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Sample records for surface antigen protein

  1. Naturally-acquired cellular immune response against Plasmodium vivax merozoite surface protein-1 paralog antigen.

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    Changrob, Siriruk; Leepiyasakulchai, Chaniya; Tsuboi, Takafumi; Cheng, Yang; Lim, Chae Seung; Chootong, Patchanee; Han, Eun-Taek

    2015-04-15

    Plasmodium vivax merozoite surface protein-1 paralog (PvMSP1P) is a glycosylphosphatidylinositol-anchored protein expressed on the merozoite surface. This molecule is a target of natural immunity, as high anti-MSP1P-19 antibody levels were detected during P. vivax infection and the antibody inhibited PvMSP1P-erythrocyte binding. Recombinant PvMSP1P antigen results in production of a significant Th1 cytokine response in immunized mice. The present study was performed to characterize natural cellular immunity against PvMSP1P-19 and PvDBP region II in acute and recovery P. vivax infection. Peripheral blood mononuclear cells (PBMCs) from acute and recovery P. vivax infection were obtained for lymphocyte proliferation assay upon PvMSP1P-19 and PvDBP region II antigen stimulation. The culture supernatant was examined for the presence of the cytokines IL-2, TNF, IFN-γ and IL-10 by enzyme-linked immunosorbent assay (ELISA). To determine whether Th1 or Th2 have a memory response against PvMSP1P-19 and PvDBPII protein antigen, PBMCs from subjects who had recovered from P. vivax infection 8-10 weeks prior to the study were obtained for lymphocyte proliferation assay. Cytokine-producing cells were analysed by flow cytometry. IL-2 was detected at high levels in lymphocyte cultures from acutely infected P. vivax patients upon PvMSP1P-19 stimulation. Analysis of the Th1 or Th2 memory response in PBMC cultures from subjects who had recovered from P. vivax infection showed significantly elevated levels of PvMSP1P-19 and PvDBPII-specific IFN-γ-producing cells (P  response of IFN-γ-producing cells in PvMSP1P stimulation was fourfold greater in recovered subjects than that in acute-infection patients. CD4(+) T cells were the major cell phenotype involved in the response to PvMSP1P-19 and PvDBPII antigen. PvMSP1P-19 strongly induces a specific cellular immune response for protection against P. vivax compared with PvDBPII as the antigen induces activation of IFN

  2. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

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    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. © 2015 by The Mycological Society of America.

  3. Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli.

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    Alerasol, Masoome; Mousavi Gargari, Seyed Latif; Nazarian, Shahram; Bagheri, Samane

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM1 binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Alexandra eIrrgang

    2015-09-01

    Full Text Available 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 analysed 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.

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

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    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; Czerkinsky, Cecil; Nandy, Ranjan K

    2016-07-01

    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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

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    Lacombe, Séverine; Bangratz, Martine; Brizard, Jean-Paul; Petitdidier, Elodie; Pagniez, Julie; Sérémé, Drissa; Lemesre, Jean-Loup; Brugidou, Christophe

    2018-01-01

    In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. [Possible Involvement of Surface Antigen Protein 2 in the Morphological Transition and Biofilm Formation of Candida albicans].

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    Okamoto-Shibayama, Kazuko; Kikuchi, Yuichiro; Kokubu, Eitoyo; Ishihara, Kazuyuki

    2017-01-01

    Surface antigen protein 2 (Csa2) is a member of the Candida albicans Common in Fungal Extracellular Membranes (CFEM) protein superfamily. We previously established its role in iron acquisition in C. albicans. However, the other roles of Csa2 remain unknown. Here, we compared growth, morphological transition, and biofilm formation among wild-type, Csa2-mutant, and complemented strains of C. albicans. Deletion of the Csa2 gene resulted in smaller and reduced colony growth, significant attenuation of the dimorphic transition under serum-inducing conditions, and reduced biofilm formation; complementation restored these levels to those of the wild-type. Our findings demonstrated that Csa2 participated in yeast-to-hyphae morphological switching under serum-inducing conditions and contributed to the biofilm formation of C. albicans. This work, therefore, provides novel insights into the potential roles of Csa2 in virulence of C. albicans.

  9. 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.

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    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.

  10. Epicutaneous sensitization with protein antigen

    Directory of Open Access Journals (Sweden)

    I-Lin Liu

    2012-12-01

    Full Text Available In the past few decades there has been a progressive understanding that epicutaneous sensitization with protein antigen is an important sensitization route in patients with atopic dermatitis. A murine protein-patch model has been established, and an abundance of data has been obtained from experiments using this model. This review discusses the characteristics of epicutaneous sensitization with protein antigen, the induced immune responses, the underlying mechanisms, and the therapeutic potential.

  11. Limited polymorphism in Plasmodium falciparum ookinete surface antigen, von Willebrand factor A domain-related protein from clinical isolates

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    Eisen Damon P

    2006-07-01

    Full Text Available Abstract Background As malaria becomes increasingly drug resistant and more costly to treat, there is increasing urgency to develop effective vaccines. In comparison to other stages of the malaria lifecycle, sexual stage antigens are under less immune selection pressure and hence are likely to have limited antigenic diversity. Methods Clinical isolates from a wide range of geographical regions were collected. Direct sequencing of PCR products was then used to determine the extent of polymorphisms for the novel Plasmodium falciparum sexual stage antigen von Willebrand Factor A domain-related Protein (PfWARP. These isolates were also used to confirm the extent of diversity of sexual stage antigen Pfs28. Results PfWARP was shown to have non-synonymous substitutions at 3 positions and Pfs28 was confirmed to have a single non-synonymous substitution as previously described. Conclusion This study demonstrates the limited antigenic diversity of two prospective P. falciparum sexual stage antigens, PfWARP and Pfs28. This provides further encouragement for the proceeding with vaccine trials based on these antigens.

  12. Limited polymorphism in Plasmodium falciparum ookinete surface antigen, von Willebrand factor A domain-related protein from clinical isolates.

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    Richards, Jack S; MacDonald, Nicholas J; Eisen, Damon P

    2006-07-05

    As malaria becomes increasingly drug resistant and more costly to treat, there is increasing urgency to develop effective vaccines. In comparison to other stages of the malaria lifecycle, sexual stage antigens are under less immune selection pressure and hence are likely to have limited antigenic diversity. Clinical isolates from a wide range of geographical regions were collected. Direct sequencing of PCR products was then used to determine the extent of polymorphisms for the novel Plasmodium falciparum sexual stage antigen von Willebrand Factor A domain-related Protein (PfWARP). These isolates were also used to confirm the extent of diversity of sexual stage antigen Pfs28. PfWARP was shown to have non-synonymous substitutions at 3 positions and Pfs28 was confirmed to have a single non-synonymous substitution as previously described. This study demonstrates the limited antigenic diversity of two prospective P. falciparum sexual stage antigens, PfWARP and Pfs28. This provides further encouragement for the proceeding with vaccine trials based on these antigens.

  13. Characterization of surface antigen protein 1 (SurA1) from Acinetobacter baumannii and its role in virulence and fitness.

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    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1

    NARCIS (Netherlands)

    Goodman, Anna L.; Epp, C.; Moss, D.; Holder, A. A.; Wilson, J. M.; Gao, G. P.; Long, C. A.; Remarque, E. J.; Thomas, A. W.; Ammendola, V.; Colloca, S.; Dicks, M. D. J.; Biswas, S.; Seibel, D.; van Duivenvoorde, L. M.; Gilbert, S. C.; Hill, A. V. S.; Draper, S. J.

    2010-01-01

    Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we

  15. Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription.

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    Qi, Pengfei; Liu, Ke; Wei, Jianchao; Li, Yuming; Li, Beibei; Shao, Donghua; Wu, Zhuanchang; Shi, Yuanyuan; Tong, Guangzhi; Qiu, Yafeng; Ma, Zhiyong

    2017-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene ( B2M ) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4. Copyright © 2017 American Society for Microbiology.

  16. Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

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    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.

  17. Monoclonal antibodies against rat leukocyte surface antigens

    NARCIS (Netherlands)

    van den Berg, T. K.; Puklavec, M. J.; Barclay, A. N.; Dijkstra, C. D.

    2001-01-01

    Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is

  18. Immunogenetic markers associated with a naturally acquired humoral immune response against an N-terminal antigen of Plasmodium vivax merozoite surface protein 1 (PvMSP-1).

    Science.gov (United States)

    Cassiano, Gustavo Capatti; Furini, Adriana A C; Capobianco, Marcela P; Storti-Melo, Luciane M; Almeida, Maria E; Barbosa, Danielle R L; Póvoa, Marinete M; Nogueira, Paulo A; Machado, Ricardo L D

    2016-06-03

    Humoral immune responses against proteins of asexual blood-stage malaria parasites have been associated with clinical immunity. However, variations in the antibody-driven responses may be associated with a genetic component of the human host. The objective of the present study was to evaluate the influence of co-stimulatory molecule gene polymorphisms of the immune system on the magnitude of the humoral immune response against a Plasmodium vivax vaccine candidate antigen. Polymorphisms in the CD28, CTLA4, ICOS, CD40, CD86 and BLYS genes of 178 subjects infected with P. vivax in an endemic area of the Brazilian Amazon were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The levels of IgM, total IgG and IgG subclasses specific for ICB2-5, i.e., the N-terminal portion of P. vivax merozoite surface protein 1 (PvMSP-1), were determined by enzyme-linked immuno assay. The associations between the polymorphisms and the antibody response were assessed by means of logistic regression models. After correcting for multiple testing, the IgG1 levels were significantly higher in individuals recessive for the single nucleotide polymorphism rs3116496 in CD28 (p = 0.00004). Furthermore, the interaction between CD28 rs35593994 and BLYS rs9514828 had an influence on the IgM levels (p = 0.0009). The results of the present study support the hypothesis that polymorphisms in the genes of co-stimulatory components of the immune system can contribute to a natural antibody-driven response against P. vivax antigens.

  19. Outer membrane protein antigens of Moraxella bovis.

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    Ostle, A G; Rosenbusch, R F

    1986-07-01

    Outer membranes were isolated from bovine isolates and type strains of Moraxella bovis, M phenylpyruvica, M lacunata, and M ovis by sodium N lauroyl sarcosinate extraction and differential centrifugation. Analysis of outer membranes from these organisms by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis revealed that all M bovis isolates shared a common polypeptide pattern that was readily distinguishable from other Moraxella spp. Nine major outer membrane protein bands were identified by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis analysis of M bovis. Immunoblotting of protein antigens of M bovis revealed several outer membrane proteins that seemed to be common antigens of all M bovis isolates.

  20. The use of outer membrane proteins as an exposure surface for foreign antigens in AIDS vaccine methodology and AIDS diagnostics

    NARCIS (Netherlands)

    Soede WWD; Hegger I

    1992-01-01

    A live recombinant bacteria or virus with HIV determinants exposed at the outermembrane is one strategy for AIDS vaccine development. Two HIV determinants that showed neutralization capacity in in- vitro experiments were tested for their expression in PhoE outer membrane protein of E coli K12.

  1. Intracellular trafficking of bio-nanocapsule-liposome complex: Identification of fusogenic activity in the pre-S1 region of hepatitis B virus surface antigen L protein.

    Science.gov (United States)

    Somiya, Masaharu; Sasaki, Yasuo; Matsuzaki, Takashi; Liu, Qiushi; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés Daniel; Kuroda, Shun'ichi

    2015-08-28

    Bio-nanocapsules (BNCs) are a hollow nanoparticle consisting of about 100-nm liposome (LP) embedding about 110 molecules of hepatitis B virus (HBV) surface antigen (HBsAg) L protein as a transmembrane protein. Owing to the human hepatocyte-recognizing domains on the N-terminal region (pre-S1 region), BNCs have recently been shown to attach and enter into human hepatic cells using the early infection mechanism of HBV. Since BNCs could form a complex with an LP containing various drugs and genes, BNC-LP complexes have been used as a human hepatic cell-specific drug and gene-delivery system in vitro and in vivo. However, the role of BNCs in cell entry and intracellular trafficking of payloads in BNC-LP complexes has not been fully elucidated. In this study, we demonstrate that low pH-dependent fusogenic activity resides in the N-terminal part of pre-S1 region (NPLGFFPDHQLDPAFG), of which the first FF residues are essential for the activity, and which facilitates membrane fusion between LPs in vitro. Moreover, BNC-LP complexes can bind human hepatic cells specifically, enter into the cells via clathrin-mediated endocytosis, and release their payloads mostly into the cytoplasm. Taken together, the BNC portion of BNC-LP complexes can induce membrane fusion between LPs and endosomal membranes under low pH conditions, and thereby facilitate the endosomal escape of payloads. Furthermore, the fusogenic domain of the pre-S1 region of HBsAg L protein may play a pivotal role in the intracellular trafficking of not only BNC-LP complexes but also of HBV. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Association of Pneumococcal Protein Antigen Serology With Age and Antigenic Profile of Colonizing Isolates.

    Science.gov (United States)

    Azarian, Taj; Grant, Lindsay R; Georgieva, Maria; Hammitt, Laura L; Reid, Raymond; Bentley, Stephen D; Goldblatt, David; Santosham, Mathuran; Weatherholtz, Robert; Burbidge, Paula; Goklish, Novalene; Thompson, Claudette M; Hanage, William P; O'Brien, Kate L; Lipsitch, Marc

    2017-03-01

    Several Streptococcus pneumoniae proteins play a role in pathogenesis and are being investigated as vaccine targets. It is largely unknown whether naturally acquired antibodies reduce the risk of colonization with strains expressing a particular antigenic variant. Serum immunoglobulin G (IgG) titers to 28 pneumococcal protein antigens were measured among 242 individuals aged - 30 days after serum collection, and the antigen variant in each pneumococcal isolate was determined using genomic data. We assessed the association between preexisting variant-specific antibody titers and subsequent carriage of pneumococcus expressing a particular antigen variant. Antibody titers often increased across pediatric groups before decreasing among adults. Individuals with low titers against group 3 pneumococcal surface protein C (PspC) variants were more likely to be colonized with pneumococci expressing those variants. For other antigens, variant-specific IgG titers do not predict colonization. We observed an inverse association between variant-specific antibody concentration and homologous pneumococcal colonization for only 1 protein. Further assessment of antibody repertoires may elucidate the nature of antipneumococcal antibody-mediated mucosal immunity while informing vaccine development. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  3. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  4. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice.

    Science.gov (United States)

    Meng, Min; He, Shenyi; Zhao, Guanghui; Bai, Yang; Zhou, Huaiyu; Cong, Hua; Lu, Gang; Zhao, Qunli; Zhu, Xing-Quan

    2012-11-26

    Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (Pmice in experimental groups was longer than control groups (Pmice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.

  5. Analysis of cell surface antigens by Surface Plasmon Resonance imaging

    NARCIS (Netherlands)

    Stojanovic, Ivan; Schasfoort, Richardus B.M.; Terstappen, Leonardus Wendelinus Mathias Marie

    2013-01-01

    Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on

  6. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

  7. 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

    Science.gov (United States)

    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...

  8. Adenovirus 5 and 35 vectors expressing Plasmodium falciparum circumsporozoite surface protein elicit potent antigen-specific cellular IFN-gamma and antibody responses in mice

    NARCIS (Netherlands)

    Shott, Joseph P.; McGrath, Shannon M.; Pau, Maria Grazia; Custers, Jerome H. V.; Ophorst, Olga; Demoitié, Marie-Ange; Dubois, Marie-Claude; Komisar, Jack; Cobb, Michelle; Kester, Kent E.; Dubois, Patrice; Cohen, Joe; Goudsmit, Jaap; Heppner, D. Gray; Stewart, V. Ann

    2008-01-01

    Falciparum malaria vaccine candidates have been developed using recombinant, replication-deficient serotype 5 and 35 adenoviruses (Ad5, Ad35) encoding the Plasmodium falciparum circumsporozoite surface protein (CSP) (Ad5.CS, Ad35.CS) (Crucell Holland BV, Leiden, The Netherlands). To evaluate the

  9. Identification and characterization of surface antigens in parasites, using radiolabelling techniques

    International Nuclear Information System (INIS)

    Ramasamy, R.

    1982-04-01

    Surface proteins of Schistosoma sp and Leishmania sp were studied using 125-Iodine as tracer. The surface proteins were labelled by the Lactoperoxidase method and the proteins then separated using SDS PAG electrophoresis and autoradiography. The possible immunogens were then separated using immunoprecipitation and Fluorescent Antibody techniques using sera from patients or from artificially immunized rabbits. Four common antigens were identified from the surfaces of male and female adult worms, cercariae and schistosomulae of S.mansoni. These antigens, which had molecular weights of 150,000, 78,000, 45,000, and 22,000 were also isolated from the surfaces of S.haematobium adults. The surface antigens on promastigotes of a Kenyan strain of Leishmania donovani were separated into three protein antigens with molecular weights of 66,000, 59,000 and 43,000 respectively. The 59,000 molecular weight antigen was a glycoprotein and was common to promastigotes of an American and Indian strain of L.donovani and to L.braziliensis mexicana. None of the isolated antigens have been shown to have a protective effect when vaccinated into mice, but the study illustrates the value of radionuclide tracers in the unravelling of the mosaic of antigens which parasites possess

  10. Paired Expression Analysis of Tumor Cell Surface Antigens

    Directory of Open Access Journals (Sweden)

    Rimas J. Orentas

    2017-08-01

    Full Text Available Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19 or antibody-based therapy (anti-CD20 in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues. We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK

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

    Science.gov (United States)

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

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 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. Chitosan-based delivery systems for protein therapeutics and antigens

    NARCIS (Netherlands)

    Amidi, M.; Mastrobattista, E.; Jiskoot, W.; Hennink, W.E.

    Therapeutic peptides/proteins and protein-based antigens are chemically and structurally labile compounds, which are almost exclusively administered by parenteral injections. Recently, non-invasive mucosal routes have attracted interest for administration of these biotherapeutics. Chitosan-based

  14. Cationic lipid-formulated DNA vaccine against hepatitis B virus: immunogenicity of MIDGE-Th1 vectors encoding small and large surface antigen in comparison to a licensed protein vaccine.

    Directory of Open Access Journals (Sweden)

    Anne Endmann

    Full Text Available Currently marketed vaccines against hepatitis B virus (HBV based on the small (S hepatitis B surface antigen (HBsAg fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.

  15. In silico Analysis of Immunologic Regions of Surface Antigens (Sags of Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Abbas Alibakhshi

    2017-06-01

    Full Text Available Background: Surface antigens (SAGs of Toxoplasma gondii are known candidates for diagnostic tests and vaccines. The present study argues about the main necessary properties for determination and prediction of T-cell agretopes and B-cell epitopes of surface antigens of Toxoplasma gondii.Materials and Methods: Primary, secondary and tertiary structures of the proteins were analyzed by different methods. The three-dimensional structures were determined by use of ab initio method for prediction of discontinues epitopes. The agretopes and epitopes were predicted via several various web servers with different methods employed.Results: The results of in silico analyses showed that the regions 129-GAPAGRNNDGSSAPT-143 for protein p22, 234-SENPWQGNASSD-245 for protein p30 and 348-PGTEGESQAGT-358 for protein p43, have the highest immunogenic potential.Conclusion: We reached to three antigenic epitopes for cloning and protein expression. In following the purified polypeptide will be applied for diagnosis of Toxoplasma gondii.

  16. Identification of Surface Exposed Elementary Body Antigens of ...

    African Journals Online (AJOL)

    This study sought to identify the surface exposed antigenic components of Cowdria ruminantium elementary body (EB) by biotin labeling, determine effect of reducing and non-reducing conditions and heat on the mobility of these antigens and their reactivity to antibodies from immunized animals by Western blotting.

  17. Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

    Science.gov (United States)

    Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

    2015-01-01

    The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

  18. Protein antigen adsorption to the DDA/TDB liposomal adjuvant

    DEFF Research Database (Denmark)

    Hamborg, Mette; Jorgensen, Lene; Bojsen, Anders Riber

    2013-01-01

    Understanding the nature of adjuvant-antigen interactions is important for the future design of efficient and safe subunit vaccines, but remains an analytical challenge. We studied the interactions between three model protein antigens and the clinically tested cationic liposomal adjuvant composed...

  19. Antigenic differences within the Cryptosporidium hominis and Cryptosporidium parvum surface proteins P23 and GP900 defined by monoclonal antibody reactivity.

    Science.gov (United States)

    Sturbaum, Gregory D; Schaefer, Deborah A; Jost, B Helen; Sterling, Charles R; Riggs, Michael W

    2008-06-01

    The biological basis for the specificity of host infectivity patterns of Cryptosporidium spp., in particular C. hominis and C. parvum, has yet to be fully elucidated. Comparison of the C. parvum and C. hominis P23 and GP900 predicted amino acid sequences revealed 3 differences in P23 and 4 and 17 differences in GP900 domains 1 and 5, respectively. Using monoclonal antibodies developed against the surface (glyco)proteins P23 and GP900 of the C. parvum Iowa isolate, solubilized glycoprotein from three C. hominis isolates was screened for reactivity using Western immunoblots. One of ten P23 MAbs and three of 21 GP900 MAbs were not reactive with any of the three C. hominis isolates. The non-reactive P23 MAb binds to a peptide epitope, while the non-reactive GP900 MAbs bind to either carbohydrate/carbohydrate-dependent or peptide epitopes of C. parvum. These results demonstrate phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion.

  20. Targeted disruption of a ring-infected erythrocyte surface antigen (RESA)-like export protein gene in Plasmodium falciparum confers stable chondroitin 4-sulfate cytoadherence capacity

    DEFF Research Database (Denmark)

    Goel, Suchi; Muthusamy, Arivalagan; Miao, Jun

    2014-01-01

    . In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb...... (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected...... erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest...

  1. Antigenic and structural conservation of herpesvirus DNA-binding proteins.

    Science.gov (United States)

    Littler, E; Yeo, J; Killington, R A; Purifoy, D J; Powell, K L

    1981-10-01

    Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

  2. Seroprevalence of hepatitis B surface antigen among pregnant ...

    African Journals Online (AJOL)

    Seroprevalence of hepatitis B surface antigen among pregnant women attending the Hospital for Women & Children in Koutiala, Mali. Brett MacLean, Rosanna F Hess, Edward Bonvillain, Joseph Kamate, Daoda Dao, Amy Cosimano, Shannon Hoy ...

  3. Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor

    Science.gov (United States)

    Su, Fengyu; Xu, Chunye; Taya, Minoru; Murayama, Kimie; Shinohara, Yasuro; Nishimura, Shin-Ichiro

    2008-01-01

    Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2nd and 3rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold. PMID:27879935

  4. Protein and antigenic heterogeneity among isolates of Bacillus piliformis.

    OpenAIRE

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

    1990-01-01

    Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels a...

  5. HIV immune evasion disruption of antigen presentation by the HIV Nef protein.

    Science.gov (United States)

    Wonderlich, Elizabeth R; Leonard, Jolie A; Collins, Kathleen L

    2011-01-01

    The Human Immunodeficiency Virus (HIV) Nef protein is necessary for high viral loads and for timely progression to AIDS. Nef plays a number of roles, but its effect on antigen presentation and immune evasion are among the best characterized. Cytotoxic T lymphocytes (CTLs) recognize and lyse virally infected cells by detecting viral antigens in complex with host major histocompatibility complex class I (MHC-I) molecules on the infected cell surface. The HIV Nef protein disrupts antigen presentation at the cell surface by interfering with the normal trafficking pathway of MHC-I and thus reduces CTL recognition and lysis of infected cells. The molecular mechanism by which Nef causes MHC-I downmodulation is becoming more clear, but some questions remain. A better understanding of how Nef disrupts antigen presentation may lead to the development of drugs that enhance the ability of the anti-HIV CTLs to control HIV disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  7. Participation of CD1 molecules in the presentation of bacterial protein antigens in humans.

    Science.gov (United States)

    Ulanova, M; Tarkowski, A; Hahn-Zoric, M; Hanson, L A

    1999-10-01

    Human CD1 molecules, expressed on the surface of professional antigen-presenting cells (including dendritic cells, Langerhans' cells, B cells and activated monocytes) are structurally homologous to major histocompatibility complex (MHC) class I and class II molecules. CD1b and CD1c have been shown to present nonpeptide bacterial antigens to T cells. We hypothesized that CD1 molecules may also be involved in the presentation of bacterial protein antigens. Human peripheral blood mononuclear cells (PBMC) were exposed to two medically important proteins, tetanus toxoid (TT) and purified protein derivative (PPD), with and without murine monoclonal antibodies (MoAbs) specific for CD1a, CD1b and CD1c. All the MoAbs substantially inhibited the proliferative responses of PBMC to TT and PPD. Simultaneous interaction of CD1 and MHC class II molecules was even more inhibitory to these antigen-specific proliferative responses. In contrast, neither mixed lymphocyte reaction nor superantigen and mitogenic responses were affected by CD1-specific antibodies, indicating a certain restriction pattern in antigen presentation. Our findings suggest that, besides MHC class I and II molecules, there is a family of nonpolymorphic cell surface molecules that is able to present certain bacterial protein antigens to T cells.

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

    Directory of Open Access Journals (Sweden)

    Robert Wilson

    2017-01-01

    Full Text Available 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.

  9. Virosome-mediated delivery of protein antigens to dendritic cells

    NARCIS (Netherlands)

    Bungener, L; Serre, K; Bijl, L; Leserman, L; Wilschut, J; Daemen, T; Machy, P

    2002-01-01

    Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and 11

  10. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

  11. 21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Comparative characteristic of the methods of protein antigens epitope mapping

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2014-08-01

    Full Text Available Comparative analysis of experimental methods of epitope mapping of protein antigens has been carried out. The vast majority of known techniques are involved in immunochemical study of the interaction of protein molecules or peptides with antibodies of corresponding specifici­ty. The most effective and widely applicable metho­dological techniques are those that use synthetic and genetically engineered peptides. Over the past 30 years, these groups of methods have travelled a notable evolutionary path up to the maximum automation and the detection of antigenic determinants of various types (linear and conformational epitopes, and mimotopes. Most of epitope searching algorithms were integrated into a computer program, which greatly facilitates the analysis of experimental data and makes it possible to create spatial models. It is possible to use comparative epitope mapping for solving the applied problems; this less time-consuming method is based on the analysis of competition between different antibodies interactions with the same antigen. The physical method of antigenic structure study is X-ray analysis of antigen-antibody complexes, which may be applied only to crystallizing­ proteins, and nuclear magnetic resonance.

  14. Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus : Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

    NARCIS (Netherlands)

    Endmann, Anne; Klunder, Katharina; Kapp, Kerstin; Riede, Oliver; Oswald, Detlef; Talman, Eduard G.; Schroff, Matthias; Kleuss, Christiane; Ruiters, Marcel H. J.; Juhls, Christiane

    2014-01-01

    Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible

  15. Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP142 administered intramuscularly with adjuvant system AS01

    Directory of Open Access Journals (Sweden)

    Otsyula Nekoye

    2013-01-01

    Full Text Available Abstract Background The development of an asexual blood stage vaccine against Plasmodium falciparum malaria based on the major merozoite surface protein-1 (MSP1 antigen is founded on the protective efficacy observed in preclinical studies and induction of invasion and growth inhibitory antibody responses. The 42 kDa C-terminus of MSP1 has been developed as the recombinant protein vaccine antigen, and the 3D7 allotype, formulated with the Adjuvant System AS02A, has been evaluated extensively in human clinical trials. In preclinical rabbit studies, the FVO allele of MSP142 has been shown to have improved immunogenicity over the 3D7 allele, in terms of antibody titres as well as growth inhibitory activity of antibodies against both the heterologous 3D7 and homologous FVO parasites. Methods Two Phase 1 clinical studies were conducted to examine the safety, reactogenicity and immunogenicity of the FVO allele of MSP142 in the adjuvant system AS01 administered intramuscularly at 0-, 1-, and 2-months: one in the USA and, after evaluation of safety data results, one in Western Kenya. The US study was an open-label, dose escalation study of 10 and 50 μg doses of MSP142 in 26 adults, while the Kenya study, evaluating 30 volunteers, was a double-blind, randomized study of only the 50 μg dose with a rabies vaccine comparator. Results In these studies it was demonstrated that this vaccine formulation has an acceptable safety profile and is immunogenic in malaria-naïve and malaria-experienced populations. High titres of anti-MSP1 antibodies were induced in both study populations, although there was a limited number of volunteers whose serum demonstrated significant inhibition of blood-stage parasites as measured by growth inhibition assay. In the US volunteers, the antibodies generated exhibited better cross-reactivity to heterologous MSP1 alleles than a MSP1-based vaccine (3D7 allele previously tested at both study sites. Conclusions Given that the primary

  16. Prevalence of Hepatitis B Surface Antigen among Women of ...

    African Journals Online (AJOL)

    This study documents the seroprevalence of hepatitis B surface antigen (HBs Ag) among women of childbearing age attending various family clinics in Lagos, Nigeria. A total of 501 women were screened with Wellcozyme ELISA technique, of which 45(8.9%) were seropositive. Women in occupations related to needle work ...

  17. Prevalence of hepatitis B surface antigen seropositivity among HIV ...

    African Journals Online (AJOL)

    Hepatitis B surface antigen (HBsAg) was tested for using a one step lateral flow rapid chromatographic immunoassay (Acumen labs and diagnostic centre, Bangalore, India) and HIV 1/2 was tested using two kits, Determine (made by Abbot, Japan for Inverness Medical, Japan). Results: A total of 2018 subjects were studied ...

  18. Prevalence of hepatitis b virus surface antigens (HBsag) and ...

    African Journals Online (AJOL)

    The prevalences of hepatitis B virus surface antigen (HBsAg) and hepatitis C virus (HCV) antibodies were determined in 560 blood donors sera using ELISA kits (DIALAB., Austria). Forty eight (8.57%) of these were positive for hepatitis B virus infection, while 33(5.89%) were positive to hepatitis C virus antibodies. The sex ...

  19. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, J.S.G.; Trust, T.J.

    1988-02-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase /sup 125/I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to /sup 125/I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.

  20. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    International Nuclear Information System (INIS)

    Dooley, J.S.G.; Trust, T.J.

    1988-01-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125 I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to 125 I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein

  1. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  2. Surface proteome analysis and characterization of surface cell antigen (Sca or autotransporter family of Rickettsia typhi.

    Directory of Open Access Journals (Sweden)

    Khandra T Sears

    Full Text Available Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca autotransporter (AT family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1-3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis. Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i to infect mammalian cells should the flea bite a host, ii to remain

  3. Cellular Cancer Vaccines: an Update on the Development of Vaccines Generated from Cell Surface Antigens

    Directory of Open Access Journals (Sweden)

    Petr G. Lokhov, Elena E. Balashova

    2010-01-01

    Full Text Available A recent advance in anti-cancer therapies has been the use of cancer cells to develop vaccines. However, immunization with cancer cell-based vaccines has not resulted in significant long-term therapeutic benefits. A possible reason for this is that while cancer cells provide surface antigens that are targets for a desired immune response, they also contain a high abundance of housekeeping proteins, carbohydrates, nucleic acids, lipids, and other intracellular contents that are ubiquitous in all mammalian cells. These ubiquitous molecules are not the intended targets of this therapy approach, and thus, the immune response generated is not sufficient to eliminate the cancer cells present. In this review, a discussion of the cell surface of cancer cells is presented in relation to the goals of improving antigen composition of cancer cell-based vaccines. Strategies to enrich vaccines for cancer-specific antigens are also discussed.

  4. Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

    Science.gov (United States)

    Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

    2017-11-01

    Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  6. Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32

    NARCIS (Netherlands)

    Wieles, B.; van Agterveld, M.; Janson, A.; Clark-Curtiss, J.; Rinke de Wit, T.; Harboe, M.; Thole, J.

    1994-01-01

    Secreted proteins may serve as major targets in the immune response to mycobacteria. To identify potentially secreted Mycobacterium leprae antigens, antisera specific for culture filtrate proteins of Mycobacterium tuberculosis were used to screen a panel of recombinant antigens selected previously

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

    DEFF Research Database (Denmark)

    Jakobsen, P H; Hviid, L; Theander, T G

    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 i...

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

    Science.gov (United States)

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

    2013-01-01

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

  9. Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

    Directory of Open Access Journals (Sweden)

    Thompson Joanne

    2007-05-01

    Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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...

  12. Protein A-containing staphylococcus aureus as an immunoglobulin-binding reagent: 1) in radioimmunoassays - 'staf-RIA' - recently also for antibiotics and microbial antigens/antibodies, and 2) in a non-radioactive surface immunoassay - 'Staph-ace ay' read by the naked eye - primarily for antibodies to antigens adsorbed to transparent surfaces

    International Nuclear Information System (INIS)

    Jonsson, S.

    1977-01-01

    This paper is intended to summarize recent developments for the use of protein A-containing staphylococci as an immunoglobulin-binding reagent in various types of radioimmunoassay and some related areas, particularly the staphylococcal surface immunoassay. The paper also presents a new process for the large scale preparation of a freeze-dried preparation of the immunoglobulin-binding, killed staphylococci, which thereby gain a much improved suspension stability. (orig.) [de

  13. Antigenic protein synthesis of Campylobacter jejuni in contact with chicken cells

    DEFF Research Database (Denmark)

    Vegge, Christina Skovgaard; Bang, Dang D.; Li, Yiping

    to the environment of the avian gastrointestinal tract. Consequently, the most important reservoir for C. jejuni is the gut of chickens, which are colonized efficiently without causing disease in the birds. Upon co-cultivation with mammalian cells, C. jejuni secrete specific Cia proteins, which are required...... the synthesis of antigenic C. jejuni proteins upon cultivation with chicken cells. Two strains of C. jejuni (the human isolate NCTC11168 and the chicken isolate DVI-SC11) were incubated with primary intestinal chicken cells and subsequently used to raise antisera in rabbits. Negative controls were carried out...... in parallel. These antisera were tested by Western blotting against C. jejuni total protein as well as periplasmic-, surface- and extracellular protein fractions. A unique antibody reaction was discovered to a protein from samples, which had been cultivated with chicken cells. The identity of this protein...

  14. Heterologous expression of carcinoembryonic antigen in Lactococcus lactis via LcsB-mediated surface displaying system for oral vaccine development.

    Science.gov (United States)

    Zhang, Xiaowei; Hu, Shumin; Du, Xue; Li, Tiejun; Han, Lanlan; Kong, Jian

    2016-12-01

    Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. CEA was cloned into an Escherichia coli/Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA-LcsB fusion was confirmed by western blot analysis. Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing the CEA-LcsB fused protein. In addition, the CEA-LcsB antigen group showed a higher spleen index compared to the CEA antigen alone or negative control, demonstrating that surface-displayed CEA antigen could induce a higher immune response. These results provided the first evidence for displaying CEA antigen on the cell surfaces of LABs as oral vaccines against cancer or infectious diseases. Copyright © 2014. Published by Elsevier B.V.

  15. Genetic variation and significance of hepatitis B surface antigen

    Directory of Open Access Journals (Sweden)

    ZHANG Zhenhua

    2013-11-01

    Full Text Available Hepatitis B virus (HBV is prone to genetic variation because there is reverse transcription in the process of HBV replication. The gene mutation of hepatitis B surface antigen may affect clinical diagnosis of HBV infection, viral replication, and vaccine effect. The current research and existing problems are discussed from the following aspects: the mechanism and biological and clinical significance of S gene mutation. Most previous studies focused on S gene alone, so S gene should be considered as part of HBV DNA in the future research on S gene mutation.

  16. Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC.

    Science.gov (United States)

    Abiko, Yuki; Nagano, Keiji; Yoshida, Yasuo; Yoshimura, Fuminobu

    2014-01-01

    Treponema denticola, a gram-negative and anaerobic spirochete, is associated with advancing severity of chronic periodontitis. In this study, we confirmed that two major antigenic proteins were Msp and TmpC, and examined their physiological and pathological roles using gene-deletion mutants. Msp formed a large complex that localized to the outer membrane, while TmpC existed as a monomer and largely localized to the inner membrane. However, TmpC was also detected in the outer membrane fraction, but its cell-surface exposure was not detected. Msp defects increased cell-surface hydrophobicity and secretion of TNF-α from macrophage-like cells, whereas TmpC defects decreased autoagglutination and chymotrypsin-like protease activities. Both mutants adhered to gingival epithelial cells similarly to the wild-type and showed slightly decreased motility. In addition, in Msp-defective mutants, the TDE1072 protein, which is a major membrane protein, was abolished; therefore, phenotypic changes in the mutant can be, at least in part, attributed to the loss of the TDE1072 protein. Thus, the major antigenic proteins, Msp and TmpC, have significant and diverse impacts on the characteristics of T. denticola, especially cell surface properties.

  17. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

    Directory of Open Access Journals (Sweden)

    Tim J Schuijt

    2011-01-01

    Full Text Available Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

  18. Proteins at surfaces

    NARCIS (Netherlands)

    Efimova, Y.M.

    2006-01-01

    Understanding protein adsorption is of vital importance in many fields of medicine and industry that can be divided into two categories: those in which it is desired to minimize adsorption, and those in which protein adsorption is desired. The first category covers materials for kidney dialysis

  19. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    International Nuclear Information System (INIS)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

    1990-01-01

    A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

  20. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum.

    Science.gov (United States)

    Cox, D L; Chang, P; McDowall, A W; Radolf, J D

    1992-01-01

    Virulent Treponema pallidum reacts poorly with the specific antibodies present in human and rabbit syphilitic sera, a phenomenon often attributed to an outer coat of host serum proteins. Here we present additional evidence that the limited antigenicity of virulent organisms actually is due to a paucity of proteins in the outer membrane. Initially, we used electron microscopy to demonstrate that the outer membrane is highly susceptible to damage from physical manipulation (i.e., centrifugation and resuspension) and nonionic detergents. Organisms with disrupted outer membranes were markedly more antigenic than intact treponemes as determined by immunoelectron microscopy (IEM) with rabbit syphilitic and antiendoflagellar antisera. Data obtained with a new radioimmunoassay, designated the T. pallidum surface-specific radioimmunoassay, corroborated these IEM findings by demonstrating that the major T. pallidum immunogens are not surface exposed; the assay also was unable to detect serum proteins, including fibronectin, on the surfaces of intact organisms. Furthermore, IEM of T. pallidum on ultrathin cryosections with monospecific anti-47-kDa-immunogen antiserum confirmed the intracellular location of the 47-kDa immunogen. On the basis of these and previous findings, we proposed a new model for T. pallidum ultrastructure in which the outer membrane contains a small number of transmembrane proteins and the major membrane immunogens are anchored by lipids to the periplasmic leaflet of the cytoplasmic membrane. This unique ultrastructure explains the remarkable ability of virulent organisms to evade the humoral immune response of the T. pallidum-infected host. Images PMID:1541522

  3. Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites of Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Hua Cong

    2013-01-01

    Full Text Available Toxoplasma gondii is a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

  4. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children

    DEFF Research Database (Denmark)

    Dodoo, D; Staalsoe, T; Giha, H

    2001-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endoth...

  5. Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions

    International Nuclear Information System (INIS)

    Jones, S.K.

    1992-01-01

    Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus. (Author)

  6. Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

    Science.gov (United States)

    Tobias, Joshua; Lebens, Michael; Wai, Sun Nyunt; Holmgren, Jan; Svennerholm, Ann-Mari

    2017-04-01

    Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2010-07-01

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

  8. Stable isotope labeling of oligosaccharide cell surface antigens

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A. [and others

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  9. Heat shock protein HSP60 and the perspective for future using as vaccine antigens

    Directory of Open Access Journals (Sweden)

    Joanna Bajzert

    2015-10-01

    Full Text Available Heat Shock Proteins (HSPs are widely spread in nature, highly conserved proteins, found in all prokaryotic and eukaryotic cells. HSPs have been classified in 10 families, one of them is the HSP60 family. HSP60 function in the cytoplasm as ATP-dependent molecular chaperones by assisting the folding of newly synthesised polypeptides and the assembly of multiprotein complexes. There is a large amount of evidence which demonstrate that HSP60 is expressed on the cell surface. Especially in bacteria the expression on the surface occurs constitutively and increases remarkably during host infection. HSP60 also play an important role in biofilm formation. In the extracellular environment, HSP60 alone or with self or microbial proteins can acts not only as a link between immune cells, but also as a coordinator of the immune system activity. This protein could influence the immune system in a different way because they act as an antigen, a carrier of other functional molecules or as a ligand for receptor. They are able to stimulate both cells of the acquired (naïve, effector, regulatory T lymphocyte, B lymphocyte and the innate (macrophages, monocytes, dendritic cells immune system. HSPs have been reported to be potent activators of the immune system and they are one of the immunodominant bacterial antigens they could be a good candidate for a subunit vaccine or as an adjuvant.

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

    International Nuclear Information System (INIS)

    Pandey, B.; Stine, K.J.; Demchenko, A.V.

    2012-01-01

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

  11. Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins

    Science.gov (United States)

    Chen, Tie; Grunert, Fritz; Medina-Marino, Andrew; Gotschlich, Emil C.

    1997-01-01

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

  12. In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

    OpenAIRE

    Chamakh-Ayari, Rym; Bras-Gonçalves, Rachel; Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb)...

  13. Identification of the Streptococcus pyogenes surface antigens recognised by pooled human immunoglobulin

    Science.gov (United States)

    Reglinski, Mark; Gierula, Magdalena; Lynskey, Nicola N.; Edwards, Robert J.; Sriskandan, Shiranee

    2015-01-01

    Immunity to common bacteria requires the generation of antibodies that promote opsonophagocytosis and neutralise toxins. Pooled human immunoglobulin is widely advocated as an adjunctive treatment for clinical Streptococcus pyogenes infection however, the protein targets of the reagent remain ill defined. Affinity purification of the anti-streptococcal antibodies present within pooled immunoglobulin resulted in the generation of an IgG preparation that promoted opsonophagocytic killing of S. pyogenes in vitro and provided passive immunity in vivo. Isolation of the streptococcal surface proteins recognised by pooled human immunoglobulin permitted identification and ranking of 94 protein antigens, ten of which were reproducibly identified across four contemporary invasive S. pyogenes serotypes (M1, M3, M12 and M89). The data provide novel insight into the action of pooled human immunoglobulin during invasive S. pyogenes infection, and demonstrate a potential route to enhance the efficacy of antibody based therapies. PMID:26508447

  14. Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

    Science.gov (United States)

    Lai, Zengzu; Schreiber, John R

    2009-05-21

    Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

  15. The promastigote surface antigen gene family of the Leishmania parasite : differential evolution by positive selection and recombination - art. no. 292

    OpenAIRE

    Devault, A.; Banuls, Anne-Laure

    2008-01-01

    Background: PSA (promastigote surface antigen) is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results: From the newly available complete genome sequences of L...

  16. Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition.

    Directory of Open Access Journals (Sweden)

    Daniel P Depledge

    2010-09-01

    Full Text Available A family of hydrophilic acylated surface (HASP proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic and intracellular (amastigote stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia have lost HASP genes from their genomes.We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia species, L. (V. braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L. mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o HASPs are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are

  17. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    International Nuclear Information System (INIS)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.-A.; Trummel, C.L.; Robertson, P.R.

    1982-01-01

    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated 45 Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis. (author)

  18. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    Energy Technology Data Exchange (ETDEWEB)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.A.; Trummel, C.L.; Robertson, P.R. (Department of Periodontology, University of Connecticut, School of Dental Medicine, Farmington, Connecticut, U.S.A.)

    1982-01-01

    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated /sup 45/Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.

  19. Effect of hepatitis B immunisation in newborn infants of mothers positive for hepatitis B surface antigen

    DEFF Research Database (Denmark)

    Lee, Chuanfang; Gong, Yan; Brok, Jesper

    2006-01-01

    To evaluate the effects of hepatitis B vaccine and immunoglobulin in newborn infants of mothers positive for hepatitis B surface antigen.......To evaluate the effects of hepatitis B vaccine and immunoglobulin in newborn infants of mothers positive for hepatitis B surface antigen....

  20. Hepatitis B Virus DNA in Blood Samples Positive for Antibodies to Core Antigen and Negative for Surface Antigen

    Science.gov (United States)

    Gutiérrez, C.; León, G.; Loureiro, C. L.; Uzcátegui, N.; Liprandi, F.; Pujol, F. H.

    1999-01-01

    Anti-hepatitis B core antigen (HBcAg)-positive hepatitis B surface antigen (HBsAg)-negative plasma samples from blood donors were tested by nested PCR. DNA positivity was more significantly associated with high levels of anti-HBcAg than with low levels of anti-HBsAg antibodies. Analysis of a dilution of anti-HBcAg antibodies might result in a more rational exclusion of anti-HBcAg-positive HBsAg-negative samples, reducing the number of donations discarded and enabling more countries to incorporate anti-HBcAg testing. PMID:10473534

  1. Identification of surface proteins of Trichinella spiralis muscle larvae using immunoproteomics.

    Science.gov (United States)

    Liu, R D; Cui, J; Wang, L; Long, S R; Zhang, X; Liu, M Y; Wang, Z Q

    2014-12-01

    Trichinella spiralis surface proteins are directly exposed to the host's immune system, making them the main target antigens which induce the immune responses and may play an important role in the larval invasion and development process. The analysis and characterization of T. spiralis surface proteins could provide useful information to elucidate the host-parasite interaction, identify the early diagnostic antigens and the targets for vaccine. The purpose of this study was to identify the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) Western-blot analysis and mass spectrometry. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Fourteen protein spots were recognized by sera of mice infected with T. spiralis at 42 dpi or at 18 dpi, and 12 spots were successfully identified by MALDI-TOF/TOF-MS, which represented 8 different proteins of T. spiralis. Out of the 8 T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity, which might be the invasion-related proteins and the targets for vaccine. The 4 proteins (deoxyribonuclease II family protein, serine protease, 53 kDa ES antigen and hypothetical protein Tsp_08444) recognized by infection sera at 18 dpi might be the early diagnostic antigens for trichinellosis.

  2. Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells

    Science.gov (United States)

    2013-01-01

    Background Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Results We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns. Conclusions This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker. PMID:24070190

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Protein modeling of apical membrane antigen-1(AMA-1) of ...

    African Journals Online (AJOL)

    Apical membrane Antigen-1(AMA-1), an asexual blood stage antigen of Plasmodium cynomolgi, is an important candidate for testing as a component of malarial vaccine. The degree of conservation of. AMA-1 sequences implies a conserved function for this molecule across different species of Plasmodium. Since the AMA-1 ...

  5. Carcinoembryonic antigen family receptor recognition by gonococcal Opa proteins requires distinct combinations of hypervariable Opa protein domains.

    Science.gov (United States)

    Bos, Martine P; Kao, David; Hogan, Daniel M; Grant, Christopher C R; Belland, Robert J

    2002-04-01

    Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore, shuffling of HV regions may result in novel HSPG receptor binding activity.

  6. Artificial neural network accurately predicts hepatitis B surface antigen seroclearance.

    Directory of Open Access Journals (Sweden)

    Ming-Hua Zheng

    Full Text Available BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg seroclearance and seroconversion are regarded as favorable outcomes of chronic hepatitis B (CHB. This study aimed to develop artificial neural networks (ANNs that could accurately predict HBsAg seroclearance or seroconversion on the basis of available serum variables. METHODS: Data from 203 untreated, HBeAg-negative CHB patients with spontaneous HBsAg seroclearance (63 with HBsAg seroconversion, and 203 age- and sex-matched HBeAg-negative controls were analyzed. ANNs and logistic regression models (LRMs were built and tested according to HBsAg seroclearance and seroconversion. Predictive accuracy was assessed with area under the receiver operating characteristic curve (AUROC. RESULTS: Serum quantitative HBsAg (qHBsAg and HBV DNA levels, qHBsAg and HBV DNA reduction were related to HBsAg seroclearance (P<0.001 and were used for ANN/LRM-HBsAg seroclearance building, whereas, qHBsAg reduction was not associated with ANN-HBsAg seroconversion (P = 0.197 and LRM-HBsAg seroconversion was solely based on qHBsAg (P = 0.01. For HBsAg seroclearance, AUROCs of ANN were 0.96, 0.93 and 0.95 for the training, testing and genotype B subgroups respectively. They were significantly higher than those of LRM, qHBsAg and HBV DNA (all P<0.05. Although the performance of ANN-HBsAg seroconversion (AUROC 0.757 was inferior to that for HBsAg seroclearance, it tended to be better than those of LRM, qHBsAg and HBV DNA. CONCLUSIONS: ANN identifies spontaneous HBsAg seroclearance in HBeAg-negative CHB patients with better accuracy, on the basis of easily available serum data. More useful predictors for HBsAg seroconversion are still needed to be explored in the future.

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

    DEFF Research Database (Denmark)

    Jakobsen, P H; Hviid, L; Theander, T G

    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...... 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....

  8. Enhanced immunogenicity of DNA fusion vaccine encoding secreted hepatitis B surface antigen and chemokine RANTES

    International Nuclear Information System (INIS)

    Kim, Seung Jo; Suh, Dongchul; Park, Sang Eun; Park, Jeong-Sook; Byun, Hyang-Min; Lee, Chan; Lee, Sun Young; Kim, Inho; Oh, Yu-Kyoung

    2003-01-01

    To increase the potency of DNA vaccines, we constructed genetic fusion vaccines encoding antigen, secretion signal, and/or chemokine RANTES. The DNA vaccines encoding secreted hepatitis B surface antigen (HBsAg) were constructed by inserting HBsAg gene into an expression vector with an endoplasmic reticulum (ER)-targeting secretory signal sequence. The plasmid encoding secretory HBsAg (pER/HBs) was fused to cDNA of RANTES, generating pER/HBs/R. For comparison, HBsAg genes were cloned into pVAX1 vector with no signal sequence (pHBs), and further linked to the N-terminus of RANTES (pHBs/R). Immunofluorescence study showed the cytoplasmic localization of HBsAg protein expressed from pHBs and pHBs/R, but not from pER/HBs and pER/HBs/R at 48 h after transfection. In mice, RANTES-fused DNA vaccines more effectively elicited the levels of HBsAg-specific IgG antibodies than pHBs. All the DNA vaccines induced higher levels of IgG 2a rather than IgG 1 antibodies. Of RANTES-fused vaccines, pER/HBs/R encoding the secreted fusion protein revealed much higher humoral and CD8 + T cell-stimulating responses compared to pHBs/R. These results suggest that the immunogenicity of DNA vaccines could be enhanced by genetic fusion to a secretory signal peptide sequence and RANTES

  9. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Dileep Tiwari

    2017-04-01

    Conclusion: Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

  10. HMME-based PDT restores expression and function of transporter associated with antigen processing 1 (TAP1) and surface presentation of MHC class I antigen in human glioma.

    Science.gov (United States)

    Zhang, Shan-Yi; Li, Jun-Liang; Xu, Xin-Ke; Zheng, Mei-Guang; Wen, Cheng-Cai; Li, Fang-Cheng

    2011-11-01

    Numerous studies have established that photodynamic therapy (PDT) can trigger tumor-specific immunity and cancer cell immunogenicity, both of which play a critical role in the long-term control of oncogenesis; however, the underlying mechanisms are largely unexplained. Deficiency of the transporter associated with antigen processing 1 (TAP1) has been observed in a variety of tumors, and the question has been raised whether the restoration of TAP1 could facilitate the activation of antitumor immunity. To elucidate the mechanisms underlying PDT-induced immunopotentiation, we examined the hypothesis that upregulating TAP1 via PDT may contribute to enhancement of antitumor immunity and cancer cell immunogenicity. In this study, we investigated the effects of PDT on the expression and function of TAP1 in glioma cells. We found that HMME-based PDT restored TAP1 expression in a rapid and transient manner. Furthermore, the newly synthesized TAP1 protein was capable of potentiating the activity of transporting antigen peptides. As a result, restoration of the expression and function of TAP1 translated into augmenting the presentation of surface MHC class I molecules. Overall, our data indicate that PDT enables glioma cells to recover both the expression of functional TAP1 and the presentation of surface MHC class I antigens, which are processes that may enhance antitumor immunity after PDT. These findings may have implications for PDT and provide new insights into the mechanisms underlying PDT-induced immunopotentiation.

  11. Simultaneous detection of Hepatitis B surface antigen and its antibody by radioimmunoassay

    International Nuclear Information System (INIS)

    Crouzat-Reynes, Gerard; Perigois, Francois; Lecureuil, Michel; Lejeune, Bernard

    1981-01-01

    The authors describe an original radioimmunoassay which allows the simultaneous detection of hepatitis B surface antigen and its antibody in a biological sample. Antigen and antibody are indiscriminately detected in a first step and then distinguished in a second step using the same reagents [fr

  12. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    Directory of Open Access Journals (Sweden)

    Tam Yew

    2012-10-01

    Full Text Available Abstract Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg from Pichia pastoris expression cells were optimized using response surface methodology (RSM based on the central composite design (CCD. The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

  13. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    Science.gov (United States)

    2012-01-01

    Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

  14. Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...... was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine...... acts on an early event in the antigen handling by accessory cells. Chloroquine is a well known inhibitor of lysosomal proteolysis, and it is likely that its effect on antigen presentation is caused by an inhibition of antigen degradation....

  15. Antigens in human glioblastomas and meningiomas: Search for tumour and onco-foetal antigens. Estimation of S-100 and GFA protein

    DEFF Research Database (Denmark)

    Dittmann, L; Axelsen, N H; Norgaard-Pedersen, B

    1977-01-01

    Extracts of glioblastomas and meningiomas were analysed by quantitative immunoelectrophoresis for the presence of foetal brain antigens and tumour-associated antigens, and levels of 2 normal brain-specific proteins were also determined. The following antibodies were used: monospecific anti-S-100......-alpha-foetoprotein; and monospecific anti-ferritin. Using the antibodies raised against the tumours, several antigens not present in foetal or adult normal brain were found in the glioblastomas and the meningiomas. These antigens cross-reacted with antigens present in normal liver and were therefore not tumour-associated. S-100...... was found in glioblastomas in approximately one tenth the amount in whole brain homogenate, whereas GFA was found 2-4 times enriched. The 2 proteins were absent in meningiomas. The possible use of the GFA protein as a marker for astroglial neoplasia is discussed. Five foetal antigens were found in foetal...

  16. Sero-prevalence of hepatitis B surface antigen among pregnant ...

    African Journals Online (AJOL)

    There were no significant differences between prevalence recorded and agegroups but these were significant associations between risk factors namely tribal marks/tattooing, types of marriages and prevalence of hepatitis infection. The prevalence of hepatitis B antigen among pregnant women suggests that vertical ...

  17. Immunochemical Investigations of Cell Surface Antigens of Anaerobic Bacteria.

    Science.gov (United States)

    1976-01-15

    has also been visulalized . With use of a radioactive anti’qen bindinq assay, antibody to this capsularpolysaccharide has been demonstrated in anti- sera...With many bacteria, serogrouning is based on cansular oolysaccharide antigens. Serogrouping has led to much valuable epidemiologic information

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

    Directory of Open Access Journals (Sweden)

    Michael Mühle

    Full Text Available 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

  19. Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory.

    Directory of Open Access Journals (Sweden)

    Marjorie S Morgan

    2017-06-01

    Full Text Available Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose.The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host's protective response.An aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies.We identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum.Thirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite's ability to modulate its host's innate and adaptive immune responses.

  20. Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory.

    Science.gov (United States)

    Morgan, Marjorie S; Rider, S Dean; Arlian, Larry G

    2017-06-01

    Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose. The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host's protective response. An aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies. We identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum. Thirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite's ability to modulate its host's innate and adaptive immune responses.

  1. Preservation of surface-dependent properties of viral antigens following immobilization on particulate ceramic delivery vehicles.

    Science.gov (United States)

    Kossovsky, N; Gelman, A; Sponsler, E; Rajguru, S; Torres, M; Mena, E; Ly, K; Festekjian, A

    1995-05-01

    B-cell stimulation for the purpose of evoking an effective neutralizing humoral immune response is a surface phenomenon that is exquisitely specific to antigen conformation. Consequently, successful delivery of antigen, such as would be desired in a vaccine, entails preservation of an antigen's apparent native surface (conformational) properties. Prior to testing the actual vaccinating efficacy of delivered antigens, the surface properties could be assessed through a variety of in vitro and in vivo assays in which the measurement standard would be the properties of the antigens in their native state (whole virus). Using surface modified nanocrystalline carbon and calcium-phosphate ceramic particulates (carbon ceramics and brushite), we evaluated the surface activity of immobilized non-nuclear material extracted from HIV-1. Physical characterization showed that the particles with immobilized antigen ("HIV decoys") measured 50 nm in diameter (HIV = 50-100 nm) and exhibited the same zeta potentials as whole (live) HIV. In vitro testing showed that the HIV decoys were recognized by both conformationally nonspecific and specific monoclonal antibodies, were recognized by human IgG from HIV antibody-positive patients, and could promote surface agglomeration among malignant T-cells similar to live HIV. Last, in vivo testing in three vaccinated animal species showed that the HIV decoys elicited humoral and cellular immune responses similar to that evoked by whole (live) HIV.

  2. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    Energy Technology Data Exchange (ETDEWEB)

    Wise K.S.; Kim, M.F.

    1987-12-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface /sup 125/I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with (/sup 35/S) methionine, /sup 14/C-amino acids, or (/sup 3/H) palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11.

  3. Major membrane surface proteins of Mycoplasma hyopneumoniae selectively modified by covalently bound lipid

    International Nuclear Information System (INIS)

    Wise, K.S.; Kim, M.F.

    1987-01-01

    Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface binding or by radioimmunoprecipitation of surface 125 I-labeled proteins with a series of monoclonal antibodies (MAbs). Radioimmunoprecipitation of TX-114-phase proteins from cells labeled with [ 35 S] methionine, 14 C-amino acids, or [ 3 H] palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein, p60) were selectively labeled with lipid. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids by amide or stable O-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural host as measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with whole organisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodies to individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspecies size variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J, ATCC 25934), which recognized a large p73 component on strain VPP11 (ATCC 25617). In addition, MAb to internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11

  4. A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

    DEFF Research Database (Denmark)

    Götz, C; Koenig, M G; Issinger, O G

    1995-01-01

    The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... of T antigen by the associated kinase is reduced whereas a p34cdc2-kinase-specific peptide has no influence. In addition, the T-antigen-associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated...

  5. Salmonella regulates polyubiquitination and surface expression of MHC class II antigens.

    Science.gov (United States)

    Lapaque, Nicolas; Hutchinson, James L; Jones, Des C; Méresse, Stéphane; Holden, David W; Trowsdale, John; Kelly, Adrian P

    2009-08-18

    Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, alphabeta dimers from the cell surface. Immature alphabetaIi complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.

  6. A novel merozoite surface antigen of Plasmodium falciparum (MSP-3 identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

    Directory of Open Access Journals (Sweden)

    Claude Oeuvray

    1994-01-01

    Full Text Available We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

  7. An immunoproteomic approach revealed antigenic proteins enhancing serodiagnosis performance of bird fancier's lung.

    Science.gov (United States)

    Rouzet, Adeline; Reboux, Gabriel; Dalphin, Jean-Charles; Gondouin, Anne; De Vuyst, Paul; Balliau, Thierry; Millon, Laurence; Valot, Benoit; Roussel, Sandrine

    2017-11-01

    Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances. Antigenic proteins of pigeon dropping extracts were investigated using 2-dimensional immunoblotting with sera from patients with BFL, asymptomatic exposed controls and healthy volunteers. We investigated the origin of these antigenic proteins by analyzing droppings, blooms and sera using a shotgun proteomic analysis. BFL-associated proteins were produced as recombinant antigens in E. coli and were assessed in ELISA with sera from patients (n=25) and subject exposed controls (n=30). These diagnostic performances were compared with those obtained by precipitin techniques (agar gel double diffusion, immunoelectrophoresis). We identified 14 antigenic proteins mainly located in droppings and blooms. These proteins were involved in either the digestive or immune systems of pigeons. Using the recombinant BFL-associated proteins: Immunoglobulin lambda-like polypeptide-1 (IGLL1: sensitivity: 76%; specificity: 100%; AUC: 0.93) and Proproteinase E (ProE: sensitivity: 84%; specificity: 80%; AUC: 0.85), the ELISA test showed better performance than precipitin assays with pigeon dropping extracts (sensitivity: 60%; specificity: 93.3%; AUC: 0.76). IGLL1 and ProE were identified as the biomarkers of the disease. The use of these highly standardized antigens discriminates BFL cases from exposed subjects in serological assays. The results of this study offer new possibilities for the serological diagnosis of the disease. ClinicalTrials.gov: Identifier NCT03056404. Copyright

  8. Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems

    DEFF Research Database (Denmark)

    Hamborg, Mette; Rose, Fabrice; Jorgensen, Lene

    2014-01-01

    is generally known about how antigens and adjuvants interact at the molecular level. The aim of this study was to elucidate the mechanisms of interactions between the equally sized, but oppositely charged model protein antigens α-lactalbumin and lysozyme, and i) the clinically tested cationic liposomal......The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little...... interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and neutralized...

  9. The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle.

    Science.gov (United States)

    Handman, E; Osborn, A H; Symons, F; van Driel, R; Cappai, R

    1995-11-01

    The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.

  10. Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen

    1998-01-01

    Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

  11. Molecular mechanisms of viral immune evasion proteins to inhibit MHC class I antigen processing and presentation.

    Science.gov (United States)

    Zhou, Fang

    2009-01-01

    Viral products inhibit MHC class I antigen processing and presentation via three major pathways: inhibition of major histocompatibility complex (MHC) class I expression on cells, blockade of peptide trafficking and loading on MHC class I molecules, and inhibition of peptide generation in host cells. Viral products also interfere with IFN-gamma -mediated JAK/STAT signal transduction in cells. These results imply that viral proteins probably inhibit the function of IFN-gamma in MHC class I antigen presentation via inactivation of JAK/STAT signal transduction in host cells. Mechanisms of viral products to inhibit IFN-gamma -mediated MHC class I antigen presentation were summarized in this literature review.

  12. Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly

    NARCIS (Netherlands)

    Hochstenbach, F.; DAVID, V.; WATKINS, S.; Brenner, M. B.

    1992-01-01

    In the endoplasmic reticulum (ER), newly synthesized subunits of the T-cell antigen receptor (TCR), membrane-bound immunoglobulin (mIg), and major histocompatibility complex (MHC) class I antigens must fold correctly and assemble completely into multimeric protein complexes prior to transport to the

  13. Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

    NARCIS (Netherlands)

    ter Borg, F.; ten Kate, F. J.; Cuypers, H. T.; Leentvaar-Kuijpers, A.; Oosting, J.; Wertheim-van Dillen, P. M.; Honkoop, P.; Rasch, M. C.; de Man, R. A.; van Hattum, J.; Chamuleau, R. A.; Reesink, H. W.; Jones, E. A.

    1998-01-01

    BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA

  14. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  15. The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive-determinants of clinical outcome of P. falciparum malaria. The evide...

  16. Surface plasmon resonance is an analytically sensitive method for antigen profiling of extracellular vesicles

    NARCIS (Netherlands)

    Gool, Elmar L.; Stojanovic, Ivan; Schasfoort, Richardus B.M.; Sturk, Auguste; Van Leeuwen, Ton G.; Nieuwland, Rienk; Terstappen, Leon W.M.M.; Coumans, Frank A.W.

    2017-01-01

    BACKGROUND: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface. METHODS: We investigated the potential of a 48- multiplex surface plasmon resonance imaging

  17. Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles

    NARCIS (Netherlands)

    Gool, Elmar L.; Stojanovic, Ivan; Schasfoort, Richard B. M.; Sturk, Auguste; van Leeuwen, Ton G.; Nieuwland, Rienk; Terstappen, Leon W. M. M.; Coumans, Frank A. W.

    2017-01-01

    Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface. We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV

  18. The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation.

    Science.gov (United States)

    Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J; Hu, Qingang; Hu, Hongming

    2017-12-01

    Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe 2 O 3 /APTS (3-aminopropyltrimethoxysilane) NPs and γFe 2 O 3 /DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe 2 O 3 /APTS NPs, but not negative charged γFe 2 O 3 /DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe 2 O 3 /APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe 2 O 3 /DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

  19. Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, L.M.; Forney, J.D.

    1984-08-01

    A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.

  20. Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia

    International Nuclear Information System (INIS)

    Epstein, L.M.; Forney, J.D.

    1984-01-01

    A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei

  1. The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation

    Science.gov (United States)

    Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J.; Hu, Qingang; Hu, Hongming

    2017-01-01

    Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

  2. Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin.

    Science.gov (United States)

    Shi, Yunnuo; Halperin, Scott A; Lee, Song F

    2018-02-01

    Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography and refolded by step-wise dialysis to give a yield of 11.8 mg/L of culture. The OVA-CD40LS was purified by a 'two-round' nickel affinity and on-column protein-refolding chromatography. The yield was 528 μg/L of culture. The purified OVA-CD40LS, but not the rOVA, was able to simulate the production of pro-inflammatory cytokines and up-regulate cell surface marker proteins in mouse bone marrow-derived dendritic cells. The purified OVA-CD40LS elicited a robust immune response when injected submucosally in the oral cavity of mice. Collectively, the results indicate that the OVA-CD40LS fusion protein was biologically active, functioning as an antigen-targeting protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The SSX Family of Cancer-Testis Antigens as Target Proteins for Tumor Therapy

    Directory of Open Access Journals (Sweden)

    Heath A. Smith

    2010-01-01

    Full Text Available Cancer-testis antigens (CTAs represent an expanding class of tumor-associated proteins defined on the basis of their tissue-restricted expression to testis or ovary germline cells and frequent ectopic expression in tumor tissue. The expression of CTA in MHC class I-deficient germline cells makes these proteins particularly attractive as immunotherapeutic targets because they serve as essentially tumor-specific antigens for MHC class I-restricted CD8+ T cells. Moreover, because CTAs are expressed in many types of cancer, any therapeutic developed to target these antigens might have efficacy for multiple cancer types. Of particular interest among CTAs is the synovial sarcoma X chromosome breakpoint (SSX family of proteins, which includes ten highly homologous family members. Expression of SSX proteins in tumor tissues has been associated with advanced stages of disease and worse patient prognosis. Additionally, both humoral and cell-mediated immune responses to SSX proteins have been demonstrated in patients with tumors of varying histological origin, which indicates that natural immune responses can be spontaneously generated to these antigens in cancer patients. The current review will describe the history and identification of this family of proteins, as well as what is known of their function, expression in normal and malignant tissues, and immunogenicity.

  4. Antigen sequence typing of outer membrane protein (fetA gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

    Directory of Open Access Journals (Sweden)

    S Dwivedi

    2014-01-01

    Full Text Available Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

  5. Protein modeling of apical membrane antigen-1(AMA-1) of ...

    African Journals Online (AJOL)

    SERVER

    2007-11-19

    Nov 19, 2007 ... available for the vast majority of the protein sequences. At this juncture, prediction methods have gained much. *Corresponding author. E-mail: drdmmamatha@gmail.com interest (Schwede et al. 2003). Genome sequencing projects continuously detect new protein sequences, this provides new information ...

  6. Programming the composition of polymer blend particles for controlled immunity towards individual protein antigens.

    Science.gov (United States)

    Zhan, Xi; Shen, Hong

    2015-05-28

    In order for a more precise control over the quality and quantity of immune responses stimulated by synthetic particle-based vaccines, it is critical to control the colloidal stability of particles and the release of protein antigens in both extracellular space and intracellular compartments. Different proteins exhibit different sizes, charges and solubilities. This study focused on modulating the release and colloidal stability of proteins with varied isoelectric points. A polymer particle delivery platform made from the blend of three polymers, poly(lactic-co-glycolic acid) (PLGA) and two random pH-sensitive copolymers, were developed. Our study demonstrated its programmability with respective to individual proteins. We showed the colloidal stability of particles at neutral environment and the release of each individual protein at different pH environments were dependent on the ratio of two charge polymers. Subsequently, two antigenic proteins, ovalbumin (OVA) and Type 2 Herpes Simplex Virus (HSV-2) glycoprotein D (gD) protein, were incorporated into particles with systematically varied compositions. We demonstrated that the level of in vitro CD8(+) T cell and in vivo immune responses were dependent on the ratio of two charged polymers, which correlated well with the release of proteins. This study provided a promising design framework of pH-responsive synthetic vaccines for protein antigens of interest. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Elvander, M.; Vilcek, S.; Baule, C.

    1998-01-01

    Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were...... of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88-100% among BRSV...

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

    Science.gov (United States)

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

    2014-11-01

    We have generated a polyclonal antibody against a novel immunomodulator, neem leaf glycoprotein (NLGP) that can react to a specific 47 kDa subunit of NLGP. Generated anti-NLGP antibody (primarily IgG2a) was tested for its anti-tumor activity in murine carcinoma (EC, CT-26), sarcoma (S180) and melanoma (B16Mel) tumor models. Surprisingly, tumor growth restriction was only observed in CT-26 carcinoma models, without any alteration in other tumor systems. Comparative examination of antigenicity between four different tumor models revealed high expression of CEA-like protein on the surface of CT-26 tumors. Subsequent examination of the cross-reactivity of anti-NLGP antibody with purified or cell bound CEA revealed prominent recognition of CEA by anti-NLGP antibody, as detected by ELISA, Western Blotting and immunohistochemistry. This recognition seems to be responsible for anti-tumor function of anti-NLGP antibody only on CEA-like protein expressing CT-26 tumor models, as confirmed by ADCC reaction in CEA(+) tumor systems where dependency to anti-NLGP antibody is equivalent to anti-CEA antibody. Obtained result with enormous therapeutic potential for CEA(+) tumors may be explained in view of the epitope spreading concept, however, further investigation is crucial. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Antibody response dynamics to the Plasmodium falciparum conserved vaccine candidate antigen, merozoite surface protein-1 C-terminal 19kD (MSP1-19kD, in Peruvians exposed to hypoendemic malaria transmission

    Directory of Open Access Journals (Sweden)

    Gamboa Dionicia

    2008-09-01

    Full Text Available Abstract Background In high-transmission areas, developing immunity to symptomatic Plasmodium falciparum infections requires 2–10 years of uninterrupted exposure. Delayed malaria-immunity has been attributed to difficult-to-develop and then short-lived antibody responses. Methods In a study area with P. falciparum infections/person/year, antibody responses to the MSP1-19kD antigen were evaluated and associations with P. falciparum infections in children and adults. In months surrounding and during the malaria seasons of 2003–2004, 1,772 participants received ≥6 active visits in one study-year. Community-wide surveys were conducted at the beginning and end of each malaria season, and weekly active visits were completed for randomly-selected individuals each month. There were 79 P. falciparum infections with serum samples collected during and approximately one month before and after infection. Anti-MSP1-19kD IgG levels were measured by ELISA. Results The infection prevalence during February-July was similar in children (0.02–0.12 infections/person/month and adults (0.03–0.14 infections/person/month and was negligible in the four-month dry season. In children and adults, the seroprevalence was maintained in the beginning (children = 28.9%, adults = 61.8% versus ending malaria-season community survey (children = 26.7%, adults = 64.6%. Despite the four-month non-transmission season, the IgG levels in Plasmodium-negative adults were similar to P. falciparum-positive adults. Although children frequently responded upon infection, the transition from a negative/low level before infection to a high level during/after infection was slower in children. Adults and children IgG-positive before infection had reduced symptoms and parasite density. Conclusion Individuals in low transmission areas can rapidly develop and maintain αMSP1-19kD IgG responses for >4 months, unlike responses reported in high transmission study areas. A greater immune

  10. 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.

    Science.gov (United States)

    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.

  11. Determinants of spontaneous surface antigen loss in hepatitis B e antigen-negative patients with a low viral load.

    Science.gov (United States)

    Tseng, Tai-Chung; Liu, Chun-Jen; Yang, Hung-Chih; Su, Tung-Hung; Wang, Chia-Chi; Chen, Chi-Ling; Kuo, Stephanie Fang-Tzu; Liu, Chen-Hua; Chen, Pei-Jer; Chen, Ding-Shinn; Kao, Jia-Horng

    2012-01-01

    Loss of hepatitis B surface antigen (HBsAg) usually indicates the cure of hepatitis B virus (HBV) infection. In spontaneous hepatitis B e antigen (HBeAg) seroconverters, lower serum HBsAg and HBV DNA levels have been shown to be associated with HBsAg loss over time. However, little is known about their impacts on HBsAg loss in HBeAg-negative patients with limited viral replication. A total of 688 HBeAg-negative patients with baseline serum HBV DNA levels loss were investigated. In a mean follow-up of 11.6 years, the average annual rate of HBsAg loss was 1.6%. Baseline HBsAg and HBV DNA levels were inversely associated with subsequent HBsAg loss. When compared to patients who had HBsAg levels >1000 IU/mL, the rates of HBsAg loss were significantly higher in patients with HBsAg levels of 100-999, 10-99, and loss was 13.2 (95% CI, 7.8-22.1) for HBsAg level loss. In HBeAg-negative patients with HBV genotype B or C infection who have HBV DNA level loss. Copyright © 2011 American Association for the Study of Liver Diseases.

  12. Rational Design of Adjuvant for Skin Delivery: Conjugation of Synthetic β-Glucan Dectin-1 Agonist to Protein Antigen.

    Science.gov (United States)

    Donadei, Agnese; Gallorini, Simona; Berti, Francesco; O'Hagan, Derek T; Adamo, Roberto; Baudner, Barbara C

    2015-05-04

    The potential benefits of skin delivery of vaccines derive from the presence of a densely connected network of antigen presenting cells in the skin layer, most significantly represented by Langerhans cells and dermal dendritic cells. Targeting these cells by adjuvant conjugated to an antigen should result in enhanced immunogenicity of a vaccine. Since one of the most widely used adjuvants is an insoluble salt of aluminum (aluminum hydroxide) that cannot be used for skin delivery due to reactogenicity, we focused our attention on agonists of receptors present on skin dendritic cells, including the Dectin-1 receptor. β-(1-3)-glucans, which are the most abundant components of the fungal surface, are known to activate the innate immune response by interaction with the C-type lectin-like Dectin-1 receptor. In this work we identified by rational design a well-defined synthetic β-(1-3)-glucan hexasaccharide as a Dectin-1 agonist and chemically conjugated it to the genetically detoxified diphtheria toxin (CRM197) protein antigen, as a means to increase the binding to Dectin-1 receptor and to target to skin dendritic cells. We demonstrated that the in vitro activation of the receptor was significantly impacted by the presentation of the glucan on the protein carrier. In vivo results in mice showed that the conjugation of the synthetic β-(1-3)-glucan when delivered intradermally resulted in higher antibody titers in comparison to intramuscular (i.m.) immunization and was not different from subcutaneous (s.c.) delivery. These findings suggest that weak receptor binders can be turned into more potent agonists by the multivalent presentation of many ligands covalently conjugated to the protein core. Moreover, this approach is particularly valuable to increase the immunogenicity of antigens administered via skin delivery.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary

    2014-01-01

    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Pla...

  15. The ultrastructural localization of keratin proteins and carcinoembryonic antigen in malignant mesotheliomas.

    Science.gov (United States)

    Warhol, M J

    1984-09-01

    The immunoultrastructural localization of keratin proteins and carcinoembryonic antigen (CEA) in mesothelioma cells was accomplished with a low-temperature embedding colloidal gold technique. The keratin antiserum labeled only intermediate filaments. These filaments surrounded the cytoplasmic organelles and were inserted into desmosomes. The CEA antiserum labeled cytoplasmic vesicles and droplets. No definite labeling of microvilli was observed.

  16. Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

    Directory of Open Access Journals (Sweden)

    Hemavathy Harikrishnan

    Full Text Available Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

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

    NARCIS (Netherlands)

    Wilson, R. (Robert); J. Cohen (Jonathan); Reglinski, M. (Mark); R.J. Jose; Chan, W.Y. (Win Yan); Marshall, H. (Helina); C.P. de Vogel (Corné); S.B. Gordon (Stephen); Goldblatt, D. (David); Petersen, F.C. (Fernanda C.); H. Baxendale (Helen); Brown, J.S. (Jeremy S.)

    2017-01-01

    textabstractNaturally 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

  18. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination

    OpenAIRE

    Bañuls Anne-Laure; Devault Alain

    2008-01-01

    Abstract Background PSA (promastigote surface antigen) is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results From the newly available complete genome sequenc...

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

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

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

  20. Hepatitis B-Surface Antigen In Ascitic Fluid Of Patients With Chronic ...

    African Journals Online (AJOL)

    A prospective evaluation of eleven consecutive cases of chronic liver disease over a twelve-month period was carried out clinically and ultrasonographically. By the use of the method of reverse passive haemagglutination, sera and ascitic fluid of the patients were tested for the presence of the Hepatitis B surface antigen.

  1. Prevalence of Hepatitis B Surface Antigen (HBsAg) Amongst Alcohol ...

    African Journals Online (AJOL)

    The prevalence of Hepatitis B surface Antigen was studied amongst alcohol consumers and non– consumers (Control subjects) in Bassa Local Government Area (LGA) of Plateau State. Three hundred and five (305) subjects comprising 255(83.61%) alcoholics and 50(16.39%) non-alcoholic control subjects were screened ...

  2. Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

    NARCIS (Netherlands)

    Schuijt, T.J.; Narasimhan, S.; Daffre, S.; Deponte, K.; Hovius, J.W.R.; van 't Veer, C.; van der Poll, T.; Bakhtiari, K.; Meijers, J.C.M.; Boder, E.T.; van Dam, A.P.; Fikrig, E.

    2011-01-01

    Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary

  3. Surface antigen-negative hepatitis B virus infection in Dutch blood donors

    NARCIS (Netherlands)

    Lieshout-Krikke, R. W.; Molenaar-de Backer, M. W. A.; van Swieten, P.; Zaaijer, H. L.

    2014-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of

  4. Prevalence of Hepatitis-B Surface Antigen among Blood Donors in ...

    African Journals Online (AJOL)

    Information is scarce on the prevalence of Hepatitis-B Virus (HBV) infection among blood donors in Taraba State. Hepatitis-B surface antigen (HBsAg) ELISA [Gudans Industrial Hong 2 Kou, China] was used to determine the prevalence of HBsAg among 804 blood donors aged between 11 and 65 years in Federal Medical ...

  5. Multiple surface antigen mutations in five blood donors with occult hepatitis B virus infection

    NARCIS (Netherlands)

    Zaaijer, H. L.; Torres, P.; Ontañón, A.; Ponte, L. González; Koppelman, M. H. G. M.; Lelie, P. N.; Hemert, F. J. van; Boot, H. J.

    2008-01-01

    Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA while the HBV surface antigen (HBsAg) remains undetectable. The HBV genomes in five asymptomatic blood donors with occult HBV infection and low viremia ( <10 to 1,000 HBV DNA copies/mL, genotype D) were studied. An

  6. Prevalence of Hepatitis-B Surface Antigen (HbsAg), Hepatitis C ...

    African Journals Online (AJOL)

    The prevalence of Hepatitis-B surface antigen (HBsAg), Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) was determined among apparently healthy male blood donors in Aminu Kano Teaching Hospital, Kano, between January and December, 2002. A total of 2,288 blood samples from the blood donors ...

  7. A sensitive immunoradiometric assay for the detection of hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Cameron, C.H.; Combridge, B.S.; Howell, D.R.; Barbara, J.A.J.

    1980-01-01

    A solid-phase immunoradiometric assay for hepatitis B surface antigen is described which has been in use since 1972. Initially it was used for reference laboratory work, but from 1974 it has also been used for screening blood and blood products. Methods for the production of reagents and their use in blood transfusion and reference work, are outlined. (Auth.)

  8. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

    DEFF Research Database (Denmark)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-01-01

    microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 oC without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies...

  9. Sero-prevalence of Hepatitis B Surface Antigen (HBsAg), in Sexually ...

    African Journals Online (AJOL)

    Sero-prevalence of Hepatitis B Surface Antigen (HBsAg), in Sexually Transmitted Disease Patients. ... partners in the past 6 months, a history of a number of episodes of STDs, history of heterosexual exposure to partners at risk, for example prostitutes; a history of symptoms of an STD at the commencement of the study.

  10. Characterizing the statistical properties of protein surfaces

    Science.gov (United States)

    Bak, Ji Hyun; Bitbol, Anne-Florence; Bialek, William

    Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of the shapes of their surfaces. We take into account the high-resolution structures of E.coli non-DNA-binding cytoplasmic proteins, retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the protein surfaces at two levels: First, we study the intrinsic dimensionality at the level of the ensemble of the surface objects. Second, at the level of the individual surfaces, we determine the scale of shape variation. We further discuss how the dimensionality of the shape space is linked to the statistical properties of individual protein surfaces. Jhb and WB acknowledge support from National Science Foundation Grants PHY-1305525 and PHY-1521553. AFB acknowledges support from the Human Frontier Science Program.

  11. Increased parasite surface antigen-2 expression in clinical isolates of Leishmania donovani augments antimony resistance.

    Science.gov (United States)

    Bhandari, Vasundhra; Kumar, Dhiraj; Verma, Sandeep; Srividya, Gurumurthy; Negi, Narendra Singh; Singh, Ruchi; Salotra, Poonam

    2013-11-01

    Resistance to sodium antimony gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of antimony resistant (n=10) and sensitive (n=4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (≥1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2(++)) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2(++) parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2(++) parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Patarroyo Manuel E

    2011-10-01

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

  13. A Survey about Protective Effect of Echinococcus Granulosus Protoscolices Surface Antigens in Preventing Secondary Hydatid Cyst

    Directory of Open Access Journals (Sweden)

    H Yousofi

    2006-10-01

    Full Text Available ABSTRACT: Introduction & Objective: Hydatid cyst is located in human and some animal visceral organs such as liver and lung. The disease is considered as a medical, veterinary and economical problem in endemic area. When the hydatid cyst is ruptured, protoscolices from inside the cyst may spread out to other parts of the body and develops a new cyst named secondary hydatid cyst. In this research in an attempt to prevent secondary hydatid cyst, protective potential of protoscolices surface antigens extracted with different detergents has been investigated in animal model. Materials & Methods: In this experimental study, groups of Balb/c mice were immunized intra-peritoneally with protoscolices homogenate and three detergent (SDS, Tween and Triton x–100 extracted protoscolices surface antigens and alum as adjuvant. These mice were then boosted two times with the same antigens fortnightly. Control mice were simultaneously injected with alum alone. Two weeks following the last injection all the mice in cases and control groups were challenged with live protoscolices. Three months afterward all the mice in case and control groups were sacrificed and their peritoneal cavities were explored for hydatid cysts. Results: The mean of developed cyst number in mice injected with protoscolices homogenate was 3±2, while in control group the mean of developed cysts number was 5.8 ± 1.7 (p< 0.02. The mean of developed cyst number in mice injected with SDS, Tween and Triton x–100 extracted protoscolices surface antigens was 3, 3.6 and 3.4, respectively, while the mean of developed cyst number in control group was 5.8. Conclusion: The mean of cyst number in cases and control groups was different and this difference was statistically significant. Results of this investigation revealed that protoscolices homogenate antigens and some detergent extracted antigens are protective against secondary hydatid cyst infection

  14. Genomic Characterization of Variable Surface Antigens Reveals a Telomere Position Effect as a Prerequisite for RNA Interference-Mediated Silencing in Paramecium tetraurelia

    Science.gov (United States)

    Baranasic, Damir; Oppermann, Timo; Cheaib, Miriam; Cullum, John; Schmidt, Helmut

    2014-01-01

    ABSTRACT Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes. PMID:25389173

  15. Antibody response to the lipopolysaccharide and protein antigens of Salmonella typhi during typhoid infection

    International Nuclear Information System (INIS)

    Tsang, R.S.W.; Chau, P.Y.; Lam, S.K.

    1981-01-01

    Serum antibody responses to the lipopolysaccharide and protein antigens of S. typhi in typhoid patients were studied using a solid-phase radioimmunoassay technique with 125 I labelled anti-immunoglobulin antibody. Sera from 24 adult typhoid patients and 20 non-typhoid adult controls were compared. As a group, sera from typhoid patients showed increased IgA, IgG and IgM immunoglobulin levels and gave significantly higher anti-LPS and anti-protein antibody titres in all three major immunoglobulin classes than did non-typhoid controls. Levels of antibodies against LPS or protein in sera of typhoid patients were highly variable with a skew distribution. A good correlation was found between antibody titres to the LPS antigen and those to a protein antigen. No correlation, however, was found between the anti-LPS antibody titres measured by radioimmunoassay and the anti-O antibody titres measured by the Widal agglutination test. Titration of anti-LPS or anti-protein antibodies by radioimmunoassay was found to be more sensitive and specific than Widal test for the serological diagnosis of typhoid fever. The advantages of measuring antibody response by radioimmunoassay over conventional Widal test are discussed. (author)

  16. Interactions between whey proteins and kaolinite surfaces

    International Nuclear Information System (INIS)

    Barral, S.; Villa-Garcia, M.A.; Rendueles, M.; Diaz, M.

    2008-01-01

    The nature of the interactions between whey proteins and kaolinite surfaces was investigated by adsorption-desorption experiments at room temperature, performed at the isoelectric point (IEP) of the proteins and at pH 7. It was found that kaolinite is a strong adsorbent for proteins, reaching the maximum adsorption capacity at the IEP of each protein. At pH 7.0, the retention capacity decreased considerably. The adsorption isotherms showed typical Langmuir characteristics. X-ray diffraction data for the protein-kaolinite complexes showed that protein molecules were not intercalated in the mineral structure, but immobilized at the external surfaces and the edges of the kaolinite. Fourier transform IR results indicate the absence of hydrogen bonding between kaolinite surfaces and the polypeptide chain. The adsorption patterns appear to be related to electrostatic interactions, although steric effects should be also considered

  17. Characterization of Monoclonal Antibody–Protein Antigen Complexes Using Small-Angle Scattering and Molecular Modeling

    Directory of Open Access Journals (Sweden)

    Maria Monica Castellanos

    2017-12-01

    Full Text Available The determination of monoclonal antibody interactions with protein antigens in solution can lead to important insights guiding physical characterization and molecular engineering of therapeutic targets. We used small-angle scattering (SAS combined with size-exclusion multi-angle light scattering high-performance liquid chromatography to obtain monodisperse samples with defined stoichiometry to study an anti-streptavidin monoclonal antibody interacting with tetrameric streptavidin. Ensembles of structures with both monodentate and bidentate antibody–antigen complexes were generated using molecular docking protocols and molecular simulations. By comparing theoretical SAS profiles to the experimental data it was determined that the primary component(s were compact monodentate and/or bidentate complexes. SAS profiles of extended monodentate complexes were not consistent with the experimental data. These results highlight the capability for determining the shape of monoclonal antibody–antigen complexes in solution using SAS data and physics-based molecular modeling.

  18. Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

    Science.gov (United States)

    Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

    1996-01-01

    Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

  19. Variants of a Leishmania surface antigen derived from a multigenic family.

    Science.gov (United States)

    Murray, P J; Spithill, T W

    1991-12-25

    The promastigote surface antigen-2 (PSA-2) complex comprises a group of immunogenic surface antigens linked to the surface of the Leishmania major promastigote with glycosylphosphatidylinositol anchors. The L. major genome contains at least 14 PSA-2 genes on a 950-kilobase chromosome and comprising approximately 20% of the length of this chromosome. The sequence of three independent, but incomplete, PSA-2 cDNAs and one genomic fragment encoding a complete PSA-2 coding sequence were compared. PSA-2 genes encode polypeptides exhibiting 22-25aa tandem repeat elements, threonine-rich segments which vary between genes, a conserved COOH-terminal cysteine-rich region, and a conserved GPI anchor signal sequence. PSA-2 genes appear to be transcribed in a complex manner with multiple RNAs. The complex genomic organization of PSA-2 genes is present in other members of the genus suggesting that PSA-2 function is important for the biology of Leishmania.

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

    Science.gov (United States)

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

    2017-07-01

    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

  1. Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

    Directory of Open Access Journals (Sweden)

    Danijela Koppers-Lalic

    2008-05-01

    Full Text Available Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1, pseudorabies virus (PRV, and equine herpesvirus 1 and 4 (EHV-1 and EHV-4 are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

  2. Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens

    International Nuclear Information System (INIS)

    Stempfer, René; Weinhäusel, Andreas; Syed, Parvez; Vierlinger, Klemens; Pichler, Rudolf; Meese, Eckart; Leidinger, Petra; Ludwig, Nicole; Kriegner, Albert; Nöhammer, Christa

    2010-01-01

    The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarrays. Protein arrays were hybridized with the serum samples from brain and lung tumour patients. Methods for the generation of microarrays were successfully established when using antigens derived from membrane-based selection. Signal patterns obtained by microarrays analysis of brain and lung tumour patients' sera were highly reproducible (R = 0.92-0.96). This provides the technical foundation for diagnostic applications on the basis of auto-antibody patterns. In this limited test set, the assay provided high reproducibility and a broad dynamic range to classify all brain and lung samples correctly. Protein microarray is an efficient means for auto-antibody-based detection when using SEREX-derived clones expressing antigenic proteins. Protein microarrays are preferred to macroarrays due to the easier handling and the high reproducibility of auto-antibody testing. Especially when using only a few microliters of patient samples protein microarrays are ideally suited for validation of auto

  3. Determinants of antigenicity and specificity in immune response for protein sequences

    Directory of Open Access Journals (Sweden)

    Li Cheng

    2011-06-01

    Full Text Available Abstract Background Target specific antibodies are pivotal for the design of vaccines, immunodiagnostic tests, studies on proteomics for cancer biomarker discovery, identification of protein-DNA and other interactions, and small and large biochemical assays. Therefore, it is important to understand the properties of protein sequences that are important for antigenicity and to identify small peptide epitopes and large regions in the linear sequence of the proteins whose utilization result in specific antibodies. Results Our analysis using protein properties suggested that sequence composition combined with evolutionary information and predicted secondary structure, as well as solvent accessibility is sufficient to predict successful peptide epitopes. The antigenicity and the specificity in immune response were also found to depend on the epitope length. We trained the B-Cell Epitope Oracle (BEOracle, a support vector machine (SVM classifier, for the identification of continuous B-Cell epitopes with these protein properties as learning features. The BEOracle achieved an F1-measure of 81.37% on a large validation set. The BEOracle classifier outperformed the classical methods based on propensity and sophisticated methods like BCPred and Bepipred for B-Cell epitope prediction. The BEOracle classifier also identified peptides for the ChIP-grade antibodies from the modENCODE/ENCODE projects with 96.88% accuracy. High BEOracle score for peptides showed some correlation with the antibody intensity on Immunofluorescence studies done on fly embryos. Finally, a second SVM classifier, the B-Cell Region Oracle (BROracle was trained with the BEOracle scores as features to predict the performance of antibodies generated with large protein regions with high accuracy. The BROracle classifier achieved accuracies of 75.26-63.88% on a validation set with immunofluorescence, immunohistochemistry, protein arrays and western blot results from Protein Atlas database

  4. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  5. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.

    1989-01-01

    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  6. Surface display of proteins by Gram-negative bacterial autotransporters

    Directory of Open Access Journals (Sweden)

    Mourez Michael

    2006-06-01

    Full Text Available Abstract Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

  7. Neospora caninum surface antigen (p40) is a potential diagnostic marker for cattle neosporosis.

    Science.gov (United States)

    He, Pengfei; Li, Jianhua; Gong, Pengtao; Liu, Chengwu; Zhang, Guocai; Yang, Ju; Tuo, Wenbin; Yang, Bintong; Zhang, Xichen

    2013-05-01

    Neospora caninum is an intracellular protozoan that infects domestic and wild canids as well as many warm-blooded animals as shown by the isolation of viable parasites. The effectiveness of diagnostic tests for detecting specific antibodies against N. caninum is hampered by potential cross-reaction with other Coccidia. So, there is currently an urgent need for a sensitive and specific diagnostic assay for detecting N. caninum in animals. The N. caninum 40-kD surface antigen (p40), similar to NcSAG1 and NcSRS2, was shown to belong to surface antigen super family and thus represents an excellent marker for the diagnosis of neosporosis. In order to test the hypothesis, recombinant Ncp40 (rNcp40) was expressed in Escherichia coli, and an indirect ELISA test was developed using recombinant NCp40 antigen for N. caninum serodiagnosis. The antigen used in this study did not have cross-reactivity with anti-Toxoplasma gondii serum. Anti-p40 antibodies were detected by ELISA in the sera of Yellow cattle and were compared with (IFAT). Optimal sensitivity and specificity (98.2 and 98.6 %) were identified by IFAT. Additionally, 37 positive sera of T. gondii were detected and there was no significant difference with the negative serum of N. caninum. The rNcp40 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.

  8. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    and that the outcome of IgG adsorption is much more sensitive to surface characteristics than the outcome of albumin adsorption. Using high concentrations of protein solution and hydrophobic polymer surfaces during adsorption can induce IgG aggregation, which is observed as extremely high IgG adsorptions. Besides......In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...

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

    Directory of Open Access Journals (Sweden)

    Langella P.

    1999-01-01

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

  10. [Expression, purification and protective antigen analysis of cell wall protein MRP of Streptococcus suis type 2].

    Science.gov (United States)

    Wang, Ping-ping; Pian, Ya-ya; Yuan, Yuan; Zheng, Yu-ling; Jiang, Yong-qiang; Xiong, Zheng-ying

    2012-02-01

    To amplify the mrp gene of Streptococcus suis type 2 05ZYH33, express it in E.coli BL21 in order to acquire high purity recombinant protein MRP, then evaluate the protective antigen of recombinant protein MRP. Using PCR technology to obtain the product of mrp gene of 05ZYH33, and then cloned it into the expression vector pET28a(+). The recombinant protein was purified by affinity chromatography, later immunized New Zealand rabbit to gain anti-serum, then test the anti-serum titer by ELISA. The opsonophagocytic killing test demonstrated the abilities of protective antigen of MRP. The truncated of MRP recombinant protein in E.coli BL21 expressed by inclusion bodies, and purified it in high purity. After immunoprotection, the survival condition of CD-1 was significantly elevated. The survival rate of wild-type strain 05ZYH33 in blood was apparently decreased after anti-serum opsonophagocyticed, but the mutant delta; MRP showed no differences. MRP represent an important protective antigen activity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, C.Y.G. (Univ. of British Columbia, Vancouver, Canada); 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.

  12. Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Wootton, S K; Nelson, E A; Yoo, D

    1998-11-01

    A collection of 12 monoclonal antibodies (MAbs) raised against porcine reproductive and respiratory syndrome (PRRS) virus was used to study the antigenic structure of the virus nucleocapsid protein (N). The full-length N gene, encoded by open reading frame 7, was cloned from the Canadian PRRS virus, PA-8. Deletions were introduced into the N gene to produce a series of nine overlapping protein fragments ranging in length from 25 to 112 amino acids. The individual truncated genes were cloned as glutathione S-transferase fusions into a eukaryotic expression vector downstream of the T7 RNA polymerase promoter. HeLa cells infected with recombinant vaccinia virus expressing T7 RNA polymerase were transfected with plasmid DNA encoding the N protein fragments, and the antigenicity of the synthesized proteins was analyzed by immunoprecipitation. Based on the immunoreactivities of the N protein deletion mutants with the panel of N-specific MAbs, five domains of antigenic importance were identified. MAbs SDOW17, SR30, and 5H2.3B12.1C9 each identified independent domains defined by amino acids 30 to 52, 69 to 123, and 37 to 52, respectively. Seven of the MAbs tested specifically recognized the local protein conformation formed in part by the amino acid residues 52 to 69. Furthermore, deletion of 11 amino acids from the carboxy terminus of the nucleocapsid protein disrupted the epitope configuration recognized by all of the conformation-dependent MAbs, suggesting that the carboxy-terminal region plays an important role in maintaining local protein conformation.

  13. Evaluation of a Major Surface Antigen of Babesia microti Merozoites as a Vaccine Candidate against Babesia Infection

    Directory of Open Access Journals (Sweden)

    Suqin Man

    2017-12-01

    Full Text Available Babesia species are tick-borne intraerythrocytic protozoa that cause babesiosis in humans worldwide. No vaccine has yet proven effective against Babesia infection. Surface antigens of merozoites are involved in the invasion of erythrocytes by Babesia. Surface antigens may be presented by both babesial sporozoites and merozoites and provide a general target for antibody-mediated inhibition of erythrocyte invasion. Here we evaluated a major surface antigen of B. microti merozoites, BMSA, as a potential vaccine to prevent babesiosis. Our data indicated that bmsa is transcribed during different phases, including ring form, amoeboid form, and merozoites, and that its expression is significantly increased in mature merozoites. The protein was found to be located in the membrane of B. microti and in the cytoplasm of infected erythrocytes. The immune response induced by BMSA had a significant inhibitory effect on parasite invasion of the host erythrocytes (83.3% inhibition of invasion and parasite growth in vivo. The levels of parasitemia significantly decreased after BMSA vaccination when mice were infected with babesia parasite. Importantly, protective immunity was significantly related to the upregulation of the Th17 cytokine interleukin-17, the Th1 cytokine interleukin-12p70 and the Th2 cytokines, such as interleukin-4, -6, and -10. Ingenuity Pathway Analysis indicated that interleukin-17 facilitated the secretion of Th2 cytokines, such as interleukin-10, -4, and -6, thereby inducing a predominately Th2 protective immune response and promoting the expression a high level of special IgG1 against Babesia infection. Further, an anti-BMSA monoclonal antibody successfully protected NOD/SCID mice from a challenge with B. microti. Taken together, our results indicated that BMSA induces a protective immune response against Babesia infection and may serve as a potential vaccine.

  14. A radioimmunoassay for antibodies against surface membrane antigens using adhering cells

    International Nuclear Information System (INIS)

    Tax, A.; Manson, L.A.

    1976-01-01

    A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125 I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface

  15. Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery.

    Directory of Open Access Journals (Sweden)

    Claude Nogues

    Full Text Available BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65 and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen

  16. Protein and Antigenic Profile among Mycoplasma bovis Field Strains Isolated in Bosnia and Herzegovina

    Directory of Open Access Journals (Sweden)

    Maid Rifatbegović

    2009-01-01

    Full Text Available Mycoplasma bovis is a serious, worldwide-spread but often overlooked pathogen causing respiratory disease, mastitis, and arthritis in cattle. In this study we characterize the protein and antigenic profiles of M. bovis field strains isolated in Bosnia and Herzegovina by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, and analyze possible variations among these strains. Greater differences occurred when comparing field strains with the reference strain PG45. One field strain isolated from lung samples of a heifer was markedly different from strains isolated from nasal swabs taken from cattle raised in another geographic region. A possible correlation may exist between protein and antigen profiles of M. bovis field strains, geographic regions and anatomical sites of isolation.

  17. The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

    DEFF Research Database (Denmark)

    Hviid, Lars

    2010-01-01

    that development of PfEMP1-based vaccines to protect specifically against severe malaria syndromes-in particular PAM-is feasible. This review summarizes the evidence that VSAs are important targets of NAI, discusses why VSA-based vaccines might be feasible despite the extensive intra- and interclonal variation...... of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria.......There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs), such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive determinants of clinical outcome of P. falciparum malaria...

  18. High hepatitis B surface antigen levels predict insignificant fibrosis in hepatitis B e antigen positive chronic hepatitis B.

    Directory of Open Access Journals (Sweden)

    Wai-Kay Seto

    Full Text Available INTRODUCTION: There is no data on the relationship between hepatitis B surface antigen (HBsAg levels and liver fibrosis in hepatitis B e antigen (HBeAg-positive patients with chronic hepatitis B (CHB. METHODS: Serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients with liver biopsies were analyzed. The upper limit of normal (ULN of alanine aminotransferase (ALT was 30 and 19 U/L for men and women respectively. Histologic assessment was based on Ishak fibrosis staging for fibrosis and Knodell histologic activity index (HAI for necroinflammation. RESULTS: 140 patients (65% male, median age 32.7 years were recruited. 56 (40% had ALT ≤2×ULN. 72 (51.4% and 42 (30% had fibrosis score ≤ 1 and necroinflammation grading ≤ 4 respectively. Patients with fibrosis score ≤ 1, when compared to patients with fibrosis score >1, had significantly higher median HBsAg levels (50,320 and 7,820 IU/mL respectively, p<0.001. Among patients with ALT ≤2×ULN, serum HBsAg levels achieved an area under receiver operating characteristic curve of 0.869 in predicting fibrosis score ≤ 1. HBsAg levels did not accurately predict necroinflammation score. HBsAg ≥ 25,000 IU/mL was independently associated with fibrosis score ≤ 1 (p=0.025, odds ratio 9.042.Using this cut-off HBsAg level in patients with ALT ≤2×ULN, positive and negative predictive values for predicting fibrosis score ≤ 1 were 92.7% and 60.0% respectively. HBV DNA levels had no association with liver histology. CONCLUSION: Among HBeAg-positive patients with ALT ≤2×ULN, high serum HBsAg levels can accurately predict fibrosis score ≤ 1, and could potentially influence decisions concerning treatment commencement and reduce the need for liver biopsy.

  19. KIR content genotypes associate with carriage of hepatitis B surface antigen, e antigen and HBV viral load in Gambians.

    Directory of Open Access Journals (Sweden)

    Louis-Marie Yindom

    Full Text Available Hepatocellular carcinoma (HCC causes over 800,000 deaths worldwide annually, mainly in low income countries, and incidence is rising rapidly in the developed world with the spread of hepatitis B (HBV and C (HCV viruses. Natural Killer (NK cells protect against viral infections and tumours by killing abnormal cells recognised by Killer-cell Immunoglobulin-like Receptors (KIR. Thus genes and haplotypes encoding these receptors may be important in determining both outcome of initial hepatitis infection and subsequent chronic liver disease and tumour formation. HBV is highly prevalent in The Gambia and the commonest cause of liver disease. The Gambia Liver Cancer Study was a matched case-control study conducted between September 1997 and January 2001 where cases with liver disease were identified in three tertiary referral hospitals and matched with out-patient controls with no clinical evidence of liver disease.We typed 15 KIR genes using the polymerase chain reaction with sequence specific primers (PCR-SSP in 279 adult Gambians, 136 with liver disease (HCC or Cirrhosis and 143 matched controls. We investigated effects of KIR genotypes and haplotypes on HBV infection and associations with cirrhosis and HCC.Homozygosity for KIR group A gene-content haplotype was associated with HBsAg carriage (OR 3.7, 95% CI 1.4-10.0 whilst telomeric A genotype (t-AA was associated with reduced risk of e antigenaemia (OR 0.2, 95% CI 0.0-0.6 and lower viral loads (mean log viral load 5.2 vs. 6.9, pc = 0.022. One novel telomeric B genotype (t-ABx2 containing KIR3DS1 (which is rare in West Africa was also linked to e antigenaemia (OR 8.8, 95% CI 1.3-60.5. There were no associations with cirrhosis or HCC.Certain KIR profiles may promote clearance of hepatitis B surface antigen whilst others predispose to e antigen carriage and high viral load. Larger studies are necessary to quantify the effects of individual KIR genes, haplotypes and KIR/HLA combinations on long

  20. KIR content genotypes associate with carriage of hepatitis B surface antigen, e antigen and HBV viral load in Gambians.

    Science.gov (United States)

    Yindom, Louis-Marie; Mendy, Maimuna; Bodimeade, Christopher; Chambion, Caroline; Aka, Peter; Whittle, Hilton C; Rowland-Jones, Sarah L; Walton, Robert

    2017-01-01

    Hepatocellular carcinoma (HCC) causes over 800,000 deaths worldwide annually, mainly in low income countries, and incidence is rising rapidly in the developed world with the spread of hepatitis B (HBV) and C (HCV) viruses. Natural Killer (NK) cells protect against viral infections and tumours by killing abnormal cells recognised by Killer-cell Immunoglobulin-like Receptors (KIR). Thus genes and haplotypes encoding these receptors may be important in determining both outcome of initial hepatitis infection and subsequent chronic liver disease and tumour formation. HBV is highly prevalent in The Gambia and the commonest cause of liver disease. The Gambia Liver Cancer Study was a matched case-control study conducted between September 1997 and January 2001 where cases with liver disease were identified in three tertiary referral hospitals and matched with out-patient controls with no clinical evidence of liver disease. We typed 15 KIR genes using the polymerase chain reaction with sequence specific primers (PCR-SSP) in 279 adult Gambians, 136 with liver disease (HCC or Cirrhosis) and 143 matched controls. We investigated effects of KIR genotypes and haplotypes on HBV infection and associations with cirrhosis and HCC. Homozygosity for KIR group A gene-content haplotype was associated with HBsAg carriage (OR 3.7, 95% CI 1.4-10.0) whilst telomeric A genotype (t-AA) was associated with reduced risk of e antigenaemia (OR 0.2, 95% CI 0.0-0.6) and lower viral loads (mean log viral load 5.2 vs. 6.9, pc = 0.022). One novel telomeric B genotype (t-ABx2) containing KIR3DS1 (which is rare in West Africa) was also linked to e antigenaemia (OR 8.8, 95% CI 1.3-60.5). There were no associations with cirrhosis or HCC. Certain KIR profiles may promote clearance of hepatitis B surface antigen whilst others predispose to e antigen carriage and high viral load. Larger studies are necessary to quantify the effects of individual KIR genes, haplotypes and KIR/HLA combinations on long

  1. An overview of serum prostatic surface antigen cut points for recommendation of prostatic biopsy

    OpenAIRE

    Patwardhan, Sujata K.; Patil, Bhushan P.; Shelke, Umesh Ravikant; Singh, Abhishek G.

    2018-01-01

    Introduction: Patients in India frequently present with prostatic surface antigen (PSA) report and request for prostatic biopsy to rule out malignancy. With fear of harboring malignancy set in patient's mind, it becomes difficult to counsel them about absolute indications and need of biopsy. Whether serum PSA has same predictability in symptomatic patients in the Indian context for advising prostatic biopsy at same reference ranges as in western countries, remains to be answered. Materials an...

  2. Characterization of a Monoclonal Antibody Specific for the Parasite Surface Antigen-2 of Leishmania major

    OpenAIRE

    "AR Khabiri; F Bagheri; SR Naddaf; M Assmar; A Hosseini Taghavi"

    2004-01-01

    The Leishmania major Parasite surface Antigen-2 (PSA-2) is a family of glycoinositol phospholipids anchored glycoprotoins expressed in both promastigotes and amastigotes. Promastigote PSA-2 comprises three polypeptides with approximate molecular weight of 96, 80 and 50 kDa. Amastigote express a distinct but closely PSA-2 polypeptide with molecular weight of 50 kDa. In this study fusion of SP2/0 myeloma cells with immunized mice spleenocytes infected with promastigotes of L. major intraperiton...

  3. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S.J.

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  4. The E5 protein of human papillomavirus type 16 perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-γ

    International Nuclear Information System (INIS)

    Zhang Benyue; Li Ping; Wang Exing; Brahmi, Zacharie; Dunn, Kenneth W.; Blum, Janice S.; Roman, Ann

    2003-01-01

    Major histocompatibility complex (MHC) class II antigens are expressed on human foreskin keratinocytes (HFKs) following exposure to interferon gamma. The expression of MHC class II proteins on the cell surface may allow keratinocytes to function as antigen-presenting cells and induce a subsequent immune response to virus infection. Invariant chain (Ii) is a chaperone protein which plays an important role in the maturation of MHC class II molecules. The sequential degradation of Ii within acidic endocytic compartments is a key process required for the successful loading of antigenic peptide onto MHC class II molecules. Since human papillomavirus (HPV) 16 E5 can inhibit the acidification of late endosomes in HFKs, the E5 protein may be able to affect proper peptide loading onto the MHC class II molecule. To test this hypothesis, HFKs were infected with either control virus or a recombinant virus expressing HPV16 E5 and the infected cells were subsequently treated with interferon-γ. ELISAs revealed a decrease of MHC class II expression on the surface of E5-expressing cells compared with control virus-infected cells after interferon treatment. Western blot analysis showed that, in cells treated with interferon gamma, E5 could prevent the breakdown of Ii and block the formation of peptide-loaded, SDS-stable mature MHC class II dimers, correlating with diminished surface MHC class II expression. These data suggest that HPV16 E5 may be able to decrease immune recognition of infected keratinocytes via disruption of MHC class II protein function

  5. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

    Science.gov (United States)

    Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

    2018-02-01

    Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

  6. Structure-based design of chimeric antigens for multivalent protein vaccines.

    Science.gov (United States)

    Hollingshead, S; Jongerius, I; Exley, R M; Johnson, S; Lea, S M; Tang, C M

    2018-03-13

    There is an urgent need to develop vaccines against pathogenic bacteria. However, this is often hindered by antigenic diversity and difficulties encountered manufacturing membrane proteins. Here we show how to use structure-based design to develop chimeric antigens (ChAs) for subunit vaccines. ChAs are generated against serogroup B Neisseria meningitidis (MenB), the predominant cause of meningococcal disease in wealthy countries. MenB ChAs exploit factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and the PorA VR2 epitope adopts an immunogenic conformation. In mice, immunisation with ChAs generates fHbp and PorA antibodies that recognise the antigens expressed by clinical MenB isolates; these antibody responses correlate with protection against meningococcal disease. Application of ChAs is therefore a potentially powerful approach to develop multivalent subunit vaccines, which can be tailored to circumvent pathogen diversity.

  7. The ability of IgY to recognize surface proteins of Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Basri A. Gani

    2009-12-01

    Full Text Available Background: Streptococcus mutans are gram positive bacteria classified into viridians group, and have a role in pathogenesis of dental caries. It’s adhesion to the tooth surface is mediated by cell surface proteins, which interact with specific receptor located in tooth pellicle. Glucan binding protein, Glukosyltransferase, and antigen I/II are basic proteins of S. mutans, which have a role in initiating the interaction. A previous study showed that chicken’s IgY can interfere the interaction. Purpose: The objective of this study was to assess the ability of IgY in recognizing the surface molecule of Streptococcus mutans expressed by various serotypes (c, d, e, f and a strain derived from IPB, Bogor. Method: Western blot was used as a method to determine such capability. Result: The result showed that IgY has a potency to recognize antigen I/II, but not the other proteins on the cell surface of all bacteria tested. Conclusion: The ability of IgY to bind the surface protein, antigen I/II, indicates that this avian antibody could be used as a candidate for anti-adhesion in preventing dental caries.

  8. Antigen cross-priming of cell-associated proteins is enhanced by macroautophagy within the antigen donor cell

    Directory of Open Access Journals (Sweden)

    Matthew eAlbert

    2012-03-01

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

  9. Roughness of the globular protein surface

    International Nuclear Information System (INIS)

    Timchenko, A.A.; Galzitskaya, O.V.; Serdyuk, I.N.

    1998-01-01

    Protein surface analysis using high resolution X ray shows that this surface has a two-level organization, on the micro- and macro-scales. On the micro-scale (2-7 Angstroem), the surface is characterized by the d = 2.1 fractal dimension which is intrinsic to surface with weak deformation and reflects the local atomic group packing. On the macro-scale the large scale surface defects are revealed which are interpreted as the result of secondary structure elements packing

  10. Evaluation the Surface Antigen of the Salmonella typhimurium ATCC 14028 Ghosts Prepared by “SLRP”

    Directory of Open Access Journals (Sweden)

    Amara A. Amro

    2014-01-01

    Full Text Available Recently, bacterial ghosts (BGs were prepared using a protocol based on critical chemical concentrations. It has been given the name “sponge like” (SL protocol and used in its reduced form “sponge like reduced protocol” (SLRP. While specific antibody for Salmonella is available on the market under the commercial names (of some kits such as Febrile Antigen Kit (N.S. BIO-TEC, we used the described Kit to investigate the validity of the SLRP. In this study, using SLRP we succeeded to prepare STGs with correct surface antigens could interact with their specific antibodies. Additionally the study has included oral vaccination with STGs with challenge test. The rats serums have been evaluated against both of the O and H antigens. The antigen-antibody interaction (agglutination results of both the SLRP and the animal experiments prove that we have correct STGs able to immunize the rats against viable Salmonella. STGs could be used as vaccine and as adjuvant and in the antibodies and in the diagnostic kits production. This study is an additional step for the establishment of correct BGs for immunological purposes.

  11. Cell surface antigens of radiation leukemia virus-induced BALB/c leukemias defined by syngeneic cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Kaneko, Yukio; Oettgen, H.F.; Obata, Yuichi; Nakayama, Eiichi.

    1989-01-01

    Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus (RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2 d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2K d , but not H-2D d . The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines. (author)

  12. Protein crystallization on polymeric film surfaces

    Science.gov (United States)

    Fermani, Simona; Falini, Giuseppe; Minnucci, Massimiliano; Ripamonti, Alberto

    2001-04-01

    Polymeric films containing ionizable groups, such as sulfonated polystyrene, cross-linked gelatin films with adsorbed poly- L-lysine or entrapped poly- L-aspartate and silk fibroin with entrapped poly- L-lysine or poly- L-aspartate, have been tested as heterogeneous nucleant surfaces for proteins. Concanavalin A from jack bean and chicken egg-white lysozyme were used as models. It was found that the crystallization of concanavalin A by the vapor diffusion technique, is strongly influenced by the presence of ionizable groups on the film surface. Both the induction time and protein concentration necessary for the crystal nucleation decrease whereas the nucleation density increases on going from the reference siliconized cover slip to the uncharged polymeric surfaces and even more to the charged ones. Non-specific attractive and local interactions between the protein and the film surface might promote molecular collisions and the clustering with the due symmetry for the formation of the crystal nuclei. The results suggest that the studied polymeric film surfaces could be particularly useful for the crystallization of proteins from solutions at low starting concentration, thus using small quantities of protein, and for proteins with very long crystallization time.

  13. Immuno disc assay for screening duck hepatitis B surface antigen in serum, liver tissue and cultured hepatocytes

    NARCIS (Netherlands)

    G.A. de Wilde (G.); R.A. Heijtink

    1993-01-01

    textabstractAn immuno disc assay (IDA) for semi-quantitative analysis of the surface antigen (DHBsAg) of duck hepatitis B virus (DHBV) is described. Unpurified antigen preparations were adsorbed onto punched-out nitrocellulose membrane discs. Rabbit antiserum raised against serum-derived

  14. Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces.

    Directory of Open Access Journals (Sweden)

    Cristiano De Michele

    2016-03-01

    Full Text Available In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion, which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts.

  15. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

    DEFF Research Database (Denmark)

    Kemp, M; Handman, E; Kemp, K

    1998-01-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune......-beta, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population...... and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen...

  16. Proteins in solution: Fractal surfaces in solutions

    Directory of Open Access Journals (Sweden)

    R. Tscheliessnig

    2016-02-01

    Full Text Available The concept of the surface of a protein in solution, as well of the interface between protein and 'bulk solution', is introduced. The experimental technique of small angle X-ray and neutron scattering is introduced and described briefly. Molecular dynamics simulation, as an appropriate computational tool for studying the hydration shell of proteins, is also discussed. The concept of protein surfaces with fractal dimensions is elaborated. We finish by exposing an experimental (using small angle X-ray scattering and a computer simulation case study, which are meant as demonstrations of the possibilities we have at hand for investigating the delicate interfaces that connect (and divide protein molecules and the neighboring electrolyte solution.

  17. Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

    Directory of Open Access Journals (Sweden)

    Alam SM

    2013-08-01

    Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

  18. Structural, antigenic, and evolutionary characterizations of the envelope protein of newly emerging Duck Tembusu Virus.

    Directory of Open Access Journals (Sweden)

    Kexiang Yu

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

  19. Vaccinia virus G8R protein: a structural ortholog of proliferating cell nuclear antigen (PCNA.

    Directory of Open Access Journals (Sweden)

    Melissa Da Silva

    Full Text Available BACKGROUND: Eukaryotic DNA replication involves the synthesis of both a DNA leading and lagging strand, the latter requiring several additional proteins including flap endonuclease (FEN-1 and proliferating cell nuclear antigen (PCNA in order to remove RNA primers used in the synthesis of Okazaki fragments. Poxviruses are complex viruses (dsDNA genomes that infect eukaryotes, but surprisingly little is known about the process of DNA replication. Given our previous results that the vaccinia virus (VACV G5R protein may be structurally similar to a FEN-1-like protein and a recent finding that poxviruses encode a primase function, we undertook a series of in silico analyses to identify whether VACV also encodes a PCNA-like protein. RESULTS: An InterProScan of all VACV proteins using the JIPS software package was used to identify any PCNA-like proteins. The VACV G8R protein was identified as the only vaccinia protein that contained a PCNA-like sliding clamp motif. The VACV G8R protein plays a role in poxvirus late transcription and is known to interact with several other poxvirus proteins including itself. The secondary and tertiary structure of the VACV G8R protein was predicted and compared to the secondary and tertiary structure of both human and yeast PCNA proteins, and a high degree of similarity between all three proteins was noted. CONCLUSIONS: The structure of the VACV G8R protein is predicted to closely resemble the eukaryotic PCNA protein; it possesses several other features including a conserved ubiquitylation and SUMOylation site that suggest that, like its counterpart in T4 bacteriophage (gp45, it may function as a sliding clamp ushering transcription factors to RNA polymerase during late transcription.

  20. Antigenic variation and the genetics and epigenetics of the PfEMP1 erythrocyte surface antigens in Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Arnot, David E; Jensen, Anja T R

    2011-01-01

    How immunity to malaria develops remains one of the great unresolved issues in bio-medicine and resolution of its various paradoxes is likely to be the key to developing effective malaria vaccines. The basic epidemiological observations are; under conditions of intense natural transmission, humans...... on the function and control of this multi-gene family of parasite variable surface antigens....

  1. Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

    NARCIS (Netherlands)

    van der Lelij, A.; Rothova, A.; Stilma, J. S.; Hoekzema, R.; Kijlstra, A.

    1990-01-01

    Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from

  2. Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

    2017-05-31

    Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Conformational Heterogeneity in Antibody-Protein Antigen Recognition IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

    Czech Academy of Sciences Publication Activity Database

    Addis, P. W.; Hall, c. J.; Bruton, S.; Veverka, Václav; Wilkinson, I. C.; Muskett, F. W.; Renshaw, P. S.; Prosser, C. E.; Carrington, B.; Lawson, A. D. G.; Griffin, R.; Taylor, R. J.; Waters, L. C.; Henry, A. J.; Carr, M. D.

    2014-01-01

    Roč. 289, č. 10 (2014), s. 7200-7210 ISSN 0021-9258 Institutional support: RVO:61388963 Keywords : NMR * antibody * protein-protein interaction * protein conformation Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  4. Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

    International Nuclear Information System (INIS)

    Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

    1986-01-01

    A radioimmunoassay that makes use of whole Schistosomula and 125 I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000

  5. A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Njoroge, Samuel M; Boinett, Christine J; Madé, Laure F; Ouko, Tom T; Fèvre, Eric M; Thomson, Nicholas R; Kariuki, Samuel

    2015-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described. © FEMS 2015.

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

    Science.gov (United States)

    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

  7. In Vitro Variant Surface Antigen Expression in Plasmodium falciparum Parasites from a Semi-Immune Individual Is Not Correlated with Var Gene Transcription

    Science.gov (United States)

    Tschan, Serena; Flötenmeyer, Matthias; Koch, Iris; Berger, Jürgen; Kremsner, Peter; Frank, Matthias

    2016-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections. PMID:27907004

  8. Effect of UV radiation on the surface of mammalian immunocompetent cells. 1. The change in expression of some antigens and receptors of murine spleen lymphocyte surface

    Energy Technology Data Exchange (ETDEWEB)

    Krylenkov, V.A.; Malygin, A.M. (AN SSSR, Leningrad. Inst. Tsitologii)

    1982-12-01

    Short-wave (254nm) and long-wave (365 nm) UV rays (ShUS and LUV rays) induce the increase in the expression of surface markers of T lymphocytes-THETA(Thy-1) antigens and B lymphocytes-MBLA-antigens and EAS receptors when affecting mouse spleen cells in nonlethal and small lethal doses. Total cell content with T and B lymphocyte characters in an irradiated suspension exceeds even the total cell quantity in non-irradiated suspension (100%) which points to the possibility of the expression of plasmatic membrane antigens and receptors not manifested on the surface of nonirradiated lymphocytes. In the isolethal dose range (LD/sup 15/-LD/sup 28/) ShUV rays suppress and LUV rays induce further increase of THETA and MBLA antigens expression. Among B lymphocytes surface markers the MBLA antigens are more resistant to ShUV an LUV radiation as compared with the EAC receptors.

  9. Cancer associated aberrant protein O-glycosylation can modify antigen processing and immune response.

    Directory of Open Access Journals (Sweden)

    Caroline B Madsen

    Full Text Available Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA-MUC1 fusion peptides (+/- glycosylation loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB-Tg(HLA-A/H2-D2Enge/J (HLA-A2 transgenic mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.

  10. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    International Nuclear Information System (INIS)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-01-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in λgt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein

  11. Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics.

    Science.gov (United States)

    Wang, Li; Cui, Jing; Hu, Dan Dan; Liu, Ruo Dan; Wang, Zhong Quan

    2014-01-22

    The excretory-secretory (ES) proteins of Trichinella spiralis muscle larvae (ML) come mainly from the excretory granules of the stichosome and the cuticles (membrane proteins), are directly exposed to the host's immune system, and are the main target antigens, which induce the immune responses. Although the ES proteins are the most commonly used diagnostic antigens for trichinellosis, their main disadvantage are the false negative results during the early stage of infection. The aim of this study was to identify early specific diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. Two-dimensional electrophoresis (2-DE) combined with Western blot were used to screen the early diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The protein spots recognized by the sera from BALB/c mice infected with T. spiralis at 18 days post-infection (dpi) were identified by MALDI-TOF/TOF-MS and putatively annotated using GO terms obtained from the InterPro databases. The ES proteins were analyzed by 2-DE, and more than 33 protein spots were detected with molecular weight varying from 40 to 60 kDa and isoelectric point (pI) from 4 to 7. When probed with the sera from infected mice at 18 dpi, 21 protein spots were recognized and then identified, and they were characterized to correlate with five different proteins of T. spiralis, including two serine proteases, one deoxyribonuclease (DNase) II, and two kinds of trypsin. The five proteins were functionally categorized into molecular function and biological process according to GO hierarchy. 2-DE and Western blot combined with MALDI-TOF/TOF-MS were used to screen the diagnostic antigens from the main components of T. spiralis muscle larval ES proteins. The five proteins of T. spiralis identified (two serine proteases, DNase II and two kinds of trypsin) might be the early specific diagnostic antigens of trichinellosis.

  12. Surface-Engineering of Red Blood Cells as Artificial Antigen Presenting Cells Promising for Cancer Immunotherapy.

    Science.gov (United States)

    Sun, Xiaoqi; Han, Xiao; Xu, Ligeng; Gao, Min; Xu, Jun; Yang, Rong; Liu, Zhuang

    2017-10-01

    The development of artificial antigen presenting cells (aAPCs) to mimic the functions of APCs such as dendritic cells (DCs) to stimulate T cells and induce antitumor immune responses has attracted substantial interests in cancer immunotherapy. In this work, a unique red blood cell (RBC)-based aAPC system is designed by engineering antigen peptide-loaded major histocompatibility complex-I and CD28 activation antibody on RBC surface, which are further tethered with interleukin-2 (IL2) as a proliferation and differentiation signal. Such RBC-based aAPC-IL2 (R-aAPC-IL2) can not only provide a flexible cell surface with appropriate biophysical parameters, but also mimic the cytokine paracrine delivery. Similar to the functions of matured DCs, the R-aAPC-IL2 cells can facilitate the proliferation of antigen-specific CD8+ T cells and increase the secretion of inflammatory cytokines. As a proof-of-concept, we treated splenocytes from C57 mice with R-aAPC-IL2 and discovered those splenocytes induced significant cancer-cell-specific lysis, implying that the R-aAPC-IL2 were able to re-educate T cells and induce adoptive immune response. This work thus presents a novel RBC-based aAPC system which can mimic the functions of antigen presenting DCs to activate T cells, promising for applications in adoptive T cell transfer or even in direct activation of circulating T cells for cancer immunotherapy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Kumar Sukhdeo

    Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

  14. Surface peptide mapping of protein I and protein III of four strains of Neisseria gonorrhoeae

    International Nuclear Information System (INIS)

    Judd, R.C.

    1982-01-01

    Whole cells and isolated outer membranes (OMs) of four strains of gonococci were surface radioiodinated with either lactoperoxidase or Iodogen (Pierce Chemical Co., Rockford, Ill.). These preparations were solubilized in sodium dodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and digested with alpha-chymotrypsin, and the resultant 125 I-peptide fragments were resolved by high-voltage electrophoresis and thin-layer chromatography (i.e., surface peptide mapping). Radioemitting peptidic fragments were visualized by autoradiography. Results demonstrated that the PI molecule of each gonococcal strain studied had unique iodinatable peptides exposed on the surface of whole cells and OMs, whereas PIIIs appeared to have the same portion of the molecule exposed on the surface of bacteria or OMs, regardless of the gonococcal strain from which they were isolated. Many more radiolabeled peptides were seen in surface peptide maps of PIs from radiolabeled OMs than in those from radioiodinated whole cells, whereas different peptidic fragments were seen in the surface peptide maps of PIIIs from radiolabeled OMs than were seen in those from radiolabeled whole cells. These data suggest that PI may contribute strain-specific antigenic determinants and PIII may contribute cross-reactive determinants and that the surface exposure of PI and PIII is different in isolated OMs than in the OM of intact gonococci

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

    Directory of Open Access Journals (Sweden)

    Nora Céspedes

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

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

    Science.gov (United States)

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

    2012-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Karen Elizabeth Kempsell

    2015-08-01

    Full Text Available A commercial Bacillus anthracis (Anthrax whole genome protein microarray has been used to identify immunogenic Anthrax proteins 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 frames were also recognised by infected or exposed groups in comparison to controls. Some of these antigens e.g. BA4182 are not recognised by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo and 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 synthesised, tested in mouse immunogenicity studies and validated in parallel using human sera from the

  18. The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen.

    Science.gov (United States)

    Murray, P J; Spithill, T W; Handman, E

    1989-12-15

    Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.

  19. Cooperative Serum Bactericidal Activity Between Human Antibodies to Meningococcal Factor H Binding Protein and Neisserial Heparin Binding Antigen

    Science.gov (United States)

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

    2011-01-01

    A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies to fHbp and/or NHba. All six subjects developed ≥4-fold increases in SBA titer against a test strain with fHbp in the variant 1 group with an amino acid sequence that matched the vaccine antigen (GMT 95 percent of the SBA was directed against fHbp. Four subjects developed ≥4-fold increases in SBA titer against a test strain with a heterologous fHbp variant 2 antigen and a homologous NHba amino acid sequence that matched the vaccine antigen (GMT bactericidal anti-fHbp variant 1 antiserum with a mouse anti-NHba antiserum also augmented the anti-NHba SBA titer against this test strain. For meningococcal vaccines that target relatively sparsely-exposed antigens such fHbp or NHba, non-bactericidal antibodies against individual antigens can cooperate and elicit SBA. PMID:21241734

  20. Study on reducing antigenic response and IgE-binding inhibitions of four milk proteins of Lactobacillus casei 1134.

    Science.gov (United States)

    Yao, Minjing; Xu, Qian; Luo, Yongkang; Shi, Jing; Li, Zheng

    2015-04-01

    Cow's milk allergy has aroused public concern. The aim of this study was to investigate the effects of fermentation by Lactobacillus casei 1134 on the antigenicity and allergenicity (IgE-binding inhibitions) of milk proteins. The effects of pH value on the antigenicity and allergenicity of four milk proteins (α-lactalbumin, β-lactoglobulin, α-casein and β-casein) were examined by indirect competitive enzyme-linked immunosorbent assay. The free amino acids which were produced in the fermentation process were analysed and the proteolysis of milk proteins was detected. Fermentation by L. casei 1134 could significantly reduce the antigenicity and allergenicity of the four proteins in reconstituted milk. The allergenicity of milk proteins was further reduced in the process of simulated gastrointestinal digestion. Moreover, we could deduce that one of the potential factors of antigenicity was lactic acid with the comparison of the antigenicity of the four proteins between L. casei 1134 fermented milk and lactic acid milk at different pH values. There are many factors which can affect the milk proteins allergen, including lactic acid and proteolytic enzymes. © 2014 Society of Chemical Industry.

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

    Directory of Open Access Journals (Sweden)

    Linnebacher Michael

    2011-07-01

    Full Text Available Abstract 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.

  2. Antigen-driven bystander effect accelerates epicutaneous sensitization with a new protein allergen

    Directory of Open Access Journals (Sweden)

    Yu Jhang-Sian

    2009-03-01

    Full Text Available Abstract Exposure to protein allergen epicutaneously, inducing a Th2-dominant immune response, sensitizes the host to the development of atopic disease. Antigen-driven bystander effect demonstrates that polarized T cells could instruct naïve T cells to differentiate into T cells with similar phenotype. In this study, we aimed to determine the contribution of antigen-driven bystander effect on epicutaneous sensitization with a newly introduced protein allergen. BALB/c mice were immunized intraperitoneally with BSA emulsified in alum, known to induce a Th2 response, three weeks before given BSA and OVA epicutaneously. Lymph node cells from these mice restimulated with OVA secreted higher levels IL-4, IL-5 and IL-13 as compared with cells from mice without BSA immunization. In addition, BALB/c mice immunized subcutaneously with BSA emulsified in complete Freund's adjuvant, known to induce a Th1-predominant response, also induced higher Th1 as well as Th2 cytokine response when restimulated with OVA as compared with mice without immunization. We demonstrated that subcutaneous immunization with BSA in CFA induced Th2 as well as Th1 response. The threshold of epicutaneous sensitization to OVA was also reduced, possibly due to increased expressions of IL-4 and IL-10 in the draining lymph nodes during the early phase of sensitization. In conclusion, antigen-driven bystander effect, whether it is of Th1- or Th2-predominant nature, can accelerate epicutaneous sensitization by a newly introduced protein allergen. These results provide a possible explanation for mono- to poly-sensitization spread commonly observed in atopic children.

  3. Identification and characterization of the interactive proteins with cytotoxic T-lymphocyte antigen-2α.

    Science.gov (United States)

    Nga, Bui Thi To; Luziga, Claudius; Yamamoto, Misa; Kusakabe, Ken Takeshi; Yamamoto, Yoshimi

    2015-01-01

    Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.

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

    Science.gov (United States)

    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.

  5. Expression of a hantavirus N protein and its efficacy as antigen in immune assays

    Directory of Open Access Journals (Sweden)

    L.T.M. Figueiredo

    2008-07-01

    Full Text Available Hantavirus cardiopulmonary syndrome (HCPS has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

  6. Cell division cycle-associated protein 1 as a new melanoma-associated antigen.

    Science.gov (United States)

    Tokuzumi, Aki; Fukushima, Satoshi; Miyashita, Azusa; Nakahara, Satoshi; Kubo, Yosuke; Yamashita, Junji; Harada, Miho; Nakamura, Kayo; Kajihara, Ikko; Jinnin, Masatoshi; Ihn, Hironobu

    2016-12-01

    Immune checkpoint inhibitors have increased the median survival of melanoma patients. To improve their effects, antigen-specific therapies utilizing melanoma-associated antigens should be developed. Cell division cycle-associated protein 1 (CDCA1), which has a specific function at the kinetochores for stabilizing microtubule attachment, is overexpressed in various cancers. CDCA1, which is a member of cancer-testis antigens, does not show detectable expression levels in normal tissues. Quantitative reverse transcription polymerase chain reaction and immunoblotting analyses revealed that CDCA1 was expressed in all of the tested melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. An immunohistochemical analysis and a Cox proportional hazards model showed that CDCA1 could be a prognostic marker in malignant melanoma (MM) patients. CDCA1-specific siRNA inhibited the cell proliferation of SKMEL2 and WM115 cells, but did not reduce the migration or invasion activity. These results suggest that CDCA1 may be a new therapeutic target of melanoma. © 2016 Japanese Dermatological Association.

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

    Directory of Open Access Journals (Sweden)

    Stefanie Hausammann

    Full Text Available 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.

  8. [Biological properties of Lactobacillus surface proteins].

    Science.gov (United States)

    Buda, Barbara; Dylus, Ewa; Górska-Frączek, Sabina; Brzozowska, Ewa; Gamian, Andrzej

    2013-04-04

    Lactobacillus, a genus of Gram-positive bacteria, includes many strains of probiotic microflora. Probiotics, by definition, are living microorganisms that exert beneficial effects on the host organism. The morphology and physiology of the Lactobacillus bacterial genus are described. The structure of the cell wall of Gram-positive bacteria is discussed. The surface S-layer of Lactobacillus composed of proteins (SLP) with low molecular mass is presented. Cell surface proteins participating in the regulation of growth and survival of the intestinal epithelium cells are characterized. The influence of stress factors such as increased temperature, pH, and enzymes of gastric and pancreatic juice on SLP expression is described. The ability of binding of heavy metal ions by S-layer proteins is discussed. The characteristics of these structures, including the ability to adhere to epithelial cells, and the inhibition of invasion of pathogenic microflora of type Shigella, Salmonella, Escherichia coli and Clostridium and their toxins, are presented. 

  9. Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg).

    Science.gov (United States)

    Kim, Jinwoon; Oh, Seo Yeong; Shukla, Shruti; Hong, Seok Bok; Heo, Nam Su; Bajpai, Vivek K; Chun, Hyang Sook; Jo, Cheon-Ho; Choi, Bong Gill; Huh, Yun Suk; Han, Young-Kyu

    2018-06-01

    This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10-15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Stem Cell Physics. Laser Manipulation of Blood Types: Laser-Stripping-Away of Red Blood Cell Surface Antigens

    Science.gov (United States)

    Stefan, V. Alexander

    2014-03-01

    A novel mechanism of importance for the transfusion medicine[2] is proposed. The interaction of ultrashort wavelength multilaser beams with the flowing blood thin films can lead to a conversion of blood types A, B, and AB into O type.[3] The stripping away of antigens is done by the scanning-multiple-lasers of a high repetition rate in the blue-purple frequency domain. The guiding-lasers are in the red-green frequency domain. The laser force, (parametric interaction with the antigen eigen-oscillation),[4] upon the antigen protein molecule must exceed its weight. Supported by Nikola Tesla Labs, La Jolla, CA.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  13. Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

    Directory of Open Access Journals (Sweden)

    Jason M Shapiro

    Full Text Available We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1, heat shock protein 90 (HSP90, and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

    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

  15. Antigenicity of fractions of Helicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies.

    Science.gov (United States)

    Stacey, A R; Hawtin, P R; Newell, D G

    1990-10-01

    The antigenicity of Helicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well defined Helicobacter pylori status. The antigenic material of Helicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding of Helicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91-100% specific, however fractions 16 to 19 showed a reduced sensitivity (78%) compared with the acid extract (95%). The urease fractions were 91% sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83% sensitive and 93.3% specific.

  16. Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I

    DEFF Research Database (Denmark)

    Cuesta, Àngel M; Suárez, Eduardo; Larsen, Martin

    2006-01-01

    Although DNA-based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour-bearing patients. Genetic technology offers strategies for promoting...... immune pathways by adding immune-activating genes to the tumour antigen sequence. In this work, we converted a model non-immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion...... generated antigen-specific T helper 1-type cellular immune responses. These results demonstrate that the incorporation of protein III into a DNA vaccine formulation can modulate the gene-mediated immune response and may thus provide a strategy for improving its therapeutic effect....

  17. Postvaccination seroconversion against the surface antigen of Hepatitis B virus, in nursing students

    Directory of Open Access Journals (Sweden)

    Gladys Amanda Mera-Urbano

    2013-09-01

    Full Text Available Objective: To determine the status of seroconversion after vaccination against the surface antigen of hepatitis B virus in nursing students, University of Cauca. Methods: Cross sectional study in students of V and VI semester. The sample was taken from 37 students, 15 of V and 22 of VI semester. The instrument used was a survey that included 11 questions of multiple selections. Records for weight, height and laboratory results were collected; blood samples for antibody titers were performed with informed consent. The data were tabulated and analyzed using SPSS, version 17.0. Results: 89.2% of students had levels of antibodies to the surface antigen. This value was greater than 10 mUI/ml, considered by the scientific community as a protector value of Hepatitis B. 10.8% of had lesser values. Regarding vaccination scheme, 24% had a dose, 19% two, 48% three and 8% had a one dose. The population with 3 doses and reinforcement seroconverted by 100%. Conclusion: This study demonstrated failings in the scheme of vaccination of the students of nursing and that 10.8 % presented lower values than 10 mIU/ml. It is necessary to apply the institutional rules with more strength as a preventive measure for hepatitis B.

  18. Protein-mediated surface structuring in biomembranes

    Directory of Open Access Journals (Sweden)

    Maggio B.

    2005-01-01

    Full Text Available The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein, integral (Folch-Lees proteolipid protein and amphitropic (c-Fos and c-Jun proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase, in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

  19. Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing.

    Science.gov (United States)

    Zahid, Maria; Lünsdorf, Heinrich; Rinas, Ursula

    2015-07-17

    The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

    NARCIS (Netherlands)

    Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    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 replaceme...

  4. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein (MTP)

    DEFF Research Database (Denmark)

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus

    2014-01-01

    microsomal triglyceride transfer protein (MTP), 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 (APCs), which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis....

  5. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.

    OpenAIRE

    Hoogenboom, H R; Griffiths, A D; Johnson, K S; Chiswell, D J; Hudson, P; Winter, G

    1991-01-01

    The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that...

  6. Recombinant forms of Leishmania amazonensis excreted/secreted promastigote surface antigen (PSA) induce protective immune responses in dogs

    OpenAIRE

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-01-01

    International audience; Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), fr...

  7. Neurofibromatosis type 2 tumor suppressor protein, NF2, induces proteasome-mediated degradation of JC virus T-antigen in human glioblastoma.

    Directory of Open Access Journals (Sweden)

    Sarah Beltrami

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

  8. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

  9. Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

    DEFF Research Database (Denmark)

    Rasmussen, A B; Zocca, M-B; Bonefeld, C M

    2004-01-01

    on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...

  10. Evaluate the efficiency of Antigen 60 (A60 protein from BCG strain of Mycobacterium bovis as a diagnostic antigen

    Directory of Open Access Journals (Sweden)

    Nafiseh Shakibamehr

    2016-01-01

    Conclusion: Results of reactions of the injected A60 and standard human tuberculin shows the effectiveness of this antigen in comparison with standard human tuberculin. Detection of antibody in the serum of patients is a rapid and repeatable method. A60 with 89% sensitivity and 94% specificity could be an appropriate matter for the diagnosis of tuberculosis. Because this method can be performed without radioactive materials or advanced and expensive equipment, it will provide results quickly.

  11. Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Bengtsson, Dominique C; Sowa, Kordai M P; Arnot, David E

    2008-01-01

    There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines...... is discussed here in the context of malaria parasite-infected cells, it can also be modified to visualize the membrane and intracellular distribution of surface and internal proteins in other eukaryotic cells....

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

    Science.gov (United States)

    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.

  13. Enhanced discrimination of malignant from benign pancreatic disease by measuring the CA 19-9 antigen on specific protein carriers.

    Directory of Open Access Journals (Sweden)

    Tingting Yue

    Full Text Available The CA 19-9 assay detects a carbohydrate antigen on multiple protein carriers, some of which may be preferential carriers of the antigen in cancer. We tested the hypothesis that the measurement of the CA 19-9 antigen on individual proteins could improve performance over the standard CA 19-9 assay. We used antibody arrays to measure the levels of the CA 19-9 antigen on multiple proteins in serum or plasma samples from patients with pancreatic adenocarcinoma or pancreatitis. Sample sets from three different institutions were examined, comprising 531 individual samples. The measurement of the CA 19-9 antigen on any individual protein did not improve upon the performance of the standard CA 19-9 assay (82% sensitivity at 75% specificity for early-stage cancer, owing to diversity among patients in their CA 19-9 protein carriers. However, a subset of cancer patients with no elevation in the standard CA 19-9 assay showed elevations of the CA 19-9 antigen specifically on the proteins MUC5AC or MUC16 in all sample sets. By combining measurements of the standard CA 19-9 assay with detection of CA 19-9 on MUC5AC and MUC16, the sensitivity of cancer detection was improved relative to CA 19-9 alone in each sample set, achieving 67-80% sensitivity at 98% specificity. This finding demonstrates the value of measuring glycans on specific proteins for improving biomarker performance. Diagnostic tests with improved sensitivity for detecting pancreatic cancer could have important applications for improving the treatment and management of patients suffering from this disease.

  14. Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli.

    Science.gov (United States)

    Kim, Tae-Geum; Huy, Nguyen-Xuan; Kim, Mi-Young; Jeong, Dong-Keun; Jang, Yong-Suk; Yang, Moon-Sik; Langridge, William H R; Lee, Jin-Yong

    2009-02-01

    The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial

  15. Longitudinal microarray analysis of cell surface antigens on peripheral blood mononuclear cells from HIV+ individuals on highly active antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Wang Bin

    2008-03-01

    Full Text Available Abstract Background The efficacy of highly active antiretroviral therapy (HAART determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6 achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6 responded intermittently. Blood samples were collected over an average of 3 years and 5–8 time points were selected for microarray assay and statistical analysis. Results Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28 for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33 were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively. Conclusion Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.

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

    Science.gov (United States)

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

    2010-01-01

    Purpose We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding PSA with radiotherapy in patients with localized prostate cancer. Here we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome. Experimental Design Pre- and post-treatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serological screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses. Results Western blotting revealed treatment-associated autoantibody responses in 15/33 (45.5%) patients treated with vaccine + EBRT versus 1/8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that seven of 33 patients (21.2%) treated with vaccine + EBRT demonstrated a vaccine-associated autoantibody response to four ubiquitously expressed self antigens: DIRC2, NDUFS1, MRFAP1 and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend towards decreased biochemical-free survival. Conclusions Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self proteins, which may influence the efficacy of vaccination. PMID:20562209

  17. In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

    Science.gov (United States)

    Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection. PMID:24786587

  18. In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

    Science.gov (United States)

    Chamakh-Ayari, Rym; Bras-Gonçalves, Rachel; Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

    2014-01-01

    PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

  19. In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Rym Chamakh-Ayari

    Full Text Available PSA (Promastigote Surface Antigen belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L. species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm or L. braziliensis (CCLb or visceral leishmaniasis due to L. donovani (CVLd and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection or non immune/naive individuals (HLR: Healthy Low Responders, depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.

  20. A molecular assembly system for presentation of antigens on the surface of HBc virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Blokhina, Elena A.; Kuprianov, Victor V. [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); Stepanova, Ludmila A.; Tsybalova, Ludmila M. [Research Institute of Influenza, Russian Federation Ministry of Health and Social Development, St. Petersburg (Russian Federation); Kiselev, Oleg I. [Research Institute of Influenza, Russian Federation Ministry of Health and Social Development, St. Petersburg (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation); Ravin, Nikolai V., E-mail: nravin@biengi.ac.ru [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation); Skryabin, Konstantin G. [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation)

    2013-01-20

    Hepatitis B virus-like particles, icosahedral structures formed by multiple core protein dimers, are promising immune-enhancing vaccine carriers for foreign antigens. Insertions into the surface-exposed immunodominant loop are especially immunogenic. However, the need to conserve the particulate structure to ensure high immunogenicity imposes restraints on the nature of the heterologous sequence that can be inserted. We propose a new approach to constructing HBc particles linked to the target epitopes that relies on non-covalent interactions between the epitope and pre-assembled unmodified HBc particles. Interaction was enabled by fusion of the epitope to the GSLLGRMKGA peptide, binding to the spike tips. This peptide may be used as a 'binding tag' allowing in vitro construction of HBc particles carrying the target peptide. Such virus-like particles carrying multiple copies of the extracellular domain of the M2 protein of different influenza strains appeared to be highly immunogenic and protected immunised mice against a lethal influenza challenge.

  1. Liver Allograft Its Use in Chronic Active Hepatitis With Macronodular Cirrhosis, Hepatitis B Surface Antigen

    Science.gov (United States)

    Corman, Jarques L.; Putnam, Charles W.; Iwatsuki, Shunzaburo; Redeker, Allan G.; Porter, K. A.; Peters, Robert L.; Schröter, Gerhard; Starzl, Thomas E.

    2010-01-01

    A patient suffering from chronic active hepatitis with macronodular cirrhosis, positive for hepatitis B surface antigen (HB,Ag), was treated with an orthoiopic liver allograft. The HB, antigenemia, as measured with several precipltation tests and by complement fixation, became negative after transplantation and remained so for about 2½ months. During the interval, very low Iters of the antigen were detectable by, radioimmunoassay. At about three months after transplantation, she had an attack of acute hepatitis, at which time HB,Ag became detectable by all tests. She recovered, but progressive liver disease developed during the remaining 1½ years of her life. She died of disseminaled nocardiosis and candidiasis with deteriorating hepatic function. The homograft at autopsy, showed no evidence of rejection, but was the site of chronic active liver disease, although of a different pathologic pattern than that affecting her native liver. The differences in histology may reflect the influence of chronic Immunosuppression on the features of chronic active hepatitis. PMID:365134

  2. Reduced third-trimester levels of soluble human leukocyte antigen G protein in severe preeclampsia.

    Science.gov (United States)

    Hackmon, Rinat; Koifman, Arie; Hyodo, Hironobu; Hyobo, Hirohito; Glickman, Hagit; Sheiner, Eyal; Geraghty, Daniel E

    2007-09-01

    Recently, lower maternal plasma human leukocyte antigen (HLA)-G protein levels in preeclampsia (PE) in the first and second trimester was reported. Thus, we sought to evaluate the levels of HLA-G protein in patients with severe PE during the third trimester. In this prospective case control study, amniotic fluid and maternal and cord blood samples were aspirated from 50 pregnant women during the third trimester. The study group included 26 pregnant women diagnosed with severe PE and 24 women without PE serving as controls. A soluble HLA-G-specific enzyme-linked immunosorbent assay was used to measure protein levels. Statistical analysis included the Student t test and simple regression analysis. Maternal serum HLA-G levels in PE pregnancies were found to be significantly lower as compared with normal pregnancies (10.97 +/- 6.55 vs 36.05 +/- 34.53 microg/mL; P = .003). A reduced level of maternal HLA-G protein was associated with severe PE during the third trimester. This finding may suggest an essential role for HLA-G in normal and preeclamptic pregnancies.

  3. The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major.

    Science.gov (United States)

    Kemp, M; Handman, E; Kemp, K; Ismail, A; Mustafa, M D; Kordofani, A Y; Bendtzen, K; Kharazmi, A; Theander, T G

    1998-03-01

    The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes. Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor-beta, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population and that these cells were the major source of interferon-gamma. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis.

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

    Directory of Open Access Journals (Sweden)

    Michal Sima

    2016-03-01

    Full Text Available 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.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.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-mediated reactions also in other host species.

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

    Science.gov (United States)

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

    2016-03-01

    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. 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. 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-mediated reactions also in other host species.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

    DEFF Research Database (Denmark)

    Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

    2012-01-01

    Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...... response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-¿ release, and antibody induction. Gal...

  9. Age-specific immunoglobulin g (IgG) and IgA to pneumococcal protein antigens in a population in coastal kenya.

    Science.gov (United States)

    Laine, Catherine; Mwangi, Tabitha; Thompson, Claudette M; Obiero, Jacktone; Lipsitch, Marc; Scott, J Anthony G

    2004-06-01

    Streptococcus pneumoniae is the primary etiological agent of community-acquired pneumonia and a major cause of meningitis and bacteremia. Three conserved pneumococcal proteins-pneumolysin, pneumococcal surface adhesin A (PsaA), and pneumococcal surface protein A (PspA)-are currently being investigated as vaccine candidates. Such protein-based vaccines, if proven effective, could provide a cheaper alternative to conjugate vaccine formulae. Few data from sub-Saharan Africa exist concerning the development of natural antibody to these antigens, however. To investigate the age-specific development of antiprotein immunoglobulin G (IgG) and IgA antibody responses, the sera of 220 persons 2 weeks to 84 years of age from coastal Kenya were assayed using enzyme-linked immunosorbent assays. IgG and IgA antibody responses to each antigen were observed in all age groups. Serum concentrations of IgG and IgA antibody responses to PspA and PdB (a recombinant toxoid derivative of pneumolysin), but not to PsaA, increased significantly with age (P < 0.001). No decline was observed in the sera of the elderly. Anti-protein IgG concentrations were only weakly correlated (0.30 < r < 0.56; P < 0.0001), as were IgA concentrations (0.24 < r < 0.54; P < 0.0001).

  10. Structural Studies on Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Malaria Antigens Using Small Angle X-Ray Scattering (SAXS)

    DEFF Research Database (Denmark)

    Christoffersen, Stig

    against placental malaria, VAR2CSA was studied extensively. By the use of SAXS on multiple truncated VAR2CSA constructs it was possible to combine the acquired information and present a structural model for the DBL and CIDR domain arrangement in VAR2CSA. This work was published in Journal of Biological...... symptoms and vaccine research is focused on trying to mimic or advance this immune acquisition. Immunity is primarily caused by acquisition of antibodies directed against a family of Plasmodium protein antigens called PfEMP1s located on the surface of infected erythrocytes. The PfEMP1 proteins are adhesive...... knowledge for these proteins is required. During the time course of this PhD thesis I have studied a number of PfEMP1 proteins using the SAXS technique. This biophysical technique has gained widespread attention during the past decade due to scientific and computational developments. SAXS is X...

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

    Science.gov (United States)

    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

  12. Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

    Science.gov (United States)

    Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

    2012-01-01

    This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

  13. A highly sensitive radioimmunoassay technique for subtyping the antibody to hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Fang, C.T.; Nath, N.; Berberian, H.; Dodd, R.Y.

    1978-01-01

    A highly sensitive technique for determining the subtype specificity of antibody to hepatitis B surface antigen (anti-HBs) is described. Immunoadsorbent consisting of controlled pore glass coated with subtype specific HBsAg was used to remove homologous antibody from the test samples before testing them for residual antibody by a commercially available radioimmunoassay (RIA). A total of 73 anti-HBs-positive samples from asymptomatic blood donors were tested. In nearly 80% of these samples the subtype reactivity could be determined by this technique. Only 67% could be typed by conventional liquid phase absorption RIA and 22% by passive hemagglutination inhibition techniques. Among the samples with low anti-HBs titer, ad and ay subtypes were found with equal frequency; however, with the increase in anti-HBs titer, considerably higher proportion of ad specificity was detected. (Auth.)

  14. Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens

    DEFF Research Database (Denmark)

    Ofori, Michael F; Dodoo, Daniel; Staalsoe, Trine

    2002-01-01

    In areas of intense Plasmodium falciparum transmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area......, clinical disease is caused mainly by parasites expressing VSA not recognized by preexisting VSA-specific antibodies in that child. Such malaria episodes are known to cause an increase in agglutinating antibodies specifically recognizing VSA expressed by the parasite isolate causing the illness, whereas...... antibody responses to other parasite isolates are relatively unaffected. However, the detailed kinetics of this VSA antibody acquisition are unknown and hence were the aim of this study. We show that P. falciparum malaria in Ghanaian children generally caused a rapid and sustained increase in variant...

  15. A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B.

    Science.gov (United States)

    Kato, J; Hasegawa, K; Torii, N; Yamauchi, K; Hayashi, N

    1996-03-01

    To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance.

  16. Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections

    DEFF Research Database (Denmark)

    Giha, H A; Staalsoe, T; Dodoo, D

    2000-01-01

    is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh...... susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm...

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

    Directory of Open Access Journals (Sweden)

    Kumar A

    2011-06-01

    Full Text Available Amit Kumar, Xinran Li, Michael A Sandoval, B Leticia Rodriguez, Brian R Sloat, Zhengrong CuiUniversity of Texas at Austin, College of Pharmacy, Pharmaceutics Division, Austin, TX, USABackground: 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.Keywords: antibody responses, safety of microneedles, transepidermal water loss

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Chiriva-Internati, Maurizio

    2011-01-01

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

  1. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    Science.gov (United States)

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

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

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  5. [Evaluation of the Recombinant Protein Tp0965 of Treponema Pallidum as Perspective Antigen for the Improved Serological Diagnosis of Syphilis].

    Science.gov (United States)

    Runina, A V; Starovoitova, A S; Deryabin, D G; Kubanov, A A

    2016-01-01

    BACKGRAUND. Treponemal tests based on the detection of antibodies against the Treponema pallidum antigens are the most specific methods for serological diagnosis of syphilis. Due to the inability to cultivate this bacterium in vitro, the most promising sources of antigens for diagnostics are recombinant proteins of T. pallidum. Evaluation of the analytical value of certain T. pallidum proteins is the approach to improve sensitivity, specificity, and reproducibility of syphilis serological tests, including possibilities of differential diagnosis of various forms of the disease. The aim of the research was to evaluate the analytical values (sensitivity and specificity) of recombinant protein Tp0965 of T. pallidum as a candidate antigen for serological diagnosis of syphilis. tp0965 gene was cloned into the expression vector pET28a and the construct was used for the transformation of E. coli BL-21 (DE3) cells and further expression and purification of the recombinant protein. The collected protein was used as T. pallidum antigen for serum analysis (ELISA) of groups of patients with various forms of syphilis (n=84) and the group of healthy donors (n = 25). High frequency of positive ELISA results was shown with serum of patients with syphilis, compared to the group of healthy donors. The sensitivity of serological reactions using recombinant protein Tp0965 was 98.8%, specificity--87.5%. The highest sensitivity (100%) was detected in the groups of patients with primary, secondary and early latent syphilis while in the group of patients with late latent syphilis it decreased to 95.2%. We concluded that due to its specificity T. pallidum recombinant protein Tp0965 can be used as a novel perspective antigen for development of syphilis serological diagnostic assays (for primary and early latent forms).

  6. Identification and characterization of the surface proteins of Clostridium difficile

    Energy Technology Data Exchange (ETDEWEB)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated.

  7. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination.

    Science.gov (United States)

    Devault, Alain; Bañuls, Anne-Laure

    2008-10-24

    PSA (promastigote surface antigen) is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. From the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance) subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes. PSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs. Seven of these are evolving relatively slowly and could

  8. The promastigote surface antigen gene family of the Leishmania parasite: differential evolution by positive selection and recombination

    Directory of Open Access Journals (Sweden)

    Bañuls Anne-Laure

    2008-10-01

    Full Text Available Abstract Background PSA (promastigote surface antigen is one of the major classes of membrane proteins present at the surface of the parasitic protozoan Leishmania. While it harbours leucine rich repeats, which are suggestive of its involvement in parasite-to-host physical interactions, its exact role is largely unknown. Furthermore, the extent of diversity of this gene family, both in copy number and sequence has not been established. Results From the newly available complete genome sequences of L. major, L. infantum and L. braziliensis, we have established the complete list of PSA genes, based on the conservation of specific domain architecture. The latter includes an array of leucine rich repeats of unique signature flanked by conserved cysteine-rich domains. All PSA genes code either for secreted or membrane-anchored surface proteins. Besides the few previously identified PSA genes, which are shown here to be part of a relatively large subclass of PSA genes located on chromosome 12, this study identifies seven other PSA subtypes. The latter, whose genes lie on chromosomes 5, 9, 21 and 31 in all three species, form single gene (two genes in one instance subfamilies, which phylogenetically cluster as highly related orthologs. On the other hand, genes found on chromosome 12 generally show high diversification, as reflected in greater sequence divergence between species, and in an extended set of divergent paralogs. Moreover, we show that the latter genes are submitted to strong positive selection. We also provide evidence that evolution of these genes is driven by intra- and intergenic recombination, thereby modulating the number of LRRs in protein and generating chimeric genes. Conclusion PSA is a Leishmania family of membrane-bound or secreted proteins, whose main signature consists in a specific LRR sequence. All PSA genes found in the genomes of three sequenced Leishmania species unambiguously distribute into eight subfamilies of orthologs

  9. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

    Directory of Open Access Journals (Sweden)

    Hadrien Peyret

    Full Text Available The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

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

    Science.gov (United States)

    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.

  11. IgM response to a human Pneumocystis carinii surface antigen in HIV-infected patients with pulmonary symptoms

    DEFF Research Database (Denmark)

    Lundgren, Bettina; Kovacs, J A; Mathiesen, Lars Reinhardt

    1993-01-01

    We have developed an ELISA to detect IgM antibodies to a major human Pneumocystis carinii surface antigen (gp95), and investigated the IgM response in 128 HIV-infected patients who underwent bronchoscopy for evaluation of pulmonary symptoms. Only 5 (4%) patients had IgM antibodies to P. carinii gp...... human P. carinii surface antigen. The increase in IgM response during the course of PCP observed in 3 patients suggests either reinfection with a new strain, or antigenic drift of an already acquired strain of P. carinii.......95. Four of the 5 patients with IgM antibodies also had IgG antibodies to gp95 and microbiologically proven P. carinii pneumonia (PCP). In 76/128 patients for whom serial samples were available, changes in antibody response were determined. In 3 patients we demonstrated an increase in IgM antibody...

  12. The Electrophoretic Mobility of Proteins near Surfaces

    Science.gov (United States)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  13. Plant bioreactors for the antigenic hook-associated flgK protein expression

    Directory of Open Access Journals (Sweden)

    Luciana Rossi

    2014-01-01

    Full Text Available Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seedspecific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

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

    Directory of Open Access Journals (Sweden)

    Maria L Knudsen

    Full Text Available 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.

  15. Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems

    NARCIS (Netherlands)

    C. D'Oliveira; A. Feenstra; H.W. Vos (Helma); A.D.M.E. Osterhaus (Albert); B.R. Shiels; A.W.C.A. Cornelissen; F. Jongejan

    1997-01-01

    textabstractAllelic forms (Tams1-1 and Tams1-2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we

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

    Directory of Open Access Journals (Sweden)

    Swapna Uplekar

    2017-01-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Mauro Bombaci

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

  18. Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.

    Science.gov (United States)

    Nosanchuk, Joshua D; Steenbergen, Judith N; Shi, Li; Deepe, George S; Casadevall, Arturo

    2003-10-01

    A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAb's were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAb's before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAb's was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice. The mAb's increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAb's bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B-like protein. This study establishes that mAb's to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

  19. Heat shock protein-90 inhibitors enhance antigen expression on melanomas and increase T cell recognition of tumor cells.

    Directory of Open Access Journals (Sweden)

    Timothy J Haggerty

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

  20. Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

    Science.gov (United States)

    Ponomarenko, Natalia A; Durova, Oxana M; Vorobiev, Ivan I; Belogurov, Alexey A; Kurkova, Inna N; Petrenko, Alexander G; Telegin, Georgy B; Suchkov, Sergey V; Kiselev, Sergey L; Lagarkova, Maria A; Govorun, Vadim M; Serebryakova, Marina V; Avalle, Bérangère; Tornatore, Pete; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

  1. Evolutionary structure of Plasmodium falciparum major variant surface antigen genes in South America: Implications for epidemic transmission and surveillance.

    Science.gov (United States)

    Rougeron, Virginie; Tiedje, Kathryn E; Chen, Donald S; Rask, Thomas S; Gamboa, Dionicia; Maestre, Amanda; Musset, Lise; Legrand, Eric; Noya, Oscar; Yalcindag, Erhan; Renaud, François; Prugnolle, Franck; Day, Karen P

    2017-11-01

    Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum . This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding " Plasmodium falciparum Erythrocyte Membrane Protein 1" ( Pf EMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.

  2. Subcloning of the 200-kDa Porphyromonas gingivalis antigen gene and inhibition of hemagglutination by an antibody against the recombinant protein.

    Science.gov (United States)

    Suyama, Tsutomu; Hayakawa, Mitsuo; Abiko, Yoshimitsu

    2004-09-01

    Porphyromonas gingivalis is a major etiologic agent of periodontitis and exhibits hemagglutinating and adherence activities. We previously succeeded in molecular cloning the 200-kDa cell-surface antigenic protein (200-k AP), designated pMD101, that is recognized in sera from periodontitis patients, and identified the 200-k AP as a hemagglutinin A (HagA) derivative. HagA is one of the hemagglutinins known to be a useful vaccine against periodontitis. HagA has four large, contiguous, direct repeats and the repeat unit is believed to contain the hemagglutinin domain. Because production of 200-k AP was low in the Escherichia coli host, it was difficult to obtain large amounts of recombinant protein. In this study, we attempt to subclone the gene encoding the useful antigen from pMD101 in an effort to obtain large quantities. A subclone, designated pMD160, encoding a fusion protein of 80-kDa HagA and maltose-binding protein was successfully constructed, and the novel clone produced relatively large amounts of recombinant protein. DNA nucleotide sequences of the pMD160 insert demonstrated that the 80-kDa protein contained a short hemagglutinin motif and a direct repeat unit region. The recombinant protein was purified to homogeneity and rabbit antiserum was raised. The antibody was capable of inhibiting the hemagglutinating activity of P. gingivalis. These findings suggest that novel 80-kDa HagA derivative proteins can be produced efficiently from E. coli hosts and these may be useful in developing immunotherapy against periodontitis infected by P. gingivalis.

  3. Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Hasman, Henrik; Schembri, Mark

    2002-01-01

    to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (alpha and beta), where the beta-subunit forms an integral outer membrane translocator to which the alpha-subunit is noncovalently attached. The simplicity of the Ag43 system...... makes it ideally suited as a surface display scaffold. Here we demonstrate that the Ag43 alpha-module can accommodate and display correctly folded inserts and has the ability to display entire functional protein domains, exemplified by the FimH lectin domain. The presence of heterologous cysteine...... bridges does not interfere with surface display, and Ag43 chimeras are correctly processed into alpha- and beta-modules, offering optional and easy release of the chimeric alpha-subunits. Furthermore, Ag43 can be displayed in many gram-negative bacteria. This feature is exploited for display of our...

  4. Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

    Science.gov (United States)

    Rashidi, Mahnaz; Galetto, Luciana; Bosco, Domenico; Bulgarelli, Andrea; Vallino, Marta; Veratti, Flavio; Marzachì, Cristina

    2015-09-30

    Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization. Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells. Artificial feeding

  5. A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

    DEFF Research Database (Denmark)

    Bengtsson, Dominique; Sowa, Kordai M; Salanti, Ali

    2008-01-01

    -ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy RESULTS: Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage...

  6. Varicellovirus UL49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

    NARCIS (Netherlands)

    Koppers-Lalic, D.; Verweij, M.C.; Lipinska, A.D.; Wang, Y.; Quinten, E.; Reits, E.A.; Koch, J.; Loch, S.; Rezende, M.M.; Daus, F.J.; Bienkowska-Szewczyk, K.; Osterrieder, N.; Mettenleiter, T.C.; Heemskerk, M.H.M.; Tampe, R.; Neefjes, J.J.; Chowdhury, S.I.; Ressing, M.E.; Rijsewijk, F.A.M.; Wiertz, E.J.H.J.

    2008-01-01

    Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing

  7. Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

    NARCIS (Netherlands)

    Koppers-Lalic, Danijela; Verweij, Marieke C.; Lipińska, Andrea D.; Wang, Ying; Quinten, Edwin; Reits, Eric A.; Koch, Joachim; Loch, Sandra; Rezende, Marisa Marcondes; Daus, Franz; Bieńkowska-Szewczyk, Krystyna; Osterrieder, Nikolaus; Mettenleiter, Thomas C.; Heemskerk, Mirjam H. M.; Tampé, Robert; Neefjes, Jacques J.; Chowdhury, Shafiqul I.; Ressing, Maaike E.; Rijsewijk, Frans A. M.; Wiertz, Emmanuel J. H. J.

    2008-01-01

    Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing

  8. The type-specific polysaccharide and the R protein antigens of the L-phase from a group B, type III Streptococcus.

    Science.gov (United States)

    Flores, A E; Ferrieri, P

    1985-04-01

    The type-specific polysaccharide and the R protein antigens from filtered culture supernatants of the bacterial phase and L-phase of the group B, type III streptococcal strain 76-043 were studied by several immunological methods. In the L-phase of growth, the two antigens were separate and distinct molecules which were found principally in the culture supernatant even on the 254th serial subculture in the cell-wall-defective state. Only trace amounts of these antigens were detected in extracts of L-phase cells. The type III polysaccharide antigens in the supernatant of cultures of the parent bacterium and the L-phase gave reactions of identity in immunodiffusion. Precipitin bands obtained by immunoelectrophoresis (IEP) revealed that the type-specific antigen of the bacterial phase of growth migrated toward the anode, whereas that of the L-phase remained near the antigen well. The R protein antigen in the L-phase supernatant was immunologically identical to the R protein of the supernatant and 1% trypsin-extracted antigens from whole cells of the parent bacterial strain, and other groups A, B and C streptococcal strains sharing a common R antigen. Immunologically, the R antigen appeared to be the species R4. The R protein of the L-phase and bacterial phase cultures was resistant to 5% trypsin but sensitive to 0.5% pepsin at 37 degrees C/2hr. Antiserum prepared in rabbits against L-phase cells contained an antibody reactive with the R protein antigens of the bacterial and L-phase cultures. The soluble, naturally released type III and R protein streptococcal antigens of the L-phase of growth permitted immunological confirmation of its bacterial origin.

  9. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  10. Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses.

    Directory of Open Access Journals (Sweden)

    Gustaf Ahlén

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

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

    Science.gov (United States)

    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

  12. IL-2/neuroantigen fusion proteins as antigen-specific tolerogens in experimental autoimmune encephalomyelitis (EAE): correlation of T cell-mediated antigen presentation and tolerance induction.

    Science.gov (United States)

    Mannie, Mark D; Clayson, Barbara A; Buskirk, Elizabeth J; DeVine, Jarret L; Hernandez, Jose J; Abbott, Derek J

    2007-03-01

    The purpose of this study was to assess whether the Ag-targeting activity of cytokine/neuroantigen (NAg) fusion proteins may be associated with mechanisms of tolerance induction. To assess this question, we expressed fusion proteins comprised of a N-terminal cytokine domain and a C-terminal NAg domain. The cytokine domain comprised either rat IL-2 or IL-4, and the NAg domain comprised the dominant encephalitogenic determinant of the guinea pig myelin basic protein. Subcutaneous administration of IL2NAg (IL-2/NAg fusion protein) into Lewis rats either before or after an encephalitogenic challenge resulted in an attenuated course of experimental autoimmune encephalomyelitis. In contrast, parallel treatment of rats with IL4NAg (IL-4/NAg fusion protein) or NAg lacked tolerogenic activity. In the presence of IL-2R(+) MHC class II(+) T cells, IL2NAg fusion proteins were at least 1,000 times more potent as an Ag than NAg alone. The tolerogenic activity of IL2NAg in vivo and the enhanced potency in vitro were both dependent upon covalent linkage of IL-2 and NAg. IL4NAg also exhibited enhanced antigenic potency. IL4NAg was approximately 100-fold more active than NAg alone in the presence of splenic APC. The enhanced potency of IL4NAg also required covalent linkage of cytokine and NAg and was blocked by soluble IL-4 or by a mAb specific for IL-4. Other control cytokine/NAg fusion proteins did not exhibit a similar enhancement of Ag potency compared with NAg alone. Thus, the IL2NAg and IL4NAg fusion proteins targeted NAg for enhanced presentation by particular subsets of APC. The activities of IL2NAg revealed a potential relationship between NAg targeting to activated T cells, T cell-mediated Ag presentation, and tolerance induction.

  13. Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

    Science.gov (United States)

    Lin, Mingqun; Bachman, Katherine; Cheng, Zhihui; Daugherty, Sean C; Nagaraj, Sushma; Sengamalay, Naomi; Ott, Sandra; Godinez, Al; Tallon, Luke J; Sadzewicz, Lisa; Fraser, Claire; Dunning Hotopp, Julie C; Rikihisa, Yasuko

    2017-07-01

    Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N. helminthoeca: a single small circular chromosome of 884 232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides and bacterioferritin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in southern California, Western blot analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain-variable surface antigen were uniformly recognized. Our finding will help understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large-scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  14. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...

  15. Crystal Structure of Neurotropism-Associated Variable Surface Protein 1 (VSP1) of Borrelia Turicatae

    Energy Technology Data Exchange (ETDEWEB)

    Lawson,C.; Yung, B.; Barbour, A.; Zuckert, W.

    2006-01-01

    Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to allow bacterial persistence in spite of an immune response. Two isogenic serotypes of Borrelia turicatae strain Oz1 differ in their Vsp sequences and in disease manifestations in infected mice: Vsp1 is associated with the selection of a neurological niche, while Vsp2 is associated with blood and skin infection. We report here crystal structures of the Vsp1 dimer at 2.7 and 2.2 Angstroms. The structures confirm that relapsing fever Vsp proteins share a common helical fold with OspCs of Lyme disease-causing Borrelia. The fold features an inner stem formed by highly conserved N and C termini and an outer 'dome' formed by the variable central residues. Both Vsp1 and OspC structures possess small water-filled cavities, or pockets, that are lined largely by variable residues and are thus highly variable in shape. These features appear to signify tolerance of the Vsp-OspC fold for imperfect packing of residues at its antigenic surface. Structural comparison of Vsp1 with a homology model for Vsp2 suggests that observed differences in disease manifestation may arise in part from distinct differences in electrostatic surface properties; additional predicted positively charged surface patches on Vsp2 compared to Vsp1 may be sufficient to explain the relative propensity of Vsp2 to bind to acidic glycosaminoglycans.

  16. FULL-LENGTH PEPTIDE ASSAY OF ANTIGENIC PROFILE OF ENVELOPE PROTEINS FROM SIBERIAN ISOLATES OF HEPATITIS C VIRUS

    Directory of Open Access Journals (Sweden)

    A. A. Grazhdantseva

    2010-01-01

    Full Text Available Antigenic profiles of envelope glycoproteins of hepatitis C virus presented by three genotypes 1b, 2a/2c and 3a, which are most widespread in the territory of Russia and, in particular, in Novosibirsk, were studied using a panel of overlapping synthetic peptides. It was shown that highly immunogenic peptide epitopes of Е1 and Е2 proteins common for all HCV genotypes, are located in amino acid positions 250-260, 315-325 (Е1 protein, 390-400 (hypervariable region 1, 430-440, and 680-690 (Е2 protein. The greatest inter-genotypic differences were recorded in positions 280-290, 410-430 and 520-540. A novel antigenic determinant was detected in the region of aa 280-290 of the Е1 protein which was typical only for HCV 2a/2c genotype. A broad variation in the boundaries for the most epitopes suggests a high variability of the Е1 and Е2 viral proteins; however, a similar repertoire of antibodies induced by different HCV genotypes indicates to an opportunity of designing a new generation of cross-reactive HCV vaccines based on mapping of the E1 and E2 antigenic regions.

  17. Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo [Seoul National Univ. Seoul (Korea, Republic of)

    2011-03-15

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA) based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA) based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R{sup 2=}0.838, P<0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R{sup 2=}0.067, P=0.316 by IRMA, and R{sup 2=}0.101, P=0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA based method in chronic hepatitis B patients.

  18. Hepatitis B Surface Antigen Seroprevalence among Children in Papua New Guinea, 2012–2013

    Science.gov (United States)

    Kitau, Russel; Sankar Datta, Siddhartha; Patel, Minal K.; Hennessey, Karen; Wannemuehler, Kathleen; Sui, Gerard; Lagani, William

    2015-01-01

    Approximately 8% of the population in Papua New Guinea (PNG) has chronic hepatitis B virus (HBV) infection. To decrease the burden of chronic HBV infection, a national 3-dose infant hepatitis B vaccination program was implemented starting in 1989, with a birth dose (BD) added to the schedule in 1992. To assess the impact of the hepatitis B vaccination program, we conducted a serosurvey among children born after vaccine introduction. During 2012–2013, a cross-sectional stratified four-stage cluster survey was conducted to estimate hepatitis B surface antigen (HBsAg) prevalence among children 4–6 years of age. We collected demographic data, vaccination history, and tested children for HBsAg. Of 2,133 participants, 2,130 children had vaccination data by either card or recall: 28% received a BD; 81% received ≥ 3 vaccine doses. Of 2,109 children providing a blood sample, 60 (2.3%) tested positive for HBsAg. This is the largest, most geographically diverse survey of hepatitis B vaccination and HBsAg seroprevalence done in PNG. Progress has been made in PNG toward the Western Pacific Regional goal to reduce the prevalence of chronic HBV infection to < 1% by 2017 among 5-year-old children. Vaccination efforts should be strengthened, including increasing BD coverage and completing the 3-dose series. PMID:25582692

  19. Sensitive prostate specific antigen quantification using dihydrolipoic acid surface-functionalized phosphorescent quantum dots.

    Science.gov (United States)

    García-Cortés, Marta; Fernández-Argüelles, María Teresa; Costa-Fernández, José M; Sanz-Medel, Alfredo

    2017-09-22

    Herein, high-quality Mn-doped ZnS quantum dots (QDs) have been synthesized using a facile approach directly in aqueous media. The surface of the obtained QDs was further modified by cap-exchange of the native cysteine shell with dihydrolipoic acid (DHLA) ligands resulting in nanocrystals with high water-stability having an intense phosphorescent signal. Covalent bioconjugation of the DHLA-coated nanoparticles with an anti-IgG antibody was then carried out. Interestingly the QD immunoprobe (QD-labelled antibodies) maintained an intense phosphorescence emission, without any significant spectral-shift (as compared to the free QDs). Coupling of an asymmetric flow field flow fractionation technique to an elemental mass spectrometry detection enabled the accurate determination of the efficiency of the bioconjugation reaction. The obtained nanoparticle-antibody bioconjugate was then applied to develop a quantitative sandwich-type phosphorescent immunoassay for Prostate Specific Antigen (PSA), and a limit of detection (LOD) of 17 pg mL -1 of PSA was achieved and allow to quantify such biomarker in samples within clinically relevant levels. Finally, the assay was validated for the quantification of PSA in the cellular media of prostate cancer cells. Obtained results proved the robustness of the proposed immunoassay based on long-lived phosphorescence measurements against eventual photoluminescent interferences significantly affecting the conventional short-lived fluorescence detection. Copyright © 2017. Published by Elsevier B.V.

  20. ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

    Science.gov (United States)

    Ahn, Hye-Jin; Kim, Sera; Kim, Dae-Yong

    2003-01-01

    An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 × His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals. PMID:12972732

  1. Reactive oxygen species modulator 1, a novel protein, combined with carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

    Science.gov (United States)

    Chen, Xianmeng; Zhang, Na; Dong, Jiahui; Sun, Gengyun

    2017-05-01

    The differential diagnosis of malignant pleural effusion and benign pleural effusion remains a clinical problem. Reactive oxygen species modulator 1 is a novel protein overexpressed in various human tumors. The objective of this study was to evaluate the diagnostic value of joint detection of reactive oxygen species modulator 1 and carcinoembryonic antigen in the differential diagnosis of malignant pleural effusion and benign pleural effusion. One hundred two consecutive patients with pleural effusion (including 52 malignant pleural effusion and 50 benign pleural effusion) were registered in this study. Levels of reactive oxygen species modulator 1 and carcinoembryonic antigen were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Results showed that the concentrations of reactive oxygen species modulator 1 both in pleural fluid and serum of patients with malignant pleural effusion were significantly higher than those of benign pleural effusion (both p carcinoembryonic antigen were 69.23% and 88.00%, respectively, at the cutoff value of 3.05 ng/mL, while serum carcinoembryonic antigen were 80.77% and 72.00% at the cutoff value of 2.60 ng/mL. The sensitivity could be raised to 88.17% in parallel detection of plural fluid reactive oxygen species modulator 1 and carcinoembryonic antigen concentration, and the specificity could be improved to 97.84% in serial detection.

  2. Hepatitis B surface antigen variants in voluntary blood donors in Nanjing, China

    Directory of Open Access Journals (Sweden)

    Yong-lin Yang

    2012-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV is still one of the serious infectious risks for the blood transfusion safety in China. One plausible reason is the emergence of the variants in the major antigenic alpha determinant within the major hydrophilic region (MHR of hepatitis B surface antigen (HBsAg, which have been assumed to evade the immune surveillance and pose a challenge to the disease diagnosis. It is well documented that some commercial ELISA kits could detect the wild-type but not the mutant viruses. The high prevalence of HBV in China also impaired the application of nucleic acid testing (NAT in the improvement of blood security. Molecular epidemiological study of HBsAg variations in China is still limited. This study was designed to identify the prevalence of mutations in the HBsAg in voluntary blood donors in Nanjing, China. Methods A total of 20,326 blood units were enrolled in this study, 39 donors were positive for HBV S gene in the nested-PCR. Mutations in the major hydrophilic region (MHR; aa 99-169 were identified by direct sequencing of S region. Results Among of 20,326 blood units in the Red Cross Transfusion Center of Nanjing from October 2008 to April 2009, 296 samples (1.46%, 296/20,326 were HBsAg positive in the 2 successive rounds of the ELISA test. In these HBsAg positive units, HBV S gene could be successfully amplified from 39 donors (13.18%, 39/296 in the nested-PCR. Sequence analysis revealed that 32 strains (82.1%, 32/39 belong to genotype B, 7 strains (17.9%, 7/39 to genotype C. Besides well known G145R, widely dispersed variations in the MHR of S region, were observed in 20 samples of all the strains sequenced. Conclusions HBV/B and HBV/C are dominant in Nanjing, China. The mutations in the MHR of HBsAg associated with disease diagnosis are common.

  3. The cathelicidin protein CRAMP is a potential atherosclerosis self-antigen in ApoE(-/- mice.

    Directory of Open Access Journals (Sweden)

    Peter M Mihailovic

    Full Text Available Auto-immunity is believed to contribute to inflammation in atherosclerosis. The antimicrobial peptide LL-37, a fragment of the cathelicidin protein precursor hCAP18, was previously identified as an autoantigen in psoriasis. Given the reported link between psoriasis and coronary artery disease, the biological relevance of the autoantigen to atherosclerosis was tested in vitro using a truncated (t form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE(-/- mice. Stimulation with tCRAMP resulted in increased CD8+ T cells with Central Memory and Effector Memory phenotypes in ApoE(-/- mice, differentially activated by feeding with normal chow or high fat diet. Immunization of ApoE(-/- with different doses of the shortened peptide (Cramp resulted in differential outcomes with a lower dose reducing atherosclerosis whereas a higher dose exacerbating the disease with increased neutrophil infiltration of the atherosclerotic plaques. Low dose Cramp immunization also resulted in increased splenic CD8+ T cell degranulation and reduced CD11b+CD11c+ conventional dendritic cells (cDCs, whereas high dose increased CD11b+CD11c+ cDCs. Our results identified CRAMP, the mouse homolog of hCAP-18, as a potential self-antigen involved in the immune response to atherosclerosis in the ApoE(-/- mouse model.

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

    Science.gov (United States)

    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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Enhancing recovery of recombinant hepatitis B surface antigen in lab-scale and large-scale anion-exchange chromatography by optimizing the conductivity of buffers.

    Science.gov (United States)

    Mojarrad Moghanloo, Gol Mohammad; Khatami, Maryam; Javidanbardan, Amin; Hosseini, Seyed Nezamedin

    2018-01-01

    In biopharmaceutical science, ion-exchange chromatography (IEC) is a well-known purification technique to separate the impurities such as host cell proteins from recombinant proteins. However, IEC is one of the limiting steps in the purification process of recombinant hepatitis B surface antigen (rHBsAg), due to its low recovery rate (rate of 82% in both lab-scale and large-scale weak anion-exchange chromatography without any harsh effect on the purity percentage of rHBsAg. The recovery enhancement via increasing the conductivity of Eq. and Wash. buffers can be explained by their roles in reducing the binding strength and aggregation of retained particles in the column. Moreover, further increase in the salt concentration of Elut. Buffer could substantially promote the ion exchange process and the elution of retained rHBsAg. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    Science.gov (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

  7. Serological versus molecular typing of surface-associated immune evading polysaccharide antigens-based phenotypes of Staphylococcus aureus.

    Science.gov (United States)

    Waryah, Charlene B; Gogoi-Tiwari, Jully; Wells, Kelsi; Costantino, Paul; Al-Salami, Hani; Sunagar, Raju; Isloor, Shrikrishna; Hegde, Nagendra; Richmond, Peter; Mukkur, Trilochan

    2014-11-01

    The aim of this study was to compare the performance of serological versus molecular typing methods to detect capsular polysaccharide (CP) and surface-associated polysaccharide antigen 336 phenotypes of Staphylococcus aureus isolates. Molecular typing of CP types 1, 5 and 8 was carried out using PCR, whereas serological typing of CP1, 2, 5, 8 and antigen 336 was carried out by slide agglutination using specific antisera. By genotyping, 14/31 strains were CP8 positive, 12/31 strains were CP5 and the remaining 6/31 isolates were non-typable (NT). One isolate was positive for both CP5 and CP8 by PCR, but was confirmed as CP8 type serologically. Detection of CP2 and type 336 by PCR was not possible because specific primers were either not available or non-specific. Using serotyping, 14/31 strains were CP8 positive, 11/31 CP5 positive and 2/31 positive for antigen 336. The remaining four S. aureus isolates were serologically NT. However, three of four NT and two 336-positive S. aureus isolates were encapsulated as determined by light microscopy after capsular staining. This discovery was surprising and warrants further investigations on the identification and characterization of additional capsular phenotypes prevalent among S. aureus clinical isolates. It was concluded that serological typing was a better method than molecular typing for use in epidemiological investigations based upon the distribution of surface-associated polysaccharide antigens-based phenotypes. © 2014 The Authors.

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

    Science.gov (United States)

    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. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. [Cloning, expression and antigenic analysis of VP1-VP4 gene encoding the structural protein of Coxsackie virus A16].

    Science.gov (United States)

    Song, Yuanbin; He, Sijie; Yu, Nan; Chen, Xinxin; Wang, Bin; Che, Xiaoyan; Zeng, Qiyi

    2012-12-01

    To clone and express VP1-VP4 genes encoding the structural proteins of Coxsackie virus A16 and analyze the antigenicity of the expressed recombinant proteins. The VP1-VP4 cDNAs were amplified with RT-PCR from the extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4 genes were inserted to the multi-cloning sites of the plasmid pQE30a, and the protein expressions in E. coli M15 were induced by IPTG. After purification by washing with 8 mol/L urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their immunogenicity against rabbit antisera of Coxsackie virus A16 and enterovirus 71, respectively. The recombinant VP1-VP4 proteins were highly expressed in E. coli M15. The purified proteins could be recognized by rabbit antiserum of Coxsackie virus A16 and showed cross reactivity with the rabbit antiserum of Enterovirus 71. The recombinant Coxsackie virus A16 VP1-VP4 proteins obtained possess good antigenicity.

  10. Quantitative analysis of rat Ig (sub)classes binding to cell surface antigens

    International Nuclear Information System (INIS)

    Nilsson, R.; Brodin, T.; Sjoegren, H.-O.

    1982-01-01

    An indirect 125 I-labeled protein A assay for detection of cell surface-bound rat immunoglobulins is presented. The assay is quantitative and rapid and detects as little as 1 ng of cell surface-bound Ig. It discriminates between antibodies belonging to different IgG subclasses, IgM and IgA. The authors describe the production and specificity control of the reagents used and show that the test can be used for quantitative analysis. A large number of sera from untreated rats are tested to evaluate the frequency of falsely positive responses and variation due to age, sex and strain of rat. With this test it is relatively easy to quantitate the binding of classes and subclasses of rat immunoglobulins in a small volume (6 μl) of untreated serum. (Auth.)

  11. Synthesis and structural insight into ESX-1 Substrate Protein C, an immunodominant Mycobacterium tuberculosis-secreted antigen.

    Science.gov (United States)

    Son, Soo Jung; Harris, Paul W R; Squire, Chris J; Baker, Edward N; Brimble, Margaret A

    2016-05-01

    Tuberculosis, the second leading cause of death from a single infectious agent, is recognized as a major threat to human health due to a lack of practicable vaccines against the disease and the widespread occurrence of drug resistance. With a pressing need for a novel protein target as a platform for new vaccine development, ESX-1 Substrate Protein C (EspC) was recently identified as a novel Mycobacterium tuberculosis-secreted antigen that is as immunodominant as the two specific immunodiagnostic T-cell antigens, CFP-10 and ESAT-6. Here, we present the first chemical total synthesis, folding conditions, and circular dichroism data of EspC. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 267-274, 2016. © 2016 Wiley Periodicals, Inc.

  12. An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1 Governs Ligand Binding Selectivity.

    Directory of Open Access Journals (Sweden)

    Michelle L Parker

    Full Text Available Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs on the parasite surface and rhoptry neck 2 (RON2 proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop. Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2 peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON

  13. Serological responses to Cryptosporidium antigens in inhabitants of Hungary using conventionally filtered surface water and riverbank filtered drinking water.

    Science.gov (United States)

    Farkas, K; Plutzer, J; Moltchanova, E; Török, A; Varró, M J; Domokos, K; Frost, F; Hunter, P R

    2015-10-01

    In this study the putative protective seroprevalence (PPS) of IgG antibodies to the 27-kDa and 15/17-kDa Cryptosporidium antigens in sera of healthy participants who were and were not exposed to Cryptosporidium oocysts via surface water-derived drinking water was compared. The participants completed a questionnaire regarding risk factors that have been shown to be associated with infection. The PPS was significantly greater (49-61%) in settlements where the drinking water originated from surface water, than in the control city where riverbank filtration was used (21% and 23%). Logistic regression analysis on the risk factors showed an association between bathing/swimming in outdoor pools and antibody responses to the 15/17-kDa antigen complex. Hence the elevated responses were most likely due to the use of contaminated water. Results indicate that waterborne Cryptosporidium infections occur more frequently than reported but may derive from multiple sources.

  14. Prevalence of Hepatitis B surface antigen in children with sickle cell anemia

    Directory of Open Access Journals (Sweden)

    Baba Jibrin

    2014-01-01

    Full Text Available Background: Hepatitis B virus is known to be endemic in Africa. The seroepidemiological studies of HBV have shown that infection commonly occurs in childhood in Africa resulting in an increased tendency to chronicity. This cross-sectional study was undertaken to determine the seroprevalence of hepatitis B surface antigen among pediatric patients with homozygous hemoglobin S. Materials and Methods: Three hundred sickle cell anemia children aged 6 months-15 years (both in steady state and in crises attending the SCA clinic and on admission in emergency pediatrics unit and pediatrics medical ward, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, were screened for hepatitis B infection using HBsAg as marker of infection. The sensitive enzyme linked immunosorbent assay method was used for detection of the marker. Three hundred children with minor illness attending pediatrics outpatient department and on admission in EPU/PMW for various treatment in the same hospital served as gender- and age-marched controls cohorts. Results: The sero-prevalence of HBsAg seropositivity for hepatitis B virus infection among SCA children was 17.3% (52/300 compared to 10.7% (32/300 of the control (P = 0.0875. The peak prevalence age group for HBV infection among SCA children was in the age group 1.1-5.0 years (6% compared to 10.1-15.0 years (4.7% in the control. Risk factors for HBV infection such as blood transfusion, traditional scarification/circumcision/uvulectomy, and tattooing did not significantly affect the prevalence of HBV infection in both SCA children and controls. Conclusion: Hepatitis B infection is common in Sokoto. The need for strict adherence to HBV immunization and further community-based studies on the risk factors are recommended.

  15. PRISM hepatitis B surface antigen detection of hepatits B virus minipool nucleic acid testing yield samples.

    Science.gov (United States)

    Linauts, Sandy; Saldanha, John; Strong, D Michael

    2008-07-01

    Hepatitis B virus (HBV) residual risk has been estimated at 1:63,000-1:205,000 and introduction of more sensitive serological tests and nucleic acid testing (NAT) would reduce that risk. Sensitivity of the recently licensed Abbott PRISM hepatitis B surface antigen (HBsAg) CLIA and minipool (MP) HBV NAT has been described as comparable and thus the need for HBV NAT has not been compelling. In this study, eight samples identified as yield samples with MP HBV NAT were tested using the PRISM test. Seven samples were identified using the Roche COBAS AmpliScreen HBV test and one additional sample was obtained from the clinical trial for the Roche cobas TaqScreen MPX test. Each of these samples was reactive by MP HBV NAT and nonreactive for HBsAg using one of three licensed enzyme immunoassay (EIA) tests. After licensure of the PRISM HBsAg, aliquots were tested with this assay, and DNA quantitation and genotyping were repeated where sample volume permitted. Three samples (2000, 2300, and 61,000 copies/mL) produced reactive results with PRISM. Four samples with viral loads less than 300 copies per mL produced nonreactive results. One sample, originally quantitated at 37,000 copies per mL (but 3850 copies/mL in repeat testing) was also nonreactive by PRISM. Genotyping of this sample indicated a type C genotype with no mutations. Adding serological sensitivity of PRISM CLIA reduced the NAT yield from the original 1: 385,555 to 1:610,488. However, MP HBV NAT still provides additional sensitivity over CLIA, even for a donation with a viral load of almost 4000 copies per mL.

  16. Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

    Science.gov (United States)

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

    2014-02-01

    Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.

  17. Higher lifetime chance of spontaneous surface antigen loss in hepatitis B carriers with genotype C infection.

    Science.gov (United States)

    Tseng, T-C; Liu, C-J; Chen, C-L; Yang, W-T; Yang, H-C; Su, T-H; Wang, C-C; Kuo, S F-T; Liu, C-H; Chen, P-J; Chen, D-S; Kao, J-H

    2015-05-01

    Clearance of hepatitis B surface antigen (HBsAg) indicates clinical control of hepatitis B virus (HBV) infection. However, little is known about the impact of viral genomic variations on HBsAg loss. We explored the association between viral genomic factors and HBsAg loss in 2121HBeAg-negative patients. HBV pre-core stop codon (1896) and basal core promoter (BCP) (1762/1764) sequences were determined in patients with HBV DNA ≥200 IU/mL (N = 1693). The effect of HBV genotype on HBsAg loss was further validated in the whole cohort of 3445 HBsAg carriers. The cumulative lifetime (age 28-75 years) incidence of HBsAg loss was 50.4% in 2121 HBeAg-negative patients. We found that genotype C, but not pre-core stop codon or BCP mutants, was associated with HBsAg loss. Compared to genotype B patients, genotype C patients had higher lifetime chance of HBsAg loss, with hazard ratio of 1.8 (95% confidence interval: 1.4-2.4). Multivariable analysis showed that male sex, elevated ALT levels, lower serum HBV DNA and HBsAg levels, and genotype C infection were associated with higher chance of HBsAg loss independently. We then performed sensitivity analysis, which re-included HBeAg-positive, cirrhotic and treatment-experienced patients, and confirmed the robustness of our results in 3445 HBsAg carriers. Genotype C infection, compared to genotype B, is associated with a higher lifetime chance of HBsAg loss in Asian HBV carriers. © 2015 John Wiley & Sons Ltd.

  18. An overview of serum prostatic surface antigen cut points for recommendation of prostatic biopsy.

    Science.gov (United States)

    Patwardhan, Sujata K; Patil, Bhushan P; Shelke, Umesh Ravikant; Singh, Abhishek G

    2018-01-01

    Patients in India frequently present with prostatic surface antigen (PSA) report and request for prostatic biopsy to rule out malignancy. With fear of harboring malignancy set in patient's mind, it becomes difficult to counsel them about absolute indications and need of biopsy. Whether serum PSA has same predictability in symptomatic patients in the Indian context for advising prostatic biopsy at same reference ranges as in western countries, remains to be answered. Symptomatic patients between 45 and 70 years of age presenting with either raised serum PSA (>4 ng/ml) reports or abnormal digital rectal examination (DRE) were considered as cases. Standard 12 core transrectal ultrasound-guided prostatic biopsy was done. Statistical analysis using optimal cut points, an R package was done to overview different PSA cut points for the recommendation of prostatic biopsy. A total of 534 patients were included. Mean age was 64 years. Malignancy was detected in total 77 patients (14.42%). Malignancy was identified in 3.59% (10/279) and 30% (63/210) patients at serum PSA ranges 4-10 ng/ml and serum PSA >10 ng/ml, respectively. Both, maximum sensitivity and specificity were found at PSA cut point 9.7 ng/ml. We evaluated these patients to identify the PSA cut point above which unnecessary biopsies will be avoided. We kept power of study maximum, i.e., 1 with confidence interval of 0.95. PSA value 9.7 ng/ml should be considered as the cut point above which prostatic biopsy should be done to avoid unnecessary biopsies. Unless accompanied by abnormal DRE finding at PSA range 4-10 ng/ml, morbidity of prostatic biopsy procedure can be avoided using this cut-point.

  19. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    Science.gov (United States)

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

  20. Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

    Directory of Open Access Journals (Sweden)

    Yakov Lomakin

    2017-07-01

    Full Text Available Multiple sclerosis (MS is an autoimmune chronic inflammatory disease of the central nervous system (CNS. Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV latent membrane protein 1 (LMP1. In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  1. Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

    Science.gov (United States)

    Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

    2017-01-01

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading. PMID:28729867

  2. Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

    Science.gov (United States)

    Lomakin, Yakov; Arapidi, Georgii Pavlovich; Chernov, Alexander; Ziganshin, Rustam; Tcyganov, Evgenii; Lyadova, Irina; Butenko, Ivan Olegovich; Osetrova, Maria; Ponomarenko, Natalia; Telegin, Georgy; Govorun, Vadim Markovich; Gabibov, Alexander; Belogurov, Alexey

    2017-01-01

    Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo . We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

  3. Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles

    DEFF Research Database (Denmark)

    Hummel, R; Nørgaard, P; Andreasen, P H

    1992-01-01

    The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically...... of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place. In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T. thermophila...... to vertebrates. Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen. The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns...

  4. Characterization of rat basophilic leukemia cell surface proteins using monoclonal antibodies

    International Nuclear Information System (INIS)

    Buonocore-Buzzelli, L.M.

    1988-01-01

    Rat basophilic leukemia (RBL) cells express both immunoglobulin E (IgE) and immunoglobulin G (IgG) receptors. In this study, mouse monoclonal antibodies were produced against the RBL cell and screened for their ability to precipitate specific bands from 125 I surface labeled cells. Fourteen hybridomas were selected and divided into five groups since many of the hybridomas precipitated bands of identical molecular weight. One or more of the hybridomas from each group, and the cell surface antigens they identified, were further characterized. Binding of all the monoclonal antibodies to the RBL-2H3 cell surface was saturable and of high affinity. In cross inhibition studies, two of the antibodies were found to bind to identical or neighboring epitopes, presumably on the same cell surface molecule. Binding studies using other cell populations demonstrated that the monoclonal antibodies react not only with commonly expressed rat cell surface molecules but also with molecules specifically expressed on rat mast cells and basophils. None of the antibodies were found to induce or inhibit serotonin release from the RBL cells. Western blotting showed most of the antibodies to react with bands whose molecular weights resembled those seen by immuno-precipitation. Antibodies number sign 8 and number sign 12, although from the same group, were found to react with different subunits of the same cell surface protein. Sequential immunoprecipitation and peptide mapping confirmed that the antigens defined by these antibodies were structurally related

  5. Prevalence of Hepatitis B Surface Antigen in US-Born and Foreign-Born Asian/Pacific Islander College Students

    Science.gov (United States)

    Quang, Yen N.; Vu, Joanne; Yuk, Jihey; Li, Chin-Shang; Chen, Moon; Bowlus, Christopher L.

    2010-01-01

    The prevalence of chronic hepatitis B (HBV) among college-age US-born Asian and Pacific Islanders (A/PI) is not well known. Objectives: To compare the prevalence of hepatitis B surface antigen (HBsAg) seropositivity in US-born to A/PI-born students at a public university. Participants: Undergraduate who self-identified themselves as A/PI. Results:…

  6. Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

    OpenAIRE

    Handman, E; Symons, F M; Baldwin, T M; Curtis, J M; Scheerlinck, J P

    1995-01-01

    Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. ...

  7. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

    OpenAIRE

    Clark, R; Peden, K; Pipas, J M; Nathans, D; Tjian, R

    1983-01-01

    We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of th...

  8. RPE cell surface proteins in normal and dystrophic rats

    International Nuclear Information System (INIS)

    Clark, V.M.; Hall, M.O.

    1986-01-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

  9. Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

    Directory of Open Access Journals (Sweden)

    Amelia M Clark

    Full Text Available Antigenic drift of the hemagglutinin (HA and neuraminidase (NA influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses accounts for the limited effectiveness (around 40% of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI and HA-specific neutralizing serum antibody (Ab titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to

  10. Conserved cysteine residues provide a protein-protein interaction surface in dual oxidase (DUOX) proteins.

    Science.gov (United States)

    Meitzler, Jennifer L; Hinde, Sara; Bánfi, Botond; Nauseef, William M; Ortiz de Montellano, Paul R

    2013-03-08

    Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.

  11. Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

    Science.gov (United States)

    Sánchez, Alemao G Carpinteyro; Virginio, Veridiana Gomes; Maschio, Vinicius José; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2016-10-01

    Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

  12. Monte Carlo Simulation of Protein Adsorption on Energetically Heterogeneous Surfaces

    OpenAIRE

    Danwanichakul, Panu

    2014-01-01

    The modified triangular-well potential model was applied to incorporate the effect of surface energy on the adsorption of particles or proteins on energetically heterogeneous surfaces. The method is convenient in simulating the adsorption on heterogeneous surface of which different region possesses different free energy. Spherical particles with attractive forces were added on the surface and underwent surface diffusion before they were quenched in place. It was seen that the ratio of surface...

  13. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

    Science.gov (United States)

    Montanaro, Jacqueline; Inic-Kanada, Aleksandra; Ladurner, Angela; Stein, Elisabeth; Belij, Sandra; Bintner, Nora; Schlacher, Simone; Schuerer, Nadine; Mayr, Ulrike Beate; Lubitz, Werner; Leisch, Nikolaus; Barisani-Asenbauer, Talin

    2015-01-01

    To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

  14. Surface Antigen Profiling of Helicobacter pylori-Infected and -Uninfected Gastric Cancer Cells Using Antibody Microarray.

    Science.gov (United States)

    Sukri, Asif; Hanafiah, Alfizah; Kosai, Nik Ritza; Mohamed Taher, Mustafa; Mohamed Rose, Isa

    2016-10-01

    Comprehensive immunophenotyping cluster of differentiation (CD) antigens in gastric adenocarcinoma, specifically between Helicobacter pylori-infected and -uninfected gastric cancer patients by using DotScan(™) antibody microarray has not been conducted. Current immunophenotyping techniques include flow cytometry and immunohistochemistry are limited to the use of few antibodies for parallel examination. We used DotScan(™) antibody microarray consisting 144 CD antibodies to determine the distribution of CD antigens in gastric adenocarcinoma cells and to elucidate the effect of H. pylori infection toward CD antigen expression in gastric cancer. Mixed leukocytes population derived from gastric adenocarcinoma patients were immunophenotyped using DotScan(™) antibody microarray. AGS cells were infected with H. pylori strains and cells were captured on DotScan(™) slides. Cluster of differentiation antigens involved in perpetuating the tolerance of immune cells to tumor cells was upregulated in gastric adenocarcinoma cells compared to normal cells. CD279 which is essential in T cells apoptosis was found to be upregulated in normal cells. Remarkably, H. pylori-infected gastric cancer patients exhibited upregulated expression of CD27 that important in maintenance of T cells. Infection of cagA+ H. pylori with AGS cells increased CD antigens expression which involved in cancer stem cell while cagA- H. pylori polarized AGS cells to express immune-regulatory CD antigens. Increased CD antigens expression in AGS cells infected with cagA+ H. pylori were also detected in H. pylori-infected gastric cancer patients. This study suggests the tolerance of immune system toward tumor cells in gastric cancer and distinct mechanisms of immune responses exploited by different H. pylori strains. © 2016 John Wiley & Sons Ltd.

  15. Effect of a novel saponin adjuvant derived from Quillaja saponaria on the immune response to recombinant hepatitis B surface antigen.

    Science.gov (United States)

    So, H S; Yoon, H S; Choi, D Y; Kwon, Y S; Sung, J H; Lee, T G; Park, E S; Cho, H S; Lee, B M; Cho, J M; Ryu, W S

    1997-04-30

    Adjuvant activity of saponins extracted from the South American tree Quillaja saponaria has been demonstrated with many antigens. Recently, four saponin fractions (designated as QS-7, QS-17, QS-18, and QS-21) with adjuvant activity were purified by reverse phase chromatography. In particular, efficacy of the less toxic QS-21 fraction has been demonstrated with several recombinant viral antigens including HIV gp120. Here, we report a novel saponin fraction (designated as QS-L1) derived from Quillaja saponaria. Unlike previously identified saponins, QS-L1 had a different chemical structure and showed adjuvant activity only when administered in the presence of alum-precipitated antigen. Interestingly, the QS-L1 greatly increased not only a humoral immune response but also cellular immune response to recombinant hepatitis B virus surface antigen (HBsAg). Furthermore, QS-L1 showed lower toxicity in vivo and in vitro than the previously identified saponin fraction, QS-21. Finally, we examined the chemical structure of the QS-L1 using mass spectroscopic analysis, carbohydrate composition analysis and NMR spectroscopic analysis. Thus, our results indicated that this novel QS-L1 saponin fraction had several desirable properties required for an effective adjuvant.

  16. Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.

    Science.gov (United States)

    Gospodarek, Adrian M; Sun, Weitong; O'Connell, John P; Fernandez, Erik J

    2014-12-05

    In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Invariant Chain Modulates HLA Class II Protein Recycling and Peptide Presentation in Nonprofessional Antigen Presenting Cells

    OpenAIRE

    Haque, Azizul; Hajiaghamohseni, Laela M.; Li, Ping; Toomy, Katherine; Blum, Janice S.

    2007-01-01

    The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecu...

  18. CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens

    OpenAIRE

    Sharma, Sharad K.; Roumanes, David; Almudevar, Anthony; Mosmann, Tim R.; Pichichero, Michael E.

    2013-01-01

    We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing...

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Developmental expression of a cell surface protein involved in sea urchin skeleton formation. [Strongylocentrotus purpuratus; Lytechinus pictus

    Energy Technology Data Exchange (ETDEWEB)

    Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

    1986-05-01

    The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with (/sup 3/H) leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts.

  1. Novel Plasmodium falciparum malaria vaccines: evidence-based searching for variant surface antigens as candidates for vaccination against pregnancy-associated malaria

    DEFF Research Database (Denmark)

    Staalsoe, Trine; Jensen, Anja T R; Theander, Thor G

    2002-01-01

    Malaria vaccine development has traditionally concentrated on careful molecular, biochemical, and immunological characterisation of candidate antigens. In contrast, evidence of the importance of identified antigens in immunity to human infection and disease has generally been limited......) in particular, to provide robust evidence of a causal link between the two in order to allow efficient and evidence-based identification of candidate antigens for malaria vaccine development....... to statistically significant co-variation with protection rather than on demonstration of causal relationships. We have studied the relationship between variant surface antigen-specific antibodies and clinical protection from Plasmodium falciparum malaria in general, and from pregnancy-associated malaria (PAM...

  2. Leishmania infantum: gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity.

    Science.gov (United States)

    Larreta, R; Soto, M; Alonso, C; Requena, J M

    2000-10-01

    The complete nucleotide sequence for the Leishmania infantum homologue to the glucose-regulated protein 94 (GRP94) gene was determined from the isolation and characterization of a genomic clone. Like the mammalian and plant GRP94s, the L. infantum GRP94 sequence possesses both an N-terminal signal peptide and a putative endoplasmic reticulum retention signal, consisting of the C-terminal tetrapeptide EDDL. Thus, L. infantum is the first protozoan organism in which GRP94 has been identified. Southern blot analysis has indicated that this protein is encoded by a single-copy gene. The L. infantum GRP94 gene was expressed in Escherichia coli and the recombinant protein used to evaluate its antigenicity and immunogenicity. Eighty-four percent of sera from dogs with visceral leishmaniasis reacted with the protein, indicating that GRP94 is a potent immunogen during Leishmania infection. Given the immunogenic and antigenic properties shown by the L. infantum GRP94, we think that this protein constitutes a valuable molecule for diagnostic purposes and a potential candidate for studies of protective immunogenicity. Copyright 2000 Academic Press.

  3. A small antigenic determinant of the Chikungunya virus E2 protein is sufficient to induce neutralizing antibodies which are partially protective in mice.

    Directory of Open Access Journals (Sweden)

    Christopher Weber

    2015-04-01

    Full Text Available The mosquito-borne Chikungunya virus (CHIKV causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses.E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L and surface-exposed parts of the E2 domain A (sA alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+ was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+ induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA, MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice.The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.

  4. Trichinella britovi human infection in Spain : antibody response to surface, excretory/secretory and somatic antigens

    Directory of Open Access Journals (Sweden)

    Rodríguez-Osorio M.

    2003-06-01

    Full Text Available A third outbreak of Trichinella britovi with 140 people involved, occurred in Granada Spain (December 1998. The source of infection was sausage made from uninspected wild boar meat. Fifty-two patients agreed to participated in this study. An elevated eosinophil level (> 5 % was detected in 59.6 % of patients, and persisted in most of these cases for two months. A moderate IgG response was observed. At the onset of symptoms, Western blot (WB test detected more positive cases than Enzyme linked immunosorbent assay (ELISA and indirect immunofluorescence (IIF. Six months from infection, ELISA revealed fewer positive cases than the other two tests. It would appear that the response to somatic antigens starts earlier than those to cuticular and excretory/secretory (ES antigens and that the response to ES antigens is the first to decrease.

  5. Mechanisms of recurrent otitis media: importance of the immune response to bacterial surface antigens.

    Science.gov (United States)

    Murphy, T F; Yi, K

    1997-12-29

    Otitis-prone children experience recurrent episodes of otitis media due to nontypeable H. influenzae (NTHI). A protective immune response occurs following infection, but this immune response is specific for the infecting strain, leaving the child susceptible to infection by other strains of NTHI. Little is known about the mechanism by which a strain-specific antibody response occurs to nonencapsulated bacteria. To explore the mechanism by which this strain-specific response occurs, animals were inoculated with whole bacterial cells and the antibody response was studied. The antibody response was predominantly directed to a highly strain-specific, immunodominant surface loop on the major outer membrane protein. This exquisitely restricted immune response leaves the host susceptible to recurrent infections by many strains of NTHI. The ability of the bacterium to direct the host to make a strain-specific antibody response has important implications in understanding the immune response to otitis media due to NTHI and in designing strategies for vaccine development.

  6. Expression of myeloid antigens on lymphoblast surface in childhood acute lymphoblastic leukemia at diagnosis and its effect on early response to treatment: a preliminary report.

    Science.gov (United States)

    Sobol-Milejska, Grazyna; Mizia-Malarz, Agnieszka; Wos, Halina

    2013-09-01

    Immunodiagnosis of acute lymphoblastic leukemia (ALL) is based on the assessment of surface antigens. There are also cases in which both lymphoid and myeloid antigens can be found on the surface of lymphoblasts. The purpose of our research was to assess the expression of myeloid and lymphoblastic antigens in children with ALL, and to determine the impact of surface antigens on early response to treatment. 58 children [33 girls (56.9 %), 25 boys (43.2 %)] with ALL were studied. Response to treatment was assessed on days 8, 15, and 33. Univariate logistic regression analysis of the effect of myeloid antigens (MyAg) on response to treatment on days 8 and 33 revealed expression of any MyAg on lymphoblast surface as a factor associated with poor response to treatment. The multivariate logistic regression analysis of treatment response on day 33, showed that the expression of CD13 antigen on lymphoblast surface is a key factor affecting delayed remission (p = 0.03; odds ratio 0.12; 95 % CI 0.01-0.81). The expression of MyAg in childhood ALL adversely affects early response to treatment. The expression of CD13 antigen on day 33 is a key factor affecting complete remission in ALL patients.

  7. The Murine Humoral Immune Response to Hepatitis B Surface Antigen: Idiotype Network Pathways.

    Science.gov (United States)

    Schick, Michael Roy

    Recognition of a wide spectrum in disease outcomes following Hepatitis B Virus (HBV) infection has led to the suggestion that individual differences may be due to characteristics of the immune response. HBV, a hepatotropic virus, is not directly cytopathic to the host hepatocytes but the cellular damage which does not occur may be due to the host's own immune response. It is this variety in immune response capabilities following natural infection or vaccination which led to the present study in which the murine humoral immune response to hepatitis B surface antigen (HBsAg) was examined. Following immunization with purified HBsAg an anti-HBs response could be detected in 19 inbred strains of mice. The response, which varied among the strains, was linked to the major histocompatibility complex (MHC). Among high responders to HBsAg were two strains in which a poor response to a single epitope could be detected. Although quantitatively serum from these strains resembled serum from other high responders, there was a major difference in the qualitative aspects. Included within this study was the role of idotype networks within the murine anti-HBs response. By directly targeting HBsAg-specific B cells within the framework of an idiotype network by an Ab-2, it was possible to circumvent T cell-dependent regulation of an immune response. In each of five inbred strains of mice immunized with a polyclonal rabbit Ab-2 an Ab-3 population with HBsAg-specificity (Ab -1^') was induced. These mice were also immunized with HBsAg resulting in a higher anti-HBs response as compared to HBsAg immunization alone in all of the strains tested except for one. The response in this strain, normally a low responder to HBsAg, indicated that the mechanisms for genetic restriction of the anti -HBs response was still active, although it was not apparent during anti-Id immunization. The effects of an anti-Id on the murine antibody response to HBsAg may lead to insights on the presence of idiotype

  8. SURF'S UP! – Protein classification by surface comparisons

    Indian Academy of Sciences (India)

    Prakash

    Surf's Up – Protein Classification by Surface Comparisons. 97. J. Biosci. 32(1), January 2007. 1. Introduction. With an increasing number of experimentally uncharacterized protein sequences and structures produced by genome sequencing or structural genomic initiatives, we often encounter large protein families with only ...

  9. Directed Supramolecular Surface Assembly of SNAP-tag Fusion Proteins

    NARCIS (Netherlands)

    Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, Hoang D.; Schenkel, J.H.; Huskens, Jurriaan; Ravoo, B.J.; Jonkheijm, Pascal; Brunsveld, Luc

    2012-01-01

    Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

  10. VASCo: computation and visualization of annotated protein surface contacts

    Directory of Open Access Journals (Sweden)

    Thallinger Gerhard G

    2009-01-01

    Full Text Available Abstract Background Structural data from crystallographic analyses contain a vast amount of information on protein-protein contacts. Knowledge on protein-protein interactions is essential for understanding many processes in living cells. The methods to investigate these interactions range from genetics to biophysics, crystallography, bioinformatics and computer modeling. Also crystal contact information can be useful to understand biologically relevant protein oligomerisation as they rely in principle on the same physico-chemical interaction forces. Visualization of crystal and biological contact data including different surface properties can help to analyse protein-protein interactions. Results VASCo is a program package for the calculation of protein surface properties and the visualization of annotated surfaces. Special emphasis is laid on protein-protein interactions, which are calculated based on surface point distances. The same approach is used to compare surfaces of two aligned molecules. Molecular properties such as electrostatic potential or hydrophobicity are mapped onto these surface points. Molecular surfaces and the corresponding properties are calculated using well established programs integrated into the package, as well as using custom developed programs. The modular package can easily be extended to include new properties for annotation. The output of the program is most conveniently displayed in PyMOL using a custom-made plug-in. Conclusion VASCo supplements other available protein contact visualisation tools and provides additional information on biological interactions as well as on crystal contacts. The tool provides a unique feature to compare surfaces of two aligned molecules based on point distances and thereby facilitates the visualization and analysis of surface differences.

  11. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

    DEFF Research Database (Denmark)

    Skjøt, R L; Oettinger, T; Rosenkrands, I

    2000-01-01

    ), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

  12. Proteome of conidial surface associated proteins of Aspergillus fumigatus reflecting potential vaccine candidates and allergens.

    Science.gov (United States)

    Asif, Abdul R; Oellerich, Michael; Amstrong, Victor W; Riemenschneider, Birgit; Monod, Michel; Reichard, Utz

    2006-04-01

    Aspergillus fumigatus is a mold causing most of the invasive fungal lung infections in the immunocompromised host. In addition, the species is the causative agent of certain allergic diseases. Both in invasive and in allergic diseases, the conidial surface mediates the first contact with the human immune system. Thus, conidial surface proteins may be reasonable vaccine candidates as well as important allergens. To broaden the list of those antigens, intact viable Aspergillus conidia were extracted with mild alkaline buffer at pH 8.5 in the presence of a 1,3-beta-glucanase. The proteome of this fraction was separated by two- dimensional gel electrophoresis (2-DE) and analyzed by liquid chromatography coupled with tandem mass spectrometry. Altogether 26 different A. fumigatus proteins were identified, twelve of which contain a signal for secretion. Among these were the known major conidial surface protein rodlet A, one acid protease PEP2, one lipase, a putative disulfide isomerase and a putative fructose-1,6-biphosphatase. The known allergen Aspf 3 was identified among the proteins without a signal for secretion. On the basis of the recently annotated A. fumigatus genome (Nature 2005, 438, 1151-1156), proteome analysis is now a powerful tool to confirm expression of hypothetical proteins and, thereby to identify additional vaccine candidates and possible new allergens of this important fungal pathogen.

  13. Immunity of Surface Layer Protein of Aeromonas ‎hydrophila in Rabbits

    Directory of Open Access Journals (Sweden)

    Lobna Adil Al-Noori

    2017-11-01

    Full Text Available In this study the Surface layer (S-layer protein was extracted from Aeromonas hydrophila bacteria ,the humoral immune response that induced by S-layer protein only or as adjuvant was investigated  by using 16 males New Zealand rabbits and divided into four groups, each group contained four rabbits, the first group was immunized with  S-layer protein only, the second group was immunized with heated killed antigen(HKAof Sallmonella typhi only, the third group was immunized with mixed antigens (S-layer+ HKA,while the fourth group considered as control group and immunized with normal saline. The HKA of S. typhi  was used to evaluate the efficiency of S-layer protein as adjuvant. After the immunization period, the humoral immune response was investigated by several tests include, tube agglutination test and passive agglutination test that used to detect the antibody titer. Biuret method was used to determine the total protein concentration in serum  samples and total protein concentration of secretory immunoglobulin that extracted form appendix samples. In addition the Radical Immunodiffution (RID  method was used to detect the concentration level of the IgG in serum samples. Moreover the concentration level of the CD4 in the serum samples was determined by enzyme linked immunosorbent assay (ELISA method .In all these tests the result revealed, both of S-layer protein only , HKA of only and mixed antigens(S-layer+ HKA were given significantly increased in comparison with control group at P<0.05. The result showed that the  concentration level of IgG with mean values (2365.5 , 3505 and 2916 mg/dl respectively  while the control group with mean value (1662mg/dl. In addition the concentration  level of CD4 molecule with mean values (9.37, 11.77 and 17.36 ng/ml respectively while the control group with mean value (6.91 ng/ml .The results showed that these three types of antigens induced the humoral immune response

  14. Prediction of antigenic sites on ALS1 and HWP1 protein sequences in vaginal isolated C. albicans of using bioinformatics analysis

    Directory of Open Access Journals (Sweden)

    Mona Pakdel

    2015-04-01

    Full Text Available Background and Aim: The ability to predict antigenic sites on proteins is of major importance for medication. The aim of this study was to predict the antigenic sites on Agglutin in Like Sequence (ALS1 and Hyphal Wall Protein Sequences (HWP1 in Candida albicans isolated of vaginal infections using Physico-Chemical Profiles server. Materials and Methods: 7 isolates were obtained from women with vaginal infection which were collected from various medical centers of Tehran in 2011 and 2012. At the first,DNA was extracted  by Phenol-Chloroform method. Multiplex PCR was performed by using specific primers. In order to do bioinformatic studies, the genes were sequenced and then translated. Antigenic sites of protein sequences were identified by Physico-Chemical Profiles program. Results: The results showed that the presence of two genes als1 and hwp1 in isolates. In ALS1 and HWP1, respectively 2 and 1 antigenic site with the most antigenicity were identified. Conclusions: According to previous studies, Serine and Threonine phosphorylation is an important mechanism in pathogenesis of ALS1 and HWP1 proteins. Results in this study showed that serine and threonine are the most amino acids in the antigenic sites with high antigenicity property.

  15. A 135-kilodalton surface antigen of Mycoplasma hominis PG21 contains multiple directly repeated sequences

    DEFF Research Database (Denmark)

    Ladefoged, Søren; Birkelund, Svend; Hauge, S

    1995-01-01

    -base substitution, C-->A, gave rise to the stop codon of lmp1. Thus, the C-terminal 945 amino acids were encoded by the 471-bp direct repeats. As evidenced by Southern blot analysis, the gene encoding the 135-kDa antigen is part of a multigene family. One of the genes, lmp2, was situated directly downstream from...

  16. Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

    Science.gov (United States)

    Luo, L; Sabara, M I; Li, Y

    2009-10-01

    Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B.

  17. Immune selection and within-host competition can structure the repertoire of variant surface antigens in Plasmodium falciparum -a mathematical model

    DEFF Research Database (Denmark)

    van Noort, Sander P; Nunes, Marta C; Weedall, Gareth D

    2010-01-01

    -studied VSA family is erythrocyte membrane protein 1 (PfEMP1). Each parasite genome encodes about 60 PfEMP1 variants, which are important virulence factors and major targets of host antibody responses. Transcriptional switching is the basis of clonal PfEMP1 variation and immune evasion. A relatively conserved......BACKGROUND: The evolutionary mechanisms structuring the expression pattern of variant surface antigen (VSA) families that allow pathogens to evade immune responses and establish chronic and repeated infections pose major challenges to theoretical research. In Plasmodium falciparum, the best...... evidence regarding VSAs, in particular PfEMP1, to formulate a mathematical model of the evolutionary mechanisms shaping VSA organization and expression patterns. The model integrates the transmission dynamics between hosts and the competitive interactions within hosts, based on the hypothesis that the VSAs...

  18. Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

    Science.gov (United States)

    Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

    2013-03-01

    A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

  19. Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

    DEFF Research Database (Denmark)

    Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

    2018-01-01

    but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...

  20. Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    Directory of Open Access Journals (Sweden)

    Montanaro J

    2015-07-01

    Full Text Available Jacqueline Montanaro,1 Aleksandra Inic-Kanada,1 Angela Ladurner,1 Elisabeth Stein,1 Sandra Belij,1 Nora Bintner,1 Simone Schlacher,1 Nadine Schuerer,1 Ulrike Beate Mayr,2 Werner Lubitz,2 Nikolaus Leisch,3 Talin Barisani-Asenbauer11Laura Bassi Centres of Expertise, OCUVAC – Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria; 2BIRD-C GmbH & Co KG, Kritzendorf, Austria; 3Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaAbstract: To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN, whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results

  1. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  2. A novel antigen-toxin chimeric protein: myelin basic protein-pseudomonas exotoxin (MBP-PE 40) for treatment of experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Brenner, T; Steinberger, I; Soffer, D; Beraud, E; Ben-Nun, A; Lorberboum-Galski, H

    1999-06-01

    Myelin basic protein (MBP), is a major component of the central nervous system (CNS) myelin. MBP can stimulate T cells that migrate into the CNS, initiating a cascade of events that result in perivascular infiltration and demyelination. EAE is an inflammatory and demyelinating autoimmune disease of the CNS that serves as a model for the human disease Multiple Sclerosis (MS). Taking advantage of the fact that EAE can be mediated by T cells, able to recognize MBP or its peptides, we developed a new approach to target anti-MBP T cells by fusing an MBP-sequence to a toxin. In the new chimeric protein, an oligonucleotide coding for the guinea pig MBP encephalitogenic moiety (residues 68-88) was fused to a cDNA encoding a truncated form of the PE gene (PE40). The chimeric gene termed MBP-PE was expressed in E. coli and highly purified. MBP-PE chimeric protein was cytotoxic to various anti-MBP T cells. Moreover, treatment with the novel MBP-toxin blocked the clinical signs of EAE as well as CNS inflammation and demyelination. A chimeric protein such as MBP-PE40 presents a novel prototype of chimeric proteins, composed of antigen/peptide-toxin, that could prove to be an efficient and specific immunotherapeutic agent for autoimmune diseases in which a known antigen is involved.

  3. Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus.

    Directory of Open Access Journals (Sweden)

    Xiaowei Ren

    Full Text Available The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.

  4. Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen.

    Science.gov (United States)

    Klose, Diana; Saunders, Ute; Barth, Stefan; Fischer, Rainer; Jacobi, Annett Marita; Nachreiner, Thomas

    2016-02-17

    In an earlier study we developed a unique strategy allowing us to specifically eliminate antigen-specific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells. The present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA') to induce apoptosis after cellular uptake. The TTC-ETA' fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27(+) memory B cells. This protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.

  5. Surface-enhanced Raman scattering study of carcinoembryonic antigen in serum from patients with colorectal cancers

    Science.gov (United States)

    Chen, Gang; Chen, Yanping; Zheng, Xiongwei; He, Cheng; Lu, Jianping; Feng, Shangyuan; Chen, Rong; Zeng, Haisan

    2013-12-01

    In this work, we developed a SERS platform for quantitative detection of carcinoembryonic antigen (CEA) in serum of patients with colorectal cancers. Anti-CEA-functionalized 4-mercaptobenzoic acid-labeled Au/Ag core-shell bimetallic nanoparticles were prepared first and then used to analyze CEA antigen solutions of different concentrations. A calibration curve was established in the range from 5 × 10-3 to 5 × 105 ng/mL. Finally, this new SERS probe was applied for quantitative detection of CEA in serum obtained from 26 colorectal cancer patients according to the calibration curve. The results were in good agreement with that obtained by electrochemical luminescence method, suggesting that SERS immunoassay has high sensitivity and specificity for CEA detection in serum. A detection limit of 5 pg/ml was achieved. This study demonstrated the feasibility and great potential for developing this new technology into a clinical tool for analysis of tumor markers in the blood.

  6. Trichomonas vaginalis surface proteins: a view from the genome

    DEFF Research Database (Denmark)

    Hirt, R. P.; Noel, C. J.; Sicheritz-Pontén, Thomas

    2007-01-01

    Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa....... However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T...

  7. Construction of bifunctional molecules specific to antigen and antibody’s Fc-fragment by fusion of scFv-antibodies with staphylococcal protein A

    Directory of Open Access Journals (Sweden)

    Kolibo D. V.

    2009-06-01

    Full Text Available Aim. To develop approach for detection of scFv and their complexes with antigens. Methods. The fusion proteins, which include sequences of scFv and staphylococcal protein A, were constructed and the obtained bifunctional molecules were immunochemically analysed. Results. It was shown, that scFv fused with protein A and their complexes with antigens are effectively recognized by labelled immunoglobulins with unrestricted antigenic specificity. Conclusions. The fusion of scFv with protein A fragment is a perspective approach to increase the efficiency of application in ELISA. The obtained scFv, fused with protein A, could be used for development of test-systems for the detection of diphtheria toxin.

  8. Seroprevalence of Hepatitis B Surface Antigen and Occupational Risk Factors Among Health Care Workers in Ekiti State, Nigeria

    Science.gov (United States)

    Alese, Oluwole Ojo; Ohunakin, Afolabi; Oluyide, Peter Olumuyiwa

    2016-01-01

    Introduction Hepatitis B virus (HBV) infection is contracted from blood and other body fluid making healthcare workers (HCW) prone to the infection especially in the developing world. Though it is a vaccine preventable disease, the level of awareness and universal precaution among HCW is low in sub-Saharan African and Asia. Aim The study was aimed at determining the seroprevalence of hepatitis B surface antigen and occupational risk factors among health care workers at Ekiti State University Teaching Hospital, Ado Ekiti. Materials and Methods One hundred and eighty-seven (187) blood samples were collected from volunteer subjects who comprised of medical doctors, nurses, health attendants, and porters who are in regular contact with blood, body fluids and patients after informed consent. Well detailed and structured questionnaires were used to obtain demographic and other relevant data from the subjects. Blood samples were tested by Enzyme Linked Immunosorbent assay (ELISA) for hepatitis B surface antigen. Results Out of the 187 HCWs there were 91 males (48.7%) and 96 (51.3%) females. Only 2 participants tested positive to hepatitis B surface antigen with a prevalence of 1.1%. Also, only 30 (16.0%) of the participants had been fully vaccinated against the infection while the remaining 157(84.0%) had no adult vaccination. Conclusion It is obvious that the awareness of the infection is low among the HCWs studied thus the need to incorporate screening for HbsAg and vaccination against HBV into the periodic/pre-employment health intervention programmes by employers to help in the protection of HCWs and control the spread of the virus. PMID:27042489

  9. Seroprevalence of Hepatitis B Surface Antigen and Occupational Risk Factors Among Health Care Workers in Ekiti State, Nigeria.

    Science.gov (United States)

    Alese, Oluwole Ojo; Alese, Margaret Olutayo; Ohunakin, Afolabi; Oluyide, Peter Olumuyiwa

    2016-02-01

    Hepatitis B virus (HBV) infection is contracted from blood and other body fluid making healthcare workers (HCW) prone to the infection especially in the developing world. Though it is a vaccine preventable disease, the level of awareness and universal precaution among HCW is low in sub-Saharan African and Asia. The study was aimed at determining the seroprevalence of hepatitis B surface antigen and occupational risk factors among health care workers at Ekiti State University Teaching Hospital, Ado Ekiti. One hundred and eighty-seven (187) blood samples were collected from volunteer subjects who comprised of medical doctors, nurses, health attendants, and porters who are in regular contact with blood, body fluids and patients after informed consent. Well detailed and structured questionnaires were used to obtain demographic and other relevant data from the subjects. Blood samples were tested by Enzyme Linked Immunosorbent assay (ELISA) for hepatitis B surface antigen. Out of the 187 HCWs there were 91 males (48.7%) and 96 (51.3%) females. Only 2 participants tested positive to hepatitis B surface antigen with a prevalence of 1.1%. Also, only 30 (16.0%) of the participants had been fully vaccinated against the infection while the remaining 157(84.0%) had no adult vaccination. It is obvious that the awareness of the infection is low among the HCWs studied thus the need to incorporate screening for HbsAg and vaccination against HBV into the periodic/pre-employment health intervention programmes by employers to help in the protection of HCWs and control the spread of the virus.

  10. Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface-enhanced Raman spectroscopy.

    Science.gov (United States)

    Zou, Kun; Gao, Zhigang; Deng, Quanfeng; Luo, Yong; Zou, Lijuan; Lu, Yao; Zhao, Weijie; Lin, Bingcheng

    2016-03-01

    Carcinoembryonic antigen (CEA) is a wide-spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10(-12) M. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Study of the Annexin A1 and Its Associations with Carcinoembryonic Antigen and Mismatch Repair Proteins in Colorectal Cancer.

    Science.gov (United States)

    Ydy, Lenuce Ribeiro Aziz; do Espírito Santo, Gilmar Ferreira; de Menezes, Ivana; Martins, Michelle Santos; Ignotti, Eliane; Damazo, Amílcar Sabino

    2016-03-01

    Annexin-A1 (ANXA1) has been implicated in various tumor types, but few studies have investigated its involvement in colorectal cancer. The study aimed to analyze ANXA1 expression in the normal margin and colorectal tumor tissues of 104 patients who underwent surgery for colorectal cancer and to associate the ANXA1 expression with predictive clinicopathological variables. Hematoxylin-eosin and immunohistochemical staining were used for the analysis. ANXA1 expression was higher in colorectal cancer than in normal margin tissue (p = 0.0001). However, no differences were observed when we analyzed the ANXA1 expression in colon and rectal tumors (p = 0.830). Also, this protein positivity was associated with increased carcinoembryonic antigen levels (p = 0.004). Our data in the DNA-mismatch repair proteins expression was in accordance to the literature. And their positivity was not associated with ANXA1 presence in colorectal cancer. The high incidence of ANXA1 positive expression in colorectal cancer and its association with carcinoembryonic antigen levels might indicate the importance of this protein in the colorectal cancer biology.

  12. Expression and characterisation of a Sarcoptes scabiei protein tyrosine kinase as a potential antigen for scabies diagnosis.

    Science.gov (United States)

    Shen, Nengxing; He, Ran; Liang, Yuqing; Xu, Jing; He, Manli; Ren, Yongjun; Gu, Xiaobin; Lai, Weimin; Xie, Yue; Peng, Xuerong; Yang, Guangyou

    2017-08-29

    Scabies is a disease that harms humans and other animals that is caused by the itch mite Sarcoptes scabiei burrowing into the stratum corneum of the skin. In the early stages of scabies, symptoms are often subclinical and there are no effective diagnostic methods. Herein, we cloned, expressed and characterised an S. scabiei protein tyrosine kinase (SsPTK) and evaluated its diagnostic value as a recombinant antigen in rabbit during the early stages of Sarcoptes infestation. The SsPTK protein is ~30 kDa, lacks a signal peptide, and shares high homology with a PTK from the rabbit ear mite Psoroptes ovis cuniculi. The protein was widely distributed at the front end of mites, particularly in the chewing mouthparts and legs. Indirect ELISA using recombinant SsPTK showed good diagnostic value, with 95.2% (40/42) sensitivity and 94.1% (48/51) specificity for detecting anti-PTK antibody in serum samples from naturally-infested rabbits. More importantly, PTK ELISA could diagnose infection in the early stages (infestation for 1 week) with an accuracy of 100% (24/24). SsPTK therefore shows potential as a sensitive antigen for the early diagnosis of parasitic mite infestation.

  13. The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP-70 Recruitment and Activation

    OpenAIRE

    Ferro, Micol; Savino, Maria Teresa; Ortensi, Barbara; Finetti, Francesca; Genovese, Luca; Masi, Giulia; Ulivieri, Cristina; Benati, Daniela; Pelicci, Giuliana; Baldari, Cosima T.

    2011-01-01

    Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neuro...

  14. Exceptionally Potent Anti-Tumor Bystander Activity of an scFv:sTRAIL Fusion Protein with Specificity for EGP2 Toward Target Antigen-Negative Tumor Cells

    Directory of Open Access Journals (Sweden)

    Edwin Bremer

    2004-09-01

    Full Text Available Previously, we reported on the target cell-restricted fratricide apoptotic activity of scFvC54:sTRAIL, a fusion protein comprising human-soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL genetically linked to the antibody fragment scFvC54 specific for the cell surface target antigen EGP2. In the present study, we report that the selective binding of scFvC54:sTRAIL to EGP2-positive target cells conveys an exceptionally potent pro-apoptotic effect toward neighboring tumor cells that are devoid of EGP2 expression (bystander cells. The anti-tumor bystander activity of scFvC54:sTRAIL was detectable at target-tobystander cell ratios as low as 1:100. Treatment in the presence of EGP2-blocking or TRAIL-neutralizing antibody strongly inhibited apoptosis in both target and bystander tumor cells. In the absence of target cells, bystander cell apoptosis induction was abrogated. The bystander apoptosis activity of scFvC54:sTRAIL did not require internalization, enzymatic conversion, diffusion, or communication (gap junctional intracellular communication between target and bystander cells. Furthermore, scFvC54:sTRAIL showed no detectable signs of innocent bystander activity toward freshly isolated blood cells. Further development of this new principle is warranted for approaches where cancer cells can escape from antibody-based therapy due to partial loss of target antigen expression.

  15. A Study of Molecular Mimicry and Immunological Cross-reactivity between Hepatitis B Surface Antigen and Myelin Mimics

    Directory of Open Access Journals (Sweden)

    Diego Vergani

    2005-01-01

    Full Text Available On the basis of the reported association between hepatitis B vaccination (HBvacc and autoimmune demyelinating complications such as multiple sclerosis (MS, we have looked for aminoacid similarities between the small hepatitis B virus surface antigen (SHBsAg, and the MS-autoantigens myelin basic protein (MBP and myelin oligodendrocyte glycoprotein (MOG that could serve as targets of immunological cross-reactivity. Twenty-mer peptides spanning 4 SHBsAg/MOG and 1 SHBsAg/MBP mimicking pairs, were constructed and tested by ELISA as targets of cross-reactive responses. A total of 147 samples from 58 adults were collected before HBvacc (58/58, and post-HBvacc (48/58 before the second and 41/58 before the third boost. Eighty-seven sera from anti-SHBsAg antibody negative patients with various diseases were tested as pathological controls. Reactivity to at least one of the SHBsAg peptides was found in 8 (14% pre-HBvacc subjects; amongst the remaining 50, reactivity to at least one of the SHBsAg peptides appeared in 47 (94% post-HBvacc. Reactivity to at least one of the MOG mimics was present in 4 (8% pre-HBvacc and in 30 (60% post-HBvacc (p < 0.001. Overall 30/50 (60% vaccinees had SHBsAg/MOG double reactivity on at least one occasion compared to none before-vaccination and in 2 (2% of the pathological controls (p < 0.001 for both. SHBsAg/MOG double reactivity was cross-reactive as confirmed by inhibition studies. At 6 months post-vaccination, 3 of the 4 anti-MOG reactive cases before vaccination and 7 of the 24 (29% of the anti-MOG reactive cases at 3 months post-vaccination had lost their reactivity to MOG5-24. There was no reactivity to the SHBsAg/MBP mimics. None of the vaccinees reported symptoms of demyelinating disorders. In view of the observed SHBsAg/MOG cross-reactivity, the vaccine's possible role as an immunomodulator of viral/self cross-reactivity must be further investigated.

  16. Cleaning of biomaterial surfaces: protein removal by different solvents.

    Science.gov (United States)

    Kratz, Fabian; Grass, Simone; Umanskaya, Natalia; Scheibe, Christian; Müller-Renno, Christine; Davoudi, Neda; Hannig, Matthias; Ziegler, Christiane

    2015-04-01

    The removal of biofilms or protein films from biomaterials is still a challenging task. In particular, for research investigations on real (applied) surfaces the reuse of samples is of high importance, because reuse allows the comparison of the same sample in different experiments. The aim of the present study was to evaluate the cleaning efficiency of different solvents (SDS, water, acetone, isopropanol, RIPA-buffer and Tween-20) on five different biomaterials (titanium, gold, PMMA (no acetone used), ceramic, and PTFE) with different wettability which were covered by layers of two different adsorbed proteins (BSA and lysozyme). The presence of a protein film after adsorption was confirmed by transmission electron microscopy (TEM). After treatment of the surfaces with the different solvents, the residual proteins on the surface were determined by BCA-assay (bicinchoninic acid assay). Data of the present study indicate that SDS is an effective solvent, but for several protein-substrate combinations it does not show the cleaning efficiency often mentioned in literature. RIPA-buffer and Tween-20 were more effective. They showed very low residual protein amounts after cleaning on all examined material surfaces and for both proteins, however, with small differences for the respective substrate-protein combinations. RIPA-buffer in combination with ultrasonication completely removed the protein layer as confirmed by TEM. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. The non-structural protein 5 and matrix protein are antigenic targets of T cell immunity to genotype 1 porcine reproductive and respiratory syndrome viruses

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    Helen eMokhtar

    2016-02-01

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

  18. Quantitative hepatitis B surface antigen combined with hepatitis B e antigen as sustained virological response predictors during extended therapy with Peginterferon alfa-2a for hepatitis B e antigen-positive chronic hepatitis B.

    Science.gov (United States)

    Chen, Jie; Zhang, Dong-Hua; Xu, Cheng-Run; Zhu, Ming-Yu; Yang, Zhi-Tao; Gong, Qi-Ming; Yu, De-Min; Zhang, Xin-Xin

    2015-11-01

    The best strategy for chronic hepatitis B patients with poor response to 48 weeks of Peginterferon-based therapy has been controversial and the predictive value of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) DNA levels for determining the sustained virological response (SVR) of these patients is uncertain. To optimize management of these patients and evaluate the use of these serobiomarkers to predict SVR. Eighty-one patients with an unsatisfactory response after 48 weeks of Peginterferon-based therapy were treated with extended Peginterferon therapy with or without nucleo(s) tide analogues (NAs), for a total of 96 weeks of Peginterferon treatment. HBsAg, HBeAg and HBV DNA levels were measured serially during the treatment and follow-up. Twenty-six of 81 patients (32.1%) attained SVR during the 72-week follow-up. The SVR rate was not statistically different between groups receiving 1-year prolongation of Peginterferon with or without NAs. The serum HBsAg cut-off of 1800IU/mL at week 48 had area under curve (AUC) of 0.727, and the serum HBsAg cut-off of 1500IU/mL, combined with HBeAg loss at week 72, had AUC of 0.753 to predict SVR during the follow-up. In conclusion, extended treatment with Peginterferon with or without NAs for patients with unsatisfactory response after 48 weeks of Peginterferon-based therapy is a promising strategy to achieve SVR, and quantitative serum HBsAg at week 48 and HBsAg level combined with HBeAg loss at week 72 of therapy can predict SVR to prolongation therapy with Peginterferon. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Arginine inhibits adsorption of proteins on polystyrene surface.

    Directory of Open Access Journals (Sweden)

    Yui Shikiya

    Full Text Available Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg, lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.

  20. Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria

    DEFF Research Database (Denmark)

    Dodoo, D; Theander, T G; Kurtzhals, J A

    1999-01-01

    The 19-kDa conserved C-terminal part of the Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is a malaria vaccine candidate antigen, and human antibody responses to PfMSP119 have been associated with protection against clinical malaria. In this longitudinal study carried out in an are...

  1. Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

    Science.gov (United States)

    Hagemann, Jürgen Benjamin; Simnacher, Ulrike; Longbottom, David; Livingstone, Morag; Maile, Julia; Soutschek, Erwin; Walder, Gernot; Boden, Katharina; Sachse, Konrad; Essig, Andreas

    2016-07-01

    The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

    Science.gov (United States)

    Zhou, J; Sun, X Y; Davies, H; Crawford, L; Park, D; Frazer, I H

    1992-08-01

    Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.

  3. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  4. Proteolytic activity of prostate-specific antigen (PSA) towards protein substrates and effect of peptides stimulating PSA activity.

    Science.gov (United States)

    Mattsson, Johanna M; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  5. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  6. Modulating Protein Adsorption on Oxygen Plasma Modified Polysiloxane Surfaces

    International Nuclear Information System (INIS)

    Marletta, G.

    2006-01-01

    In the present paper we report the study on the adsorption behaviour of three model globular proteins, Human Serum Albumin, Lactoferrin and Egg Chicken Lysozyme onto both unmodified surfaces of a silicon-based polymer and the corresponding plasma treated surfaces. In particular, thin films of hydrophobic polysiloxane (about 90 degree of static water contact angle, WCA) were converted by oxygen plasma treatment at reduced pressure into very hydrophilic phases of SiOx (WCA less than 5 degree). The kinetics of protein adsorption processes were investigated by QCM-D technique, while the chemical structure and topography of the protein adlayer have been studied by Angular resolved-XPS and AFM respectively. It turned out that Albumin and Lysozyme exhibited the opposite preferential adsorption respectively onto the hydrophobic and hydrophilic surfaces, while Lactoferrin did not exhibit significant differences. The observed protein behaviour are discussed both in terms of surface-dependent parameters, including surface free energy and chemical structure, and in terms of protein-dependent parameters, including charge as well as the average molecular orientation in the adlayers. Finally, some examples of differential adsorption behaviour of the investigated proteins are reported onto nanopatterned polysiloxane surfaces consisting of hydrophobic nanopores surrounded by hydrophilic (plasma-treated) matrix and the reverse

  7. Natural selection promotes antigenic evolvability.

    Science.gov (United States)

    Graves, Christopher J; Ros, Vera I D; Stevenson, Brian; Sniegowski, Paul D; Brisson, Dustin

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

  8. Dynamics of hydration water and coupled protein sidechains around a polymerase protein surface

    Science.gov (United States)

    Qin, Yangzhong; Yang, Yi; Wang, Lijuan; Zhong, Dongping

    2017-09-01

    Water-protein coupled interactions are essential to the protein structural stability, flexibility and dynamic functions. The ultimate effects of the hydration dynamics on the protein fluctuations remain substantially unexplored. Here, we investigated the dynamics of both hydration water and protein sidechains at 13 different sites around the polymerase β protein surface using a tryptophan scan with femtosecond spectroscopy. Three types of hydration-water relaxations and two types of protein sidechain motions were determined, reflecting a highly dynamic water-protein interactions fluctuating on the picosecond time scales. The hydration-water dynamics dominate the coupled interactions with higher flexibility.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-05-03

    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.

  10. Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation.

    Science.gov (United States)

    Bullido, M J; Muñoz-Fernandez, M A; Recuero, M; Fresno, M; Valdivieso, F

    1996-08-21

    A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimer's disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

  11. The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

    Directory of Open Access Journals (Sweden)

    Luciana Galetto

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

  12. Role of sperm surface proteins in reproduction

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Maňásková, Pavla; Davidová, Nina; Tichá, M.; Pěknicová, Jana

    2009-01-01

    Roč. 30, Supplement (2009), s. 63-64 ISSN 0196-3635. [9th International Congress of Andrology. 07.03.2009-10.03.2009, Barcelona] R&D Projects: GA MŠk(CZ) 1M06011; GA ČR(CZ) GA523/08/H064; GA ČR(CZ) GA303/06/0895 Institutional research plan: CEZ:AV0Z50520701 Keywords : boar seminal plasma proteins * spermadhesins * proteinase inhibitor * DQH * boar spermatozoa Subject RIV: CE - Biochemistry

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

    Science.gov (United States)

    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

  14. Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum.

    Science.gov (United States)

    Bras-Gonçalves, Rachel; Petitdidier, Elodie; Pagniez, Julie; Veyrier, Renaud; Cibrelus, Prisca; Cavaleyra, Mireille; Maquaire, Sarah; Moreaux, Jérôme; Lemesre, Jean-Loup

    2014-06-01

    We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiESAp-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs from the vaccinated group developed a selective IgG2 response against an immunodominant antigen of 45 kDa of Leishmania amazonensis ESA promastigotes (LaESAp). In order to identify and characterize these immunodominant antigens, a mouse monoclonal antibody (mAb F5) was produced by immunization against LaESAp. It was found to recognize the major antigenic targets of both LaESAp and LiESAp. Analysis with mAb F5 of L. amazonensis amastigote and promastigote cDNA expression libraries enabled the identification of clones encoding proteins with significant structural homology to the promastigote surface antigens named PSA-2/gp-46. Among them, one clone presented a full-length cDNA and encoded a novel L. amazonensis protein of 38.6 kDa calculated molecular mass (LaPSA-38S) sharing an amino acid sequence consistent with that of the PSA polymorphic family and a N-terminal signal peptide, characteristic of a secreted protein. We then screened a L. infantum promastigote DNA cosmid library using a cDNA probe derived from the LaPSA-38S gene and identified a full-length clone of a novel excreted/secreted protein of L. infantum with a calculated molecular mass of 49.2 kDa and named LiPSA-50S. The fact that a significant immunological reactivity was observed against PSA, suggests that these newly identified proteins could have an important immunoregulatory influence on the immune response. This hypothesis is supported by the fact that (i) these proteins were naturally excreted/secreted by viable

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

    Science.gov (United States)

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

    2016-08-01

    To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P nanoparticles showed good immunoadjuvant potential. Env23 antigen was a better candidate for vaccination against HTLV-I, as it induced higher cellular immune responses, compared with env13. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

    DEFF Research Database (Denmark)

    Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

    2018-01-01

    but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

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

    Science.gov (United States)

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

    2016-02-01

    Hendra virus (HeV) is an emerging zoonotic paramyxovirus within the genus Henipavirus that has caused severe morbidity and mortality in humans and horses in Australia since 1994. HeV infection of host cells is mediated by the membrane bound attachment (G) and fusion (F) glycoproteins, that are essential for receptor binding and fusion of viral and cellular membranes. The eukaryotic unicellular parasite Leishmania tarentolae has recently been established as a powerful tool to express recombinant proteins with mammalian-like glycosylation patterns, but only few viral proteins have been expressed in this system so far. Here, we describe the purification of a truncated, Strep-tag labelled and soluble version of the HeV attachment protein (sHeV G) expressed in stably transfected L. tarentolae cells. After Strep-tag purification the identity of sHeV G was confirmed by immunoblotting and mass spectrometry. The functional binding of sHeV G to the HeV cell entry receptor ephrin-B2 was confirmed in several binding assays. Generated polyclonal rabbit antiserum against sHeV G reacted with both HeV and Nipah virus (NiV) G proteins in immunofluorescence assay and efficiently neutralised NiV infection, thus further supporting the preserved antigenicity of the purified protein. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

    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 (Phyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. The biological lavelling and 75Se of protein antigens of Fasciola hepatica

    Science.gov (United States)

    Mulligan, W.; Cuperlović, K.; Borojević, Dragica; Lalić, R.

    1972-01-01

    Adult liver flukes were incubated for several hours at 37° in a culture medium containing 75Se-L-selenomethionine. Analysis of homogenates of the parasite showed that significant amounts of isotope had become incorporated into parasite proteins. Separation of the fluke proteins on Sephadex G-100 demonstrated the highest specific activity in the most serologically active protein fractions. PMID:4648855

  2. The Role of Borrelia burgdorferi Outer Surface Proteins

    Science.gov (United States)

    Kenedy, Melisha R.; Lenhart, Tiffany R.; Akins, Darrin R.

    2012-01-01

    Human pathogenic spirochetes causing Lyme disease belong to the Borrelia burgdorferi sensu lato complex. B. burgdorferi organisms are extracellular pathogens transmitted to humans through the bite of Ixodes spp. ticks. These spirochetes are unique in that they can cause chronic infection and persist in the infected human, even though a robust humoral and cellular immune response is produced by the infected host. How this extracellular pathogen is able to evade the host immune response for such long periods of time is currently unclear. To gain a better understanding of how this organism persists in the infected human, many laboratories have focused on identifying and characterizing outer surface proteins of B. burgdorferi. Since the interface between B. burgdorferi and its human host is its outer surface, proteins localized to the outer membrane must play an important role in dissemination, virulence, tissue tropism, and, immune evasion. Over the last two decades numerous outer surface proteins from B. burgdorferi have been identified and more recent studies have begun to elucidate the functional role(s) of many borrelial outer surface proteins. This review summarizes the outer surface proteins identified in B. burgdorferi to date and provides detailed insight into the functions of many of these proteins as they relate to the unique parasitic strategy of this spirochetal pathogen. PMID:22540535

  3. Identification and characterization of Vibrio cholerae surface proteins by radioiodination

    International Nuclear Information System (INIS)

    Richardson, K.; Parker, C.D.

    1985-01-01

    Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride- lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides

  4. Enhanced bone morphogenetic protein-2 performance on hydroxyapatite ceramic surfaces.

    Science.gov (United States)

    Schuessele, A; Mayr, H; Tessmar, J; Goepferich, A

    2009-09-15

    The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces. Copyright 2008 Wiley Periodicals, Inc.

  5. Analysis of Borrelia burgdorferi surface proteins as determinants in establishing host cell interactions

    Directory of Open Access Journals (Sweden)

    Virginia L Schmit

    2011-07-01

    Full Text Available Borrelia burgdorferi infection causes Lyme borreliosis in humans, a condition which can involve a systemic spread of the organism to colonize various tissues and organs. If the infection is left untreated by antimicrobials, it can lead to manifestations including, arthritis, carditis, and/or neurological problems. Identification and characterization of B. burgdorferi outer membrane proteins that facilitate cellular attachment and invasion to establish infection continue to be investigated. In this study, we sought to further define putative cell binding properties of surface-exposed B. burgdorferi proteins by observing whether cellular adherence could be blocked by antibodies. B. burgdorferi mixed separately with monoclonal antibodies against outer surface protein (Osp A, OspC, decorin-binding protein (Dbp A, BBA64, and RevA antigens were incubated with human umbilical vein endothelial cells (HUVEC and human neuroglial cells (H4. B. burgdorferi treated with anti-OspA, -DbpA, and –BBA64 monoclonal antibodies showed a significant decrease in cellular association compared to controls, whereas B. burgdorferi treated with anti-OspC and anti-RevA showed no reduction in cellular attachment. Additionally, temporal transcriptional analyses revealed upregulated expression of bba64, ospA, and dbpA during coincubation with cells. Together, the data provide evidence that OspA, DbpA, and BBA64 function in host cell adherence and infection mechanisms.

  6. Evaluation of recombinant porin (rOmp2a) protein as a potential antigen candidate for serodiagnosis of Human Brucellosis.

    Science.gov (United States)

    Pathak, Prachi; Kumar, Ashu; Thavaselvam, Duraipandian

    2017-07-11

    Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). The rOmp2a was found to be

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

    Czech Academy of Sciences Publication Activity Database

    Chatchen, S.; Juříček, Miloslav; Rueda, P.; Kertbundit, Sunee

    2006-01-01

    Roč. 39, č. 1 (2006), s. 16-21 ISSN 1225-8687 Grant - others:Thai Research Fund(TH) BT-B-06-PG-14-4503 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : antigen presentation * canine parvo virus * epitope * papaya ringspot virus Subject RIV: EF - Botanics Impact factor: 1.465, year: 2006 http://www.jbmb.or.kr/view_article.php3?cont=jbmb&kid=174&mid=3& pid =3

  8. Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

    Science.gov (United States)

    Ichthyophthirius multifiliis, a ciliated protozoan parasite of fish, expresses surface antigens (i-antigens), which react with host antibodies that render them immobile. The nucleotide sequence of an i-antigen gene of Ichthyophthirius multifiliis strain ARS-6 was deduced. The predicted protein of 47...

  9. Purification and characterization of a major human Pneumocystis carinii surface antigen

    DEFF Research Database (Denmark)

    Lundgren, B; Lipschik, G Y; Kovacs, J A

    1991-01-01

    . The carbohydrate composition of the rat P. carinii glycoprotein was distinct from the human isolate; glucose, mannose, galactose, and glucosamine occurred in approximately equimolar ratios in the human P. carinii protein, whereas glucose and mannose were the predominant sugars of the rat P. carinii protein...

  10. Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

    Science.gov (United States)

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

  11. A vaccine prepared from the 22 nm particles of surface hepatitis B antigen (HBsAg)

    International Nuclear Information System (INIS)

    Karelin, V.P.; Babaeva, E.E.; Gubenko, E.F.; Kaulen, D.K.; Zhdanov, V.M.

    1980-01-01

    A method for obtaining a subunit inactivated vaccine preparation from the 22-nm particles of HBsAg is proposed. For inactivation of the residual infectious hepatitis B virus (HBV) the preparations were successively treated with 1% sodium dodecyl sulfate (SDS) and nucleases. In addition, thermal denaturation and ultraviolet irradiation of HBV DNA were used. As a control the biologic activity of a reference virus (SV40) was tested after the same treatment. The effectiveness of DNA inactivation was monitored by adding 3H-thymidine labeled reference virus to the vaccine preparations. The purified and inactivated HBsAg was adsorbed on Al2O3. Antigenicity was calculated on the basis of the determination of antibody in guinea pigs immunized with various doses of the vaccine, and the release of 125 I- HBsAg from blood and kidneys in immunized and control mice was analyzed. Possible methods of inactivation and control of HBV vaccine is discussed

  12. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

    Science.gov (United States)

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-01-01

    Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates. PMID:27223609

  13. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

    Science.gov (United States)

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-05-01

    Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

  14. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA Induce Protective Immune Responses in Dogs.

    Directory of Open Access Journals (Sweden)

    Elodie Petitdidier

    2016-05-01

    Full Text Available Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA, from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA or its carboxy terminal part LaPSA-12S (Cter-rPSA, combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates.

  15. Merozoite proteins from Babesia sp. BQ1 (Lintan) as potential antigens for serodiagnosis by ELISA.

    Science.gov (United States)

    Guan, G Q; Chauvin, A; Rogniaux, H; Luo, J X; Yin, H; Moreau, E

    2010-05-01

    Babesia sp. BQ1 (Lintan) is a Babesia isolated from sheep infested with Haemaphysalis qinghaiensis in China, and is closely related to B. motasi based on the 18S rRNA gene sequence. In the present study, an ELISA was developed with merozoite antigens of Babesia sp. BQ1 (Lintan) (BQMA) purified from in vitro culture. When the positive threshold was chosen as 30% of the antibodies rate, evaluated with 198 negative sera, the specificity was 95.5%. Except for Babesia sp. Tianzhu, there was no cross-reaction between BQMA and positive sera from Babesia sp. BQ1 (Ningxian)-, Babesia sp. Hebei-, Babesia sp. Xinjiang-, Theileria luwenshuni-, T. uilenbergi-, or Anaplasma ovis-infected sheep, which are the dominant haemoparasites of small ruminants in China. Specific antibodies against Babesia sp. BQ1 (Lintan) were produced 1 or 2 weeks post-infection and a high level of antibodies persisted for more than 8 months in experimentally infected sheep. This ELISA was tested on 974 sera collected from field-grazing sheep in 3 counties of Gansu province, northwestern China to evaluate the seroprevalence of Babesia sp. BQ1 (Lintan) infection and the average positive rate was 66.84%. The feasibility of increasing the specificity of this BQMA-based ELISA, by using some BQMA antigens for serodiagnosis is discussed.

  16. Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

    Directory of Open Access Journals (Sweden)

    George V. Hillyer

    1995-04-01

    Full Text Available Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44 identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

  17. Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis

    OpenAIRE

    Coscoy, Laurent; Ganem, Don

    2000-01-01

    Down-regulation of the cell surface display of class I MHC proteins is an important mechanism of immune evasion by human and animal viruses. Herpesviruses in particular encode a variety of proteins that function to lower MHC I display by several mechanisms. These include binding and retention of MHC I chains in the endoplasmic reticulum, dislocation of class I chains from the ER, inhibition of the peptide transporter (TAP) involved in antigen presentation, and shunting of newly assembled chai...

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

    Science.gov (United States)

    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.

  19. Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity.

    Science.gov (United States)

    Sayadmanesh, Ali; Ebrahimi, Firouz; Hajizade, Abbas; Rostamian, Mosayeb; Keshavarz, Hani

    2013-01-01

    Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

  20. Identification of a rare point mutation at C-terminus of merozoite surface antigen-1 gene of Plasmodium falciparum in eastern Indian isolates.

    Science.gov (United States)

    Raj, Dipak Kumar; Das, Bibhu Ranjan; Dash, A P; Supakar, Prakash C

    2004-01-01

    Merozoite surface antigen-1 (MSA-1) of Plasmodium falciparum is highly immunogenic in human. Several studies suggest that MSA-1 protein is an effective target for a protective immune response. Attempt has been made to find new point mutations by analyzing 244 bp [codon 1655(R) to 1735 (I)] relatively conserved C-terminus region of MSA-1 gene in 125 isolates. This region contains two EGF like domains, which are involved in generating protective immune response in human. Point mutations in this region are very much important in view of vaccine development. Searching of mutational hot spots in MSA-1 protein by sequencing method in a representative number of isolates is quite critical and expensive. Therefore, in this study slot blot and PCR-SSCP method have been used to find out new mutations in the individual isolates showing alterations in the mobility of DNA fragment. Sequencing of the altered bands from the SSCP gel shows a rare non-synonymous point mutation in 7 (5.6%) of the 125 isolates at amino acid position 1704 of MSA-1 gene where isoleucine is replaced by valine.

  1. A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

    Science.gov (United States)

    Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

    2015-10-01

    Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor

  2. Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men.

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  4. Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections

    DEFF Research Database (Denmark)

    Giha, H A; Staalsoe, T; Dodoo, D

    2000-01-01

    is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh......, eastern Sudan, before and after the malaria season from individuals who had (susceptible) or did not have malaria (protected) during the season, were tested for reactivity against variant antigens on the surface of nine parasite isolates by flow cytometry. Both protected and susceptible individuals...... susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm...

  5. [Characterization of antigenic types of circulating rotaviruses in Mendoza, Argentina based on typing of the external VP7 capsid protein].

    Science.gov (United States)

    Espul, C; Cuello, H; Navarta, L M; Mamani, N; O'Ryan, M; O'Ryan, M

    1993-01-01

    Rotavirus is one of the most common etiologic agents of acute diarrhea in childhood. Understanding the immunologic mechanisms involved in rotavirus diseases, including knowledge on seasonal and geographic antigenic variations may be crucial for vaccine development. A monoclonal antibody based ELISA specific for antigenic domains on the outer capsid protein VP7 has been developed and used widely in the past years. We studied the rotavirus VP7-serotype epidemiology causing diarrhea in children who consulted at two main hospitals of Mendoza, Argentina over a 20 month period. A total of 227 cases of diarrhea were identified, 45 of which (20%) were rotavirus positive. We're able to serotype 43 viruses (96%), 42 VP7-type 1 and one VP7-type 3. The VP7-type 3 was detected towards the end of the second year, possibly representing a new incoming VP7-type. Three electropherotype patterns were identified, two corresponding to VP7-type epidemiology in Mendoza, Argentina seems to be characterized by a relatively homogeneous pattern of circulation with a strong predominance of VP7-type 1 viruses, at least during the 20 month period studied, in contrast to what has been reported in larger, more cosmopolitan cities like Buenos Aires.

  6. Detection of the circulating antigen 14-3-3 protein of Schistosoma japonicum by time-resolved fluoroimmunoassay in rabbits

    Directory of Open Access Journals (Sweden)

    Wang Jie

    2011-05-01

    Full Text Available Abstract Background Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum. Results The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection. Conclusions A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

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

    Science.gov (United States)

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

    2013-05-03

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. The Staphylococcus aureus Cell Wall-Anchored Protein Clumping Factor A Is an Important T Cell Antigen.

    Science.gov (United States)

    Lacey, Keenan A; Leech, John M; Lalor, Stephen J; McCormack, Niamh; Geoghegan, Joan A; McLoughlin, Rachel M

    2017-12-01

    Staphylococcus aureus has become increasingly resistant to antibiotics, and vaccines offer a potential solution to this epidemic of antimicrobial resistance. Targeting of specific T cell subsets is now considered crucial for next-generation anti- S. aureus vaccines; however, there is a paucity of information regarding T cell antigens of S. aureus This study highlights the importance of cell wall-anchored proteins as human CD4 + T cell activators capable of driving antigen-specific Th1 and Th17 cell activation. Clumping factor A (ClfA), which contains N1, N2, and N3 binding domains, was found to be a potent human T cell activator. We further investigated which subdomains of ClfA were involved in T cell activation and found that the full-length ClfA N123 and N23 were potent Th1 and Th17 activators. Interestingly, the N1 subdomain was capable of exclusively activating Th1 cells. Furthermore, when these subdomains were used in a model vaccine, N23 and N1 offered Th1- and Th17-mediated systemic protection in mice upon intraperitoneal challenge. Overall, however, full-length ClfA N123 is required for maximal protection both locally and systemically. Copyright © 2017 Lacey et al.

  9. The hemochromatosis protein HFE 20 years later: An emerging role in antigen presentation and in the immune system.

    Science.gov (United States)

    Reuben, Alexandre; Chung, Jacqueline W; Lapointe, Réjean; Santos, Manuela M

    2017-09-01

    Since its discovery, the hemochromatosis protein HFE has been primarily defined by its role in iron metabolism and homeostasis, and its involvement in the genetic disease termed hereditary hemochromatosis (HH). While HH patients are typically afflicted by dysregulated iron levels, many are also affected by several immune defects and increased incidence of autoimmune diseases that have thereby implicated HFE in the immune response. Growing evidence has supported an immunological role for HFE with recent studies describing HFE specifically as it relates to MHC I antigen presentation. Here, we present a comprehensive overview of the relationship between iron metabolism, HFE, and the immune system to better understand the origin and cause of immune defects in HH patients. We further describe the role of HFE in MHC I antigen presentation and its potential to impair autoimmune responses in homeostatic conditions, a mechanism which may be exploited by tumors to evade immune surveillance. Overall, this increased understanding of the role of HFE in the immune response sets the stage for better treatment and management of HH and other iron-related diseases, as well as of the immune defects related to this condition. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

  10. An autoclave treatment reduces the solubility and antigenicity of an allergenic protein found in buckwheat flour.

    Science.gov (United States)

    Tomotake, Hiroyuki; Yamazaki, Rikio; Yamato, Masayuki

    2012-06-01

    The effects of an autoclave treatment of buckwheat flour on a 24-kDa allergenic protein were investigated by measuring reduction in solubility and antibody binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the intensity of the major bands, including that of the 24-kDa allergen, was reduced by the autoclave treatment. The protein solubility in buckwheat flour was variably decreased by the autoclave treatment. Enzyme-linked immunosorbent assay analysis using a monoclonal antibody specific for buckwheat 24-kDa protein showed that the reactivity of protein extracts (10 μg/ml) from buckwheat flour was lowered by the autoclave treatment. The autoclave treatment may reduce the major allergen content of buckwheat. Future studies