonso; Moraes, Maria Elisa; Hamerschlak, Nelson; Kutner, José Mauro

    2015-01-01

    Background Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. Methods A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. Results Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group), partial compatible (one cross-reactive group) or less compatible (two cross-reactive group) donors, respectively. Conclusion The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services. PMID:26969768

  4. C-Reactive Protein and Cognition Are Unrelated to Leukoaraiosis

    PubMed Central

    Marques, Fabricio Correia; Rosset, Idiane; Moriguchi, Emilio Hideyuki; Picon, Paulo Dornelles; Chaves, Marcia Lorena Fagundes; Roriz-Cruz, Matheus

    2014-01-01

    Elevated serum levels of C-reactive protein (CRP) have been associated with leukoaraiosis in elderly brain. However, several studies indicate that leukoaraiosis is associated with an increased risk of cognitive impairment. It is unknown how the effect of CRP on cognition is mediated by leukoaraiosis. The purpose of this study is to assess the relationship between serum levels of CRP, the presence of leukoaraiosis, and cognitive impairment in a population of coronary patients over 50 years old. CRP levels explained 7.18% (P: 0.002) of the variance of the MMSE. The adjustment for the presence of leukoaraiosis little changed this variance (5.98%, P: 0.005), indicating that only a small portion of the CRP influence on cognition was mediated via leukoaraiosis. Patients with CRP levels ≥5.0 had 2.9 (95% CI: 1.26–6.44) times more chance to present cognitive impairment (P: 0.012). We found that elevated serum levels of CRP were associated with increased risk of cognitive impairment in elderly and it was not mediated by presence of leukoaraiosis. PMID:24587705

  5. Immune recognition of protein antigens

    SciTech Connect

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

    1985-01-01

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

  6. Allogeneic hematopoietic stem cell transplant for hematological malignancies from mismatched 9/10 human leukocyte antigen unrelated donors: comparison with transplants from 10/10 unrelated donors and human leukocyte antigen identical siblings.

    PubMed

    Michallet, Mauricette; Sobh, Mohamad; Serrier, Caroline; Morisset, Stéphane; Labussière, Hélène; Ducastelle, Sophie; Barraco, Fiorenza; Gilis, Lila; Thomas, Xavier; Nicolini, Franck E

    2015-04-01

    We studied the outcome of 213 patients who received allo-HSCT for hematological malignancies, 121 (57%) from HLA identical siblings, 63 (29%) from 10/10 HLA identical unrelated donors and 29 (14%) from 9/10 HLA mismatched unrelated donors. Engraftment was lower in the 9/10 HLA group (90%) than in the 10/10 HLA group (95%) than in the sibling group (99%); 3 months CI of aGVHD ≥ 2 was 32% (23-41), 20% (15-26) and 27% (23-32) respectively; the one year CI of extensive cGVHD was 21% (13-30), 9% (5-13) and 17% (14-21) respectively. The median OS was 10 months (5-21), 18 months (11-NR) and 60 months (31-NR) respectively with 2-years probability of 19% (8-44), 43% (31-59) and 63% (54-74) respectively. TRM was significantly higher in the 9/10 HLA group with 1 year CI of 45% (35-55), compared to 33% (27-39) in the unrelated 10/10 HLA group and 12% (9-15) in the identical siblings group (p < 0.001).

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. Impact of Human Leukocyte Antigen Allele Mismatch in Unrelated Bone Marrow Transplantation with Reduced-Intensity Conditioning Regimen.

    PubMed

    Yokoyama, Hisayuki; Kanda, Junya; Fuji, Shigeo; Kim, Sung-Won; Fukuda, Takahiro; Najima, Yuho; Ohno, Hitoshi; Uchida, Naoyuki; Ueda, Yasunori; Eto, Tetsuya; Iwato, Koji; Kobayashi, Hikaru; Ozawa, Yukiyasu; Kondo, Tadakazu; Ichinohe, Tatsuo; Atsuta, Yoshiko; Kanda, Yoshinobu

    2017-02-01

    The impact of HLA mismatch in hematopoietic stem cell transplantation with reduced-intensity conditioning (RIC) has not been fully examined. We analyzed a total of 1130 cases to examine the effects of HLA allele mismatch in unrelated bone marrow transplantation (BMT) with RIC in the Japan Marrow Donor Program registry cohort. Compared with HLA 8/8-allele match (n = 720, 8/8 match), both 1 (n = 295, 7/8 match) and 2 allele mismatches (n = 115, 6/8 match) were associated with significant reduction of overall survival (OS) (hazard ratio [HR],  1.34; P = .0024 and HR, 1.33; P = .035 for 7/8 and 6/8 match, respectively). The incidence of grades 2 to 4 acute graft-versus-host disease (aGVHD) increased with increasing number of mismatched alleles (HR, 1.36 and HR, 2.08 for 7/8 and 6/8 match, respectively). Nonrelapse mortality showed a similar tendency to aGVHD (HR, 1.35 for 7/8 and HR, 1.63 for 6/8). One-allele mismatches at the HLA-A or -B and HLA-C loci were significantly associated with inferior OS compared with 8/8 match (HR, 1.64 for A or B mismatch and HR, 1.41 for C mismatch), whereas HLA-DRB1 allele mismatch was not (HR, 1.16; P = .30). However, the effect of HLA-A or -B and -C mismatch on OS was not observed in those who received RIC BMT since 2010, in contrast to recipients before 2010. These results suggested that in unrelated RIC BMT, 1-allele mismatch is associated with poorer outcome, and the impact of HLA mismatch may differ depending on the HLA locus, although these HLA mismatch effects may be different in recent cases.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. Limited HLA sequence variation outside of antigen recognition domain exons of 360 10 of 10 matched unrelated hematopoietic stem cell transplant donor-recipient pairs.

    PubMed

    Hou, L; Vierra-Green, C; Lazaro, A; Brady, C; Haagenson, M; Spellman, S; Hurley, C K

    2017-01-01

    Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.

  12. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    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

    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 PMID:2659529

  13. [Common epitopes of protein antigens in Neisseria and Moraxella].

    PubMed

    Goncharenko, A V; Filatova, T N; Padiukov, L N; Baturo, A P

    2000-01-01

    Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.

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

    PubMed Central

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

    2012-01-01

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

  15. Changes in structural and antigenic properties of proteins by radiation

    NASA Astrophysics Data System (ADS)

    Kume, Tamikazu; Matsuda, Tsukasa

    1995-08-01

    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 [ θ] 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 [ θ] 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.

  16. Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

    NASA Astrophysics Data System (ADS)

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

    1990-04-01

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

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

    PubMed

    To, Ho; Nagai, Shinya

    2007-07-01

    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.

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

    PubMed

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

    2015-10-22

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

  19. Demonstration of heterogeneity among the antigenic proteins of Mobiluncus species.

    PubMed Central

    Schwebke, J R; Hillier, S L; Fohn, M J; Lukehart, S A

    1990-01-01

    The protein and antigenic profiles of the American Type Culture Collection type strains of Mobiluncus species and those of 114 clinical isolates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting with homologous polyvalent antisera. The majority of isolates (82%) possessed characteristic protein profiles and could be identified to the species level by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The major protein bands were also antigenic, and some antigenic cross-reactivity was noted between the two Mobiluncus species. All of the isolates were examined for reactivity with a panel of 12 monoclonal antibodies previously prepared against the type strains. While 56 of 60 clinical isolates of Mobiluncus curtisii (93%) reacted with one or more of the monoclonal antibodies, only 23 of 54 clinical isolates which were identified as Mobiluncus mulieris by biochemical methods (48%) reacted with one or more of the monoclonal antibodies. One of the 4 M. curtisii isolates (25%) and 11 of the 31 M. mulieris isolates (35%) which did not react with the monoclonal antibodies also had atypical protein profiles. These results demonstrate a high degree of heterogeneity in the protein and antigenic profiles of Mobiluncus isolates and suggest that further taxonomic division may be appropriate. Images PMID:1691207

  20. Antigenic differences in the H proteins of canine distemper viruses.

    PubMed

    Iwatsuki, K; Tokiyoshi, S; Hirayama, N; Nakamura, K; Ohashi, K; Wakasa, C; Mikami, T; Kai, C

    2000-02-01

    Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.

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

    PubMed

    Sampson, H A; Cooke, S

    1992-10-01

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

  2. Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

    SciTech Connect

    Pesando, J.M.; Hoffman, P.; Abed, M.

    1986-12-01

    Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, the authors identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate the 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the ..mu.. chain of immunoglobulin is B cell specific. Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.

  3. Surface proteins, ERAD and antigenic variation in Trypanosoma brucei.

    PubMed

    Tiengwe, Calvin; Muratore, Katherine A; Bangs, James D

    2016-11-01

    Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2-3 fold more is synthesized than required, the excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.

  4. Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae.

    PubMed

    Bowtell, D D; Mitchell, G F; Anders, R F; Lightowlers, M W; Rickard, M D

    1983-12-01

    Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

    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. PMID:20416366

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

    SciTech Connect

    Shanklin, J.; Somerville, C. )

    1991-03-15

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

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

    PubMed Central

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

    2016-01-01

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

  10. Retinal S Antigen Identified as the 48K Protein Regulating Light-Dependent Phosphodiesterase in Rods

    NASA Astrophysics Data System (ADS)

    Pfister, C.; Chabre, M.; Plouet, J.; Tuyen, V. V.; de Kozak, Y.; Faure, J. P.; Kuhn, H.

    1985-05-01

    Retinal S antigen chromatographically purified from whole retina, induces experimental autoimmune uveoretinitis in laboratory animals. The 48K protein, a soluble protein found in rod outer segments, is purified through its specific binding to photoexcited rhodopsin and is involved in the quenching of light-induced guanosine 3',5'-monophosphate-phosphodiesterase activity. Biochemical, immunological, functional, and pathological tests showed that retinal S antigen and the 48K protein are identical.

  11. Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

    PubMed

    Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

    2017-03-31

    We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

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

    SciTech Connect

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

    1987-03-01

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

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

    SciTech Connect

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

    2008-01-01

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

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

    PubMed

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

    2016-01-01

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

  15. Protein sorting within the MHC class II antigen-processing pathway.

    PubMed

    Marks, M S

    1998-01-01

    Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.

  16. Antibody specificity and antigen characterization of rat monoclonal antibodies against Streptococcus mutans cell wall-associated protein antigens.

    PubMed Central

    Ackermans, F; Klein, J P; Cormont, F; Bazin, H; Ogier, J A; Frank, R M; Vreven, J

    1985-01-01

    Monoclonal antibodies to Streptococcus mutans OMZ175 (serotype f) cell wall-associated antigens (wall-extracted antigens [WEA]) were derived from the fusion of Lou C plasmocytoma rat cells (IR 983 F) and spleen cells from Wistar R inbred rats immunized with WEA. Four cell lines producing monoclonal antibodies directed against a component of S. mutans WEA have been established. All four monoclonal antibodies reacted only with two antigens of WEA from S. mutans OMZ175 by Western blotting and immunoprecipitation techniques, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot analysis of WEA showed that the four monoclonal antibodies recognized two related cell wall-associated proteins with apparent molecular weights of 125,000 and 76,000. Immunoprecipitation of whole cells with the monoclonal antibodies confirmed the surface localization of the two antigens. The ELISA and competitive ELISA were used to analyze the distribution of the epitopes on seven S. mutans serotypes. All S. mutans serotypes were found to express the recognized epitopes; however, different reactivity patterns could be distinguished among the various strains tested, and the four monoclonal antibodies reacted only weakly with S. mutans serotypes d and g. Images PMID:2410364

  17. Murine T-Cell Response to Native and Recombinant Protein Antigens of Rickettsia Tsutsugamushi

    DTIC Science & Technology

    1993-02-01

    Wright, and J. Sadoff. 1985. 18-kilodalton protein of Mycobacterium leprae recognized by Immunoenzymatic analysis by monoclonal antibodies of bacte- Vo...determinants and closely resembles T-cell antigenic determinants, Rothbard and Taylor, by the GroEL homolog (65 kDa) of Mycobacterium tuberculo- analysis of...not be completely present in protein that is recognized by 20% of the mycobacterium - peptide 91-110. If this were the core of the antigenic deter

  18. Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells.

    PubMed Central

    Vapalahti, O; Lundkvist, A; Kallio-Kokko, H; Paukku, K; Julkunen, I; Lankinen, H; Vaheri, A

    1996-01-01

    Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes. PMID:8748286

  19. Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

    PubMed

    Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

    2014-03-15

    Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research.

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

    SciTech Connect

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

    1985-04-01

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

  1. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    PubMed Central

    Reglinski, Mark; Jose, Ricardo J.; Marshall, Helina; de Vogel, Corné; Gordon, Stephen; Petersen, Fernanda C.; Baxendale, Helen

    2017-01-01

    Naturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG), representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule. PMID:28135322

  2. Permeability of milk protein antigens across the intestinal epithelium in vitro.

    PubMed

    Marcon-Genty, D; Tomé, D; Dumontier, A M; Kheroua, O; Desjeux, J F

    1989-01-01

    Degradations by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer of food protein antigens to blood. These steps were studied in vitro for the milk protein antigens beta-lactoglobulin (beta-Lg), alpha-Lactalbumin (alpha-La) and beta-casein (beta-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin-trypsin hydrolysis showed an increasing resistance in the order beta-cas less than alpha-La less than beta-Lg. The rate of absorption of the antigenic proteins by isolated rabbit ileum was in the same order, and the rate of absorption of the whole proteins (degraded and antigenic forms) was significantly higher for beta-Lg than for alpha-La and beta-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity is probably important in the mechanism of food protein sensitization via the oral route.

  3. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis

    PubMed Central

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-01-01

    Background & objectives: Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Methods: Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. Results: For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Interpretation & conclusions: Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB. PMID:23287127

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

    PubMed

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

    2015-07-01

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

  5. The composed antigenic structure of the adenovirus hexon protein.

    PubMed

    Nász, I

    1988-01-01

    Three panels of MAbs were prepared against AV1, AV35, and BAV2 hexons, respectively. It was shown, that in all cases, antibodies are developed against the genus specific determinant of the adenovirus hexon. The other MAbs specified numerous identical or overlapping epitopes on the hexon types studied. The epitopes could be characterized as intertype-, intersubgenus-, subgenus- and type-specific ones beside the genus-specific determinant based on the RPs of the MAbs from indirect ELISA and passive HA results. The identical epitopes are present in more than one copy on the trimeric form of the hexon capsomer. The epitopes on the hexon molecule could be separated into three antigenic sites, of which one antigenic site is characterized by seven epitope clusters (antigenic site II). Monoclonal antibodies were able to precipitate different hexon types in gel diffusion tests by which the differentiation of the distinct epitopes seemed to be possible. With the help of monoclonal antibodies to AV1 hexon, 17 hours after the infection, hexons (or epitopes) were detected in the cytoplasm and in the nucleus of the infected cells showing different distribution patterns in indirect immunofluorescence assay.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Implication of antigenic conversion of Helicobacter pylori lipopolysaccharides that involve interaction with surfactant protein D.

    PubMed

    Yokota, Shin-ichi; Amano, Ken-ichi; Nishitani, Chiaki; Ariki, Shigeru; Kuroki, Yoshio; Fujii, Nobuhiro

    2012-08-01

    We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

  8. Protease digestion of hepatitis A virus: disparate effects on capsid proteins, antigenicity, and infectivity.

    PubMed Central

    Lemon, S M; Amphlett, E; Sangar, D

    1991-01-01

    High concentrations of either trypsin or chymotrypsin caused nearly complete cleavage of capsid protein VP2 of hepatitis A virus but did not significantly reduce the infectivity, thermostability, or antigenicity of the virus. Chymotrypsin also had a lesser effect on VP1. These findings indicate the presence of a protease-accessible VP2 surface site which neither contributes significantly to the dominant antigenic site nor plays a role in the attachment of the virus to putative cell receptors. Images PMID:1654460

  9. Blood Group Antigen Recognition via the Group A Streptococcal M Protein Mediates Host Colonization

    PubMed Central

    De Oliveira, David M. P.; Hartley-Tassell, Lauren; Everest-Dass, Arun; Day, Christopher J.; Dabbs, Rebecca A.; Ve, Thomas; Kobe, Bostjan; Nizet, Victor; Packer, Nicolle H.; Walker, Mark J.; Jennings, Michael P.

    2017-01-01

    ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally diverse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. PMID:28119471

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

    PubMed Central

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

    2015-01-01

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

  11. Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG.

    PubMed Central

    De Bruyn, J; Bosmans, R; Turneer, M; Weckx, M; Nyabenda, J; Van Vooren, J P; Falmagne, P; Wiker, H G; Harboe, M

    1987-01-01

    An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt.) Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two- and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization. Images PMID:3539805

  12. Biotechnology approaches to produce potent, self-adjuvanting antigen-adjuvant fusion protein subunit vaccines.

    PubMed

    Moyle, Peter Michael

    Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their

  13. Protein expression in yeast as an approach to production of recombinant malaria antigens.

    PubMed

    Bathurst, I C

    1994-01-01

    The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical. However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines. Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases. For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production. Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins. Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models. In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful. Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria.

  14. Capsid-Incorporation of Antigens into Adenovirus Capsid Proteins for a Vaccine Approach

    PubMed Central

    Matthews, Qiana L.

    2010-01-01

    Some viral vectors are potent inducers of cellular and humoral responses; therefore, viral vectors can be used to vaccinate against cancer or infectious diseases. This report will focus on adenovirus (Ad)-based vectors. Traditional viral-vector vaccination embodies the concept that the vector uses the host-cell machinery to express antigens that are encoded as transgenes within the viral vector. Several preclinical successes have used this approach in animal model systems. However, in some instances, these conventional Ad-based vaccines have yielded suboptimal clinical results. These suboptimal results are ascribed, in part, to preexisting Ad serotype 5 (Ad5) immunity. To address this issue, the “antigen capsid-incorporation” strategy has been developed to circumvent the drawbacks associated with conventional transgene expression of antigens by Ad vectors. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. Incorporating immunogenic peptides into the Ad capsid offers potential advantages. Importantly, vaccination by means of the antigen capsid-incorporated approach results in a strong humoral response, similar to the response generated by native Ad capsid proteins. This strategy also allows for the boosting of antigenic specific responses. This strategy may be the way forward for improved vaccine schemes, especially for those infections requiring a strong humoral antigenic response. PMID:21047139

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2002-11-01

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

  17. Identification of a 68-kilodalton protective protein antigen from Bordetella bronchiseptica.

    PubMed Central

    Montaraz, J A; Novotny, P; Ivanyi, J

    1985-01-01

    A 68-kilodalton (kd) outer membrane protein antigen of Bordetella bronchiseptica has been identified by using monoclonal antibodies that recognized two nonoverlapping determinants. Antibody BB05 also reacted with homologous proteins from Bordetella pertussis and Bordetella parapertussis but not with another 12 organisms from various bacterial genera. Passive injection of BB05 antibody protected mice from aerosol infection with B. bronchiseptica as shown by reduced mortality and reduced pathology of turbinate bones. The 68-kd B. bronchiseptica antigen was purified by BB05-based affinity chromatography and evaluated for its potency to immunize mice actively against either intraperitoneal or aerosol challenge with B. bronchiseptica. Immunization with the 68-kd antigen in incomplete Freund adjuvant significantly reduced the levels of mortality in intraperitoneally challenged mice. In the aerosol infection model, injection of the 68-kd antigen with complete or incomplete Freund adjuvant or saponin reduced the bacterial counts in the lungs of infected mice. These results suggest that the 68-kd protein may represent a potential "protective" antigen of B. bronchiseptica. Images PMID:3972452

  18. Western blot analysis of antibody response to pneumococcal protein antigens in a murine model of pneumonia.

    PubMed Central

    Mouneimne, H; Juvin, M; Beretti, J L; Azoulay-Dupuis, E; Vallee, E; Geslin, P; Petitpretz, P; Berche, P; Gaillard, J L

    1997-01-01

    To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcal protein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera were tested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulin G-type response against a small number of polypeptides. The antibody response was strain dependent: sera from individuals infected with the same strain gave similar banding patterns on immunoblots. The banding patterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by all reactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved among the different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective of the capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to 38-kDa protein antigen may be of value for the development of a serological test for humans. PMID:9384307

  19. The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity.

    PubMed

    Park, S K; Yun, D H; Chung, J Y; Kong, Y; Cho, S Y

    2000-09-01

    Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.

  20. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products

    PubMed Central

    1990-01-01

    A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well- characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as

  1. [Hyperprolactinemia unrelated to prolactinoma].

    PubMed

    Krysiak, Robert; Okopień, Bogusław

    2014-01-01

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

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2002-06-05

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

    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

    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. PMID:19666533

  7. Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

    PubMed

    Larsson, K; Hofström, C; Lindskog, C; Hansson, M; Angelidou, P; Hökfelt, T; Uhlén, M; Wernérus, H; Gräslund, T; Hober, S

    2011-07-29

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

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

    PubMed Central

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

    2016-01-01

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

  9. Antigenic Structure of Outer Membrane Protein E of Moraxella catarrhalis and Construction and Characterization of Mutants

    PubMed Central

    Murphy, Timothy F.; Brauer, Aimee L.; Yuskiw, Norine; Hiltke, Thomas J.

    2000-01-01

    Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella catarrhalis which possesses several characteristics indicating that the protein will be an effective vaccine antigen. To study the antigenic structure of OMP E, eight monoclonal antibodies were developed and characterized. Three of the antibodies recognized epitopes which are present on the bacterial surface. Fusion peptides corresponding to overlapping regions of OMP E were constructed, and immunoblot assays were performed to localize the areas of the molecule bound by the monoclonal antibodies. These studies identified a surface-exposed epitope in the region of amino acids 80 through 180. To further study the protein, two mutants which lack OMP E were constructed. In bactericidal assays, the mutants were more readily killed by normal human serum compared to the isogenic parent strains. These results indicate that OMP E is involved in the expression of serum resistance of M. catarrhalis. PMID:11035732

  10. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    PubMed

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

  11. Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

    PubMed

    Mokhtarian, Kobra; Akhlaghi, Lame; Meamar, Ahmad Reza; Razmjou, Elham; Manouchehri Naeini, Kourosh; Gholami, Samaneh; Najafi Samei, Masoomeh; Falak, Reza

    2016-08-01

    In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

  12. Purification, characterization, and localization of a protein antigen shared by thermophilic campylobacters.

    PubMed Central

    Dubreuil, J D; Kostrzynska, M; Logan, S M; Harris, L A; Austin, J W; Trust, T J

    1990-01-01

    A protein antigen with an apparent molecular weight (Mr) of 31,000 was isolated from 0.2 M glycine hydrochloride (pH 2.2) extracts of a typical human fecal isolate, Campylobacter jejuni VC74. The protein was purified to homogeneity on a preparative scale by immunoaffinity chromatography followed by molecular sieving with a Superose 12 column. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.3, and amino acid composition analysis showed that the protein was unusually rich in lysine, containing 14.9 mol% of this basic amino acid. Cysteine and tryptophan were absent. The protein also contained approximately 35% hydrophobic amino acid residues, and N-terminal amino acid analysis showed that 17 of the first 38 residues were hydrophobic. This amino-terminal sequence to residue 22 was virtually identical to that of an antigenically cross-reactive 31,000-Mr protein isolated from another C. jejuni strain belonging to a different heat-labile serogroup. Western blotting (immunoblotting) of glycine extracts of other C. jejuni, Campylobacter coli, and Campylobacter laridis strains belonging to different thermolabile and thermostable serotypes, as well as Campylobacter fetus, with a rabbit polyclonal antiserum raised against the purified C. jejuni VC74 protein showed that all C. jejuni, C. coli, and C. laridis strains tested contained a 31,000-Mr protein with epitopes which were antigenically cross-reactive with the C. jejuni VC74 protein. The antigenically cross-reactive epitopes of this protein were also readily detected by immunodot blot assay of glycine extracts of C. jejuni, C. coli, and C. laridis with monospecific polyclonal antisera to the 31,000-Mr protein, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of these important pathogens. Slide agglutination reactions, immunofluorescence assay, and immunogold electron microscopy with antisera to purified 31,000-Mr protein and

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

    PubMed

    Kim, Namsoo; Cho, Yong-Jin

    2013-03-01

    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.

  14. Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering.

    PubMed

    Dalgliesh, Ailsa J; Liu, Zhi Zhao; Griffiths, Leigh G

    2017-03-10

    Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens

    PubMed Central

    1992-01-01

    The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3- expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial

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

    PubMed Central

    2014-01-01

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

  20. Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection.

    PubMed

    Kalenda, Yombo Dan Justin; Kato, Kentaro; Goto, Yasuyuki; Fujii, Yoshito; Hamano, Shinjiro

    2015-12-01

    The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for sero-diagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1990-01-01

    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 PMID:2117568

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

    SciTech Connect

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

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1982-06-01

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

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

    PubMed

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

    2015-09-01

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

  8. Purification, partial characterization, and seroreactivity of a genuswide 60-kilodalton Legionella protein antigen.

    PubMed Central

    Pau, C P; Plikaytis, B B; Carlone, G M; Warner, I M

    1988-01-01

    A genuswide protein antigen extracted from Legionella pneumophila serogroup 1 (strain Philadelphia 1) cells was enriched by differential pelleting and ammonium sulfate precipitation and subsequently purified with a combination of high-performance size-exclusion and ion-exchange chromatography. The protein has an apparent molecular weight of 650,000 before and 63,000 after urea (5 M) treatment, as determined by size-exclusion chromatography. These proteins resolved to a single band of 60,000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The urea-treated protein had an isoelectric point of 5.8. This purified 60-kilodalton protein reacted with a convalescent-phase serum sample from a patient with legionellosis and rabbit immune sera prepared against each of 23 Legionella species. The 60-kilodalton protein may be useful in developing diagnostic tests for legionellosis. Images PMID:3343316

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

    PubMed

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

    2014-01-01

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

  10. Contribution of Redox Status to Hepatitis C Virus E2 Envelope Protein Function and Antigenicity*

    PubMed Central

    Fenouillet, Emmanuel; Lavillette, Dimitri; Loureiro, Silvia; Krashias, George; Maurin, Guillemette; Cosset, François-Loïc; Jones, Ian M.; Barbouche, Rym

    2008-01-01

    Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5′-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design. PMID:18667425

  11. Food allergy and the potential allergenicity-antigenicity of microparticulated egg and cow's milk proteins.

    PubMed

    Sampson, H A; Cooke, S K

    1990-08-01

    Approximately 3-4 million Americans experience food allergic reactions at some time in their lives. In the pediatric population, eggs and milk are most frequently implicated in food allergic reactions. The most well-understood adverse reactions to foods are secondary to the development of IgE antibodies to specific food antigens. Once an individual becomes sensitized (i.e., makes specific IgE antibodies), ingestion of the food may lead to a variety of cutaneous, respiratory, and/or gastrointestinal symptoms, and anaphylactic shock. The use of SDS-PAGE and immunoblot analyses with sera from documented food allergic patients provide a very sensitive indicator of the antigenic/allergic composition of various foods. As demonstrated in a study of infant formulas of hydrolyzed cow's milk protein, the absence of demonstrable bands on SDS-PAGE gels and immunoblots correlates with an inability to provoke an allergic response. In addition, it was demonstrated that SDS-PAGE with silver staining could detect protein fractions at a concentration of 50-100 ng/ml, a concentration below which allergic individuals are unlikely to react. These studies confirmed that patients clinically allergic to egg and/or cow's milk possess IgE and IgG antibodies to protein fractions in egg and cow's milk, as well as the microparticulated egg/cow's milk proteins, Simplesse and Beta IL. Compared to egg and cow's milk, there is no evidence that the Simplesse or Beta IL test materials possess any "novel" protein fractions or antigens. In addition, there is no evidence that these microparticulated proteins result in increased immunologic activity, as determined by the intensity of protein band staining.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2005-06-01

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

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

    PubMed

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

    2015-12-01

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

  14. Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

    PubMed

    Chan, Kun-Wei; Hsieh, Hsien-Hua; Wang, Hsien-Chi; Lee, Ya-Jane; Sung, Ming-Hua; Wong, Min-Liang; Hsu, Wei-Li

    2009-01-01

    Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

  15. Functional variation of the antigen I/II surface protein in Streptococcus mutans and Streptococcus intermedius.

    PubMed

    Petersen, F C; Assev, S; van der Mei, H C; Busscher, H J; Scheie, A A

    2002-01-01

    Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

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

    PubMed

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

    2007-09-01

    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

  17. Cellular proteins that associate with the middle and small T antigens of polyomavirus.

    PubMed

    Pallas, D C; Cherington, V; Morgan, W; DeAnda, J; Kaplan, D; Schaffhausen, B; Roberts, T M

    1988-11-01

    We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.

  18. Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

    PubMed

    Kästner, Julia; Saluz, Hans Peter; Hänel, Frank

    2015-05-01

    Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1986-03-05

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

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

    PubMed

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

    2004-12-01

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

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

    PubMed

    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

    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.

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

    PubMed Central

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

    2015-01-01

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

  4. Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71.

    PubMed

    Zhang, Jianhua; Dong, Min; Jiang, Bingfu; Dai, Xing; Meng, Jihong

    2012-08-01

    The complete VP1 protein of enterovirus 71 (EV71) and a series of truncations were expressed in Escherichia coli and their antigenic characteristics were studied. Immunoblot analysis showed the major immunoreactive region of the VP1 protein was located in the N-terminal portion at position of amino acid (aa) 1-100. The complete VP1 possessed strong cross-reactivity with antisera against coxsackievirus A16 (CA16) and echovirus 6 (Echo6), while the truncated fragment at position 1-100 aa only had weak cross-reactivity. Moreover, an EV71-specific linear epitope at position 94-105 aa was identified using two EV71-specific mAbs (2B9 and 5B7) with indirect ELISA, but could not be recognized by antibodies against EV71 virus particles. The complete and all of truncated VP1 proteins except His-VP1(202-297) and GST-VP1(202-248) failed to elicit a significant neutralizing antibody response in mice. His-VP1(202-297) and GST-VP1(202-248) containing neutralizing epitope(s) could be recognized only by anti-EV71 mouse sera but not rabbit or human sera. These findings may contribute to a further understanding of antigenic characteristics of the capsid protein VP1 and may be helpful to the development of diagnostic reagents and vaccines.

  5. Cross-linking analysis of antigenic outer membrane protein complexes of Neisseria meningitidis.

    PubMed

    Sánchez, Sandra; Abel, Ana; Arenas, Jesús; Criado, María Teresa; Ferreirós, Carlos M

    2006-03-01

    Polysaccharide-based approaches have not enabled the development of effective vaccines against meningococci of serogroup B, and the most promising current research is focused on the use of outer membrane vesicles. Due to the toxicity of the outer membrane oligosaccharides, new vaccines based on purified proteins are being sought, but despite the application of advanced techniques, they remain elusive, perhaps due to the fact that standard techniques for analysis of antigens overlook conformational epitopes located in membrane complexes. Membrane complex antigens have been analyzed in Neisseria gonorrhoeae, and a study published on Neisseria meningitidis has reported the in vitro formation of 800-kD complexes by deposition of a purified protein (MSP63) onto synthetic lipid layers; however, no studies to date have attempted to identify membrane complexes present in vivo in N. meningitidis. In the present study, cross-linking with formaldehyde was used to identify outer membrane protein associations in various N. meningitidis and Neisseria lactamica strains. In N. meningitides, complexes of about 450 kD (also present in N. lactamica), 165 and 95 kD were detected and shown to be made up of the proteins MSP63, PorA/PorB/RmpM/FetA, and PorA/PorB/RmpM, respectively. In western blots, the 450-kD complex was identified by mouse antibodies raised against outer membrane vesicles, but not by antibodies raised against the purified complex, demonstrating the importance of conformational epitopes, and thus suggesting that the analysis of antigens in their native conformation may be useful or even essential for the design of effective vaccines against meningococci.

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

    PubMed

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

    2006-02-22

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

  7. Influenza virus-like particles engineered by protein transfer with tumor-associated antigens induces protective antitumor immunity.

    PubMed

    Patel, Jaina M; Vartabedian, Vincent F; Kim, Min-Chul; He, Sara; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-06-01

    Delivery of antigen in particulate form using either synthetic or natural particles induces stronger immunity than soluble forms of the antigen. Among naturally occurring particles, virus-like particles (VLPs) have been genetically engineered to express tumor-associated antigens (TAAs) and have shown to induce strong TAA-specific immune responses due to their nano-particulate size and ability to bind and activate antigen-presenting cells. In this report, we demonstrate that influenza VLPs can be modified by a protein transfer technology to express TAAs for induction of effective antitumor immune responses. We converted the breast cancer HER-2 antigen to a glycosylphosphatidylinositol (GPI)-anchored form and incorporated GPI-HER-2 onto VLPs by a rapid protein transfer process. Expression levels on VLPs depended on the GPI-HER-2 concentration added during protein transfer. Vaccination of mice with protein transferred GPI-HER-2-VLPs induced a strong Th1 and Th2-type anti-HER-2 antibody response and protected mice against a HER-2-expressing tumor challenge. The Soluble form of GPI-HER-2 induced only a weak Th2 response under similar conditions. These results suggest that influenza VLPs can be enriched with TAAs by protein transfer to develop effective VLP-based subunit vaccines against cancer without chemical or genetic modifications and thus preserve the immune stimulating properties of VLPs for easier production of antigen-specific therapeutic cancer vaccines.

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

    PubMed

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

    2016-01-01

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

  9. Stabilization of a model formalinized protein antigen encapsulated in poly(lactide-co-glycolide)-based microspheres.

    PubMed

    Jiang, W; Schwendeman, S P

    2001-10-01

    A formaldehyde-mediated aggregation pathway (FMAP) has been shown to be primarily responsible for the solid-state aggregation of lyophilized formalinized protein antigens [e.g., tetanus toxoid (TT) and formalinized bovine serum albumin (f-BSA)] in the presence of moisture and physiological temperature. Coincorporation of the formaldehyde-interacting amino acid, histidine, strongly inhibits the FMAP. The purpose of this study was to test whether previous solid-state data are applicable toward the stabilization of formalinized antigens encapsulated in poly(lactide-co-glycolide) (PLGA)-based microspheres. Formaldehyde-treated bovine serum albumin (f-BSA) and BSA were selected as a model formalinized protein antigen and a nonformalinized control, respectively. As in the solid state, we found that the FMAP was dominant in the aggregation of f-BSA encapsulated in PLGA 50/50 microspheres, whereas the aggregation mechanism of encapsulated BSA was mostly converted from thiol-disulfide interchange to an acid-catalyzed noncovalent pathway. The lack of noncovalent aggregation in encapsulated f-BSA could be explained by its higher thermodynamic stability after formalinization, which inhibits protein unfolding. Targeting the FMAP, coencapsulation of histidine and trehalose successfully inhibited the aggregation of f-BSA in microspheres. By combining the use of an optimized oil-in-oil (o/o) encapsulation method, coencapsulation of histidine and trehalose, and use of low-acid-content poly(D,L-lactide) (PLA) and poly(ethylene glycol) (PEG) blends, a 2-month continuous release of f-BSA was achieved with the absence of aggregation.

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

    PubMed

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

    2005-09-01

    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.

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

    PubMed

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

    2015-05-19

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

  12. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    PubMed

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

    2013-01-01

    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.

  13. Multidrug-resistance proteins are weak tumor associated antigens for colorectal carcinoma

    PubMed Central

    2011-01-01

    Background Multidrug resistance (MDR) is a clinically, highly relevant phenomenon. Under chemotherapy many tumors show an increasing resistance towards the applied substance(s) and to a certain extent also towards other agents. An important molecular cause of this phenomenon is an increased expression of transporter proteins. The functional relationship between high expression levels and chemotherapy resistance makes these MDR and MRP (MDR related protein) proteins to interesting therapeutic targets. We here wanted to systematically analyze, whether these proteins are tumor specific antigens which could be targeted immunologically. Results Using the reverse immunology approach, 30 HLA-A2.1 restricted MDR and MRP derived peptides (MDP) were selected. Stimulated T cell lines grew well and mainly contained activated CD8+ cells. Peptide specificity and HLA-A2.1 restriction were proven in IFN-γ-ELISpot analyses and in cytotoxicity tests against MDP loaded target cells for a total of twelve peptides derived from MDR-1, MDR-3, MRP-1, MRP-2, MRP-3 and MRP-5. Of note, two of these epitopes are shared between MDR-1 and MDR-3 as well as MRP-2 and MRP-3. However, comparably weak cytotoxic activities were additionally observed against HLA-A2.1+ tumor cells even after upregulation of MDR protein expression by in vitro chemotherapy. Conclusions Taken together, these data demonstrate that human T cells can be sensitised towards MDPs and hence, there is no absolute immunological tolerance. However, our data also hint towards rather low endogenous tumor cell processing and presentation of MDPs in the context of HLA-A2.1 molecules. Consequently, we conclude that MDR and MRP proteins must be considered as weak tumor specific antigens-at least for colorectal carcinoma. Their direct contribution to therapy-failure implies however, that it is worth to further pursue this approach. PMID:21740599

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

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

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

  15. The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis.

    PubMed

    Mukaigasa, Katsuki; Hanasaki, Akira; Maéno, Mitsugu; Fujii, Hiroshi; Hayashida, Shin-ichiro; Itoh, Mari; Kobayashi, Makoto; Tochinai, Shin; Hatta, Masayuki; Iwabuchi, Kazuya; Taira, Masanori; Onoé, Kazunori; Izutsu, Yumi

    2009-10-27

    Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.

  16. Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

    PubMed Central

    Beck, M A; Tracy, S M

    1989-01-01

    Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens. PMID:2476566

  17. 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

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

    1990-01-01

    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 PMID:2108930

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

    PubMed Central

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

    2005-01-01

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

  19. Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.

    PubMed

    Lewis, J B; Thompson, Y G; Feng, X; Holden, V R; O'Callaghan, D; Caughman, G B

    1997-04-14

    The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

  20. Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies.

    PubMed

    Shin, Gee-Wook; Nho, Seong-Won; Park, Seong-Bin; Jang, Ho-Bin; Cha, In-Seok; Ha, Mi-Ae; Kim, Young-Rim; Dalvi, Rishikesh S; Joh, Seong-Joon; Jung, Tae-Sung

    2009-10-20

    Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

  1. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

    PubMed Central

    Wieczorek, Marek; Abualrous, Esam T.; Sticht, Jana; Álvaro-Benito, Miguel; Stolzenberg, Sebastian; Noé, Frank; Freund, Christian

    2017-01-01

    Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

  2. Protective activity of the purified protein antigen of Erysipelothrix rhusiopathiae in pigs.

    PubMed

    Yamazaki, Y; Sato, H; Sakakura, H; Shigeto, K; Nakano, K; Saito, H; Maehara, N

    1999-02-01

    We purified the protein antigen (P64), which contains 66 and 64 kDa proteins, from the alkaline extract (AE) of whole cells of Erysipelothrix rhusiopathiae strain Agata (serovar 5) to determine the protective activity of the antigen against E. rhusiopathiae infection in pigs. The serum titre of antibody against P64 rapidly increased in pigs immunized with 500 and 100 micrograms of P64 and reached maximum values at 3 weeks after the first immunization (1 week after the second immunization). However, the serum antibody titres were not increased in pigs immunized with 20 micrograms of P64 and in nonimmunized pigs. In the pigs immunized with live cell vaccine (acriflavin-fast attenuated strain Koganei 65-0.15), the serum titres of antibody against P64 also increased at 1-2 weeks after immunization. In a pig challenge test performed on immunized and nonimmunized pigs, all nonimmunized pigs showed typical clinical signs of swine erysipelas (fever, erysipeloid, arthritis), while all pigs immunized with 500 and 100 micrograms of P64 and live cell vaccine showed no clinical signs of this disease. In Western blot analysis, sera from pigs immunized with P64 and live cell vaccine strongly reacted with the 64 kDa protein. In contrast, the serum from nonimmunized pigs did not react with any proteins. From these results, it was suggested that a specific antibody against the 64 kDa protein could be increased in pigs immunized with P64 or live cell vaccine and that this anti-P64 antibody has a strong protective effect against E. rhusiopathiae infection in pigs.

  3. N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

    PubMed Central

    Puddington, L; Bevan, M J; Rose, J K; Lefrançois, L

    1986-01-01

    The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed. Images PMID:3022003

  4. Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division.

    PubMed

    Barrôco, R M; De Veylder, L; Magyar, Z; Engler, G; Inzé, D; Mironov, V

    2003-02-01

    Although the majority of cyclin-dependent kinases (CDKs) play a key role in cell cycle progression, recent evidence has shown that CDKs are also implicated in transcription regulation. Here, we describe two Arabidopsis CDKs designated Arath;CDKC;1 and Arath; CDKC;2. These CDKs share a PITAIRE signature in the cyclin-binding domain and the structural characteristics of mammalian CDK9. Yeast two-hybrid screens and immunoprecipitation assays identified CDKC-interacting proteins with homology to the animal cyclin T/cyclin K group. We suggest that these Arabidopsis CDKCs may be part of a kinase complex similar to the animal positive transcription elongation factor b, whose activity is essential for transcription control. Expression studies showed that Arath; CDKC transcripts are mainly confined to epidermal tissues and are most abundant in flower tissues. No expression was detected in actively dividing Arabidopsis tissues, suggesting a role for the CDKC proteins in differentiated cells.

  5. 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

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

    1992-01-01

    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. PMID:1537894

  6. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  7. Synthesis of protein-loaded hydrogel particles in an aqueous two-phase system for coincident antigen and CpG oligonucleotide delivery to antigen-presenting cells.

    PubMed

    Jain, Siddhartha; Yap, Woon Teck; Irvine, Darrell J

    2005-01-01

    Materials that effectively deliver protein antigens together with activating ligands to antigen-presenting cells are sought for improved nonviral vaccines. To this end, we synthesized protein-loaded poly(ethylene glycol) (PEG)-based hydrogel particles by cross-linking PEG within the polymer-rich phase of an emulsion formed by a poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) triblock copolymer in saturated aqueous salt solution. These particles (500-nm diameter) contained high levels of encapsulated protein (approximately 75% of dry mass), which was selectively released by proteolytic enzymes normally present in the phagosomal/endosomal compartments of dendritic cells (DCs). For co-delivery of cellular activation signals, gel particles were surface-modified by sequential adsorption of poly(l-arginine) and CpG oligonucleotides. DCs pulsed with protein-loaded particles activated naïve T cells in vitro approximately 10-fold more efficiently than DCs incubated with soluble protein. This organic solvent-free strategy for protein encapsulation within submicron-sized hydrophilic particles is attractive for macromolecule delivery to a variety of phagocytic and nonphagocytic cells.

  8. Characterisation and differentiation of pathogenic and non-pathogenic Acanthamoeba strains by their protein and antigen profiles.

    PubMed

    Walochnik, J; Sommer, K; Obwaller, A; Haller-Schober, E-M; Aspöck, H

    2004-03-01

    Free-living amoebae of the genus Acanthamoeba are the causative agents of Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis. Acanthamoebae occur ubiquitously in the environment and are thus a constant cause of antigenic stimulation. In a previous study we have shown that compared to control sera, AK patients exhibit markedly lower immunoreactivities to whole cell antigen of Acanthamoeba spp. As the pathogenicity of acanthamoebae primarily relies on the excretion of proteins, it was the aim of the present study to investigate the immunoreactivity of metabolic antigen from different Acanthamoeba strains of varying pathogenicity. Three Acanthamoeba strains, one highly pathogenic, one non-pathogenic but thermophilic and one non-thermophilic non-pathogenic, were used for antigen extraction. The antigen was harvested before and after contact with human cells and all strains were tested with AK sera and with sera from healthy individuals. It was shown that the somatic protein profiles of the Acanthamoeba strains correlated to the morphological groups, and that within morphological group II-the group associated with AK-the profiles of the metabolic antigens correlated to strain pathogenicity. Moreover, it was shown that the control sera showed markedly higher immunoreactivities than the sera of the AK patients and that this immunoreactivity was generally higher to the non-pathogenic strains than to the pathogenic strain. Altogether our results once again raise the question of whether there is an immunological predisposition in AK. To our knowledge this is the first study on the immunoreactivity of metabolic antigen of acanthamoebae.

  9. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii.

    PubMed Central

    Saavedra, R; Becerril, M A; Dubeaux, C; Lippens, R; De Vos, M J; Hérion, P; Bollen, A

    1996-01-01

    The ROP2 protein of Toxoplasma gondii possesses immunological and biological properties which have led to its proposal as a vaccine candidate. To identify epitopes recognized by human T cells in the ROP2 antigen, we submitted the sequence of this protein to three reported T-cell epitope prediction algorithms. Three sequences that were predicted by all three methods were selected (sequences 197 to 216, 393 to 410, and 501 to 524), and the corresponding peptides were synthesized. The peptides were first tested in a proliferation assay with a DPw4-restricted, ROP2-specific human T-cell clone, and the peptide corresponding to residues 197 to 216 was shown to stimulate the T-cell clone. The three peptides were further tested in proliferation assays with peripheral blood mononuclear cells from a panel of T. gondii-seropositive and -seronegative individuals. We found that cells from a high proportion of the seropositive donors (64%) recognized at least one of the three peptides. The most frequently recognized ones were peptides 197 to 216 (45%) and 501 to 524 (36%). None of the seronegative donors responded to any peptide. These results show that the ROP2 antigen of T. gondii contains T-cell epitopes recognized by a high percentage of the immune population and further strengthen its potential as a vaccine candidate. PMID:8751939

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

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

    SciTech Connect

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

    1986-08-01

    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.

  12. Positive and negative regulation of antigen receptor signaling by the Shc family of protein adapters.

    PubMed

    Finetti, Francesca; Savino, Maria Teresa; Baldari, Cosima T

    2009-11-01

    The Shc adapter family includes four members that are expressed as multiple isoforms and participate in signaling by a variety of cell-surface receptors. The biological relevance of Shc proteins as well as their variegated function, which relies on their highly conserved modular structure, is underscored by the distinct and dramatic phenotypic alterations resulting from deletion of individual Shc isoforms both in the mouse and in two model organisms, Drosophila melanogaster and Caenorhabditis elegans. The p52 isoform of ShcA couples antigen and cytokine receptors to Ras activation in both lymphoid and myeloid cells. However, the recognition of the spectrum of activities of p52ShcA in the immune system has been steadily expanding in recent years to other fundamental processes both at the cell and organism levels. Two other Shc family members, p66ShcA and p52ShcC/Rai, have been identified recently in T and B lymphocytes, where they antagonize survival and attenuate antigen receptor signaling. These developments reveal an unexpected and complex interplay of multiple Shc proteins in lymphocytes.

  13. TSOL18/HP6-Tsol, an immunogenic Taenia solium oncospheral adhesion protein and potential protective antigen.

    PubMed

    Parkhouse, R Michael E; Bonay, Pedro; González, Luis Miguel; Ferrer, Elizabeth; Gárate, Teresa; Aguilar, Cruz M; Cortez A, Milagros M; Harrison, Leslie J S

    2008-04-01

    In this study, we employed Taenia solium mRNA extracted from a tapeworm of Venezuelan origin to clone express and test the recombinant protein of the T. solium homologue of the 18-kDa oncospheral adhesion molecule of Taenia saginata (HP6-Tsag/TSA18). We first confirm the conserved nature of the sequence of the T. solium homologue (TSOL18/HP6-Tsol) and demonstrate that the recombinant protein, which, as with its T. saginata homologue, is characterised by a fibronectin type III homology region, functions as an adhesion molecule. This emphasises the possible importance of TSOL18/HP6-Tsol in tissue invasion, thus providing a rational explanation for its efficacy as a vaccine. As protection against Taenia spp., oncospheres is antibody mediated, logically, therefore, TSOL18/HP6-Tsol may also serve as a diagnostic antigen, as is indeed the case for recombinant HP6-Tsag/TSA18.

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

    PubMed

    Vzorov, A N; Compans, R W

    2016-01-01

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

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

    PubMed

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

    2002-12-01

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

  16. MISTIC-fusion proteins as antigens for high quality membrane protein antibodies

    PubMed Central

    Alves, Natalia Silva; Astrinidis, Susanne Adina; Eisenhardt, Nathalie; Sieverding, Cornelia; Redolfi, Josef; Lorenz, Michael; Weberruss, Marion; Moreno-Andrés, Daniel; Antonin, Wolfram

    2017-01-01

    Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this “antibody bottleneck”. We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens. PMID:28148968

  17. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

    PubMed Central

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  18. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    PubMed

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner; Denner, Joachim

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  19. Characterization of protein and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

    PubMed

    Reiss, E; Huppert, M; Cherniak, R

    1985-01-01

    detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS)

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

    PubMed

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

    2016-07-19

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

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

    PubMed

    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

    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.

  2. Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70

    PubMed Central

    DONG, LEI; ZHANG, XIAOPENG; YU, CHANGMING; REN, JUN; HOU, LIHUA; FU, LING; YI, SHAOQIONG; CHEN, WEI

    2013-01-01

    The aim of this study was to express and purify recombinant proteins based on human prostate stem cell antigen (PSCA) and heat shock protein-70 (HSP70). The PSCA gene and various structural domains of HSP70 were amplified by polymerase chain reaction (PCR) with the respective primers. Then, the PSCA was cloned into the prokaryotic expression vector pET21a(+) with the amino-terminus, carboxyl-terminus and overall length of HSP70, by enzyme digestion to construct the recombinant plasmids pET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP, respectively. After being expressed in Escherichia coli (E. coli) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, recombinant fusion proteins were purified. Western blotting was performed to confirm the expression of the recombinant proteins. The results revealed that recombinant plasmids were successfully constructed. The PSCA-HSPC and PSCA-HSP expressed in E. coli existed in soluble form, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the recombinant proteins PSCA-HSPC and PSCA-HSP reached >95% following purification with the nickel-nitrilotriacetic acid (Ni-NTA) resin, Phenyl-Sepharose Fast Flow and Superdex 75, which lays a foundation for the development of vaccines for prostate cancer. PMID:23596484

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  5. Safety and immunogenicity of the tetravalent protein-conjugated meningococcal vaccine (MCV4) in recipients of related and unrelated allogeneic hematopoietic stem cell transplantation.

    PubMed

    Mahler, Michelle B; Taur, Ying; Jean, Raymond; Kernan, Nancy A; Prockop, Susan E; Small, Trudy N

    2012-01-01

    Given the high morbidity and mortality associated with meningococcal disease, in 2007 the Advisory Committee of Immunization Practices recommended immunization of all children ages 11-18 with a protein-conjugated meningococcal vaccine. There are limited data on the immunogenicity of this vaccine after allogeneic hematopoietic stem cell transplantation (allo-HCT). Since 2007, we have immunized 48 patients with the MCV4 vaccine. Two vaccinated patients who lacked follow-up titers were excluded from this analysis. Stem cells were derived from an HLA-identical sibling (n = 17) or an alternative donor (n = 29). The median time to vaccination was 2.34 years after allo-HCT. Only 7 patients responded to all 4 serogroups, and 16 patients responded to none of the serogroups. The response to serogroups A, C, Y, and W-135 was 52%, 30%, 46%, and 33%, respectively. The ability to respond to 2 or more serogroups was not affected by age, diagnosis, time to vaccination, or history of graft-versus-host disease. Receipt of a T cell-depleted graft was associated with a poorer response (P = .044). Eight of 16 patients who received a second MCV4 vaccination responded to all 4 serogroups. This retrospective study suggests that response to a single MCV4 vaccination is poor after allo-HCT. Administration of a 2-dose series, as currently recommended for patients with asplenia, complement deficiency, and HIV infection, should be evaluated in this patient population.

  6. Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

    PubMed Central

    Reversat, Anne; Yuseff, Maria-Isabel; Lankar, Danielle; Malbec, Odile; Obino, Dorian; Maurin, Mathieu; Penmatcha, Naga Venkata Gayathri; Amoroso, Alejandro; Sengmanivong, Lucie; Gundersen, Gregg G.; Mellman, Ira; Darchen, François; Desnos, Claire; Pierobon, Paolo; Lennon-Duménil, Ana-Maria

    2015-01-01

    B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells. PMID:25631815

  7. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    PubMed Central

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP. PMID:27141962

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

    PubMed Central

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

    2014-01-01

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

  9. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    DOE PAGES

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; ...

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain amore » canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.« less

  10. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Here, proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  11. A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp

    SciTech Connect

    Altieri, Amanda S.; Ladner, Jane E.; Li, Zhuo; Robinson, Howard; Sallman, Zahur F.; Marino, John P.; Kelman, Zvi

    2016-05-03

    Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL. A small protein, called TIP, binds to PCNA and inhibits PCNA-dependent activities although it does not contain a canonical PIP motif. The X-ray crystal structure of TIP bound to PCNA reveals that TIP binds to the canonical PIP interaction site, but also extends beyond it through a helix that relocates the IDCL. TIP alters the relationship between domains I and II within the PCNA monomer such that the trimeric ring structure is broken, while the individual domains largely retain their native structure. Small angle X-ray scattering (SAXS) confirms the disruption of the PCNA trimer upon addition of the TIP protein in solution and together with the X-ray crystal data, provides a structural basis for the mechanism of PCNA inhibition by TIP.

  12. Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

    PubMed

    Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

    2016-08-01

    Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection.

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

    NASA Astrophysics Data System (ADS)

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

    2010-07-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2002-01-01

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

  16. Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets.

    PubMed

    Granger, D L; Yamamoto, K I; Ribi, E

    1976-02-01

    A myocardial glycolipid (P3) mixed with protein antigens in oil-in-water emulsion induced lasting delayed hypersensitivity (DH) and granulomatous inflammation after intradermal injection into guinea pigs. This did not occur when P3 and bovine serum albumin (BSA) were given in Freund's incomplete adjuvant. The oil-in-water emulsions consisted of microscopic oil droplets suspended in aqueous medium. By separating oil and aqueous phases from BSA + P3 emulsion it was shown that antigen retained with oil droplets led to DH and granuloma formation. The association of antigen with oil droplets was P3 dependent and was quantitated with 125I-labeled BSA. The same phenomenon occurred with 125I-labeled rabbit gamma-globulin (RGG) + P3 emulsion. Fluorescein-conjugated RGG was observed in a particulate state within or on oil droplets in emulsion containing P3. These physical characteristics of antigen + P3 emulsion appeared to be important for immunogenicity.

  17. Screening and characterization of apical membrane antigen 1 interacting proteins in Eimeria tenella.

    PubMed

    Han, Hongyu; Xue, Pu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Huang, Bing

    2016-11-01

    Avian coccidiosis is a widespread and economically significant disease of poultry. It is an enteric disease caused by several protozoan Eimeria species. Eimeria belongs to the phylum Apicomplexa, which exhibits an unusual mechanism of host cell invasion. During invasion of host cells, the protein apical membrane antigen 1 (AMA1) is essential for invasion of Toxoplasma gondii and Plasmodium. Contrary to the roles of AMA1 during host cell invasion in T. gondii and Plasmodium, the precise functions of Eimeria AMA1 (EtAMA1) are unclear. In order to study the functions of EtAMA1, a yeast two-hybrid cDNA library was constructed from E. tenella sporozoites. The EtAMA1 ectodomain was cloned into the pGBKT7 vector to construct the bait plasmid pGBKT7- EtAMA1. Autoactivation and toxicity of the bait protein in yeast cells were tested by comparison with the pGBKT7 empty vector. Expression of the bait protein was detected by western blots. The bait plasmid pGBKT7-EtAMA1 was used to screen yeast two-hybrid cDNA library from E. tenella sporozoites. After multiple screenings with high-screening-rate medium and exclusion of false-positive plasmids, positive preys were sequenced and analyzed using BLAST. We obtained 14 putative EtAMA1-interacting proteins including E. tenella acidic microneme protein2 (EtMIC2), E. tenella putative cystathionine beta-synthase, E. tenella Eimeria-specific protein, four E. tenella conserved hypothetical proteins (one in the serine/threonine protein kinase family) and seven unknown proteins. Gene Ontology analysis indicated that two known proteins were associated with metabolic process, pyridoxal phosphate binding and protein phosphorylation. Functional analysis indicated EtMIC2 was implicated in parasite motility, migration, recognition and invasion of host cells. The data suggested that EtAMA1 may be important during host cell invasion, but also involved in other biological processes.

  18. MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's disease.

    PubMed

    Bannantine, John P; Lingle, Cari K; Stabel, Judith R; Ramyar, Kasra X; Garcia, Brandon L; Raeber, Alex J; Schacher, Pascal; Kapur, Vivek; Geisbrecht, Brian V

    2012-07-01

    The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.

  19. A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein

    PubMed Central

    1992-01-01

    Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10- kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae- specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection. PMID:1730920

  20. Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

    PubMed

    Galay, Remil Linggatong; Miyata, Takeshi; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2016-01-01

    Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

  1. Management of psychiatric complications in unrelated donor before unrelated peripheral hematopoietic stem cell collections

    PubMed Central

    Hequet, Olivier; Mialou, Valerie; Audat, Francoise; Wattel, Eric; Chapel, Valerie; Revesz, Damiela; Jouet, Jean-Piere; Fisseaux, Brigitte; Saoud, Mohamed; Michallet, Mauricette

    2017-01-01

    Allogeneic hematopoietic stem cell transplantation can efficiently treat patients with severe hematological diseases. A human leukocyte antigen-compatible donor is required for performing transplantation. The occurrence of unexpected acute severe diseases in a donor can compromise the feasibility of allogeneic hematopoietic stem cell transplantation. However, when a severe health problem occurs in a donor while the recipient has already received a conditioning regimen, hematologists have to find the best solutions for the recipient, while the team in charge of the donor has to find the best medical solutions for the donor. We describe here the occurrence of psychiatric acute complications in an unrelated donor while the myeloablative conditioning regimen had already been given to the recipient. We report the successive decisions that were made in an emergency based upon the expertise of physicians specialized in hematology, apheresis, cell therapy, and psychiatry to preserve the donor’s health and recipient’s life. PMID:28115876

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

    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

    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.

  4. A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

    PubMed

    de Oliveira, Natasha Rodrigues; Jorge, Sérgio; Gomes, Charles Klazer; Rizzi, Caroline; Pacce, Violetta Dias; Collares, Thais Farias; Monte, Leonardo Garcia; Dellagostin, Odir Antônio

    2017-03-01

    Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.

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

    PubMed

    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

    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.

  6. Preliminary protective capacity study of a Dicrocoelium dendriticum antigenic protein in hamsters.

    PubMed

    González-Lanza, C; Manga-González, M Y; Revilla-Nuín, B

    2006-11-01

    The purpose of this study was to evaluate the protective capacity of 130 kDa Dicrocoelium dendriticum protein in hamsters experimentally infected with this parasite. Forty hamsters divided into four groups of ten animals each were used: G1 (control), G2 (infected), G3 (immunized with Freund's adjuvant and infected), G4 (130 kDa protein vaccinated + adjuvant and infected). Infection with 40 metacercariae/hamster was carried out 4 weeks after the last immunization. Parasitological studies [number of eggs per gram (epg) and worm burden] and biochemical parameters (total proteins, albumin, and total bilirubin), hepatic enzymes [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)], and total IgG levels were determined. A reduction in epg in G3 and G4 was observed 16 weeks postinfection with the higher reduction percentage in the latter (25.2%). No statistically significant differences were detected in the number of recovered worms among groups, although the mean was slightly less in G4 (12.2 +/- 2.08, mean +/- SE) than in G2 (15.4 +/- 2.90). In G4, global protection was 20.9% and an increase in AST and ALT levels was observed. Total IgG levels were similar in the three infected groups. The protection obtained was inadequate, so the antigen dose, immunization-infection period, adjuvants, and immunization route must be optimized.

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

    SciTech Connect

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

    2013-01-18

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2013-01-01

    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 (χ2 =  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. PMID:23516396

  10. MAPkinase: a second site of G-protein regulation of B-cell activation via the antigen receptors.

    PubMed Central

    Deehan, M R; Klaus, G G; Holman, M J; Harnett, W; Harnett, M M

    1998-01-01

    Ligation of the antigen receptors on B cells transduces transmembrane signals leading to the induction of DNA synthesis. We now show that a pertussis toxin-sensitive heterotrimeric G-protein(s) of the Gi class plays a key role in the regulation of surface immunoglobulin (sIg)-mediated DNA synthesis in B cells. This site of G-protein regulation is distinct from that we have previously reported to govern the coupling of the antigen receptors on B cells to the phospholipase C-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate. We have, moreover, identified a candidate target for this new G-protein regulation by showing that mitogen-activating protein kinase (MAPkinase) activity, which plays a key role in the transduction of sIg-mediated proliferative signals in B cells, is abrogated by pre-exposure to pertussis toxin that covalently modifies and inactivates heterotrimeric G-proteins of the Gi class. Furthermore, our data suggest that this pertussis toxin-sensitive G-protein couples the antigen receptors to MAPkinase activation, at least in part, by regulating sIg-coupling to Lyn, Syk and perhaps Blk and Fyn activity, results consistent with studies in other systems which show that classical G-protein-coupled receptors recruit such protein tyrosine kinases to tranduce MAPkinase activation. Interestingly, however, this G-protein plays no apparent role in the control of up-regulation of major histocompatibility complex class II expression on B cells, suggesting that such G-protein-regulated-tyrosine kinase and MAPkinase activation is not required for the induction of this biological response following antigen receptor ligation. Images Figure 5 PMID:9824472

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2009-11-01

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

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

    SciTech Connect

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

    2009-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2000-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

    PubMed

    Sullender, W M; Britt, W J

    1996-04-01

    The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

  17. Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

    PubMed Central

    Knudsen, Maria L.; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose. PMID:25643354

  18. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

    PubMed

    Knudsen, Maria L; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  19. Determination of antigenicity-altering patches on the major surface protein of human influenza A/H3N2 viruses.

    PubMed

    Kratsch, Christina; Klingen, Thorsten R; Mümken, Linda; Steinbrück, Lars; McHardy, Alice C

    2016-01-01

    Human influenza viruses are rapidly evolving RNA viruses that cause short-term respiratory infections with substantial morbidity and mortality in annual epidemics. Uncovering the general principles of viral coevolution with human hosts is important for pathogen surveillance and vaccine design. Protein regions are an appropriate model for the interactions between two macromolecules, but the currently used epitope definition for the major antigen of influenza viruses, namely hemagglutinin, is very broad. Here, we combined genetic, evolutionary, antigenic, and structural information to determine the most relevant regions of the hemagglutinin of human influenza A/H3N2 viruses for interaction with human immunoglobulins. We estimated the antigenic weights of amino acid changes at individual sites from hemagglutination inhibition data using antigenic tree inference followed by spatial clustering of antigenicity-altering protein sites on the protein structure. This approach determined six relevant areas (patches) for antigenic variation that had a key role in the past antigenic evolution of the viruses. Previous transitions between successive predominating antigenic types of H3N2 viruses always included amino acid changes in either the first or second antigenic patch. Interestingly, there was only partial overlap between the antigenic patches and the patches under strong positive selection. Therefore, besides alterations of antigenicity, other interactions with the host may shape the evolution of human influenza A/H3N2 viruses.

  20. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein.

    PubMed

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus; Broekema, Marjoleine F; de Haar, Colin; Schipper, Henk S; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-08-08

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.

  1. CD1d-mediated Presentation of Endogenous Lipid Antigens by Adipocytes Requires Microsomal Triglyceride Transfer Protein*

    PubMed Central

    Rakhshandehroo, Maryam; Gijzel, Sanne M. W.; Siersbæk, Rasmus; Broekema, Marjoleine F.; de Haar, Colin; Schipper, Henk S.; Boes, Marianne; Mandrup, Susanne; Kalkhoven, Eric

    2014-01-01

    Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and –δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis. PMID:24966328

  2. Discovery of GAMA, a Plasmodium falciparum merozoite micronemal protein, as a novel blood-stage vaccine candidate antigen.

    PubMed

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

    2011-11-01

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

  3. Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays.

    PubMed

    Uplekar, Swapna; Rao, Pavitra Nagesh; Ramanathapuram, Lalitha; Awasthi, Vikky; Verma, Kalpana; Sutton, Patrick; Ali, Syed Zeeshan; Patel, Ankita; G, Sri Lakshmi Priya; Ravishankaran, Sangamithra; Desai, Nisha; Tandel, Nikunj; Choubey, Sandhya; Barla, Punam; Kanagaraj, Deena; Eapen, Alex; Pradhan, Khageswar; Singh, Ranvir; Jain, Aarti; Felgner, Philip L; Davies, D Huw; Carlton, Jane M; Das, Jyoti

    2017-01-01

    Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.

  4. Characterizing Antibody Responses to Plasmodium vivax and Plasmodium falciparum Antigens in India Using Genome-Scale Protein Microarrays

    PubMed Central

    Awasthi, Vikky; Verma, Kalpana; Sutton, Patrick; Ali, Syed Zeeshan; Patel, Ankita; G., Sri Lakshmi Priya; Ravishankaran, Sangamithra; Desai, Nisha; Tandel, Nikunj; Choubey, Sandhya; Barla, Punam; Kanagaraj, Deena; Eapen, Alex; Pradhan, Khageswar; Singh, Ranvir; Jain, Aarti; Felgner, Philip L.; Davies, D. Huw; Das, Jyoti

    2017-01-01

    Understanding naturally acquired immune responses to Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world. PMID:28118367

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

    PubMed

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

    2012-12-01

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

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

    PubMed Central

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

    2009-01-01

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

  7. Multiple antigen peptide dendrimer elicits antibodies for detecting rat and mouse growth hormone binding proteins

    PubMed Central

    Aguilar, Roberto M.; Talamantes, Frank J.; Bustamante, Juan J.; Muñoz, Jesus; Treviño, Lisa R.; Martinez, Andrew O.; Haro, Luis S.

    2009-01-01

    The membrane-bound rat growth hormone receptor (GH-R) and an alternatively spliced isoform, the soluble rat GH binding protein (GH-BP), are comprised of identical N-terminal GH binding domains, however, their C-terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent multiple antigen peptide (MAP) dendrimer with four identical branches of a C-terminal peptide sequence of the rat GH-BP (GH-BP263-279) was synthesized and used as an immunogen in rabbits. Solid-phase peptide synthesis of four GH-BP263-279 segments onto a tetravalent Lys2-Lys-β-Ala-OH core peptide was carried out using N-(9-fluorenyl)methoxycarbonyl chemistry. The mass of the RP-HPLC purified synthetic product, 8398 Da, determined by ESI-MS, was identical to expected mass. Three anti-rat GH-BP263-279 MAP antisera, BETO-8039, BETO-8040 and BETO-8041, at dilutions of 10-3, recognized both the rat GH-BP263-279 MAP and recombinant mouse GH-BP with ED50s within a range of 5-10 fmol but did not cross-react with BSA in dot blot analyses. BETO-8041 antisera (10-3 dilution) recognized GH-BPs of rat serum and liver having Mrs ranging from 35-130 kDa but did not recognize full-length rat GH-Rs. The antisera also detected recombinant mouse GH-BPs. In summary, the tetravalent rat GH-BP263-279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C-termini of both rat and mouse GH-BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH-BPs. PMID:19089805

  8. Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

    PubMed

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

    2009-06-01

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

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

    PubMed Central

    Neipel, F; Ellinger, K; Fleckenstein, B

    1992-01-01

    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. Images PMID:1374813

  10. Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

    PubMed

    de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

    2017-01-18

    We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KD(eff). Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

  11. [Isolation and identification in Senegal of the most immunogenic protein soluble antigen of a Clostridium chauvoei strain].

    PubMed

    Mbengue, M B

    2008-02-01

    Clostridium chauvoei is the pathogenic agent for blackleg, a toxinfection disease in bovine and small ruminants, always lethal and involving considerable economic losses. Some bacteriological, biochemical, immunological studies permitted to isolate identify the major soluble antigenic protein of this bacteria. It's a protein fragment of 70 kDa weight, the 19 fraction, excreted by the bacteria in a suitable culture medium. The 19 fraction of extracellular medium leads to antibodies production on guinea pigs revealed by the ELISA/antibody test.

  12. Identification of surface proteins and antigens from larval stages of Ascaris suum by two-dimensional electrophoresis.

    PubMed

    Kasuga-Aoki, H; Tsuji, N; Suzuki, K; Isobe, T; Yoshihara, S

    2000-12-01

    An understanding of the biology of the cuticle in the larval stages of Ascariodea is of importance since the cuticle molecules not only possess a variety of functions related to survival but also have a potential role as a target for immunoprophylaxis. Thus, we made a preliminary characterization of surface proteins and antigens from 3rd-stage larvae (L3) and lung-stage larvae of Ascaris suum using two biotin-derivatives and two-dimen sional polyacrylamide gel electrophoresis (2D-PAGE). The proteins labelled with biotin comprised a total of 37 and 32 spots, with molecular weights (Air) ranging from 15 to 101 kDa and isoelectric points (pI) from 3.8 to 7.6, in L3 and lung-stage larvae, respectively. The profiles revealed that the individual spots bound to one or both biotin derivatives. In addition, stage-common and stage-specific spots were found in L3 and lung-stage larvae. 2D-PAGE/immunoblotting analysis was performed with antisera from rabbits repeatedly inoculated with A. suum L3. Nineteen spots were recognized as surface antigens, with Mr ranging from 32 to 66 kDa and pI from 4.9 to 7.6, from L3 and lung-stage larvae after alignment of the immunoblots with the profile of the surface proteins. These spots were found to include stage-common and stage-specific antigens. Identification of surface proteins by biotin labelling combined with 2D-PAGE allows a substantial shortening of sample preparation time for the target proteins, and will be a viable method for protein analysis of surface proteins and antigens of A. suum L3 and lung-stage larvae.

  13. Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins.

    PubMed

    Léonetti, M; Galon, J; Thai, R; Sautès-Fridman, C; Moine, G; Ménez, A

    1999-04-19

    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.

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

    PubMed

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

    2007-09-01

    This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100 degrees C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50 degrees C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (beta-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of beta-lactoglobulin, alpha-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4 x 7 H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of

  15. A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

    PubMed

    Baranov, Konstantin; Volkova, Olga; Chikaev, Nikolai; Mechetina, Ludmila; Laktionov, Pavel; Najakshin, Alexander; Taranin, Alexander

    2008-03-20

    We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

  16. Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

    PubMed Central

    Beckhove, Philipp; Warta, Rolf; Lemke, Britt; Stoycheva, Diana; Momburg, Frank; Schnölzer, Martina; Warnken, Uwe; Schmitz-Winnenthal, Hubertus; Ahmadi, Rezvan; Dyckhoff, Gerhard; Bucur, Mariana; Jünger, Simone; Schueler, Thomas; Lennerz, Volker; Woelfel, Thomas; Unterberg, Andreas; Herold-Mende, Christel

    2010-01-01

    Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele. PMID:20458140

  17. Dephosphorylation of JC virus agnoprotein by protein phosphatase 2A: Inhibition by small t antigen

    PubMed Central

    Sariyer, Ilker K.; Khalili, Kamel; Safak, Mahmut

    2009-01-01

    Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein–protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82–124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of

  18. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  19. Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii.

    PubMed Central

    Schuenke, K W; Walker, D H

    1994-01-01

    Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses. Images PMID:8112862

  20. Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.

    PubMed

    Wang, Lizhen; Sun, Yani; Du, Taofeng; Wang, Chengbao; Xiao, Shuqi; Mu, Yang; Zhang, Gaiping; Liu, Lihong; Widén, Frederik; Hsu, Walter H; Zhao, Qin; Zhou, En-Min

    2014-12-01

    The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

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

    SciTech Connect

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

    1991-10-01

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

  2. A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.

    PubMed

    Arnaiz, Blanca; Madrigal-Estebas, Laura; Todryk, Stephen; James, Tharappel C; Doherty, Derek G; Bond, Ursula

    2006-04-08

    The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-gamma (IFN-gamma) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  6. Elastin, a novel extracellular matrix protein adhering to mycobacterial antigen 85 complex.

    PubMed

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

    2013-02-08

    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 (K(D) = 0.13 ± 0.006 μm), 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.

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

    PubMed Central

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

    2016-01-01

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

  8. Antigen 85A and mycobacterial DNA-binding protein 1 are targets of immunoglobulin G in individuals with past tuberculosis.

    PubMed

    Osada-Oka, Mayuko; Tateishi, Yoshitaka; Hirayama, Yukio; Ozeki, Yuriko; Niki, Mamiko; Kitada, Seigo; Maekura, Ryoji; Tsujimura, Kunio; Koide, Yukio; Ohara, Naoya; Yamamoto, Taro; Kobayashi, Kazuo; Matsumoto, Sohkichi

    2013-01-01

    Development of accurate methods for predicting progression of tuberculosis (TB) from the latent state is recognized as vitally important in controlling TB, because a majority of cases develop from latent infections. Past TB that has never been treated has a higher risk of progressing than does latent Mycobacterium tuberculosis infection in patients who have previously received treatment. Antibody responses against 23 kinds of M. tuberculosis proteins in individuals with past TB who had not been medicated were evaluated. These individuals had significantly higher concentrations of antibodies against Antigen 85A and mycobacterial DNA-binding protein 1 (MDP1) than did those with active TB and uninfected controls. In addition, immunohistochemistry revealed colocalization of tubercle bacilli, antigen 85 and MDP1 inside tuberculous granuloma lesions in an asymptomatic subject, showing that M. tuberculosis in lesions expresses both antigen 85 and MDP1. Our study suggests the potential usefulness of measuring antibody responses to antigen 85A and MDP1 for assessing the risk of TB progression.

  9. Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

    PubMed

    Rocco, C J; Davey, M E; Bakaletz, L O; Goodman, S D

    2017-04-01

    Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.

  10. Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins

    PubMed Central

    Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M. H.; Idrees, Muhammad

    2016-01-01

    The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV. PMID:27934901

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

    PubMed

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

    2015-08-07

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

  12. SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

    PubMed

    Larbolette, O; Wollscheid, B; Schweikert, J; Nielsen, P J; Wienands, J

    1999-02-01

    Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

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

    PubMed

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

    2014-08-01

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

  14. Protein-bound 4-hydroxy-2-nonenal: an endogenous triggering antigen of antI-DNA response.

    PubMed

    Toyoda, Kazuyo; Nagae, Ritsuko; Akagawa, Mitsugu; Ishino, Kosuke; Shibata, Takahiro; Ito, Sohei; Shibata, Noriyuki; Yamamoto, Tomoko; Kobayashi, Makio; Takasaki, Yoshinari; Matsuda, Tsukasa; Uchida, Koji

    2007-08-31

    Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti-DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti-HNE titer than healthy individuals. To determine if a specific anti-DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti-DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i.e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.

  15. Epstein-Barr virus latent membrane protein 2A exacerbates experimental autoimmune encephalomyelitis and enhances antigen presentation function

    PubMed Central

    Chang, Rhoda A.; Miller, Stephen D.; Longnecker, Richard

    2012-01-01

    Multiple sclerosis (MS) is an inflammatory, autoimmune disease of the central nervous system. The cause of MS is still unknown but epidemiological and immunological studies have implicated Epstein-Barr virus (EBV), which infects B cells, as a possible etiological agent involved in disease. Of particular interest is EBV latent membrane protein 2A (LMP2A) because previous studies have demonstrated that LMP2A enhances the expansion and differentiation of B cells upon antigen stimulation, revealing a potential contribution of this protein in autoimmunity. Since B cells are thought to contribute to MS, we examined the role of LMP2A in the animal model experimental autoimmune encephalomyelitis (EAE). In this model, transgenic mice in which B cells express LMP2A show increased severity and incidence of disease. This difference was not due to lymphocyte recruitment into the CNS or differences in T cell activation, rather, we show that LMP2A enhances antigen presentation function. PMID:22616025

  16. Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

    PubMed Central

    Briles, D E; Forman, C; Horowitz, J C; Volanakis, J E; Benjamin, W H; McDaniel, L S; Eldridge, J; Brooks, J

    1989-01-01

    Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay. PMID:2707854

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

    PubMed

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

    2015-12-01

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

  18. HLA class I is most tightly linked to levels of tapasin compared with other antigen-processing proteins in glioblastoma

    PubMed Central

    Thuring, Camilla; Follin, Elna; Geironson, Linda; Freyhult, Eva; Junghans, Victoria; Harndahl, Mikkel; Buus, Søren; Paulsson, Kajsa M

    2015-01-01

    Background: Tumour cells can evade the immune system by dysregulation of human leukocyte antigens (HLA-I). Low quantity and/or altered quality of HLA-I cell surface expression is the result of either HLA-I alterations or dysregulations of proteins of the antigen-processing machinery (APM). Tapasin is an APM protein dedicated to the maturation of HLA-I and dysregulation of tapasin has been linked to higher malignancy in several different tumours. Methods: We studied the expression of APM components and HLA-I, as well as HLA-I tapasin-dependency profiles in glioblastoma tissues and corresponding cell lines. Results: Tapasin displayed the strongest correlation to HLA-I heavy chain but also clustered with β2-microglobulin, transporter associated with antigen processing (TAP) and LMP. Moreover, tapasin also correlated to survival of glioblastoma patients. Some APM components, for example, TAP1/TAP2 and LMP2/LMP7, showed variable but coordinated expression, whereas ERAP1/ERAP2 displayed an imbalanced expression pattern. Furthermore, analysis of HLA-I profiles revealed variable tapasin dependence of HLA-I allomorphs in glioblastoma patients. Conclusions: Expression of APM proteins is highly variable between glioblastomas. Tapasin stands out as the APM component strongest correlated to HLA-I expression and we proved that HLA-I profiles in glioblastoma patients include tapasin-dependent allomorphs. The level of tapasin was also correlated with patient survival time. Our results support the need for individualisation of immunotherapy protocols. PMID:26313662

  19. 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

    DTIC Science & Technology

    1990-05-01

    on the cell envelopes of Rickettsia 29. Messing, J. 1983. New M13 vectors for cloning. Methods prowazekii, Rickettsia rickettsii , and Rickettsia ...gene of Rickettsia tsu sugamushi:Sequence homology and antigenic comparison to the 60-kilodalton family of stresproteins. 12. PERSONAL AUTHOR(S...IuwRnuiy dy "jmber FIELD GROUP S ROUP Rickettsia tsutsugamushi, antigens, molecular cloning,. FIED_ GROU__ SUB-GROUP scrub typhus, heat-shock proteins

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2012-01-01

    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. PMID:22232548

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

    PubMed Central

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

    1988-01-01

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

  3. Identification of an immunodominant antigenic site involving the capsid protein VP3 of hepatitis A virus.

    PubMed Central

    Ping, L H; Jansen, R W; Stapleton, J T; Cohen, J I; Lemon, S M

    1988-01-01

    Hepatitis A virus, an hepatotropic picornavirus, is a common cause of acute hepatitis in man for which there is no available vaccine. Competitive binding studies carried out in solid phase suggest that neutralizing monoclonal antibodies to hepatitis A virus recognize a limited number of epitopes on the capsid surface, although the polypeptide locations of these epitopes are not well defined. Neutralization-escape mutants, selected for resistance to monoclonal antibodies, demonstrate broad cross-resistance to other monoclonal antibodies. Sequencing of virion RNA from several of these mutants demonstrated that replacement of aspartic acid residue 70 of capsid protein VP3 (residue 3070) with histidine or alanine confers resistance to neutralization by monoclonal antibody K2-4F2 and prevents binding of this antibody and other antibodies with similar solid-phase competition profiles. These results indicate that residue 3070 contributes to an immunodominant antigenic site. Mutation at residue 102 of VP1 (residue 1102) confers partial resistance against antibody B5-B3 and several other antibodies but does not prevent antibody attachment. Both VP3 and VP1 sites align closely in the linear peptide sequences with sites of neutralization-escape mutations in poliovirus and human rhinovirus, suggesting conservation of structure among these diverse picornaviruses. However, because partial neutralization resistance to several monoclonal antibodies (2D2, 3E1, and B5-B3) was associated with mutation at either residue 3070 or residue 1102, these sites appear more closely related functionally in hepatitis A virus than in these other picornaviruses. PMID:2460866

  4. In vivo neutralization of eosinophil-derived major basic protein inhibits antigen-induced bronchial hyperreactivity in sensitized guinea pigs.

    PubMed Central

    Lefort, J; Nahori, M A; Ruffie, C; Vargaftig, B B; Pretolani, M

    1996-01-01

    This study examines the effect of purified rabbit antiguinea pig eosinophil-derived major basic protein (MBP) Ig on antigen-induced bronchial hyperreactivity to inhaled acetylcholine in aerosol-sensitized guinea pigs. Ovalbumin inhalation by sensitized guinea pigs induced a rise in the numbers of eosinophils and in the levels of MBP in the bronchoalveolar lavage fluid, which peaked at 24 h and resolved at 72 h. Antigen-challenged animals exhibited bronchial hyperreactivity to inhale acetylcholine at 72 h, but not at 6 or 24 h. The intranasal administration of 200 microliter of purified rabbit anti-guinea pig MBP Ig, at 2.5 mg/ml, but not of the control preimmune rabbit Ig, 1 h before and 5 h after ovalbumin inhalation suppressed bronchial hyperreactivity to acetylcholine at 72 h without affecting the number of eosinophils accumulating in the bronchoalveolar lavage fluid. These findings indicate that antigen challenge in sensitized guinea pigs is followed by early eosinophil infiltration and activation within the airways and by late bronchial hyperreactivity. Neutralization of endogenously secreted MBP by a specific antiserum prevented antigen-induced bronchial hyperreactivity, suggesting that eosinophil degranulation plays an important role in the alterations of bronchopulmonary function in the guinea pig. PMID:8613536

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

    PubMed

    Zav'yalov, V P; Abramov, V M; Cherepanov, P G; Spirina, G V; Chernovskaya, T V; Vasiliev, A M; Zav'yalova, G A

    1996-05-01

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

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

    PubMed

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

    2016-01-20

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

  7. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    PubMed

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection.

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

    PubMed

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

    2014-08-06

    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.

  9. Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology

    PubMed Central

    Welly, Bryan T.; Miller, Michael R.; Stott, Jeffrey L.; Blanchard, Myra T.; Islas-Trejo, Alma D.; O’Rourke, Sean M.; Young, Amy E.; Medrano, Juan F.; Van Eenennaam, Alison L.

    2016-01-01

    Epizootic bovine abortion (EBA), or “foothill abortion,” is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus, or “pajaroello tick,” was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis. This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis. This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice. PMID:28040777

  10. Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner

    PubMed Central

    Huang, Pengwei; Xia, Ming; Zhong, Weiming; Wei, Chao; Wang, Leyi; Morrow, Ardythe

    2012-01-01

    Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells. PMID:22345472

  11. Expression and functional properties of the Streptococcus intermedius surface protein antigen I/II.

    PubMed

    Petersen, F C; Pasco, S; Ogier, J; Klein, J P; Assev, S; Scheie, A A

    2001-07-01

    Streptococcus intermedius is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in S. intermedius. The S. intermedius antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the S. intermedius NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the S. intermedius antigen I/II is involved in adhesion to human receptors and in IL-8 induction.

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

    PubMed

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

    2007-12-01

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

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

    PubMed

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

    2012-04-01

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

  14. Host proteins that bind to or mimic SV40 large T antigen: using antibodies to look at protein interactions and their significance

    PubMed Central

    Mole, S. E.; Gannon, J. V.; Anton, I. A.; Ford, M. J.; Lane, D. P.

    1989-01-01

    The papovavirus SV40 is able to induce tumours in susceptible hosts and will transform cells in vitro. Its major early protein, large T antigen, is required for viral DNA synthesis, both in vivo and in vitro, and is also responsible for the oncogenic action of the virus. We have made use of an extensive library of anti-T monoclonal antibodies to investigate the cellular effects of T. Large T shares an antigenic determinant with a growth-regulated host protein, p68, which is a member of an expanding super-family of helicases with particular homology to the translation initiation factor elF-4A. We have also studied the binding and interaction of large T with two particular host components: the replicative enzyme DNA polymerase α and the proto-oncogene p53. These two proteins bind to similar regions of T and exert similar effects on its antigenic structure. We found that p53 can block the binding of DNA polymerase α to T as well as co-existing with DNA polymerase α in a trimeric complex with T. This suggests that these interactions may be important in the oncogenic and replicative action of large T.

  15. Proteins of Human Immunodeficiency Virus that Cross-React with Human ’Self’ Antigens

    DTIC Science & Technology

    1991-11-20

    evaluated a number of experimental conditions in order to have high M22 antigen expression. We found that scrapie agent caused, by several fold, the...highest expression of M22 antigen ( scrapie > Theiler’s = EAE). Lastly, we evaluated different regions in the CNS of SWR/J and C57BI/6 mice for M22...analysis of the quality and GFAP expression in total RNA isolated from control (-) and scrapie -infected (+) brain regions in C57B1/6 mice. A large

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Exemptions from Unrelated Business Tax: Rental Income

    ERIC Educational Resources Information Center

    Reed, George E.

    1975-01-01

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

  19. Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry.

    PubMed

    Díez, Soraya; Gómez, Beatriz L; Restrepo, Angela; Hay, Rod J; Hamilton, Andrew J

    2002-02-01

    The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.

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

    PubMed Central

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

    1989-01-01

    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 PMID:2553610

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-08-05

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

  3. Murine Monoclonal Antibodies for Antigenic Discrimination of HIV-1 Envelope Proteins

    PubMed Central

    Sealy, Robert E.; Jones, Bart G.; Surman, Sherri L.; Branum, Kristen; Howlett, Nanna M.; Flynn, Patricia M.

    2016-01-01

    Abstract In the influenza virus field, antibody reagents from research animals have been instrumental in the characterization of antigenically distinct hemagglutinin and neuraminidase membrane molecules. These small animal reagents continue to support the selection of components for inclusion in human influenza virus vaccines. Other cocktail vaccines against variant pathogens (e.g., polio virus, pneumococcus) are similarly designed to represent variant antigens, as defined by antibody reactivity patterns. However, a vaccine cocktail comprising diverse viral membrane antigens defined in this way has not yet been advanced to a clinical efficacy study in the HIV-1 field. In this study, we describe the preparation of mouse antibodies specific for HIV-1 gp140 or gp120 envelope molecules. Our experiments generated renewable reagents able to discriminate HIV-1 envelopes from one another. Monoclonals yielded more precise discriminatory capacity against their respective immunogens than did a small panel of polyclonal human sera derived from recently HIV-1-infected patients. Perhaps these and other antibody reagents will ultimately support high-throughput cartography studies with which antigenically-distinct envelope immunogens may be formulated into a successful HIV-1 envelope cocktail vaccine. PMID:26544795

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

    PubMed

    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

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

    PubMed Central

    Rodríguez, Luis E.; Curtidor, Hernando; Ocampo, Marisol; Garcia, Javier; Puentes, Alvaro; Valbuena, John; Vera, Ricardo; López, Ramses; Patarroyo, Manuel E.

    2005-01-01

    Receptor–ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented α-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%–85%, suggesting that MSP-3 protein’s role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens. PMID:15987906

  7. Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein.

    PubMed

    Mousa, Jarrod J; Sauer, Marion F; Sevy, Alexander M; Finn, Jessica A; Bates, John T; Alvarado, Gabriela; King, Hannah G; Loerinc, Leah B; Fong, Rachel H; Doranz, Benjamin J; Correia, Bruno E; Kalyuzhniy, Oleksandr; Wen, Xiaolin; Jardetzky, Theodore S; Schief, William R; Ohi, Melanie D; Meiler, Jens; Crowe, James E

    2016-11-01

    Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

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

    PubMed

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

    2011-01-01

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

  9. c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.

    PubMed Central

    Iguchi-Ariga, S M; Itani, T; Yamaguchi, M; Ariga, H

    1987-01-01

    Replicating activity of SV40 origin-containing plasmid was tested in human cells as well as in monkey CosI cells. All the plasmids possessing SV40 ori sequences could replicate, even in the absence of SV40 T antigen, in human HL-60 and Raji cells which are expressing c-myc gene at high level. The copy numbers of the replicated plasmids in these human cells were 1/100 as high as in monkey CosI cells which express SV40 T antigen constitutively. Exactly the same plasmids as the transfected original ones were recovered from the Hirt supernatant of the transfected HL-60 cells. Furthermore, replication of the SV40 ori-containing plasmids in HL-60 cells was inhibited by anti-c-myc antibody co-transfected into the cells. These results indicate that the c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication. Images PMID:3037484

  10. Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

    PubMed

    Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

    2015-05-19

    A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 μL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

  11. Mycobacterium avium ssp. paratuberculosis recombinant heat shock protein 70 interaction with different bovine antigen-presenting cells.

    PubMed

    Langelaar, M F M; Hope, J C; Rutten, V P M G; Noordhuizen, J P T M; van Eden, W; Koets, A P

    2005-03-01

    Abstract Heat shock proteins (Hsp) can deliver antigen into the major histocompatibility complex class I presentation pathway of antigen-presenting cells (APC), a process called cross priming, thus stimulating antigen-specific CD8+ T-cell reactions. Hsp were shown to elicit proinflammatory responses in APC. Both processes require interaction of Hsp with APC via specific receptors. This study describes the interaction of recombinant Hsp70 (rHsp70) of Mycobacterium avium subspecies paratuberculosis with bovine peripheral blood mononuclear cells that was restricted to CD14+ cells. Characterized monocyte-derived macrophages, monocyte-derived dendritic cells (DC) and BoMac, an immortalized bovine macrophage cell line, were used to investigate the interaction of rHsp70 with different bovine APC. Saturation of immature DC with high concentrations of rHsp70 is demonstrated, and it was found that interaction of rHsp70 with DC was related to the maturation stage of the DC. Involvement of CD91 as a cellular receptor for rHsp70 was demonstrated; however, competition studies with immature DC demonstrated that other receptors exist on bovine APC. These data suggest that rHsp70-based vaccines may be useful for the successful immunization of cattle.

  12. Antigenic and immunogenic properties of truncated VP28 protein of white spot syndrome virus in Procambarus clarkii.

    PubMed

    Du, Hua-Hua; Hou, Chong-Lin; Wu, Xiao-Guo; Xie, Rong-hui; Wang, Yi-Zhen

    2013-01-01

    Previous studies identify VP28 envelope protein of white spot syndrome virus (WSSV) as its main antigenic protein. Although implicated in viral infectivity, its functional role remains unclear. In the current study, we described the production of polyclonal antibodies to recombinant truncated VP28 proteins including deleted N-terminal (rVP28ΔN), C-terminal (rVP28ΔC) and middle (rVP28ΔM). In antigenicity assays, antibodies developed from VP28 truncations lacking the N-terminal or middle regions showed significantly lowered neutralization of WSSV in crayfish, Procambarus clarkii. Further immunogenicity analysis showed reduced relative percent survival (RPS) in crayfish vaccinating with these truncations before challenge with WSSV. These results indicated that N-terminal (residues 1-27) and middle region (residues 35-95) were essential to maintain the neutralizing linear epitopes of VP28 and responsible in eliciting immune response. Thus, it is most likely that these regions are exposed on VP28, and will be useful for rational design of effective vaccines targeting VP28 of WSSV.

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

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

    1985-01-01

    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

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

    PubMed Central

    Chen, Wuguo; Dittmer, Dirk P.

    2011-01-01

    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. PMID:21734034

  16. Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein.

    PubMed

    Gomez Barroso, Juan Arturo; Aguilar, Carlos Fernando

    2014-09-01

    Ribosomal P proteins form a "stalk" complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1α, TcP1β, TcP2α and TcP2β. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0-TcP1α-TcP1β-TcP2α-TcP2β have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca(+2)/K(+) channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an α/β globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1-P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein.

  17. Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum.

    PubMed Central

    Jakobsen, P H; Hviid, L; Theander, T G; Afare, E A; Ridley, R G; Heegaard, P M; Stuber, D; Dalsgaard, K; Nkrumah, F K

    1993-01-01

    The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living in an area with a high rate of transmission of malaria. Lymphocytes from a large proportion of the Ghanaian blood donors proliferated in response to the RAP-1 peptide, unlike those of Danish control blood donors, indicating that this sequence contains a malaria-specific T-cell epitope broadly recognized by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides. PMID:8418048

  18. Eliciting Epitope-Specific CD8+ T Cell Response by Immunization with Microbial Protein Antigens Formulated with α-Galactosylceramide: Theory, Practice, and Protocols.

    PubMed

    Gilchuk, Pavlo; Knight, Frances C; Wilson, John T; Joyce, Sebastian

    2017-01-01

    CD8+ cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8+ T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. To induce a robust antigen-specific CD8+ T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8+ T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant.

  19. The global distribution and diversity of protein vaccine candidate antigens in the highly virulent Streptococcus pnuemoniae serotype 1.

    PubMed

    Cornick, Jennifer E; Tastan Bishop, Özlem; Yalcin, Feyruz; Kiran, Anmol M; Kumwenda, Benjamin; Chaguza, Chrispin; Govindpershad, Shanil; Ousmane, Sani; Senghore, Madikay; du Plessis, Mignon; Pluschke, Gerd; Ebruke, Chinelo; McGee, Lesley; Sigaùque, Beutel; Collard, Jean-Marc; Bentley, Stephen D; Kadioglu, Aras; Antonio, Martin; von Gottberg, Anne; French, Neil; Klugman, Keith P; Heyderman, Robert S; Alderson, Mark; Everett, Dean B

    2017-02-07

    Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (<2%). The vast majority of the observed variation was predicted to have no impact on the efficaciousness of a protein vaccine incorporating a single variant of SP0148, SP1912 and/or SP2108 from S. pneumoniae TIGR4. Our findings emphasise the importance of taking geographic differences into account when designing global vaccine interventions and support the continued development of SP0148, SP1912 and SP2108 as protein vaccine candidates against this

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-07-01

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

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

    SciTech Connect

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

    2013-05-03

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

  3. Circulating Gut-Homing (α4β7+) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

    PubMed Central

    Sinha, Anuradha; Dey, Ayan; Saletti, Giulietta; Samanta, Pradip; Chakraborty, Partha Sarathi; Bhattacharya, M. K.; Ghosh, Santanu; Ramamurthy, T.; Kim, Jae-Ouk; Yang, Jae Seung; Kim, Dong Wook

    2016-01-01

    Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, pan-Shigella surface protein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α4β7+) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α4β7+ IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α4β7+ ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude. PMID:27193041

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

    PubMed Central

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

    1983-01-01

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

  5. The consequences of the intracellular retention of pathogen-derived T-cell-independent antigens on protein presentation to T cells.

    PubMed

    Leyva-Cobián, F; Outschoorn, I M; Carrasco-Marín, E; Alvarez-Domínguez, C

    1997-10-01

    Intracellular pathogens can be considered as particulate antigens chemically composed of a complex mixture of T-cell-dependent antigens (TD) (peptides and proteins) and T-cell-independent antigens (TI) (glycolipids and complex polysaccharides). A large range of saccharides (from oligosaccharides to complex polysaccharides) derived from pathogenic microorganisms are being isolated and characterized. They are currently implicated in signaling systems and concomitant host-parasite relationships. However, there are not many structure-function relationships described for these pathogens. This is particularly true of polysaccharides. In this report we have reviewed the role of defined TI antigens in the processing and presentation of defined TD antigens to specific T cells by antigen-presenting cells (APC). We also considered the importance of some of the chemical characteristics shared by different carbohydrates implicated in the inhibition of antigen presentation. These findings are discussed in relation to the clear immunopathological consequences of long retention periods of complex carbohydrate molecules derived from intracellular parasites inside certain APC and the absence of antigen presentation impairment in physiological situations such as the removal of senescent or damaged red blood cells by splenic macrophages or intracellular accumulation of carbohydrates in colostrum and milk macrophages during lactation.

  6. Antibody-producing cell responses to an isolated outer membrane protein and to complexes of this antigen with lipopolysaccharide or with vesicles of phospholipids from Proteus mirabilis.

    PubMed Central

    Karch, H; Nixdorff, K

    1981-01-01

    Antibody-producing cell responses of mice to a protein isolated from the outer membrane of Proteus mirabilis were typical of the responses to a thymus-dependent antigen. The immunoglobulin G antibody-producing cell responses to the protein were increased after administration of the antigen complexed with either lipopolysaccharide or with vesicles of phospholipids extracted from P. mirabilis. The protein in turn significantly increased the immune response to lipopolysaccharide and also converted this response from predominantly immunoglobulin M to predominantly immunoglobulin G. PMID:6164651

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

    PubMed

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

    2016-01-01

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

  8. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

    PubMed

    Folle, Ana Maite; Kitano, Eduardo S; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K; Rosenzvit, Mara; Batthyány, Carlos; Ferreira, Ana María

    2017-01-01

    The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in

  9. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype

    PubMed Central

    Folle, Ana Maite; Kitano, Eduardo S.; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K.; Rosenzvit, Mara; Batthyány, Carlos

    2017-01-01

    The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in

  10. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

    PubMed Central

    de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

  11. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin.

    PubMed

    de Virgilio, Maddalena; De Marchis, Francesca; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

  12. In vivo selection for Neisseria gonorrhoeae opacity protein expression in the absence of human carcinoembryonic antigen cell adhesion molecules.

    PubMed

    Simms, Amy N; Jerse, Ann E

    2006-05-01

    The neisserial opacity (Opa) proteins are phase-variable, antigenically distinct outer membrane proteins that mediate adherence to and invasion of human cells. We previously reported that Neisseria gonorrhoeae Opa protein expression appeared to be selected for or induced during experimental murine genital tract infection. Here we further defined the kinetics of recovery of Opa variants from the lower genital tracts of female mice and investigated the basis for this initial observation. We found that the recovery of different Opa phenotypes from mice appears cyclical. Three phases of infection were defined. Following intravaginal inoculation with primarily Opa- gonococci, the majority of isolates recovered were Opa+ (early phase). A subsequent decline in the percentage of Opa+ isolates occurred in a majority of mice (middle phase) and was followed by a reemergence of Opa+ variants in mice that were infected for longer than 8 days (late phase). We showed the early phase was due to selection for preexisting Opa+ variants in the inoculum by constructing a chloramphenicol-resistant (Cm(r)) strain and following Cm(r) Opa+ populations mixed with a higher percentage of Opa- variants of the wild-type (Cm(s)) strain. Reciprocal experiments (Opa- Cm(r) gonococci spiked with Opa+ Cm(s) bacteria) were consistent with selection of Opa+ variants. Based on the absence in mice of human carcinoembryonic antigen cell adhesion molecules, the major class of Opa protein adherence receptors, we conclude the observed selection for Opa+ variants early in infection is not likely due to a specific adherence advantage and may be due to Opa-mediated evasion of innate defenses.

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

    SciTech Connect

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

    1994-10-01

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

  14. Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.

    PubMed Central

    Jindal, S; Dudani, A K; Singh, B; Harley, C B; Gupta, R S

    1989-01-01

    The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases. Images PMID:2568584

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

    PubMed Central

    Alves, Rúbens Prince dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta

    2016-01-01

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

  16. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    PubMed

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

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

    PubMed

    Futami, Junichiro; Nonomura, Hidenori; Kido, Momoko; Niidoi, Naomi; Fujieda, Nao; Hosoi, Akihiro; Fujita, Kana; Mandai, Komako; Atago, Yuki; Kinoshita, Rie; Honjo, Tomoko; Matsushita, Hirokazu; Uenaka, Akiko; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-10-21

    Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

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

    PubMed

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

    2016-11-01

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

  19. The cellular proteins which can associate specifically with polyomavirus middle T antigen in human 293 cells include the major human 70-kilodalton heat shock proteins.

    PubMed

    Pallas, D C; Morgan, W; Roberts, T M

    1989-11-01

    We compared the proteins which associate with middle T antigen (MT) of polyomavirus in human cells infected with Ad5(pymT), a recombinant adenovirus which directs the overexpression of MT, with the MT-associated proteins (MTAPs) previously identified in murine fibroblasts expressing MT. MTAPs of 27, 29, 36, and 63 kilodaltons (kDa) appeared to be fairly well conserved between the two species, as judged by comigration on two-dimensional gels. Several 61-kDa MTAP species detected in MT immunoprecipitates from both cell sources also comigrated on these gels. However, no protein comigrating precisely with the murine 85-kDa MTAP could be detected in the human cells. Furthermore, two proteins of 72 and 74 kDa associated with wild-type MT in the infected human cells but not in murine fibroblasts expressing MT. It had been previously reported for murine cells that the 70-kDa heat shock protein associates with a particular mutant MT but not with wild-type MT (G. Walter, A. Carbone, and W.J. Welch, J. Virol. 61:405-410, 1987). By the criteria of comigration on two-dimensional gels, tryptic peptide mapping, and immunoblotting, we showed that the 72- and 74-kDa proteins that associate with wild-type MT in human cells are the major human 70-kDa heat shock proteins.

  20. Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein

    PubMed Central

    O’Connor, Kevin C; McLaughlin, Katherine A; De Jager, Philip L; Chitnis, Tanuja; Bettelli, Estelle; Xu, Chenqi; Robinson, William H; Cherry, Sunil V; Bar-Or, Amit; Banwell, Brenda; Fukaura, Hikoaki; Fukazawa, Toshiyuki; Tenembaum, Silvia; Wong, Susan J; Tavakoli, Norma P; Idrissova, Zhannat; Viglietta, Vissia; Rostasy, Kevin; Pohl, Daniela; Dale, Russell C; Freedman, Mark; Steinman, Lawrence; Buckle, Guy J; Kuchroo, Vijay K; Hafler, David A; Wucherpfennig, Kai W

    2012-01-01

    The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS. PMID:17237795

  1. Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.

    PubMed

    O'Connor, Kevin C; McLaughlin, Katherine A; De Jager, Philip L; Chitnis, Tanuja; Bettelli, Estelle; Xu, Chenqi; Robinson, William H; Cherry, Sunil V; Bar-Or, Amit; Banwell, Brenda; Fukaura, Hikoaki; Fukazawa, Toshiyuki; Tenembaum, Silvia; Wong, Susan J; Tavakoli, Norma P; Idrissova, Zhannat; Viglietta, Vissia; Rostasy, Kevin; Pohl, Daniela; Dale, Russell C; Freedman, Mark; Steinman, Lawrence; Buckle, Guy J; Kuchroo, Vijay K; Hafler, David A; Wucherpfennig, Kai W

    2007-02-01

    The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

  2. Pharmaceutical proteins in plants. A strategic genetic engineering approach for the production of tuberculosis antigens.

    PubMed

    Frutos, Roger; Denise, Hubert; Vivares, Christian; Neuhaus, Jean-Marc; Vitale, Sandro; Pedrazzini, Emmanuela; Ma, Julian; Dix, Phil; Gray, John; Pezzotti, Mario; Conrad, Udo; Robinson, David

    2008-12-01

    Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy.

  3. Permeation of antigen protein-conjugated nanoparticles and live bacteria through microneedle-treated mouse skin

    PubMed Central

    Kumar, Amit; Li, Xinran; Sandoval, Michael A; Rodriguez, B Leticia; Sloat, Brian R; Cui, Zhengrong

    2011-01-01

    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. PMID:21753877

  4. Antibodies to Mycobacterium paratuberculosis-specific protein antigens in Crohn's disease.

    PubMed Central

    Elsaghier, A; Prantera, C; Moreno, C; Ivanyi, J

    1992-01-01

    The possible role of infection with Mycobacterium paratuberculosis (MAP) for the etiopathogenesis of Crohn's disease (CD) has been a matter of long-term controversy. In addition to similarities with the pathology of ruminant paratuberculosis, DNA fingerprinting confirmed the organism isolated from gut tissue, but the specificity of the immune repertoire has not as yet been evaluated. We report here on a serological study of 29 patients with CD, 20 patients with ulcerative colitis and 18 healthy control subjects, using three antigens attributed with species-specificity and selective immunogenicity following MAP infection. Antibodies binding to the 38-kD band of MAP extract were demonstrable by the Western blot technique in 57% of CD patients. Antibody levels to the 24-kD (p24BCD) cathodic bands, determined by competition ELISA using a monospecific murine antiserum, and to the 18-kD protease-resistant purified bacterioferritin, detected by standard ELISA, were significantly elevated in 53% of CD patients. However, these three antibody specificities tested in individual CD patients did not show any correlation with each other. Thus, 18% of patients were positive for all three specificities, whilst 84% had antibodies to at least one of the specific antigens. Although the exact proportion of affected patients is yet to be defined, the serological results obtained support the view that MAP infection may play an etiological role in Crohn's disease. Images Fig. 1 Fig. 2 Fig. 3 PMID:1281056

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

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  6. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-03

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  7. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    PubMed

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination.

  8. Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus.

    PubMed

    Li, J; Kasper, D L; Ausubel, F M; Rosner, B; Michel, J L

    1997-11-25

    The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the alpha C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the alpha C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the alpha antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of alpha-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, alpha antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of alpha-specific antibodies. In addition, antibodies to the alpha C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the alpha antigen as expressed from the bca gene. Therefore, the alpha C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

  9. Translational control of beta2-adrenergic receptor mRNA by T-cell-restricted intracellular antigen-related protein.

    PubMed

    Kandasamy, Karthikeyan; Joseph, Kusumam; Subramaniam, Kothandharaman; Raymond, John R; Tholanikunnel, Baby G

    2005-01-21

    Cellular expression of the beta(2)-adrenergic receptor (beta(2)-AR) is suppressed at the translational level by 3'-untranslated region (UTR) sequences. To test the possible role of 3'-UTR-binding proteins in translational suppression of beta(2)-AR mRNA, we expressed the full-length 3'-UTR or the adenylate/uridylate-rich (A+U-rich element (ARE)) RNA from the 3'-UTR sequences of beta(2)-AR in cell lines that endogenously express this receptor. Reversal of beta(2)-adrenergic receptor translational repression by retroviral expression of 3'-UTR sequences suggested that ARE RNA-binding proteins are involved in translational suppression of beta(2)-adrenergic receptor expression. Using a 20-nucleotide ARE RNA from the receptor 3'-UTR as an affinity ligand, we purified the proteins that bind to these sequences. T-cell-restricted intracellular antigen-related protein (TIAR) was one of the strongly bound proteins identified by this method. UV-catalyzed cross-linking experiments using in vitro transcribed 3'-UTR RNA and glutathione S-transferase-TIAR demonstrated multiple binding sites for this protein on beta(2)-AR 3'-UTR sequences. The distal 340-nucleotide region of the 3'-UTR was identified as a target RNA motif for TIAR binding by both RNA gel shift analysis and immunoprecipitation experiments. Overexpression of TIAR resulted in suppression of receptor protein synthesis and a significant shift in endogenously expressed beta(2)-AR mRNA toward low molecular weight fractions in sucrose gradient polysome fractionation. Taken together, our results provide the first evidence for translational control of beta(2)-AR mRNA by TIAR.

  10. Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

    SciTech Connect

    Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M.

    2012-05-08

    Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

  11. Serodiagnosis of pulmonary tuberculosis in Argentina by enzyme-linked immunosorbent assay (ELISA) of IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative

    PubMed Central

    Balestrino, E. A.; Daniel, T. M.; de Latini, M. D. S.; Latini, O. A.; Ma, Y.; Scocozza, J. B.

    1984-01-01

    IgG antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) was measured, by enzyme-linked immunosorbent assay (ELISA), in serum samples from 86 patients with active pulmonary tuberculosis and 91 non-tuberculous control subjects from Santa Fé, Argentina. The geometric mean titre for the tuberculosis patients was 74.6 with antigen 5 and 99.5 with PPD. In 91 control subjects the geometric mean titres were 3.6 and 15.6 respectively. Titres were not related to tuberculin reactor status or prior BCG vaccination. At a serum dilution end-point of 1:40, ELISA with antigen 5 had a sensitivity of 81.4% and a specificity of 93.4% for tuberculosis. At 1:40, ELISA with PPD showed a sensitivity of 82.6% and a specificity of 54.9% for tuberculosis. Applied at a serum dilution of 1:40 to a hypothetical model population with a tuberculosis prevalence of 2%, ELISA using antigen 5 would correctly classify 93.2% of persons and ELISA with PPD, 55.5%. At a dilution of 1:80, accuracy is increased to 99.3% with antigen 5 and 83.3% with PPD, but sensitivity decreases to 64.0% with antigen 5 and 72.1% with PPD. Thus, antigen 5 is more accurate than PPD for the diagnosis of tuberculosis using ELISA. PMID:6439426

  12. Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

    PubMed

    Babij, Konrad; Bajzert, Joanna; Dąbrowska, Anna; Szołtysik, Marek; Zambrowicz, Aleksandra; Lubec, Gert; Stefaniak, Tadeusz; Willak-Janc, Ewa; Chrzanowska, Józefa

    2015-11-01

    In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

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

    SciTech Connect

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

    1988-07-01

    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 the primary structure of CALLA, the authors purified the protein to homogeneity, obtained the NH{sub 2}-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 NM{sub 2}-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA{sup +} 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.

  14. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R.

    PubMed

    Kraushar, Matthew L; Thompson, Kevin; Wijeratne, H R Sagara; Viljetic, Barbara; Sakers, Kristina; Marson, Justin W; Kontoyiannis, Dimitris L; Buyske, Steven; Hart, Ronald P; Rasin, Mladen-Roko

    2014-09-09

    Precise spatiotemporal control of mRNA translation machinery is essential to the development of highly complex systems like the neocortex. However, spatiotemporal regulation of translation machinery in the developing neocortex remains poorly understood. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stage-dependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of initiation and elongation factors in the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some HuR-dependent proteins, the association with polysomes likewise depends on the eukaryotic initiation factor 2 alpha kinase 4, which associates with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR before embryonic day 10 disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity.

  15. Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis.

    PubMed

    Raynaud, Cécile; Sozzani, Rosangela; Glab, Nathalie; Domenichini, Séverine; Perennes, Claudette; Cella, Rino; Kondorosi, Eva; Bergounioux, Catherine

    2006-08-01

    The proliferating cell nuclear antigen (PCNA) functions as a sliding clamp for DNA polymerase, and is thus a key actor in DNA replication. It is also involved in DNA repair, maintenance of heterochromatic regions throughout replication, cell cycle regulation and programmed cell death. Identification of PCNA partners is therefore necessary for understanding these processes. Here we identify two Arabidopsis SET-domain proteins that interact with PCNA: ATXR5 and ATXR6. A truncated ATXR5Deltaex2, incapable of interacting with PCNA, also occurs in planta. ATXR6, upregulated during the S phase, is upregulated by AtE2F transcription factors, suggesting that it is required for S-phase progression. The two proteins differ in their subcellular localization: ATXR5 has a dual localization in plastids and in the nucleus, whereas ATXR6 is solely nuclear. This indicates that the two proteins may play different roles in plant cells. However, overexpression of either ATXR5 or ATXR6 causes male sterility because of the degeneration of defined cell types. Taken together, our results suggest that both proteins may play a role in the cell cycle or DNA replication, and that the activity of ATXR5 may be regulated via its subcellular localization.

  16. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    PubMed

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans.

  17. Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men.

    PubMed

    Shetty, Jagathpala; Bronson, Richard A; Herr, John C

    2008-01-01

    Anti-sperm antibodies (ASA) are an important cause of immunological infertility. The objective of this study was to identify immunodominant sperm antigens recognized by anti-sperm antibodies (ASA) in serum samples of infertile men, women and vasectomized men. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE), followed by PAGE and Western blotting. Serum samples from five infertile male and five infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT), were analyzed by Western blotting using NEPHGE gels followed by enhanced chemiluminescence (ECL) to identify the basic sperm antigens reactive to the sera. Serum samples from five fertile male and five fertile female subjects that were ASA-negative by IBT were used as controls. Serum samples from six vasectomized men collected before vasectomy and at different time intervals until 6 months after vasectomy were analyzed by Western blotting using IEF gels. The ECL blots were analyzed to compare immunoreactivity between serum samples from fertile and infertile subjects and identify antigens unique to sera of the infertile subjects. Similarly, immunoreactivity between serum samples from pre- and post-vasectomy was compared to identify antigens unique to sera collected following vasectomy. Five allo-antigenic basic protein spots were recognized by sera from infertile males but not from fertile subjects. Five sperm iso-antigenic basic spots were recognized by infertile female subjects. Two among six of the vasectomized men's sera showed a difference in the Western blot profile 6 months after vasectomy, recognizing at least one new protein spot in each case when compared to pre-vasectomy sera. The acrosomal protein SP-10 was identified as an alloantigen recognized by a post-vasectomy serum. Molecular identities of the known allo- and iso-antigens

  18. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-11-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes ( IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon ( IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  19. Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

    NASA Astrophysics Data System (ADS)

    Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

    2016-03-01

    We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

  20. Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

    PubMed Central

    Biedroń, Rafał; Konopiński, Maciej K.; Marcinkiewicz, Janusz; Józefowski, Szczepan

    2015-01-01

    The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect

  1. A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein.

    PubMed

    Fetterer, R H; Miska, K B; Jenkins, M C; Barfield, R C

    2004-12-01

    A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

  2. Identification of three antigen epitopes on the nucleocapsid protein of the genotype C of bovine parainfluenza virus type 3.

    PubMed

    Ren, Jian-Le; Zhu, Yuan-Mao; Zhou, Yue-Hui; Lv, Chuang; Yan, Hao; Ma, Lei; Shi, Hong-Fei; Xue, Fei

    2015-07-09

    Bovine parainfluenza virus type 3 (BPIV3) is an important respiratory tract pathogen for both young and adult cattle. So far, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis. But fine mapping of epitopes of BPIV3 is scant and the antigenic variations among the three genotypes of BPIV3 have not been reported. Nucleocapsid protein (NP) is the most abundant protein in the virion and highly conserved in BPIV3, which is crucial for the induction of protective immunity in host. To identify antigenic determinants of BPIV3 NP, a panel of monoclonal antibodies (mAbs) was tested against a series of overlapping recombinant NP fragments expressed in Escherichia coli. Firstly, six monoclonal antibodies (mAbs) against NP of the genotype C of BPIV3 (BPIV3c) were generated by using the purified BPIV3c strain SD0835 as immunogen and the recombinant NP of SD0835 as screening antigen. Then three antigen epitopes were identified with the six mAbs. One epitope (91)GNNADVKYVIYM(102) was recognized by mAb 5E5. The mAbs 7G5, 7G8, 7G9, and 7H5 were reactive with another epitope (407)FYKPTGG(413). The third epitope (428)ESRGDQDQ(435) was reactive with mAb 6F8. Further analysis showed that the epitope (91-102 amino acids [aa]) was the most conserved and reactive with mAb 5E5 for all three genotypes of BPIV3 and HPIV3. The epitope (407-413 aa) was relatively conserved and reactive with mAbs 7G5, 7G8, 7G9, and 7H5 for BPIV3a, BPIV3c and HPIV3, but not reactive with BPIV3b. The epitope (428-435 aa) was less conserved and was reactive only with mAb 6F8 for BPIV3a and BPIV3c. These results suggested that there were evident antigenic variations among the three genotypes of BPIV3 and HPIV3. The mAb 6F8 could be used to detect BPIV3a and BPIV3c. The mAbs 7G5, 7G8, 7G9, and 7H5 might be used for differentiate BPIV3a, BPIV3c and HPIV3 from BPIV3b. The mAb 5E5 might be used for detecting all three types of BPIV3 and HPIV3. The results in this

  3. Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

    PubMed Central

    Roy, S; Das, A B; Ghosh, A N; Biswas, T

    1994-01-01

    The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study. Images PMID:7927692

  4. Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

    PubMed Central

    2010-01-01

    Introduction Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. Methods IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. Results Circulating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. Conclusions Citrullinated fibrinogenβ and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin

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

    PubMed Central

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

    2014-01-01

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

  6. Adult Schistosoma mansoni worms positively modulate soluble egg antigen-induced inflammatory hepatic granuloma formation in vivo. Stereological analysis and immunophenotyping of extracellular matrix proteins, adhesion molecules, and chemokines.

    PubMed Central

    Jacobs, W.; Bogers, J.; Deelder, A.; Wéry, M.; Van Marck, E.

    1997-01-01

    Synchronized liver granulomas were induced by injecting Sepharose beads to which SEA soluble egg antigen (SEA) or the concanavalin A binding fraction of SEA had been coupled into a mesenteric vein in naive, single-sex (35 days) and bisexually (28 days) Schistosoma mansoni-infected and Plasmodium berghei-immunized mice. Stereological analysis revealed that peak granuloma formation was already reached 8 days after injection in single-sex infected mice compared with 16 days in naive animals. No difference in granuloma formation between naive and P. berghei-immunized animals and between unisexually and bisexually S. mansoni-infected mice was observed. This suggests that the positive immunomodulatory effect on the granulomogenesis is worm specific and not likely to be due to arousal of the immune system by unrelated factors, nor is it influenced by the gender or degree of maturation of female worms. At all stages in time, the concanavalin A binding-fraction-induced granulomas reached only 65 to 70% of the volume of SEA-induced granulomas. Immunophenotyping of extracellular matrix proteins around deposited heads revealed that fibronectin was the dominant extracellular matrix protein and that also type I and IV collagen and laminin were deposited. Temporal analysis of the expression of the adhesion molecules ICAM-1, LFA-1, VLA-4, and VLA-6 was performed. Morphological evidence is presented for the role of adhesion molecules in the initiation and maintenance of hepatic granuloma formation. The chemokine monocyte chemoattractant protein-1 was expressed in the granuloma and in hepatic artery branches. From these data, it is concluded that adult S. mansoni worms positively modulate schistosomal hepatic granuloma formation in vivo. Adhesion molecules and chemokines play important roles in schistosomal granuloma formation. Images Figure 1 Figure 2 Figure 3 PMID:9176396

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

    PubMed Central

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

    2013-01-01

    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 towards Escherichia coli. When the PspA nanoparticle formulations were administered subcutaneously to mice, the animals 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

  8. Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

    PubMed

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-09-01

    The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

  9. The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen

    PubMed Central

    Hofbauer, Anna; Melnik, Stanislav; Tschofen, Marc; Arcalis, Elsa; Phan, Hoang T.; Gresch, Ulrike; Lampel, Johannes; Conrad, Udo; Stoger, Eva

    2016-01-01

    Zein is a water-insoluble polymer from maize seeds that has been widely used to produce carrier particles for the delivery of therapeutic molecules. We encapsulated a recombinant model vaccine antigen in newly formed zein bodies in planta by generating a fusion construct comprising the ectodomain of hemagglutinin subtype 5 and the N-terminal part of γ-zein. The chimeric protein was transiently produced in tobacco leaves, and H5-containing protein bodies (PBs) were used to immunize mice. An immune response was achieved in all mice treated with H5-zein, even at low doses. The fusion to zein markedly enhanced the IgG response compared the soluble H5 control, and the effect was similar to a commercial adjuvant. The co-administration of adjuvants with the H5-zein bodies did not enhance the immune response any further, suggesting that the zein portion itself mediates an adjuvant effect. While the zein portion used to induce protein body formation was only weakly immunogenic, our results indicate that zein-induced PBs are promising production and delivery vehicles for subunit vaccines. PMID:26909090

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

    PubMed

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

    2010-07-14

    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.

  11. The Epstein-Barr virus lytic protein BZLF1 as a candidate target antigen for vaccine development1

    PubMed Central

    Hartlage, Alex S.; Liu, Tom; Patton, John T.; Garman, Sabrina L.; Zhang, Xiaoli; Kurt, Habibe; Lozanski, Gerard; Lustberg, Mark E.; Caligiuri, Michael A.; Baiocchi, Robert A.

    2015-01-01

    The Epstein-Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. While most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life threatening diseases such as Hodgkin’s lymphoma and post-transplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8(+) T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases. PMID:25735952

  12. Epstein–Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1

    PubMed Central

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L.; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-01-01

    The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. PMID:25092922

  13. The large tumor antigen: a "Swiss Army knife" protein possessing the functions required for the polyomavirus life cycle.

    PubMed

    Topalis, D; Andrei, G; Snoeck, R

    2013-02-01

    The SV40 large tumor antigen (L-Tag) is involved in the replication and cell transformation processes that take place during the polyomavirus life cycle. The ability of the L-Tag to interact with and to inactivate the tumor suppressor proteins p53 and pRb, makes this polyfunctional protein an interesting target in the search for compounds with antiviral and/or antiproliferative activities designed for the management of polyomavirus-associated diseases. The severe diseases caused by polyomaviruses, mainly in immunocompromised hosts, and the absence of licensed treatments, make the discovery of new antipolyomavirus drugs urgent. Parallels can be made between the SV40 L-Tag and the human papillomavirus (HPV) oncoproteins (E6 and E7) as they are also able to deregulate the cell cycle in order to promote cell transformation and its maintenance. In this review, a presentation of the SV40 L-Tag characteristics, regarding viral replication and cellular transformation, will show how similar these two processes are between the polyoma- and papillomavirus families. Insights at the molecular level will highlight similarities in the binding of polyoma- and papillomavirus replicative helicases to the viral DNA and in their disruptions of the p53 and pRb tumor suppressor proteins.

  14. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins

    PubMed Central

    1993-01-01

    The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins. PMID:8227122

  15. Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity.

    PubMed

    Shin, Gu-Choul; Chung, Yoon-Seok; Kim, In-Soo; Cho, Hae-Wol; Kang, Chun

    2007-07-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is involved in the pathological reaction to SARS and is a key antigen for the development of a sensitive diagnostic assay. However, the antigenic properties of this N protein are largely unknown. To facilitate the studies on the function and antigenicity of the SARS-CoV N protein, 6x histidine-tagged recombinant SARS-CoV N (rSARS-N) with a molecular mass of 46 and 48kDa was successfully produced using the recombinant baculovirus system in insect cells. The rSARS-N expressed in insect cells (BrSARS-N) showed remarkably higher specificity and immunoreactivity than rSARS-N expressed in E. coli (ErSARS-N). Most of all, BrSARS-N proteins were expressed as a highly phosphorylated form with a molecular mass of 48kDa, but ErSARS-N was a nonphosphorylated protein. In further analysis to determine the correlation between the phosphorylation and the antigenicity of SARS-N protein, dephosphorylated SARS-N protein treated with protein phosphatase 1 (PP1) remarkably enhanced the cross-reactivity against SARS negative serum and considerably reduced immunoreactivity with SARS-N mAb. These results suggest that the phosphorylation plays an important role in the immunoreactivity and specificity of SARS-N protein. Therefore, the BrSARS-N protein may be useful for the development of highly sensitive and specific assays to determine SARS infection and for further research of SARS-N pathology.

  16. Major Trypanosoma cruzi antigenic determinant in Chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

    PubMed Central

    Mesri, E A; Levitus, G; Hontebeyrie-Joskowicz, M; Dighiero, G; Van Regenmortel, M H; Levin, M J

    1990-01-01

    A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease. PMID:1696282

  17. T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

    PubMed Central

    Sánchez-Jiménez, Carmen; Izquierdo, José M.

    2013-01-01

    Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress. PMID:24086455

  18. T-cell intracellular antigen (TIA)-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy.

    PubMed

    Sánchez-Jiménez, Carmen; Izquierdo, José M

    2013-01-01

    Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress.

  19. Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

    PubMed

    Chen, Austen Y; Fry, Scott R; Daggard, Grant E; Mukkur, Trilochan K S

    2008-08-12

    Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

  20. Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

    PubMed

    Kim, Jong-Hyun; Yang, Ae-Hee; Sohn, Hae-Jin; Kim, Daesik; Song, Kyoung-Ju; Shin, Ho-Joon

    2009-11-01

    Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

  1. HIV-1 matrix protein p17: a candidate antigen for therapeutic vaccines against AIDS.

    PubMed

    Fiorentini, Simona; Giagulli, Cinzia; Caccuri, Francesca; Magiera, Anna K; Caruso, Arnaldo

    2010-12-01

    The success in the development of anti-retroviral therapies (HAART) that contain human immunodeficiency virus type 1 (HIV-1) infection is challenged by the cost of this lifelong therapy and by its toxicity. Immune-based therapeutic strategies that boost the immune response against HIV-1 proteins or protein subunits have been recently proposed to control virus replication in order to provide protection from disease development, reduce virus transmission, and help limit the use of anti-retroviral treatments. HIV-1 matrix protein p17 is a structural protein that is critically involved in most stages of the life cycle of the retrovirus. Besides its well established role in the virus life cycle, increasing evidence suggests that p17 may also be active extracellularly in deregulating biological activities of many different immune cells that are directly or indirectly involved in AIDS pathogenesis. Thus, p17 might represent a promising target for developing a therapeutic vaccine as a contribution to combating AIDS. In this article we review the biological characteristics of HIV-1 matrix protein p17 and we describe why a synthetic peptide representative of the p17 functional epitope may work as a vaccine molecule capable of inducing anti-p17 neutralizing response against p17 derived from divergent HIV-1 strains.

  2. Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

    PubMed

    Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

    2016-12-01

    We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients.

  3. GBP-5 splicing variants: New guanylate-binding proteins with tumor-associated expression and antigenicity.

    PubMed

    Fellenberg, Friederike; Hartmann, Tanja B; Dummer, Reinhard; Usener, Dirk; Schadendorf, Dirk; Eichmüller, Stefan

    2004-06-01

    We have identified a new gene, gbp-5, with high homology to the guanylate binding proteins (GBP) belonging to the GTPase superfamily including the ras gene. gbp-5 is transcribed at least into three splicing variants (gbp-5a, -5b, and -5ta) leading to two different proteins (GBP-5a/b, GBP-5ta). GBP-5ta is C-terminally truncated by 97aa and has therefore lost its isoprenylation site. Although RT-PCR results indicated expression of GBP-5 members in selected normal tissues, western blotting using two newly generated antibodies revealed that expression of both proteins is restricted to peripheral blood monocytes with GBP-5ta at lower levels. In contrast, cutaneous T-cell lymphoma (CTCL) tumor tissues (seven of seven) were positive solely for GBP-5ta, and four of four CTCL cell lines expressed both proteins. Eight of nine melanoma cell lines expressed GBP-5a/b and four of nine additionally low levels of GBP-5ta. SEREX retesting using CTCL sera indicated a higher immunogenicity for GBP-5ta (nine of 16) than for GBP-5a/b (two of 11). Treatment of CTCL cell lines with interferon-gamma did not alter protein expression of GBP-5ta or GBP-5a/b. The restricted expression pattern of both GBP-5ta and GBP-5a/b and the pivotal role of many known members of the GTP-binding proteins in proliferation and differentiation suggest possible cancer-related functions of gbp-5.

  4. Immunogenic protein variations of Clostridium chauvoei cellular antigens associated with the culture growth phase.

    PubMed

    Mattar, María Aída; Cortiñas, Teresa Inés; de Guzmán, Ana María Stefanini

    2002-03-25

    The immunoprotective capacity of four Clostridium chauvoei strains at different growth stages is reported. In all the strains tested, the cells coming from the stationary phase were those with the highest immunoprotective capacity and, depending on the strain, this protective capacity diminished or even disappeared in other phases. Protein profiles were similar in all the strains and few proteins were differentially expressed during growth as shown by SDS-PAGE. For strain 17, a local strain, a clear relationship was observed between the diminution of immunogenicity and the total loss of protective capacity of sonicated cells at late stationary phase.

  5. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

    PubMed Central

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  6. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    PubMed

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-11-13

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

  7. Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.

    PubMed Central

    Evans, C M; Fryer, A D; Jacoby, D B; Gleich, G J; Costello, R W

    1997-01-01

    In antigen-challenged guinea pigs there is recruitment of eosinophils into the lungs and to airway nerves, decreased function of inhibitory M2 muscarinic autoreceptors on parasympathetic nerves in the lungs, and airway hyperresponsiveness. A rabbit antibody to guinea pig eosinophil major basic protein was used to determine whether M2 muscarinic receptor dysfunction, and the subsequent hyperresponsiveness, are due to antagonism of the M2 receptor by eosinophil major basic protein. Guinea pigs were sensitized, challenged with ovalbumin and hyperresponsiveness, and M2 receptor function tested 24 h later with the muscarinic agonist pilocarpine. Antigen-challenged guinea pigs were hyperresponsive to electrical stimulation of the vagus nerves compared with controls. Likewise, loss of M2 receptor function was demonstrated since the agonist pilocarpine inhibited vagally-induced bronchoconstriction in control but not challenged animals. Pretreatment with rabbit antibody to guinea pig eosinophil major basic protein prevented hyperresponsiveness, and protected M2 receptor function in the antigen-challenged animals without inhibiting eosinophil accumulation in the lungs or around the nerves. Thus, hyperresponsiveness is a result of inhibition of neuronal M2 muscarinic receptor function by eosinophil major basic protein in antigen-challenged guinea pigs. PMID:9410903

  8. The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...

  9. Analysis of the Cross-Reactivity of Various 56 kDa Recombinant Protein Antigens with Serum Samples Collected after Orientia tsutsugamushi Infection by ELISA

    DTIC Science & Technology

    2011-01-01

    has increased the yield of Orientia , 28 considerable difficulties still exist in mass production of pure Orientia and in retaining its stability...Rickettsia tsutsugamushi Boryong with partial 56-kilodalton recombinant antigen fused with the maltose - binding protein MBP-Bor56 . Infect Immun 65

  10. Specific nongluten proteins of wheat are novel target antigens in celiac disease humoral response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Celiac disease is an immune-mediated enteropathy that is generally understood to be triggered by the ingestion of gluten proteins of wheat and related cereals. The skin manifestation of the condition is known as dermatitis herpetiformis. Antibody response to native and deamidated seque...

  11. Antigenic features of prion proteins of sheep and of other mammalian species.

    PubMed

    Groschup, M H; Harmeyer, S; Pfaff, E

    1997-08-22

    Pathological prion protein (PrPSc) which is a conformational isoform of a host-encoded protein designated (PrPC) serves as a specific marker protein for the immunochemical diagnosis of transmissible spongiform encephalopathies (TSE). The generation of suitable antibodies to PrPSc therefore underlies the specificity and sensitivity of diagnostic assays. However, most antibodies reported to date are directed to a limited number of epitopes only. PrPC is a highly conserved cell membrane protein in all mammalian species studied to date. In an attempt to generate antibodies to further regions of PrP we raised antisera in rabbits and chicken against sixteen synthetic peptides which represent the complete aminoacid sequence of ovine PrP. By this approach immunotolerance was overcome and immunoblot-reactive antibodies were stimulated to epitopes at almost any site of ovine PrPC and PrPSc. A large number of different antibodies cross-reacted also with affinity-purified PrPCs from other mammalian species including cow, goat, pig, man, dog, cat, mink, mouse, hamster and guinea pig. No epitope, however, was recognized exclusively on the pathological or cellular isoform of PrP indicating that both isoforms occur in highly denatured conformations on the immunoblots. Antibodies to the amino-terminus are suitable for immunoprecipitation of PrP. The availability of rabbit and chicken anti-peptide antibodies to PrP will greatly improve immunochemical diagnosis and pathogenetic studies on these diseases.

  12. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  13. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of...

  14. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  15. Characterization and Antigenicity of Recombinant Campylobacter jejuni Flagellar Capping Protein FliD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 microorganism. Bacterial flagella, composed of more than 35 proteins, play important roles in c...

  16. Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs.

    PubMed Central

    Garcia, M M; Lutze-Wallace, C L; Denes, A S; Eaglesome, M D; Holst, E; Blaser, M J

    1995-01-01

    Campylobacter fetus subsp. venerealis isolated from a case of human vaginosis was inoculated into the uterus of a C. fetus-negative heifer. Isolates obtained weekly from the vaginal mucus exhibited variations in high-molecular-mass-protein profiles from that of the original inoculum, which had a dominant 110-kDa S-layer protein. Immunoblots of the weekly isolates with monoclonal antibody probes against the 110-kDa S-layer protein and other C. fetus S-layer proteins demonstrated antigenic shifts. Genomic digests of the isolates probed with a 75-mer oligonucleotide of the conserved sapA region also indicated that antigenic variation of the S-layer is accompanied by DNA rearrangement. PMID:7721688

  17. Tubulointerstitial nephritis antigen: an extracellular matrix protein that selectively regulates tubulogenesis vs. glomerulogenesis during mammalian renal development.

    PubMed

    Kanwar, Y S; Kumar, A; Yang, Q; Tian, Y; Wada, J; Kashihara, N; Wallner, E I

    1999-09-28

    Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix protein and is expressed in the renal tubular basement membranes. Its role in metanephric development was investigated. TIN-ag cDNA, isolated from the newborn mouse library, had an ORF of 1,425 nucleotides, a putative signal sequence, and an ATP/GTP-binding site. The translated sequence had approximately 80% identity with rabbit TIN-ag. Among various tissues, TIN-ag mRNA was primarily expressed in the newborn kidney. In the embryonic metanephros, TIN-ag expression was confined to the distal convolution or pole of the S-shaped body, the segment of the nascent nephron that is the progenitor of renal tubules. Treatment with TIN-ag antisense oligodeoxynucleotide induced dysmorphogenesis of the embryonic metanephroi, malformation of the S-shaped body, and a decrease in the tubular population, whereas the glomeruli were unaffected. Treatment also led to a decrease of TIN-Ag mRNA, de novo synthesis of TIN-ag protein, and its antibody reactivity. The mRNA expression of glomerular epithelial protein 1 (a marker for renal podocytes), anti-heparan-sulfate-proteoglycan antibody reactivity, and wheat germ agglutinin lectin staining of the metanephros were unaffected. The anti-TIN-ag antibody treatment also caused deformation of the S-shaped body and a reduction in the tubular population, whereas the glomeruli were unchanged. The data suggest that the TIN-ag, unlike other basement membrane proteins, selectively regulates tubulogenesis, whereas glomerulogenesis is largely unaffected.

  18. Extent and effects of recurrent shortages of purified-protein derivative tuberculin skin test antigen solutions - United States, 2013.

    PubMed

    2013-12-13

    Two purified-protein derivative (PPD) tuberculin skin test (TST) antigen solutions are approved by the U.S. Food and Drug Administration (FDA): Tubersol (Sanofi Pasteur Limited) and Aplisol (JHP Pharmaceuticals, LLC). Tubersol was out of production in late 2012 through April 2013. Shortages of Aplisol have resulted from increased demand as practitioners have sought a substitute for Tubersol. Tubersol production resumed in May 2013, and supplies had been nearly restored by early June. However, in mid-July, state tuberculosis (TB) control officials notified CDC of difficulty obtaining Tubersol and Aplisol. Sanofi Pasteur notified FDA of a temporary delay in the availability of tuberculin in the 10-dose and 50-dose presentations. In mid-October, the 10-dose presentation was being returned to market, on allocation, which means that historical purchasing practices determine the amount that customers are allotted. In late October, the 50-dose presentation was being returned to market, also on allocation, one vial per historical customer per month. Supplies are forecast to approach normal during January 2014, after distributors have restored their supply chains. A compensatory surge in testing after deferment of testing during the periods of shortage might cause further temporary instability of supplies. In mid-August 2013, officials in 29 of 52 U.S. jurisdictions noted a shortage of at least one PPD TST antigen solution in health departments to the extent that it interrupted activities. This report includes a summary of the extent and effects of the shortages and a reiteration of advice on how to adapt to them.

  19. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  20. Replication protein A and proliferating cell nuclear antigen coordinate DNA polymerase selection in 8-oxo-guanine repair.

    PubMed

    Maga, Giovanni; Crespan, Emmanuele; Wimmer, Ursula; van Loon, Barbara; Amoroso, Alessandra; Mondello, Chiara; Belgiovine, Cristina; Ferrari, Elena; Locatelli, Giada; Villani, Giuseppe; Hübscher, Ulrich

    2008-12-30

    The adenine misincorporated by replicative DNA polymerases (pols) opposite 7,8-dihydro-8-oxoguanine (8-oxo-G) is removed by a specific glycosylase, leaving the lesion on the DNA. Subsequent incorporation of C opposite 8-oxo-G on the resulting 1-nt gapped DNA is essential for the removal of the 8-oxo-G to prevent G-C to T-A transversion mutations. By using model DNA templates, purified DNA pols beta and lambda and knockout cell extracts, we show here that the auxiliary proteins replication protein A and proliferating cell nuclear antigen act as molecular switches to activate the DNA pol lambda- dependent highly efficient and faithful repair of A:8-oxo-G mismatches in human cells and to repress DNA pol beta activity. By using an immortalized human fibroblast cell line that has the potential to induce cancer in mice, we show that the development of a tumoral phenotype in these cells correlated with a differential expression of DNA pols lambda and beta.

  1. Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis.

    PubMed

    Ghosal, Gargi; Leung, Justin Wai-Chung; Nair, Binoj C; Fong, Ka-Wing; Chen, Junjie

    2012-10-05

    DNA damage-induced proliferating cell nuclear antigen (PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves either translesion synthesis (TLS) or damage avoidance via template switching. In this study, we have identified and characterized C1orf124 as a regulator of TLS. C1orf124 co-localizes and interacts with unmodified and mono-ubiquitinated PCNA at UV light-induced damage sites, which require the PIP box and UBZ domain of C1orf124. C1orf124 also binds to the AAA-ATPase valosin-containing protein via its SHP domain, and cellular resistance to UV radiation mediated by C1orf124 requires its interactions with valosin-containing protein and PCNA. Interestingly, C1orf124 binds to replicative DNA polymerase POLD3 and PDIP1 under normal conditions but preferentially associates with TLS polymerase η (POLH) upon UV damage. Depletion of C1orf124 compromises PCNA monoubiquitination, RAD18 chromatin association, and RAD18 localization to UV damage sites. Thus, C1orf124 acts at multiple steps in TLS, stabilizes RAD18 and ubiquitinated PCNA at damage sites, and facilitates the switch from replicative to TLS polymerase to bypass DNA lesion.

  2. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    NASA Astrophysics Data System (ADS)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  3. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  4. Immunohistochemical distribution of heat shock protein 70 and proliferating cell nuclear antigen in mouse placenta at different gestational stages.

    PubMed

    Ozaydin, Tugba; Sur, Emrah; Oznurlu, Yasemin; Celik, Ilhami; Uluisik, Deniz

    2016-04-01

    The aim of the present study was to investigate immunohistochemical distribution of heat shock protein 70 (Hsp70) and proliferating cell nuclear antigen (PCNA) in the mouse placenta at different gestational stages. For this purpose a total of 18 Swiss albino female mice at 12-14 weeks of age were used. Females were sacrificed on days 3 (early), 10 (mid-), and 17 (late) of pregnancy and the implantation sites of the pregnant uterus were sampled. The sections were made transversely through the central region of the implantation site and stained with hematoxylin and eosin for histological examination. PCNA and Hsp70 was stained immunohistochemically. Since the definitive placenta was not still formed on day 3 of pregnancy, Hsp70 and PCNA positivity were evaluated in only luminal epithelium and decidual-stromal cells. On days 10 and 17 of pregnancy, Hsp70 and PCNA positivity were evaluated in labyrinth zone, junctional zone and decidual layer of placenta. Hsp70 expression was observed trophoblast cells and decidual cells and was relatively constant throughout the pregnancy. This protein was strongly labeled in the trophoblast cells; while decidual cells were displayed moderate staining. In early pregnant mouse uteri, PCNA was mainly localized in decidual-stromal cells. The trophoblast cells and decidual cells displayed highly proliferative activity at the midgestational period. However there was a significant decrease in the percentage of PCNA positive cells in late gestation.

  5. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

  6. Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

    PubMed Central

    Jónsson, Z O; Hindges, R; Hübscher, U

    1998-01-01

    The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis. PMID:9545252

  7. Large, detergent-resistant complexes containing murine antigens Thy-1 and Ly-6 and protein tyrosine kinase p56lck.

    PubMed

    Bohuslav, J; Cinek, T; Horejsí, V

    1993-04-01

    A number of human and mouse leukocyte surface (glyco)proteins anchored in a membrane via glycosylphosphatidylinositol (GPI) moiety have been previously shown to be noncovalently associated with protein tyrosine kinases (Science 1991. 254: 1016; J. Biol. Chem. 1992. 267: 12317). Here we show that two murine antigens of this group, Thy-1 and Ly-6, implicated in the activation of the T cells, are associated with each other, with the kinase p56lck and with several of potential kinase substrates in very large, detergent-resistant complexes, the size of which is between 50 and 200 nm, as determined by ultrafiltration and gel chromatography. Experiments on simultaneous solubilization of mixed human and mouse cells rule out that the observed complexes are artifacts induced by the detergent. Complexes of similar composition and properties were obtained when either detergents Brij-58, Nonidet-P40 or 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane-sulfonate (Chaps) were used for solubilization of the cells, while octylglucoside at least partially dissociated them. These "GPI-complexes" may be essential for the well-known signal-transducing capacity of Thy-1 and Ly-6.

  8. Antigenicity of recombinant maltose binding protein-Mycobacterium avium subsp. paratuberculosis fusion proteins with and without factor Xa cleaving

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that MAP expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such prot...

  9. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens

    PubMed Central

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway. PMID:26752992

  10. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.

    PubMed

    Golshani, Maryam; Rafati, Sima; Jahanian-Najafabadi, Ali; Nejati-Moheimani, Mehdi; Siadat, Seyed Davar; Shahcheraghi, Fereshteh; Bouzari, Saeid

    2015-01-01

    Globally, Brucella melitensis and B. abortus are the most common cause of human brucellosis. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens which are considered as potential vaccine candidates. We aimed to design the fusion protein from Brucella L7/L12 and truncated Omp31proteins, in silico, clone the fusion in pET28a vector, and express it in Escherichia coli host. Two possible fusion forms, L7/L12-TOmp31 and TOmp31-L7/L12 were subjected to in silico modeling and analysis. Analysis and validation of the fusion proteins with three dimensional (3D) models showed that both models are in the range of native proteins. However, L7/L12-Tomp31 structure was more valid than the TOmp31-L7/L12 model and subjected to in vitro production. The major histocompatibility complex (MHC II) epitope mapping using IEDB database indicated that the model contained good MHC II binders. The L7/L12-TOmp31 coding sequence was cloned in pET28a vector. The integrity of the construct was confirmed by polymerase chain reaction, restriction enzyme mapping, and sequencing. The fusion was successfully expressed in E. coli BL21 (DE3) by induction with isopropyl β-D-thiogalactopyranoside. The rL7/L12-TOmp31 was purified with Ni-NTA column. The yield of the purified rL7/L12-TOmp31 was estimated by Bradford method and found to be 40 mg/L of the culture. Western blotting with anti-His antibody revealed a specific reactivity with purified rL7/L12-TOmp31 produced in E. coli and showed the functional expression in the prokaryotic system. In this study, a new protein vaccine candidate against brucellosis was constructed with the help of bioinformatics tools and the construct was expressed in the bacterial host. Studies evaluating the immunogenicity and cross-protection of this fusion protein against B. melitensis and B. abortus are underway.

  11. Plasmodium falciparum: Exported Protein-1, A Blood Stage Antigen is Expressed in Liver Stage Parasites.

    DTIC Science & Technology

    1994-01-01

    protein of Plasmodium falci- Meihods in Enzymology 185. 6"-9. parum is synthesized as an integral membrane pro- tein. Molecular and Biochemical ...1, Tower 12 DN244530 8901 Wisconsin Avenue Bethesda, Maryland 20889-5606 11. SUPPLEMENTARY NOTES Reprinted from: Experimental Parasitology 1994;vol.79...THIS PAGE I OF ABSTRACT Unclassified I Unclassified I Unclassified Unlimited NSN ,7.ui-01au.SSOO ~n~t am26~~25 EXPERIMENTAL PARASITOLOGY 79, 59-62 (1994

  12. Identification, Purification and Characterization of Major Antigenic Proteins of Campylobacter jejuni

    DTIC Science & Technology

    1991-01-01

    QUT Z-i - .. . Campylobacter, Vaccines, Biotechnology, ID, Diseases, RADI, lab animals , Volunteers Unclassified Unclassified Unclassfied Unlimited...frequently present in the intestinal tracts amino-terminal sequence. Two of the four proteins are com- of the animals we use for food processing, and...and human and animal populations would be desirable, also may be of value in serological assays for the diagnosis of Several lines of evidence

  13. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

    PubMed Central

    Barber, E K; Dasgupta, J D; Schlossman, S F; Trevillyan, J M; Rudd, C E

    1989-01-01

    Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex. Images PMID:2470098

  14. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis

    PubMed Central

    Goo, Leslie; VanBlargan, Laura A.; Dowd, Kimberly A.; Diamond, Michael S.

    2017-01-01

    The structural flexibility or ‘breathing’ of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing. PMID:28207910

  15. Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2.

    PubMed

    Mason, Lyndel; Tribolet, Leon; Simon, Anne; von Gnielinski, Natascha; Nienaber, Lisa; Taylor, Paul; Willis, Charlene; Jones, Malcolm K; Sternberg, Paul W; Gasser, Robin B; Loukas, Alex; Hofmann, Andreas

    2014-05-01

    Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity. Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.

  16. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens

    SciTech Connect

    Bradley, M.K.; Smith, T.F.; Lathrop, R.H.; Livingston, D.M.; Webster, T.A.

    1987-06-01

    The location and sequence composition of a consensus element of the nucleotide binding site in both simian virus 40 (SV40) and polyomavirus (PyV) large tumor antigens (T antigens) can be predicted with the assistance of a computer-based pattern-matching system, ARIADNE. The latter was used to optimally align elements of T antigen primary sequence and predicted secondary structure with a descriptor for a mononucleotide binding fold. Additional consensus elements of the nucleotide binding site in these two proteins were derived from comparisons of T antigen primary and predicted secondary structures with x-ray structures of the nucleotide binding sites in four otherwise unrelated proteins. Each of these elements was predicted to be encompassed within a 110-residue segment that is highly conserved between the two T antigens residues 418-528 in SV 40 T antigen and residues 565-675 in PyV. Results of biochemical and immunologic experiments on the nucleotide binding behavior of these proteins using (/sup 32/P)-Amp-labeled SV40 T antigen, were found to be consistent with these predictions. Taken together, the latter have resulted in a topological model of the ATP binding site in these two oncogene products.

  17. Effect of context and adjuvant on the immunogenicity of recombinant proteins and peptide conjugates derived from the polymorphic malarial surface antigen MSA2.

    PubMed

    Jones, G L; Spencer, L; Lord, R; Saul, A J

    1996-01-01

    We have identified a 51 kDa glycosylated myristylated merozoite surface antigen (MSA2) as the target of a number of monoclonal antibodies which inhibit in vitro invasion of the human malarial parasite Plasmodium falciparum. This antigen has been shown to exist in a limited number of strain specific forms but despite wide variation in the sequences of the internal repeat regions both N and C terminal elements of the protein are almost totally conserved. Accordingly, we prepared a large number of overlapping peptide constructs and demonstrated that one peptide SNTFINNA (E71) from the N terminus and two peptides, QHGHMHGS (G5) and NTSDSQKE (G12) from the C terminus could, when suitably conjoined to the carrier protein diphtheria toxoid (DT), elicit antibodies reactive with MSA2 from diverse strains of P. falciparum. Here we compare the immunogenicity of these peptide constructs with two recombinant proteins containing the entire amino acid sequence of MSA2 from the FCQ-27/PNG strain (1609) and the 3D7 strain (1623). We have formulated these recombinant and peptide antigens with Freund's adjuvant, Alum and Algammulin. Both recombinant and peptide antigens elicit high titre antibodies when tested by ELISA against the immunogens themselves. Although both recombinant proteins include the constant region peptide sequences E71, G5 and G12, the extent of ELISA cross reaction between antibody raised against recombinant and peptide antigen or antibody raised against peptide and recombinant antigen is small and sporadic, and depends to an extent on the adjuvant employed. Antisera against both recombinant proteins 1609 and 1623 detected either recombinant on Western blots, as well as detecting native MSA2 in whole protein extracts from both FCQ-27/PNG and 3D7 strains. Antisera against peptide construct E71 recognized recombinant 1609 but not 1623 but recognized the native MSA2 in both strains studied. Antisera against peptide construct G5 showed a similar pattern of recognition

  18. Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

    PubMed

    Hua, Chun-Zhen; Hu, Wei-Lin; Shang, Shi-Qiang; Li, Jian-Ping; Hong, Li-Quan; Yan, Jie

    2015-12-16

    Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae.

  19. Unrelated business income tax: an update.

    PubMed

    Fama, A J

    1984-02-01

    To meet spiraling costs, tax-exempt hospitals increasingly are operating businesses unrelated to direct patient care. Knowing which activities may be open to challenge by the Internal Revenue Service (IRS) is essential to avoid the unrelated business income (UBI) tax. Three criteria must be met for an activity to be taxable as UBI: It must constitute a trade or business; It must be regularly carried on; and It must be unrelated to the organization's exempt purpose. The Internal Revenue Code and IRS rulings clearly exclude the following areas from UBI taxation: Activities performed by unpaid volunteers (e.g., hospital auxiliaries' fund-raising dinners and bazaars and the operation of thrift stores); Operations conducted for the convenience of the organization's members, students, patients, or employees (e.g., gift shops, cafeterias, coffee shops, parking lots, lounges, vending machines, pharmaceutical sales to inpatients and emergency room outpatients, and research activities for students' benefit; The sale of merchandise that has been received by gift (e.g., flea markets, baked goods sales, book sales, and rummage sales); Investment income such as dividends, interest, annuities, royalties, certain rents, and capital gains from the sale of investment assets; Gifts or contributions made directly to the facility; and Bingo games that are conducted commercially. Areas which may be subject to UBI taxation, or in which there have been controversial or contradictory court rulings, include: Pharmaceutical sales to the public or private physicians' patients; and Laboratory services provided to private physicians for treating their patients. IRS private letter rulings, though not precedential, have excluded from UBI taxation the x-ray income from a hospital's branch facility and rental income from property leased for use as a clinic or medical office building that is substantially related to the hospital's exempt functions. Private letter rulings have subjected to UBI

  20. Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate*

    PubMed Central

    Sneha Priya, Rangasamy; Veena, Menon; Kalisz, Irene; Whitney, Stephen; Priyanka, Dhopeshwarkar; LaBranche, Celia C.; Sri Teja, Mullapudi; Montefiori, David C.; Pal, Ranajit; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines. PMID:25691567

  1. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  2. Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.

    PubMed

    Setoh, Y X; Hobson-Peters, J; Prow, N A; Young, P R; Hall, R A

    2011-07-01

    Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.

  3. Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron‐specific antigens

    PubMed Central

    Vojdani, Aristo

    2016-01-01

    Abstract Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co‐occurrence of anti‐MBP and anti‐MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA‐human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. PMID:27610592

  4. Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59

    PubMed Central

    1994-01-01

    Schistosomiasis is a parasitic disease affecting approximately 200 million people, primarily in the third world. Schistosoma mansoni, one of the causative agents of this disease, parasitize the human mesenteric and portal blood systems while successfully evading host immune responses. During parasite penetration into the mammalian host and shortly afterwards, the larvae rapidly convert from being sensitive to being resistant to C-mediated killing. Treatment of the C-resistant parasitic forms with trypsin renders the parasite susceptible to C attack, thus indicating the presence of C inhibitory protein(s) on the parasite surface. We describe here an intrinsic schistosome C inhibitory protein (SCIP-1) that exhibits antigenic and functional similarities with the human C-inhibitor CD59. Like CD59, SCIP-1 is capable of inhibiting formation of the C membrane attack complex (MAC), probably by binding to C8 and C9 of the C terminal pathway. In addition, SCIP-1 is apparently also membrane-anchored via glycosyl phosphatidylinositol as it can be specifically released with phosphatidylinositol-specific phospholipase C. Soluble SCIP-1, partially purified from Nonidet P-40 extracts of schistosome tegument is capable of inhibiting hemolysis of sensitized sheep erythrocytes and of rabbit erythrocytes by human C. Anti-human CD59 antibodies block this activity of SCIP-1 and in addition, upon binding to intact parasites, render them vulnerable to killing by human and guinea pig C. SCIP-1 is located on the surface of C-resistant forms of the parasite, i.e., 24-h cultured mechanical schistosomula and in vivo-derived adult worms as revealed by immunofluorescence and immunogold electron microscopy studies. These results identify one of the mechanisms schistosomes use to escape immune attack. PMID:7513011

  5. Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.

    PubMed

    Liu, Wen-xing; Hu, Sen; Qiao, Zu-jian; Chen, Wei-ye; Liu, Lin-tao; Wang, Fang-kun; Hua, Rong-hong; Bu, Zhi-gao; Li, Xiang-rui

    2011-01-01

    Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

  6. Ki67 antigen contributes to the timely accumulation of protein phosphatase 1γ on anaphase chromosomes.

    PubMed

    Takagi, Masatoshi; Nishiyama, Yuko; Taguchi, Atsuko; Imamoto, Naoko

    2014-08-15

    Ki67 is a protein widely used as cell-proliferation marker, with its cellular functions being hardly unveiled. In this paper, we present the direct interaction between Ki67 and PP1γ, a protein phosphatase showing characteristic accumulation on anaphase chromosomes via the canonical PP1-binding motif within Ki67. In cells depleted of Ki67, PP1γ is targeted to anaphase chromosomes less efficiently. Additionally, overexpression of Ki67, but not a mutant form without the ability to bind PP1γ, induced ectopic localization of PP1γ οn metaphase chromosomes. These observations demonstrate that Ki67 is one factor that defines the cellular behavior of PP1γ in anaphase. To explore the specific roles of the subset of PP1γ recruited on chromosome via its interaction with Ki67 (PP1γ-Ki67), endogenous Ki67 was replaced with a Ki67 mutant deficient in its ability to interact with PP1γ. Although no obvious defects in the progression of mitosis were observed, the timing of dephosphorylation of the mutant Ki67 in anaphase was delayed, indicating that Ki67 itself is one of the substrates of PP1γ-Ki67.

  7. Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

    SciTech Connect

    Tilley, M.; Upton, S.J. )

    1990-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and {sup 125}I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.

  8. Identification of Antigens of Pathogenic Free-Living Amoebae by Protein Immunoblotting with Rabbit Immune and Human Sera

    DTIC Science & Technology

    1994-09-01

    or more broad bands of less than 1.85 kDa were prominent features in three different Acanthamoeba species. Few cross-reactive antibodies could be...detected between representative species of the three subgroups of Acanthamoeba . Naegleria antigen was likewise serologically distinct, as were...antigens. A prominent band of less than 18.5 kDa was identified in the Acanthamoeba culbertsoni antigen lane in 2 of the 10 human serum specimen pools. When

  9. Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

    PubMed

    Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

    2017-03-15

    Although tumor protein D52 (TPD) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than TPD53 and 54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3' end of a reporter green fluorescence protein gene. RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RNA immunoprecipitation assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3' end of the 78-280 fragment. Stimulation of TGF-b and EGF decreased the binding ability of these factors, resulted in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we herein report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.

  10. Protein A radio-assay of H-Y antigen on human leukocytes using mouse and rat antisera and monoclonal antibodies.

    PubMed

    Savikurki, H; Andersson, L C; Wachtel, S S; de la Chapelle, A

    1983-01-01

    The presence of H-Y antigen on human leukocytes was investigated using a protein A radio-assay. H-Y antigen could be demonstrated on male cells using either conventional H-Y antisera produced in mice and rats, or monoclonal H-Y antibodies. With mouse antiserum and IgG-type monoclonal antibody the reaction was male-specific using a single antibody. The reaction obtained with rat antiserum was enhanced by the application of a second antibody (rabbit anti-mouse IgG). This technique provides a rapid, simple, objective, and semiquantitative method for the determination of cellular H-Y antigen, the results being expressed as radioactivity bound to the test cells and thus being independent of human observation. It requires only 10-20 ml of blood and small quantities of antiserum or antibody.

  11. Antigenicity of the pollen proteins of various cultivars of Olea europaea.

    PubMed

    Waisel, Y; Geller-Bernstein, C; Keynan, N; Arad, G

    1996-11-01

    The incidence of positive skin test responses among atopic subjects with suspected respiratory allergy was investigated with commercial and autochthonous pollen extracts of various cultivars of Olea europaea. Pollen was collected from olive trees of well-defined cultivars, extracted, and separated by SDS-PAGE. Immunoblots were used to identify the various IgE-binding proteins of the pollen extracts of the various cultivars. The results revealed six predominant IgE-binding bands, some of which appear in all the cultivars examined. The 18-20-kDa band (Ole e 1) appeared in only eight of the cultivars, but not in the nine others. The presence of specific IgE-binding bands in the various pollen extracts and their correlation with the incidence of positive skin tests are discussed.

  12. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    NASA Astrophysics Data System (ADS)

    Zhou, Changhua; Mao, Mao; Yuan, Hang; Shen, Huaibin; Wu, Feng; Ma, Lan; Li, Lin Song

    2013-09-01

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 °C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  13. WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis.

    PubMed Central

    Klein, B S; Sondel, P M; Jones, J M

    1992-01-01

    A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response. PMID:1383148

  14. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  15. A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.

    PubMed

    Patra, Kailash P; Saito, Mayuko; Atluri, Vidya L; Rolán, Hortensia G; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N; Gotuzzo, Eduardo; Gilman, Robert H; Tsolis, Renee M; Vinetz, Joseph M

    2014-06-01

    Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

  16. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  17. Herpes simplex virus 2 (HSV-2) infected cell proteins are among the most dominant antigens of a live-attenuated HSV-2 vaccine.

    PubMed

    Geltz, Joshua J; Gershburg, Edward; Halford, William P

    2015-01-01

    Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.

  18. Characterization of the interface of the bone marrow stromal cell antigen 2-Vpu protein complex via computational chemistry.

    PubMed

    Zhou, Jinming; Zhang, Zhixin; Mi, Zeyun; Wang, Xin; Zhang, Quan; Li, Xiaoyu; Liang, Chen; Cen, Shan

    2012-02-14

    Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of enveloped viruses from the cell surface. Various viral counter measures have been discovered, which allow viruses to escape BST-2 restriction. Human immunodeficiency virus type 1 (HIV-1) encodes viral protein U (Vpu) that interacts with BST-2 through their transmembrane domains and causes the downregulation of cell surface BST-2. In this study, we used a computer modeling method to establish a molecular model to investigate the binding interface of the transmembrane domains of BST-2 and Vpu. The model predicts that the interface is composed of Vpu residues I6, A10, A14, A18, V25, and W22 and BST-2 residues L23, I26, V30, I34, V35, L41, I42, and T45. Introduction of mutations that have been previously reported to disrupt the Vpu-BST-2 interaction led to a calculated higher binding free energy (MMGBSA), which supports our molecular model. A pharmacophore was also generated on the basis of this model. Our results provide a precise model that predicts the detailed interaction occurring between the transmembrane domains of Vpu and BST-2 and should facilitate the design of anti-HIV agents that are able to disrupt this interaction.

  19. Characterization of surface antigen protein 1 (SurA1) from Acinetobacter baumannii and its role in virulence and fitness.

    PubMed

    Liu, Dong; Liu, Zeng-Shan; Hu, Pan; Cai, Ling; Fu, Bao-Quan; Li, Yan-Song; Lu, Shi-Ying; Liu, Nan-Nan; Ma, Xiao-Long; Chi, Dan; Chang, Jiang; Shui, Yi-Ming; Li, Zhao-Hui; Ahmad, Waqas; Zhou, Yu; Ren, Hong-Lin

    2016-04-15

    Acinetobacter baumannii is a Gram-negative bacillus that causes nosocomial infections, such as bacteremia, pneumonia, and meningitis and urinary tract and wound infections. In the present study, the surface antigen protein 1 (SurA1) gene of A. baumannii strain CCGGD201101 was identified, cloned and expressed, and then its roles in fitness and virulence were investigated. Virulence was observed in the human lung cancer cell lines A549 and HEp-2 at one week after treatment with recombinant SurA1. One isogenic SurA1 knock-out strain, GR0015, which was derived from the A. baumannii strain CCGGD201101 isolated from diseased chicks in a previous study, highlighted the effect of SurA1 on fitness and growth. Its growth rate in LB broth and killing activity in human sera were significantly decreased compared with strain CCGGD201101. In the Galleria mellonella insect model, the isogenic SurA1 knock-out strain exhibited a lower survival rate and decreased dissemination. These results suggest that SurA1 plays an important role in the fitness and virulence of A. baumannii.

  20. Glucose-Regulated Protein 78-Induced Myeloid Antigen-Presenting Cells Maintained Tolerogenic Signature upon LPS Stimulation

    PubMed Central

    Yang, Muyang; Zhang, Fan; Qin, Kai; Wu, Min; Li, Heli; Zhu, Huifen; Ning, Qin; Lei, Ping; Shen, Guanxin

    2016-01-01

    The 78-kDa glucose-regulated protein (Grp78) is stress-inducible chaperone that mostly reside in the endoplasmic reticulum. Grp78 has been described to be released at times of cellular stress and as having extracellular properties that are anti-inflammatory or favor the resolution of inflammation. As antigen-presenting cells (APCs) play a critical role in both the priming of adaptive immune responses and the induction of self-tolerance, herein, we investigated the effect of Grp78 on the maturation of murine myeloid APCs (CD11c+ cells). Results showed that CD11c+ cells could be bound by AF488-labeled Grp78 and that Grp78 treatment induced a tolerogenic phenotype comparable to immature cells. Furthermore, when exposed to lipopolysaccharide, Grp78-treated CD11c+ cells (DCGrp78) did not adopt a mature dendritic cell phenotype. DCGrp78-primed T cells exhibited reduced proliferation along with a concomitant expansion of CD4+CD25+FoxP3+ cells in pancreaticoduodenal lymph nodes and induction of T cell apoptosis in vitro and ex vivo. The above work suggests that Grp78 is an immunomodulatory molecule that could aid resolution of inflammation. It may thus contribute to induce durable tolerance to be of potential therapeutic benefit in transplanted allogeneic grafts and autoimmune diseases such as type I diabetes. PMID:27990144

  1. Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators

    PubMed Central

    Schnabel, Renate B.; Baumert, Jens; Barbalic, Maja; Dupuis, Josée; Ellinor, Patrick T.; Durda, Peter; Dehghan, Abbas; Bis, Joshua C.; Illig, Thomas; Morrison, Alanna C.; Jenny, Nancy S.; Keaney, John F.; Gieger, Christian; Tilley, Cathy; Yamamoto, Jennifer F.; Khuseyinova, Natalie; Heiss, Gerardo; Doyle, Margaret; Blankenberg, Stefan; Herder, Christian; Walston, Jeremy D.; Zhu, Yanyan; Vasan, Ramachandran S.; Klopp, Norman; Boerwinkle, Eric; Larson, Martin G.; Psaty, Bruce M.; Peters, Annette; Ballantyne, Christie M.; Witteman, Jacqueline C. M.

    2010-01-01

    To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10−323). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10−13). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications. PMID:20040767

  2. Antigenic polymorphism of human very late activation protein-2 (platelet glycoprotein Ia-IIa). Platelet alloantigen Hca.

    PubMed Central

    Woods, V L; Pischel, K D; Avery, E D; Bluestein, H G

    1989-01-01

    We have found evidence for a human alloantigenic system on the very late activation protein -2 (VLA-2) heterodimer (platelet GPIa/IIa). Sera from two patients with systemic lupus erythematosus (SLE) contained antibodies that immunoprecipitated surface molecules from platelets and fibroblasts that comigrated on SDS-PAGE and two-dimensional O'Farrell gels with platelet GPIa (VLA-alpha2 chain) and platelet GPIIa (VLA-beta chain). These SLE antibodies were alloreactive as they precipitated VLA molecules from only 5 of 22 normal donors' platelets and did not react with the lupus patients' own platelets, despite the expression of apparently normal amounts of VLA on the donors' cells. Two-dimensional O'Farrell analysis demonstrated no differences in the molecular weight or isoelectric point of GPIa and GPIIa obtained from platelets of alloantibody reactive or unreactive donors. Sequential immunoprecipitation experiments with VLA chain-specific monoclonal antibodies, and the pattern of immunoprecipitation of several different VLA heterodimers demonstrated that the alloantibody-reactive determinant was present on the VLA-2 heterodimer, and not other VLA molecules. Thus, these SLE sera demonstrate a previously unrecognized antigenic polymorphism of the VLA-2 (platelet GPIa/IIa) heterodimer, platelet alloantigen Hca. Images PMID:2646323

  3. Detection of antibodies to hepatitis C virus (HCV) structural proteins in anti-HCV-positive sera by an enzyme-linked immunosorbent assay using synthetic peptides as antigens.

    PubMed Central

    Ishida, C; Matsumoto, K; Fukada, K; Matsushita, K; Shiraki, H; Maeda, Y

    1993-01-01

    We have defined 10 linear immunogenic regions encoded by the putative hepatitis C virus (HCV) structural proteins (core and envelope) by employing an enzyme-linked immunosorbent assay (ELISA) and by using 17 sequential synthetic peptides covering the N-terminal 330 amino acids of the structural polyproteins as antigens. These peptides correspond to amino acids 1 to 24, 21 to 44, 42 to 68, 64 to 91, and 100 to 120 of the putative core protein and amino acids 192 to 212, 223 to 238, 236 to 258, 250 to 266, and 307 to 330 of the putative envelope protein. In particular, the peptide covering amino acids 21 to 44 of the core protein was reactive with all but one (40 of 41) of the serum samples giving a positive signal in the passive hemagglutination assay (PHA) using the core and nonstructural proteins (NS 3/4) of the virus as antigens. We detected the HCV genome in 25 (61%) of 41 PHA-positive serum samples by the polymerase chain reaction (PCR) test. Of 25 PCR-positive serum samples, 17 serum samples had reactivity to the peptides derived from the envelope protein. On the other hand, only 1 of the 16 PCR-negative serum samples had reactivity to the peptides derived from the envelope protein. Interestingly, we often observed high serum alanine aminotransferase levels in PCR-positive individuals bearing antibodies to the envelope protein. PMID:7681849

  4. A Peptide mimicking a region in proliferating cell nuclear antigen specific to key protein interactions is cytotoxic to breast cancer.

    PubMed

    Smith, Shanna J; Gu, Long; Phipps, Elizabeth A; Dobrolecki, Lacey E; Mabrey, Karla S; Gulley, Pattie; Dillehay, Kelsey L; Dong, Zhongyun; Fields, Gregg B; Chen, Yun-Ru; Ann, David; Hickey, Robert J; Malkas, Linda H

    2015-02-01

    Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo.

  5. Female tick Hyalomma marginatum marginatum salivary glands: preliminary study on protein changes during feeding process and antigens recognized by repeatedly infested cattle.

    PubMed

    Tikki, N; Rhalem, A; Sadak, A; Sahibi, H

    1999-12-01

    Proteins extracted from salivary glands of unfed, three days and five days fed adult Hyalomma marginatum marginatum were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We have noticed changes during the three feeding steps. Some proteins disappeared during feeding process (23, 38, 39, 40 to 50, 95 and 112 kDa), they might be proteins which were converted in other substances and are secreted. Other antigens (13 to 14, 20, 25, 29, 165 and 210 kDa) were synthesized as a result of tick attachment and feeding. They may be related to growth and development or are the ciment which fixed the adult. Also, three Holstein calves were infested five times with 100 pairs of adult ticks of the same species. The five infestations were performed two weeks from the previous infestation. The sera before infestations and after each infestation were used in western-blot analyses to identify antigens from five days salivary gland extracts of the primary infestation of ticks. Three antigens (18.7, 50 and 80 kDa) were revealed weakly after the first and the second infestations by sera samples but not at infestation onward. Others (13.5, 17 to 18.5, 25, 30, 70, 133, 176 and 193 kDa) were revealed only by sera taken after manifestation of resistance (third infestation). A 13.5 kDa antigen was particularly revealed when resistance had appeared and became more evident after the fourth and fifth infestations. The late antigens recognized might be associated with establishment of calves resistance against ticks.

  6. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System.

    PubMed

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo.

  7. Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

    PubMed

    Peron, G; Fernandes, F F; Landgraf, T N; Martinez, R; Panunto-Castelo, A

    2017-04-03

    Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

  8. Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System

    PubMed Central

    Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

    2015-01-01

    Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

  9. Antigenic proteins of Lactobacillus acidophilus that are recognised by serum IgG antibodies in children with type 1 diabetes and coeliac disease.

    PubMed

    Prangli, Anna-Liisa; Utt, Meeme; Talja, Ija; Sepp, Epp; Mikelsaar, Marika; Rajasalu, Tarvo; Uibo, Oivi; Tillmann, Vallo; Uibo, Raivo

    2010-06-01

    Immune responses to lactobacilli have been so far insufficiently investigated in patients with autoimmune diseases. We used whole-cell lysate of an indigenous Lactobacillus acidophilus strain isolated from an Estonian child to study serum IgG antibodies in children groups with type 1 diabetes [insulin dependent diabetes mellitus (IDDM)] (n = 21, age 4-18 yr) and with acute coeliac disease (CD) (n = 20, age 0.6-15 yr) and to compare the results with the controls (n = 24, age 2-17 yr). We found that our developed 1-D immunoblot assay readily enables to reveal antibodies against 28 L. acidophilus antigenic proteins in patients' and controls' sera. As verified by immunoproteomics analysis with 2-D and LC ESI-MS/MS the antigens of L. acidophilus were mainly common cytoplasmic proteins GroEL (HSP60), enolase, transcription factor EF-Ts and EF-Tu. However, in addition we identified formyl-CoA transferase being target for antibodies in every tested IDDM patients' serum. We have characterized for the first time the antigenic profile of L. acidophilus whole-cell lysate using sera from children with IDDM, CD, and controls. The different prevalence of reactions against tested antigens in patients and controls sera may indicate significant differences in immune system and commensal bacteria cross-talk in these groups.

  10. Eimeria tenella heat shock protein 70 enhances protection of recombinant microneme protein MIC2 subunit antigen vaccination against E. tenella challenge.

    PubMed

    Zhang, Lei; Ma, Liping; Liu, Renqiang; Zhang, Yunfei; Zhang, Shouping; Hu, Chunmei; Song, Meng; Cai, Jianping; Wang, Ming

    2012-09-10

    Heat shock proteins have been reported to stimulate the immune system via innate receptors. Our study found that the novel immunopotentiator, Eimeria tenella (E. tenella) heat shock protein 70 (HSP70), enhanced protective immunity elicited by E. tenella antigen microneme protein 2 (EtMIC2) against avian coccidiosis. It demonstrated that the expression of TLR2 and TLR4 were strongly upregulated in EtHSP70 and EtMIC2 plus EtHSP70 stimulated chicken embryo fibroblasts (CEF) compared with untreated controls and EtMIC2 alone. In addition, the same treatment induced high levels of interleukin (IL)-12 and interferon (IFN)-γ that are critical cytokines of innate immunity. In vivo experiments involved using broiler chickens subcutaneously immunized with EtMIC2 alone or EtMIC2 plus EtHSP70 at 7 and 14 days post-hatch, which were then orally challenged with live E. tenella at 7 days following secondary immunization. Body weight gains, cecal lesion scores, fecal oocyst shedding, serum antibody responses against MIC2, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with EtMIC2 plus EtHSP70 showed increased body weight gains, decreased oocyst shedding, increased serum antibody responses, and high levels of IL-12, IFN-γ, and IL-17 compared with the EtMIC2 only or control groups. Moreover, chickens immunized with EtHSP70 alone showed significantly protective effect against E. tenella infection. In summary, this study provides the first evidence of the immunoenhancing activities of EtHSP70 in poultry.

  11. Isolation and characterization of a human liver and kidney-specific protein: the hepato-renal (H-R) antigen.

    PubMed Central

    Nerenberg, S T; Prasad, R; Inboriboon, P; Biskup, N; Pedersen, L; Faiferman, I

    1980-01-01

    This paper reports the isolation and characterization of a soluble antigen shared by the liver and kidney of human and some other animal species. Homogenates of human liver in saline were centrifugated at 27,000 g and the supernatants were fractionated by preparative polyacrylamide gel electrophoresis. The gels were divided in sections and each was injected into rabbits; after absorption with polymerized normal human serum, the antiserum obtained by injecting one of the sections reacted only with saline extracts of human liver and kidney when tested against a variety of human tissue extracts. The absorbed antiserum, polymerized and insolubilized with glutaraldehyde, was used to purify the antigen by affinity chromatography. The purified antigen proved to be a glycoprotein containing 19 percent carbohydrate, had a molecular weight of 5.8-6.0 x 10(4) Daltons and a pI of 7.2-7.4. The antigen, relatively thermostable, was precipitated by 35-55 percent ammonium sulphate; its antigenic activity was not affected by extraction with 0.6 N perchloric acid or by incubation with ribonuclease, deoxyribonuclease or neuraminidase but was destroyed by incubation with ttypsin or chymotrypsin. Immunoperoxidase studies showed that the antigen appeared concentrated in the neclei of liver and kidney glomerular epithelial and tubular epithelial cells in humans and rats. The antigen could not be detected in human hepatomas or hypernephromas or in the rat Morris hepatoma 5123. Images FIG. 1 FIG. 7 PMID:6155231

  12. Intracellular Delivery of a Protein Antigen with an Endosomal-Releasing Polymer Enhances CD8 T-Cell Production and Prophylactic Vaccine Efficacy

    PubMed Central

    Foster, Suzanne; Duvall, Craig L.; Crownover, Emily F.; Hoffman, Allan S.; Stayton, Patrick S.

    2010-01-01

    Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their ability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with the cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized compared to control conjugates and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5 fold increase in the longevity of tumor free survival relative to controls. Mechanistically this response was attributed to the 8-fold increase in production of ovalbumin-specific CD8+ T-lymphocytes and an 11-fold increase in production of anti-ovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications. PMID:21043513

  13. Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

  14. Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

    PubMed

    Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

    2014-04-01

    The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

  15. Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model.

    PubMed

    Tsolaki, Anthony G; Nagy, Judit; Leiva, Sergio; Kishore, Uday; Rosenkrands, Ida; Robertson, Brian D

    2013-07-01

    In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.

  16. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

    PubMed Central

    Vodkin, M H; Williams, J C

    1988-01-01

    A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen. Images PMID:3343219

  17. Transcription analysis of the major antigenic protein 1 multigene family of three in vitro-cultured Ehrlichia ruminantium isolates.

    PubMed

    Bekker, Cornelis P J; Postigo, Milagros; Taoufik, Amar; Bell-Sakyi, Lesley; Ferraz, Conchita; Martinez, Dominique; Jongejan, Frans

    2005-07-01

    Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.

  18. Disease-specific analyses of unrelated cord blood transplantation compared with unrelated bone marrow transplantation in adult patients with acute leukemia.

    PubMed

    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

    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.

  19. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.

    PubMed

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C; Steinman, Ralph M

    2002-12-16

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

  20. Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

    PubMed

    Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

    2014-09-01

    The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients.

  1. Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance

    PubMed Central

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C.; Steinman, Ralph M.

    2002-01-01

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation. PMID:12486105

  2. Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter

    PubMed Central

    1992-01-01

    We have previously shown that postnatal expression of the viral oncoprotein SV40 T antigen in rod photoreceptors (transgene MOT1), at a time when retinal cells have withdrawn from the mitotic cycle, leads to photoreceptor cell death (Al-Ubaidi et al., 1992. Proc. Natl. Acad. Sci. USA. 89:1194-1198). To study the effect of the specificity of the promoter, we replaced the mouse opsin promoter in MOT1 by a 1.3-kb promoter fragment of the human IRBP gene which is expressed in both rod and cone photoreceptors during embryonic development. The resulting construct, termed HIT1, was injected into mouse embryos and five transgenic mice lines were established. Mice heterozygous for HIT1 exhibited early bilateral retinal and brain tumors with varying degrees of incidence. Histopathological examination of the brain and eyes of three of the families showed typical primitive neuroectodermal tumors. In some of the bilateral retinal tumors, peculiar rosettes were observed, which were different from the Flexner-Wintersteiner rosettes typically associated with human retinoblastomas. The ocular and cerebral tumors, however, contained Homer-Wright rosettes, and showed varying degrees of immunoreactivity to antibodies against the neuronal specific antigens, synaptophysin and Leu7, but not to antibodies against photoreceptor specific proteins. Taken together, the results indicate that the specificity of the promoter used for T antigen and/or the time of onset of transgene expression determines the fate of photoreceptor cells expressing T antigen. PMID:1334963

  3. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    PubMed

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  4. Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

    PubMed

    Nilo, Alberto; Morelli, Laura; Passalacqua, Irene; Brogioni, Barbara; Allan, Martin; Carboni, Filippo; Pezzicoli, Alfredo; Zerbini, Francesca; Maione, Domenico; Fabbrini, Monica; Romano, Maria Rosaria; Hu, Qi-Ying; Margarit, Immaculada; Berti, Francesco; Adamo, Roberto

    2015-07-17

    Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design

  5. Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

    PubMed Central

    Fan, Yi-Chin; Chiu, Hsien-Chung; Chen, Li-Kuang; Chang, Gwong-Jen J.; Chiou, Shyan-Song

    2015-01-01

    Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines. PMID:26495991

  6. Living unrelated donor kidney transplantation between spouses.

    PubMed

    Haberal, M; Gulay, H; Tokyay, R; Oner, Z; Enunlu, T; Bilgin, N

    1992-01-01

    From November 3, 1975 to November 3, 1990, 874 kidney transplants were performed at out centers. Of these, 675 (77.2%) were from living donors and 199 (22.8%) were from cadaver donors. Five hundred eighty (66.4%) of the living donors were first degree related while 99 (11.3%) were unrelated or second degree related donors, 29 of which were spouses. All donor recipient pairs were ABO-compatible, with the exception of one pair. Donor recipient relations were wife to husband in 25 cases and husband to wife in 4 cases. All were first grafts and started functioning during surgery. In this series, the follow-up for the recipients was 4 to 64 months (mean 33.5 +/- 4.5 months). One-year patient survival and graft survival rates were 92.4% and 81.9%, respectively. Two-year patient survival and graft survival rates were 92.4% and 78.2%, respectively. The single ABO-incompatible case is also doing well, 21 months postoperatively. This study demonstrates that the interspouse kidney transplantation may be used when cadaver organ shortage is a problem. While providing the couple with a better quality of life, interspouse kidney transplantation also enables the couple to share the joy of giving and receiving the "gift of life" from one another.

  7. Isolated metaphyseal injury influences unrelated bones

    PubMed Central

    Tätting, Love; Sandberg, Olof; Bernhardsson, Magnus; Ernerudh, Jan; Aspenberg, Per

    2017-01-01

    Background and purpose Fracture healing involves different inflammatory cells, some of which are not part of the traditional bone field, such as B-cells and cytotoxic T-cells. We wanted to characterize bone healing by flow cytometry using 15 different inflammatory cell markers in a mouse model of metaphyseal injury, and incidentally discovered a previously unknown general skeletal reaction to trauma. Material and methods A bent needle was inserted and twisted to traumatize the cancellous bone in the proximal tibia of C57/Bl6 female mice. This is known to induce vivid bone formation locally in the marrow compartment. Cells were harvested from the injured region, the uninjured contralateral tibia, and the humerus. The compositions of the immune cell populations were compared to those in untraumatized control animals. Results Tibial metaphyseal injury led to substantial changes in the cell populations over time. Unexpectedly, similar changes were also seen in the contralateral tibia and in the humerus, despite the lack of local trauma. Most leukocyte subsets were affected by this generalized reaction. Interpretation A relatively small degree of injury to the proximal tibia led to systemic changes in the immune cell populations in the marrow of unrelated bones, and probably in the entire skeleton. The few changes that were specific for the injury site appeared to relate to modulatory functions. PMID:28128005

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

    PubMed Central

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

    2010-01-01

    Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution. PMID:20363948

  9. Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

    PubMed Central

    Wang, Anqi; Welch, Rene; Zhao, Bo; Ta, Tram; Keleş, Sündüz

    2015-01-01

    ABSTRACT Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for

  10. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

    NASA Astrophysics Data System (ADS)

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

    2016-06-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

  11. Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients with type-1 autoimmune hepatitis

    PubMed Central

    Costa, M; Rodríguez-Sánchez, J L; Czaja, A J; Gelpí, C

    2000-01-01

    We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti‐tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec. PMID:10931155

  12. Effects of simian virus 40 large and small tumor antigens on mammalian target of rapamycin signaling: small tumor antigen mediates hypophosphorylation of eIF4E-binding protein 1 late in infection.

    PubMed

    Yu, Yongjun; Kudchodkar, Sagar B; Alwine, James C

    2005-06-01

    We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.

  13. Effects of Simian Virus 40 Large and Small Tumor Antigens on Mammalian Target of Rapamycin Signaling: Small Tumor Antigen Mediates Hypophosphorylation of eIF4E-Binding Protein 1 Late in Infection

    PubMed Central

    Yu, Yongjun; Kudchodkar, Sagar B.; Alwine, James C.

    2005-01-01

    We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen. PMID:15890927

  14. Development and Application of an ELISA Assay Using Excretion/Secretion Proteins from Epimastigote Forms of T. cruzi (ESEA Antigens) for the Diagnosis of Chagas Disease

    PubMed Central

    Berrizbeitia, Mariolga; Figueroa, Milagros; Ward, Brian J.; Rodríguez, Jessicca; Jorquera, Alicia; Figuera, Maria A.; Romero, Leomerys; Ndao, Momar

    2012-01-01

    An indirect enzyme-linked immunoabsorbent assay (ELISA) for Trypanosoma cruzi was developed using epimastigote secretion/excretion proteins (ESEA antigens) obtained from axenic culture supernatants. A panel of 120 serum samples from subjects with confirmed Chagas disease (n = 50), healthy controls (n = 50), and patients with other parasitic diseases (n = 20) was used to evaluate the new ESEA-based ELISA (ELISAESEA). This new test had excellent sensitivity (98%) and acceptable specificity (88%). Cross-reactivity was observed largely in sera from subjects with Leishmania and Ascaris infections. Using Western blotting and epimastigotes from two distinct T. cruzi isolates, several polypeptide bands with molecular masses ranging from 50 to 220 kDa were detected in pooled chagasic sera. However, the band pattern for each isolate was different. These data suggest that an inexpensive and technically simple ELISA based on ESEA antigens is a promising new tool for the diagnosis of Chagas disease. PMID:23049572

  15. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product.

    PubMed Central

    Gassmann, G S; Jacobs, E; Deutzmann, R; Göbel, U B

    1991-01-01

    The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis. Images PMID:1704884

  16. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

    PubMed Central

    Rihs, H P; Jans, D A; Fan, H; Peters, R

    1991-01-01

    We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport. Images PMID:1848177

  17. Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection.

    PubMed

    Bai, Xiaofei; Shaozhou, Wulin; Zhang, Qingshan; Li, Chenxi; Qiu, Na; Meng, Runzhe; Liu, Ming; Zhang, Yun

    2015-03-01

    The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds.

  18. Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

    PubMed Central

    Azoitei, M.L.; Ban, Y.A.; Kalyuzhny, O.; Guenaga, J.; Schroeter, A.; Porter, J.; Wyatt, R.; Schief, W.R.

    2015-01-01

    Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of pre-defined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of HIV-1 neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with sub-nanomolar affinity (KD = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. PMID:25043744

  19. Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R.

    PubMed

    Izquierdo, José M

    2010-12-01

    T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

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

    PubMed Central

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

    2015-01-01

    Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ∼80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens. DOI: http://dx.doi.org/10.7554/eLife.07467.001 PMID:26252514

  1. Comparison of immune responses induced by rat RT-1 antigens presented as inserts into liposomes, as protein micelles and as intact cells.

    PubMed

    Hedlund, G; Jansson, B; Sjögren, H O

    1984-09-01

    Partially purified rat transplantation antigens (RT-1) were inserted into liposomes composed of various types of lipids and used for immunization. The immune responses induced by the liposomes were compared with responses induced by RT-1 as protein micelles, alone or emulsified in Freund's incomplete adjuvant, or intact cells. Liposomes gave generally a higher humoral response than protein micelles. Each type of RT-1 immunization gave a particular pattern of specific Ig (sub)class responses. Freund's incomplete adjuvant was not only lacking in potentiating effect on low protein dose immunization but had a significant inhibitory effect. Besides intact cells only distearoyl-phosphatidylcholine liposomes had the potential to induce a cell-mediated cytotoxic response.

  2. A pre-clinical model of double versus single unit unrelated cord blood transplantation

    PubMed Central

    Georges, George E.; Lesnikov, Vladimir; Baran, Szczepan W.; Aragon, Anna; Lesnikova, Marina; Jordan, Robert; Yang, Ya-Ju Laura; Yunusov, Murad Y.; Zellmer, Eustacia; Heimfeld, Shelly; Venkataraman, Gopalakrishnan M.; Harkey, Michael A.; Graves, Scott S.; Storb, Rainer; Storer, Barry E.; Nash, Richard A.

    2010-01-01

    Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5×107/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained ≤ 1.7×107 TNC/kg. Recipients were given 9.2 Gy total body irradiation and DLA-matched unrelated cord blood with post-grafting cyclosporine and mycophenolate mofetil. After double-unit CBT, 5 dogs engrafted and 4 survived long term with one dominant engrafting unit and prompt immune reconstitution. In contrast, 0 of 5 dogs given single-unit CBT survived beyond 105 days (p=0.03, log-rank test); neutrophil and platelet recovery was delayed (both p=0.005) and recipients developed fatal infections. This new large animal model showed that outcomes were improved after double-unit compared to single-unit CBT. After double-unit CBT, the non-engrafted unit facilitates engraftment of the dominant unit. PMID:20304085

  3. Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

    PubMed

    Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

    2017-01-01

    Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10(6) to approximately 10(8) cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

  4. Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein.

    PubMed

    Li, F; Riddell, M A; Seow, H F; Takeda, N; Miyamura, T; Anderson, D A

    2000-04-01

    A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats. Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses. Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation. Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 [Li et al. (1997) Journal of Medical Virology 52:289-300], a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients. Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition). Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection.

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

    PubMed

    Glaser, R W; Hausdorf, G

    1996-01-16

    The interaction between HIV core protein p24 and the murine monoclonal antibody CB-4/1 or its Fab fragment showed unusual kinetics. Recombinant p24 was immobilised in a hydrophilic carboxymethyldextran matrix. At high concentration of CB-4/1 Fab the association of the antigen-antibody complex proceeds in two phases, while dissociation is mono-exponential. The antigen has a 'memory', i.e. shortly after dissociation of Fab-antigen complex the fast association phase is enhanced. Biphasic association was also found in solution. Experiments suggest a reversible change of binding properties in the epitope region with an overall time constant of about 100 s at room temperature. Intermediate steps with faster time constants must be involved. Slow conformational changes of p24 seem to be the most probable explanation. A simple model that provides a quantitative description of this process could not be found. Real-time analysis of antibody binding by surface plasmon resonance is a powerful method for studying such changes in the time domain of a few seconds to a few minutes.

  6. Using human sera to identify a 52-kDa exoantigen of Penicillium chrysogenum and implications of polyphasic taxonomy of anamorphic ascomycetes in the study of antigenic proteins.

    PubMed

    Wilson, Aaron M; Luo, Wen; Miller, J David

    2009-11-01

    We are interested in isolating and identifying antigenic fungal proteins from species that grow on damp building materials. The indoor clade of Penicillium chrysogenum, the so-called Fleming clade, is the most common species of Penicillium on moldy building materials. We have identified a 52-kDa marker protein for the indoor clade of P. chrysogenum not present in a taxonomically diverse selection of fungi. It is found in high concentrations in protein extracted from the fungus grown on paper-faced gypsum wallboard. During this process, we illuminated the variability in response to patient sera and of strains of the fungus collected over a wide geographic area. From a collection of sera from all over the USA, 25 of the 48 patients reacted to the 52-kDa protein from this prescreened collection of sera. Most strain/antibody combinations had proportionate ELISA response associated with the presence of the target. However, approximately 25% of the strain/patient serum combinations included people who responded to many common allergens from the Penicillia. All the P. chrysogenum strains tested produced the target protein. However, there was considerable variability in patient IgG response to 32-, 30-, and 18-kDa antigens and in their production by the various clade 4 strains. The target protein was not found in spores or culture extracts of a wide selection of relevant fungi. It appears that the previous studies have been conducted on strains of the fungus from the three clades not those associated with the built environment.

  7. Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

    PubMed

    Moriguchi, K; Mitamura, Y; Iwami, J; Hasegawa, Y; Higuchi, N; Murakami, Y; Maeda, H; Yoshimura, F; Nakamura, H; Ohno, N

    2012-11-01

    Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

  8. A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G.

    PubMed

    Neng, Jing; Harpster, Mark H; Zhang, Hao; Mecham, James O; Wilson, William C; Johnson, Patrick A

    2010-11-15

    A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interrogation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also confirmed the formation of antigen-antibody-protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced electromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA.

  9. Broadly distributed T cell reactivity, with no immunodominant loci, to the pre-erythrocytic antigen thrombospondin-related adhesive protein of Plasmodium falciparum in West Africans.

    PubMed

    Flanagan, K L; Plebanski, M; Akinwunmi, P; Lee, E A; Reece, W H; Robson, K J; Hill, A V; Pinder, M

    1999-06-01

    Protective immunity to malaria has been achieved in human volunteers utilizing the pre-erythrocytic Plasmodium falciparum antigen, the circumsporozoite protein (CS). However, T cell reactivity to CS is focused on several highly polymorphic T cell epitope regions, potentially limiting the efficacy of any vaccine to specific malaria strains. Another important pre-erythrocytic malaria antigen, the thrombospondin-related adhesive protein (TRAP), can induce protection in animal models of malaria, but knowledge of human T cell responses is limited to the identification of CD8 T cell epitopes, with no CD4 epitopes identified to date. This comprehensive study assessed reactivity to overlapping peptides spanning almost the whole of P. falciparum TRAP (PfTRAP), as well as peptides selected on the basis of HLA class II-binding motifs. A total of 50 naturally exposed Gambian adults were assessed to define 26 T cell epitopes in PfTRAP capable of inducing rapid IFN-gamma or IL-4 production, as assessed by enzyme-linked immunospot assays. In contrast to the CS protein, this reactivity was broadly distributed along the length of TRAP. Moreover, of the 26 epitopes identified, 10 were found to be conserved in West Africa.

  10. Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

    PubMed

    Kashino, S S; Abeijon, C; Qin, L; Kanunfre, K A; Kubrusly, F S; Silva, F O; Costa, D L; Campos, D; Costa, C H N; Raw, I; Campos-Neto, A

    2012-07-01

    Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

  11. The use of hybrid phage displaying antigen epitope and recombinant protein in the diagnosis of systemic Candida albicans infection in rabbits and cancer patients.

    PubMed

    Quanping, Su; Yanyan, Huai; Yicun, Wang; Zhigang, Ju; Yuling, Geng; Li, Wang

    2010-12-01

    Hsp90 and Sap2 are 2 immunodominant antigens of Candida albicans. Both of them can induce the production of antibody. In this article, systemically infected rabbits were used to study the Hsp90 and Sap2 antibody production. Also, pET28a-Hsp90 protein, pET28a-Sap2 protein, hybrid phage displaying LKVIRK epitope, and hybrid phage displaying VKYTS epitope were used for diagnosis of the antibody in cancer patients. The results showed that the Sap2 antibody appeared earlier than Hsp90 antibody in systemically infected rabbits. Meanwhile, both of the antibodies can perform protection in rabbits. The conclusion is that Sap2 antibody, which appears at early stage in systemic candidiasis, may be better than Hsp90 antibody for the diagnosis of invasive candidiasis. For 141 sera of cancer patients, 52 sera were detected Sap2 antibody and 57 sera were detected Hsp90 antibody. Only 14 sera contained both the 2 antibodies. Although recombinant protein was slightly more sensitive than hybrid phage, there was no significant difference between them. For its easy preparation, less expensive hybrid phage displaying antigen epitope may be a better agent for diagnosis of candidiasis.

  12. Application and expression of Toxoplasma gondii surface antigen 2 (SAG2) and rhoptry protein 2 (ROP2) from recombinant Escherichia coli strain.

    PubMed

    Yan, Hua; Yan, Huishen; Tao, Yong; Chen, Hongju; Li, Guocai; Gong, Weijuan; Jiao, Hongmei; Tian, Fang; Ji, Mingchun

    2012-06-01

    The gene encoding surface antigen 2 (SAG2) or rhoptry protein 2 (ROP2) of Toxoplasma gondii was cloned into the plasmid pGEX-4T-1 and subsequently expressed in Escherichia coli as a glutathione-s-transferase (GST) fusion protein. The characteristics of purified GST-SAG2 or GST-ROP2 were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. The specific IgG of a panel of serum samples provided by the National Institute for the Control of Pharmaceutical and Biological Products were tested with commercial ELISA and the lateral flow immunoassay (LFIA) based on GST-SAG2, GST-ROP2 or GST-SAG2+ROP2. A total of 1096 sera and saliva samples from pregnant women were tested by GST-SAG2+ROP2-LFIA. In total, 20 T. gondii IgM positive sera (1.82%), 81 T. gondii IgG positive sera (7.4%) and 23 T. gondii IgA positive saliva (2.1%) were finally confirmed. The SAG2+ROP2 specific IgG and IFN-γ producing CD8+ T cells were induced in mice immunised with GST-SAG2+ROP2. The results indicate that GST-SAG2+ROP2 protein can be used as an antigen for diagnosing T. gondii infection and provide a strategy for development of subunit vaccines for protection against T. gondii infection.

  13. Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.

    PubMed

    Hodgson, Susanne H; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Rampling, Thomas W; Biswas, Sumi; Poulton, Ian D; Miura, Kazutoyo; Douglas, Alexander D; Alanine, Daniel Gw; Illingworth, Joseph J; de Cassan, Simone C; Zhu, Daming; Nicosia, Alfredo; Long, Carole A; Moyle, Sarah; Berrie, Eleanor; Lawrie, Alison M; Wu, Yimin; Ellis, Ruth D; Hill, Adrian V S; Draper, Simon J

    2014-12-01

    The development of effective vaccines against difficult