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1
Fernández-Quintero, Monica L., Johannes R. Loeffler, Franz Waibl, Anna S. Kamenik, Florian Hofer, and Klaus R. Liedl. "Conformational selection of allergen-antibody complexes—surface plasticity of paratopes and epitopes." Protein Engineering, Design and Selection 32, no. 11 (November 2019): 513–23. http://dx.doi.org/10.1093/protein/gzaa014.
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Abstract Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.
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Mavenyengwa, Rooyen T., Johan A. Maeland, and Sylvester R. Moyo. "Putative Novel Surface-Exposed Streptococcus agalactiae Protein Frequently Expressed by the Group B Streptococcus from Zimbabwe." Clinical and Vaccine Immunology 16, no. 9 (July 8, 2009): 1302–8. http://dx.doi.org/10.1128/cvi.00133-09.
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ABSTRACTGroup B streptococci (GBS) express a variety of surface-exposed and strain-variable proteins which function as phenotypic markers and as antigens which are able to induce protective immunity in experimental settings. Among these proteins, the chimeric and immunologically cross-reacting alpha-like proteins are particularly important. Another protein, R3, which has been less well studied, occurred at a frequency of 21.5% in GBS from Zimbabwe and, notably, occurred in serotype V strains at a frequency of 75.9%. Working with rabbit antiserum raised against the R3 reference strain ATCC 49447 (strain 10/84; serotype V/R3) to detect the expression of the R3 protein, we recorded findings which suggested that strain 10/84 expressed a strain-variable protein antigen, in addition to R3. The antigen was detected by various enzyme-linked immunosorbent assay-based tests by using acid extract antigens or GBS whole-cell coats and by whole-cell-based Western blotting. We named the putative novel antigen the Z antigen. The Z antigen was a high-molecular-mass antigen that was susceptible to degradation by pepsin and trypsin but that was resistant tom-periodate oxidation and failed to show immunological cross-reactivity with any of a variety of other GBS protein antigens. The Z antigen was expressed by 33/121 (27.2%) of strains of a Zimbabwean GBS strain collection and by 64.2% and 72.4% of the type Ib and type V strains, respectively, and was occasionally expressed by GBS of other capsular serotypes. Thus, the putative novel GBS protein named Z showed distinct capsular antigen associations and presented as an important phenotypic marker in GBS from Zimbabwe. It may be an important antigen in GBS from larger areas of southern Africa. Its prevalence in GBS from Western countries is not known.
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Elkon, K. B., and P. W. Jankowski. "Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting." Journal of Immunology 134, no. 6 (June 1, 1985): 3819–24. http://dx.doi.org/10.4049/jimmunol.134.6.3819.
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Abstract Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.
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Delaney, Kristen N., and Steven B. Mizel. "A vaccine containing recombinant poxvirus proteins and flagellin promotes protective immunity against vaccinia virus (132.17)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 132.17. http://dx.doi.org/10.4049/jimmunol.182.supp.132.17.
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Abstract Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore, we evaluated the response to these antigens when flagellin was used as an adjuvant. We found that flagellin promoted a robust antigen-specific humoral response to poxvirus antigens delivered intranasally and intramuscularly, but intramuscular immunization was more efficient. Flagellin/poxvirus antigen fusion proteins promoted humoral responses that were greater in magnitude than antigen and flagellin as separate proteins. However, there was competition when more than one fusion protein was administered. At least three immunizations with flagellin/poxvirus fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Although mice were protected, they still exhibited significant, but reversible weight loss. This work was supported by a grant from the National Institutes of Health, P01 AI 60642 (to S.B.M.)
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Lichtenwalner, Anne B., Dorothy L. Patton, Wesley C. Van Voorhis, Yvonne T. Cosgrove Sweeney, and Cho-Chou Kuo. "Heat Shock Protein 60 Is the Major Antigen Which Stimulates Delayed-Type Hypersensitivity Reaction in the Macaque Model of Chlamydia trachomatis Salpingitis." Infection and Immunity 72, no. 2 (February 2004): 1159–61. http://dx.doi.org/10.1128/iai.72.2.1159-1161.2004.
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ABSTRACT Chlamydial delayed-type hypersensitivity antigens were analyzed by using the subcutaneous salpingeal autotransplant model of Macaca nemestrina infected with Chlamydia trachomatis serovar E. Heat shock protein 60 was the only antigen shown to induce delayed-type hypersensitivity among other antigens tested, including UV-inactivated organisms, recombinant major outer membrane protein, purified outer membrane proteins, and heat shock protein 10.
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Yu, Hong, Karuna P. Karunakaran, Xiaozhou Jiang, Caixia Shen, Peter Andersen, and Robert C. Brunham. "Chlamydia muridarum T Cell Antigens and Adjuvants That Induce Protective Immunity in Mice." Infection and Immunity 80, no. 4 (January 30, 2012): 1510–18. http://dx.doi.org/10.1128/iai.06338-11.
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ABSTRACTMajor impediments to aChlamydiavaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model againstChlamydiainfection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for aChlamydiavaccine, we evaluated 10 newChlamydiaT cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6′-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720–CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4+T cells coexpressing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13ChlamydiaT cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly definedChlamydiavaccine.
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Rennert, Paul, Lan Wu, Lihe Su, Roy Lobb, and Christine Ambrose. "160 Evaluation and development of dual and triple antigen targeting CAR-T Engager proteins for Her2-positive CNS metastases and solid tumors." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A170. http://dx.doi.org/10.1136/jitc-2021-sitc2021.160.
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BackgroundSolid tumors display pronounced antigen heterogeneity and clinical studies have shown that antigen escape from therapy occurs rapidly, limiting the persistence and efficacy of CAR T cells. Here we present dual and triple-antigen binding proteins that bridge CAR T cells to multiple antigens, allowing a simultaneous attack on tumor antigens by a single CAR T antibody domain. We call these CAR T Engager proteins. CAR T Engager proteins can be encoded into lentiviral vectors for secretion from CAR T cells, can be encoded into oncolytic viral vectors for secretion from transduced tumor cells, or can be engineered as biologics for injection.MethodsCAR T Engagers contain a protein target for a CAR T cell, eg. an anti-CD19 CAR T cell. We have previously presented a Her2-binding CAR T Engager protein with potent in vivo activity against solid tumors. We used this CAR T Engager as the basis for building dual and triple antigen binding proteins. Specifically, we mapped antigen expression for Her2-positive solid tumors, Her2-positive metastases, and primary CNS tumors. Our analysis identified expression patterns of two and three antigens that would essentially saturate the cellular composition of specific solid tumors, greatly reducing the chance of antigen escape from therapy. We created the corresponding CAR T Engagers and have developed single, dual and triple antigen expression cells lines to model the activity and potency of these novel proteins, administered alongside CAR T cells.ResultsCAR T cells plus dual antigen CAR T Engagers that recognize and target Her2 and B7H3 demonstrate potent cytotoxicity against either antigen alone, and synergistic potency (2 pM) if both antigens are expressed. Similarly, triple antigen CART Engagers show single antigen binding and potent cytotoxicity which is enhanced when multiple antigens are expressed on a target cell. All of the cytotoxicity is mediated through one CAR domain expressed on the primary T cells. T cells can be pre-loaded with multi-antigen CAR T Engagers and retain cytotoxic activity. Because the underlying CAR is an anti-CD19 CAR, cell persistence and fitness is further enhanced in the presence of normal B cells.ConclusionsThe CAR T Engager platform is a robust and modular solution for the multi-antigen targeting of solid tumors. Diverse antigens can be readily targeted for diverse indications. Examples of other functional modalities that can be added will be presented.AcknowledgementsWe thank Cancer Research UK for their ongoing support.
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Kim, Ae, Isamu Hartman, and Scheherazade Sadegh-Nasseri. "A cell free antigen processing system identifies immunodominant epitopes (78.19)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 78.19. http://dx.doi.org/10.4049/jimmunol.182.supp.78.19.
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Abstract T cell-mediated responses to protein antigen involve recognition of selected antigenic peptides bound to Major Histocompatibility Complex (MHC) molecules. Proteolytic digestion of protein antigens during antigen processing generates many peptide fragments that can potentially bind to MHC molecules. However, only few selected antigenic epitopes induce T cell responses, defined as "immunodominant". Identifying immunodominant epitopes from a given antigen has wide applications in development of peptide vaccines and therapies against infectious diseases, cancer, autoimmune diseases, and allergy. Currently available methods are not efficient in elucidating CD4+ T cell epitopes. Here, we provide a novel approach for identifying the immunodominant epitopes from protein antigens using a cell-free MHC class II antigen-processing assay. Two different novel antigens, Hemagglutinin (HA1) of the A/Vietnam/1203/2004 (H5N1) virus, and a recombinant form of Plasmodium falciparum liver-stage antigen 1 (LSA-NRC) were used to evaluate our assay. HLA-DR1 restricted epitopes from both proteins were identified by mass spectrometry and were tested for their ability to activate T cells in HLA-DR1 tg mice. We observed that the identified epitopes were indeed immunodominant as CD4+ T cells from mice immunized with the proteins proliferated and produced cytokines upon challenge with the identified epitopes at levels comparable to the whole protein. This cell-free assay provides a useful tool for identification of CD4+ T cell epitopes for specific MHC class II alleles.
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Zou, Jin-Tao, Hai-Ming Jing, Yue Yuan, Lang-Huan Lei, Zhi-Fu Chen, Qiang Gou, Qing-Shan Xiong, et al. "Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens." PLOS Pathogens 17, no. 7 (July 21, 2021): e1009752. http://dx.doi.org/10.1371/journal.ppat.1009752.
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Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.
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Arribillaga, Laura, Maika Durantez, Teresa Lozano, Francesc Rudilla, Federico Rehberger, Noelia Casares, Lorea Villanueva, et al. "A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell ResponsesIn Vivo." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/864720.
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The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with aKd~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβby TLR4-expressing cells, as well as the production of TNF-αby the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
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Venditti, Paola, Paolo Bergamo, Riccardo Talevi, Giovanni sansone, and Paolo Abrescia. "Localisation and capacitation-dependent loss of buffalo sperm-coating antigens shared with rat sperm." Zygote 2, no. 1 (February 1994): 5–13. http://dx.doi.org/10.1017/s0967199400001714.
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SummaryThe heterodimeric sperm-coating protein CFS was previously localised on the middle-piece region of rat spermatozoa by anti-CFS rabbit antibodies. CFS-immunorelated antigens were detected in the secretion of the water buffalo seminal vesicle by protein electrophoresis and Western blotting. Spermatozoa from buffalo epididymal cauda were incubated with the rat antigen and, upon immunostaining with anti-CFS antibodies and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgGs, CFS was found attached on both the post-acrosomal region and the tail. Indirect immunofluorescence analysis permitted the localisation of CFS-related antigens on the same domains of buffalo ejaculated spermatozoa. These results suggest that the buffalo antigens not only share some epitopes with the homologous rat antigen but may also have some of its functional properties. Ejaculated spermatozoa were capacitated in vitro and then assayed for their content of CFS-like antigens. An inverse relationship was found between the levels of capacitation and the amounts of antigens detected, thus suggesting that the in vitro treatment was effective at removing CFS-related proteins from the cell surface. Titration of these proteins to proteins to monitor plasma membrane changes during sperm manipulation or to evaluate sperm quality is proposed.
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O’Rourke, Sara M., Giora I. Morozov, Jacob T. Roberts, Adam W. Barb, and Nikolaos G. Sgourakis. "Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR." Protein Engineering, Design and Selection 32, no. 12 (December 2019): 525–32. http://dx.doi.org/10.1093/protein/gzaa015.
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Abstract Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.
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Geiger, Rebekka, Thomas Duhen, Antonio Lanzavecchia, and Federica Sallusto. "Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells." Journal of Experimental Medicine 206, no. 7 (June 29, 2009): 1525–34. http://dx.doi.org/10.1084/jem.20090504.
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The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen dose–response of human naive CD4+ T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 106 naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.
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Simmons, Ryan S., Erik Cram, Amy Palmer, and Brian Dolan. "Chlamydial infection and the efficiency of MHC class I self-antigen presentation in a lymphoblastoid cell line." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 116.20. http://dx.doi.org/10.4049/jimmunol.196.supp.116.20.
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Abstract The direct MHC class I antigen presentation pathway presents cytosolic proteins for immune surveillance. While this pathway is an attractive target for therapeutics, we must first understand the efficiency of antigen presentation. Previous studies have shown the presentation efficiency from pathogen-derived antigens ranges from 0.03–25%. The intracellular lifestyle of Chlamydia makes them an attractive model to study class I presentation. It is likely that Chlamydia have developed mechanisms to evade CD8+ T cells through manipulation of this pathway. Here, we investigate the impact of Chlamydial infection on a recombinant fusion protein expressed in a human lymphoblastoid cell line (JY). The fusion protein contains a destabilization domain, an antigenic peptide, and a reporter. Chlamydial infection led to decreased accumulation of the model protein and increased presentation of model protein-derived antigens. This enhanced self-peptide presentation was only observed when antigen presentation was restricted to the DRiP form of the protein. We also investigated the antigen presentation efficiency of different model recombinant proteins in JY cells. We calculated the antigen presentation efficiency to be 0.35%, much lower than was previously observed in murine cells with a similar protein. The skewed antigen presentation phenotype observed following Chlamydial infection was not statistically different from the efficiency observed in the absence of infection. These data suggest that Chlamydia have evolved a mechanism to skew the host antigen presentation machinery toward presentation of DRiPs, and that the efficiency of direct class I presentation may be mechanistically unique among common immune signaling pathways.
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Kim, Deuk-Su, Yang Joo Kang, Kyung Jin Lee, Lu Qiao, Kinarm Ko, Dae Heon Kim, Soon Chul Myeung, and Kisung Ko. "A Plant-Derived Antigen–Antibody Complex Induces Anti-Cancer Immune Responses by Forming a Large Quaternary Structure." International Journal of Molecular Sciences 21, no. 16 (August 5, 2020): 5603. http://dx.doi.org/10.3390/ijms21165603.
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The antigen–antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production.
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McWilliam, Hamish E. G., Jeffrey Y. W. Mak, Wael Awad, Matthew Zorkau, Sebastian Cruz-Gomez, Hui Jing Lim, Yuting Yan, et al. "Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens." Proceedings of the National Academy of Sciences 117, no. 40 (September 21, 2020): 24974–85. http://dx.doi.org/10.1073/pnas.2011260117.
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The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.
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van der Put, Robert M. F., Bernard Metz, and Roland J. Pieters. "Carriers and Antigens: New Developments in Glycoconjugate Vaccines." Vaccines 11, no. 2 (January 19, 2023): 219. http://dx.doi.org/10.3390/vaccines11020219.
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Glycoconjugate vaccines have proven their worth in the protection and prevention of infectious diseases. The introduction of the Haemophilus influenzae type b vaccine is the prime example, followed by other glycoconjugate vaccines. Glycoconjugate vaccines consist of two components: the carrier protein and the carbohydrate antigen. Current carrier proteins are tetanus toxoid, diphtheria toxoid, CRM197, Haemophilus protein D and the outer membrane protein complex of serogroup B meningococcus. Carbohydrate antigens have been produced mainly by extraction and purification from the original host. However, current efforts show great advances in the development of synthetically produced oligosaccharides and bioconjugation. This review evaluates the advances of glycoconjugate vaccines in the last five years. We focus on developments regarding both new carriers and antigens. Innovative developments regarding carriers are outer membrane vesicles, glycoengineered proteins, new carrier proteins, virus-like particles, protein nanocages and peptides. With regard to conjugated antigens, we describe recent developments in the field of antimicrobial resistance (AMR) and ESKAPE pathogens.
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Ciccotelli, Jo Erika, Helene Toussaint, Geza Erdos, Cara Carey, Simon Watkins, and Louis Falo. "Intradermal immunization with polyguanine conjugated antigens enables targeted and sustained delivery of protein antigens to dendritic cells in vivo. (APP3P.111)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 111.12. http://dx.doi.org/10.4049/jimmunol.192.supp.111.12.
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Abstract Effective vaccine design depends on the ability to target specific antigens to antigen presenting cells (APCs), including dendritic cells (DCs). Conjugation of proteins antigens to polyguanine (Poly(dG)) molecules is a novel immunization approach capable of transforming soluble antigens into aggregated particulates with exposed scavenger receptor (SR) ligands. We have previously shown that Poly(dG) conjugation to protein antigen results in increased antigen-specific helper and cytotoxic T cell responses, memory T cell induction, and antibody titers. Here, we specifically investigate mechanisms of Poly(dG)-conjugated antigen delivery and internalization. Compared to soluble OVA, Poly(dG)-OVA is rapidly and more efficiently internalized by bone marrow derived DCs in vitro, and this internalization is inhibited by scavenger receptor blockade. Importantly, in a mouse model intradermally injected Poly(dG)-OVA results in antigen persistence in the skin for up to 7 days. This is accompanied by increased antigen uptake by skin resident DCs and persistent migration of antigen loaded DCs to the draining lymph nodes. DCs exposed to Poly(dG)-OVA had increased expression of CCR7 and secretion of MCP-1, TNF-α, and IL-6. These results suggest that coupling Poly(dG) to protein antigens enables efficient DC targeting through SRs, prolonged delivery of antigens in vivo, and activation of innate immunity. This approach may be used to design more efficient antiviral and antitumor vaccines.
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Elkon, K. B., A. P. Parnassa, and C. L. Foster. "Lupus autoantibodies target ribosomal P proteins." Journal of Experimental Medicine 162, no. 2 (August 1, 1985): 459–71. http://dx.doi.org/10.1084/jem.162.2.459.
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All nine SLE (systemic lupus erythematosus) sera with antiribosomal antibody activity targeted the same three ribosomal protein antigens, of molecular masses 38 and 17/19 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. One serum reacted with an additional protein of approximately kD. Ribosomal subunit fractionation by composite gel electrophoresis and sucrose density ultracentrifugation showed that these proteins were part of the large subunit. Isoelectric focusing in agarose, and two-dimensional polyacrylamide gel electrophoresis revealed that the antigens had pI between 4.5 and 6.5, but that the 17/19 kD antigens were more acidic than the 38 kD antigen. Similarities in the molecular masses, charges, as well as the presence of highly conserved crossreactive epitopes, failure to bind to carboxymethylcellulose at pH 4.2, and extractability of the 17/19 kD proteins by 400 mM NH4Cl-ethanol at 0 degrees C indicated that these antigens were analogous to the proteins P0 (38 kD) and P1/P2 (17/19 kD) described previously (25, 36). Co-identity was confirmed using reference antibodies and antigen. Although antibodies to these proteins were only found in 5-10% of more than 50 sera screened by radioimmunoassay or Western blotting, the selective production of antibodies to epitopes on three (out of a total of more than 80) ribosomal proteins may provide further clues to autoantibody induction of SLE.
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Beare, Paul A., Chen Chen, Timo Bouman, Jozelyn Pablo, Berkay Unal, Diane C. Cockrell, Wendy C. Brown, et al. "Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray." Clinical and Vaccine Immunology 15, no. 12 (October 8, 2008): 1771–79. http://dx.doi.org/10.1128/cvi.00300-08.
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ABSTRACT Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.
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Murata, Makoto, Edus H. Warren, and Stanley R. Riddell. "A Human Minor Histocompatibility Antigen Resulting from Differential Expression due to a Gene Deletion." Journal of Experimental Medicine 197, no. 10 (May 12, 2003): 1279–89. http://dx.doi.org/10.1084/jem.20030044.
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Minor histocompatibility antigens (minor H antigens) are targets of graft-versus-host disease and graft-versus-leukemia responses after allogeneic human leukocyte antigen identical hematopoietic stem cell transplantation. Only a few human minor H antigens have been molecularly characterized and in all cases, amino acid differences between homologous donor and recipient proteins due to nucleotide polymorphisms in the respective genes were responsible for immunogenicity. Here, we have used cDNA expression cloning to identify a novel human minor H antigen encoded by UGT2B17, an autosomal gene in the multigene UDP-glycosyltransferase 2 family that is selectively expressed in liver, intestine, and antigen-presenting cells. In contrast to previously defined human minor H antigens, UGT2B17 is immunogenic because of differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion in the donor. Deletion of individual members of large gene families is a common form of genetic variation in the population and our results provide the first evidence that differential protein expression as a consequence of gene deletion is a mechanism for generating minor H antigens in humans.
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Li, Dan, Haozhi Song, Jialei Li, Xingjian Liu, Xintao Gao, Tong Wu, Zhifang Zhang, and Yinü Li. "Expression and Evaluation of a Novel PPRV Nanoparticle Antigen Based on Ferritin Self-Assembling Technology." Pharmaceutics 14, no. 9 (September 8, 2022): 1902. http://dx.doi.org/10.3390/pharmaceutics14091902.
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Peste des Petits Ruminants (PPR) is a highly pathogenic disease that is classified as a World Organization for Animal Health (OIE)-listed disease. PPRV mainly infects small ruminants such as goats and sheep. In view of the global and high pathogenicity of PPRV, in this study, we proposed a novel nanoparticle vaccine strategy based on ferritin (Fe) self-assembly technology. Using Helicobacter pylori (H. pylori) ferritin as an antigen delivery vector, a PPRV hemagglutinin (H) protein was fused with ferritin and then expressed and purified in both Escherichia coli (E. coli) and silkworm baculovirus expression systems. Subsequently, the nanoparticle antigens’ expression level, immunogenicity and protective immune response were evaluated. Our results showed that the PPRV hemagglutinin–ferritin (H-Fe) protein was self-assembled in silkworms, while it was difficult to observe the correctly folded nanoparticle in E. coli. Meanwhile, the expression level of the H-Fe protein was higher than that of the H protein alone. Furthermore, the immunogenicity and protective immune response of H-Fe nanoparticle antigens expressed by silkworms were improved compared with the H antigen alone. Particularly, the protective immune response of H-Fe antigens expressed in E. coli did not change, as opposed to the H antigen, which was probably due to the incomplete nanoparticle structure in E. coli. This study indicated that the use of ferritin nanoparticles as antigen delivery carriers could increase the expression of antigen proteins and improve the immunogenicity and immune effect of antigens.
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Cornillez-Ty, Cromwell T., and David W. Lazinski. "Determination of the Multimerization State of the Hepatitis Delta Virus Antigens In Vivo." Journal of Virology 77, no. 19 (October 1, 2003): 10314–26. http://dx.doi.org/10.1128/jvi.77.19.10314-10326.2003.
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ABSTRACT Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.
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Rapoport, Basil, Hideshi Hirayu, Pui Seto, and Ronald P. Magnusson. "Molecular cloning of antigens to thyroid autoantibodies using the expression vector lambda gt 11." Acta Endocrinologica 116, no. 1_Suppl (August 1987): S139—S145. http://dx.doi.org/10.1530/acta.0.114s139.
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Abstract. The molecular cloning of certain antigens to thyroid autoantibodies present in patients with autoimmune thyroid disease would be of great value in further understanding the pathogenesis of these diseases, and in devising new approaches for their treatment. The ideal method for molecular cloning is to first obtain a partial amino acid sequence of a purified protein and to construct an oligonucleotide probe for screening an appropriate cDNA library. Unfortunately in the case if thyroid autoimmunity, important antigens such as the TSH receptor and the microsomal antigen have not been purified. An alternate approach devised by Young & Davis (1983) is to construct a cDNA library in a vector that expresses the encoded proteins. The library can then be screened with an antibody as a probe. We have constructed cDNA libraries in gt11 using mRNA purified from human, pig and rat thyroid cells. Our experiences in constructing and screening these libraries will be described. The advantages of this system are 1) the protein does not have to be purified, 2) previously unknown antigens may be identified. The disadvantages are 1) lack of specificity with antibody selection, 2) because the cDNA is inserted in the β-galactosidase gene in the vector the antigen is expressed as a fusion protein. This may disturb the tertiary structure of the antigen and alter its antigenicity, 3) cDNA inserts frequently only contain part of the antigen molecule, and may therefore lack important epitopes; polyclonal antibody may therefore be preferable to monoclonal, 4) only 1 in 6 cDNA inserts will be in the correct reading frame for antigen expression, 5) the expressed protein is not glycosylated, 6) antibody absorption or purification is necessary to reduce the high background of antibody binding to bacterial host protein products. Despite these disadvantages the system has been used to clone numerous proteins. Thus far we have been successful in cloning 3 newly recognized autoimmune thyroid disease-related antigens that are of potential importance in further understanding the nature of these diseases.
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Reeder, Sophia M., Emma L. Reuschel, Mamadou A. Bah, Kun Yun, Nicholas J. Tursi, Kevin Y. Kim, Jacqueline Chu, et al. "Synthetic DNA Vaccines Adjuvanted with pIL-33 Drive Liver-Localized T Cells and Provide Protection from Plasmodium Challenge in a Mouse Model." Vaccines 8, no. 1 (January 10, 2020): 21. http://dx.doi.org/10.3390/vaccines8010021.
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The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%–88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.
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Li, Charles, Hyun Lillehoj, Xianghe Yan, Zhifeng Sun, Mingmin Lu, Baohong Yuan, and Liheng Liu. "Immunization with a subunit of housekeeping proteins provide partial protection against experimental necrotic enteritis in broiler chickens." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 168.25. http://dx.doi.org/10.4049/jimmunol.204.supp.168.25.
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Abstract Necrotic enteritis (NE) due to Clostridium perfringens (CP) infections is a prevalent enteric infectious disease accounting for $6 billion economical loss in global poultry industry. The increasing incidence of NE has been associated with the reduction and withdrawal of antibiotic growth promoters from animal feed during recent years. Therefore, the development of effective vaccines specific for NE assumes a priority for poultry industry. By far, no any effective vaccines are commercially available in broiler chickens. Our aim was to identify the potential CP proteins as vaccine targets for NE. We expressed 4 different recombinant CP proteins targeting 6 antigens: 3 major virulence-related toxins (alpha-toxin, NetB and TpeL), and 3 pilus subunit or enzymes (Collagen adhesion protein (Cna), Fructose-1,6-bisphosphate aldolase (FBA) and a Hypothetic protein (HP)), and their protection efficacies were evaluated with a severe Coccidiosis/NE challenge model using netB+tpeL+ CP strain. Young chicks were immunized twice subcutaneously with adjuvanted CP proteins on days 4, and 15. Prior to challenge, the chickens immunized with related antigens had much higher serum antibody titers for Cna and FBA/HP antigens than those with any of 3 toxin antigen-immunized groups. Following the challenge, the Cna-immunized group showed the least mortality and lowest lesion score among the individual antigen-vaccinated groups, while the pooled antigens-immunized group demonstrated no mortality against virulent challenges. The results indicate that the immunization with CP housekeeping protein, Cna, conferred better protection than other conventional virulence-related toxin proteins. Mucosal delivery system is being investigated next.
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Newcombe, Jane, Tom A. Mendum, Chuan-peng Ren, and Johnjoe McFadden. "Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS." Microbiology 160, no. 2 (February 1, 2014): 429–38. http://dx.doi.org/10.1099/mic.0.071829-0.
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Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.
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Lajoie, Jason M., Yong Ku Cho, Dustin Frost, Samantha Bremner, Lingjun Li, and Eric V. Shusta. "A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes." Protein Engineering, Design and Selection 32, no. 5 (May 2019): 219–30. http://dx.doi.org/10.1093/protein/gzz035.
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Abstract Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.
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Rao, N., CF Whitsett, SM Oxendine, and MJ Telen. "Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype." Blood 81, no. 3 (February 1, 1993): 815–19. http://dx.doi.org/10.1182/blood.v81.3.815.815.
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Abstract The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.
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Rao, N., CF Whitsett, SM Oxendine, and MJ Telen. "Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype." Blood 81, no. 3 (February 1, 1993): 815–19. http://dx.doi.org/10.1182/blood.v81.3.815.bloodjournal813815.
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The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.
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Fouda, Genevieve G., Rose F. G. Leke, Carole Long, Pierre Druilhe, Ainong Zhou, Diane Wallace Taylor, and Armead H. Johnson. "Multiplex Assay for Simultaneous Measurement of Antibodies to Multiple Plasmodium falciparum Antigens." Clinical and Vaccine Immunology 13, no. 12 (October 11, 2006): 1307–13. http://dx.doi.org/10.1128/cvi.00183-06.
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ABSTRACTAntibodies toPlasmodium falciparumare classically measured using the enzyme-linked immunosorbent assay (ELISA). Although highly sensitive, this technique is labor-intensive when large numbers of samples must be screened against multiple antigens. The suspension array technology (SAT) might be an alterative to ELISA, as it allows measurement of antibodies against multiple antigens simultaneously with a small volume of sample. This study sought to adapt the new SAT multiplex system for measuring antibodies against nine malarial vaccine candidate antigens, including recombinant proteins from two variants of merozoite surface protein 1, two variants of apical merozoite antigen 1, erythrocyte binding antigen 175, merozoite surface protein 3, and peptides from the circumsporozoite protein, ring erythrocyte surface antigen, and liver-stage antigen 1. Various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Cameroonian adults were screened by SAT in mono- and multiplex formats and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10- to 100-fold less than that needed for ELISA. Excellent agreement was found between the single and multiplex formats (R≥ 0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 μl of plasma, and had a good correlation with ELISA. Thus, SAT provides an important new tool for studying the immune response to malaria rapidly and efficiently in large populations, even when the amount of plasma available is limited, e.g., in studies of neonates or finger-prick blood.
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Terkawi, Mohamad Alaa, Nguyen Xuan Huyen, Putut Eko Wibowo, Faasoa Junior Seuseu, Mahmoud Aboulaila, Akio Ueno, Youn-Kyoung Goo, Naoaki Yokoyama, Xuenan Xuan, and Ikuo Igarashi. "Spherical Body Protein 4 Is a New Serological Antigen for Global Detection ofBabesia bovisInfection in Cattle." Clinical and Vaccine Immunology 18, no. 2 (December 1, 2010): 337–42. http://dx.doi.org/10.1128/cvi.00388-10.
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ABSTRACTFiveBabesia bovisrecombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentallyB. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentallyB. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions ofB. bovisendemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies toB. bovisin cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.
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Doyle, Hester A., Jing Zhou, Martin J. Wolff, Bohdan P. Harvey, Robert M. Roman, Renelle J. Gee, Raymond A. Koski, and Mark J. Mamula. "Isoaspartyl Post-translational Modification Triggers Anti-tumor T and B Lymphocyte Immunity." Journal of Biological Chemistry 281, no. 43 (September 1, 2006): 32676–83. http://dx.doi.org/10.1074/jbc.m604847200.
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A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.
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Ogun, Solabomi A., Laurence Dumon-Seignovert, Jean-Baptiste Marchand, Anthony A. Holder, and Fergal Hill. "The Oligomerization Domain of C4-Binding Protein (C4bp) Acts as an Adjuvant, and the Fusion Protein Comprised of the 19-Kilodalton Merozoite Surface Protein 1 Fused with the Murine C4bp Domain Protects Mice against Malaria." Infection and Immunity 76, no. 8 (May 12, 2008): 3817–23. http://dx.doi.org/10.1128/iai.01369-07.
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ABSTRACT Highly purified protein antigens are usually poor immunogens; in practice, adjuvants are needed to obtain satisfactory immune responses. Plasmodium yoelii 19-kDa merozoite surface protein 1 (MSP119) is a weak antigen, but mice vaccinated with this antigen in strong adjuvants can survive an otherwise lethal parasite challenge. Fusion proteins comprising this antigen fused to the oligomerization domain of the murine complement inhibitor C4-binding protein (C4bp) and a series of homologues have been produced. These C4bp domains acted as adjuvants for the fused antigen; the MSP119-murine C4bp fusion protein induced protective immunity in BALB/c mice. Because this fusion protein also induced antibodies against circulating murine C4bp, distantly related C4bp oligomerization domains fused to the same antigen were tested. These homologous domains did not induce antibodies against murine C4bp and, surprisingly, induced higher antibody titers against the antigen than the murine C4bp domain induced. These results demonstrate a new adjuvantlike effect of C4bp oligomerization domains.
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Velaz-Faircloth, Maria, Alison J. Cobb, Amanda L. Horstman, Stanley C. Henry, and Richard Frothingham. "Protection against Mycobacterium aviumby DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein." Infection and Immunity 67, no. 8 (August 1, 1999): 4243–50. http://dx.doi.org/10.1128/iai.67.8.4243-4250.1999.
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ABSTRACT Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, theMycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination againstM. avium.
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Sinitsyn, B. F. "Detecting a psoriatic antigen analogous to infectious prion proteins." Russian Journal of Infection and Immunity 9, no. 3-4 (November 15, 2019): 589–94. http://dx.doi.org/10.15789/2220-7619-2019-3-4-589-594.
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Until now, psoriatic antigen as a specific antigen derived from some infectious agent potentially related to origin of psoriasis has not been identified, thereby strongly arguing against infectious theory of psoriasis. However, the lack of specific psoriasis-associated antigens may be theoretically accounted for by an idea that psoriatic antigen could be analogous to infectious prion proteins (PrPSc analogue). It might be identical to some epidermal protein in psoriasis-free subjects that might differ antigenically from related normal protein by resistance to digestive enzymes similarly to PrPSc. Since PrPSc cytopathogenic effect is associated with its location within cell structures of affected body tissue, by analogy with PrPSc we aimed at identifying a potential carrier of psoriatic antigen by examining peptic hydrolysates of psoriatic squamous elements. Psoriatic squamous elements and epidermal stratum corneum were collected from heel area of healthy subjects with a metal grater for further examination. Squamous elements were homogenized in isotonic saline followed by centrifugation to separate them from soluble components. Homogenates of squamous elements structures and their homogenate supernatants underwent peptic hydrolysis reaction. Antigens and their identity were analysed by Ouchterlony immunoprecipitation in agar with antiserum obtained after immunizing rabbits with homogenate of the psoriatic squamous element structures, before and after hydrolysis, using peptic hydrolysates of squamous element structures and relevant supernatants. It was found that two antigens were precipitated in peptic hydrolysates of homogenates of psoriatic squamous cell structures, underlying acidin-pepsin resistance of antigen carriers. However, compared to the remainder of antigens precipitated in examined substrates only one out of two antigens might be considered as a psoriatic antigen being some PrPSc analogue that was identical to one of supernatant antigens derived from epidermal stratum corneum homogenates collected from healthy subjects. Similar to PrPSc, it differed from antigenically related normal protein by acidin-pepsin resistance. Albeit confirming available reports revealing no psoriasis-associated pathogen-specific antigens, this, nonetheless, might not be sufficient for rejecting infectious theory of psoriasis, as psoriatic antigen exhibits some properties similar to those of PrPSc.
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van der Kant, Rob, Joschka Bauer, Anne R. Karow-Zwick, Sebastian Kube, Patrick Garidel, Michaela Blech, Frederic Rousseau, and Joost Schymkowitz. "Adaption of human antibody λ and κ light chain architectures to CDR repertoires." Protein Engineering, Design and Selection 32, no. 3 (March 2019): 109–27. http://dx.doi.org/10.1093/protein/gzz012.
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Abstract Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.
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Thanawastien, Ann, Robert T. Cartee, Thomas J. Griffin, Kevin P. Killeen, and John J. Mekalanos. "Conjugate-like immunogens produced as protein capsular matrix vaccines." Proceedings of the National Academy of Sciences 112, no. 10 (February 19, 2015): E1143—E1151. http://dx.doi.org/10.1073/pnas.1425005112.
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Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell–independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a “carrier protein” antigen. Such “conjugate vaccines” efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.
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Holec-Gąsior, Lucyna, Bartłomiej Ferra, Dorota Drapała, Dariusz Lautenbach, and Józef Kur. "A New MIC1-MAG1 Recombinant Chimeric Antigen Can Be Used Instead of the Toxoplasma gondii Lysate Antigen in Serodiagnosis of Human Toxoplasmosis." Clinical and Vaccine Immunology 19, no. 1 (November 23, 2011): 57–63. http://dx.doi.org/10.1128/cvi.05433-11.
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ABSTRACTThis study presents an evaluation of the MIC1 (microneme protein 1)-MAG1 (matrix antigen 1)Toxoplasma gondiirecombinant chimeric antigen for the serodiagnosis of human toxoplasmosis for the first time. The recombinant MIC1-MAG1 antigen was obtained as a fusion protein containing His tags at the N- and C-terminal ends using anEscherichia coliexpression system. After purification by metal affinity chromatography, the chimeric protein was tested for usefulness in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-T. gondiiimmunoglobulin G (IgG). One hundred ten sera from patients at different stages of infection and 40 sera from seronegative patients were examined. The results obtained for the MIC1-MAG1 chimeric antigen were compared with those of IgG ELISAs using aToxoplasmalysate antigen (TLA), a combination of recombinant antigens (rMIC1ex2-rMAG1) and single recombinant proteins (rMIC1ex2 and rMAG1). The sensitivity of the IgG ELISA calculated from all of the positive serum samples was similar for the MIC1-MAG1 chimeric antigen (90.8%) and the TLA (91.8%), whereas the sensitivities of the other antigenic samples used were definitely lower, at 69.1% for the mixture of antigens, 75.5% for the rMIC1ex2, and 60% for rMAG1. This study demonstrates that the MIC1-MAG1 recombinant chimeric antigen can be used instead of the TLA in the serodiagnosis of human toxoplasmosis.
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Chen, Shuxiong, Natalie A. Parlane, Jason Lee, D. Neil Wedlock, Bryce M. Buddle, and Bernd H. A. Rehm. "New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli." Applied and Environmental Microbiology 80, no. 8 (February 14, 2014): 2526–35. http://dx.doi.org/10.1128/aem.04168-13.
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ABSTRACTThe tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared fromMycobacterium bovisare present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenicMycobacterium tuberculosiscomplex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinantEscherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected withM. boviswith no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.
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Kopylov, P. Kh, and S. V. Dentovskaya. "Direct Quantification of Protein Antigens in Subunit Plague and Rickettsial Vaccine Preparations." Problems of Particularly Dangerous Infections, no. 4 (February 11, 2023): 69–74. http://dx.doi.org/10.21055/0370-1069-2022-4-69-74.
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The aim of the work was to put forward the methods for direct quantitative determination of the content of Yersinia pestis and Rickettsia raoultii protein antigens in preparations and various prototypes of subunit vaccines. Materials and methods. Y. pestis LcrV and Caf1 antigens enclosed in the substance of the molecular microencapsulated plague vaccine (MMPV) and separately, in microcrystals of amino acids co-precipitated with plague proteins were used as model antigens. R. raoultii Adr2, OmpB24, and YbgF antigens were adsorbed on the prototype substance of the rickettsia vaccine. The release of plague antigens from MMPV microcapsules was carried out through successive treatment of the latter with organic solvents, methylene chloride and methanol, respectively; the carrier microcrystals were dissolved in 0.1 M sodium citrate buffer at pH 6.0. The antigen content in the prototype substance of the rickettsial vaccine was determined by measuring the amount of proteins not bound to the alumogel. Quantitative parameters characterizing the content of antigens in the substances and prototypes of vaccine preparations were calculated by processing digital images of polyacrylamide gels obtained by electrophoresis of protein antigen fractions extracted from carriers. Results and discussion. Methods for direct extraction and subsequent quantitative analysis of Y. pestis LcrV and Caf1 antigens from subunit vaccine preparations based on amino acid microcrystals and polylactide microcapsules that do not cause protein degradation have been studied. A different nature of the binding of LcrV and Caf1 in the substances of microcrystals has been established, while the proportion of antigens released from microcrystals has been quantified only in case of their complete dissolution. It was found that at low concentrations of LcrV and Caf1 proteins extracted from microcrystals, it is necessary to concentrate the extracts with subsequent removal of salts for their reliable visualization. It has been confirmed that 10 μg of plague antigens and proteins of R. raoultii in a dose volume of 200 μl of suspension is sufficient for quantitative analysis using electrophoretic method. The prospects of other physicochemical methods alternative to direct extraction of antigens for evaluating the composition and quality of vaccine preparations are discussed.
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Hisham, Yasmin, and Yaqoub Ashhab. "Identification of Cross-Protective Potential Antigens against PathogenicBrucellaspp. through Combining Pan-Genome Analysis with Reverse Vaccinology." Journal of Immunology Research 2018 (December 9, 2018): 1–15. http://dx.doi.org/10.1155/2018/1474517.
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Brucellosis is a zoonotic infectious disease caused by bacteria of the genusBrucella.Brucella melitensis,Brucella abortus, andBrucella suisare the most pathogenic species of this genus causing the majority of human and domestic animal brucellosis. There is a need to develop a safe and potent subunit vaccine to overcome the serious drawbacks of the live attenuatedBrucellavaccines. The aim of this work was to discover antigen candidates conserved among the three pathogenic species. In this study, we employed a reverse vaccinology strategy to compute the core proteome of 90 completed genomes: 55B. melitensis, 17B. abortus, and 18B. suis. The core proteome was analyzed by a metasubcellular localization prediction pipeline to identify surface-associated proteins. The identified proteins were thoroughly analyzed using variousin silicotools to obtain the most potential protective antigens. The number of core proteins obtained from analyzing the 90 proteomes was 1939 proteins. The surface-associated proteins were 177. The number of potential antigens was 87; those with adhesion score ≥ 0.5 were considered antigen with “high potential,” while those with a score of 0.4–0.5 were considered antigens with “intermediate potential.” According to a cumulative score derived from protein antigenicity, density of MHC-I and MHC-II epitopes, MHC allele coverage, and B-cell epitope density scores, a final list of 34 potential antigens was obtained. Remarkably, most of the 34 proteins are associated with bacterial adhesion, invasion, evasion, and adaptation to the hostile intracellular environment of macrophages which is adjusted to depriveBrucellaof required nutrients. Our results provide a manageable list of potential protective antigens for developing a potent vaccine against brucellosis. Moreover, our elaborated analysis can provide further insights into novelBrucellavirulence factors. Our next step is to test some of these antigens using an appropriate antigen delivery system.
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Rennert, Paul, Alyssa Birt, Lihe Su, Lan Wu, Fay Dufort, Roy Lobb, Christine Ambrose, and Paul Rennert. "133 Development of novel cellular therapeutics for metastatic and primary CNS malignancies." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A146. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0133.
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BackgroundTreatment of solid tumors with cell therapeutics will require optimal T cell persistence, fitness, and trafficking. Heterogeneous solid tumors will also have to be attacked through multiple antigens simultaneously in order to prevent resistance linked to loss of antigen expression. Here we use chimeric antigen receptor (CAR) T cells that secrete bridging proteins that act as CAR-T engagers to create an optimal platform for attacking solid tumors in the CNS.MethodsLentiviral vectors encoding an anti-CD19 CAR and secreted bridging proteins were created. The bridging proteins contained the CD19 extracellular domain, which is the target for the CAR, and anti-tumor antigen binding domains derived from antibodies (scFv and llama VH). The resulting anti-CD19 CAR T cells secrete the bridging proteins. These candidate cell therapeutics were evaluated for antigen binding and induction of antigen-specific cytotoxicity. An anti-CD19 CAR that secretes a CD19-anti-Her2 bridging protein has moved into development. Using the CD19-anti-Her2 bridging protein as a core module, we have begun evaluating a series of multi-antigen bridging proteins.ResultsCAR-CD19 T cells that secrete bridging proteins have potent cytotoxic activity against single- and multi-antigen-positive cells. ALETA-002 is the lead candidate lentiviral vector construct encoding the anti-CD19 CAR domain and the CD19-anti-Her2 bridging protein, and has entered a GMP viral particle development campaign. This therapeutic will be systemically administered to Her2-positive breast cancer patients who are relapsing with CNS metastases. Next, multi-antigen bridging proteins encoding an anti-Her2 scFv and anti-B7H3, anti-B7H6 or anti-IL13Ra2 llama VH were assayed for potency. Lead candidates for development for the treatment of primary CNS malignancies were identified and are being manufactured at pilot-scale in 4-plasmid lentivirus production runs.ConclusionsThe use of anti-CD19 CAR T cells that can expand off of the normal CD19-positive B cell pool enables tumor-antigen independent persistence, fitness and robust trafficking into the CNS. The use of small, modular bridging proteins allows us to leverage anti-CD19 CAR T cells and use these to attack solid tumor antigens that are present on CNS resident cancers and on CNS metastatic lesions. Novel cell therapeutics for the treatment of Her2-positive CNS metastases and heterogeneous primary CNS malignancies including glioblastoma and the pediatric gliomas have been developed.
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Dimitrov, Ivan, and Mariana Atanasova. "AllerScreener – A Server for Allergenicity and Cross-Reactivity Prediction." Cybernetics and Information Technologies 20, no. 6 (December 1, 2020): 175–84. http://dx.doi.org/10.2478/cait-2020-0071.
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Abstract Allergenicity of proteins is a subtle property encoded in their structures. The prediction of allergenicity of novel proteins saves time and resources for subsequent experimental work. In the host antigen-presenting cells, the allergens are processed as antigens by the means of Human Leukocyte Antigens (HLA) class II proteins. Sometimes, people allergic to a given protein show allergic reaction to a different protein, even when the two proteins have different routes of exposure. This phenomenon is termed cross-reactivity. Here, we describe a server for allergenicity and cross-reactivity prediction based on the abilities of allergenic proteins to generate binders to HLA class II proteins. The generated peptides are compared to HLA binders originating from known allergens. As a result, the server returns a list of common binders, origin proteins, and species. Different species generate common HLA binders and this determines their cross-reactivity. The server is named AllerScreener and is freely accessible at: http://www.ddg-pharmfac.net/AllerScreener .
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Barlow, Avlin K., Xin He, and Charles Janeway. "Exogenously Provided Peptides of a Self-antigen Can Be Processed into Forms that Are Recognized by Self–T Cells." Journal of Experimental Medicine 187, no. 9 (May 4, 1998): 1403–15. http://dx.doi.org/10.1084/jem.187.9.1403.
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Major histocompatibility complex (MHC) class II molecules can present peptides derived from two different sources. The predominant source of peptide in uninfected antigen presenting cells (APCs) is from self-proteins that are synthesized within the cell and traffic through the MHC class II compartment. The other source of antigen is endocytosed proteins, which includes both self- and foreign proteins. Foreign protein antigens generate adaptive immune responses, whereas self-peptides stabilize the MHC class II heterodimer on the cell surface, allowing positive and negative selection of thymocytes. Therefore, self-antigens play an important normal role in shaping the T cell receptor repertoire as well as a pathological role in autoimmunity. To determine whether processing and presentation of self-antigens by MHC class II molecules differs depending on whether the antigen is supplied through synthesis within the cell or by endocytosis, we used a T cell clone against an Eα peptide presented by I-Ab to show that processing through these two routes can differ. We also show that mice can be tolerant to the epitope formed through the endogenous route, but responsive to the epitope that can be formed through endocytosis. This suggests that negative selection occurs primarily against antigens that are synthesized within the APC, and that endocytosed self-antigens could serve as autoantigens. Finally, we also demonstrate that lipopolysaccharide-activated B cells are defective for uptake, processing, and presentation of this self-antigen, and that this correlates with the increased expression of the costimulatory molecules B7.1 and B7.2. This may provide a model for studying the onset of an autoimmune response.
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Igwe, Emeka I., Gernot Geginat, and Holger Rüssmann. "Concomitant Cytosolic Delivery of Two Immunodominant Listerial Antigens by Salmonella enterica Serovar Typhimurium Confers Superior Protection against Murine Listeriosis." Infection and Immunity 70, no. 12 (December 2002): 7114–19. http://dx.doi.org/10.1128/iai.70.12.7114-7119.2002.
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ABSTRACT During its interaction with host cells, Salmonella enterica serovar Typhimurium employs a type III secretion system for cytosolic targeting of virulence factors. This protein translocation mechanism is a useful tool for heterologous antigen delivery by attenuated Salmonella vaccine carrier strains. In the present study, we used the Yersinia outer protein E (YopE) as a carrier molecule for Salmonella type III-dependent cytosolic delivery of the immunodominant CD8 T-cell antigens listeriolysin O (LLO) and p60 of Listeria monocytogenes. It is shown that concomitant translocation of hybrid YopE/LLO and YopE/p60 proteins by Salmonella led to antigen presentation and CD8 T-cell priming efficacies comparable to those of translocation of single listerial antigens. However, simultaneous translocation of LLO and p60 significantly surpassed single cytosolic antigen delivery in the ability to protect against Listeria. For the first time, this study demonstrates that concomitant expression of two independent antigens via the same recombinant plasmid leads to superior protection against a challenge with an intracellular bacterial pathogen. In conclusion, these findings emphasize the versatility of Salmonella type III-mediated heterologous antigen delivery for the induction of cytotoxic T-lymphocyte-mediated immunity.
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Lazzarino, Deborah A., Peter Blier, and Ira Mellman. "The Monomeric Guanosine Triphosphatase rab4 Controls an Essential Step on the Pathway of Receptor-mediated Antigen Processing in B Cells." Journal of Experimental Medicine 188, no. 10 (November 16, 1998): 1769–74. http://dx.doi.org/10.1084/jem.188.10.1769.
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Each member of the rab guanosine triphosphatase protein family assists in the regulation of a specific step within the biosynthetic or endocytic pathways. We have found that the early endosome-associated rab4 protein controls a step critical for receptor-mediated antigen processing in a murine A20 B cell line. Expression of the dominant negative rab4N121I mutant dramatically inhibited the processing and presentation of ovalbumin, λ cI repressor, or rabbit immunoglobulin G internalized as antigens by B cell antigen receptors or transfected Fc receptors. This defect did not reflect a block in antigen endocytosis or degradation, and transfected cells remained completely capable of presenting exogenously added ovalbumin and λ repressor peptides. Most remarkably, rab4N121I-expressing cells were undiminished in their ability to present each of these antigens when whole proteins were internalized at high concentration by fluid-phase endocytosis. Thus, expression of the rab4N121I selectively inactivated a portion of the endocytic pathway required for the processing of receptor-bound, but not nonspecifically internalized, antigens. These results suggest that elements of the early endosome-recycling pathway play an important and selective role in physiologically relevant forms of antigen processing in B cells.
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Lim, Seah H., Zhiqing Wang, Maurizio Chiriva-Internati, and Yuying Xue. "Sperm protein 17 is a novel cancer-testis antigen in multiple myeloma." Blood 97, no. 5 (March 1, 2001): 1508–10. http://dx.doi.org/10.1182/blood.v97.5.1508.
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Various studies have demonstrated the aberrant expression of normal testicular proteins in neoplastic cells. These proteins collectively form the new class of tumor antigens called cancer-testis (CT) antigens. Their selective normal tissue expression makes them ideal antigens for immune targeting of the malignant disease. In this study, the expression of a spermatozoa protein, Sp17, in multiple myeloma was investigated. It was found that Sp17 is detectable in tumor cells from 12 of 47 (26%) myeloma patients. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis detected Sp17 transcripts and proteins, respectively. Northern blot analysis and RT-PCR demonstrated that Sp17 transcripts were detected only in normal testis, supporting its tissue specificity. Since a high proportion of normal individuals develop antibodies against Sp17 following vasectomy, Sp17 is likely to be a highly immunogenic protein in vivo. Sp17 is therefore a novel member of the CT antigen family and should be an ideal target for immunotherapy of multiple myeloma.
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Sock, Elisabeth, Janna Enderich, and Michael Wegner. "The J Domain of Papovaviral Large Tumor Antigen Is Required for Synergistic Interaction with the POU-Domain Protein Tst-1/Oct6/SCIP." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 2455–64. http://dx.doi.org/10.1128/mcb.19.4.2455.
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ABSTRACT Large T antigens from polyomaviruses are multifunctional proteins with roles in transcriptional regulation, viral DNA replication, and cellular transformation. They have been shown to enhance the activity of various cellular transcription factors. In the case of the POU protein Tst-1/Oct6/SCIP, this enhancement involves a direct physical interaction between the POU domain of the transcription factor and the amino-terminal region of large T antigen. Here we have analyzed the structural requirements for synergistic interaction between the two proteins in greater detail. Tst-1/Oct6/SCIP and the related POU protein Brn-1 were both capable of direct physical interaction with large T antigen. Nevertheless, only Tst-1/Oct6/SCIP functioned synergistically with large T antigen. This differential behavior was due to differences in the amino-terminal regions of the proteins, as evident from chimeras between Tst-1/Oct6/SCIP and Brn-1. Synergy was specifically observed for constructs containing the amino-terminal region of Tst-1/Oct6/SCIP. Large T antigen, on the other hand, functioned synergistically with Tst-1/Oct6/SCIP only when the integrity of its J-domain-containing amino terminus was maintained. Mutations that disrupted the J domain concomitantly abolished the ability to enhance the function of Tst-1/Oct6/SCIP. The J domain of T antigen was also responsible for the physical interaction with Tst-1/Oct6/SCIP and could be replaced in this property by other J domains. Intriguingly, a heterologous J domain from a human DnaJ protein partially substituted for the amino terminus of T antigen even with regard to the synergistic enhancement of Tst-1/Oct6/SCIP function. Given the general role of J domains, we propose chaperone activity as the underlying mechanism for synergy between Tst-1/Oct6/SCIP and large T antigens.
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Murray, R. J., M. G. Kurilla, J. M. Brooks, W. A. Thomas, M. Rowe, E. Kieff, and A. B. Rickinson. "Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies." Journal of Experimental Medicine 176, no. 1 (July 1, 1992): 157–68. http://dx.doi.org/10.1084/jem.176.1.157.
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Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins.