Toward a Network Model of MHC Class II-Restricted Antigen Processing

PubMed Central

Miller, Michael A.; Ganesan, Asha Purnima V.; Eisenlohr, Laurence C.

2013-01-01

The standard model of Major Histocompatibility Complex class II (MHCII)-restricted antigen processing depicts a straightforward, linear pathway: internalized antigens are converted into peptides that load in a chaperone dependent manner onto nascent MHCII in the late endosome, the complexes subsequently trafficking to the cell surface for recognition by CD4+ T cells (TCD4+). Several variations on this theme, both moderate and radical, have come to light but these alternatives have remained peripheral, the conventional pathway generally presumed to be the primary driver of TCD4+ responses. Here we continue to press for the conceptual repositioning of these alternatives toward the center while proposing that MHCII processing be thought of less in terms of discrete pathways and more in terms of a network whose major and minor conduits are variable depending upon many factors, including the epitope, the nature of the antigen, the source of the antigen, and the identity of the antigen-presenting cell. PMID:24379819

Antigen Loss Variants: Catching Hold of Escaping Foes.

PubMed

Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

2017-01-01

Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

PubMed Central

Abdizadeh, Rahman; Maraghi, Sharif; Ghadiri, Ata A.; Tavalla, Mehdi; Shojaee, Saeedeh

2015-01-01

Background: Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. Objectives: The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Materials and Methods: Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB). Results: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. Conclusions: The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development. PMID:25861441

Antigen I/II encoded by integrative and conjugative elements of Streptococcus agalactiae and role in biofilm formation.

PubMed

Chuzeville, Sarah; Dramsi, Shaynoor; Madec, Jean-Yves; Haenni, Marisa; Payot, Sophie

2015-11-01

Streptococcus agalactiae (i.e. Group B streptococcus, GBS) is a major human and animal pathogen. Genes encoding putative surface proteins and in particular an antigen I/II have been identified on Integrative and Conjugative Elements (ICEs) found in GBS. Antigens I/II are multimodal adhesins promoting colonization of the oral cavity by streptococci such as Streptococcus gordonii and Streptococcus mutans. The prevalence and diversity of antigens I/II in GBS were studied by a bioinformatic analysis. It revealed that antigens I/II, which are acquired by horizontal transfer via ICEs, exhibit diversity and are widespread in GBS, in particular in the serotype Ia/ST23 invasive strains. This study aimed at characterizing the impact on GBS biology of proteins encoded by a previously characterized ICE of S. agalactiae (ICE_515_tRNA(Lys)). The production and surface exposition of the antigen I/II encoded by this ICE was examined using RT-PCR and immunoblotting experiments. Surface proteins of ICE_515_tRNA(Lys) were found to contribute to GBS biofilm formation and to fibrinogen binding. Contribution of antigen I/II encoded by SAL_2056 to biofilm formation was also demonstrated. These results highlight the potential for ICEs to spread microbial adhesins between species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

PubMed Central

Owen, Peter; Salton, Milton R. J.

1977-01-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions. Images PMID:144722

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

PubMed

Owen, P; Salton, M R

1977-12-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants in Salmonella Vaccine Strains

PubMed Central

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B.; Lattemann, Claus T.

2003-01-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp6074-86 was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains. PMID:12654812

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

PubMed

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B; Lattemann, Claus T

2003-04-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.

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

PubMed Central

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

2016-01-01

Human influenza viruses are rapidly evolving RNA viruses that cause short-term respiratory infections with substantial morbidity and mortality in annual epidemics. Uncovering the general principles of viral coevolution with human hosts is important for pathogen surveillance and vaccine design. Protein regions are an appropriate model for the interactions between two macromolecules, but the currently used epitope definition for the major antigen of influenza viruses, namely hemagglutinin, is very broad. Here, we combined genetic, evolutionary, antigenic, and structural information to determine the most relevant regions of the hemagglutinin of human influenza A/H3N2 viruses for interaction with human immunoglobulins. We estimated the antigenic weights of amino acid changes at individual sites from hemagglutination inhibition data using antigenic tree inference followed by spatial clustering of antigenicity-altering protein sites on the protein structure. This approach determined six relevant areas (patches) for antigenic variation that had a key role in the past antigenic evolution of the viruses. Previous transitions between successive predominating antigenic types of H3N2 viruses always included amino acid changes in either the first or second antigenic patch. Interestingly, there was only partial overlap between the antigenic patches and the patches under strong positive selection. Therefore, besides alterations of antigenicity, other interactions with the host may shape the evolution of human influenza A/H3N2 viruses. PMID:27774294

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

PubMed

Kawa, Diane E; Stephens, Richard S

2002-05-15

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

Reactivity of commercially available monoclonal antibodies to human CD antigens with peripheral blood leucocytes of dromedary camels (Camelus dromedarius)

PubMed Central

Hussen, Jamal; Shawaf, Turke; Al-herz, Abdulkareem Imran; Alturaifi, Hussain R.; Alluwaimi, Ahmed M.

2017-01-01

Monoclonal antibodies (mAbs) to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present study two-parameter flow cytometry was used to screen existing commercially available mAbs to human leukocyte antigens and major histocompatibility molecules (MHC) for their reactivity with camel leukocytes. The comparison of patterns of reactivity obtained after labelling human and camel leukocytes have shown that mAbs specific to human cluster of differentiation (CD) 18, CD11a, CD11b and CD14 are predicted to be cross-reactive with homologous camel antigens. PMID:28652982

Protein-scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density and Ratio.

PubMed

Smith, Mason R; Tolbert, Stephanie V; Wen, Fei

2018-05-07

Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was co-assembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein-scaffold directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

A T-Cell Receptor Breaks the Rules | Center for Cancer Research

Cancer.gov

Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their

Extended low-resolution structure of a Leptospira antigen offers high bactericidal antibody accessibility amenable to vaccine design

PubMed Central

Tseng, Andrew; Suguiura, Igor Massahiro de Souza; McDonough, Sean P; Sritrakul, Tepyuda; Li, Ting; Lin, Yi-Pin; Gillilan, Richard E

2017-01-01

Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen’s structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines. PMID:29210669

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

PubMed

Schofield, L; McConville, M J; Hansen, D; Campbell, A S; Fraser-Reid, B; Grusby, M J; Tachado, S D

1999-01-08

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

Plasmid and surface antigen markers of endemic and epidemic Legionella pneumophila strains.

PubMed Central

Brown, A; Vickers, R M; Elder, E M; Lema, M; Garrity, G M

1982-01-01

Environmental and clinical isolates of Legionella pneumophila obtained from the Pittsburgh Veterans Administration Medical Center were studied for the presence of plasmids and for unique surface antigens. The majority of environmental isolates contained a single 80-megadalton plasmid. After an epidemic of nosocomial Legionnaires disease subsided in the Spring of 1981, plasmid-bearing environmental isolates persisted in the environment. Whereas L. pneumophila could not be reisolated from most sites with plasmidless isolates. During this epidemic the attack rate was highest on wards with plasmidless isolates. All clinical isolates were plasmidless. Strains were serotyped by the indirect immunofluorescence method with serum from a single immunized rat which was used both without absorption and after absorption with various plasmid-bearing and plasmidless isolates. These studies suggested that a plasmid-associated surface antigen was present and that the most common plasmidless environmental serotype was similar to the epidemic clinical serotype. Images PMID:7119096

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

USDA-ARS?s Scientific Manuscript database

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...

Inference of Genotype–Phenotype Relationships in the Antigenic Evolution of Human Influenza A (H3N2) Viruses

PubMed Central

Steinbrück, Lars; McHardy, Alice Carolyn

2012-01-01

Distinguishing mutations that determine an organism's phenotype from (near-) neutral ‘hitchhikers’ is a fundamental challenge in genome research, and is relevant for numerous medical and biotechnological applications. For human influenza viruses, recognizing changes in the antigenic phenotype and a strains' capability to evade pre-existing host immunity is important for the production of efficient vaccines. We have developed a method for inferring ‘antigenic trees’ for the major viral surface protein hemagglutinin. In the antigenic tree, antigenic weights are assigned to all tree branches, which allows us to resolve the antigenic impact of the associated amino acid changes. Our technique predicted antigenic distances with comparable accuracy to antigenic cartography. Additionally, it identified both known and novel sites, and amino acid changes with antigenic impact in the evolution of influenza A (H3N2) viruses from 1968 to 2003. The technique can also be applied for inference of ‘phenotype trees’ and genotype–phenotype relationships from other types of pairwise phenotype distances. PMID:22532796

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

PubMed

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

PubMed

Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

2016-02-29

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking.

PubMed

Ramakrishna, V; Eisenthal, A; Skornick, Y; Shinitzky, M

1993-05-01

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2',3'-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.

Blood Types

MedlinePlus

... typing and cross-matching. There are four major blood groups determined by the presence or absence of two ... A and B – on the surface of red blood cells: Group A Has only the A antigen on red ...

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

PubMed Central

Tremblay, Jacqueline M.; Oliveira, Sergio C.; Da’dara, Akram A.; Skelly, Patrick J.

2017-01-01

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development. PMID:28095417

Major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagel, S.D.; Boothroyd, J.C.

1989-04-05

P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with (/sup 3/H)palmitic acid and with myo-(2-/sup 3/H)inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological cross-reacting determinant first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with (/sup 35/S)methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purifiedmore » (/sup 3/H) palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.« less

Enteric trimethyl chitosan nanoparticles containing hepatitis B surface antigen for oral delivery.

PubMed

Farhadian, Asma; Dounighi, Naser Mohammadpour; Avadi, Mohammadreza

2015-01-01

Oral vaccination is the preferred route of immunization. However, the degradative condition of the gastrointestinal tract and the higher molecular size of peptides pose major challenges in developing an effective oral vaccination system. One of the most excellent methods used in the development of oral vaccine delivery system relies on the entrapment of the antigen in polymeric nanoparticles. In this work, trimethyl chitosan (TMC) nanoparticles were fabricated using ionic gelation teqnique by interaction hydroxypropyl methylcellulose phthalate (HPMCP), a pH-sensitive polymer, with TMC and the utility of the particles in the oral delivery of hepatitis B surface antigen (HBsAg) was evaluated employing solutions that simulated gastric and intestinal conditions. The particle size, morphology, zeta potential, loading capacity, loading efficiency, in vitro release behavior, structure, and morphology of nanoparticles were evaluated, and the activity of the loaded antigen was assessed. Size of the optimized TMC/HPMCP nanoparticles and that of the antigen-loaded nanoparticles were 85 nm and 158 nm, respectively. Optimum loading capacity (76.75%) and loading efficiency (86.29%) were achieved at 300 µg/mL concentration of the antigen. SEM images revealed a spherical shape as well as a smooth and near-homogenous surface of nanoparticles. Results of the in vitro release studies showed that formulation with HPMCP improved the acid stability of the TMC nanoparticles as well as their capability to preserve the loaded HBsAg from gastric destruction. The antigen showed good activity both before and after loading. The results suggest that TMC/HPMCP nanoparticles could be used in the oral delivery of HBsAg vaccine.

[The occurrence of Escherichia coli with K1 surface antigen in pregnant women and in newborns].

PubMed

Kaczmarek, Agnieszka; Budzyńska, Anna; Gospodarek, Eugenia

2010-01-01

The aim of the study was to determine the frequency of occurrence of K1 surface antigen in Escherichia coli strains isolated from the pregnant women and newborns. A total of 425 of E. coli strains isolated from the faecal samples, 67 strains isolated from the vagina of pregnant women and 40 strains isolated from the newborns' nasal cavity were included into the study. All strains were collected between June and September of 2008. Identification of isolates was followed by the assessment of presence of K1 surface antigen in E. coli strains. The presence of K1 antigen was found in 17,6% of E. coli strains isolated from the faecal samples, 20,9% of E. coli strains isolated from the vagina of pregnant women and in 17,5% of E. coli strains isolated from the newborns' nasal cavity. Routine screening of E. coli K1 colonization gives an opportunity to identify women with the risk of E. coli K1 transmission to neonates during delivery and thereby with major probability of perinatal infections. Latex agglutination test Pastorex Meningitis (Bio-Rad) provides fast identification of E. coli K1 strains.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

PubMed Central

Lee, John K.; Bangayan, Nathanael J.; Chai, Timothy; Smith, Bryan A.; Pariva, Tiffany E.; Yun, Sangwon; Vashisht, Ajay; Zhang, Qingfu; Park, Jung Wook; Corey, Eva; Huang, Jiaoti; Wohlschlegel, James; Witte, Owen N.

2018-01-01

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting. PMID:29686080

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

PubMed Central

Cram, Erik D.; Simmons, Ryan S.; Palmer, Amy L.; Hildebrand, William H.; Rockey, Daniel D.

2015-01-01

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8+ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8+ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8+ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. PMID:26597986

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 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...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 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...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 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...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true 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...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

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...

Comprehensive Analysis and Characterization of Linear Antigenic Domains on HN Protein from Genotype VII Newcastle Disease Virus Using Yeast Surface Display System

PubMed Central

Li, Tao; Wang, Gaoling; Shi, Bingtian; Liu, Peixin; Si, Wei; Wang, Bin; Jiang, Li; Zhou, Lunjiang; Xiu, Jinsheng; Liu, Henggui

2015-01-01

Circulation of genotype VII Newcastle disease virus (NDV) has posed a great threat for the poultry industry worldwide. Antibodies against Hemagglutinin-neuraminidase (HN), a membrane protein of NDV with critical roles in NDV infection, have been reported to provide chickens protection from NDV infection. In this study, we comprehensively analyzed the in vivo antibody responses against the linear antigenic domains of the HN protein from genotype VII NDV using a yeast surface display system. The results revealed four distinct regions of HN, P1 (1-52aa), P2 (53-192aa), P3 (193-302aa) and P4 (303-571aa), respectively, according to their antigenic potency. Analysis by FACS and ELISA assay indicated P2 to be the dominant linear antigenic domain, with the immunogenic potency to protect the majority of chickens from NDV challenge. In contrast, the P1, P3 and P4 domains showed weak antigenicity in vivo and could not protect chickens from NDV challenge. These results provide important insight into the characteristic of humoral immune responses elicited by HN of NDV in vivo. PMID:26121247

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

Proteome Analysis and Serological Characterization of Surface-Exposed Proteins of Rickettsia heilongjiangensis

PubMed Central

Qi, Yong; Xiong, Xiaolu; Wang, Xile; Duan, Changsong; Jia, Yinjun; Jiao, Jun; Gong, Wenping; Wen, Bohai

2013-01-01

Background Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. Methods R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA). Results Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. Conclusions Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease. PMID:23894656

Blocking Blood Supply to Breast Carcinoma With a DNA Vaccine Encoding VEGF Receptor-2

DTIC Science & Technology

2006-03-01

recognize antigens in the form of 8 to 10 amino acid long peptides, presented to T- cell receptors (TCRs) on the cell surface as complexes with major... receptor , and providing tumor- associated antigens , our DNA vaccine can efficiently activate DCs, NK cells , and CTLs, presumably in Peyer’s patches. The... immunoreceptor in immune cell activation and natural killing. Immunity. 2002;17:19-29. (5) Snyder MR, Weyand CM, Goronzy JJ. The double life of NK receptors

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

PubMed

Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens. Copyright © 2017 Schmid-Siegert et al.

Regions of recognition by blocking antibodies on the light chain of botulinum neurotoxin A: antigenic structure of the entire toxin.

PubMed

Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair

2011-06-01

The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1-453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed. Copyright © 2010 Elsevier GmbH. All rights reserved.

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display.

PubMed

Leung, Carol S; Haigh, Tracey A; Mackay, Laura K; Rickinson, Alan B; Taylor, Graham S

2010-02-02

Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBV-infected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4(+) T cell epitopes. Using CD4(+) T cell clones against three indicator epitopes, we find that two epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface.

Surface-exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis.

PubMed Central

Murphy, T F; Bartos, L C

1989-01-01

The outer membrane proteins (OMPs) of Branhamella catarrhalis were studied in an effort to identify surface-exposed determinants that are conserved among strains of the bacterium. Aliquots of polyclonal antiserum were absorbed individually by strains of B. catarrhalis. The absorbed antisera were tested in comparison with unabsorbed antiserum in an immunoblot assay against OMPs of the homologous strain. The absence of a band recognized by antibodies in the absorbed antiserum compared with the unabsorbed antiserum indicated that surface-exposed determinants of the absorbing strain cross-reacted with determinants on the homologous strain. Two antisera were absorbed individually by 20 strains of B. catarrhalis, and the absorbed sera were studied in this way in immunoblot assays. OMP E (molecular weight, ca. 56,000) expresses surface-exposed determinants that are shared among 17 of the 20 strains studied. Antibodies to OMP G (molecular weight, 28,000) were absorbed from both antisera by 14 of the 20 strains. These studies demonstrate that OMP E and OMP G express determinants that are exposed on the surface of the intact bacterium. Furthermore, these determinants are antigenically conserved among a majority of strains of B. catarrhalis. On the basis of these observations, OMPs E and G should be considered when bacterial antigens are evaluated as potential vaccine candidates. Images PMID:2476393

Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol).

PubMed

Murad, K L; Mahany, K L; Brugnara, C; Kuypers, F A; Eaton, J W; Scott, M D

1999-03-15

We previously showed that the covalent modification of the red blood cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW approximately 5 kD] could significantly attenuate the immunologic recognition of surface antigens. However, to make these antigenically silent RBC a clinically viable option, the mPEG-modified RBC must maintain normal cellular structure and functions. To this end, mPEG-derivatization was found to have no significant detrimental effects on RBC structure or function at concentrations that effectively blocked antigenic recognition of a variety of RBC antigens. Importantly, RBC lysis, morphology, and hemoglobin oxidation state were unaffected by mPEG-modification. Furthermore, as shown by functional studies of Band 3, a major site of modification, PEG-binding does not affect protein function, as evidenced by normal SO4- flux. Similarly, Na+ and K+ homeostasis were unaffected. The functional aspects of the mPEG-modified RBC were also maintained, as evidenced by normal oxygen binding and cellular deformability. Perhaps most importantly, mPEG-derivatized mouse RBC showed normal in vivo survival ( approximately 50 days) with no sensitization after repeated transfusions. These data further support the hypothesis that the covalent attachment of nonimmunogenic materials (eg, mPEG) to intact RBC may have significant application in transfusion medicine, especially for the chronically transfused and/or allosensitized patient.

Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

PubMed

Dauvillée, David; Delhaye, Stéphane; Gruyer, Sébastien; Slomianny, Christian; Moretz, Samuel E; d'Hulst, Christophe; Long, Carole A; Ball, Steven G; Tomavo, Stanislas

2010-12-15

Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true 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...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 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...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 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...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 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...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

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...

Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

NASA Astrophysics Data System (ADS)

Sharma, Shobhona; Godson, G. Nigel

1985-05-01

The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

Mouse HLA-DPA homologue H2-Pa: A pseudogene that maps between H2-Pb and H2-Oa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arimura, Y.; Koda, T.; Kishi, M.

1996-12-31

The major histocompatibility complex (MHC) class II subregion contains several subclasses of genes. The classical class II genes, HLA-DP, DQ, and DR homologues, present antigens directly to CD4{sup +} T cells. HLA-DM homologues facilitate the efficacy and transport of antigens to the cell surface by removing the CLIP peptides from the classical class II molecules. HLA-DNA/DOB homologues show unusual expression patterns and limited polymorphism, but their function is yet to be elucidated. 15 refs., 2 figs.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Natural killer T cells in health and disease

PubMed Central

Wu, Lan; Van Kaer, Luc

2013-01-01

Natural killer T (NKT) cells are a subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semiinvariant T cell receptor that reacts with glycolipid antigens presented by the major histocompatibility complex class I-related protein CD1d on the surface of antigen-presenting cells. NKT cells become activated during a variety of infections and inflammatory conditions, rapidly producing large amounts of immunomodulatory cytokines. NKT cells can influence the activation state and functional properties of multiple other cell types in the immune system and, thus, modulate immune responses against infectious agents, autoantigens, tumors, tissue grafts and allergens. One attractive aspect of NKT cells is that their immunomodulatory activities can be readily harnessed with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid alpha-galactosylceramide. These properties of NKT cells are being exploited for therapeutic intervention to prevent or treat cancer, infections, and autoimmune and inflammatory diseases. PMID:21196373

Cross-specificity of protective human antibodies against Klebsiella pneumoniae LPS O-antigen.

PubMed

Rollenske, Tim; Szijarto, Valeria; Lukasiewicz, Jolanta; Guachalla, Luis M; Stojkovic, Katarina; Hartl, Katharina; Stulik, Lukas; Kocher, Simone; Lasitschka, Felix; Al-Saeedi, Mohammed; Schröder-Braunstein, Jutta; von Frankenberg, Moritz; Gaebelein, Gereon; Hoffmann, Peter; Klein, Sabrina; Heeg, Klaus; Nagy, Eszter; Nagy, Gabor; Wardemann, Hedda

2018-06-01

Humoral immune responses to microbial polysaccharide surface antigens can prevent bacterial infection but are typically strain specific and fail to mediate broad protection against different serotypes. Here we describe a panel of affinity-matured monoclonal human antibodies from peripheral blood immunoglobulin M-positive (IgM + ) and IgA + memory B cells and clonally related intestinal plasmablasts, directed against the lipopolysaccharide (LPS) O-antigen of Klebsiella pneumoniae, an opportunistic pathogen and major cause of antibiotic-resistant nosocomial infections. The antibodies showed distinct patterns of in vivo cross-specificity and protection against different clinically relevant K. pneumoniae serotypes. However, cross-specificity was not limited to K. pneumoniae, as K. pneumoniae-specific antibodies recognized diverse intestinal microbes and neutralized not only K. pneumoniae LPS but also non-K. pneumoniae LPS. Our data suggest that the recognition of minimal glycan epitopes abundantly expressed on microbial surfaces might serve as an efficient humoral immunological mechanism to control invading pathogens and the large diversity of the human microbiota with a limited set of cross-specific antibodies.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

PubMed

Talmadge, J E; Talmadge, C B; Zbar, B; McEwen, R; Meeker, A K; Tribble, H

1987-06-01

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment.

PubMed

Wang, Zhenguang; Guo, Yelei; Han, Weidong

2017-12-01

Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

[Production, specificity and structure of immunoglobulins].

PubMed

Goujard, C; Delfraissy, J F

1991-03-21

Immunoglobulin is a key factor of the immune response resulting from B-cell activation and associated with T-cell stimulation. Because of its structure, this antibody has a dual function: it specifically recognizes the inducer antigen in the variable region and eliminates it by a constant portion which is responsible for effector properties. Surface immunoglobulin, therefore, is the B-cell antigen receptor; it differs from the T-cell receptor in that it recognizes the antigen unbound to the major istocompatibility complex; binding the antigen results in direct signal transduction first in the cytoplasm, then in the nucleus. This receptor can be secreted in the body: it is made up of circulating immunoglobulins. Human immunoglobulins are divided into 5 classes, each of them with its own response kinetics, distribution and functions. The variability of the antibody response accounts for a genetic organization involving numerous genes which may be associated with each other, or mutate, or recombine during maturation of the lymphocytes. Altogether, this system has a theoretical capacity of response to three hundred million different antigens.

The Density Code for the Development of a Vaccine?

PubMed Central

Cheng, Wei

2016-01-01

The development of prophylactic vaccines remains largely empirical in nature and rarely have general rules been applied in the strategic decision and the formulation of a viral vaccine. Currently there are a total of 15 virus agents from 12 unique virus families with vaccines licensed by the US Food and Drug Administration. Extensive structural information on these viral particles and potential mechanisms of protection are available for the majority of these virus pathogens and their respective vaccines. Here I review the quantitative features of these viral surface antigens in relation to the molecular mechanisms of B cell activation, and point out a potential correlation between the density of immunogenic proteins displayed on the surface of the vaccine antigen carrier and the success of a vaccine. These features help us understand the humoral immunity induced by viral vaccines on a quantitative ground and re-emphasize the importance of antigen density on the activation of the immune system. Although the detailed mechanisms behind this phenomenon remain to be explored, it implies that both the size of antigen carriers and the density of immunogenic proteins displayed on these carriers are important parameters that may need to be optimized for the formulation of a vaccine. PMID:27649885

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

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

PubMed

Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

2009-12-01

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 development 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.

T-cell receptor revision: friend or foe?

PubMed

Hale, J Scott; Fink, Pamela J

2010-04-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

PubMed Central

Fung, Erik; Esposito, Laura; Todd, John A.; Wicker, Linda S.

2010-01-01

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (dendritic cells, monocytes, B lymphocytes) in minimally manipulated whole blood. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, to demonstrate the quantification of surface expression levels of molecules involved in chemotaxis (e.g. CX3CR1, CCR2), adhesion (e.g. CD11b, CD62L), antigen presentation (e.g. CD83, CD86, CD209) and immune regulation (e.g. CD101) on circulating antigen-presenting cells. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood, no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h. PMID:20134434

Mobile DNA in the pathogenic Neisseria

PubMed Central

Obergfell, Kyle P.; Seifert, H. Steven

2015-01-01

The genus Neisseria contains two pathogenic species of notable public health concern: Neisseria gonorrhoeae and Neisseria meningitidis. These pathogens display a notable ability to undergo frequent programmed recombination events. The recombination mediated pathways of transformation and pilin antigenic variation in the Neisseria are well studied systems that are critical for pathogenesis. Here we will detail the conserved and unique aspects of transformation and antigenic variation in the Neisseria. Transformation will be followed from initial DNA binding through recombination into the genome with consideration to the factors necessary at each step. Additional focus is paid to the unique type IV secretion system that mediates donation of transforming DNA in the pathogenic Neisseria. The pilin antigenic variation system uses programed recombinations to alter a major surface determinant which allows immune avoidance and promotes infection. We discuss the trans- and cis- acting factors which facilitate pilin antigenic variation and present the current understanding of the mechanisms involved in the process. PMID:25866700

A T-Cell Receptor Breaks the Rules | Center for Cancer Research

Cancer.gov

Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their parents; and the antigen fragment, called a peptide epitope, is excised from one of thousands of possible proteins—originally part of an invading pathogen or a cancer cell—that T cells are capable of identifying and attacking. The framework of an MHC molecule holding a centrally displayed or “presented” peptide is what engages the TCR and triggers T-cell action. This role of MHC molecules presenting antigens to the TCR is a central tenet of immunology, with the fit between a TCR and the MHC framework actually “hardwired” into their three-dimensional structures.

Genomic analysis of Bacteroides fragilis reveals extensive DNA inversions regulating cell surface adaptation

PubMed Central

Kuwahara, Tomomi; Yamashita, Atsushi; Hirakawa, Hideki; Nakayama, Haruyuki; Toh, Hidehiro; Okada, Natsumi; Kuhara, Satoru; Hattori, Masahira; Hayashi, Tetsuya; Ohnishi, Yoshinari

2004-01-01

Bacteroides are predominant human colonic commensals, but the principal pathogenic species, Bacteroides fragilis (BF), lives closely associated with the mucosal surface, whereas a second major species, Bacteroides thetaiotaomicron (BT), concentrates within the colon. We find corresponding differences in their genomes, based on determination of the genome sequence of BF and comparative analysis with BT. Both species have acquired two mechanisms that contribute to their dominance among the colonic microbiota: an exceptional capability to use a wide range of dietary polysaccharides by gene amplification and the capacity to create variable surface antigenicities by multiple DNA inversion systems. However, the gene amplification for polysaccharide assimilation is more developed in BT, in keeping with its internal localization. In contrast, external antigenic structures can be changed more systematically in BF. Thereby, at the mucosal surface, where microbes encounter continuous attack by host defenses, BF evasion of the immune system is favored, and its colonization and infectious potential are increased. PMID:15466707

Conservation of myeloid surface antigens on primate granulocytes.

PubMed

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Genetic diversity of K-antigen gene clusters of Escherichia coli and their molecular typing using a suspension array.

PubMed

Yang, Shuang; Xi, Daoyi; Jing, Fuyi; Kong, Deju; Wu, Junli; Feng, Lu; Cao, Boyang; Wang, Lei

2018-04-01

Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.

Mapping epitopes and antigenicity by site-directed masking

NASA Astrophysics Data System (ADS)

Paus, Didrik; Winter, Greg

2006-06-01

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets*

PubMed Central

Rhoden, John J.; Dyas, Gregory L.

2016-01-01

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. PMID:27022022

Adsorption of virus-like particles on ion exchange surface: Conformational changes at different pH detected by dual polarization interferometry.

PubMed

Yang, Yanli; Mengran Yu; Zhang, Songping; Ma, Guanghui; Su, Zhiguo

2015-08-21

Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The "lateral" protein-protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein-surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism for the disassembling of VLPs on the solid-liquid interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Fya/Fyb antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria

PubMed Central

King, Christopher L.; Adams, John H.; Xianli, Jia; Grimberg, Brian T.; McHenry, Amy M.; Greenberg, Lior J.; Siddiqui, Asim; Howes, Rosalind E.; da Silva-Nunes, Monica; Ferreira, Marcelo U.; Zimmerman, Peter A.

2011-01-01

Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fya or Fyb, resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fya, compared with Fyb, significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fya had 41–50% lower binding compared with Fyb cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fya+b− phenotype demonstrated a 30–80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fya+b− phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes. PMID:22123959

Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria.

PubMed

King, Christopher L; Adams, John H; Xianli, Jia; Grimberg, Brian T; McHenry, Amy M; Greenberg, Lior J; Siddiqui, Asim; Howes, Rosalind E; da Silva-Nunes, Monica; Ferreira, Marcelo U; Zimmerman, Peter A

2011-12-13

Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fy(a) or Fy(b), resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fy(a), compared with Fy(b), significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fy(a) had 41-50% lower binding compared with Fy(b) cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fy(a+b-) phenotype demonstrated a 30-80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fy(a+b-) phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

PubMed Central

Worth, Danielle; Huang, Sherri

2018-01-01

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host. PMID:29718996

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis.

PubMed

Radke, Joshua B; Worth, Danielle; Hong, David; Huang, Sherri; Sullivan, William J; Wilson, Emma H; White, Michael W

2018-05-01

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host.

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica.

PubMed

Hiriart, Yanina; Serradell, Maria; Martínez, Araci; Sampaolesi, Sofia; Maciel, Dolores Gonzalez; Chabalgoity, Jose Alejandro; Yim, Lucía; Algorta, Gabriela; Rumbo, Martin

2013-01-01

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.

Salmonella O48 Serum Resistance is Connected with the Elongation of the Lipopolysaccharide O-Antigen Containing Sialic Acid

PubMed Central

Pawlak, Aleksandra; Rybka, Jacek; Dudek, Bartłomiej; Krzyżewska, Eva; Rybka, Wojciech; Kędziora, Anna; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-01-01

Complement is one of the most important parts of the innate immune system. Some bacteria can gain resistance against the bactericidal action of complement by decorating their outer cell surface with lipopolysaccharides (LPSs) containing a very long O-antigen or with specific outer membrane proteins. Additionally, the presence of sialic acid in the LPS molecules can provide a level of protection for bacteria, likening them to human cells, a phenomenon known as molecular mimicry. Salmonella O48, which contains sialic acid in the O-antigen, is the major cause of reptile-associated salmonellosis, a worldwide public health problem. In this study, we tested the effect of prolonged exposure to human serum on strains from Salmonella serogroup O48, specifically on the O-antigen length. After multiple passages in serum, three out of four tested strains became resistant to serum action. The gas-liquid chromatography/tandem mass spectrometry analysis showed that, for most of the strains, the average length of the LPS O-antigen increased. Thus, we have discovered a link between the resistance of bacterial cells to serum and the elongation of the LPS O-antigen. PMID:28934165

The future for blood-stage vaccines against malaria.

PubMed

Richards, Jack S; Beeson, James G

2009-07-01

Malaria is a leading cause of mortality and morbidity globally, and effective vaccines are urgently needed. Malaria vaccine approaches can be broadly grouped as pre-erythrocytic, blood stage and transmission blocking. This review focuses on blood-stage vaccines, and considers the evidence supporting the development of blood-stage vaccines, the advantages and challenges of this approach, potential targets, human vaccine studies and future directions. There is a strong rationale for the development of vaccines based on antigens of blood-stage parasites. Symptomatic malaria is caused by blood-stage parasitemia and acquired immunity in humans largely targets blood-stage antigens. Several candidate vaccines have proved efficacious in animal models and at least one vaccine showed partial efficacy in a clinical trial. At present, all leading candidate blood-stage antigens are merozoite proteins, located on the merozoite surface or within the apical organelles. Major challenges and priorities include overcoming antigenic diversity, identification of protective epitopes, understanding the nature and targets of protective immune responses, and defining antigen combinations that give the greatest efficacy. Additionally, objective criteria and approaches are needed to prioritize the large number of candidate antigens, and strong candidates need to be tested in clinical trials as quickly as possible.

Salmonella O48 Serum Resistance is Connected with the Elongation of the Lipopolysaccharide O-Antigen Containing Sialic Acid.

PubMed

Pawlak, Aleksandra; Rybka, Jacek; Dudek, Bartłomiej; Krzyżewska, Eva; Rybka, Wojciech; Kędziora, Anna; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-09-21

Complement is one of the most important parts of the innate immune system. Some bacteria can gain resistance against the bactericidal action of complement by decorating their outer cell surface with lipopolysaccharides (LPSs) containing a very long O-antigen or with specific outer membrane proteins. Additionally, the presence of sialic acid in the LPS molecules can provide a level of protection for bacteria, likening them to human cells, a phenomenon known as molecular mimicry. Salmonella O48, which contains sialic acid in the O-antigen, is the major cause of reptile-associated salmonellosis, a worldwide public health problem. In this study, we tested the effect of prolonged exposure to human serum on strains from Salmonella serogroup O48, specifically on the O-antigen length. After multiple passages in serum, three out of four tested strains became resistant to serum action. The gas-liquid chromatography/tandem mass spectrometry analysis showed that, for most of the strains, the average length of the LPS O-antigen increased. Thus, we have discovered a link between the resistance of bacterial cells to serum and the elongation of the LPS O-antigen.

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

PubMed Central

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

PubMed

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Expression of hepatitis B surface antigen in transgenic plants.

PubMed Central

Mason, H S; Lam, D M; Arntzen, C J

1992-01-01

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems. Images PMID:1465391

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles.

PubMed

Wacker, Ron; Ceyhan, Buelent; Alhorn, Petra; Schueler, Dirk; Lang, Claus; Niemeyer, Christof M

2007-06-01

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

Endoplasmic reticulum aminopeptidase 1 function and its pathogenic role in regulating innate and adaptive immunity in cancer and major histocompatibility complex class I-associated autoimmune diseases.

PubMed

Fruci, D; Romania, P; D'Alicandro, V; Locatelli, F

2014-08-01

Major histocompatibility complex (MHC) class I molecules present antigenic peptides on the cell surface to alert natural killer (NK) cells and CD8(+) T cells for the presence of abnormal intracellular events, such as virus infection or malignant transformation. The generation of antigenic peptides is a multistep process that ends with the trimming of N-terminal extensions in the endoplasmic reticulum (ER) by aminopeptidases ERAP1 and ERAP2. Recent studies have highlighted the potential role of ERAP1 in reprogramming the immunogenicity of tumor cells in order to elicit innate and adaptive antitumor immune responses, and in conferring susceptibility to autoimmune diseases in predisposed individuals. In this review, we will provide an overview of the current knowledge about the role of ERAP1 in MHC class I antigen processing and how its manipulation may constitute a promising tool for cancer immunotherapy and treatment of MHC class I-associated autoimmune diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Adaptor protein-3 is required in dendritic cells for optimal Toll-like receptor signaling from phagosomes and antigen presentation to CD4+ T cells

PubMed Central

Mantegazza, Adriana R.; Guttentag, Susan H.; El-Benna, Jamel; Sasai, Miwa; Iwasaki, Akiko; Shen, Hao; Laufer, Terri M.; Marks, Michael S.

2012-01-01

SUMMARY Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4+ T cell activation and Th1 effector function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores. PMID:22560444

Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

PubMed

Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

2014-12-01

Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1. © 2014 John Wiley & Sons Ltd.

A biochip-based combined immunoassay for detection of serological status of Borrelia burgdorferi in Lyme borreliosis.

PubMed

Huang, Na-Li; Ye, Lei; Lv, Hui; Du, Yi-Xin; Schneider, Marion; Fan, Li-Bin; Du, Wei-Dong

2017-09-01

Dithiobis (succinimidyl undecanoate) modified gold surface biochip were used as a combined immunoassay platform for concurrently detecting immune responses to Borrelia burgdorferi (B. burgdorferi) sensu lato antigens, flagellin, outer surface protein C, variable major protein-like sequence proteins, and 3 VlsE protein IR 6 peptides. The peptides represented intrinsic Borrelia genospecies: B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. Fourier transform infrared spectroscopy was utilized to validate the surface chemical characteristics on the modified gold surface. The limits in detection of IgG antibody on the biochips were as little as 0.39μg/ml for anti-VlsE and 0.78μg/ml for anti-flagellin and anti-OspC, respectively. Samples from 56 neuroborreliosis (NB) patients and 114 healthy individuals were analyzed by the combined biochip. We found that the seroprevalences of IgM or IgG antibody against the 6 antigens were contributed to increased overall sensitivity by the multiplex immunobiochip assay. Serum combined positive rates of the 6 antigens in the patients were 92.86% for IgM antibody and 91.07% for IgG antibody. Part of the patients bore antibody responses against the 3 VlsE IR 6 variant peptides, indicating that Lyme borreliosis would attribute to consequence of multiple infections by one or more Borrelia burgdorferi strains. Concurrent assessment for both IgM and IgG antibodies against the protein antigens and B. burgdorferi IR 6 peptides in the sera of NB patients was beneficial from the biochip format, enabling detection of expanded serologic infection status and therapy strategy-making more efficiently. The combined biochip-based immunoassay, as a potential substitution of ELISA, provided a promising approach to extend the detection spectrum of infectious antibodies against a panel of Borrelia antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of density and epitopes of D antigen on the surface of erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele.

PubMed

Gu, Juan; Sun, An-Yuan; Wang, Xue-Dong; Shao, Chao-Peng; Li, Zheng; Huang, Li-Hua; Pan, Zhao-Lin; Wang, Qing-Ping; Sun, Guang-Ming

2014-04-01

The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population. A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

PubMed

Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

2005-09-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Detection of low-level environmental chemical allergy by a long-term sensitization method.

PubMed

Fukuyama, Tomoki; Ueda, Hideo; Hayashi, Koichi; Tajima, Yukari; Shuto, Yasufumi; Saito, Toru R; Harada, Takanori; Kosaka, Tadashi

2008-07-30

Multiple chemical sensitivity (MCS) is characterized by various signs, including neurological disorders and allergy. Exposure may occur through a major event, such as a chemical spill, or from long-term contact with chemicals at low levels. We are interested in the allergenicity of MCS and the detection of low-level chemical-related hypersensitivity. We used long-term sensitization followed by low-dose challenge to evaluate sensitization by well-known Th2 type sensitizers (trimellitic anhydride (TMA) and toluene diisocyanate (TDI)) and a Th1 type sensitizer (2,4-dinitrochlorobenzene (DNCB)). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them with TMA, TDI or DNCB, we assayed their auricular lymph nodes (LNs) for number of lymphocytes, surface antigen expression of B cells, and local cytokine production, and measured antigen-specific serum IgE levels. TMA and TDI induced marked increases in levels of antigen-specific serum IgE and of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) produced by ex vivo restimulated lymph node cells. DNCB induced a marked increase in Th1 cytokine (IL-2, IFN-gamma, and TNF-alpha) levels, but antigen-specific serum IgE levels were not elevated. All chemicals induced significant increases in number of lymphocytes and surface antigen expression of B cells. Our mouse model enabled the identification and characterization of chemical-related allergic reactions at low levels. This long-term sensitization method would be useful for detecting environmental chemical-related hypersensitivity.

Classification of infectious bursal disease virus into genogroups.

PubMed

Michel, Linda O; Jackwood, Daral J

2017-12-01

Infectious bursal disease virus (IBDV) causes infectious bursal disease (IBD), an immunosuppressive disease of poultry. The current classification scheme of IBDV is confusing because it is based on antigenic types (variant and classical) as well as pathotypes. Many of the amino acid changes differentiating these various classifications are found in a hypervariable region of the capsid protein VP2 (hvVP2), the major host protective antigen. Data from this study were used to propose a new classification scheme for IBDV based solely on genogroups identified from phylogenetic analysis of the hvVP2 of strains worldwide. Seven major genogroups were identified, some of which are geographically restricted and others that have global dispersion, such as genogroup 1. Genogroup 2 viruses are predominately distributed in North America, while genogroup 3 viruses are most often identified on other continents. Additionally, we have identified a population of genogroup 3 vvIBDV isolates that have an amino acid change from alanine to threonine at position 222 while maintaining other residues conserved in this genogroup (I242, I256 and I294). A222T is an important mutation because amino acid 222 is located in the first of four surface loops of hvVP2. A similar shift from proline to threonine at 222 is believed to play a role in the significant antigenic change of the genogroup 2 IBDV strains, suggesting that antigenic drift may be occurring in genogroup 3, possibly in response to antigenic pressure from vaccination.

Use of existing data for public health planning: a study of the prevalence of hepatitis B surface antigen and core antibody in Al Ain Medical District, United Arab Emirates.

PubMed Central

al-Owais, A.; al-Suwaidi, K.; Amiri, N.; Carter, A. O.; Hossain, M. M.; Sheek-Hussein, M. M.

2000-01-01

INTRODUCTION: Hepatitis B is of major public health importance. Accurate information on its occurrence, with particular reference to the prevalence of immunity and chronic infection (marked by the presence of hepatitis B core antibody and surface antigen, respectively, in serum), is essential for planning public health programmes for the control of the disease. The generation of marker prevalence data through serological surveys is costly and time-consuming. The present study in Al Ain Medical District, United Arab Emirates, investigated the possibility of obtaining sufficiently accurate marker prevalence estimates from existing data to plan public health programmes. METHODS: Two antenatal screening databases, one student serological survey database, one immunization programme database and one pre-marriage screening database containing information on marker prevalence were identified. Epidemiological data were abstracted from these databases and analysed. RESULTS: The data showed that the prevalence of hepatitis B surface antigen and the prevalence of core antibody in young citizens in 1998 were approximately 2% and 14% respectively, that any immunization campaign aimed at citizens of the United Arab Emirates should target teenagers as they had the highest risk of acquiring the disease, and that pre-immunization screening of young adults would be wasteful. However, the data did not yield information on the prevalence of hepatitis B surface antigen and core antibody in other population subgroups of public health significance. DISCUSSION: While data generated by the study are sufficient to support a hepatitis B immunization programme targeted at teenaged citizens, more accurate data, generated by a well-designed serological survey, would be essential for optimal public health planning. PMID:11143192

Rabies virus glycoprotein as a carrier for anthrax protective antigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Mary Ellen; Koser, Martin; Xiao Sa

2006-09-30

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen wasmore » also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.« less

Sweat Allergy.

PubMed

Hiragun, Takaaki; Hide, Michihiro

2016-01-01

For many years, sweat has been recognized as an exacerbation factor in all age groups of atopic dermatitis (AD) and a trigger of cholinergic urticaria (CholU). Recently, we reported the improvement of AD symptoms by spray with tannic acid, which suppresses basophil histamine release by semipurified sweat antigens in vitro, and showering that removes antigens in sweat from the skin surface. We finally identified MGL_1304 secreted by Malassezia globosa as a major histamine-releasing antigen in human sweat. MGL_1304 is detected as a 17-kDa protein in sweat and exhibits almost the highest histamine-release ability from basophils of patients with AD and CholU among antigens derived from Malassezia species. Moreover, serum levels of anti-MGL_1304 IgE of patients with AD and CholU were significantly higher than those of normal controls. Desensitization therapy using autologous sweat or MGL_1304 purified from culture of M. globosa or its cognates might be beneficial for patients with intractable CholU due to sweat allergy. © 2016 S. Karger AG, Basel.

B-cell acquisition of antigen: Sensing the surface.

PubMed

Knight, Andrew M

2015-06-01

B-cell antigen receptor (BCR) recognition and acquisition of antigen by B cells is the essential first step in the generation of effective antibody responses. As B-cell-mediated antigen presentation is also believed to play a significant role in the activation of CD4(+) Th-cell responses, considerable effort has focused on clarifying the nature of antigen/BCR interactions. Following earlier descriptions of interactions of soluble antigens with the BCR, it is now clear that B cells also recognize, physically extract and present antigens that are tethered to, or integral components of, the surfaces or extracellular matrix of other cells. In this issue of the European Journal of Immunology, Zeng et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] examine how the physical property or "stiffness" of the surface displaying antigens to B cells influences the B-cell response. This commentary reports that antigen tethered on "less stiff" surfaces induces increased B-cell activation and antibody responses. I then infer how "sensing the surface" by B cells may represent a new component of the immune system's ability to detect "damage," and how this understanding may influence approaches to clinical therapies where immune activity is either unwanted or desired. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

T-cell receptor revision: friend or foe?

PubMed Central

Hale, J Scott; Fink, Pamela J

2010-01-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue. PMID:20201984

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center for... used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen. [40 FR...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

A Molecular Simulation Study of Antibody-Antigen Interactions on Surfaces for the Rational Design of Next-Generation Antibody Microarrays

NASA Astrophysics Data System (ADS)

Bush, Derek B.

Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

Binding of HBGA-expressing bacteria does not protect Tulane virus from acute heat stress

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the major cause of gastroenteritis outbreaks worldwide. Human noroviruses can interact with histo-blood group antigens (HBGAs) on the surface of mammalian cells as well as bacterial cells. HBGAs have been considered as putative receptors or co-receptors for HuNoVs in m...

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody.

PubMed

Shanker, Sreejesh; Czakó, Rita; Sapparapu, Gopal; Alvarado, Gabriela; Viskovska, Maria; Sankaran, Banumathi; Atmar, Robert L; Crowe, James E; Estes, Mary K; Prasad, B V Venkataram

2016-10-04

Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.

Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

PubMed Central

Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

2014-01-01

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

PubMed Central

Davoodi-Semiromi, Abdoreza; Schreiber, Melissa; Nallapali, Samson; Verma, Dheeraj; Singh, Nameirakpam D.; Banks, Robert K.; Chakrabarti, Debopam; Daniell, Henry

2009-01-01

Summary Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10+ T cell but not Foxp3+ regulatory T cells, suppression of interferon-γ and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity. PMID:20051036

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

NASA Astrophysics Data System (ADS)

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.

O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa

PubMed Central

Rapicavoli, Jeannette N.; Kinsinger, Nichola; Perring, Thomas M.; Backus, Elaine A.; Shugart, Holly J.; Walker, Sharon

2015-01-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. PMID:26386068

O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

PubMed

Rapicavoli, Jeannette N; Kinsinger, Nichola; Perring, Thomas M; Backus, Elaine A; Shugart, Holly J; Walker, Sharon; Roper, M Caroline

2015-12-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies

PubMed Central

Neauport-Sautes, Catherine; Silvestre, Daniele; Niccolai, Marie-Gabrielle; Kourilsky, F. M.; Levy, J. P.

1972-01-01

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed. ImagesFIG. 2FIG. 3FIG. 4FIG. 5 PMID:5063188

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

PubMed

Lyon, J A; Haynes, J D; Diggs, C L; Chulay, J D; Pratt-Rossiter, J M

1986-03-15

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients.

PubMed

Zhao, Yongxing; Qiao, Hailing

2003-12-01

To investigate the mechanism(s) of penicillins allergic reaction. The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test. Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P < 0.05). The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

IgGs are made for walking on bacterial and viral surfaces

NASA Astrophysics Data System (ADS)

Preiner, Johannes; Kodera, Noriyuki; Tang, Jilin; Ebner, Andreas; Brameshuber, Mario; Blaas, Dieter; Gelbmann, Nicola; Gruber, Hermann J.; Ando, Toshio; Hinterdorfer, Peter

2014-07-01

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit ‘bipedal’ stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer1

PubMed Central

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-01-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informativemodel for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction. PMID:18030364

Enhanced expression of interferon-gamma-induced antigen-processing machinery components in a spontaneously occurring cancer.

PubMed

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-11-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informative model for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.

Cloning and analysis of the gene for a major surface antigen of Mycoplasma gallisepticum.

PubMed

Spencer, Denise L; Kurth, Kathy Toohey; Menon, Sreekumar A; VanDyk, Tina; Minion, F Chris

2002-01-01

Myplasma gallisepticum infects a wide variety of gallineaceous birds including chickens, turkeys, and pheasants. Infection occurs both horizontally and vertically. Thus, control of the spread of M. gallisepticum to noninfected flocks is difficult. Continual monitoring is necessary to identify infected flocks even under the most stringent infectious control practices. Monitoring, however, is usually performed by measuring hemagglutination activity (HA) in serum, an insensitive and variable test. Variability in the HA test arises differences in agglutination antigen, changes in antigenic profiles of the M. gallisepticum strain, and variability in reading the agglutination reaction. Enzyme-linked immunosorbent assays (ELISAs) are the preferred method of testing because of the ease in obtaining sera and the sensitivity and reproducibility of the assays, but the ELISA suffers from a lack of standardization in the test antigen. The ELISA test will be more easily accepted once the test antigen has been standardized. To this end, we have identified, cloned, and characterized the gene for an antigen that has potential as a species-specific antigen for M. gallisepticum The gene codes for a 75-kD protein, P75, that is recognized during natural infections. Recombinant P75 is not recognized in immunoblots by convalescent sera produced in chickens infected with Mycoplasma synoviae, Mycoplasma gallinarum, and Mycoplasma gallinaceum or in turkeys infected with Mycoplasma meleagridis.

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo

2006-02-05

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. Thismore » defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.« less

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, L.H.; Calabi, F.; Lefebvre, F.A.

1987-12-01

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have not been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure ofmore » CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the ..beta../sub 2/-microglobulin-binding-domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.« less

A pathway of costimulation that prevents anergy in CD28- T cells: B7- independent costimulation of CD1-restricted T cells

PubMed Central

1995-01-01

A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B- LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7- independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells. PMID:7500046

Imaging of blood antigen distribution on blood cells by thermal lens microscopy

NASA Astrophysics Data System (ADS)

Kimura, Hiroko; Sekiguchi, Kazuya; Nagao, Fumiko; Mukaida, Masahiro; Kitamori, Takehiko; Sawada, Tsuguo

2000-05-01

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.

Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.

2012-02-07

Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals.more » The 3.2-{angstrom} X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.« less

Bayesian multivariate Poisson abundance models for T-cell receptor data.

PubMed

Greene, Joshua; Birtwistle, Marc R; Ignatowicz, Leszek; Rempala, Grzegorz A

2013-06-07

A major feature of an adaptive immune system is its ability to generate B- and T-cell clones capable of recognizing and neutralizing specific antigens. These clones recognize antigens with the help of the surface molecules, called antigen receptors, acquired individually during the clonal development process. In order to ensure a response to a broad range of antigens, the number of different receptor molecules is extremely large, resulting in a huge clonal diversity of both B- and T-cell receptor populations and making their experimental comparisons statistically challenging. To facilitate such comparisons, we propose a flexible parametric model of multivariate count data and illustrate its use in a simultaneous analysis of multiple antigen receptor populations derived from mammalian T-cells. The model relies on a representation of the observed receptor counts as a multivariate Poisson abundance mixture (m PAM). A Bayesian parameter fitting procedure is proposed, based on the complete posterior likelihood, rather than the conditional one used typically in similar settings. The new procedure is shown to be considerably more efficient than its conditional counterpart (as measured by the Fisher information) in the regions of m PAM parameter space relevant to model T-cell data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immunological properties of Micrococcus lysodeikticus membranes.

PubMed

Fukui, Y; Nachbar, M S; Salton, M R

1971-01-01

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Age-Related Buildup of Humoral Immunity against Epitopes for Rosette Formation and Agglutination in African Areas of Malaria Endemicity

PubMed Central

Barragan, Antonio; Kremsner, Peter G.; Weiss, Walter; Wahlgren, Mats; Carlson, Johan

1998-01-01

In this report, we show an age-related buildup of agglutinating activity as well as serum activity against rosette formation in children living in areas of Kenya and Gabon where malaria is endemic. Sera from Kenyans in general exhibited a stronger and wider immune response toward the epitopes, probably reflecting a difference in transmission patterns between the two areas. Thus, our results indicate that repeated malaria attacks in areas of endemicity, and consequently exposure to different isolate-specific antigens, will elicit an antibody-mediated response eventually enabling recognition of the majority of rosetting and agglutinating antigens. The correlation between antirosetting and agglutinating capacity was poor in individual cases, indicating that the rosetting epitopes are only a minor part of the highly diverse surface-exposed antigens (mainly PfEMP1) on the surface of parasitized erythrocytes toward which antibodies may react. These data together with our previous findings that the protection against cerebral malaria correlates with presence of antirosetting antibodies shed new light on our understanding of the gradual acquisition of immunity toward severe complications of malarial infection which children reared in areas of endemicity attain. PMID:9746579

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

PubMed

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

PubMed

Nakano, Satoshi; Nagao, Miki; Yamasaki, Tomomi; Morimura, Hiroyuki; Hama, Natsuki; Iijima, Yoshio; Shinomiya, Hiroto; Tanaka, Michio; Yamamoto, Masaki; Matsumura, Yasufumi; Miyake, Shiro; Ichiyama, Satoshi

2018-06-01

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 10 6 and 17.6 × 10 6  CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid.

PubMed

Pozzuoli, R; Piantelli, M; Perucci, C; Arru, E; Musiani, P

1975-11-01

This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.

Immunoelectrophoretic study of cell surface antigens from different Streptococcus mutans serotypes and Streptococcus sanguis.

PubMed

Ogier, J A; Klein, J P; Niddam, R; Frank, R M

1985-06-01

Antigens prepared from culture supernatants or whole cells of several cariogenic strains were examined by immunoelectrophoresis for their crossed antigenicity, with reference to Streptococcus mutans OMZ175, serotype f. Crossed immunoelectrophoresis revealed a crossreactivity between soluble extracellular and wall associated antigens of six strains of Streptococcus mutans and one strain of Streptococcus sanguis. Protease destroyed the immunoreactivity of crossreactive antigens. One of them was shown to be localized on the bacterial surface.

Correlation of the cell surface antigens with stage and grade in cancer of the bladder.

PubMed

Emmott, R C; Javadpour, N; Bergman, S M; Soares, T

1979-01-01

We examined 76 bladder tumors of various stages and grades for the presence of the ABO (H) cell surface antigen, using the specific red cell adherence technique. Of the grade I lesions studied 70 per cent were positive for the cell surface antigen and none of the 26 grade III tumors retained the antigens. When correlated with clinical stage the tumors showed no antigens for those of stages B1 to D, while 12 of 16 stage A lesions were positive for the antigen. When stage A lesions were studied and the findings were correlated with recurrence and metastasis/invasion rates the cell surface antigen was present on the initial tumor in only 1 lesion that recurred at an invasive stage. The findings of this study show that the specific red cell adherence technique may be valuable for predicting malignant potential in low grade, low stage cancer of the bladder. If supported by further investigation this technique may offer the capability of selecting low grade, low stage bladder tumors that are destined to invade or metastasize while they are at curable stages.

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

PubMed

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Immunoliposomes containing Soluble Leishmania Antigens (SLA) as a novel antigen delivery system in murine model of leishmaniasis.

PubMed

Eskandari, Faeze; Talesh, Ghazal Alipour; Parooie, Maryam; Jaafari, Mahmoud Reza; Khamesipour, Ali; Saberi, Zahra; Abbasi, Azam; Badiee, Ali

2014-11-01

Development of new generation of vaccines against leishmaniasis requires adjuvants to elicit the type and intensity of immune response needed for protection. The coupling of target-specific antibodies to the liposomal surface to create immunoliposomes has appeared as a promising way in achieving a liposome active targeting. In this study, immunoliposomes were prepared by grafting non-immune mouse IgG onto the liposomal surface. The influence of active targeted immunoliposomes on the type and intensity of generated immune response against Leishmania was then investigated and compared with that of liposomes and control groups which received either SLA or HEPES buffer alone. All formulations contained SLA and were used to immunize the mice in the left hind footpad three times in 3-week intervals. Evaluation of lesion development and parasite burden in the foot and spleen after challenge with Leishmania major, evaluation of Th1 cytokine (IFN-γ), and titration of IgG isotypes were carried out to assess the type of generated immune response and the extent of protection. The results indicated that liposomes might be effective adjuvant systems to induce protection against L. major challenge in BALB/c mice, but stronger cell mediated immune responses were induced when immunoliposomes were utilized. Thus, immune modulation using immunoliposomes might be a practical approach to improve the immunization against L. major. Copyright © 2014 Elsevier Inc. All rights reserved.

Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

PubMed Central

Tamura, A; Ohashi, N; Urakami, H; Takahashi, K; Oyanagi, M

1985-01-01

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera. Images PMID:3922893

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

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

PubMed Central

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

2015-01-01

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

Immune activation with peptide assemblies carrying Lewis y tumor-associated carbohydrate antigen.

PubMed

Yamazaki, Yuji; Watabe, Naoki; Obata, Hiroaki; Hara, Eri; Ohmae, Masashi; Kimura, Shunsaku

2017-02-01

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Le y ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine) m -block-poly(L-lactic acid) 30 (m = 15 or 50, Lactosome), poly(sarcosine) m -block-(D/L-Leu-Aib) n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Le y on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Le y and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Le y IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Le y IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Le y on the molecular assembly was required for anti-Le y IgM production. The antigenicity of Le y is therefore dependent on the molecular assemblies on which Le y is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo.

PubMed

Pestel, J; Dissous, C; Dessaint, J P; Louis, J; Engers, H; Capron, A

1985-06-01

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell clones here studied were restimulated either with IPSm or with schistosomulum Ag, it appears that such IPSm-specific T cell clones could be involved in the concomitant immunity mechanisms.

A Novel Protective Vaccine Antigen from the Core Escherichia coli Genome

PubMed Central

Moriel, Danilo G.; Tan, Lendl; Goh, Kelvin G. K.; Ipe, Deepak S.; Lo, Alvin W.; Peters, Kate M.

2016-01-01

ABSTRACT Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains—an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCE E. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify putative broadly protective vaccine antigens. One such antigen was identified that was highly immunogenic and induced protection in a mouse model of bacteremia. Overall, our study provides a genomic and proteomic framework for the selection of novel vaccine antigens that could mediate broad protection against pathogenic E. coli. PMID:27904885

Trypanosoma cruzi. Surface antigens of blood and culture forms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nogueira, N.; Chaplan, S.; Tydings, J.D.

1981-03-01

The surface polypeptides of both cultured and blood forms of Trypanosoma cruzi were iodinated by the glucose oxidase-lactoperoxidase technique. Blood-form trypomastigotes (BFT) isolated form infected mice displayed a major 90,000-Mr component. In contrast, both epimastigotes and trypomastigotes obtained form acellular cultures expressed a smaller 75,000-Mr peptide. Both major surface components were presumably glycoproteins in terms of their binding to concanavalin A-Sepharose 4B. Within a 3-h period, both blood and culture forms synthesized their respective surface glycoproteins (90,000 Mr and 75,000 Mr, respectively in vitro. (/sub 35/S)methionine-labeled surface peptides were immunoprecipitated with immune sera of both human and murine origin. Amore » panel of sera form patients with chronic Chagas' disease and hyperimmunized mice recognized similar surface peptides. These immunogens were the same components as the major iodinated species. The major BFT surface peptide was readily removed by trypsin treatment of the parasites, although the procedure did not affect the 75,000-Mr peptide from the culture forms. Two-dimensional polyacrylamide gel electrophoresis revealed that the 90,000-Mr peptide found on BFT was an acidic protein of isoelectric point (pI) 5.0, whereas, the 75,000-Mr peptide form culture-form trypomastigotes has a pI of 7.2. The 90,000-Mr component is thought to be responsible for the anti-phagocytic properties of the BFT (1).« less

Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.

PubMed

Bien, Christian G; Vincent, Angela; Barnett, Michael H; Becker, Albert J; Blümcke, Ingmar; Graus, Francesc; Jellinger, Kurt A; Reuss, David E; Ribalta, Teresa; Schlegel, Jürgen; Sutton, Ian; Lassmann, Hans; Bauer, Jan

2012-05-01

Classical paraneoplastic encephalitis syndromes with 'onconeural' antibodies directed to intracellular antigens, and the recently described paraneoplastic or non-paraneoplastic encephalitides and antibodies against both neural surface antigens (voltage-gated potassium channel-complexes, N-methyl-d-aspartate receptors) and intracellular antigens (glutamic acid decarboxylase-65), constitute an increasingly recognized group of immune-mediated brain diseases. Evidence for specific immune mechanisms, however, is scarce. Here, we report qualitative and quantitative immunopathology in brain tissue (biopsy or autopsy material) of 17 cases with encephalitis and antibodies to either intracellular (Hu, Ma2, glutamic acid decarboxylase) or surface antigenic targets (voltage-gated potassium channel-complex or N-methyl-d-aspartate receptors). We hypothesized that the encephalitides with antibodies against intracellular antigens (intracellular antigen-onconeural and intracellular antigen-glutamic acid decarboxylase groups) would show neurodegeneration mediated by T cell cytotoxicity and the encephalitides with antibodies against surface antigens would be antibody-mediated and would show less T cell involvement. We found a higher CD8/CD3 ratio and more frequent appositions of granzyme-B(+) cytotoxic T cells to neurons, with associated neuronal loss, in the intracellular antigen-onconeural group (anti-Hu and anti-Ma2 cases) compared to the patients with surface antigens (anti-N-methyl-d-aspartate receptors and anti-voltage-gated potassium channel complex cases). One of the glutamic acid decarboxylase antibody encephalitis cases (intracellular antigen-glutamic acid decarboxylase group) showed multiple appositions of GrB-positive T cells to neurons. Generally, however, the glutamic acid decarboxylase antibody cases showed less intense inflammation and also had relatively low CD8/CD3 ratios compared with the intracellular antigen-onconeural cases. Conversely, we found complement C9neo deposition on neurons associated with acute neuronal cell death in the surface antigen group only, specifically in the voltage-gated potassium channel-complex antibody patients. N-methyl-d-aspartate receptors-antibody cases showed no evidence of antibody and complement-mediated tissue injury and were distinguished from all other encephalitides by the absence of clear neuronal pathology and a low density of inflammatory cells. Although tissue samples varied in location and in the stage of disease, our findings strongly support a central role for T cell-mediated neuronal cytotoxicity in encephalitides with antibodies against intracellular antigens. In voltage-gated potassium channel-complex encephalitis, a subset of the surface antigen antibody encephalitides, an antibody- and complement-mediated immune response appears to be responsible for neuronal loss and cerebral atrophy; the apparent absence of these mechanisms in N-methyl-d-aspartate receptors antibody encephalitis is intriguing and requires further study.

Structural analysis of determinants of histo-blood group antigen binding specificity in genogroup I noroviruses.

PubMed

Shanker, Sreejesh; Czako, Rita; Sankaran, Banumathi; Atmar, Robert L; Estes, Mary K; Prasad, B V Venkataram

2014-06-01

Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. Human NoVs are classified into two major genogroups (genogroup I [GI] and GII), with each genogroup further divided into several genotypes. GII NoVs are more prevalent and exhibit periodic emergence of new variants, suggested to be driven by altered HBGA binding specificities and antigenic drift. Recent epidemiological studies show increased activity among GI NoVs, with some members showing the ability to bind nonsecretor HBGAs. NoVs bind HBGAs through the protruding (P) domain of the major capsid protein VP1. GI NoVs, similar to GII, exhibit significant sequence variations in the P domain; it is unclear how these variations affect HBGA binding specificities. To understand the determinants of possible strain-specific HBGA binding among GI NoVs, we determined the structure of the P domain of a GI.7 clinical isolate and compared it to the previously determined P domain structures of GI.1 and GI.2 strains. Our crystallographic studies revealed significant structural differences, particularly in the loop regions of the GI.7 P domain, altering its surface topography and electrostatic landscape and potentially indicating antigenic variation. The GI.7 strain bound to H- and A-type, Lewis secretor, and Lewis nonsecretor families of HBGAs, allowing us to further elucidate the structural determinants of nonsecretor HBGA binding among GI NoVs and to infer several contrasting and generalizable features of HBGA binding in the GI NoVs. Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Recent epidemiological studies have shown increased prevalence of genogroup I (GI) NoVs. Although secretor-positive status is strongly correlated with NoV infection, cases of NoV infection associated with secretor-negative individuals are reported. Biochemical studies have shown that GI NoVs exhibit genotype-dependent binding to nonsecretor histo-blood group antigens (HBGAs). From our crystallographic studies of a GI.7 NoV, in comparison with previous studies on GI.1 and GI.2 NoVs, we show that genotypic differences translate to extensive structural changes in the loop regions that significantly alter the surface topography and electrostatic landscape of the P domain; these features may be indicative of antigenic variations contributing to serotypic differentiation in GI NoVs and also differential modulation of the HBGA binding characteristics. A significant finding is that the threshold length and the structure of one of the loops are critical determinants in the binding of GI NoVs to nonsecretor HBGAs.

Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

PubMed

Henry, Kevin A; Tanha, Jamshid

2018-05-01

Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT.

PubMed

Shen, Chuanlai; Xu, Tao; Wu, You; Li, Xiaoe; Xia, Lingzhi; Wang, Wei; Shahzad, Khawar Ali; Zhang, Lei; Wan, Xin; Qiu, Jie

2017-11-27

Conventional peptide-major histocompatibility complex (pMHC) multimer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay cannot concurrently determine the frequency and reactivity of antigen-specific T cells (AST) in a single assay. In this report, pMHC multimer, magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a 96-well microplate which pre-coated with cytokine-capture antibodies. This method, termed AAPC-microplate, allows the enumeration and local cytokine production of AST cells in a single assay without using flow cytometry or fluorescence intensity scanning, thus will be widely applicable. Here, ovalbumin 257-264 -specific CD8 + T cells from OT-1 T cell receptor (TCR) transgenic mice were measured. The methodological accuracy, specificity, reproducibility, and sensitivity in enumerating AST cells compared well with conventional pMHC multimer staining. Furthermore, the AAPC-microplate was applied to detect the frequency and reactivity of Hepatitis B virus (HBV) core antigen 18-27 - and surface antigen 183-191 -specific CD8 + T cells for the patients, and was compared with conventional method. This method without the need of high-end instruments may facilitate the routine analysis of patient-specific cellular immune response pattern to a given antigen in translational studies.

Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.

1990-01-01

Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surfacemore » by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs.« less

Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection

PubMed Central

Contreras, Marinela; Alberdi, Pilar; Mateos-Hernández, Lourdes; Fernández de Mera, Isabel G.; García-Pérez, Ana L.; Vancová, Marie; Villar, Margarita; Ayllón, Nieves; Cabezas-Cruz, Alejandro; Valdés, James J.; Stuen, Snorre; Gortazar, Christian; de la Fuente, José

2017-01-01

Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4) and Heat shock protein 70 (HSP70) were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens. PMID:28725639

Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

PubMed Central

Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

2005-01-01

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

Human Antibodies to a Mr 155,000 Plasmodium falciparum Antigen Efficiently Inhibit Merozoite Invasion

NASA Astrophysics Data System (ADS)

Wahlin, Birgitta; Wahlgren, Mats; Perlmann, Hedvig; Berzins, Klavs; Bjorkman, Anders; Patarroyo, Manuel E.; Perlmann, Peter

1984-12-01

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 μ g/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.

Anaplasma phagocytophilum MSP4 and HSP70 Proteins Are Involved in Interactions with Host Cells during Pathogen Infection.

PubMed

Contreras, Marinela; Alberdi, Pilar; Mateos-Hernández, Lourdes; Fernández de Mera, Isabel G; García-Pérez, Ana L; Vancová, Marie; Villar, Margarita; Ayllón, Nieves; Cabezas-Cruz, Alejandro; Valdés, James J; Stuen, Snorre; Gortazar, Christian; de la Fuente, José

2017-01-01

Anaplasma phagocytophilum transmembrane and surface proteins play a role during infection and multiplication in host neutrophils and tick vector cells. Recently, A. phagocytophilum Major surface protein 4 (MSP4) and Heat shock protein 70 (HSP70) were shown to be localized on the bacterial membrane, with a possible role during pathogen infection in ticks. In this study, we hypothesized that A. phagocytophilum MSP4 and HSP70 have similar functions in tick-pathogen and host-pathogen interactions. To address this hypothesis, herein we characterized the role of these bacterial proteins in interaction and infection of vertebrate host cells. The results showed that A. phagocytophilum MSP4 and HSP70 are involved in host-pathogen interactions, with a role for HSP70 during pathogen infection. The analysis of the potential protective capacity of MSP4 and MSP4-HSP70 antigens in immunized sheep showed that MSP4-HSP70 was only partially protective against pathogen infection. This limited protection may be associated with several factors, including the recognition of non-protective epitopes by IgG in immunized lambs. Nevertheless, these antigens may be combined with other candidate protective antigens for the development of vaccines for the control of human and animal granulocytic anaplasmosis. Focusing on the characterization of host protective immune mechanisms and protein-protein interactions at the host-pathogen interface may lead to the discovery and design of new effective protective antigens.

Effective antigen presentation to helper T cells by human eosinophils.

PubMed

Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach

PubMed Central

Vicente, Sara; Diaz-Freitas, Belen; Peleteiro, Mercedes; Sanchez, Alejandro; Pascual, David W.; Gonzalez-Fernandez, Africa; Alonso, Maria J.

2013-01-01

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier's surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC−), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg. PMID:23614052

Translation and Clinical Development of Bispecific T‐cell Engaging Antibodies for Cancer Treatment

PubMed Central

Yuraszeck, T; Kasichayanula, S

2017-01-01

Bispecific T‐cell Engagers (BiTE®) antibody constructs enable a polyclonal T‐cell response to cell‐surface tumor‐associated antigens, bypassing the narrow specificities of T‐cell receptors and the need for antigen presentation through the major histocompatibility complex pathways. Blinatumomab, a CD19xCD3 BiTE® antibody construct, received accelerated approval for the treatment of relapsed/refractory Philadelphia chromosome negative acute lymphoblastic leukemia. Herein we review the pharmacology, safety, and efficacy observed in studies of blinatumomab and other BiTE® antibody constructs. Quantitative systems pharmacology is envisioned as a means to optimize dosing decisions for trials in which BiTE® antibody constructs are administered as monotherapy or in combination with other immunotherapies. PMID:28182247

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population

PubMed Central

Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C. Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B.; Nauck, Markus; Kaminski, Wolfgang E.

2017-01-01

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its “a” determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the “a” determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of “a” determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated. PMID:28472040

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

PubMed

Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-Suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B; Nauck, Markus; Kaminski, Wolfgang E

2017-01-01

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

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

PubMed

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

1997-11-25

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

SPM for functional identification of individual biomolecules

NASA Astrophysics Data System (ADS)

Ros, Robert; Schwesinger, Falk; Padeste, Celestino; Plueckthun, Andreas; Anselmetti, Dario; Guentherodt, Hans-Joachim; Tiefenauer, Louis

1999-06-01

The identification of specific binding molecules is of increasing interest in the context of drug development based on combinatorial libraries. Scanning Probe Microscopy (SPM) is the method of choice to image and probe individual biomolecules on a surface. Functional identification of biomolecules is a first step towards screening on a single molecule level. As a model system we use recombinant single- chain Fv fragment (scFv) antibody molecules directed against the antigen fluorescein. The scFv's are covalently immobilized on a flat gold surface via the C-terminal cysteine, resulting in a high accessibility of the binding site. The antigen is immobilized covalently via a long hydrophilic spacer to the silicon nitride SPM-tip. This arrangement allows a direct measurement of binding forces. Thus, closely related antibody molecules differing in only one amino acid at their binding site could be distinguished. A novel SPM-software has been developed which combines imaging, force spectroscopic modes, and online analysis. This is a major prerequisite for future screening methods.

Antibody response to Ehrlichia risticii and antibody reactivity to the component antigens in horses with induced Potomac horse fever.

PubMed

Dutta, S K; Mattingly, B L; Shankarappa, B

1989-10-01

The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type antibody response was detected in 30% of the horses by ELISA. In the primary antibody response, a distinct titer was observed at 2 weeks postinoculation (PI), when the immunoglobulin M (IgM)/IgG ratio was 2 to 5, and the overall antibody titer peaked at 6 to 8 weeks PI. The secondary-type antibody response exhibited a characteristic titer at 1 week PI, the IgM and IgG titers were about equal at 2 weeks PI, and the overall antibody titer peaked at 6 weeks PI. A transient depression in the IgG response at 4 weeks PI was observed in both response types. The antibody was maintained at a high titer for over a year in all horses. Western immunoblot reactivity showed that the antisera collected from these infected horses at 4 to 5 weeks PI recognized some or all of the six major E. risticii component antigens (70, 55, 51, 44, 33, and 28 kilodaltons), all of which were apparent surface components. The 6- to 8-week PI antisera recognized up to 16 component antigens, including 9 major antigens (110, 86, 70, 55, 51, 49, 44, 33, and 28 kilodaltons). However, the PrI sera of these horses showed reactivity at various intensities with one to seven of the component antigens. There was no apparent correlation between this reactivity pattern and the subsequent antibody response types.

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

PubMed Central

MacKay, P; Pasek, M; Magazin, M; Kovacic, R T; Allet, B; Stahl, S; Gilbert, W; Schaller, H; Bruce, S A; Murray, K

1981-01-01

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals. PMID:6170067

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

PubMed Central

Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

2014-01-01

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLellan, Jason S.; Chen, Man; Chang, Jung-San

Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling ofmore » 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.« less

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuhki, Naoya; O'Brien, S.J.

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. The authors present here a quantitative analysis of restriction fragmentmore » length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations.« less

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history.

PubMed Central

Yuhki, N; O'Brien, S J

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. We present here a quantitative analysis of restriction fragment length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations. Images PMID:1967831

Impact of Human Immunodeficiency Virus Infection in Pregnant Women on Variant-Specific Immunity to Malaria▿

PubMed Central

Dembo, Edson G.; Mwapasa, Victor; Montgomery, Jacqui; Craig, Alister G.; Porter, Kimberly A.; Meshnick, Steven R.; Molyneux, Malcolm E.; Rogerson, Stephen J.

2008-01-01

Human immunodeficiency virus (HIV) increases susceptibility to Plasmodium falciparum infection, and this has most clearly been demonstrated in pregnant women. Variant surface antigens on the surfaces of erythrocytes infected with P. falciparum are major targets of protective immunity. We studied the impact of HIV infection on pregnant women's humoral immunity to variant surface antigens expressed by placental and pediatric isolates of P. falciparum. By flow cytometry, sera from HIV-infected women more frequently lacked antibodies to these antigens than sera from HIV-uninfected women. This difference was similar in magnitude for pediatric isolates (unadjusted odds ratio [OR] = 6.36; 95% confidence interval [CI] = 1.14, 35.32; P < 0.05) and placental isolates (unadjusted OR = 6.47; 95% CI = 0.75, 55.64; P < 0.10). We divided women into high and low responders on the basis of their antibody levels. After adjustment for CD4 count, maternal age, and gravidity, we found that HIV-infected women more frequently had low responses to both pediatric isolates (OR = 5.34; 95% CI = 1.23, 23.16; P = 0.025) and placental isolates (OR = 4.14; 95% CI = 1.71, 10.02; P = 0.002). The relative quantity of antibodies to both pediatric isolates (P = 0.035) and placental isolates (P = 0.005) was lower in HIV-infected women than in HIV-uninfected women. HIV infection has a broad impact on variant-specific immunity, which may explain the susceptibility of infected individuals to clinical malaria episodes. PMID:18199738

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes

PubMed Central

Ruwona, Tinashe B; Mcbride, Ryan; Chappel, Rebecca; Head, Steven R; Ordoukhanian, Phillip; Burton, Dennis R.; Law, Mansun

2014-01-01

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research. PMID:24269751

Antibodies to dendritic neuronal surface antigens in opsoclonus myoclonus ataxia syndrome

PubMed Central

Panzer, Jessica A.; Anand, Ronan; Dalmau, Josep; Lynch, David R.

2015-01-01

Opsoclonus myoclonus ataxia syndrome (OMAS) is an autoimmune disorder characterized by rapid, random, conjugate eye movements (opsoclonus), myoclonus, and ataxia. Given these symptoms, autoantibodies targeting the cerebellum or brainstem could mediate the disease or be markers of autoimmunity. In a subset of patients with OMAS, we identified such autoantibodies, which bind to non-synaptic puncta on the surface of live cultured cerebellar and brainstem neuronal dendrites. These findings implicate autoimmunity to a neuronal surface antigen in the pathophysiology of OMAS. Identification of the targeted antigen(s) could elucidate the mechanisms underlying OMAS and provide a biomarker for diagnosis and response to therapy. PMID:26298330

The Role of FcRn in Antigen Presentation

PubMed Central

Baker, Kristi; Rath, Timo; Pyzik, Michal; Blumberg, Richard S.

2014-01-01

Immunoglobulins are unique molecules capable of simultaneously recognizing a diverse array of antigens and themselves being recognized by a broad array of receptors. The abundance specifically of the IgG subclass and the variety of signaling receptors to which it binds render this an important immunomodulatory molecule. In addition to the classical Fcγ receptors that bind IgG at the cell surface, the neonatal Fc receptor (FcRn) is a lifelong resident of the endolysosomal system of most hematopoietic cells where it determines the intracellular fate of both IgG and IgG-containing immune complexes (IgG IC). Cross-linking of FcRn by multivalent IgG IC within antigen presenting cells such as dendritic cells initiates specific mechanisms that result in trafficking of the antigen-bearing IgG IC into compartments from which the antigen can successfully be processed into peptide epitopes compatible with loading onto both major histocompatibility complex class I and II molecules. In turn, this enables the synchronous activation of both CD4+ and CD8+ T cell responses against the cognate antigen, thereby bridging the gap between the humoral and cellular branches of the adaptive immune response. Critically, FcRn-driven T cell priming is efficient at very low doses of antigen due to the exquisite sensitivity of the IgG-mediated antigen delivery system through which it operates. FcRn-mediated antigen presentation has important consequences in tissue compartments replete with IgG and serves not only to determine homeostatic immune activation at a variety of sites but also to induce inflammatory responses upon exposure to antigens perceived as foreign. Therapeutically targeting the pathway by which FcRn enables T cell activation in response to IgG IC is thus a highly attractive prospect not only for the treatment of diseases that are driven by immune complexes but also for manipulating local immune responses against defined antigens such as those present during infections and cancer. PMID:25221553

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

PubMed

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

PubMed

Chan, Jo-Anne; Stanisic, Danielle I; Duffy, Michael F; Robinson, Leanne J; Lin, Enmoore; Kazura, James W; King, Christopher L; Siba, Peter M; Fowkes, Freya Ji; Mueller, Ivo; Beeson, James G

2017-12-01

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

PubMed

Wun, Kwok S; Reijneveld, Josephine F; Cheng, Tan-Yun; Ladell, Kristin; Uldrich, Adam P; Le Nours, Jérôme; Miners, Kelly L; McLaren, James E; Grant, Emma J; Haigh, Oscar L; Watkins, Thomas S; Suliman, Sara; Iwany, Sarah; Jimenez, Judith; Calderon, Roger; Tamara, Kattya L; Leon, Segundo R; Murray, Megan B; Mayfield, Jacob A; Altman, John D; Purcell, Anthony W; Miles, John J; Godfrey, Dale I; Gras, Stephanie; Price, David A; Van Rhijn, Ildiko; Moody, D Branch; Rossjohn, Jamie

2018-04-01

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

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

PubMed Central

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

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

PubMed

Sebra, Robert P; Masters, Kristyn S; Bowman, Christopher N; Anseth, Kristi S

2005-11-22

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

PubMed

Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

2009-02-24

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

[Research advances of genomic GYP coding MNS blood group antigens].

PubMed

Liu, Chang-Li; Zhao, Wei-Jun

2012-02-01

The MNS blood group system includes more than 40 antigens, and the M, N, S and s antigens are the most significant ones in the system. The antigenic determinants of M and N antigens lie on the top of GPA on the surface of red blood cells, while the antigenic determinants of S and s antigens lie on the top of GPB on the surface of red blood cells. The GYPA gene coding GPA and the GYPB gene coding GPB locate at the longarm of chromosome 4 and display 95% homologus sequence, meanwhile both genes locate closely to GYPE gene that did not express product. These three genes formed "GYPA-GYPB-GYPE" structure called GYP genome. This review focuses on the molecular basis of genomic GYP and the variety of GYP genome in the expression of diversity MNS blood group antigens. The molecular basis of Miltenberger hybrid glycophorin polymorphism is specifically expounded.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T-cell activation.

PubMed

Kitzmüller, C; Wallner, M; Deifl, S; Mutschlechner, S; Walterskirchen, C; Zlabinger, G J; Ferreira, F; Bohle, B

2012-11-01

BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1-like fold and displays negligible IgE-binding but strong T cell-activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation. Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T-cell clones were compared. Fluorescently labeled BM4 and Bet v 1 were used to study surface binding, endocytosis, and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T-cell clones and the polarization of naïve T cells. BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 showed increased surface binding and internalization as well as faster endolysosomal degradation compared with Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T-cell clones and promoted less IL-5 production in T cells than Bet v 1-pulsed mdDC. The loss of the Bet v 1-fold changes the protein's interaction with the human immune system at the level of antigen-presenting cells resulting in altered T-cell responses. By combining low IgE-binding with strong and modulating T cell-activating capacity, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy. © 2012 John Wiley & Sons A/S.

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

PubMed Central

Kitzmüller, Claudia; Zulehner, Nora; Roulias, Anargyros; Briza, Peter; Ferreira, Fatima; Faé, Ingrid; Fischer, Gottfried F.; Bohle, Barbara

2015-01-01

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing. PMID:25670010

In vivo selection of populations of Plasmodium chabaudi chabaudi AS resistant to a monoclonal antibody that reacts with the precursor to the major merozoite surface antigen.

PubMed Central

Wood, J C; Sales de Aguiar, J C; Jarra, W; Ogun, S A; Snounou, G; Brown, K N

1989-01-01

Mice bearing a hybridoma secreting a monoclonal antibody (MAb), MAb-3, which significantly delays the onset of a Plasmodium chabaudi chabaudi AS, but not P. chabaudi chabaudi CB, challenge parasitemia in a passive transfer assay and which is specific for the precursor to the major merozoite surface antigen (PMMSA) of P. chabaudi chabaudi AS, were challenged intravenously with 10(3) P. chabaudi chabaudi AS-parasitized erythrocytes. The resultant parasitemia was very similar to that in normal mice except that initially the parasitemia was sometimes slightly delayed. Parasites derived from cryopreserved stabilates isolated from MAb-3 hybridoma mice with an unmodified parasitemia, or with a delayed parasitemia, were found to have lost their susceptibility to MAb-3 in the passive transfer assay. A number of anti-PMMSA MAb were used to immunoprecipitate lysates of parasite populations isolated directly from hybridoma-bearing mice. In some instances and with certain of the MAb, immunoprecipitation patterns were modified, but other isolates were not detectably different when compared with unselected P. chabaudi chabaudi AS parasites. Using a panel of MAb reacting with the PMMSA of P. chabaudi chabaudi AS, immunoprecipitation patterns of parasites derived from cryopreserved stabilates isolated from hybridoma-bearing mice were determined at 2-h intervals through the appropriate part of the parasite maturation cycle. In these derived populations, resistance to MAb-3 was not associated with a change in the immunoprecipitation reaction with the MAb used. These results are discussed in the context of current knowledge of genotypic and phenotypic antigenic diversity of malaria parasites and other protozoa. Images PMID:2731986

Plasmodium falciparum Malaria in the Peruvian Amazon, a Region of Low Transmission, Is Associated with Immunologic Memory

PubMed Central

Clark, Eva H.; Silva, Claudia J.; Weiss, Greta E.; Li, Shanping; Padilla, Carlos; Crompton, Peter D.; Hernandez, Jean N.

2012-01-01

The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119 IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+ CD27+ CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions. PMID:22252876

Plasmodium falciparum malaria in the Peruvian Amazon, a region of low transmission, is associated with immunologic memory.

PubMed

Clark, Eva H; Silva, Claudia J; Weiss, Greta E; Li, Shanping; Padilla, Carlos; Crompton, Peter D; Hernandez, Jean N; Branch, OraLee H

2012-04-01

The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP1(19)). After observing a more robust antibody response to MSP1(19), we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP1(19) IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19(+) CD27(+) CD38(high)) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

PubMed Central

Maeland, Johan A.; Bevanger, Lars; Lyng, Randi Valsoe

2004-01-01

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. PMID:15539502

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Gold nanoparticles paper as a SERS bio-diagnostic platform.

PubMed

Ngo, Ying Hui; Then, Whui Lyn; Shen, Wei; Garnier, Gil

2013-11-01

Bioactive papers are usually challenged by four major limitations: sensitivity, selectivity, simplicity and strength (4S). Gold nanoparticles (AuNPs) treated paper has previously been demonstrated as a Surface Enhanced Raman Scattering (SERS) active substrate, capable of addressing the 4S issues. In this study, AuNPs on paper substrate were functionalized by a series of biomolecules to develop a generic SERS platform for antibody-antigen detection. The functionalization steps were performed by taking advantage of the high affinity association between Streptomyces avidinii-derived protein, streptavidin, and biotin. Streptavidin was firstly bound onto the AuNPs treated paper using biotinylated-thiol. Subsequently, desired biotinylated-antibody was bound onto the streptavidin. SERS spectra of each functionalization step were obtained to ensure specific adsorption of the bio-molecules. The binding interaction of the antibody with its specific antigen was detected using SERS. Shifts of Raman band associated with α-helix and β-sheet structures indicated structural modification of the antibody upon interaction with its antigen. Predominant tryptophan and tyrosine residue bands were also detected, confirming the presence of antigen. Reproducible spectral features were quantified as AuNP papers were subjected to different concentrations of antigen; the spectra intensity increased as a function of the antigen concentration. The retention of AuNPs on paper remained constant after all the consecutive washing and functionalization steps. The feasibility of AuNPs paper as a low-cost and generic SERS platform for bio-diagnostic applications was demonstrated. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Immunohistochemical detection and correlation between MHC antigen and cell-mediated immune system in recurrent glioma by APAAP method.

PubMed

Miyagi, K; Ingram, M; Techy, G B; Jacques, D B; Freshwater, D B; Sheldon, H

1990-09-01

As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor-infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration.

The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells.

PubMed

Altomonte, M; Pucillo, C; Maio, M

1999-06-01

Besides their "classical" antigenic peptide-presenting activity, major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune and nonimmune cells. However, this "nonclassical" role and its functional consequences are still substantially overlooked. In this review, we will focus on these alternative functional properties of MHC class II antigens, to reawaken attention to their present and foreseeable immunobiologic and pathogenetic implications. The main issues that will be addressed concern 1) the role of MHC class II molecules as basic components of exchangeable oligomeric protein complexes with intracellular signaling ability; 2) the nonclassical functions of MHC class II antigens in immune cells; 3) the pathogenetic role of MHC class II antigens in inflammatory/autoimmune and infectious disease; and 4) the functional role of MHC class II antigens in solid malignancies.

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

PubMed

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.

Invariant Chain Complexes and Clusters as Platforms for MIF Signaling

PubMed Central

Lindner, Robert

2017-01-01

Invariant chain (Ii/CD74) has been identified as a surface receptor for migration inhibitory factor (MIF). Most cells that express Ii also synthesize major histocompatibility complex class II (MHC II) molecules, which depend on Ii as a chaperone and a targeting factor. The assembly of nonameric complexes consisting of one Ii trimer and three MHC II molecules (each of which is a heterodimer) has been regarded as a prerequisite for efficient delivery to the cell surface. Due to rapid endocytosis, however, only low levels of Ii-MHC II complexes are displayed on the cell surface of professional antigen presenting cells and very little free Ii trimers. The association of Ii and MHC II has been reported to block the interaction with MIF, thus questioning the role of surface Ii as a receptor for MIF on MHC II-expressing cells. Recent work offers a potential solution to this conundrum: Many Ii-complexes at the cell surface appear to be under-saturated with MHC II, leaving unoccupied Ii subunits as potential binding sites for MIF. Some of this work also sheds light on novel aspects of signal transduction by Ii-bound MIF in B-lymphocytes: membrane raft association of Ii-MHC II complexes enables MIF to target Ii-MHC II to antigen-clustered B-cell-receptors (BCR) and to foster BCR-driven signaling and intracellular trafficking. PMID:28208600

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

PubMed

Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

2015-01-01

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

PubMed

Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

2017-07-28

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4 + and CD8 + T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

PubMed

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

A new technique to detect antibody-antigen reaction (biological interactions) on a localized surface plasmon resonance (LSPR) based nano ripple gold chip

NASA Astrophysics Data System (ADS)

Saleem, Iram; Widger, William; Chu, Wei-Kan

2017-07-01

We demonstrate that the gold nano-ripple localized surface plasmon resonance (LSPR) chip is a low cost and a label-free method for detecting the presence of an antigen. A uniform stable layer of an antibody was coated on the surface of a nano-ripple gold pattern chip followed by the addition of different concentrations of the antigen. A red shift was observed in the LSPR spectral peak caused by the change in the local refractive index in the vicinity of the nanostructure. The LSPR chip was fabricated using oblique gas cluster ion beam (GCIB) irradiation. The plasmon-resonance intensity of the scattered light was measured by a simple optical spectroscope. The gold nano ripple chip shows monolayer scale sensitivity and high selectivity. The LSPR substrate was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen).

Surface antigens of Plasmodium falciparum gametocytes--a new class of transmission-blocking vaccine targets?

PubMed

Sutherland, Colin J

2009-08-01

The re-establishment of elimination and eradication on the malaria control agenda has led to calls for renewed effort in the development of parasite transmission-blocking interventions. Vaccines are ideally suited to this task, but progress towards an anti-gamete transmission-blocking vaccine, designed to act on parasites in blood-fed mosquitoes, has been slow. Recent work has confirmed that the surface of the gametocyte-infected erythrocyte presents antigens to the host immune system, and elicits specific humoral immune responses to these antigens, termed gametocyte surface antigens (GSAs). Likely candidate molecules, including antigens encoded by sub-telomeric multi-gene families, are discussed, and a hypothetical group of parasite molecules involved in spatial and temporal signal transduction in the human host is proposed, the tropins and circadins. The next steps for development of anti-gametocyte transmission-blocking vaccines for P. falciparum and the other human malaria species are considered.

Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

PubMed Central

Xing, Dongxia; Li, Sufang; Robinson, Simon N.; Yang, Hong; Steiner, David; Komanduri, Krishna V.; Shpall, Elizabeth J.

2009-01-01

In the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these “Th-1 DCs” exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8+ T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies. PMID:19171878

A STING-activating nanovaccine for cancer immunotherapy

NASA Astrophysics Data System (ADS)

Luo, Min; Wang, Hua; Wang, Zhaohui; Cai, Haocheng; Lu, Zhigang; Li, Yang; Du, Mingjian; Huang, Gang; Wang, Chensu; Chen, Xiang; Porembka, Matthew R.; Lea, Jayanthi; Frankel, Arthur E.; Fu, Yang-Xin; Chen, Zhijian J.; Gao, Jinming

2017-07-01

The generation of tumour-specific T cells is critically important for cancer immunotherapy. A major challenge in achieving a robust T-cell response is the spatiotemporal orchestration of antigen cross-presentation in antigen-presenting cells with innate stimulation. Here, we report a minimalist nanovaccine, comprising a simple physical mixture of an antigen and a synthetic polymeric nanoparticle, PC7A NP, which generates a strong cytotoxic T-cell response with low systemic cytokine expression. Mechanistically, the PC7A NP achieves efficient cytosolic delivery of tumour antigens to antigen-presenting cells in draining lymph nodes, leading to increased surface presentation while simultaneously activating type I interferon-stimulated genes. This effect is dependent on stimulator of interferon genes (STING), but not the Toll-like receptor or the mitochondrial antiviral-signalling protein (MAVS) pathway. The nanovaccine led to potent tumour growth inhibition in melanoma, colon cancer and human papilloma virus-E6/E7 tumour models. The combination of the PC7A nanovaccine and an anti-PD-1 antibody showed great synergy, with 100% survival over 60 days in a TC-1 tumour model. Rechallenging of these tumour-free animals with TC-1 cells led to complete inhibition of tumour growth, suggesting the generation of long-term antitumour memory. The STING-activating nanovaccine offers a simple, safe and robust strategy in boosting anti-tumour immunity for cancer immunotherapy.

Isolation and purification of antigenic components of Cryptococcus.

PubMed

Wozniak, Karen L; Levitz, Stuart M

2009-01-01

The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species.

Protamine-based nanoparticles as new antigen delivery systems.

PubMed

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Dry eye syndrome: developments and lifitegrast in perspective

PubMed Central

Lollett, Ivonne V; Galor, Anat

2018-01-01

Dry eye (DE) is a chronic ocular condition with high prevalence and morbidity. It has a complex pathophysiology and is multifactorial in nature. Chronic ocular surface inflammation has emerged as a key component of DE that is capable of perpetuating ocular surface damage and leading to symptoms of ocular pain, discomfort, and visual phenomena. It begins with stress to the ocular surface leading to the production of proinflammatory mediators that induce maturation of resident antigen-presenting cells which then migrate to the lymph nodes to activate CD4 T cells. The specific antigen(s) targeted by these pathogenic CD4+ T cells remains unknown. Two emerging theories include self-antigens by autoreactive CD4 T cells or harmless exogenous antigens in the setting of mucosal immunotolerance loss. These CD4 T cells migrate to the ocular surface causing additional inflammation and damage. Lifitegrast is the second topical anti-inflammatory agent to be approved by the US Food and Drug Administration for the treatment of DE and the first to show improvement in DE symptoms. Lifitegrast works by blocking the interaction between intercellular adhesion molecule-1 and lymphocyte functional associated antigen-1, which has been shown to be critical for the migration of antigen-presenting cells to the lymph nodes as well as CD4+ T cell activation and migration to the ocular surface. In four large multicenter, randomized controlled trials, lifitegrast has proven to be effective in controlling both the signs and symptoms of DE with minimal side effects. Further research should include comparative and combination studies with other anti-inflammatory therapies used for DE. PMID:29391773

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

PubMed

Dagenais, P; Desprez, B; Albert, J; Escher, E

1994-10-01

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

PubMed Central

Dazzo, F B; Hubbell, D H

1975-01-01

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin. Images PMID:55100

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

In vitro antigen-induced, antigen-specific antibody production in man. Specific and polyclonal components, kinetics, and cellular requirements

PubMed Central

1981-01-01

A highly specific and reproducible antigen-induced, antigen-specific culture and assay system for antibody production by human peripheral blood B lymphocytes has been developed. The system is clearly T cell and monocyte dependent and is independent of exogenous mitogens. The major factors in our ability to trigger specific antibody production with antigen alone have been the use of extremely low concentrations of antigen in vitro (doses several orders of magnitude below those inducing a peak blastogenic response), careful attention to in vitro cell density and culture vessel geometry, and appreciation of the kinetics of the circulating antigen-inducible B cell repertoire. A dichotomy and overlap between antigen-induced, antigen-specific and antigen-induced, polyclonal responses was observed in the study of doubly immunized individuals. Whereas antibody responses highly specific for the antigen in culture were observed under one set of culture conditions (flat-bottomed vessels, 1.5 x 10(6) cells), switching to another culture system (round-bottomed vessels, 5 x 10(5) cells) resulted in polyclonal responses to antigen. Despite these culture condition-related differences in the induction of antibody synthesis, the suppression of specific antibody production that occurred at high concentrations of antigen was specific only for the antigen in culture. The capability to easily and reproducibly look at truly antigen-induced, antigen specific antibody production should be a major tool in furthering the understanding of human B cell activation and immunoregulation. PMID:6169778

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

USDA-ARS?s Scientific Manuscript database

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

SNSAG5 IS AN ALTERNATIVE SURFACE ANTIGEN OF SARCOCYSTIS NEURONA STRAINS THAT IS MUTUALLY EXCLUSIVE TO SNSAG1

USDA-ARS?s Scientific Manuscript database

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore can...

Recombinant poxviruses as mucosal vaccine vectors.

PubMed

Gherardi, M Magdalena; Esteban, Mariano

2005-11-01

The majority of infections initiate their departure from a mucosal surface, such as Human immunodeficiency virus (HIV), a sexually transmitted virus. Therefore, the induction of mucosal immunity is a high priority in the development of vaccines against mucosal pathogens. The selection of an appropriate antigen delivery system is necessary to induce an efficient mucosal immune response. Poxvirus vectors have been the most intensively studied live recombinant vector, and numerous studies have demonstrated their ability to induce mucosal immune responses against foreign expressed antigens. Previous studies have demonstrated that recombinants based on the attenuated modified vaccinia virus Ankara (MVA) vector were effective in inducing protective responses against different respiratory viruses, such as influenza and respiratory syncytial virus, following immunization via mucosal routes. Recent studies performed in the murine and macaque models have shown that recombinant MVA (rMVA) does not only stimulate HIV-specific immunity in the genital and rectal tracts following mucosal delivery, but can also control simian/human immunodeficiency viraemia and disease progression. In addition, a prime-boost vaccination approach against tuberculosis emphasized the importance of the intranasal rMVA antigen delivery to induce protective immunity against Mycobacterium tuberculosis. The aim of this review is to summarize the studies employing recombinant poxviruses, specifically rMVA as a mucosal delivery vector. The results demonstrate that rMVAs can activate specific immune responses at mucosal surfaces, and encourage further studies to characterize and improve the MVA mucosal immunogenicity of poxvirus vectors.

Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles.

PubMed

Srivastava, Atul; Gowda, Devegowda Vishakante; Madhunapantula, SubbaRao V; Shinde, Chetan G; Iyer, Meenakshi

2015-04-01

Mucosal immune responses are the first-line defensive mechanisms against a variety of infections. Therefore, immunizations of mucosal surfaces from which majority of infectious agents make their entry, helps to protect the body against infections. Hence, vaccinization of mucosal surfaces by using mucosal vaccines provides the basis for generating protective immunity both in the mucosal and systemic immune compartments. Mucosal vaccines offer several advantages over parenteral immunization. For example, (i) ease of administration; (ii) non-invasiveness; (iii) high-patient compliance; and (iv) suitability for mass vaccination. Despite these benefits, to date, only very few mucosal vaccines have been developed using whole microorganisms and approved for use in humans. This is due to various challenges associated with the development of an effective mucosal vaccine that can work against a variety of infections, and various problems concerned with the safe delivery of developed vaccine. For instance, protein antigen alone is not just sufficient enough for the optimal delivery of antigen(s) mucosally. Hence, efforts have been made to develop better prophylactic and therapeutic vaccines for improved mucosal Th1 and Th2 immune responses using an efficient and safe immunostimulatory molecule and novel delivery carriers. Therefore, in this review, we have made an attempt to cover the recent advancements in the development of adjuvants and delivery carriers for safe and effective mucosal vaccine production. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Recent advances in Major Histocompatibility Complex (MHC) class I antigen presentation: Plastic MHC molecules and TAPBPR-mediated quality control

PubMed Central

van Hateren, Andy; Bailey, Alistair; Elliott, Tim

2017-01-01

We have known since the late 1980s that the function of classical major histocompatibility complex (MHC) class I molecules is to bind peptides and display them at the cell surface to cytotoxic T cells. Recognition by these sentinels of the immune system can lead to the destruction of the presenting cell, thus protecting the host from pathogens and cancer. Classical MHC class I molecules (MHC I hereafter) are co-dominantly expressed, polygenic, and exceptionally polymorphic and have significant sequence diversity. Thus, in most species, there are many different MHC I allotypes expressed, each with different peptide-binding specificity, which can have a dramatic effect on disease outcome. Although MHC allotypes vary in their primary sequence, they share common tertiary and quaternary structures. Here, we review the evidence that, despite this commonality, polymorphic amino acid differences between allotypes alter the ability of MHC I molecules to change shape (that is, their conformational plasticity). We discuss how the peptide loading co-factor tapasin might modify this plasticity to augment peptide loading. Lastly, we consider recent findings concerning the functions of the non-classical MHC I molecule HLA-E as well as the tapasin-related protein TAPBPR (transporter associated with antigen presentation binding protein-related), which has been shown to act as a second quality-control stage in MHC I antigen presentation. PMID:28299193

Selection of tumor antigens as targets for immune attack using immunohistochemistry: protein antigens.

PubMed

Zhang, S; Zhang, H S; Cordon-Cardo, C; Ragupathi, G; Livingston, P O

1998-11-01

The relative expression of mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 and glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta on different cancers and normal tissues is difficult to determine from available reports. We have compared the distribution of these antigens by immunohistology on a broad range of malignant and normal tissues. MUC1 expression was most intense in cancers of breast, lung, ovarian, and endometrial origin; MUC2 was most intense in cancers of colon and prostate origin; and MUC5AC was most intense in cancers of breast and gastric origin. MUC4 was intensely expressed in 50% of cancers of colon and pancreas origin, and MUC3, MUC5B, and MUC7 were expressed in a variety of epithelial cancers, but not so intensely. KSA was intensely and uniformly expressed on all epithelial cancers; carcinoembryonic antigen was expressed in most cancers of breast, lung, colon, pancreas, and gastric origin; and PSMA was expressed only in cancers of prostate origin. Human chorionic gonadotropin-beta was expressed on the majority of sarcomas and cancers of breast, lung, and pancreas origin, although intense staining was not seen. Staining on normal tissues was restricted to one or many normal epithelial tissues ranging from MUC3, MUC4, and PSMA, which were expressed only on epithelia of pancreas, stomach, and prostate origin, respectively, to MUC1 and KSA, which were expressed on most normal epithelia. Expression was restricted to the secretory borders of these epithelia while stroma and other normal tissues were completely negative. These results plus the results of the two previous papers (S. Zhang et al, Int. J. Cancer, 73: 42-49, 1997; S. Zhang et al., Int. J. Cancer, 73: 50-56, 1997) in this series provide the basis for selection of multiple cell surface antigens as targets for antibody-mediated attack against these cancers.

Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a.

PubMed

Theillet, François-Xavier; Saul, Frederick A; Vulliez-Le Normand, Brigitte; Hoos, Sylviane; Felici, Franco; Weintraub, Andrej; Mulard, Laurence A; Phalipon, Armelle; Delepierre, Muriel; Bentley, Graham A

2009-05-15

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

PubMed

Zheng, Li; Pang, Wei; Qi, Zanmei; Luo, Enjie; Cui, Liwang; Cao, Yaming

2016-08-08

Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Antigenic differences in the surfaces of hyphae and rhizoids in allomyces.

PubMed

Fultz, S A; Sussman, A S

1966-05-06

Immunofluorescent techniques have demonstrated a difference in surface components of hyphae and rhizoids of Allomyces macrogynus. An antigenic component detected on the rhizoidal surface may be present, but masked, in the hyphal-wall matrix material. The system also allows visualization of the hyphal wall during aging, when changes from a smooth to a fissured surface are noted, and differences in adsorptive properties occur.

Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil

PubMed Central

de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

2013-01-01

Background American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. Methods A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. Results The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. Interpretation ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil. PMID:23922884

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

DTIC Science & Technology

1990-05-01

Sta58 antigen and the Sta56 strain- GroES, C. burnetii HtpA, Mycobacterium tuberculosis 12- specific major antigen of R. tsutsugamushi (strain Karp...kb HindlIl fragment carrying the gene for the Sta58 tuberculosis, and Mycobacterium smegmatis (65-kDa anti- protein was subjected to DNA sequence...the Hsp6O and HsplO proteins. R. tsu., R. isutsugamushi; M. lep., Mvtcobacteriutn leprae : C. bur., C. burneiii; Synech.. Synechococcus strain 6301; T

Isolation and Purification of Antigenic Components of Cryptococcus

PubMed Central

Wozniak, Karen L.; Levitz, Stuart M.

2012-01-01

The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species. PMID:19089377

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

PubMed Central

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

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt, as an inactivator of hepatitis B surface antigen.

PubMed Central

Sugimoto, Y; Toyoshima, S

1979-01-01

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt (CAE), exhibited a strong inactivating effect on hepatitis B surface antigen. Concentrations of CAE required for 50 and 100% inactivation of the antigen were 0.01 to 0.025% and 0.025 to 0.05% respectively. CAE completely inactivated hepatitis B surface antigen at the lowest concentration compared with various compounds including about 500 amino acid derivatives, sodium hypochlorite, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and some detergents. Furthermore, CAE inactivated vaccinia virus, herpes simplex virus, and influenza virus, whereas poliovirus was not inactivated at all. The results suggest that the inactivating effects of CAE are related to interaction with lipid-containing viral envelopes. PMID:228595

Stable expression of the hepatitis B virus surface antigen containing pre-S2 protein in mouse cells using a bovine papillomavirus vector.

PubMed

Yoneyama, T; Akatsuka, T; Miyamura, T

1988-08-01

The large BglII fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

Structure of Adeno-Associated Virus Type 4

PubMed Central

Padron, Eric; Bowman, Valorie; Kaludov, Nikola; Govindasamy, Lakshmanan; Levy, Hazel; Nick, Phillip; McKenna, Robert; Muzyczka, Nicholas; Chiorini, John A.; Baker, Timothy S.; Agbandje-McKenna, Mavis

2005-01-01

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology. PMID:15795290

Emergence of new genotype and diversity of Theileria orientalis parasites from bovines in India.

PubMed

George, Neena; Bhandari, Vasundhra; Reddy, D Peddi; Sharma, Paresh

2015-12-01

Bovine theileriosis is a serious threat to livestock worldwide. Uncertainty around species prevalence, antigenic diversity and genotypes of strains make it difficult to assess the impact of this parasite and to provide necessary treatment. We aimed to characterize genotypic diversity, phylogeny and prevalence of Theileria orientalis parasites from the states of Telangana and Andhra Pradesh, India by collecting bovine blood samples from the major districts of the two states. Bioinformatic analysis identified antigenic diversity among the prevalent parasite strains using major piroplasm surface protein (MPSP) gene. Our study revealed a prevalence rate of 4.8% (n=41/862) of T. orientalis parasites in bovine animals and a new genotype of T. orientalis parasite which was not previously reported in India. The emergence of these new genotypes could be an explanation for the frequent outbreaks of bovine theileriosis. Further, whole genome sequencing of T. orientalis strains will help to elucidate the genetic factors relevant for transmissibility and virulence as well as vaccine and new drug development. Copyright © 2015 Elsevier B.V. All rights reserved.

Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape.

PubMed

Peacock, Thomas; Reddy, Kolli; James, Joe; Adamiak, Beata; Barclay, Wendy; Shelton, Holly; Iqbal, Munir

2016-01-07

H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide use of vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants. To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopes of the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodies were generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2 viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique 'escape' mutants with substitutions across nine different amino acid residues in hemagglutinin including seven that have not been described as antigenic determinants for H9N2 viruses before. Competition assays and structural mapping revealed two novel, discrete antigenic sites "H9-A" and "H9-B". Additionally, a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptor binding site. This constitutes a novel method of escape for group 1 hemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guide surveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection and vaccine design.

Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape

PubMed Central

Peacock, Thomas; Reddy, Kolli; James, Joe; Adamiak, Beata; Barclay, Wendy; Shelton, Holly; Iqbal, Munir

2016-01-01

H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide use of vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants. To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopes of the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodies were generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2 viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique ‘escape’ mutants with substitutions across nine different amino acid residues in hemagglutinin including seven that have not been described as antigenic determinants for H9N2 viruses before. Competition assays and structural mapping revealed two novel, discrete antigenic sites “H9-A” and “H9-B”. Additionally, a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptor binding site. This constitutes a novel method of escape for group 1 hemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guide surveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection and vaccine design. PMID:26738561

The WT hemochromatosis protein HFE inhibits CD8⁺ T-lymphocyte activation.

PubMed

Reuben, Alexandre; Phénix, Mikaël; Santos, Manuela M; Lapointe, Réjean

2014-06-01

MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8(+) T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8(+) T-lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFEWT ), but not C282Y-mutated HFE, inhibits secretion of MIP-1β from antigen-specific CD8(+) T lymphocytes. HFEWT expression also resulted in major decreases in CD8(+) T-lymphocyte activation as measured by 4-1BB expression. We further demonstrate that inhibition of CD8(+) T-lymphocyte activation was independent of MHC I surface levels, β2-m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1-2 domains of HFEWT as being responsible for inhibiting CD8(+) T-lymphocyte activation. Our data imply a new role for HFEWT in altering CD8(+) T-lymphocyte reactivity, which could modulate antigen immunogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

PubMed

Tipper, Donald J; Szomolanyi-Tsuda, Eva

2016-01-01

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

Cross-presentation of IgG-containing immune complexes

PubMed Central

Baker, Kristi; Rath, Timo; Lencer, Wayne I.; Fiebiger, Edda

2012-01-01

IgG is a molecule that functionally combines facets of both innate and adaptive immunity and therefore bridges both arms of the immune system. On the one hand, IgG is created by adaptive immune cells, but can be generated by B cells independently of T cell help. On the other hand, once secreted, IgG can rapidly deliver antigens into intracellular processing pathways, which enable efficient priming of T cell responses towards epitopes from the cognate antigen initially bound by the IgG. While this process has long been known to participate in CD4+ T cell activation, IgG-mediated delivery of exogenous antigens into a major histocompatibility complex (MHC) class I processing pathway has received less attention. The coordinated engagement of IgG with IgG receptors expressed on the cell-surface (FcγR) and within the endolysosomal system (FcRn) is a highly potent means to deliver antigen into processing pathways that promote cross-presentation of MHC class I and presentation of MHC class II-restricted epitopes within the same dendritic cell. This review focuses on the mechanisms by which IgG-containing immune complexes mediate such cross-presentation and the implications that this understanding has for manipulation of immune-mediated diseases that depend upon or are due to the activities of CD8+ T cells. PMID:22847331

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Paulson, James C.

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which representmore » the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.« less

αβ T cell receptors as predictors of health and disease

PubMed Central

Attaf, Meriem; Huseby, Eric; Sewell, Andrew K

2015-01-01

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR ‘signatures' raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome. PMID:25619506

Immunotherapy Targets in Pediatric Cancer

PubMed Central

Orentas, Rimas J.; Lee, Daniel W.; Mackall, Crystal

2011-01-01

Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer. PMID:22645714

Phenotypically heterogeneous deletion of the ABH antigen from the transformed bladder urothelium. A scanning electron microscope study.

PubMed

De Harven, E; He, S; Hanna, W; Bootsma, G; Connolly, J G

1987-10-01

The deletion of ABH blood group antigens from the luminal surface of the bladder mucosa in cases of well differentiated transitional cell carcinomata, and the formation of pleomorphic microvilli have both been associated with aggressive biological behaviour and invasiveness of the tumors. We have studied cold cup biopsies from 8 normal mucosae and 17 papillary transitional cell carcinomata of the urinary bladder. The aim of our study was to correlate the formation of uniform or pleomorphic microvilli with the extent of deletion of the ABH blood group antigens on the surface of normal and transformed bladder urothelium. Immunogold scanning electron microscopy (SEM) in the backscattered electron (BE) imaging mode was used for this purpose. In the normal urothelium, uniform labeling of the luminal cells was demonstrated. In well differentiated tumors, the superficial cells exhibited uniform microvilli and a heterogeneous expression of the ABH antigens, giving characteristic 'mosaic' patterns of the antigenic labeling across the mucosal surface. These patterns were sharply delimitated at cell junctions when viewed by SEM; these observations were confirmed by transmission electron microscopy. In higher grade tumors, decreased ABH antigen expression, pleomorphic microvilli and/or featureless luminal cells were observed. In the transformed urothelium, the formation of uniform microvilli appeared to precede the loss of ABH antigen in most cases.

Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors.

PubMed

Abken, Hinrich

2015-01-01

Recent spectacular success in the adoptive cell therapy of leukemia and lymphoma with chimeric antigen receptor (CAR)-modified T cells raised the expectations that this therapy may be efficacious in a wide range of cancer entities. The expectations are based on the predefined specificity of CAR T cells by an antibody-derived binding domain that acts independently of the natural T-cell receptor, recognizes targets independently of presentation by the major histocompatibility complex and allows targeting toward virtually any cell surface antigen. We here discuss that targeting CAR T cells toward solid tumors faces certain circumstances critical for the therapeutic success. Targeting tumor stroma and taking advantage of TRUCK cells, in other words, CAR T cells with inducible release of a transgenic payload, are some strategies envisaged to overcome current limitations in the near future.

Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors.

PubMed

Aldoss, I; Bargou, R C; Nagorsen, D; Friberg, G R; Baeuerle, P A; Forman, S J

2017-04-01

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r/r ALL setting.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Qiudong; Yi, Yao; Guo, Minzhuo

Highlights: •The conformational HBV neutralization antigen domain was successfully displayed on the surface of truncated HBc particles. •Appropriate dialysis procedures to support the renaturing environment for the protein refolding. •Efficient purification procedures to obtain high purity and icosahedral particles of mosaic HBV antigen. •Strong immune responses not only including neutralization antibody response but also Th1 cell response were induced in mice. -- Abstract: Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenicmore » determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp{sup 78} and Pro{sup 79}. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH{sub 4}){sub 2}SO{sub 4} precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.« less

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

PubMed

Lempereur, Laetitia; Larcombe, Stephen D; Durrani, Zeeshan; Karagenc, Tulin; Bilgic, Huseyin Bilgin; Bakirci, Serkan; Hacilarlioglu, Selin; Kinnaird, Jane; Thompson, Joanne; Weir, William; Shiels, Brian

2017-06-05

Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen

PubMed Central

Choudhary, Alok; Honnen, William; Lai, Zhong; Gennaro, Maria Laura; Garcia-Viveros, Moncerrato; Sahloul, Kamar; Spencer, John S.; Chatterjee, Delphi

2018-01-01

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1–coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM. PMID:29610143

Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia (Oreochromis niloticus): Insight into vaccine development.

PubMed

Li, Wei; Wang, Hai-Qing; He, Run-Zhen; Li, Yan-Wei; Su, You-Lu; Li, An-Xing

2016-08-01

Streptococcus agalactiae is a major piscine pathogen that is responsible for huge economic losses to the aquaculture industry. Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against S. agalactiae. The proteins of S. agalactiae exposed to the environment, including surface proteins and secretory proteins, are important targets for the immune system and they are likely to be good vaccine candidates. To obtain a precise profile of its surface proteins, S. agalactiae strain THN0901, which was isolated from tilapia (Oreochromis niloticus), was treated with proteinase K to cleave surface-exposed proteins, which were identified by liquid chromatography-tandem spectrometry (LC-MS/MS). Forty surface-associated proteins were identified, including ten proteins containing cell wall-anchoring motifs, eight lipoproteins, eleven membrane proteins, seven secretory proteins, three cytoplasmic proteins, and one unknown protein. In addition, culture supernatant proteins of S. agalactiae were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all of the Coomassie-stained bands were subsequently identified by LC-MS/MS. A total of twenty-six extracellular proteins were identified, including eleven secretory proteins, seven cell wall proteins, three membrane proteins, two cytoplasmic proteins and three unknown proteins. Of these, six highly expressed surface-associated and secretory proteins are putative to be vaccine candidate of piscine S. agalactiae. Moreover, immunogenic secreted protein, a highly expressed protein screened from the secretome in the present study, was demonstrated to induce high antibody titer in tilapia, and it conferred protection against S. agalactiae, as evidenced by the relative percent survival (RPS) 48.61± 8.45%. The data reported here narrow the scope of screening protective antigens, and provide guidance in the development of a novel vaccine against piscine S. agalactiae. Copyright © 2016 Elsevier Ltd. All rights reserved.

Strategies to induce broadly protective antibody responses to viral glycoproteins.

PubMed

Krammer, F

2017-05-01

Currently, several universal/broadly protective influenza virus vaccine candidates are under development. Many of these vaccines are based on strategies to induce protective antibody responses against the surface glycoproteins of antigenically and genetically diverse influenza viruses. These strategies might also be applicable to surface glycoproteins of a broad range of other important viral pathogens. Areas covered: Common strategies include sequential vaccination with divergent antigens, multivalent approaches, vaccination with glycan-modified antigens, vaccination with minimal antigens and vaccination with antigens that have centralized/optimized sequences. Here we review these strategies and the underlying concepts. Furthermore, challenges, feasibility and applicability to other viral pathogens are discussed. Expert commentary: Several broadly protective/universal influenza virus vaccine strategies will be tested in humans in the coming years. If successful in terms of safety and immunological readouts, they will move forward into efficacy trials. In the meantime, successful vaccine strategies might also be applied to other antigenically diverse viruses of concern.

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

PubMed Central

Yamanaka, Nobuko; Wong, Christine J.; Gertsenstein, Marina; Casper, Robert F.; Nagy, Andras; Rogers, Ian M.

2009-01-01

Background Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice. PMID:20046883

Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12.

PubMed Central

Palermo, D A; Evans, T M; Clark, V L

1987-01-01

A gonococcal gene bank maintained in Escherichia coli K-12 was screened by colony immunoblotting, and a transformant expressing a surface antigen reactive to anti-gonococcal outer membrane antiserum was isolated. The isolate carried a recombinant plasmid, pTME6, consisting of approximately 9 kilobases of Neisseria gonorrhoeae DNA inserted into the BamHI site of pBR322. Surface labeling of E. coli HB101(pTME6) confirmed that the antigen was expressed on the E. coli cell surface. The antigenic material was resistant to proteinase K digestion and sensitive to periodate oxidation, indicating that the material was carbohydrate. Purified lipopolysaccharide (LPS) from HB101(pTME6) produced a unique band on silver-stained polyacrylamide gels that contained immunoreactive material as seen on Western blots of LPS samples. Only two of three E. coli LPS mutant strains carrying pTME6 reacted with the antigonococcal antiserum, suggesting that a certain E. coli core structure is necessary for antigen expression. We conclude that pTME6 contains one or more gonococcal genes encoding an LPS core biosynthetic enzyme(s) which can modify E. coli core LPS to produce a gonococcuslike epitope(s). Images PMID:3117695

Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA.

PubMed

Xiang, Kuan-Hui; Michailidis, Eleftherios; Ding, Hai; Peng, Ya-Qin; Su, Ming-Ze; Li, Yao; Liu, Xue-En; Dao Thi, Viet Loan; Wu, Xian-Fang; Schneider, William M; Rice, Charles M; Zhuang, Hui; Li, Tong

2017-02-01

As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Modulation of alloimmune response by commensal gut microbiota and potential new avenues to influence the outcome of allogeneic transplantation by modification of the 'gut culture'.

PubMed

Kanangat, S

2017-02-01

Host defence response against microbial infections was the foundation for the Science of Immunology. Now, we know the mechanisms of such host defence which include innate immune responses that is generally nonspecific but effective in many cases and lead to more specific responses called adaptive immune response. The gene loci of class I, II and III of the major histocompatibility complex (MHC) play a major role in directing the adaptive immune responses by presenting processed antigens to T and B cells to induce appropriate antigen-specific cellular and or humoral immune responses. In humans, these are commonly referred to as human leucocyte antigens class I/II-HLA I/II). The class III region, the gamma region in the MHC complex, is mostly associated with regulation of immune responses along with genes associated with complement activation. The adaptive immune responses are orchestrated by T and B cells that are tuned to respond to antigens that are normally foreign to the body, because these cells are educated to avoid self-antigens by a process of thymic education and selection of the T cells that are mostly non-self-reactive which also helps the B cells in eliciting specific immune responses to non-self-antigens. A by-product of this is the ability of the T and B cells to elicit strong immune responses to foreign HLA/MHC (alloimmune response), which developed into the field of histocompatibility testing for allogeneic transplantation of stem cells and organs. Now, we are beginning to learn that such alloimmune responses can be influenced by the microbiota that symbiotically live in our body especially on the mucosal surfaces and on the skin. This review deals with new and emerging data on how the commensal mucosal and skin microbiota influence the immune homeostasis, and how manipulating the commensal microbiota of the mucosa and skin could influence the survival and long-term functions of the allografts. Also, alterations of the microbiota by the inevitable immunosuppression prior to and following allogeneic transplantation could contribute towards the outcome of the allografts by alloimmune responses generated due to microbial antigen vs HLA cross-reactivity. © 2017 John Wiley & Sons Ltd.

A novel interdigitated capacitor based biosensor for detection of cardiovascular risk marker.

PubMed

Quershi, Anjum; Gurbuz, Yasar; Kang, Weng P; Davidson, Jimmy L

2009-12-15

C-reactive protein (CRP) is a potential biomarker whose elevated levels in humans determine cardiovascular disease risk and inflammation. In this study, we have developed a novel capacitive biosensor for detection of CRP-antigen using capacitor with interdigitated gold (GID) electrodes on nanocrystalline diamond (NCD) surface. The NCD surface served as a dielectric layer between the gold electrodes. GID-surface was functionalized by antibodies and the immobilization was confirmed by Fourier transform spectroscopy (FT-IR) and contact angle measurements. The CRP-antigen detection was performed by capacitive/dielectric-constant measurements. The relaxation time and polarizability constants were estimated using Cole-Cole model. Our results showed that the relaxation time constant (tau) of only CRP-antibody was within 10(-16)-10(-13)s, which was increased to 10(-11)s after the incubation with CRP-antigen, suggesting that the CRP-antigen was captured by the antibodies on GID-surface. In addition, polarizability constant (m) of CRP was also increased upon incubation with increasing concentration of CRP-antigen. Our results showed that the response of GID-NCD-based capacitive biosensor for CRP-antigen was dependent on both concentration (25-800ng/ml) as well as frequency (50-350MHz). Furthermore, using optimized conditions, the GID-NCD based capacitive biosensor developed in this study can potentially be used for detection of elevated levels of protein risk markers in suspected subjects for early diagnosis of disease.

Rh blood phenotyping (D, E, e, C, c) microarrays using multichannel surface plasmon resonance imaging.

PubMed

Pipatpanukul, Chinnawut; Takeya, Sasaki; Baba, Akira; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

2018-04-15

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of variant-specific surface proteins in Giardia muris trophozoites.

PubMed

Ropolo, Andrea S; Saura, Alicia; Carranza, Pedro G; Lujan, Hugo D

2005-08-01

Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

Mechanisms of Chemical Modulation and Toxicity of the Immune System.

DTIC Science & Technology

1988-05-15

expressing the L3T4 surface antigens and suppressor/cytotoxic cells bearing Lyt-2 surface antigens. Autoimmune or immunodeficient diseases have been...Ill. Written Publications (cumulative list) A. Suppression of mitogen-induced blastogenesis of feline lymphocytes by in vitro incubation with

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

Piezoelectric cantilever sensors

NASA Technical Reports Server (NTRS)

Shih, Wan Y. (Inventor); Shih, Wei-Heng (Inventor); Shen, Zuyan (Inventor)

2008-01-01

A piezoelectric cantilever with a non-piezoelectric, or piezoelectric tip useful as mass and viscosity sensors. The change in the cantilever mass can be accurately quantified by monitoring a resonance frequency shift of the cantilever. For bio-detection, antibodies or other specific receptors of target antigens may be immobilized on the cantilever surface, preferably on the non-piezoelectric tip. For chemical detection, high surface-area selective absorbent materials are coated on the cantilever tip. Binding of the target antigens or analytes to the cantilever surface increases the cantilever mass. Detection of target antigens or analytes is achieved by monitoring the cantilever's resonance frequency and determining the resonance frequency shift that is due to the mass of the adsorbed target antigens on the cantilever surface. The use of a piezoelectric unimorph cantilever allows both electrical actuation and electrical sensing. Incorporating a non-piezoelectric tip (14) enhances the sensitivity of the sensor. In addition, the piezoelectric cantilever can withstand damping in highly viscous liquids and can be used as a viscosity sensor in wide viscosity range.

Immunization with SV40-transformed cells yields mainly MHC-restricted monoclonal antibodies

PubMed Central

1986-01-01

Recognition of antigens on cell surfaces only in the context of the MHC- encoded alloantigens of the presenting cell (self + X) has classically been considered the province of T cells. However, evidence from several sources has indicated that B cells and antibodies can exhibit self + X- restricted recognition as well. This report concerns the mAb response to SV40-transformed H-2b fibroblast cell lines. The specificities of the antibodies obtained have been analyzed for binding to a panel of SV40-transformed H-2-syngeneic, H-2-allogeneic, and H-2b mutant fibroblast cell lines, as well as cell lines not bearing cell surface SV40 transformation-associated antigens. A large proportion of primary C57BL/6 (71%) and BALB/c (68%) splenic B cells responding to in vitro stimulation with SV40-transformed H-2b cells recognize cell surface antigens associated with SV40 transformation only when coexpressed with MHC antigens of the immunizing cell, particularly the Kb molecule, on transformed cells. To extensively define the nature of antigen recognition by these antibodies, we have generated and characterized nine hybridoma antibodies specific for SV40-transformed H-2-syngeneic cell lines. Seven of these hybridoma antibodies recognize SV40- associated transformation antigens in the context of H-2b molecules. Six of these are restricted by the Kb molecule and discriminate among a panel of SV40-transformed Kb mutant cell lines, thus confirming the participation of class I MHC-encoded molecules in the recognition by B cells of cell surface antigens. PMID:3014034

Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

PubMed Central

Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

2015-01-01

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

Proteomic Mapping of the Immune Response to Gluten in Children with Autism

DTIC Science & Technology

2015-10-01

report. 15. SUBJECT TERMS autism, immune response, gastrointestinal symptoms, antigen, gluten, IgG, antibody, microbiota 16. SECURITY...gastrointestinal symptoms, antigen, gluten, IgG, antibody, microbiota 3. ACCOMPLISHMENTS: 3.1. What were the major goals of the project? The major goals of

Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

PubMed Central

Seed, Kimberley D.; Faruque, Shah M.; Mekalanos, John J.; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

2012-01-01

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage. PMID:23028317

Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

NASA Astrophysics Data System (ADS)

Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

1995-02-01

The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus

PubMed Central

Abente, Eugenio J.; Santos, Jefferson; Lewis, Nicola S.; Gauger, Phillip C.; Stratton, Jered; Skepner, Eugene; Rajao, Daniela S.

2016-01-01

ABSTRACT Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, and 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to the wild type had a >4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had virus shedding titers and transmissibility comparable to those of the wild type, it caused a significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV has important implications for understanding IAV evolution within pigs as well as for improved vaccine development and control strategies in swine. IMPORTANCE A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control the circulation of influenza in pigs and reduce the potential for zoonotic viruses to emerge. PMID:27384658

[Differentiation of nonspecific serological reactions in brucellosis].

PubMed

Khristoforov, L

1979-01-01

Differentiation of non-specific agglutination was performed by the complement binding reaction, Coombs' reaction, Hajdu reaction, the surface fixation and agglutination reaction and the reaction of complement binding with heterologic antigens. For that purpose the following were used: 1) Serums--antiglobulin against cattle globulin, 5720 serum of various animals which had manifested non-specific agglutination with brucella antigen and brucella serums of experimentally infected sheep, of naturally infected swine and of cattle--received from abroad. 2) Antigens--of Br. abortus 99, of bacteria heterologic to brucellae: Proteus vulgaris, Listeria monocytogenes, Staphylococcus albus, Escherichia coli, Streptococcus pyogenes, S. abortus ovis, for O and OH agglutination, water extraction antigens--for complement binding and concentrated suspensions of all bacteria used in brucellose and non-brucellose serum absorption. Highest number of non-specific reactions were observed in cattle serums and lowest--in goat serums. Titers with heterologic antigens were higher than these with brucella antigens. Often the serum having non-specific agglutiantion reacted not only with one, but with more heterologic antigens. Non-specific complement binding reactions were not produced in complete antibodies with the brucella antigen. Heterologic brucella antigens were exhausted more fully than heterologic complement binding antibodies. In their effectiveness (differentiation of non-specific agglutination with brucella antigen in cattle serum) the serological reactions studied rank as follows: complement binding reaction, slow agglutination with serums absorbed by heterologic antigens, surface fixation reaction, Coombs' reaction, and Hadju agglutination.

Prevalence of Hepatitis B surface antigen among biomedical students of African descent in Usmanu Danfodiyo University, Sokoto, Nigeria.

PubMed

Okwesili, A N; Onuigwe, F U; Ibrahim, K; Buhari, H; Ibrahim, A; Jafaru, H; Erhabor, O; Onuigwe, F U; Isaac, Z; Ahmed, M H; Mainasara, M Y; Adias, T C; Yeldu, M H; Uko, E K; Udoma, F

2015-12-23

Hepatitis B (HB) is a serious global public health problem that put health professionals particularly at risk. The aim of this study was to investigate the prevalence of Hepatitis B surface antigen (HBsAg) among Biomedical Students of African descent attending Usmanu Danfodiyo University Sokoto in North-Western Nigeria. The Onsite HBsAg (CTK Biotech, USA) was used to detect the presence of hepatitis B surface antigen. We tested 186 consecutively-recruited students consisting of 147 males and 39 females aged 18-35 years (mean age 26 ± 2.0 years). Of the 186 students tested, 25 (13.4%) were positive for HBsAg. The prevalence of HBsAg was significantly higher among students in the 21-25 years age group. Hepatitis B vaccination uptake among students was 7%. Majority of subjects were single 173(93.1%) compared to married 13 (6.9%). Ethnic distribution of the subjects indicated that 104(55.9%) were Hausa compared to Yoruba 32 (17.2%), other ethnic groups 21(11.3%), Fulani 20(10.8%) and Igbo 9(4.8%). This study indicates a high prevalence of hepatitis B virus infection among Biomedical students in Sokoto, North Western, Nigeria. Finding from this study is enough justification for the implementation of a policy to routinely test students entering into the biomedical professions for Hepatitis B virus infection. There is the need to provide hepatitis B vaccination universally to all those who are found negative prior to commencement of their biomedical training. There is also need to educate students entering biomedical professions and healthcare workers on the modes of transmission and prevention, importance of being compliant with protective vaccination as well as the need to observe universal precaution and infection control guidelines during their training and future professional practice.

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

PubMed Central

Boyd, Mary A.; Wang, Jin Y.; Tulapurkar, Mohan E.; Pasetti, Marcela F.; Levine, Myron M.; Simon, Raphael

2016-01-01

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity. PMID:26998925

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

21 CFR 660.43 - Potency test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.43 Potency test. To be satisfactory for release, each filling of Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Antiserum Panel and shall be sufficiently potent to be able to detect the...

Sarcocystis neurona Merozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of the Toxoplasma gondii Surface Antigens (SAGs) and SAG-Related Sequences†

PubMed Central

Howe, Daniel K.; Gaji, Rajshekhar Y.; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-01-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s). PMID:15664946

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-02-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

Identification of Variant-Specific Surface Proteins in Giardia muris Trophozoites

PubMed Central

Ropolo, Andrea S.; Saura, Alicia; Carranza, Pedro G.; Lujan, Hugo D.

2005-01-01

Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia. PMID:16041041

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

PubMed

Nhan, Nguyen Thanh; Gonzalez de Valdivia, Ernesto; Gustavsson, Martin; Hai, Truong Nam; Larsson, Gen

2011-04-11

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Activation of bone marrow-derived mesenchymal stromal cells-a new mechanism of defocused low-energy shock wave in regenerative medicine.

PubMed

Zhao, Yong; Wang, Jinbang; Wang, Muwen; Sun, Peng; Chen, Ji; Jin, Xunbo; Zhang, Haiyang

2013-12-01

Defocused low-energy shock wave (DLSW) therapy has shown effectiveness in regenerative medicine. The mechanism of action was mainly focused on the pathophysiological improvement at the wound tissues. In this study, the activation of stem cells treated by DLSW was first examined as an important pathway during the healing process. Cultured rat bone marrow-derived mesenchymal stromal cells (BMSC) were treated by DLSW before each passage. The untreated BMSC served as a control. The secretions of vascular endothelial growth factor (VEGF) and CXC ligand 5 (CXCL5) were tested by means of enzyme-linked immunoassay. Flow cytometry was performed to analyze the BMSC (passage 4) surface antigen expressions (CD166, CD44 and CD34). The expressions of proliferating cell nuclear antigen and Ki67 were analyzed by means of Western blot. The healing abilities of conditioned media of shocked and unshocked BMSC were examined by Matrigel-based capillary-like tube formation assay and rat major pelvic ganglia culture test. The shocked BMSC secreted more VEGF and CXCL5 than did those of unshocked BMSC. The expressions of CD166, CD44 and CD34 showed no significant differences (P > 0.05) between the shocked and unshocked BMSC. The shocked BMSC demonstrated higher expressions of proliferating cell nuclear antigen (P < 0.01) and Ki67 (P < 0.01) than did those of unshocked BMSC. The shocked BMSC conditioned medium showed higher ability to enhance the growth of major pelvic ganglia neurites (P < 0.05) and Matrigel-based endothelial tube-like formation (P < 0.05). DLSW did not interfere with the expressions of cell surface markers. DLSW enhanced the secretion and proliferation of BMSC and promoted angiogenesis and nerve regeneration in vitro. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Cytoadherence and Genotype of Plasmodium falciparum Strains from Symptomatic Children in Franceville, Southeastern Gabon

PubMed Central

Touré, Fousseyni S.; Ouwe-Missi-Oukem-Boyer, Odile; Mezui-Me-Ndong, Jérôme; Ndong-Atome, Guy Roger; Bisvigou, Ulrick; Mazier, Dominique; Bisser, Sylvie

2007-01-01

Background: Plasmodium falciparum causes severe clinical manifestations by sequestering parasitized red blood cells (PRBC) in the microvasculature of major organs such as the brain. This sequestration results from PRBC adherence to vascular endothelial cells via erythrocyte membrane protein 1, a variant parasite surface antigen. Objective: To determine whether P. falciparum multiple genotype infection (MGI) is associated with stronger PRBC cytoadherence and greater clinical severity. Methods: Nested polymerase chain reaction was used to genotype P. falciparum isolates from symptomatic children and to distinguish between single genotype infection (SGI) and MGI. PRBC cytoadhesion was studied with cultured human lung endothelial cells. Results: Analysis of two highly polymorphic regions of the merozoite surface antigen (MSP)-1 and MSP-2 genes and a dimorphic region of the erythrocyte binding antigen-175 gene showed that 21.4% and 78.6% of the 42 children had SGI and MGI, respectively. It also showed that 37 (89%) of the 42 PRBC samples expressed MSP-1 allelic family K1. Cytoadherence values ranged from 58 to 1811 PRBC/mm2 of human lung endothelial cells monolayer in SGI and from 5 to 5744 PRBC/mm2 in MGI. MGI was not associated with higher cytoadherence values or with more severe malaria. Conclusions: These results suggested that infection of the same individual by multiple clones of P. falciparum does not significantly influence PRBC cytoadherence or disease severity and confirmed the predominance of the MSP-1 K1 genotype in southeastern Gabon. PMID:17607045

Design and immunological evaluation of anti-CD205-tailored PLGA-based nanoparticulate cancer vaccine.

PubMed

Jahan, Sheikh Tasnim; Sadat, Sams Ma; Haddadi, Azita

2018-01-01

The aim of this research was to develop a targeted antigen-adjuvant assembled delivery system that will enable dendritic cells (DCs) to efficiently mature to recognize antigens released from tumor cells. It is important to target the DCs with greater efficiency to prime T cell immune responses. In brief, model antigen, ovalbumin (OV), and monophosphoryl lipid A adjuvant were encapsulated within the nanoparticle (NP) by double emulsification solvent evaporation method. Targeted NPs were obtained through ligand incorporation via physical adsorption or chemical conjugation process. Intracellular uptake of the NPs and the maturation of DCs were evaluated with flow cytometry. Remarkably, the developed delivery system had suitable physicochemical properties, such as particle size, surface charge, OV encapsulation efficiency, biphasic OV release pattern, and safety profile. The ligand modified formulations had higher targeting efficiency than the non-tailored NPs. This was also evident when the targeted formulations expressed comparatively higher fold increase in surface activation markers such as CD40, CD86, and major histocompatibility complex class II molecules. The maturation of DCs was further confirmed through secretion of extracellular cytokines compared to control cells in the DC microenvironment. Physicochemical characterization of NPs was performed based on the polymer end groups, their viscosities, and ligand-NP bonding type. In conclusion, the DC stimulatory response was integrated to develop a relationship between the NP structure and desired immune response. Therefore, the present study narrates a comparative evaluation of some selected parameters to choose a suitable formulation useful for in vivo cancer immunotherapy.

Altered Expression of TAP-1 and Major Histocompatibility Complex Class I in Laryngeal Papillomatosis: Correlation of TAP-1 with Disease

PubMed Central

Vambutas, Andrea; Bonagura, Vincent R.; Steinberg, Bettie M.

2000-01-01

Recurrent respiratory papillomatosis (RRP) is an insidious disease caused by human papillomavirus (HPV) infection. It is characterized by a variable clinical course that can include frequent disease recurrence, significant morbidity, and occasional mortality. The mechanisms responsible for the variability in the clinical course and the persistence of latent HPV infection remain unknown. Effective T-cell-mediated clearance of HPV-infected cells may be defective in patients with RRP, leading to recurrent disease and failure to suppress latent HPV reactivation. This study describes the down-regulation of the transporter associated with antigen presentation (TAP-1) and the major histocompatibility complex (MHC) class I protein expression in laryngeal papilloma tissue biopsies and cell culture of primary explants. There was a statistically significant correlation between reduction of TAP-1 expression in biopsy tissues and rapid recurrence of disease. Patients with RRP had less frequent recurrence if their papillomas expressed TAP-1 at levels close to that of normal tissue, compared with those with very low expression of TAP-1, who had frequent recurrence (32 versus 5 weeks to the next surgical intervention). These findings suggest that HPV may evade immune recognition by down-regulating class I MHC cell surface expression via decreased TAP-1 levels. Expression of TAP-1 could be used for prognostic evaluation of disease severity. Gamma interferon was able to restore class I MHC expression at the surfaces of laryngeal papilloma cells in culture. This up-regulation of class I MHC antigen at the cell surface potentially allows the infected cell to become a target for the immune system again. This finding provides some promise for nonsurgical treatment of laryngeal papillomas. PMID:10618282

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PubMed

Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

2017-01-15

The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

Antigen Potency and Maximal Efficacy Reveal a Mechanism of Efficient T Cell Activation

PubMed Central

Wheeler, Richard J.; Zhang, Hao; Cordoba, Shaun-Paul; Peng, Yan-Chun; Chen, Ji-Li; Cerundolo, Vincenzo; Dong, Tao; Coombs, Daniel; van der Merwe, P. Anton

2014-01-01

T cell activation, a critical event in adaptive immune responses, follows productive interactions between T cell receptors (TCRs) and antigens, in the form of peptide-bound major histocompatibility complexes (pMHCs) on the surfaces of antigen-presenting-cells. Upon activation, T cells can lyse infected cells, secrete cytokines, such as interferon-γ (IFN-γ), and perform other effector functions with various efficiencies that directly depend on the binding parameters of the TCR-pMHC complex. The mechanism that relates binding parameters to the efficiency of activation of the T cell remains controversial; some studies suggest that the dissociation constant (KD) determines the response (the “affinity model”), whereas others suggest that the off-rate (koff) is critical (the “productive hit rate model”). Here, we used mathematical modeling to show that antigen potency, as determined by the EC50, the functional correlate that is used to support KD-based models, could not be used to discriminate between the affinity and productive hit rate models. Our theoretical work showed that both models predicted a correlation between antigen potency and KD, but only the productive hit rate model predicted a correlation between maximal efficacy (Emax) and koff. We confirmed the predictions made by the productive hit rate model in experiments with cytotoxic T cell clones and a panel of pMHC variants. Therefore, we suggest that the activity of an antigen is determined by both its potency and maximal efficacy. We discuss the implications of our findings to the practical evaluation of T cell activation, for example in adoptive immunotherapies, and relate our work to the pharmacological theory of dose-response. PMID:21653229

T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

PubMed

Bell, Catherine C; Faulkner, Lee; Martinsson, Klara; Farrell, John; Alfirevic, Ana; Tugwood, Jonathan; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

2013-05-20

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

Antigenic Drift in H5N1 Avian Influenza Virus in Poultry Is Driven by Mutations in Major Antigenic Sites of the Hemagglutinin Molecule Analogous to Those for Human Influenza Virus▿†

PubMed Central

Cattoli, Giovanni; Milani, Adelaide; Temperton, Nigel; Zecchin, Bianca; Buratin, Alessandra; Molesti, Eleonora; Aly, Mona Meherez; Arafa, Abdel; Capua, Ilaria

2011-01-01

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses. PMID:21734057

Molecular characterization of the pL40 protein in Leptospira interrogans.

PubMed

Zhao, Wei; Chen, Chun-Yan; Zhang, Xiang-Yan; Lai, Wei-Qiang; Hu, Bao-Yu; Zhao, Guo-Ping; Qin, Jin-Hong; Guo, Xiao-Kui

2009-06-01

Leptospirosis is a widespread zoonotic disease caused by pathogenic leptospires. The identification of outer membrane proteins (OMPs) conserved among pathogenic leptospires, which are exposed on the leptospiral surface and expressed during mammalian infection, has become a major focus of leptospirosis research. pL40, a 40 kDa protein coded by the LA3744 gene in Leptospira interrogans, was found to be unique to Leptospira. Triton X-114 fractionation and flow cytometry analyses indicate that pL40 is a component of the leptospiral outer membrane. The conservation of pL40 among Leptospira strains prevalent in China was confirmed by both Western blotting and PCR screening. Furthermore, the pL40 antigen could be recognized by sera from guinea pigs and mice infected with low-passage L. interrogans. These findings indicate that pL40 may serve as a useful serodiagnostic antigen and vaccine candidate for L. interrogans.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.

PubMed

Kwan, David H; Constantinescu, Iren; Chapanian, Rafi; Higgins, Melanie A; Kötzler, Miriam P; Samain, Eric; Boraston, Alisdair B; Kizhakkedathu, Jayachandran N; Withers, Stephen G

2015-05-06

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

Direct measurement of IgM-Antigen interaction energy on individual red blood cells.

PubMed

Yeow, Natasha; Tabor, Rico F; Garnier, Gil

2017-07-01

Most blood grouping tests rely on the principle of red blood cells (RBCs) agglutination. Agglutination is triggered by the binding of specific blood grouping antibodies to the corresponding RBC surface antigen on multiple cells. The interaction energies between blood grouping antibodies and antigens have been poorly defined in immunohaematology. Here for the first time, we functionalized atomic force microscope (AFM) cantilevers with the IgM form of blood grouping antibodies to probe populations of individual RBCs of different groups under physiological conditions. The force-mapping mode of AFM allowed us to measure specific antibody - antigen interactions, and simultaneously localize and quantify antigen sites on the scanned cell surface. This study provides a new insight of the interactions between IgM antibodies and its corresponding antigen. The technique and information can be translated to develop better blood typing diagnostics and optimize target-specific drug delivery for medical applications. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed Central

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-01-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells. PMID:3855866

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-02-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope.

PubMed

Metz, Stefan W; Thomas, Ashlie; White, Laura; Stoops, Mark; Corten, Markus; Hannemann, Holger; de Silva, Aravinda M

2018-04-02

The 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form "rafts" that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion. A large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics. VLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1-4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations. This study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

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

PubMed Central

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

2013-01-01

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-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented. PMID:23935216

Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen

PubMed Central

Lynch, Heather E.; Stewart, Shelley M.; Kepler, Thomas B.; Sempowski, Gregory D.; Alam, S. Munir

2014-01-01

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development. PMID:24316020

Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

PubMed Central

Standring, D N; Ou, J H; Rutter, W J

1986-01-01

Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-S1) additional NH2-terminal amino acids. We show here that Xenopus oocytes injected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-S1 and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin. Images PMID:3467308

Signet-ring cell lymphoma of T-cell origin. An immunocytochemical and ultrastructural study relating giant vacuole formation to cytoplasmic sequestration of surface membrane.

PubMed

Grogan, T M; Richter, L C; Payne, C M; Rangel, C S

1985-09-01

In contrast to previous accounts of signet-ring lymphoma as a B-cell neoplasm, we report a case of signet-ring, large-cell lymphoma of T-cell lineage. Immunologic and ultrastructural studies were performed on a subcutaneous mass noted initially, as well as on an enlarged lymph node that developed later, in a 69-year-old man. Immunologic assessment indicated strong expression of T-helper antigen (Leu 3a + b), universal T-antigens (Leu 1, 5), and Ia. There was an absence of T-suppressor/cytotoxic antigen (Leu 2a), universal T-antigens (Leu 4, 9), and immunoglobulin light and heavy chains. Collectively, these findings indicate a mature T-cell lymphoma of T-helper type in an activated (Ia+) state. In contrast to previous reports of T-cell and Ia occurring solely as surface antigens, we demonstrated pools of cytoplasmic Leu 1, 3, 5 and Ia that displaced the nucleus. The ultrastructure of the giant cytoplasmic vacuoles was identical to the microvesicle-containing vacuoles reported in signet-ring cell lymphomas of B-cell lineage. In our case of T-cell lineage, we found substantial evidence of endocytosis by the neoplastic cells and numerous giant multivesicular bodies. The pools of cytoplasmic T and Ia antigens may result from abnormal internalization of surface T-antigens or the sequestration of T-antigen-containing Golgi-derived vesicles. Our combined immunologic and ultrastructural findings suggest that aberrant membrane recycling may be the common denominator of signet-ring formation in both B- and T-cell signet-ring lymphomas.

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

PubMed Central

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. Images PMID:6092921

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

PubMed

Tomás, A L; Cardoso, F; Esteves, F; Matos, O

2016-11-08

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii

PubMed Central

Tomás, A. L.; Cardoso, F.; Esteves, F.; Matos, O.

2016-01-01

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis. PMID:27824115

Sweat allergy: Extrinsic or intrinsic?

PubMed

Hiragun, Takaaki; Hiragun, Makiko; Ishii, Kaori; Kan, Takanobu; Hide, Michihiro

2017-07-01

Sweat is an exacerbation factor in atopic dermatitis (AD) in all age groups. A body core temperature elevation with sweating triggers cholinergic urticaria (CholU). We recently reported that AD symptoms are improved by tannic acid-containing spray, which suppresses the basophil histamine release induced by semi-purified sweat antigen in vitro, and by showering, which removes antigens in sweat from the skin surface. Sweat contains small amount of proteins including proteases, protease inhibitors, and anti-microbial peptides. We finally identified MGL_1304 secreted by Malassezia (M.) globosa as a major histamine - releasing antigen in human sweat. MGL_1304 is a 17-kDa protein in sweat that elicits almost the highest histamine - release activity from basophils of patients with AD and CholU among antigens derived from Malassezia species. Moreover, serum levels of anti-MGL_1304 IgE were significantly higher in patients with AD and CholU than in normal controls. The recombinant protein produced by Pichia pastoris possessed comparable allergenicity to native MGL_1304. We found a monoclonal IgE antibody against MGL_1304 which did not elicit histamine release from sensitized mast cells. Desensitization therapy using autologous sweat, or MGL_1304 purified from culture of M. globosa or its cognates might be beneficial for patients with intractable CholU due to sweat allergy. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Potency test. 660.4 Section 660.4 Food and Drugs... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen...

The Prevalence of Hepatitis B Surface Antigen and Antibody in Home-Reared Individuals with Down Syndrome.

ERIC Educational Resources Information Center

Pueschel, Siegfried M.; And Others

1991-01-01

This study found no significant difference in the prevalence of hepatitis B surface antigens or antibodies between 180 noninstitutionalized subjects (ages 1-29) with Down's syndrome and 155 subjects without Down's syndrome. This finding contrasts with previous findings with institutionalized Down's syndrome subjects. (Author/DB)

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Production and Characterization of a Monoclonal Antibody Raised Against Surface Antigens from Mycelium of Gaeumannomyces graminis var. tritici: Evidence for an Extracellular Polyphenol Oxidase.

PubMed

Thornton, C R; Dewey, F M; Gilligan, C A

1997-01-01

ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L-DOPA, and secretion by C. globosum in response to the phenolic amino acid was significantly less compared to G. graminis var. tritici.

The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

PubMed

Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

2007-02-23

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.

Human leukocyte antigen E in human cytomegalovirus infection: friend or foe?

PubMed

Gong, Fang; Song, Shengli; Lv, Guozhong; Pan, Yuhong; Zhang, Dongqing; Jiang, Hong

2012-07-01

Human cytomegalovirus (HCMV) is a well-studied β-herpesvirus virus, which adopts a variety of strategies to evade immune surveillance. It has been reported that in HCMV-infected cells, classical major histocompatibility (MHC) class I molecules are down-regulated, but the MHC class Ib molecule human leukocyte antigen (HLA)-E is normally expressed or even overexpressed on the cell surface. HLA-E has been first described to interact with CD94/NKG2 receptors expressed mainly on the surface of natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. The engagement of CD94/NKG2A with HLA-E, with a signal peptide of the HCMV glycoprotein UL40, usually induces inhibitory signals. However, HLA-E also serves as a ligand for the TCR expressed by αβCD8(+) T cells. Recognition of peptides presented by HLA-E may result in CD8(+) effector T-cell activation. These findings will help to understand more on both pathogenic and protective roles of HLA-E in HCMV infection. In this review, we discussed recent studies about the roles of HLA-E in HCMV infection.

The leukocyte common antigen (CD45): a putative receptor-linked protein tyrosine phosphatase.

PubMed Central

Charbonneau, H; Tonks, N K; Walsh, K A; Fischer, E H

1988-01-01

A major protein tyrosine phosphatase (PTPase 1B) has been isolated in essentially homogeneous form from the soluble and particulate fractions of human placenta. Unexpectedly, partial amino acid sequences displayed no homology with the primary structures of the protein Ser/Thr phosphatases deduced from cDNA clones. However, the sequence is strikingly similar to the tandem C-terminal homologous domains of the leukocyte common antigen (CD45). A 157-residue segment of PTPase 1B displayed 40% and 33% sequence identity with corresponding regions from cytoplasmic domains I and II of human CD45. Similar degrees of identity have been observed among the catalytic domains of families of regulatory proteins such as protein kinases and cyclic nucleotide phosphodiesterases. On this basis, it is proposed that the CD45 family has protein tyrosine phosphatase activity and may represent a set of cell-surface receptors involved in signal transduction. This suggests that the repertoire of signal transduction mechanisms may include the direct control of an intracellular protein tyrosine phosphatase, offering the possibility of a regulatory balance with those protein tyrosine kinases that act at the internal surface of the membrane. Images PMID:2845400

Immunological and biochemical analysis of glycosylated surface antigens and lipophosphoglycan of Tritrichomonas foetus.

PubMed

Singh, B N; BonDurant, R H; Campero, C M; Corbeil, L B

2001-08-01

Immunoaffinity-purified TF1.17 adhesin antigen was compared biochemically and antigenically to Tritrichomonas foetus (TF) lipophosphoglycan (LPG) and a soluble glycosylated antigen (SGA) released from T. foetus and implicated in pathogenesis and immunity. The monoclonal antibodies (Mabs TF1.15 and TF1.17) specific for a glycosylated TF1.17 antigen were previously shown to prevent adhesion of the T. foetus parasites to bovine vaginal epithelial cells and to mediate killing by bovine complement. SGA was isolated from T. foetus-conditioned buffer and purified by octyl-Sepharose hydrophobic column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of SGA showed a major SGA1 component (approximately 190 kDa) and a minor SGA2 component (50-70 kDa), which migrated close to TF-LPG and TF1.17. The carbohydrate and lipid compositional analyses of affinity-purified TF1.17 and SGA2 by high-performance liquid chromatography (HPLC) and gas-liquid chromatography revealed the presence of monosaccharides and fatty acids as found in TF-LPG. All antigens contained terminal fucose as determined by alpha-fucosidase digestion followed by HPLC. ELISA and western blots were used to further characterize these glycosylated antigens and to analyze their relationships. The Mabs TF1.15 and TF1.17 reacted very strongly to TF-LPG and SGA2. as well as TF1.17 antigen, indicating that these molecules share common epitopes. These Mabs did not react with the SGA1 component either in ELISA and western blot analyses. Also, the monosaccharide composition of SGA1 was very different from the other three antigen, suggesting SGA1 was different from LPG, SGA2 and TF1.17. Although LPG reacted with Mabs to native TF1.17 antigen, LPG did not induce an immune response in cattle with the same route and adjuvant used to produce strong antibody responses to the native antigen. The latter response suggests that the tightly bound peptide present in the immunoaffinity-purified antigen is necessary for induction of a response to (an) epitope(s) in TF-LPG and TF1.17. Furthermore, vaginal fluid from T. foetus-infected heifers and serum from a cow with a T. foetus-associated pyometra recognized both TF1.17 and TF-LPG in western blots. These results suggest that T. foetus LPG and SGA2 are related to TF1.17 antigen, which was previously shown to play an important role in the pathogenesis and host response in bovine trichomoniasis.

Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volarevic, S.; Burns, C.M.; Sussman, J.J.

1990-09-01

Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculationsmore » based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.« less

Canine leishmaniosis caused by Leishmania major and Leishmania tropica: comparative findings and serology.

PubMed

Baneth, Gad; Yasur-Landau, Daniel; Gilad, Matan; Nachum-Biala, Yaarit

2017-03-13

Infection and clinical disease associated with Leishmania major and Leishmania tropica, two common agents of human cutaneous leishmaniosis, have rarely been reported in dogs. This study describes dogs infected with these Leishmania spp. prevalent in the Middle East and North Africa, and compares the serological response of dogs infected with Leishmania infantum, L. major or L. tropica to whole promastigote antigen enzyme-linked immunosorbent assay (ELISA) of each species and to rK39 dipstick. Leishmania major infection in a 5-month-old male dog was associated with alopecic and ulcerative periocular and limb skin lesions which responded to allopurinol treatment. Infection was detected by skin and blood polymerase chain reaction (PCR) and confirmed by DNA sequencing but the dog was seronegative. Leishmania tropica infection was detected in a 3-month-old female dog co-infected with Babesia vogeli and Anaplasma platys and with no skin lesions. PCR and DNA sequencing of the blood and parasite culture were positive for L. tropica. Sera from 11 dogs infected with L. infantum, L. major or L. tropica were reactive with all three Leishmania spp. antigens except for sera from a dog with L. major infection. No significant differences were found between reactivity of dog sera to the antigen of the infecting species, or to the other Leishmania spp. antigens. Sera from dogs infected with L. infantum and L. tropica were positive with the rK39 antigen kit, while dogs with L. major infection were seronegative. Skin lesions in L. major infected dogs from this study and previous reports (n = 2) were ulcerative and located on the muzzle, feet and foot pads and not associated with generalized lymphadenomegaly and splenomegaly. In previous L. tropica infections, skin lesions were proliferative mucocutaneous in young dogs (n = 2), or associated with widespread dermatitis, lymphadenomegaly and splenomegaly in older dogs with similarity to L. infantum infection (n = 2). This study suggests that ELISA serology with whole promastigote antigen is not distinctive between L. infantum, L. major and L. tropica canine infections and that some L. major infections are not seropositive. PCR with DNA sequencing should be used to discriminate between canine infections with these three species.

Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

PubMed

Dorrell, Craig; Abraham, Stephanie L; Lanxon-Cookson, Kelsea M; Canaday, Pamela S; Streeter, Philip R; Grompe, Markus

2008-09-01

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

Impairment of Immunoproteasome Function by Cigarette Smoke and in Chronic Obstructive Pulmonary Disease.

PubMed

Kammerl, Ilona E; Dann, Angela; Mossina, Alessandra; Brech, Dorothee; Lukas, Christina; Vosyka, Oliver; Nathan, Petra; Conlon, Thomas M; Wagner, Darcy E; Overkleeft, Hermen S; Prasse, Antje; Rosas, Ivan O; Straub, Tobias; Krauss-Etschmann, Susanne; Königshoff, Melanie; Preissler, Gerhard; Winter, Hauke; Lindner, Michael; Hatz, Rudolf; Behr, Jürgen; Heinzelmann, Katharina; Yildirim, Ali Ö; Noessner, Elfriede; Eickelberg, Oliver; Meiners, Silke

2016-06-01

Patients with chronic obstructive pulmonary disease (COPD) and in particular smokers are more susceptible to respiratory infections contributing to acute exacerbations of disease. The immunoproteasome is a specialized type of proteasome destined to improve major histocompatibility complex (MHC) class I-mediated antigen presentation for the resolution of intracellular infections. To characterize immunoproteasome function in COPD and its regulation by cigarette smoke. Immunoproteasome expression and activity were determined in bronchoalveolar lavage (BAL) and lungs of human donors and patients with COPD or idiopathic pulmonary fibrosis (IPF), as well as in cigarette smoke-exposed mice. Smoke-mediated alterations of immunoproteasome activity and MHC I surface expression were analyzed in human blood-derived macrophages. Immunoproteasome-specific MHC I antigen presentation was evaluated in spleen and lung immune cells that had been smoke-exposed in vitro or in vivo. Immunoproteasome and MHC I mRNA expression was reduced in BAL cells of patients with COPD and in isolated alveolar macrophages of patients with COPD or IPF. Exposure of immune cells to cigarette smoke extract in vitro reduced immunoproteasome activity and impaired immunoproteasome-specific MHC I antigen presentation. In vivo, acute cigarette smoke exposure dynamically regulated immunoproteasome function and MHC I antigen presentation in mouse BAL cells. End-stage COPD lungs showed markedly impaired immunoproteasome activities. We here show that the activity of the immunoproteasome is impaired by cigarette smoke resulting in reduced MHC I antigen presentation. Regulation of immunoproteasome function by cigarette smoke may thus alter adaptive immune responses and add to prolonged infections and exacerbations in COPD and IPF.

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

PubMed

Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

2013-10-01

Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells

PubMed Central

Oka, Tatsuya; Rios, Eon J.; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J.

2013-01-01

Background Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. Objectives We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. Methods C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti–2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl–human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Results Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Conclusions Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. PMID:23810240

Controlled Release of Antigens for One Dose Immunization

DTIC Science & Technology

1983-01-01

microencapsulation of antigen coated alum or by microencapsulating clusters of smaller (᝺ microns) microcapsules . Microcapsules under 10 microns in... microencapsulation were studied to determine what criteria must be satisfied to provide a protective immune response to hepatitis B surface antigen... microencapsulated in poly (DL-lactide-co- glycolide) in a form that was too large to be phagocytized and had an antigen release profile similar to that achieved with

Blood Type Biochemistry and Human Disease

PubMed Central

Ewald, D Rose; Sumner, Susan CJ

2016-01-01

Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determinants and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. PMID:27599872

Leukemia-associated antigens in man.

PubMed

Brown, G; Capellaro, D; Greaves, M

1975-12-01

Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

77 FR 9663 - Agency Information Collection Activities; Proposed Collection; Comment Request; Request for...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-17

... specific licensed biological products: Sec. Sec. 660.6 (21 CFR 660.6) (Antibody to Hepatitis B Surface Antigen); 660.36 (21 CFR 660.36) (Reagent Red Blood Cells); and 660.46 (21 CFR 660.46) (Hepatitis B... each lot of Antibody to Hepatitis B Surface Antigen product, and Sec. 660.6(b) provides the...

Hepatitis B surface antigen and polymerized albumin binding activity in sheep serum.

PubMed Central

Franklin, S G; Millman, I; Blumberg, B S

1984-01-01

Sera from sheep and other domestic animals contain a substance that gives a strongly positive test for antibody to hepatitis B virus surface antigen by the accepted radioimmunoassay procedure. We have purified this substance from sheep serum to near homogeneity by ion-exchange, affinity, and molecular exclusion chromatography and have identified it to be an IgM. We present evidence that this sheep IgM is an antibody to polymerized sheep albumin. This antibody may arise due to infection by hepatitis B virus, hepatitis B virus-like viruses, or other pathological agents and may react with hepatitis B virus surface antigen by combining with polymerized albumin bound to the hepatitis B virus receptor for this polymer. Images PMID:6582511

Surface antigen in early differentiation.

PubMed Central

Kemler, R; Babinet, C; Eisen, H; Jacob, F

1977-01-01

Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A. Images PMID:270688

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence.

PubMed

Nakano, Kazuhiko; Nomura, Ryota; Matsumoto, Michiyo; Ooshima, Takashi

2010-01-01

Streptococcus mutans is generally known as a pathogen of dental caries, and it is also considered to cause bacteremia and infective endocarditis (IE). S. mutans was previously classified into 3 serotypes, c, e, and f, due to the different chemical compositions of the serotype-specific polysaccharides, which are composed of a rhamnose backbone and glucose side chains. We recently designated non-c/e/f serotype S. mutans strains as novel serotype k, which is characterized by a drastic reduction in the amount of the glucose side chain. A common biological feature of novel serotype-k strains is a lower level of cariogenicity due to alterations of several major cell surface protein antigens. As for virulence in blood, these strains survive in blood for a longer duration due to lower antigenicity, while the detection rate of all strains carrying the gene encoding collagen-binding adhesin has been shown to be high. Furthermore, molecular biological analyses of infected heart valve specimens obtained from IE patients revealed a high detection rate of serotype-k S. mutans. Together, these findings suggest that serotype-k S. mutans strains show low cariogenicity but high virulence in blood as compared to the other serotypes, due to alterations of several cell surface structures.

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Epileptic syndrome in systemic lupus erythematosus and neuronal autoantibody associations.

PubMed

Kampylafka, E I; Alexopoulos, H; Fouka, P; Moutsopoulos, H M; Dalakas, M C; Tzioufas, A G

2016-10-01

We investigated systemic lupus erythematosus (SLE) patients with epilepsy, a major and organic neurological symptom. Our aim was to test patients for the autoimmune epilepsy-associated antibodies anti-GAD, anti-NMDAR, anti-AMPAR1/2, anti-GABABR and anti-VGKC. We tested sera from ten SLE patients with current or previous episodes of epileptic seizures. In addition, sera were tested for staining on primary hippocampal neurons. The patients' clinical and neuroimaging profile, disease activity and accumulated damage scores and therapeutic regimens administered were recorded, and correlations were evaluated. Patients were negative for all anti-neuronal autoantibodies tested, and showed no staining on primary hippocampal cells, which suggests the absence of autoantibodies against neuronal cell surface antigens. Epileptic seizures were all tonic-clonic, and all patients had high disease activity (mean SLE Damage Acticity Index score 19.3 ± 7.3). Six patients had minor or no brain magnetic resonance imaging findings, and three had major findings. 9/10 patients received immunosuppression for 5 ± 4 months, while anti-convulsive treatment was administered to all patients (4.2 ± 3 years). Our results suggest that the majority of SLE-related epileptic seizures cannot be attributed to the action of a single antibody against neuronal antigens. Studies with larger neuropsychiatric SLE populations and stricter inclusion criteria are necessary to verify these findings. © The Author(s) 2016.

Functional Macroautophagy Induction by Influenza A Virus without a Contribution to Major Histocompatibility Complex Class II-Restricted Presentation▿†

PubMed Central

Comber, Joseph D.; Robinson, Tara M.; Siciliano, Nicholas A.; Snook, Adam E.; Eisenlohr, Laurence C.

2011-01-01

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigen-processing pathways have been reported, little is known about their relative contributions to global CD4+ T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4+ T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4+ response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen. PMID:21525345

Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients.

PubMed

Hiragun, Takaaki; Ishii, Kaori; Hiragun, Makiko; Suzuki, Hidenori; Kan, Takanobu; Mihara, Shoji; Yanase, Yuhki; Bartels, Joachim; Schröder, Jens-M; Hide, Michihiro

2013-09-01

Sweat is a major aggravating factor of atopic dermatitis (AD) and approximately 80% of patients with AD show type I hypersensitivity against sweat. To identify and characterize an antigen in sweat that induces histamine release from basophils of patients with AD. Basophil histamine-releasing activity in sweat was purified by a combination of chromatographies, and proteins were analyzed with mass spectrometry. Recombinant proteins of the sweat antigen were generated, and their biological characteristics were studied by immunoblots, histamine release tests, and neutralization assays. We identified a fungal protein, MGL_1304, derived from Malassezia globosa (M globosa) in the purified sweat antigen. Recombinant MGL_1304 induced histamine release from basophils of most of the patients with AD, in accordance with the semi-purified sweat antigen. Moreover, recombinant MGL_1304 abolished the binding of serum IgE of patients with AD to the semi-purified sweat antigen, or vice versa in immunoblot analysis, and attenuated the sensitization of RBL-48 mast cells expressing human FcɛRI by serum IgE. Studies of truncated mutants of MGL_1304 indicated that IgE of patients with AD recognized the conformational structure of MGL_1304 rather than short peptide sequences. Western blot analysis of the whole lysate, the culture supernatant of M globosa, and the semi-purified sweat antigen showed that MGL_1304 was produced as a minor immunological antigen of M globosa with posttranslational modification, cleaved, and secreted as a 17-kDa major histamine-releasing sweat antigen. MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Kinetics of antigen binding to arrays of antibodies in different sized spots

NASA Technical Reports Server (NTRS)

Sapsford, K. E.; Liron, Z.; Shubin, Y. S.; Ligler, F. S.

2001-01-01

A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 x 10(5) M(-1) s(-1). Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 microm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.

Ocular surface epithelium induces expression of human mucosal lymphocyte antigen (HML-1) on peripheral blood lymphocytes

PubMed Central

Gomes, J A P; Dua, H S; Rizzo, L V; Nishi, M; Joseph, A; Donoso, L A

2004-01-01

Background/aims: Peripheral blood CD8+ lymphocytes that home to mucosal surfaces express the human mucosal lymphocyte antigen (HML-1). At mucosal surfaces, including the ocular surface, only intraepithelial CD8+ lymphocytes express HML-1. These lymphocytes are retained in the intraepithelial compartment by virtue of the interaction between HML-1 and its natural ligand, E-cadherin, which is expressed on epithelial cells. The purpose of this study was to determine whether ocular surface epithelial cells (ocular mucosa) could induce the expression of human mucosal lymphocyte antigen on peripheral blood lymphocytes. Methods: Human corneal and conjunctival epithelial cells were co-cultured with peripheral blood lymphocytes. Both non-activated and activated lymphocytes were used in the experiments. After 7 days of incubation, lymphocytes were recovered and analysed for the antigens CD8/HML-1, CD4/HML-1, CD3/CD8, CD3/CD4, CD3/CD25, CD8/CD25, and CD4/CD25 by flowcytometry. Results: Significant statistical differences were observed in the CD8/HML-1 expression when conjunctival epithelial cells were co-cultured with non-activated and activated lymphocytes (p = 0.04 for each) and when corneal epithelial cells were co-cultured with non-activated lymphocytes (p = 0.03). Significant statistical difference in CD4/HML-1 expression was observed only when conjunctival epithelial cells were co-cultured with activated lymphocytes (p = 0.02). Conclusion: Ocular surface epithelial cells can induce the expression of human mucosal lymphocyte antigen on CD8+ (and to some extent on CD4+) lymphocytes. This may allow the retention of CD8+ and CD4+ lymphocytes within the epithelial compartment of the conjunctiva and play a part in mucosal homing of lymphocytes. PMID:14736792

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

PubMed

Xia, Z; Goldsmith, H L; van de Ven, T G

1994-04-01

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.

Direct electrical control of IgG conformation and functional activity at surfaces

NASA Astrophysics Data System (ADS)

Ghisellini, Paola; Caiazzo, Marialuisa; Alessandrini, Andrea; Eggenhöffner, Roberto; Vassalli, Massimo; Facci, Paolo

2016-11-01

We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with Fab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically Fab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs’ charged aminoacids modulating the accessibility of the specific recognition regions of Fab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.

Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin.

PubMed

Lu, Pengfei; Chen, Jijun; He, Lixiazi; Ren, Jiangtao; Chen, Haide; Rao, Lingjun; Zhuang, Qinggang; Li, Hui; Li, Lei; Bao, Lei; He, Ji; Zhang, Wei; Zhu, Faming; Cui, Chun; Xiao, Lei

2013-12-01

Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (β2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of β2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.

Targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites.

PubMed

Rigano, M Manuela; Sala, Francesco; Arntzen, Charles J; Walmsley, Amanda M

2003-01-30

Most pathogenic microorganisms enter their host via the mucosal surfaces lining the digestive, respiratory and urino-reproductive tracts of the body. The most efficient means of protecting these surfaces is through mucosal immunization. Transgenic plants are safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with targeting proteins, such as the B subunit of the Escherichia coli heat labile enterotoxin (LTB), could increase the effectiveness of such antigens.

Genetic diversity of pneumococcal surface protein A in invasive pneumococcal isolates from Korean children, 1991-2016.

PubMed

Yun, Ki Wook; Choi, Eun Hwa; Lee, Hoan Jong

2017-01-01

Pneumococcal surface protein A (PspA) is an important virulence factor of pneumococci and has been investigated as a primary component of a capsular serotype-independent pneumococcal vaccine. Thus, we sought to determine the genetic diversity of PspA to explore its potential as a vaccine candidate. Among the 190 invasive pneumococcal isolates collected from Korean children between 1991 and 2016, two (1.1%) isolates were found to have no pspA by multiple polymerase chain reactions. The full length pspA genes from 185 pneumococcal isolates were sequenced. The length of pspA varied, ranging from 1,719 to 2,301 base pairs with 55.7-100% nucleotide identity. Based on the sequences of the clade-defining regions, 68.7% and 49.7% were in PspA family 2 and clade 3/family 2, respectively. PspA clade types were correlated with genotypes using multilocus sequence typing and divided into several subclades based on diversity analysis of the N-terminal α-helical regions, which showed nucleotide sequence identities of 45.7-100% and amino acid sequence identities of 23.1-100%. Putative antigenicity plots were also diverse among individual clades and subclades. The differences in antigenicity patterns were concentrated within the N-terminal 120 amino acids. In conclusion, the N-terminal α-helical domain, which is known to be the major immunogenic portion of PspA, is genetically variable and should be further evaluated for antigenic differences and cross-reactivity between various PspA types from pneumococcal isolates.

Novel Protective Antigens Expressed by Trypanosoma cruzi Amastigotes Provide Immunity to Mice Highly Susceptible to Chagas' Disease▿

PubMed Central

Silveira, Eduardo L. V.; Claser, Carla; Haolla, Filipe A. B.; Zanella, Luiz G.; Rodrigues, Mauricio M.

2008-01-01

Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

Novel protective antigens expressed by Trypanosoma cruzi amastigotes provide immunity to mice highly susceptible to Chagas' disease.

PubMed

Silveira, Eduardo L V; Claser, Carla; Haolla, Filipe A B; Zanella, Luiz G; Rodrigues, Mauricio M

2008-08-01

Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium*

PubMed Central

Kodali, Srinivas; Vinogradov, Evgeny; Lin, Fiona; Khoury, Nancy; Hao, Li; Pavliak, Vilo; Jones, C. Hal; Laverde, Diana; Huebner, Johannes; Jansen, Kathrin U.; Anderson, Annaliesa S.; Donald, Robert G. K.

2015-01-01

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection. PMID:26109072

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

PubMed Central

1978-01-01

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis. PMID:102651

The T cell antigen receptor: the Swiss army knife of the immune system

PubMed Central

Attaf, M; Legut, M; Cole, D K; Sewell, A K

2015-01-01

The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These ‘unconventional’ T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors. PMID:25753381

Determination by enzyme-linked immunosorbent assay of immunoglobulin G (IgG), IgM, and IgA to Brucella melitensis major outer membrane proteins and whole-cell heat-killed antigens in sera of patients with brucellosis.

PubMed Central

Araj, G F; Kaufmann, A F

1989-01-01

An enzyme-linked immunosorbent assay was used to compare Brucella melitensis major outer membrane proteins (MOMP) and whole-cell heat-killed antigens (HK) in measuring antibrucella immunoglobulin G (IgG), IgM, and IgA in sera of brucellosis patients and controls. Antibodies to MOMP were generally similar to those against HK, and the correlation coefficients between the two antigens and IgG, IgM, and IgA in patients varied between 0.73 and 0.94. Both antigens are comparably suitable in detecting antibrucella immunoglobulin isotypes for the serologic diagnosis of patients with brucellosis, with high (greater than or equal to 95%) sensitivity and specificity. PMID:2768476

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores.

PubMed

Hinc, Krzysztof; Isticato, Rachele; Dembek, Marcin; Karczewska, Joanna; Iwanicki, Adam; Peszyńska-Sularz, Grazyna; De Felice, Maurilio; Obuchowski, Michał; Ricca, Ezio

2010-01-18

The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections. We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed. UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

Blood Group Antigens on HeLa Cells shown by Mixed Agglutination

PubMed Central

Kelus, A.; Gurner, B. W.; Coombs, R. R. A.

1959-01-01

The mixed agglutination reaction has been used for investigating the presence of blood group antigens on the surface of human cervical carcinoma cells (HeLa) cultured for eight years in vitro. The H antigen was demonstrated in the absence of A and B. The MN-type antigen has been found as well as Tja. Treatment of HeLa cells with ficin greatly enhanced the reaction of anti-H and anti-Tja with the corresponding antigens on HeLa cells. The authors failed to show the following antigens: Rh(D) and Rh(c), S, P, Lea, Leb, Lua and Lub. ImagesFIG. 1FIG. 2 PMID:14405338

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

PubMed Central

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

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

PubMed

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.

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

PubMed

Karch, Christopher P; Doll, Tais A P F; Paulillo, Sara M; Nebie, Issa; Lanar, David E; Corradin, Giampietro; Burkhard, Peter

2017-09-06

The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response

PubMed Central

Bashir, Mohamed Elfatih H.; Lui, Jan Hsi; Palnivelu, Ravishankar; Naclerio, Robert M.; Preuss, Daphne

2013-01-01

Background/Aim Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins. Methodology/Principal Findings We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture. Conclusions/Significance Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules that have stimulatory or regulatory effects on immune responses. PMID:23469025

Genetically encoded pH sensor for tracking surface proteins through endocytosis.

PubMed

Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D; Watkins, Simon C; Waggoner, Alan S; Bruchez, Marcel P

2012-05-14

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biotin avidin amplified magnetic immunoassay for hepatitis B surface antigen detection using GoldMag nanoparticles

NASA Astrophysics Data System (ADS)

Yu, An; Geng, Tingting; Fu, Qiang; Chen, Chao; Cui, Yali

2007-04-01

Using GoldMag (Fe3O4/Au) nanoparticles as a carrier, a biotin-avidin amplified ELISA was developed to detect hepatitis B surface antigen (HBsAg). A specific antibody was labeled with biotin and then used to detect the antigen with an antibody coated on GoldMag nanoparticles by a sandwich ELISA assay. The results showed that 5 mol of biotin were surface bound per mole of antibody. The biotin-avidin amplified ELISA assay has a higher sensitivity than that of the direct ELISA assay. There is 5-fold difference between HBsAg positive and negative serum even at dilution of 1:10000, and the relative standard deviation of the parallel positive serum at dilution of 1:4000 is 5.98% (n=11).

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.

PubMed

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.

Mice completely lacking immunoproteasomes display major alterations in antigen presentation

PubMed Central

Kincaid, Eleanor Z; Che, Jenny W; York, Ian; Escobar, Hernando; Reyes-Vargas, Eduardo; Delgado, Julio C.; Welsh, Raymond M; Karow, Margaret L.; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rock, Kenneth L

2011-01-01

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought. PMID:22197977

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

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.

Acanthocheilonema viteae: Vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lucius, R.; Textor, G.; Kern, A.

1991-08-01

Jirds (Meriones unguiculatus) were immunized with irradiated (35 krad) stage-3 larvae (L3) of Acanthocheilonema viteae. The induced resistance against homologous challenge infection and the antibody response of the animals were studied. Immunization with 3, 2, or 1 dose of 50 irradiated L3 induced approximately 90% resistance. Immunization with a single dose of only 5 irradiated L3 resulted in 60.8% protection while immunization with a single dose of 25 L3 induced 94.1% protection. The protection induced with 3 doses of 50 irradiated L3 did not decrease significantly during a period of 6 months. Sera of a proportion, but not all resistantmore » jirds, contained antibodies against the surface of vector derived L3 as defined by IFAT. No surface antigens of microfilariae or adult worms were recognized by the sera. Vaccinated animals had antibody responses against antigens in the inner organs of L3 and in the cuticle and reproductive organs of adult worms as shown by IFAT. Immunoblotting with SDS-PAGE-separated L3 antigens and L3-CSN revealed that all sera contained antibodies against two exported antigens of 205 and 68 kDa, and against a nonexported antigen of 18 kDa. The 205-kDa antigen easily degraded into fragments of 165, 140, 125, and 105 kDa which were recognized by resistant jird sera. Various antigens of adult worms, but relatively few antigens of microfilariae, were also recognized. To test the relevance of exported antigens of L3 to resistance, jirds were immunized with L3-CSN together with a mild adjuvant. This immunization induced 67.7% resistance against challenge infection and sera of the immunized animals recognized the 205- and 68-kDa antigens of L3.« less

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

PubMed

Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery.

PubMed

Paliwal, Shivani Rai; Paliwal, Rishi; Vyas, Suresh P

2015-05-01

The pH-sensitive liposomes have been extensively used as an alternative to conventional liposomes in effective intracellular delivery of therapeutics/antigen/DNA/diagnostics to various compartments of the target cell. Such liposomes are destabilized under acidic conditions of the endocytotic pathway as they usually contain pH-sensitive lipid components. Therefore, the encapsulated content is delivered into the intracellular bio-environment through destabilization or its fusion with the endosomal membrane. The therapeutic efficacy of pH-sensitive liposomes enables them as biomaterial with commercial utility especially in cancer treatment. In addition, targeting ligands including antibodies can be anchored on the surface of pH-sensitive liposomes to target specific cell surface receptors/antigen present on tumor cells. These vesicles have also been widely explored for antigen delivery and serve as immunological adjuvant to enhance the immune response to antigens. The present review deals with recent research updates on application of pH-sensitive liposomes in chemotherapy/diagnostics/antigen/gene delivery etc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi

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 themore » 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.« less

Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens.

PubMed

Sakhatskyy, Pavlo; Wang, Shixia; Zhang, Chuanyou; Chou, Te-Hui; Kishko, Michael; Lu, Shan

2008-02-05

The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

USDA-ARS?s Scientific Manuscript database

Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8positive T-cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presenta...

Identification of Streptococcus mitis321A vaccine antigens based on reverse vaccinology

PubMed Central

Zhang, Qiao; Lin, Kexiong; Wang, Changzheng; Xu, Zhi; Yang, Li; Ma, Qianli

2018-01-01

Streptococcus mitis (S. mitis) may transform into highly pathogenic bacteria. The aim of the present study was to identify potential antigen targets for designing an effective vaccine against the pathogenic S. mitis321A. The genome of S. mitis321A was sequenced using an Illumina Hiseq2000 instrument. Subsequently, Glimmer 3.02 and Tandem Repeat Finder (TRF) 4.04 were used to predict genes and tandem repeats, respectively, with DNA sequence function analysis using the Basic Local Alignment Search Tool (BLAST) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Groups of proteins (COG) databases. Putative gene antigen candidates were screened with BLAST ahead of phylogenetic tree analysis. The DNA sequence assembly size was 2,110,680 bp with 40.12% GC, 6 scaffolds and 9 contig. Consequently, 1,944 genes were predicted, and 119 TRF, 56 microsatellite DNA, 10 minisatellite DNA and 154 transposons were acquired. The predicted genes were associated with various pathways and functions concerning membrane transport and energy metabolism. Multiple putative genes encoding surface proteins, secreted proteins and virulence factors, as well as essential genes were determined. The majority of essential genes belonged to a phylogenetic lineage, while 321AGL000129 and 321AGL000299 were on the same branch. The current study provided useful information regarding the biological function of the S. mitis321A genome and recommends putative antigen candidates for developing a potent vaccine against S. mitis. PMID:29620181

Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells.

PubMed

Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R

2009-07-22

The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

PubMed Central

Kleiveland, Charlotte R.; Minic, Rajna; Moen, Lars F.; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Eijsink, Vincent G. H.

2016-01-01

ABSTRACT Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. PMID:27815271

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

PubMed

Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

2017-01-15

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. Copyright © 2016 American Society for Microbiology.

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

PubMed

Velay, A; Jeulin, H; Eschlimann, M; Malvé, B; Goehringer, F; Bensenane, M; Frippiat, J-P; Abraham, P; Ismail, A M; Murray, J M; Combet, C; Zoulim, F; Bronowicki, J-P; Schvoerer, E

2016-05-01

For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence. © 2016 John Wiley & Sons Ltd.

Immunizations with chimeric hepatitis B virus-like particles to induce potential anti-hepatitis C virus neutralizing antibodies.

PubMed

Vietheer, Patricia T K; Boo, Irene; Drummer, Heidi E; Netter, Hans-Jürgen

2007-01-01

Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses. The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained. A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

PubMed

Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

2008-09-01

A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

The Role of Dendritic Cell Maturation in the Induction of Insulin-Dependent Diabetes Mellitus.

PubMed

Mbongue, Jacques C; Nieves, Hector A; Torrez, Timothy W; Langridge, William H R

2017-01-01

Dendritic cells (DCs) are the dominant class of antigen-presenting cells in humans and are largely responsible for the initiation and guidance of innate and adaptive immune responses involved in maintenance of immunological homeostasis. Immature dendritic cells (iDCs) phagocytize pathogens and toxic proteins and in endosomal vesicles degrade them into small fragments for presentation on major histocompatibility complex (MHC) II receptor molecules to naïve cognate T cells (Th0). In addition to their role in stimulation of immunity, DCs are involved in the induction and maintenance of immune tolerance toward self-antigens. During activation, the iDCs become mature. Maturation begins when the DCs cease taking up antigens and begin to migrate from their location in peripheral tissues to adjacent lymph nodes or the spleen where during their continued maturation the DCs present stored antigens on surface MHCII receptor molecules to naive Th0 cells. During antigen presentation, the DCs upregulate the biosynthesis of costimulatory receptor molecules CD86, CD80, CD83, and CD40 on their plasma membrane. These activated DC receptor molecules bind cognate CD28 receptors presented on the Th0 cell membrane, which triggers DC secretion of IL-12 or IL-10 cytokines resulting in T cell differentiation into pro- or anti-inflammatory T cell subsets. Although basic concepts involved in the process of iDC activation and guidance of Th0 cell differentiation have been previously documented, they are poorly defined. In this review, we detail what is known about the process of DC maturation and its role in the induction of insulin-dependent diabetes mellitus autoimmunity.

In Utero Exposure to Exosomal and B-Cell Alloantigens Lessens Alloreactivity of Recipients' Lymphocytes Rather than Confers Allograft Tolerance.

PubMed

Chen, Jeng-Chang; Ou, Liang-Shiou; Chan, Cheng-Chi; Kuo, Ming-Ling; Tseng, Li-Yun; Chang, Hsueh-Ling

2018-01-01

According to actively acquired tolerance, antigen exposure before full immune development in fetal or early neonatal life will cause tolerance to this specific antigen. In this study, we aimed to examine whether allogeneic tolerance could be elicited by in utero exposure to surface MHC antigens of allogenic cells or soluble form of MHC exosomes. Gestational day 14 FVB/N fetuses were subjected to intraperitoneal injection of allogeneic major histocompatibility complex (MHC) exosomes or highly enriched B-cells. Postnatally, the recipients were examined for the immune responses to donor alloantigens by lymphocyte proliferative reactions and skin transplantation. In utero exposure to allogeneic MHC exosomes abolished the alloreactivity of recipients' lymphocytes to the alloantigens, but could not confer skin allograft tolerance. In utero transplantation of highly enriched allogeneic B-cells generated low-level B-cell chimerism in the recipients. However, it only extended the survivals of skin allograft by a few days despite the lack of donor-specific alloreactivity of recipients' lymphocyte. Thus, an early in utero contact with exosomal or B-cell alloantigens did not lead to full skin tolerance but rather, at best, only to delayed skin rejection in the presence of microchimerism made by B-cell inocula. These results argued against the theory of actively acquired tolerance, and implicated that in utero exposure to marrow cells in previous studies was a unique model of allo-tolerance induction that involved the establishment of significant hematopoietic chimerism. Taken together with the discovery of in utero sensitization to ovalbumin in our previous studies, the immunological consequences of fetal exposure to foreign antigens might vary according to the type or nature of antigens introduced.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

PubMed

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

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

PubMed

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.

Prevalence of Hepatitis B Surface Antigen in US-Born and Foreign-Born Asian/Pacific Islander College Students

ERIC Educational Resources Information Center

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:…

77 FR 41408 - Agency Information Collection Activities; Submission for Office of Management and Budget Review...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... protocols for specific licensed biological products: Sec. Sec. 660.6 (21 CFR 660.6) (Antibody to Hepatitis B Surface Antigen); 660.36 (21 CFR 660.36) (Reagent Red Blood Cells); and 660.46 (21 CFR 660.46) (Hepatitis... samples from each lot of Antibody to Hepatitis B Surface Antigen product, and Sec. 660.6(b) provides the...

SPR platform based on image acquisition for HER2 antigen detection

NASA Astrophysics Data System (ADS)

Monteiro, Johny P.; Predabon, Sheila M.; Bonafé, Elton G.; Martins, Alessandro F.; Brolo, Alexandre G.; Radovanovic, Eduardo; Girotto, Emerson M.

2017-01-01

HER2 antigen is a marker used for breast cancer diagnosis and prevention. Its determination has great importance since breast cancer is one of the most insidious types of cancer in women. HER2 antigen assessment in human serum is traditionally achieved by enzyme-linked immunosorbent assay (ELISA method), but it has some disadvantages, such as suppressing the thermodynamic-kinetic studies regarding the antibody-antigen interaction, and the use of labeled molecules that can promote false positive responses. Biosensors based on surface plasmon resonance (SPR) are sensitive optical techniques widely applied on bioassays. The plasmonic devices do not operate with labeled molecules, overcoming conventional immunoassay limitations, and enabling a direct detection of target analytes. In this way, a new SPR biosensor to assess HER2 antigen has been proposed, using nanohole arrays on a gold thin film by signal transduction of transmitted light measurements from array image acquisitions. These metallic nanostructures may couple the light directly on surface plasmons using a simple collinear arrangement. The proposed device reached an average sensitivity for refractive index (RI) variation on a metal surface of 4146 intensity units/RIU (RIU = RI units). The device feasibility on biomolecular assessment was evaluated. For this, 3 ng ml-1 known HER2 antigen concentration was efficiently flowed (using a microfluidic system) and detected from aqueous solutions. This outcome shows that the device may be a powerful apparatus for bioassays, particularly toward breast cancer diagnosis and prognosis.

Characterization of Flagella Produced by Clinical Strains of Stenotrophomonas maltophilia

PubMed Central

de Oliveira-Garcia, Doroti; Dall'Agnol, Monique; Rosales, Mónica; Azzuz, Ana C.G.S.; Martinez, Marina B.; Girón, Jorge A.

2002-01-01

Stenotrophomonas maltophilia is an emerging nosocomial pathogen associated with opportunistic infections in patients with cystic fibrosis, cancer, and HIV. Adherence of this organism to abiotic surfaces such as medical implants and catheters represents a major risk for hospitalized patients. The adhesive surface factors involved in adherence of these bacteria are largely unknown, and their flagella have not yet been characterized biochemically and antigenically. We purified and characterized the flagella produced by S. maltophilia clinical strains. The flagella filaments are composed of a 38-kDa subunit, SMFliC, and analysis of its N-terminal amino acid sequence showed considerable sequence identity to the flagellins of Serratia marcescens (78.6%), Escherichia coli, Proteus mirabilis, Shigella sonnei (71.4%), and Pseudomonas aeruginosa (57.2%). Ultrastructural analysis by scanning electron microscopy of bacteria adhering to plastic showed flagellalike structures within the bacterial clusters, suggesting that flagella are produced as the bacteria spread on the abiotic surface. PMID:12194767

Surface-functionalized, pH-responsive poly(lactic-co-glycolic acid)-based microparticles for intranasal vaccine delivery: Effect of surface modification with chitosan and mannan.

PubMed

Li, Ze; Xiong, Fangfang; He, Jintian; Dai, Xiaojing; Wang, Gaizhen

2016-12-01

In the present study, surface-functionalized, pH-responsive poly(lactic-co-glycolic acid) (PLGA) microparticles were investigated for nasal delivery of hepatitis B surface Antigen (HBsAg). pH-responsive PLGA, chitosan modified PLGA (CS-PLGA), mannan modified PLGA (MN-PLGA), mannan and chitosan co-modified PLGA (MN-CS-PLGA) microparticles were prepared utilizing a double-emulsion method. Antigen was released rapidly from four types of microparticles at pH5.0 and pH 6.0, but slowly released at pH 7.4. Mannan and chitosan surface modification enhanced intracellular microparticle uptake by macrophages. Following intracellular macrophage antigen uptake, antigen release occurred in three different patterns: fast release from PLGA and MN-PLGA microparticles in endosomes/lysosomes, slow release from CS-PLGA microparticles in cytoplasm and a combination of fast release and slow release patterns from MN-CS-PLGA microparticles. Furthermore, chitosan coating modification increased the residence time of CS-PLGA and MN-CS-PLGA microparticles in the nasal cavity. In vivo immunogenicity studies indicated that MN-CS-PLGA microparticles induced stronger humoral and cell-mediated immune responses compared with PLGA, MN-PLGA and CS-PLGA microparticles. These results suggest that surface modification of pH-responsive PLGA microparticles with mannan and chitosan is a promising tool for nasal delivery of HBsAg. Copyright © 2016. Published by Elsevier B.V.

Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells.

PubMed

Ou-Yang, P; Chiang, B L; Hwang, L H; Chen, Y G; Yang, P M; Chi, W K; Chen, P J; Chen, D S

1999-04-01

The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

PubMed Central

Peyret, Hadrien; Gehin, Annick; Thuenemann, Eva C.; Blond, Donatienne; El Turabi, Aadil; Beales, Lucy; Clarke, Dean; Gilbert, Robert J. C.; Fry, Elizabeth E.; Stuart, David I.; Holmes, Kris; Stonehouse, Nicola J.; Whelan, Mike; Rosenberg, William; Lomonossoff, George P.; Rowlands, David J.

2015-01-01

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. PMID:25830365

Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon.

PubMed

Gachara, George; Magoro, Tshifhiwa; Mavhandu, Lufuno; Lum, Emmaculate; Kimbi, Helen K; Ndip, Roland N; Bessong, Pascal O

2017-03-08

Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI.

Sialidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype.

PubMed

Walz, Lisa; Kays, Sarah-Katharina; Zimmer, Gert; von Messling, Veronika

2018-06-20

Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the level of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of a subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strain and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with similar efficacy as the matched HA protein, illustrating the potential of the NA protein as vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive sialidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype. IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen not only conferred protection of mice and ferrets to the matched influenza strains, but also against viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype. Copyright © 2018 American Society for Microbiology.

Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects.

PubMed

Di, Shengmeng; Li, Zonghai

2016-04-01

Chimeric antigen receptors (CARs) are artificial recombinant receptors that generally combine the antigen-recognition domain of a monoclonal antibody with T cell activation domains. Recent years have seen great success in clinical trials employing CD19-specific CAR-T cell therapy for B cell leukemia. Nevertheless, solid tumors remain a major challenge for CAR-T cell therapy. This review summarizes the preclinical and clinical studies on the treatment of solid tumors with CAR-T cells. The major hurdles for the success of CAR-T and the novel strategies to address these hurdles have also been described and discussed.

Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli

NASA Astrophysics Data System (ADS)

Küpper, Hans; Keller, Walter; Kurz, Christina; Forss, Sonja; Schaller, Heinz

1981-02-01

Double-stranded DNA copies of the single-stranded genomic RNA of foot and mouth disease virus have been cloned into the Escherichia coli plasmid pBR322. A restriction map of the viral genome was established and aligned with the biochemical map of foot and mouth disease virus. The coding sequence for structural protein VP1, the major antigen of the virus, was identified and inserted into a plasmid vector where the expression of this sequence is under control of the phage λ PL promoter. In an appropriate host the synthesis of antigenic polypeptide can be demonstrated by radioimmunoassay.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-06-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method.

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

PubMed Central

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-01-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method. PMID:28761820

Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

DOE PAGES

Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

2014-11-07

The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the converved aspartate, whichmore » coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.« less

Prevalence of antibodies to Sarcocystis neurona and Neospora hughesi in horses from Mexico

PubMed Central

Yeargan, Michelle R.; Alvarado-Esquivel, Cosme; Dubey, Jitender P.; Howe, Daniel K.

2013-01-01

Equine protozoal myeloencephalitis (EPM) is a debilitating disease of horses caused by Sarcocystis neurona and Neospora hughesi. Sera from 495 horses in Durango State, Mexico were tested for anti-protozoal antibodies using enzyme-linked immunosorbent assays (ELISAs) based on major surface antigens of these two parasites. Antibodies to S. neurona were detected in 240 (48.5%) of the 495 horse sera tested with the rSnSAG2/4/3 trivalent ELISA. Multivariate analysis showed that exposure to S. neurona was associated with age, feeding grains and crops, and small herd size. Antibodies to N. hughesi were found in 15 (3.0%) of the 495 horse sera tested with the rNhSAG1 ELISA and confirmed by Western blot of N. hughesi tachyzoite antigen. This is the first report of S. neurona and N. hughesi exposure in horses in Mexico, and it affirms that EPM should be in the differential diagnosis for horses exhibiting signs of neurologic disease in this country. PMID:24016396

Early T-cell activation biophysics

PubMed Central

Henry, Nelly; Hivroz, Claire

2009-01-01

The T-cell is one of the main players in the mammalian immune response. It ensures antigen recognition at the surface of antigen-presenting cells in a complex and highly sensitive and specific process, in which the encounter of the T-cell receptor with the agonist peptide associated with the major histocompatibility complex triggers T-cell activation. While signaling pathways have been elucidated in increasing detail, the mechanism of TCR triggering remains highly controversial despite active research published in the past 10 years. In this paper, we present a short overview of pending questions on critical initial events associated with T-cell triggering. In particular, we examine biophysical approaches already in use, as well as future directions. We suggest that the most recent advances in fluorescence super-resolution imaging, coupled with the new classes of genetic fluorescent probes, will play an important role in elucidation of the T-cell triggering mechanism. Beyond this aspect, we predict that exploration of mechanical cues in the triggering process will provide new clues leading to clarification of the entire mechanism. PMID:20514131

The Emerging Importance of Non-HLA Autoantibodies in Kidney Transplant Complications.

PubMed

Cardinal, Héloise; Dieudé, Mélanie; Hébert, Marie-Josée

2017-02-01

Antibodies that are specific to organ donor HLA have been involved in the majority of cases of antibody-mediated rejection in solid organ transplant recipients. However, recent data show that production of non-HLA autoantibodies can occur before transplant in the form of natural autoantibodies. In contrast to HLAs, which are constitutively expressed on the cell surface of the allograft endothelium, autoantigens are usually cryptic. Tissue damage associated with ischemia-reperfusion, vascular injury, and/or rejection creates permissive conditions for the expression of cryptic autoantigens, allowing these autoantibodies to bind antigenic targets and further enhance vascular inflammation and renal dysfunction. Antiperlecan/LG3 antibodies and antiangiotensin II type 1 receptor antibodies have been found before transplant in patients with de novo transplants and portend negative long-term outcome in patients with renal transplants. Here, we review mounting evidence suggesting an important role for autoantibodies to cryptic antigens as novel accelerators of kidney dysfunction and acute or chronic allograft rejection. Copyright © 2017 by the American Society of Nephrology.

Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

PubMed

Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2017-11-01

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

USDA-ARS?s Scientific Manuscript database

Cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and criticism of current tests for the detection of paratuberculosis. In the present study, host immune responses to antigen preparations of Mycobacterium avium subsp. paratuberculosis (MAP), consis...

Antigenic Distance Measurements for Seasonal Influenza Vaccine Selection

PubMed Central

Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

2011-01-01

Influenza vaccination is one of the major options to counteract the effects of influenza diseases. Selection of an effective vaccine strain is the key to the success of an effective vaccination program since vaccine protection can only be achieved when the selected influenza vaccine strain matches the antigenic variants causing future outbreaks. Identification of an antigenic variant is the first step to determine whether vaccine strain needs to be updated. Antigenic distance derived from immunological assays, such as hemagglutination inhibition, is commonly used to measure the antigenic closeness between circulating strains and the current influenza vaccine strain. Thus, consensus on an explicit and robust antigenic distance measurement is critical in influenza surveillance. Based on the current seasonal influenza surveillance procedure, we propose and compare three antigenic distance measurements, including Average antigenic distance (A-distance), Mutual antigenic distance (M-distance), and Largest antigenic distance (L-distance). With the assistance of influenza antigenic cartography, our simulation results demonstrated that M-distance is a robust influenza antigenic distance measurement. Experimental results on both simulation and seasonal influenza surveillance data demonstrate that M-distance can be effectively utilized in influenza vaccine strain selection. PMID:22063385

Venom allergen-like protein 28 in Clonorchis sinensis: four epitopes on its surface and the potential role of Cys124 for its conformational stability.

PubMed

Lee, Myoung-Ro; Yoo, Won Gi; Kim, Yu Jung; Chung, Eun Ju; Cho, Shin-Hyeong; Ju, Jung-Won

2018-06-06

Venom allergen-like (VAL) proteins are important to host-parasite interactions. We previously demonstrated that a Clonorchis sinensis VAL (CsVAL) protein-derived synthetic peptide suppresses allergic and inflammatory responses. However, little is known regarding the physicochemical and antigenic properties of CsVAL proteins. Here, we identified a novel 194 amino acid VAL protein, named C. sinensis VAL 28 (CsVAL28), and characterized its functional motifs and structural details as a new member of the CAP superfamily. Unlike members of the Schistosoma mansoni VAL (SmVAL) family, CsVAL28 has a single CAP1 motif and six highly conserved disulfide bond-forming cysteines. Tertiary models of wild-type CsVAL28 and mutants were built using SmVAL4 as template via homology modeling. Normal mode analysis predicted that disulfide bond breaking by mutation of cysteine 124 to serine would greatly affect protein mobility. Four major immunoreactive linear epitopes were identified in the surface-exposed region or its vicinity via epitope mapping, using sera from clonorchiasis patients and healthy controls. Our findings provide in-depth knowledge on the structure-function properties of VAL proteins and may help determine highly antigenic regions for developing new diagnostic approaches.

Elicitation of anti-vesicular stomatitis virus cytotoxic T lymphocytes by using purified viral and cellular antigens incorporated into phospholipid vesicles.

PubMed

Ruebush, M J; Hale, A H; Harris, D T

1981-05-01

We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K(k) glycoproteins elicited specific H-2K(k)-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1(-),2(+). However, both Ly 1(+),2(-) and Ly1(-),2(+) T-cell subpopulations were shown to be required for elicitation of these CTL.

First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, F.H.; Zavala, F.; Graves, P.M.

1984-07-01

An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst.

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Ratliff, T L; Wilson, R

2002-01-01

Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

Dynamics of glycoprotein charge in the evolutionary history of human influenza.

PubMed

Arinaminpathy, Nimalan; Grenfell, Bryan

2010-12-30

Influenza viruses show a significant capacity to evade host immunity; this is manifest both as large occasional jumps in the antigenic phenotype of viral surface molecules and in gradual antigenic changes leading to annual influenza epidemics in humans. Recent mouse studies show that avidity for host cells can play an important role in polyclonal antibody escape, and further that electrostatic charge of the hemagglutinin glycoprotein can contribute to such avidity. We test the role of glycoprotein charge on sequence data from the three major subtypes of influenza A in humans, using a simple method of calculating net glycoprotein charge. Of all subtypes, H3N2 in humans shows a striking pattern of increasing positive charge since its introduction in 1968. Notably, this trend applies to both hemagglutinin and neuraminidase glycoproteins. In the late 1980s hemagglutinin charge reached a plateau, while neuraminidase charge started to decline. We identify key groups of amino acid sites involved in this charge trend. To our knowledge these are the first indications that, for human H3N2, net glycoprotein charge covaries strongly with antigenic drift on a global scale. Further work is needed to elucidate how such charge interacts with other immune escape mechanisms, such as glycosylation, and we discuss important questions arising for future study.

Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

PubMed

Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

1997-06-01

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

PubMed Central

Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

2011-01-01

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable ‘UU172 element’ from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses. PMID:21255110

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

PubMed

Johnston, Christopher D; Bannantine, John P; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D

2014-01-01

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

Myelin-oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pham-Dinh, D.; Dautigny, A.; Mattei, M.G.

1993-09-01

Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. The authors have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) andmore » B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. The authors colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC. 38 refs., 6 figs.« less

Targeted Delivery of Toxoplasma gondii Antigens to Dendritic Cells Promote Immunogenicity and Protective Efficiency against Toxoplasmosis

PubMed Central

Lakhrif, Zineb; Moreau, Alexis; Hérault, Bruno; Di-Tommaso, Anne; Juste, Matthieu; Moiré, Nathalie; Dimier-Poisson, Isabelle; Mévélec, Marie-Noëlle; Aubrey, Nicolas

2018-01-01

Toxoplasmosis is a major public health problem and the development of a human vaccine is of high priority. Efficient vaccination against Toxoplasma gondii requires both a mucosal and systemic Th1 immune response. Moreover, dendritic cells play a critical role in orchestrating the innate immune functions and driving specific adaptive immunity to T. gondii. In this study, we explore an original vaccination strategy that combines administration via mucosal and systemic routes of fusion proteins able to target the major T. gondii surface antigen SAG1 to DCs using an antibody fragment single-chain fragment variable (scFv) directed against DEC205 endocytic receptor. Our results show that SAG1 targeting to DCs by scFv via intranasal and subcutaneous administration improved protection against chronic T. gondii infection. A marked reduction in brain parasite burden is observed when compared with the intranasal or the subcutaneous route alone. DC targeting improved both local and systemic humoral and cellular immune responses and potentiated more specifically the Th1 response profile by more efficient production of IFN-γ, interleukin-2, IgG2a, and nasal IgA. This study provides evidence of the potential of DC targeting for the development of new vaccines against a range of Apicomplexa parasites. PMID:29515595

Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

1986-01-01

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

ISOANTIGENS OF THE H-2 AND Tla LOCI OF THE MOUSE

PubMed Central

Boyse, Edward A.; Stockert, Elisabeth; Old, Lloyd J.

1968-01-01

H-2 and TL isoantigens of the mouse are specified by the closely linked genetic loci H-2 and Tla. A. study of their representation on thymocytes was performed in order to reveal any interactions between the determinant genes or their products affecting the synthesis or disposition of these components of the thymocyte surface. The method employed was quantitative absorption of cytotoxic antibody by viable thymocytes. The phenotypic expression of TL antigens was found to reduce the demonstrable amount of certain H-2 antigens to as little as 34% of the quantity demonstrable on TL- thymocytes. A reduction was observed in all three H-2 types tested, (H-2b, H-2a, and H-2k). As antigenic modulation (change of TL phenotype from TL+ to TL-, produced by TL antibody) is known to entail a compensatory increase in H-2(D) antigen, it is concluded that the TL phenotype, rather than the Tla genotype, influences the surface representation of H-2 antigens. The two known TL+ phenotypes of thymocytes (TL.2 and TL.1,2,3) depress H-2 equally. The H-2 specificities affected are those determined by the D end of the E-2 locus, which is adjacent to Tla; antigens of the K end, which is distal to Tla, are not depressed. The reduction of demonstrable H-2 antigen on the thymocytes of TL+ x TL- progeny is half that of thymocytes of TL+ x TL+ progeny and the reduction affects equally the products of both H-2 alleles (cis and trans in relation to Tla), indicating that the mechanism of H-2 reduction by TL is extrachromosomal. Whether it involves diminished synthesis of H-2 or steric masking by TL at the cell membrane is unknown, but in either case the reciprocal relation of TL and H-2(D) antigens implies that they probably occupy adjacent positions on thymocytes and that the gene order, H-2(K): H-2(D):Tla is reflected in cell surface structure. Extrachromosomal interaction, apparently involving control of synthesis, occurs also within the TL system of antigens. Thymocytes of TL.2 x TL.1,2,3 progeny express the full homozygous quantity of antigens TL.1 and TL.3 (but not of TL.2), in contrast to the half-quantity present in thymocytes of TL- x TL.1,2,3 progeny. Another example of interaction is implicit in the finding that thymocytes of TL-1,2,3 x TL.1,2,3 progeny have more TL.2 antigen than thymocytes of TL.2 x TL.2 progeny, but in this instance there is nothing to indicate whether the mechanism is chromosomal or extrachromosomal. Thus the quantitative surface representation of at least some H-2 and TL antigens is influenced by the cellular complement of H-2:Tla genes as a whole. Comparison of H-2 heterozygous thymocytes with H-2 homozygous thymocytes in quantitative absorption tests shows (a) more than the expected 50% of each parental-type H-2 antigen on heterozygous cells, and (b) a greater suppression of H-2 by TL in H-2 heterozygotes in comparison with H-2 homozygotes. Both results may be explained on the basis of differences in the density of H-2 antigenic sites and consequent differences in the efficiency of absorption of H-2 antibody. These considerations may be useful in other contexts, e.g. in estimating the representation of Rh antigens on the red cells of human subjects homozygous and heterozygous for Rh components. PMID:5662018

Antigenic switching of TSA 417, a trophozoite variable surface protein, following completion of the life cycle of Giardia lamblia.

PubMed Central

Meng, T C; Hetsko, M L; Gillin, F D

1993-01-01

Expression of TSA 417, the predominant cysteine-rich variable surface protein of Giardia lamblia WB clone C6 trophozoites, did not change during encystation in vitro. However, in vitro excystation of cysts derived in vitro or in vivo consistently produced TSA 417 nonexpressing trophozoite populations, suggesting that completion of the life cycle leads to antigenic switching. Images PMID:8225614

Easy-to-Fabricate and High-Sensitivity LSPR Type Specific Protein Detection Sensor Using AAO Nano-Pore Size Control

PubMed Central

Kim, Sae-Wan; Lee, Jae-Sung; Lee, Sang-Won; Kang, Byoung-Ho; Kwon, Jin-Beom; Kim, Ok-Sik; Kim, Ju-Seong; Kim, Eung-Soo; Kwon, Dae-Hyuk; Kang, Shin-Won

2017-01-01

In this study, we developed a pore size/pore area-controlled optical biosensor-based anodic aluminum oxide (AAO) nanostructure. As the pore size of AAO increases, the unit cell of AAO increases, which also increases the non-pore area to which the antibody binds. The increase in the number of antibodies immobilized on the surface of the AAO enables effective detection of trace amounts of antigen, because increased antigen-antibody bonding results in a larger surface refractive index change. High sensitivity was thus achieved through amplification of the interference wave of two vertically-incident reflected waves through the localized surface plasmon resonance phenomenon. The sensitivity of the fabricated sensor was evaluated by measuring the change in wavelength with the change in the refractive index of the device surface, and sensitivity was increased with increasing pore-size and non-pore area. The sensitivity of the fabricated sensor was improved and up to 11.8 ag/mL serum amyloid A1 antigen was detected. In addition, the selectivity of the fabricated sensor was confirmed through a reaction with a heterogeneous substance, C-reactive protein antigen. By using hard anodization during fabrication of the AAO, the fabrication time of the device was reduced and the AAO chip was fabricated quickly and easily. PMID:28406469

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

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

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. PMID:23695536

Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression.

PubMed

Lu, Wanlu; Lu, Libing; Feng, Yun; Chen, Jiao; Li, Yan; Kong, Xiangli; Chen, Sixiu; Li, Xiaoyu; Chen, Qianming; Zhang, Ping

2013-05-01

The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8 + T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment.

Inflammation promotes oral squamous carcinoma immune evasion via induced programmed death ligand-1 surface expression

PubMed Central

LU, WANLU; LU, LIBING; FENG, YUN; CHEN, JIAO; LI, YAN; KONG, XIANGLI; CHEN, SIXIU; LI, XIAOYU; CHEN, QIANMING; ZHANG, PING

2013-01-01

The association between inflammation and cancer provides a new target for tumor biotherapy. The inflammatory cells and molecules within the tumor microenvironment have decisive dual roles in antitumor immunity and immune evasion. In the present study, phytohemagglutinin (PHA) was used to stimulate peripheral blood mononuclear cells (PBMCs) to simulate the tumor inflammatory microenvironment. The effect of immune cells and inflammatory cytokines on the surface expression of programmed cell death-1 ligand 1 (PD-L1) and tumor immune evasion was investigated using flow cytometry (FCM) and an in vivo xenotransplantation model. Based on the data, PHA-activated, but not resting, immune cells were able to promote the surface expression of PD-L1 in Tca8113 oral squamous carcinoma cells via the secretion of inflammatory cytokines, but not by cell-cell contact. The majority of the inflammatory cytokines had no significant effect on the proliferation, cell cycle progression and apoptosis of the Tca8113 cells, although they each induced the expression of PD-L1 in a dose-dependent manner. In total, 99% of the Tca8113 cells expressed PD-L1 following treatment with the supernatant of PHA-stimulated PBMCs. The PHA-supernatant pretreated Tca8113 cells unusually induced Tca8113 antigen-specific CD8+ T cell apoptosis in vitro and the evasion of antigen-specific T cell attraction in a nude mouse tumor-bearing model. These results indicate a new mechanism for the promotion of tumor immune evasion by the tumor inflammatory microenvironment PMID:23761816

Peptide/protein vaccine delivery system based on PLGA particles.

PubMed

Allahyari, Mojgan; Mohit, Elham

2016-03-03

Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted.

Peptide/protein vaccine delivery system based on PLGA particles

PubMed Central

Allahyari, Mojgan; Mohit, Elham

2016-01-01

abstract Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

High Cell Surface Expression of CD4 Allows Distinction of CD4+CD25+ Antigen-specific Effector T Cells from CD4+CD25+ Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

PubMed Central

Li, Jinzhu; Ridgway, William; Fathman, C. Garrison; Tse, Harley Y.; Shaw, Michael K.

2008-01-01

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up regulate cell surface expression of CD4. The CD4high sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25+ sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses. PMID:17920698

In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters.

PubMed

Hu, Yun; Ehrich, Marion; Fuhrman, Kristel; Zhang, Chenming

2014-01-01

Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

PubMed

Neng, Jing; Li, Yina; Driscoll, Ashley J; Wilson, William C; Johnson, Patrick A

2018-06-06

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

Partial purification and characterization of protection-inducing antigens from the muscle larva of Trichinella spiralis by molecular sizing chromatography and preparative flatbed isoelectric focusing.

PubMed

Despommier, D D

1981-01-01

The soluble portion of a large particle fraction which was derived from the muscle larva of T. spiralis was subjected to molecular sizing column chromatography using Sephacryl S-200. Five major peaks of 280 nm absorbing material were obtained. Analysis by immunoelectrophoresis revealed that each peak contained antigens, with the majority of them occurring in peaks 3, 4 and 5. Preliminary studies indicated that peak 4(mol. wt range 20 000--10 000) contained protection-inducing antigens. Crossed-immunoelectrophoretic and single-dimension electrophoretic analysis of peak 4 revealed a minimum of 10 antigens, while analytical isoelectric focusing demonstrated the presence of proteins with widely different pl, ranging from 4.0 to 9.0. Peak 4 was fractionated by preparative flatbed isoelectric focusing (PIEF) using two gradients: one from 3.5 to 9.5 and the other from 3.5 to 5.5. Fused rocket immunoelectrophoretic (FRIEP) analysis of both runs indicated that several antigens were separated from the others: one at pl 4.0 and the other at pl 9.0. The remaining antigens focused between pl 4.3 and 4.9. One hundred micrograms of whole peak 4, pl 9.0 antigen and the group of antigens at pl 4.3--4.9 were each separately injected, along with Freund's complete adjuvant, into mice. In addition, a portion of the pl 4.0 antigen was also assayed for protection. All antigenic preparations induced significant levels of protection. The pl 4.0 was further analysed on high-performance liquid chromatography (HPLC). Two sharp peaks of antigen, as detected by FRIEP, were eluted isocratically with 65% acetonitrile from a C-18 (aliphatic) column. Both peaks of antigen showed complete cross-reactivity on FRIEP and absorbed at 220 nm. Amino acid analysis of each HPLC peak revealed no detectable differences in composition. Each peak contained predominance of aspartic (13 mol%) and glutamic (18 mol%) acid. This antigen did not contain significant quantities of aromatic amino acids, and absorbed strongly at 206 nm. Neither the pl 4.0 or pl 9.0 antigen stained positively with the PAS reaction.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

PubMed Central

Choi, Min-Ho; Ryu, Jin-Sook; Lee, Mejeong; Li, Shunyu; Chung, Byung-Suk; Chai, Jong-Yil; Sithithaworn, Paiboon; Tesana, Smarn

2003-01-01

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection. PMID:12972729

Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses

PubMed Central

Opperman, Pamela A.; Rotherham, Lia S.; Esterhuysen, Jan; Charleston, Bryan; Juleff, Nicholas; Capozzo, Alejandra V.; Theron, Jacques

2014-01-01

ABSTRACT Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided. PMID:24829347

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

PubMed Central

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

2010-01-01

Nanoparticles are an attractive vaccine carrier with potent adjuvant activity. Data from our previous studies showed that immunization of mice with lecithin/glyceryl monostearate-based nanoparticles with protein antigens conjugated onto their surface induced a strong, quick, and long-lasting antigen-specific immune response. In the present study, we evaluated the feasibility of preserving the immunogenicity of protein antigens carried by nanoparticles without refrigeration using these antigen-conjugated nanoparticles as a model. The nanoparticles were lyophilized, and the immunogenicity of the antigens was evaluated in a mouse model using bovine serum albumin or the Bacillus anthracis protective antigen protein as model antigens. With proper excipients, the nanoparticles can be lyophilized while maintaining the immunogenicity of the antigens. Moreover, the immunogenicity of the model antigen conjugated onto the nanoparticles was undamaged after a relatively extended period of storage at room temperature or under accelerated conditions (37°C) when the nanoparticles were lyophilized with 5% mannitol plus 1% polyvinylpyrrolidone. To our knowledge, the present study represents an early attempt to preserve the immunogenicity of the protein antigens carried by nanoparticles without refrigeration. PMID:20416366

Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria

PubMed Central

Cahoon, Laty A.; Seifert, H. Steven

2011-01-01

Summary Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyze dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes. PMID:21812841

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

Radioimmunoassays of hidden viral antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neurath, A.R.; Strick, N.; Baker, L.

1982-07-01

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis Bmore » virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.« less

Novel Treponema pallidum Recombinant Antigens for Syphilis Diagnostics: Current Status and Future Prospects

PubMed Central

Kubanov, Aleksey; Runina, Anastassia

2017-01-01

The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized. PMID:28523273

Trypanosoma cruzi Subverts Host Cell Sialylation and May Compromise Antigen-specific CD8+ T Cell Responses*

PubMed Central

Freire-de-Lima, Leonardo; Alisson-Silva, Frederico; Carvalho, Sebastião T.; Takiya, Christina M.; Rodrigues, Maurício M.; DosReis, George A.; Mendonça-Previato, Lucia; Previato, José O.; Todeschini, Adriane R.

2010-01-01

Upon activation, cytotoxic CD8+ T lymphocytes are desialylated exposing β-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8+ T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8+ T cell surface, thereby dampening antigen-specific CD8+ T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8+ T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8+ T cell surface. The cytotoxic activity of antigen-experienced CD8+ T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase- mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8+ T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8+ T cell interactions with peptide-major histocompatibility complex class I complexes. CD8+ T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism. PMID:20106975

Immunological evaluation of colonic delivered Hepatitis B surface antigen loaded TLR-4 agonist modified solid fat nanoparticles.

PubMed

Sahu, Kantrol Kumar; Pandey, Ravi Shankar

2016-10-01

Hepatitis B is one of the leading liver diseases and remains a major global health problem. Currently available vaccines provide protection but often results in weaker/minimal mucosal immunity. Thus the present study is devoted to the development and in-vivo exploration of the colonically delivered biomimetic nanoparticles which capably enhance humoral as well as cellular immune response. In present work, Hepatitis B surface antigen (HBsAg) entrapped nanoparticles containing Monophosphoryl lipid A (MPLA) (HB+L-NP) were prepared by solvent evaporation method and characterized for particle size (~210nm), shape, zeta potential (-24mV±0.68), entrapment efficiency (58.45±1.68%), in-vitro release and antigen integrity. Dose escalation study was done to confirm prophylactic immune response following defined doses of prepared nanoparticulate formulations with or without MPLA. Intramuscular administered alum based marketed HBsAg (Genevac B) was used as standard (10μg) and were able to induce significant systemic (IgG) but remarkably low mucosal immune (IgA) response. Notably, HB+L-NP (0.5ml-10μg) induced strong systemic and robust mucosal immunity (510 and 470 mIU/ml respectively, p<0.001) from which mucosal was more significant due to the involvement of Common Mucosal Immune System (CMIS). Likewise, significant cellular immune response was elicited by HB+L-NP through T-cell activation (mixed Th1 and Th2) as confirmed by significantly increased cytokines level (IL-2 and Interferon-γ) in spleen homogenates. This study supports that delivery of HBsAg to the colon may open new vista in designing oral vaccines later being one of most accepted route for potential vaccines in future. Copyright © 2016 Elsevier B.V. All rights reserved.

Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine.

PubMed

An, So Jung; Woo, Joo Sung; Chae, Myung Hwa; Kothari, Sudeep; Carbis, Rodney

2015-03-24

The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

IFNγ producing CD8+ T cells modified to resist major immune checkpoints induce regression of MHC class I-deficient melanomas

PubMed Central

Buferne, Michel; Chasson, Lionel; Grange, Magali; Mas, Amandine; Arnoux, Fanny; Bertuzzi, Mélanie; Naquet, Philippe; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2015-01-01

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFβ1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients. PMID:25949872

β2-Microglobulin-mediated signaling as a target for cancer therapy.

PubMed

Nomura, Takeo; Huang, Wen-Chin; Zhau, Haiyen E; Josson, Sajni; Mimata, Hiromitsu; Chung, Leland W K

2014-03-01

β2-microglobulin (β2-m) has become the focus of intense scrutiny since the discovery of its undesirable roles promoting osteomimicry and cancer progression. β2-m is a well-known housekeeping protein that forms complexes with the heavy chain of major histocompatibility complex class I molecules, which are heterodimeric cell surface proteins that present antigenic peptides to cytotoxic T cells. On recognition of foreign peptide antigens on cell surfaces, T cells actively bind and lyse antigen-presenting cancer cells. In addition to its roles in tumor immunity, β2-m has two different functions in cancer cells, either tumor promoting or tumor suppressing, in cancer cell context-dependent manner. Our studies have demonstrated that β2-m is involved extensively in the functional regulation of growth, survival, apoptosis, and even metastasis of cancer cells. We found that β2-m is a soluble growth factor and a pleiotropic signaling molecule which interacts with its receptor, hemochromatosis protein, to modulate epithelial-to-mesenchymal transition (EMT) through iron-responsive pathways. Specific antibodies against β2-m have remarkable tumoricidal activity in cancer, through β2-m action on iron flux, alterations of intracellular reactive oxygen species, DNA damage and repair enzyme activities, β-catenin activation and cadherin switching, and tumor responsiveness to hypoxia. These novel functions of β2-m and β2-m signaling may be common to several solid tumors including human lung, breast, renal, and prostate cancers. Our experimental results could lead to the development of a novel class of antibody-based pharmaceutical agents for cancer growth control. In this review, we briefly summarize the recent data regarding β2-m as a promising new cancer therapeutic target and discuss antagonizing this therapeutic target with antibody therapy for the treatment of localized and disseminated cancers.

β2-Microglobulin-mediated Signaling as a Target for Cancer Therapy

PubMed Central

Nomura, Takeo; Huang, Wen-Chin; Zhau, Haiyen E.; Josson, Sajni; Mimata, Hiromitsu; Kaur, Mandeep

2014-01-01

β2-microglobulin (β2-m) has become the focus of intense scrutiny since the discovery of its undesirable roles promoting osteomimicry and cancer progression. β2-m is a well-known housekeeping protein that forms complexes with the heavy chain of major histocompatibility complex class I molecules, which are heterodimeric cell surface proteins that present antigenic peptides to cytotoxic T cells. On recognition of foreign peptide antigens on cell surfaces, T cells actively bind and lyse antigen-presenting cancer cells. In addition to its roles in tumor immunity, β2-m has two different functions in cancer cells, either tumor promoting or tumor suppressing, in cancer cell context-dependent manner. Our studies have demonstrated that β2-m is involved extensively in the functional regulation of growth, survival, apoptosis, and even metastasis of cancer cells. We found that β2-m is a soluble growth factor and a pleiotropic signaling molecule which interacts with its receptor, hemochromatosis protein, to modulate epithelial-to-mesenchymal transition (EMT) through iron-responsive pathways. Specific antibodies against β2-m have remarkable tumoricidal activity in cancer, through β2-m action on iron flux, alterations of intracellular reactive oxygen species, DNA damage and repair enzyme activities, β-catenin activation and cadherin switching, and tumor responsiveness to hypoxia. These novel functions of β2-m and β2-m signaling may be common to several solid tumors including human lung, breast, renal, and prostate cancers. Our experimental results could lead to the development of a novel class of antibody-based pharmaceutical agents for cancer growth control. In this review, we briefly summarize the recent data regarding β2-m as a promising new cancer therapeutic target and discuss antagonizing this therapeutic target with antibody therapy for the treatment of localized and disseminated cancers. PMID:23848204

Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

PubMed

Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

2007-02-01

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.

Association of chitosan and aluminium as a new adjuvant strategy for improved vaccination.

PubMed

Lebre, F; Bento, D; Ribeiro, J; Colaço, M; Borchard, G; de Lima, M C Pedroso; Borges, O

2017-07-15

The use of particulate adjuvants offers an interesting possibility to enhance and modulate the immune responses elicited by vaccines. Aluminium salts have been extensively used as vaccine adjuvants, but they lack the capacity to induce a strong cellular and mucosal immune response. Taking this into consideration, in this study we designed a new antigen delivery system combining aluminium salts with chitosan. Chitosan-aluminium nanoparticles (CH-Al NPs) exhibited a mean diameter of 280nm and a positive surface charge. The newly developed CH-Al NPs are more stable at physiological environment than classical CH NPs, showing no cytotoxic effects and revealing potential as a delivery system for a wide range of model antigens. In vivo studies showed that mice immunized with hepatitis B surface antigen (HBsAg)-containing CH NPs display high anti-HBsAg IgG titers in the serum, as well as the highest antigen-specific IgG on vaginal washes. Furthermore, in contrast to mice receiving antigen alone, mice immunized with the particulate adjuvant were able to elicit IgG2c antibody titers and exhibited higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we established that CH-Al NPs, combining two immunostimulants to enhance both humoral and cellular immune responses, are a safe and promising system for antigen delivery. Our findings point towards their potential in future vaccination approaches. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene expression of cell surface antigens in the early phase of murine influenza pneumonia determined by a cDNA expression array technique.

PubMed

Sakai, Shinya; Mantani, Naoki; Kogure, Toshiaki; Ochiai, Hiroshi; Shimada, Yutaka; Terasawa, Katsutoshi

2002-12-01

Influenza virus is a worldwide health problem with significant economic consequences. To study the gene expression pattern induced by influenza virus infection, it is useful to reveal the pathogenesis of influenza virus infection; but this has not been well examined, especially in vivo study. To assess the influence of influenza virus infection on gene expression in mice, mRNA levels in the lung and tracheal tissue 48 h after infection were investigated by cDNA array analysis. Four-week-old outbred, specific pathogen free strain, ICR female mice were infected by intra-nasal inoculation of a virus solution under ether anesthesia. The mice were sacrificed 48 h after infection and the tracheas and lungs were removed. To determine gene expression, the membrane-based microtechnique with an Atlas cDNA expression array (mouse 1.2 array II) was performed in accordance with the manual provided. We focused on the expression of 46 mRNAs for cell surface antigens. Of these 46 mRNAs that we examined, four (CD1d2 antigen, CD39 antigen-like 1, CD39 antigen-like 3, CD68 antigen) were up-regulated and one (CD36 antigen) was down-regulated. Although further studies are required, these data suggest that these molecules play an important role in influenza virus infection, especially the phase before specific immunity.

Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA.

PubMed

Calcedo, Roberto; Ramirez-Garcia, Andoni; Abad, Ana; Rementeria, Aitor; Pontón, José; Hernando, Fernando Luis

2012-01-01

Candida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor. To determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA). Under various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing. Five antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36 kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively. These glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection. Copyright © 2011 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Tomatine adjuvantation of protective immunity to a major pre-erythrocytic vaccine candidate of malaria is mediated via CD8+ T cell release of IFN-gamma.

PubMed

Heal, Karen G; Taylor-Robinson, Andrew W

2010-01-01

The glycoalkaloid tomatine, derived from the wild tomato, can act as a powerful adjuvant to elicit an antigen-specific cell-mediated immune response to the circumsporozoite (CS) protein, a major pre-erythrocytic stage malaria vaccine candidate antigen. Using a defined MHC-class-I-restricted CS epitope in a Plasmodium berghei rodent model, antigen-specific cytotoxic T lymphocyte activity and IFN-gamma secretion ex vivo were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunized mice. Further, through lymphocyte depletion it is demonstrated that antigen-specific IFN-gamma is produced exclusively by the CD8(+) T cell subset. We conclude that the processing of the P. berghei CS peptide as an exogenous antigen and its presentation via MHC class I molecules to CD8(+) T cells leads to an immune response that is an in vitro correlate of protection against pre-erythrocytic malaria. Further characterization of tomatine as an adjuvant in malaria vaccine development is indicated.

Immunogenicity and Protection Efficacy of Subunit-based Smallpox Vaccines Using Variola Major Antigens

PubMed Central

Sakhatskyy, Pavlo; Wang, Shixia; Zhang, Chuanyou; Chou, Te-Hui; Kishko, Michael; Lu, Shan

2008-01-01

The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on the VARV antigen sequences to induce immunity against poxvirus infection. PMID:17950773

Co-delivery of antigen and a lipophilic anti-inflammatory drug to cells via a tailorable nanocarrier emulsion.

PubMed

Chuan, Yap Pang; Zeng, Bi Yun; O'Sullivan, Brendan; Thomas, Ranjeny; Middelberg, Anton P J

2012-02-15

Nanotechnology promises new drug carriers that can be tailored to specific applications. Here we report a new approach to drug delivery based on tailorable nanocarrier emulsions (TNEs), motivated by a need to co-deliver a protein antigen and a lipophilic drug for specific inhibition of nuclear factor kappa B (NF-κB) in antigen presenting cells (APCs). Co-delivery for NF-κB inhibition holds promise as a strategy for the treatment of rheumatoid arthritis. We used a highly surface-active peptide (SAP) to prepare a nanosized emulsion having defined surface properties predictable from the SAP sequence. Incorporating the lipophilic drug into the oil phase at the time of emulsion formation enabled its facile packaging. The SAP is depleted from bulk during emulsification, allowing simple subsequent addition of the drug-loaded oil-in-water emulsion to a solution of protein antigen. Decoration of emulsion surface with antigen was achieved via electrostatic deposition. In vitro data showed that the TNE prepared this way was internalized and well-tolerated by model APCs, and that good suppression of NF-κB expression was achieved. This work reports a new type of nanotechnology-based carrier, a TNE, which can potentially be tailored for co-delivery of multiple therapeutic components, and can be made using simple methods using only biocompatible materials. Copyright © 2011 Elsevier Inc. All rights reserved.

Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells.

PubMed

Nacharaju, Parimala; Boctor, Fouad N; Manjula, Belur N; Acharya, Seetharama A

2005-03-01

The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.

Effects of spaceflight on levels and activity of immune cells

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Berry, Wallace D.; Mandel, Adrian D.; Konstantinova, Irena V.; Taylor, Gerald R.

1990-01-01

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of speceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell, and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

Nonclassical T Cells and Their Antigens in Tuberculosis

PubMed Central

De Libero, Gennaro; Singhal, Amit; Lepore, Marco; Mori, Lucia

2014-01-01

T cells that recognize nonpeptidic antigens, and thereby are identified as nonclassical, represent important yet poorly characterized effectors of the immune response. They are present in large numbers in circulating blood and tissues and are as abundant as T cells recognizing peptide antigens. Nonclassical T cells exert multiple functions including immunoregulation, tumor control, and protection against infections. They recognize complexes of nonpeptidic antigens such as lipid and glycolipid molecules, vitamin B2 precursors, and phosphorylated metabolites of the mevalonate pathway. Each of these antigens is presented by antigen-presenting molecules other than major histocompatibility complex (MHC), including CD1, MHC class I–related molecule 1 (MR1), and butyrophilin 3A1 (BTN3A1) molecules. Here, we discuss how nonclassical T cells participate in the recognition of mycobacterial antigens and in the mycobacterial-specific immune response. PMID:25059739

[Biochemical characteristics and antigenic structures of Chlamydia].

PubMed

Puy, H; Fuentes, V; Eb, F; Orfila, J

1989-01-01

New biotechnology in immunology and molecular biology has enabled the identification and definition of the structure of glycolipids and especially membrane proteins of Chlamydia. Chlamydia antigen lipopolysaccharide, major outer membrane protein, protein 74 kDa, eukaryotic cell binding protein and cysteine rich proteins are all carriers of antigenic determinants, genus, species or type specific. They are very usefull for diagnosis of Chlamydial infections and epidemiological studies. These membranous antigens have an important role in the pathogenesis of these bacteries. Finally these studies have contributed to the isolation of a new species: C. pneumoniae (TWAR strains).

PHENOTYPIC EXPRESSIONS OF THE MAJOR HISTOCOMPATIBILITY LOCUS IN MAN (HL-A): LEUKOCYTE ANTIGENS AND MIXED LEUKOCYTE CULTURE REACTIVITY

PubMed Central

Amos, D. Bernard; Bach, Fritz H.

1968-01-01

The evidence is reviewed that a single genetic system, the major histocompatibility locus in man, HL-A, determines most of the antigens measured by presently available leukocyte isoantisera, and also controls reactivity in one-way mixed leucocyte culture tests. Studies in 12 families are presented to support this conclusion. Some interesting exceptions to the general typing—MLC tests correlation are presented and discussed. PMID:5675436

Key residues of a major cytochrome P4502D6 epitope are located on the surface of the molecule.

PubMed

Ma, Yun; Thomas, Mark G; Okamoto, Manabu; Bogdanos, Dimitrios P; Nagl, Sylvia; Kerkar, Nanda; Lopes, Agnel R; Muratori, Luigi; Lenzi, Marco; Bianchi, Francesco B; Mieli-Vergani, Giorgina; Vergani, Diego

2002-07-01

Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1-193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267-337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6(316-327) is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.

Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys® HBsAg II assay.

PubMed

Kim, Hyon Suk; Chen, Xinyue; Xu, Min; Yan, Cunling; Liu, Yali; Deng, Haohui; Hoang, Bui Huu; Thuy, Pham Thi Thu; Wang, Terry; Yan, Yiwen; Zeng, Zhen; Gencay, Mikael; Westergaard, Gaston; Pabinger, Stephan; Kriegner, Albert; Nauck, Markus; Seffner, Anja; Gohl, Peter; Hübner, Kirsten; Kaminski, Wolfgang E

2018-06-01

To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys ® HBsAg II Qualitative assay. We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys ® HBsAg II Qualitative assay. Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys ® HBsAg II Qualitative assay. We observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys ® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia. Copyright © 2018 Roche Diagnostics International Ltd. Published by Elsevier B.V. All rights reserved.

Evaluation of Recombinant Attenuated Salmonella Vaccine Strains for Broad Protection against Extraintestinal Pathogenic Escherichia coli.

PubMed

Maddux, Jacob T; Stromberg, Zachary R; Curtiss Iii, Roy; Mellata, Melha

2017-01-01

Antibiotic-resistant bacterial infections are difficult to treat, producing a burden on healthcare and the economy. Extraintestinal pathogenic Escherichia coli (ExPEC) strains frequently carry antibiotic resistance genes, cause infections outside of the intestine, and are causative agents of hospital-acquired infections. Developing a prevention strategy against this pathogen is challenging due to its antibiotic resistance and antigenic diversity. E. coli common pilus (ECP) is frequently found in ExPEC strains and may serve as a common antigen to induce protection against several ExPEC serotypes. In addition, live recombinant attenuated Salmonella vaccine (RASV) strains have been used to prevent Salmonella infection and can also be modified to deliver foreign antigens. Thus, the objective of this study was to design a RASV to produce ECP on its surface and assess its ability to provide protection against ExPEC infections. To constitutively display ECP in a RASV strain, we genetically engineered a vector (pYA4428) containing aspartate-β-semialdehyde dehydrogenase and E. coli ecp genes and introduced it into RASV χ9558. RASV χ9558 containing an empty vector (pYA3337) was used as a control to assess protection conferred by the RASV strain without ECP. We assessed vaccine efficacy in in vitro bacterial inhibition assays and mouse models of ExPEC-associated human infections. We found that RASV χ9558(pYA4428) synthesized the major pilin (EcpA) and tip pilus adhesin (EcpD) on the bacterial surface. Mice orally vaccinated with RASV χ9558(pYA3337) without ECP or χ9558(pYA4428) with ECP, produced anti- Salmonella LPS and anti- E. coli EcpA and EcpD IgG and IgA antibodies. RASV strains showed protective potential against some E. coli and Salmonella strains as assessed using in vitro assays. In mouse sepsis and urinary tract infection challenge models, both vaccines had significant protection in some internal organs. Overall, this work showed that RASVs can elicit an immune response to E . coli and Salmonella antigens in some mice, provide significant protection in some internal organs during ExPEC challenge, and thus this study is a promising initial step toward developing a vaccine for prevention of ExPEC infections. Future studies should optimize the ExPEC antigens displayed by the RASV strain for a more robust immune response and enhanced protection against ExPEC infection.

Neuronal surface antigen antibodies in limbic encephalitis

PubMed Central

Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

2008-01-01

Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels; WBC = white blood cells. PMID:18794496

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo

PubMed Central

Hassan, Hatem A.F.M.; Smyth, Lesley; Rubio, Noelia; Ratnasothy, Kulachelvy; Wang, Julie T.-W.; Bansal, Sukhvinder S.; Summers, Huw D.; Diebold, Sandra S.; Lombardi, Giovanna; Al-Jamal, Khuloud T.

2016-01-01

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~ 386 nm), bearing net positive charge (5.8 mV), or short MWNTs-OVA (~ 122 nm) of increasing negative charges (− 23.4, − 35.8 or − 39 mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system. PMID:26802552

Natural selection promotes antigenic evolvability.

PubMed

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.

Natural Selection Promotes Antigenic Evolvability

PubMed Central

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. PMID:24244173

IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules.

PubMed

Knolle, P A; Uhrig, A; Hegenbarth, S; Löser, E; Schmitt, E; Gerken, G; Lohse, A W

1998-12-01

Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-gamma) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2 (PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver.

Preparation of protein loaded poly(D,L-lactide-co-glycolide) microparticles for the antigen delivery to dendritic cells using a static micromixer.

PubMed

Wischke, Christian; Lorenzen, Dirk; Zimmermann, Julian; Borchert, Hans-Hubert

2006-04-01

The cellular immune response against tumors, viruses, or intracellular bacteria requires adequate antigen delivery to professional phagocytes, their processing and the presentation of antigenic peptides to T-cells. Biodegradable microparticles to enhance antigen phagocytosis and the response of cytotoxic lymphocytes have been proposed. The aim of the present study was to formulate poly(lactide-co-glycolide) (PLGA) microparticles using a w/o/w solvent evaporation procedure in order to obtain suitable vehicles for vaccination. Bovine serum albumin bearing fluorescein isothiocyanate (FITC-BSA) was used as a model antigen. For microparticle preparation a static micromixer was employed. Microparticles of 2-3 microm can be produced with good reproducibility by applying high flow rates at the micromixer. Microparticles with a smooth surface and only one pore were observed using scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) allowed localisation of the FITC-BSA near the surface of the microparticle. Microencapsulation of FITC-BSA did not altered the polymer characteristics, as determined by measuring the glass transition temperature. Additionally we could determine residual methylene chloride, employed as solvent in microparticle preparation, to be less than 1/1000 of the USP and Ph. Eur. limit. The microparticles described herein were able to deliver the model antigen to human dendritic cells (DC).

Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion

PubMed Central

1993-01-01

Allograft rejection is a T cell-dependent process. Productive T cell activation by antigen requires antigen engagement of the T cell receptor as well as costimulatory signals delivered through other T cell surface molecules such as CD28. Engagement of CD28 by its natural ligand B7 can be blocked using a soluble recombinant fusion protein, CTLA4Ig. Administration of CTLA4Ig blocks antigen-specific immune responses in vitro and in vivo, and we have shown that treatment of rats with a 7-d course of CTLA4Ig at the time of transplantation leads to prolonged survival of cardiac allografts (median 30 d), although most grafts are eventually rejected. Here, we have explored additional strategies employing CTLA4Ig in order to achieve long-term allograft survival. Our data indicate that donor-specific transfusion (DST) plus CTLA4Ig can provide effective antigen-specific immunosuppression. When DST is administered at the time of transplantation followed by a single dose of CTLA4Ig 2 d later, all animals had long-term graft survival (> 60 d). These animals had delayed responses to donor-type skin transplants, compared with normal rejection responses to third-party skin transplants. Furthermore, donor-matched second cardiac allografts were well tolerated with minimal histologic evidence of rejection. These data indicate that peritransplant use of DST followed by subsequent treatment with CTLA4Ig can induce prolonged, often indefinite, cardiac allograft acceptance. These results may be clinically applicable for cadaveric organ and tissue transplantation in humans. PMID:8228826

Variability and repertoire size of T-cell receptor V alpha gene segments.

PubMed

Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells

PubMed Central

1993-01-01

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD- deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell- independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen- driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens. PMID:8418208

Activation status of mucosal-associated invariant T cells reflects disease activity and pathology of systemic lupus erythematosus.

PubMed

Chiba, Asako; Tamura, Naoto; Yoshikiyo, Kazunori; Murayama, Goh; Kitagaichi, Mie; Yamaji, Ken; Takasaki, Yoshinari; Miyake, Sachiko

2017-03-14

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes constituting a large proportion of peripheral blood T cells expressing αβ T-cell receptor in humans. In this study, we aimed to investigate their involvement in systemic lupus erythematosus (SLE). Peripheral blood MAIT cells from patients with SLE were assessed for their frequency, activation markers, and cell death by flow cytometry. The correlation between plasma cytokine levels and CD69 expression on MAIT cells was analyzed. The major histocompatibility complex class I-related protein MR1-restricted antigen-presenting capacity of antigen-presenting cells was investigated. Cytokine-mediated activation of MAIT cells in the absence of exogenous antigens was also examined. The frequency of MAIT cells was markedly reduced in SLE. The reduced number of MAIT cells was not attributable to the downregulation of surface markers, but it was partially due to the enhanced cell death of MAIT cells, possibly by activation-induced cell death. The CD69 expression levels on MAIT cells in SLE correlated with disease activity. Moreover, monocytes from patients with SLE exhibited increased ability to induce MAIT cell activation. The plasma concentration of interleukin (IL)-6, IL-18, and interferon (IFN)-α positively correlated with the expression levels of CD69 on MAIT cells in SLE. MAIT cells were activated by cytokines, including IFN-α, IL-15, and IL-12 plus IL-18, in the absence of exogenous antigens. These results suggest that MAIT cells reflect the pathological condition of SLE and that their activated status correlates with presence of disease.

Hypoxia and hypoxia-inducible factor (HIF) downregulate antigen-presenting MHC class I molecules limiting tumor cell recognition by T cells

PubMed Central

Nguyen, Thao; Hatfield, Stephen M.; Ohta, Akio; Sitkovsky, Michail V.

2017-01-01

Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME in vivo and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures in vitro; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME. PMID:29155844

Mechanisms of Chemical Modulation and Toxicity of the Immune System

DTIC Science & Technology

1989-12-15

antigens and suppressor/cytotoxic cells bearing Lyt-2 surface antigens. Autoimmune or immunodeficient diseases have been characte.ized by alterations...Written Publication (cumulative list) A. Suppression of mitogen-induced blastogenesis of feline lymphocytes by in Yi incubation with carcinogenic

Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

PubMed

Heydari Zarnagh, Hafez; Ravanshad, Mehrdad; Pourfatollah, Ali Akbar; Rasaee, Mohammad Javad

2015-04-01

Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3) cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

Collective Genetic Interaction Effects and the Role of Antigen Presenting Cells in Autoimmune Diseases

DTIC Science & Technology

2017-01-12

RESEARCH ARTICLE Collective Genetic Interaction Effects and the Role of Antigen-Presenting Cells in Autoimmune Diseases Hyung Jun Woo*, Chenggang Yu...autoimmunity. Genetic predispositions center around the major histocompatibility complex (MHC) class II loci involved in antigen presentation, the key...helper and regulatory T cells showing strong dis- ease-associated interactions with B cells. Our results provide direct genetic evidence point- ing to

Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

PubMed Central

MacInnes, J I; Rosendal, S

1987-01-01

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus. Images PMID:3298061

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II

PubMed Central

Rehm, Kristina E; Connor, Ramsey F; Jones, Gwendolyn J B; Yimbu, Kenneth; Mannie, Mark D; Roper, Rachel L

2009-01-01

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-γ, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC. PMID:20067538

Dosimetric model for intraperitoneal targeted liposomal radioimmunotherapy of ovarian cancer micrometastases

NASA Astrophysics Data System (ADS)

Syme, A. M.; McQuarrie, S. A.; Middleton, J. W.; Fallone, B. G.

2003-05-01

A simple model has been developed to investigate the dosimetry of micrometastases in the peritoneal cavity during intraperitoneal targeted liposomal radioimmunotherapy. The model is applied to free-floating tumours with radii between 0.005 cm and 0.1 cm. Tumour dose is assumed to come from two sources: free liposomes in solution in the peritoneal cavity and liposomes bound to the surface of the micrometastases. It is assumed that liposomes do not penetrate beyond the surface of the tumours and that the total amount of surface antigen does not change over the course of treatment. Integrated tumour doses are expressed as a function of biological parameters that describe the rates at which liposomes bind to and unbind from the tumour surface, the rate at which liposomes escape from the peritoneal cavity and the tumour surface antigen density. Integrated doses are translated into time-dependent tumour control probabilities (TCPs). The results of the work are illustrated in the context of a therapy in which liposomes labelled with Re-188 are targeted at ovarian cancer cells that express the surface antigen CA-125. The time required to produce a TCP of 95% is used to investigate the importance of the various parameters. The relative contributions of surface-bound radioactivity and unbound radioactivity are used to assess the conditions required for a targeted approach to provide an improvement over a non-targeted approach during intraperitoneal radiation therapy. Using Re-188 as the radionuclide, the model suggests that, for microscopic tumours, the relative importance of the surface-bound radioactivity increases with tumour size. This is evidenced by the requirement for larger antigen densities on smaller tumours to affect an improvement in the time required to produce a TCP of 95%. This is because for the smallest tumours considered, the unbound radioactivity is often capable of exerting a tumouricidal effect before the targeting agent has time to accumulate significantly on the tumour surface.

[Cellular immunophenotypes in 97 adults with acute leukemia].

PubMed

Piedras, J; López-Karpovitch, X; Cárdenas, M R

1997-01-01

To analyze hematopoietic cell surface antigen reactivity in acute leukemia (AL) by flow cytometry and identify acute mixed-lineage leukemias (AMLL) employing the most widely accepted criteria. Ninety seven patients with de novo AL were studied. Cell surface antigens were investigated with monoclonal antibodies directed to: B lymphoid (CD10, CD19, CD20, CD21, CD22); T lymphoid (CD2, CD3, CD5, CD7); and myeloid (CD13, CD14, CD15, CD33, CD41) cell lineages. Maturation cell-associated antigens (CD34, HLA-DR and TdT) were also studied. Twelve patients unclassified by cytomorphology could be classified by immunophenotype. Using cytomorphologic, cytochemical and immunophenotypic data, 54 cases corresponded to acute lymphoblastic leukemia (ALL) and 43 were acute myeloblastic leukemia (AML). In All there were 63% B lineage, 15% T, 7% T/B, 6% undifferentiated and 9% mixed-lineage (coexpression of two or more myeloid-associated antigens). In AML, myeloid immunophenotype was observed in 86% undifferentiated in 2%, and mixed-lineage in 12% (coexpression of two or more lymphoid-associated antigens). In addition, 26% of ALL cases and 12% of AML cases expressed a single myeloid and lymphoid antigen respectively. The most common aberrant antigens in ALL and AML were CD13 and CD7 respectively. The highest frequency of CD34 antigen expression (90%) was detected in patients with AMLL. Flow cytometric immunophenotypic analysis allowed to: a) establish diagnosis in cytomorphologically unclassified cases; b) identify AMLL with a frequency similar to that reported in other series; and c) confirm the heterogeneity of AL.

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

PubMed

Brinãs, Raymond P; Sundgren, Andreas; Sahoo, Padmini; Morey, Susan; Rittenhouse-Olson, Kate; Wilding, Greg E; Deng, Wei; Barchi, Joseph J

2012-08-15

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Plant-made oral vaccines against human infectious diseases—Are we there yet?

PubMed Central

Chan, Hui-Ting; Daniell, Henry

2016-01-01

Summary Although the plant-made vaccine field started three decades ago with the promise of developing low-cost vaccines to prevent infectious disease outbreaks and epidemics around the globe, this goal has not yet been achieved. Plants offer several major advantages in vaccine generation, including low-cost production by eliminating expensive fermentation and purification systems, sterile delivery and cold storage/transportation. Most importantly, oral vaccination using plant-made antigens confers both mucosal (IgA) and systemic (IgG) immunity. Studies in the past 5 years have made significant progress in expressing vaccine antigens in edible leaves (especially lettuce), processing leaves or seeds through lyophilization and achieving antigen stability and efficacy after prolonged storage at ambient temperatures. Bioencapsulation of antigens in plant cells protects them from the digestive system; the fusion of antigens to transmucosal carriers enhances efficiency of their delivery to the immune system and facilitates successful development of plant vaccines as oral boosters. However, the lack of oral priming approaches diminishes these advantages because purified antigens, cold storage/transportation and limited shelf life are still major challenges for priming with adjuvants and for antigen delivery by injection. Yet another challenge is the risk of inducing tolerance without priming the host immune system. Therefore, mechanistic aspects of these two opposing processes (antibody production or suppression) are discussed in this review. In addition, we summarize recent progress made in oral delivery of vaccine antigens expressed in plant cells via the chloroplast or nuclear genomes and potential challenges in achieving immunity against infectious diseases using cold-chain-free vaccine delivery approaches. PMID:26387509

Changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum in syphilis patients after treatment.

PubMed

Kim, D K; Lee, M G; Lee, J B

1989-06-01

The changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum after treatment of syphilis were observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Until 9 to 12 months after treatment, it was seen that there was a loss of several antibodies and some diminution in their reactivity in primary, secondary and early latent syphilis, but no changes occurred in late latent and reinfected syphilis. In primary syphilis, there was a significant loss of two IgG antibodies to the treponemal antigens of molecular weights 68,500 and 47,000 at 11 months after treatment. According to our previous study, the treponemal antigen of molecular weight 68,500 was T. pallidum specific and appeared only in primary syphilis, and that of molecular weight 47,000 was one of the major antigens of T. pallidum. The reaction between serum IgG antibodies of 14 patients who had been treated for secondary, early latent and late latent syphilis 2 to 14 years ago and major antigens of T. pallidum was observed and any loss or decrease in reactivity was not discovered. From the results obtained, it was concluded that the observation of serum IgG antibody reactivity to protein antigens of T. pallidum is not helpful in evaluating the efficacy of treatment in secondary, early latent, late latent and reinfected syphilis. However, serum IgG antibodies to treponemal antigens of molecular weights 68,500 and 47,000 could possibly be useful in the assessment of the efficacy of treatment in primary syphilis.

Changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum in syphilis patients after treatment.

PubMed Central

Kim, D. K.; Lee, M. G.; Lee, J. B.

1989-01-01

The changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum after treatment of syphilis were observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Until 9 to 12 months after treatment, it was seen that there was a loss of several antibodies and some diminution in their reactivity in primary, secondary and early latent syphilis, but no changes occurred in late latent and reinfected syphilis. In primary syphilis, there was a significant loss of two IgG antibodies to the treponemal antigens of molecular weights 68,500 and 47,000 at 11 months after treatment. According to our previous study, the treponemal antigen of molecular weight 68,500 was T. pallidum specific and appeared only in primary syphilis, and that of molecular weight 47,000 was one of the major antigens of T. pallidum. The reaction between serum IgG antibodies of 14 patients who had been treated for secondary, early latent and late latent syphilis 2 to 14 years ago and major antigens of T. pallidum was observed and any loss or decrease in reactivity was not discovered. From the results obtained, it was concluded that the observation of serum IgG antibody reactivity to protein antigens of T. pallidum is not helpful in evaluating the efficacy of treatment in secondary, early latent, late latent and reinfected syphilis. However, serum IgG antibodies to treponemal antigens of molecular weights 68,500 and 47,000 could possibly be useful in the assessment of the efficacy of treatment in primary syphilis. PMID:2688687

Cationic liposomes as vaccine adjuvants.

PubMed

Christensen, Dennis; Korsholm, Karen S; Rosenkrands, Ida; Lindenstrøm, Thomas; Andersen, Peter; Agger, Else Marie

2007-10-01

Cationic liposomes are lipid-bilayer vesicles with a positive surface charge that have re-emerged as a promising new adjuvant technology. Although there is some evidence that cationic liposomes themselves can improve the immune response against coadministered vaccine antigens, their main functions are to protect the antigens from clearance in the body and deliver the antigens to professional antigen-presenting cells. In addition, cationic liposomes can be used to introduce immunomodulators to enhance and modulate the immune response in a desirable direction and, thereby, represent an efficient tool when designing tailor-made adjuvants for specific disease targets. In this article we review the recent progress on cationic liposomes as vehicles, enhancing the effect of immunomodulators and the presentation of vaccine antigens.

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

NASA Technical Reports Server (NTRS)

Knisley, Keith A.; Rodkey, L. Scott

1986-01-01

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

PubMed

Wu, Julie H; Simonette, Rebecca A; Nguyen, Harrison P; Rady, Peter L; Tyring, Stephen K

2016-06-01

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host

PubMed Central

Marques, Lucas M.; Rezende, Izadora S.; Barbosa, Maysa S.; Guimarães, Ana M. S.; Martins, Hellen B.; Campos, Guilherme B.; do Nascimento, Naíla C.; dos Santos, Andrea P.; Amorim, Aline T.; Santos, Verena M.; Farias, Sávio T.; Barrence, Fernanda Â. C.; de Souza, Lauro M.; Buzinhani, Melissa; Arana-Chavez, Victor E.; Zenteno, Maria E.; Amarante-Mendes, Gustavo P.; Messick, Joanne B.; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)—Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis. PMID:27603136

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host.

PubMed

Marques, Lucas M; Rezende, Izadora S; Barbosa, Maysa S; Guimarães, Ana M S; Martins, Hellen B; Campos, Guilherme B; do Nascimento, Naíla C; Dos Santos, Andrea P; Amorim, Aline T; Santos, Verena M; Farias, Sávio T; Barrence, Fernanda  C; de Souza, Lauro M; Buzinhani, Melissa; Arana-Chavez, Victor E; Zenteno, Maria E; Amarante-Mendes, Gustavo P; Messick, Joanne B; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.

Exploiting the pliability and lateral mobility of Pickering emulsion for enhanced vaccination

NASA Astrophysics Data System (ADS)

Xia, Yufei; Wu, Jie; Wei, Wei; Du, Yiqun; Wan, Tao; Ma, Xiaowei; An, Wenqi; Guo, Aiying; Miao, Chunyu; Yue, Hua; Li, Shuoguo; Cao, Xuetao; Su, Zhiguo; Ma, Guanghui

2018-02-01

A major challenge in vaccine formulations is the stimulation of both the humoral and cellular immune response for well-defined antigens with high efficacy and safety. Adjuvant research has focused on developing particulate carriers to model the sizes, shapes and compositions of microbes or diseased cells, but not antigen fluidity and pliability. Here, we develop Pickering emulsions--that is, particle-stabilized emulsions that retain the force-dependent deformability and lateral mobility of presented antigens while displaying high biosafety and antigen-loading capabilities. Compared with solid particles and conventional surfactant-stabilized emulsions, the optimized Pickering emulsions enhance the recruitment, antigen uptake and activation of antigen-presenting cells, potently stimulating both humoral and cellular adaptive responses, and thus increasing the survival of mice upon lethal challenge. The pliability and lateral mobility of antigen-loaded Pickering emulsions may provide a facile, effective, safe and broadly applicable strategy to enhance adaptive immunity against infections and diseases.

Towards preserving the immunogenicity of protein antigens carried by nanoparticles while avoiding the cold chain.

PubMed

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

2010-06-30

Nanoparticles are an attractive vaccine carrier with potent adjuvant activity. Data from our previous studies showed that immunization of mice with lecithin/glyceryl monostearate-based nanoparticles with protein antigens conjugated onto their surface induced a strong, quick, and long-lasting antigen-specific immune response. In the present study, we evaluated the feasibility of preserving the immunogenicity of protein antigens carried by nanoparticles without refrigeration using these antigen-conjugated nanoparticles as a model. The nanoparticles were lyophilized, and the immunogenicity of the antigens was evaluated in a mouse model using bovine serum albumin or the Bacillus anthracis protective antigen protein as model antigens. With proper excipients, the nanoparticles can be lyophilized while maintaining the immunogenicity of the antigens. Moreover, the immunogenicity of the model antigen conjugated onto the nanoparticles was undamaged after a relatively extended period of storage at room temperature or under accelerated conditions (37 degrees C) when the nanoparticles were lyophilized with 5% mannitol plus 1% polyvinylpyrrolidone. To our knowledge, the present study represents an early attempt to preserve the immunogenicity of the protein antigens carried by nanoparticles without refrigeration. 2010 Elsevier B.V. All rights reserved.