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Dissertations / Theses on the topic 'Viral antigen'
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Clayton, Anne-Louise. "Immunoassays for viral antigen detection in clinical specimens." Thesis, Open University, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.254677.
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Kučinskaitė-, Kodzė Indrė. "Production, Characterization And Application Of New Monoclonal Antibodies Against Viral Antigens." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2011. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2011~D_20110630_104940-67693.
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The dissertation describes development and characterization of monoclonal
antibodies against recombinant yeast-expressed antigens: nucleocapsid (N)
proteins of human parainfluenza virus type 3, Menangle virus, hantavirus and
rabies virus. The newly developed antibodies were investigated by different
immunochemical assays for their specificity, affinity and ability to recognize
native viruses in infected cells. It was determined that the antibodies raised
against recombinant yeast-expressed viral proteins are suitable to identify virusinfected
cells. These data confirmed that recombinant yeast-expressed viral N
proteins possess antigenic properties similar to that of native viral nucleocapsids.
The monoclonal antibodies were also used to study the antigenic structure of
viral N proteins and localize their immunodominant regions. The obtained
results may have impact on the development of new immunodiagnostic test
systems for the detection of viral infections.
The dissertation consists of the introduction, three sections, references, and
the list of author’s publications.
In the Introductory Chapter, the research topic, the actuality, the aim and
tasks, scientific novelty and practical value of the dissertation are discussed.
Author’s publications and conference reports are also presented. The first
Chapter of the dissertation provides literature overview on the genome
organization, structural proteins, pathogenesis and epidemiology of
parainfluenza viruses, Menangle virus... [to full text]Disertacijoje aprašomi monokloniniai antikūnai, sukurti prieš rekombinantinius mielėse susintetintus antigenus: žmogaus paragripo treciojo tipo viruso, Menangle viruso, hantavirusų bei pasiutligės viruso nukleokapsidės (N) baltymus. Sukurtieji antikūnai buvo visapusiškai charakterizuoti įvairiais imunocheminės analizės metodais, įvertintas jų specifiškumas, afiniškumas, sugebėjimas atpažinti natyvius virusus infekuotų ląstelių kultūrose. Buvo nustatyta, kad antikūnai, sukurti prieš rekombinantinius mielėse susintetintus virusų baltymus, tinka virusų nustatymui infekuotose ląstelėse. Šie tyrimai patvirtino, kad rekombinantiniai mielėse susintetinti virusu N baltymai turi panašias antigenines savybes, kaip natyvūs virusų N baltymai, formuojantys nukleokapsides. Sukurtieji monokloniniai antikūnai taip pat buvo panaudoti išsamiems minėtų virusų N baltymų antigeninės struktūros tyrimams bei imunodominuojančių sekų nustatymui. Disertaciniame darbe gauti duomenys svarbūs, kuriant naujas imunodiagnostikos sistemas, skirtas virusų infekcijoms nustatyti. Disertacija sudaro įvadas, trys skyriai, naudotos literatūros sąrašas ir autorės publikacijų sąrašas. Įvadiniame skyriuje aptariama tiriamoji problema, darbo aktualumas, formuluojamas darbo tikslas bei uždaviniai, darbo mokslinis naujumas ir praktinė reikšmė, pristatomos paskelbtos publikacijos ir pranešimai konferencijose. Pirmasis disertacijos skyrius skirtas literatūros apžvalgai: jame apibūdinamos paragripo virusų, Menangle viruso... [toliau žr. visą tekstą]
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Nastke, Maria-Dorothea. "T-cell epitopes from viral and tumor associated antigens induction and analysis of antigen-specific T cells /." [S.l. : s.n.], 2005. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB12168223.
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Chen, Yun-Chi. "Control of immune response and pathogenesis by antigen during viral infection." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413974.
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Glew, Elizabeth Jane. "The interaction of bovine viral diarrhoea virus with antigen presenting cells." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312578.
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Biswas, Sumi. "Prime boost vaccination with viral vectors targeting apical membrane antigen 1." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:a17ab4e4-9b81-4ab3-b02f-41d9da36c6ab.
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Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood stage malaria and several clinical trials using mostly protein-in-adjuvant vaccines have shown limited success. This thesis describes the development of recombinant adenoviral (AdHu5) and poxviral (MVA) vectors encoding AMA1 from Plasmodium chabaudi murine parasites. In this murine malaria model, AdHu5 and MVA encoding AMA1 when used in a heterologous prime boost regime showed excellent immunogenicity, both humoral and cellular. The vaccination regime was protective against blood stage challenge and both antibodies and CD4+ T cells found to be important for vaccine induced blood stage protection. In parallel to this novel P. falciparum vaccines encoding AMA1 were also developed and administered in a similar prime boost regime to mice and rabbits. The vaccination regime induced cellular immune response and high titre antibodies against AMA1 and these antibodies showed growth inhibitory activity against the homologous parasite strain. In an effort to overcome the issue of antigenic polymorphism and to circumvent pre-existing immunity to human adenovirus, biallelic simian and human adenoviral vectors and MVA encoding AMA1 vaccines were also developed and administered to mice and macaques. These vectors also induced high titre antibodies and the serum from macaques was found to have growth inhibitory activity. These vaccine candidates are now being taken forward to Phase I/II clinical trials in Oxford. This work also described the attempt to improve MVA as a antibody inducing vector to allow better antibody mediated immunity to blood stage malaria.
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Wong, Hiu-ling Beatrice. "Development of antibody and antigen detection assays and vaccines for SARS associated coronavirus." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B39634024.
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Wong, Hiu-ling Beatrice, and 黃曉靈. "Development of antibody and antigen detection assays and vaccines for SARS associated coronavirus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39634024.
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Bhati, Anubhuti. "EXPRESSION OF HEPATITIS C VIRAL NON-STRUCTURAL 3 ANTIGEN IN TRANSGENIC CHLOROPLASTS." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3076.
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Hepatitis C viral infection is the major cause of acute hepatitis and chronic liver disease and remains the leading cause of liver transplants (NIH). An estimated 180 million people are infected globally (WHO). There is no vaccine available to prevent hepatitis C. The treatment with antiviral drugs is expensive, accompanied with various side effects and is limited only to those at risk of developing advanced liver disease. The treatment is also effective in only about 30% to 50% of treated patients and still a high percentage of patients are resistant to therapy. Therefore, there is an urgent need for the development of effective vaccine antigens and an efficacious HCV vaccine. The non-structural 3 protein of the hepatitis C virus is a multifunctional protein of the virus required for virus polyprotein processing and replication. Vaccine antigen production via chloroplast transformation system usually results in high expression levels and eliminates the possibility of contamination with vector sequences,human or animal pathogens. The HCV NS3 antigen was expressed in the chloroplast of Nicotiana tabacum var. Petit havana and LAMD-609. The 1.9kb NS3 gene was cloned into a chloroplast expression vector, pLD-Ct containing the 16S rRNA promoter, aadA gene coding for the spectinomycin selectable marker, psbA 5' untranslated region to enhance translation in the light and 3' untranslated region for transcript stability and trnI & trnA homologous flanking sequences for site specific integration into the chloroplast genome. Chloroplast integration of the NS3 gene was first confirmed by PCR. Southern blot analysis further confirmed site-specific gene integration and homoplasmy. The NS3 protein was detected in transgenic chloroplasts by immunoblot analysis. The NS3 protein was further quantified by ELISA. Maximum expression levels of NS3 up to 2% in the total soluble protein were observed even in old leaves, upon 3-day continuous illumination. These results demonstrate successful expression of the HCV non-structural 3 antigen in transgenic tobacco chloroplasts. Animal studies to test the immunogenecity of the chloroplast derived HCV NS3 will be performed using chloroplast derived NS3 antigen.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Lee, Sang-Ryul. "Bovine viral diarrhea virus infections affect professional antigen presentation in bovine monocytes." Diss., Mississippi State : Mississippi State University, 2007. http://library.msstate.edu/etd/show.asp?etd=etd-10222007-092315.
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Rao, Ankit Rohit. "Engineering an improved dendritic cell vaccine expressing whole antigen following non-viral transfection." Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3622/.
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Dendritic cells are efficient antigen-presenting-cells that can be used in tumour-antigen specific vaccination for malignant disease. Melanoma patients were recently treated with a dendritic cell vaccine expressing gp100 and Melan-A antigens after non-viral (CL22 peptide) transfection. Although clinical and immunological responses were noted, there was no correlation between responses and whole antigen expression levels in the vaccine cells that varied widely. Here, it is established that patient cells expressed detectable levels of Class I restricted epitopes from both antigens, although there was no correlation with whole antigen detection. CL22 transfected dendritic cells could simultaneously present a viral antigen (EBNA1) to CD8 and CD4 T-cells, which had not previously been demonstrated. Using RNA transfection, it was demonstrated that early after transfection cells are whole Melan-A positive yet negative for Class I epitopes and with time Melan-A antigen levels fall whilst Class I epitopes are generated. Loss of whole antigen expression seemed related to lysosomal function and, unlike the viral antigen EBNA1, Class I presentation from Melan-A was lysosome-dependent. For Class II presentation of EBNA1, cellular localisation seems to determine access to the Class II pathway although this depends on the time-scale over which epitope presentation is assessed.
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Umami, Rifqiyah Nur. "Effect of Viral and Host Factors on Hepatitis B Virus Surface Antigen Secretion." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29753.
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Hepatitis B virus (HBV) is the etiological cause of chronic hepatitis B (CHB), a leading cause of liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Despite the availability of prophylactic vaccines, CHB remains incurable due to the persistence of covalently closed circular DNA (cccDNA) which continuously produces viral transcripts. Therefore, new strategies for CHB treatment are needed. One of these strategies is to reduce HBsAg, a hallmark of CHB. High levels of HBsAg inhibit the antiviral immune response. Thus, reduction of HBsAg levels in blood will ultimately allow recovery of the immune response and achieve functional cure, an end point marked by sustainable elimination of HBsAg with or without HBsAg seroconversion. This thesis is focused on finding factors that affect HBsAg secretion, which we hypothesised may include both viral and host factors. In Chapter 3, HBV cell culture model was established by transfecting HBV DNA plasmids into Huh7 hepatoma cells. This system provides robust production of both infectious HBV virus and subviral particles containing HBsAg, allowing examination of the effects of viral and host factors on the secretion of HBsAg. In Chapter 4, the effects of viral basal core promoter (BCP) mutations on the expression, translation, and secretion of HBsAg were examined. In the clinical setting, mutations in the BCP region of the HBV genome are associated with late stages of CHB when HBsAg levels are usually low. Mutant HBV clones harbouring clinically relevant mutations in the BCP region (G1764T/C1766G, G1757A/G1764T/C1766G, or T1753C/A1762T/G1764A) were generated by site directed mutagenesis. No significant difference in the expression, translation, or secretion of HBsAg was observed in cells transfected with BCP mutant viruses compared to wild type virus, suggesting that BCP mutations do not directly alter the secretion levels of HBsAg. In Chapter 5, we shifted our focus to transmembrane 6 superfamily member 2 (TM6SF2), a potential host factor involved in HBsAg secretion. TM6SF2, regulates lipid secretion through the endoplasmic reticulum (ER)/Golgi pathway, the same pathway used by HBsAg for its secretion. Moreover, in a clinical cohort study we found that CHB patient harbouring the TM6SF2 E167K variant (which causes reduced TM6SF2 protein levels in the liver) was associated with lower blood HBsAg levels (approximately ~25% reduction) compared to those expressing wild type TM6SF2. Therefore, the effect of TM6SF2 on the secretion of HBsAg was examined in our cell culture models. Inhibition of TM6SF2 by siRNA knock down in Huh7 cells reduced the secretion of HBsAg approximately ~50%, likely by altering intracellular HBsAg trafficking in the same pathway used for lipoprotein secretion. In Chapter 6, additional host factors were identified using targeted mass-spectrometry screening of ER proteins. ER-protein enrichment was carried out on HepAD38 cells (which stably expressing viral and subviral particles) and HepG2 cells (the parental cell line control) and submitted for proteomics analysis. Ten candidate genes were identified including DPP4, ANXA6, SERPINA3, GK, SERPINF1, EPB41L2, AFP, IQGAP1, ITGA2, and LPCAT1. Functional studies were performed in our Huh7 HBV transfection model. siRNA mediated knockdown of candidate genes increased HBsAg levels in the culture supernatant, suggesting their involvement in HBsAg secretion. In conclusion, we identified several host factors that affect HBsAg secretion, but found that potential viral factors (BCP mutations) did not appear to play a role. In ongoing work we are developing antiviral therapies targeting TM6SF2, with the aim of achieving functional cure in people with CHB. In the future we will explore the potential for targeting other novel candidate genes we identified, by exploring their functions and role in the HBV replication cycle. These findings will inform future drug discovery work to improve the lives of people living with hepatitis B.
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Janbazian, Loury. "Effect of antigen load and viral sequence diversification on HIV-specific CD8+ T cells." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86806.
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Virus-specific CD8+ T cells have been implicated in the control of acute HIV and SIV infections. Although present in chronic HIV-1 infection, CD8+ T cells exhibit impaired functions due to unidentified molecular signatures. This has urged researchers to revisit parameters that define efficacious CD8+ T cell responses in HIV-1. To this date, polyfunctionality has emerged as the key feature of CD8+ T cell efficacy in chronic infection. Moreover, it is established that T-cell Receptor (TCR) diversity in HIV-specific CD8+ T cells plays a critical role in controlling viremia. Although TCR repertoire studies have been performed in the context of several acute and persistent viral infections including HIV-1, longitudinal studies that aim to study the turnover of the HIV-specific CD8+ TCR repertoire and link HIV-specific CD8+ T cell clonotypes to functional profiles have been limited. We therefore aimed to 1) define a molecular signature of CD8+ T cell exhaustion in HIV-1 infection, 2) study the effect of antigen load and, 3) the effect of viral sequence diversification on the clonality, functionality, and phenotype of HIV-specific CD8+ T cells. Our first set of results identified PD-1 as a molecule of exhaustion on HIV-specific CD8+ T cells and showed a positive correlation with viral load. Interestingly, blocking PD-1 interaction with its ligand alleviated the functional dysfunction of HIV-specific CD8+ T cells. This set of data prompted us to further examine the effect that "antigenic absence" has on the fate of HIV-specific CD8+ T cells. For this, we chose two circumstances; the institution of HAART and emergence of HIV-specific CD8+ T cell epitope escape. Our second set of data provided clear evidence of a HAART-mediated functional reconstitution of the HIV-specific CD8+ T cell compartment on the clonal level. This was attributed by different mechanisms, namely the improvement of function of persisting clonotypes and the recruitment of new clonotypes which were polyLes cellules T CD8+ sont impliquées dans le contrôle des infections virales aiguës tels que les virus VIS et VIH. Bien que ces cellules T CD8+ spécifiques au VIH persistent durant la phase chronique de l'infection, leur fonctionnalité semble être altérée par un mécanisme non identifié. Cela a exhorté les chercheurs à revoir les paramètres qui définissent l'efficacité réelle des lymphocytes T CD8+ en générale et surtout lors d'infection par le VIH. À date, il est admis que la polyfonctionnalité des cellules T CD8+ soit l'élément clé de l'efficacité de la réponse immunitaire durant l'infection chronique. De plus, il est établi que la diversité du récepteur de cellule T (RCT) occupe un rôle important et crucial dans le contrôle de la virémie. Bien que plusieurs études sur le répertoire des RCT aient été menés par plusieurs groupes dans le contexte des infections aigues et chronique par le VIH, il n'existe néanmoins pas des données combinant la fluctuation et la fonction du répertoire spécifique au VIH au cours de la progression de la maladie. De ce fait, il était important de réaliser des études longitudinales qui ont pour but principal l'étude de la régénération du répertoire T CD8+ spécifiques au VIH durant les différentes phases de l'infection. Dans mes travaux de doctorat, nous avons exploré les mécanismes responsables des l'inefficacité de cellules CD8+ cours de l'infection par le VIH. En utilisant plusieurs techniques immunologiques et génétiques, nos objectifs visaient à : 1) Définir une signature moléculaire d'épuisement, 2) Étudier l'effet de la charge virale et, 3) Caractériser l'effet de la diversification des séquences virales, sur la clonalité, la fonctionnalité, et le phénotype des cellules T CD8+ spécifiques au VIH. Nous avons en premier lieu identifié le rôle de la molécule PD-1, une des molécules régulatrices qui joue un rôle critique dans le contrôle de la réponse immunitai
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Liu, Mengya. "NanoAPC deliver antigen, IL-2 and co-stimulatory molecules to antigen specific T cells and activate viral specific T cells in chronic infections." Thesis, Brunel University, 2011. http://bura.brunel.ac.uk/handle/2438/6413.
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The study of the immune system has provided insight in the mechanism of protection induced by vaccination; primarily that most clinically protective vaccines are potent in generating neutralizing antibody responses. However, vaccination fails to protect against a wide range of acquired chronic infections caused by viruses, such as HIV, HBV and HCV. One of the major reasons for weak responses to therapeutic vaccine is the impaired function of effector T cells resulting from viral persistence. Although IL-2 can potently increase effect function of viral specific T cells, systemic administration of IL-2 induces organ pathology and expansion of Treg cells. In this study, we have now developed a novel vaccine delivery system IL-2-nanoAPC delivering antigen-MHC complexes (pMHC), co-stimulatory molecules and IL-2 to antigen specific T cells. NanoAPC are derived from the endoplasmic reticulum (ER) membranes of human B cell line 721.221 engineered with selected HLA allele and IL-2 as the ER retention proteins. The IL-2-nanoAPC interacted with antigen specific T cells, induced immune synapses and expression of high affinity IL-2 receptor and enhanced effector function of antigen specific T cells, but did not affect bystander T cells and Foxp3+ Treg cells. Together with pMHC, co-stimulatory molecules, the selective delivery of IL-2 not only increased the CD4 and CD8 T cell responses to viral antigens but also enhanced TCR proximal signalling and suppressed expression of PD1 molecules on IFNγ producing effector CD8 T cells. We also found that the co-induction of T helper responses by IL-2-nanoAPC in a mixed culture could increase CD8 T cell responses to viral antigen. The IL-2-nanoAPC effectively induced responses of CD4 and CD8 T cells from chronic HBV patients. The results demonstrate that selective delivery of IL-2, together with pMHC and co-stimulatory molecules, by nanoAPC to antigen specific T cells has potential to recover anti-viral immune responses in chronic HBV patients.
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Wang, Weiping. "SV40 large T antigen helicase roles of the hydrophilic channels and a newly identified unwinding activity /." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 152 p, 2009. http://proquest.umi.com/pqdweb?did=1833646471&sid=7&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Peng, Yu-Cai. "Interactions between polyomavirus large T antigen and the viral replication origin DNA, how and why." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0028/NQ50235.pdf.
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Peng, Yu-Cai 1965. "Interactions between polyomavirus large T antigen and the viral replication origin DNA : how and why." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35927.
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Polyomavirus large T antigen is the major regulatory protein in the polyomavirus life cycle. It binds to multiple G(A/G)GGC pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. The nature of interactions between large T antigen and the viral origin of DNA replication is not fully understood. We set out to produce large T antigen protein in the methylotropic yeast Pichia pastoris by placing the large T antigen gene downstream of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immuno-affinity chromatography by using a monoclonal antibody.While optimizing the conditions for binding of large T antigen to viral origin DNA, we discovered that binding was substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. We showed that increased binding at low pH is due to increased stability of protein-DNA complexes, and that large T antigen molecules self-associated at low pH, forming massive complexes. ATP increased binding of large T antigen to origin DNA by about 2-fold at pH 7.8, but had no detectable effect at pH 7 or below.
Enhanced, stable DNA binding by large T antigen to viral origin DNA at pH 6 enabled us to develop a novel gel mobility shift assay using unfixed protein-DNA complexes. We demonstrated that this assay is very sensitive and highly specific. This method can be used both for detection of large T antigen in crude cell lysates and for quantitation of binding of purified large T antigen to target DNAs under various conditions.
Using a series of point and deletion mutants in the viral origin of DNA replication, we demonstrated that binding of large T antigen to sites 1/2, A, B, and C is cooperative. Binding of large T antigen to one site stimulated binding to other sites 20 to 100 bp distant, and binding to inherently weak sites was strengthened if two or more such sites were present on the same DNA molecule. These findings suggest that large T antigen molecules bound to DNA interact with each other to mutually stabilise their binding.
ATP was shown to stabilise large T antigen-DNA complexes against dissociation only if the DNA contained site 1/2. ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, where hexamers are believed to form and begin unwinding DNA. We propose a model in which large T antigen molecules bound to sites 1/2, A, B, and C on origin DNA form a compact protein-DNA complex via mutual interactions; large T antigen molecules bound to sites A, B, and C are mobilised and "handed over" to site 1/2, where ATP stimulates their assembly into hexamers.
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Dubosq, Ming-Celine. "The non-viral production of Chimeric Antigen Receptor T-cells for B-cell haematological malignancies." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23170.
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Chimeric antigen receptor (CAR) T-cells are genetically engineered to express a synthetic receptor which redirects their specificity and effector functions to a tumour associated antigen. They have induced unsurpassed clinical responses in patients with relapsed and refractory CD19-positive haematological malignancies who hitherto had limited treatment options. Currently, all commercially available CAR T-cell products are manufactured using viral vectors for transduction, an efficient but highly costly and complex process. Thus, access to CAR T-cell therapy is inequitable. Historically, the use of lower-cost non-viral plasmid-based transduction methods have been limited by the high toxicity and low transfection efficiencies of the electroporation process required for gene transfer and integration. This thesis furthers previous work using the super piggyBac plasmid-based system to generate CAR T-cells. Chapter 3 outlines the optimisation of an electroporation system with available current Good Manufacturing Practice reagents. It describes the effect of altering pre-electroporation, electroporation and post-electroporation variables on transfection efficiency and evaluates the feasibility of CAR T-cell production using the methodology described. Multiple myeloma is an incurable B-cell haematological malignancy without uniform CD19 expression. Its considerable heterogeneity poses both opportunities and challenges for CAR T-cell immunotherapy. In Chapter 4, design modifications to a novel CAR construct created with the piggyBac transposon/transposase system and directed against kappa myeloma antigen (KMA) to improve its clinical applicability for multiple myeloma are described. The non-viral superPiggyBac plasmid-based system is a feasible, cost-effective alternative for the production of CAR T-cells. It is envisaged that its use will improve access to CAR T-cells for patients with CD19-positive and CD19-negative haematological malignancies.
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張紀忠 and Jizhong Zhang. "Conformational antigenic determinants of the HEV CAPSID." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31241360.
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Zhang, Jizhong. "Conformational antigenic determinants of the HEV CAPSID /." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2207918X.
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McMurtrey, Curtis Paul. "Human leukocyte antigen class I presentation and immune recognition of West Nile virus peptide epitopes." Oklahoma City : [s.n.], 2009.
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Rodríguez, Plata Mª Teresa. "Dendritic Cells and HIV-1: Molecular Mechanisms Involved in Viral Capture, Trans-infection and Antigen Presentation." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/120537.
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Les cèl·lules dendrítiques (CD) són les cèl·lules presentadores d’antigen més potents del sistema immuntari, enllaçant la immunitat innata i la immunitat adquirida. No obstant això, les CD poden contribuir a la disseminació del Virus de la Immuno-deficiència Humana tipus 1 (VIH-1) mentre intenten induir una resposta adaptativa contra la infecció viral. El paradigma immunològic clàssic sobre la funció de les CD indica que les CD immadures principalment capturen patògens, mentre que les CD madures indueixen una resposta adaptativa contra el patogen capturat. Però, la maduració de les CD amb lipopolisacàrid (LPS) augmenta la seva abilitat per capturar el VIH-1, que, al seu torn, resulta en una potent transmissió infecciosa a cèl·lules diana. L’objectiu d’aquesta tesi és el d’analitzar els mecanismes involucrats en la captura, la trans-infecció i la presentació antigènica del VIH-1 per part de las CD. Amb aquesta finalitat i utilitzant tècniques de biologia cel·lular i molecular, s’ha identificat el lligand viral i el receptor en les CD responsable del mecanisme de captura del VIH-1 independent de l’envolta viral, el destí del VIH-1 capturat per les CD, i els determinants per a una transmissió infecciosa eficient d’aquestes partícules virals als limfòcits T CD4+.
Els resultats obtinguts proporcionen nova informació sobre la contribució de les CD a la patogènesi del VIH-1. La maduració de les CD amb LPS augmenta l’expressió de Siglec-1 a la membrana plasmàtica de les CD. Aquest receptor actua reconeixen la sialillactosa dels gangliòsids de la membrana del VIH-1, constituint el mecanisme d’unió i captura del VIH-1 en la CD independent de l’envolta viral. Però, aquest mecanisme tant eficient de captura viral en les CD no representa una font d’antigen per a la presentació antigènica del VIH-1, ja que les partícules virals capturades són retingudes en un compatiment intracel·lular, protegides de la degradació i preservant la seva infectivitat. Per tant, en el cas de les CD madurades amb LPS, hi ha una dissociació entre la captura del VIH-1 i la seva presentació antigènica. Per contra, la captura del VIH-1 per Siglec-1 es redirigeix cap la transmissió infecciosa a cèl·lules diana, però sense que el VIH-1 infecti les CD. Atès que les CD interaccionen contínuament amb les cèl·lules T CD4+ durant la seva tasca immunològica, ja sigui per induir respostes immunes adaptatives o per a mantenir l’homeostasi de les cèl·lules T, el VIH-1 pot aprofitar-se d’aquests contactes, sense modular-los, per a ser transmès. D’aquesta manera, el VIH-1 passa desapercebut a través de la sinapsi CD–cèl·lula T, escapant del control immunològic i incrementant la propagació viral, que està marcadament afavorida per l’activació immune induïda pels contactes CD–cèl·lula T. Per tant, determinar la contribució de les CD en la patogènesi de la infecció pel VIH-1 pot ser de gran importància pel desenvolupament d’estrategies terapèutiques dirigides a bloquejar la disseminació del VIH-1.Las células dendrítcas (CD) son las células presentadoras de antígeno más potentes del sistema inmunitario, enlazando la inmunidad innata y la inmunidad adquirida. Sin embargo, las CD pueden contribuir a la diseminación del Virus de la Inmunodeficiencia Humana tipo 1 (VIH-1) mientras intentan inducir una respuesta adaptativa contra la infección viral. El paradigma inmunológico clásico sobre la función de las CD indica que las CD inmaduras principalmente capturan patógenos, mientras que las CD maduras inducen una respuesta adaptativa frente al patógeno capturado. No obstante, la maduración de las CD con lipopolisacárido (LPS) incrementa su abilidad para capturar VIH-1, que, a su vez, resulta en una potente transmisión infecciosa a células diana. El objetivo de esta tesis es el de analizar los mecanismos involucrados en la captura, la trans-infección y la presentación antigénica del VIH-1 por parte de las CD. Con esta finalidad y utilizando técnicas de biología celular y molecular, se ha identificado el ligando viral y el receptor en las CD responsables del mecanismo de captura del VIH-1 independiente de la envuelta viral, el destino del VIH-1 capturado por las CD, y los determinantes para una transmisión infecciosa eficiente de estas partículas virales a los linfocitos T CD4+. Los resultados obtenidos proporcionan nueva información acerca de la contri-bución de las CD en la patogénesis del VIH-1. La maduración de las CD con LPS incrementa la expresión de Siglec-1 en la membrana plasmática de las CD. Este receptor reconoce la sialillactosa de los gangliósido de la membrana del VIH-1, mediando el mecanismo de unión y captura del VIH-1 en las CD independiente de la envuelta viral. Sin embargo, este mecanismo tan eficiente de captura viral en las CD no representa una fuente de antigéno para la presentación antigénica del VIH-1, dado que las partículas virales capturas son retenidas en un compartimento intracelular, protegidas de la degradación y preservando su infectividad. Por consiguiente, en el caso de las CD maduradas con LPS, existe una disociación entre la captura del VIH-1 y su presentación antigénica. Por el contrario, la captura de VIH-1 mediada por Siglec-1 se redirige hacia la transmisión infecciosa a células diana, pero sin que el VIH-1 infecte las CD. Dado que las CD interaccionan continuamente con los célula T CD4+ durante su labor inmunológica, tanto para inducir respuestas inmunes adaptativas como para mantener la homeostasis de las células T, el VIH-1 puede aprovecharse de estos contactos, sin modularlos, para ser transmitido. De esta manera, el VIH-1 pasa desapercibido a través de la sinapsis CD–célula T, escapando del control inmunológico e incrementando la propagación viral, que está marcadamente favorecida por la activación immune inducida por los contactos CD–célula T. Por lo tanto, determinar la contribución de las CD en la patogénesis de la infección por VIH-1 puede ser de gran importancia para el desarrollo de estrategias terapéuticas dirigidas para bloquear la diseminación del VIH-1.
Dendritic cells (DC) are the most potent antigen-presenting cells in the immune system, linking innate and adaptive immune responses. However, it has been suggested a dual role of DC in Human Immunodeficiency Virus type 1 (HIV-1) infection by increasing the spread of HIV-1 while trying to trigger an adaptive response against viral infection. The classical immunological paradigm affirms that immature DC mainly mediate pathogen uptake while mature DC launch adaptive immune responses against the captured pathogen. Nevertheless, DC maturation with lipopolysaccharide (LPS) increases their ability of capturing HIV-1 particles resulting in a potent infectious transmission to target cells. The aim of this thesis is to analyze the mechanisms involved in DC-mediated HIV-1 capture, trans-infection and antigen presentation. To that end, we used methods of cellular and molecular biology, reporting the viral ligand and the DC receptor responsible for an HIV-1 Env-independent uptake mechanism, the fate of the captured HIV-1 particles, and the determinants for an efficient infectious transmission to CD4+ T lymphocytes. Our results provide new insights into contribution of DC to HIV-1 pathogenesis. Maturation of DC with LPS increases the expression of Siglec-1 on DC membrane. This receptor acts as the attachment factor in DC for sialyllactose-containing gangliosides in HIV-1 membrane, mediating the Env-independent mechanism of HIV-1 binding and uptake by DC. However, this efficient mechanism of HIV-1 capture does not represent a source of viral antigen for HLA loading and T-cell activation, given that captured virions are retained within an intracellular compartment, away from degradation, thus preserving their infectivity. Consequently, in the case of HIV-1 and DC matured with LPS, there is dissociation between pathogen uptake and antigen presentation. On the contrary, Siglec-1-mediated HIV-1 capture by DC is redirected to infectious viral transmission to susceptible target cells, without infecting host DC. Since DC continuously interact with CD4+ T lymphocytes during their immunological labor, either to elicit adaptive immune responses or to maintain T-cell homeostasis, HIV-1 can take advantage of these contacts, without modulating them, to be transmitted. As a result, HIV-1 can go unnoticed across the DC–T-cell synapses, bypassing the immunological control and increasing viral dissemination, which is markedly favored by immune activation driven by DC–T-cell contacts. Therefore, determining the contribution of DC to the pathogenesis of HIV-1 infection may be important for the development of therapeutic strategies aiming to block HIV-1 spread.
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McLean, Brendan Norbert. "The detection of viral antigen in the cerebrospinal fluid of patients with herpes simplex virus encephalitis." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/26759.
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A highly sensitive capture sandwich antibody assay was developed for the detection of Herpes Simplex Virus alkaline nuclease in cerebrospinal fluid. Chemiluminescence, using Isoluminol labelled antibody and p-Iodophenol enhanced Horse-Radish Peroxidase catalysed Luminol, was compared with Horse-Radish Peroxidase colorimetric detection in the assay. Luminol was superior to Isoluminol based chemiluminescence, but this was no more sensitive than colorimetric detection. In vitro the final optimised colorimetric assay had a sensitivity of 20 plaque forming units of Herpes Simplex Virus. A method for dissociation of immune complexed alkaline nuclease, using Hydrochloric acid as a chaotropic agent, was optimised for use with the assay. The assay was applied to 12 cerebrospinal fluid samples from 9 cases of Herpes Simplex Virus Encephalitis. All cases conformed to accepted criteria for the diagnosis of the disease in the absence of brain biopsy. All the tested samples were positive for alkaline nuclease, which was present in either free or immune complexed form or both. The earliest positive sample was day 1 of the clinical illness, and samples were positive up to day 56, which was the longest interval from onset of symptoms to lumbar puncture in these patients. Free alkaline nuclease was present in the early stages of the disease, being replaced by immune complexed alkaline nuclease after day 13, a time when locally synthesised anti-HSV1 IgG was also detected. A total of 35 control patients were also studied. Five out of 6 patients with suspected Herpes Simplex Virus Encephalitis or myelitis, and one case of definite herpes myelitis were positive. Two of the remaining 28 cases were positive: both of these were patients with Subacute Sclerosing Panencephalitis. No demyelinating, inflammatory, other infectious or non-neurological disorders were positive by the assay.
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Mahauad, Fernandez Wadie Daniel. "Role of bone marrow stromal antigen 2 (BST-2) in viral pathogenesis and breast cancer progression." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/6191.
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Bone marrow stromal antigen 2 (BST-2/tetherin) is a type II transmembrane protein that plays various roles, including protective and detrimental roles in the host. Cellular responses to BST-2 expression or the lack thereof, may be cell type and context-dependent and may vary with time. When protective, BST-2 functions as an antiviral factor, renowned for its ability to tether budding enveloped viruses to the membrane of infected cells. Tethering of budding virions prevents their release into the extracellular milieu limiting infection of naïve cells. The antiviral role of BST-2 has been predominantly studied using cultured cells. Insight into the role of BST-2 in inhibition of viral infection in vivo came from our study of the alphavirus Chikungunya virus (CHIKV) and the retrovirus mouse mammary tumor virus, (MMTV). BST-2 prevents the release of CHIKV and MMTV virions from infected cells and limits the replication of both viruses in mice. In the context of CHIKV infection, BST-2 protects the host in a tissue-type dependent manner. In lymphoid and most non-lymphoid tissues, expression of BST-2 limits CHIKV replication. In addition, BST-2 regulates CHIKV-induced inflammatory responses in mice, an indication that BST-2 may function to initiate and amplify innate immune responses. Host response to MMTV infection depends on the stage of the infection and disease sequela. Acute infection of immune cells with MMTV results in an initial increase in BST-2 expression followed by a sharp decline. In contrast, in MMTV-induced mammary tumors, BST-2 mRNA and protein are elevated, so is the viral load. This is an indication that the antiviral role of BST-2 is not operative once mammary tumors have developed. These data provided the initial evidence that BST-2 may promote breast cancer progression. Indeed, data from two mouse models of breast cancer show that expression of BST-2 is necessary for cell to cell and cell to extracellular matrix interactions. Thus, BST-2 expression in breast cancer cells enhances cancer cell adhesion, anchorage-independency, migration, and invasion, culminating in increased tumor mass, increased metastases, and reduced host survival. Structurally, BST-2 homodimerization is important for its cancer-promoting role as dimers of BST-2 regulate anchorage-independency, resistance to anoikis, and enhanced adhesion between cancer cells and components (proteins and cells) of the tumor microenvironment. How BST-2 is enriched in breast cancer cells was elusive until our in silico analyses of a large human breast cancer dataset that revealed the involvement of epigenetic regulation of BST-2 in breast tumors. In highly aggressive breast cancers, specific CpG sites in and at close proximity to the BST-2 promoter are hypomethylated. This is in sharp contrast to non-aggressive luminal cancers and normal breast epithelial cells. These data suggest that a progressive loss of methylation on the BST-2 gene may contribute to constitutive overexpression of BST-2 in tumors. Overall, these findings show that 1) BST-2 contributes to the emergence and progression of breast malignancies and may be used as a therapeutic target or as a biomarker for aggressive breast cancers; and, 2) BST-2 acts as a viral sensor to initiate antiviral inflammatory responses and could be exploited therapeutically to treat viral infections. We highlight the need for additional research on the antiviral and cancer-promoting roles of BST-2 to reconcile both functions for the purpose of therapeutics.
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Nastke, Maria-Dorothea [Verfasser], and Hans-Georg [Akademischer Betreuer] Rammensee. "T-cell epitopes from viral and tumor associated antigens : induction and analysis of antigen-specific T cells / Maria-Dorothea Nastke ; Betreuer: Hans-Georg Rammensee." Tübingen : Universitätsbibliothek Tübingen, 2005. http://d-nb.info/1162199148/34.
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Meei-Yun, Lin. "Evolution of the T Cell Receptor Repertoire during and after Viral Infection: a Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/244.
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The overall goal of this thesis is to examine how the T cell receptor (TCR) repertoire evolves during and after viral infections. Previous studies had examined TCR usage of selected virus-specific T cell clones, but little was known about how a diverse T cell repertoire changes during the transition between an acute infection and a memory response. It was also unclear how the T cell repertoire evolves under conditions of persistent infections associated with clonal exhaustion. To address these issues I used as a model system the lymphocytic choriomeningitis virus (LCMV) infection of mice, for which the T cell response is well-characterized. LCMV, strain Armstrong (LCMV-ARM), infection induces a strong CD8+ T cell response, which clears the virus and converts to a memory response. In contrast, high doses of LCMV clone 13 leads to persistent infections associated with T cell clonal exhaustion. These two extremes of T cell responses enable one to compare the evolution of the TCR repertoire under conditions where an acute T cell response converts to a memory response with that of T cell clonal exhaustion. In this thesis I analyzed the TCR repertoire usage directly ex vivo by the technique of CDR3 length spectratyping throughout the acute LCMV infection, into memory, after modulation by subsequent heterologous and homologous viral infections, and under conditions of T cell clonal exhaustion.
Kinetic studies on the frequencies of precursor cytotoxic T lymphocytes (PCTL) to the three LCMV immunodominant peptides had suggested that the virus-specific T cell repertoire becomes fixed by day 7 postinfection, when the virus is cleared. The pCTL data also showed that a high frequency of the LCMV-specific memory T cells remained stable throughout the lifetime of the mouse. To examine any changes of the TCR repertoire usage that may develop during the acute LCMV infection and into memory, the Vβ8 population was subjected to spectratype analyses, because Vβ8 represented a substantial amount of the LCMV-induced CD8+ T cells recognizing several LCMV-encoded peptides. Analyses of the Vβ8.1 spectratype showed that genetically identical mice generated remarkably different T cell responses, as reflected by different spectratypes and different TCR sequences in same-sized spectratype bands; a conserved CDR3 motif was, however, found within some same-sized bands. This indicated that meaningful studies on the evolution of the T cell repertoire required longitudinal studies within individual mice instead of comparisons between mice. Such longitudinal studies with peripheral blood (PB) samples showed that the virus-induced T cell repertoire changed little after viral clearance and during the silencing phase of the T cell response and that dominant spectratype peaks were preserved into long term memory. To determine the effect of secondary LCMV infection on the spectratype, the recalled LCMV-induced spectratypes were analyzed. Most of the dominant peaks detected in the primary infections remained present in the secondary infection. Some new peaks were also detected for the first time in the secondary infection, suggesting a further selection of the virus-induced T cell repertoire. The spectratype data support the concepts that the LCMV-induced T cell repertoire remains unchanged during the silencing phase after clearance of the virus and that the LCMV infection dramatically skews the host T cell repertoire in the memory state long after the virus is cleared.
Studies had shown that high doses of LCMV clone 13 induce a transient anti-viral CTL response followed by clonal exhaustion of T cells. To determine how the TCR repertoire evolves under conditions of persistent infections associated with T cell clonal exhaustion, the Vβ8.1 spectratypes were analyzed at various time points after the infection. In contrast to the stable LCMV-induced spectratype after viral clearance, continuous selection of the T cell repertoire occurred under conditions of persistent infections, as the T cell clones appeared and disappeared at different rates. The T cell repertoire ultimately returned to a Gaussian distribution under conditions of clonal exhaustion, indicating that clonal deletion occurs in the great majority of the virus-induced T cells.
To test the stability of the LCMV-induced TCR repertoire under conditions of subsequent heterologous viral infections, the recalled LCMV-induced spectratypes were examined in the presence or absence of intervening heterologous viruses. The results showed that the intervening heterologous viruses disrupted the recalled Vβ8.1-Jβ1.3 spectratype on secondary LCMV infection; this otherwise remained stable in the absence of intervening heterologous viruses. This result supports the hypothesis that subsequent heterologous viral infections disrupt the stable LCMV-induced T cell repertoire. To detennine whether a subset of the memory T cells was deleted by the IFN-induced apoptosis of memory T cells, the LCMV-immune spectratypes were analyzed before and after the injection of the IFN inducer, poly I:C. The LCMV-immune spectratypes remained relatively stable after poly I:C injection, suggesting that there is no selective protection or deletion of discrete memory T cell clones during the IFN-induced apoptosis.
In summary, the data in this thesis show that (i) the virus-induced T cell repertoire changes little after viral clearance and during the silencing phase of the T cell response, (ii) the LCMV infection dramatically skews the host T cell repertoire in the memory state, (iii) the evolution of the T cell repertoire occurs during secondary infections and under conditions of clonal exhaustion associated with persistent infections, (iv) genetically identical hosts generate different T cell responses to the same virus, and (v) intervening heterologous viral infections disrupt the recalled LCMV-induced T cell repertoire, but the LCMV-immune repertoire remained relatively stable upon the treatment of the IFN inducer, poly I:C.
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Cho, Uhn-Soo. "Structural studies of protein phosphatase 2A, simian virus 40 small t antigen, and the PhoQ sensor domain /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/5677.
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Wang, Xiaoting Z. "Organ-Dependent and Epitope-Dependent Repertoire Usage and Apoptosis of Antigen-Specific T Cells in Viral Infections: a Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/285.
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During virus infections, activation of CD8 T cells takes place in secondary lymphoid organs including spleen and lymph nodes. The kinetics of the T cell response in lymphoid tissues has been clearly studied. However, a large number of virus-specific T cells disseminate into various nonlymphoid tissues. As reservoirs for effector and memory cells, nonlymphoid organs play an important role for defending against infections. T cell responses in nonlymphoid organs may differ from lymphoid organs.
T cell repertoire usage in lymphoid and nonlymphoid tissues was studied in an acute lymphocytic choriomeningitis virus (LCMV)-infected murine model. The hierarchy of CD8 T cell specificities was examined with cytotoxic T lymphocyte (CTL) sodium 51 chromate (51Cr) release assays and intracellular interferon (IFN)γ assays. T cell receptor (TCR) repertoire usage was determined by complementarity determining region (CDR)3 length spectratyping analysis. Both T cell specificity and TCR repertoire usage revealed some similarities and differences between several organs. Within an epitope-specific CD8 T cell population, the TCR repertoire usage was similar in different organs of the same mouse, but highly heterogeneous between individual mice with genetically identical backgrounds.
A very restricted CD4 TCR repertoire was observed in BALB/c mice after secondary respiratory syncytial virus (RSV) infection. Most of the CD4 T cells of BALB/c mice pre-immunized with RSV glycoprotein (GP) predominantly express Vβ14 TCR with discrete oligoclonal CDR3 regions. Depletion of Vβ14 CD4 T cells dramatically reduced immunopathology.
The apoptotic phenotype of LCMV-specific CD8 T cells was studied in various lymphoid and nonlymphoid tissues during acute and memory stages of infections. Peripheral tissues (peritoneal cavity (PEC), fat pad, and lung) reacted with a much lower frequency with the early apoptotic marker Annexin V than those in spleen and lymph nodes. This was not due to a TCR-based selection because similar TCR spectratypes were seen in different organs. Activated lymphoid and nonlymphoid T cells from LCMV GP33 transgenic mice, which have identical TCR α and β chains on all T cells, had differential Annexin V binding. When incubated shortly in vitro, most Annexin V+ T cells rapidly fragmented their DNA and became terminal transferase-mediated dUTP nick end-labeling positive (TUNEL+), while much fewer Annexin V- cells became TUNEL+. Therefore, those Annexin-V+ cells were truly in a pre-apoptotic stage. The differential spontaneous apoptosis in different tissues is independent of several death/survival-related molecules, including Fas/Fas ligand (FasL), turner necrosis factor (TNF)α, interleukin (IL-15), perforin, B cell lymphoma (Bcl)-2 and independent of virus tropism.
I further investigated the significance of the high Annexin V reactivity of lymphoid T cells. Pre-apoptotic cells were prevented from fragmenting their DNA by anti-CD3 or IL-2 stimulation in vitro. However, this pre-apoptotic phenotype precluded generation of memory. Annexin V reactive cells did not give rise to long-lived memory after being transferred into naïve hosts. The pre-apoptotic phenotype is also an intrinsic property of the epitope. Different proportions of apoptotic cells were found in LCMV effector and, memory T cells specific to two different epitopes, nucleoprotein (NP)396 and GP33. Higher Annexin V reactivity of NP396-specific CD8 T cells was independent of virus tropism and duration of encounter with antigen. Higher expression of IL-7R was found in peripheral, Annexin V- and GP33-specific CD8 T cells, indicating that IL-7-dependent signals may inhibit apoptosis.
Nonlymphoid T cells were more resistant than lymphoid T cells to activation-induced cell death (AICD). When stimulated with anti-CD3 in vitro for 40 hours (hr), a significantly reduced number of splenic transgenic T cells were recovered with much higher frequency of Annexin V reactivity and TUNEL staining than transgenic T cells from PEC. Consistent with the finding that Fas and FasL regulates AICD, a much lower expression of Fas and FasL was observed in PEC and lung transgenic T cells than spleen and lymph nodes after short time stimulation. FasL blockage largely increased cell-number recovery and reduced Annexin V and TUNEL staining of spleen transgenic T cells.
Interestingly, the leukocyte environment played an important role of deciding the fate of transgenic T cells. When placing activated spleen transgenic T cells with excess infected PEC cells, spleen transgenic cells rapidly reduced their Annexin V staining and TUNEL staining and were recovered with greater number after stimulation. Vice versa, PEC transgenic T cells became Annexin V and TUNEL positive with lower numbers of cells recovered when placed with excess splenocytes. Less detection of Annexin V+ cells in peripheral tissues was not due to rapid phagocytosis by macrophages, because Cytochalasin D, which can inhibit phagocytosis, did not induce equal amount of pre-apoptotic cells in spleen and PEC. This reduced death in the periphery may contribute to the long-term maintenance of nondividing nonlymphoid memory T cells, enabling them to efficiently function without being driven into apoptosis.
Overall, this study characterizes in detail the different T cell repertoire usage and apoptosis of virus-specific T cells based on their organ localization and specificities and helps to better understand T cell immunity after infections and vaccine design.
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Novak, Erik Joseph. "Tracking antigen-specific immune responses in human infection and disease /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/5084.
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Zarozinski, Christopher C. "T Cell Receptor-Dependent and Independent Events During Potent Anti-Viral T Cell Responses." eScholarship@UMMS, 1998. http://escholarship.umassmed.edu/gsbs_diss/175.
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The relative contribution of T cell receptor-dependent stimulation versus TcR-independent bystander stimulation in the massive increase in the number of activated proliferating CD8+ T cells seen during acute many acute viral infections is unclear. To determine if this increase was the result of TcR-independent bystander activation and proliferation, anti-viral cytotoxic T lymphocytes were induced in vivo via DNA immunization so that the anti-viral immune response could be examined in the absence of the high levels of cytokines generated during acute infection. After a single immunization with a plasmid encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV) a nearly 2 log10 reduction in viral titers in the spleen was observed 3 days after LCMV infection. After 2 or 3 immunizations a greater that 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. After intracerebral challenge vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. By limiting dilution analysis LCMV-specific CTL precursors were detected in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at days 4 after LCMV challenge. However, the numbers of CTL precursors elicited by DNA vaccination was too low to determine if cytokine-mediated TcR-independent bystander activation and proliferation had taken place.
HY-specific TcR-transgenic mice, which have a restricted TcR repertoire, and LCMV-carrier mice, which are tolerant to LCMV, were used to determine the extent of TcR-independent bystander activation and proliferation during acute LCMV infection. LCMV infection of C57BL/6 mice induced CTL that lysed uninfected H-2k and H-2d allogeneic targets, but, LCMV-induced CTL from HY- transgenic mice lysed only the H-2k-expressing cells. The HY-mice generated both anti-H-2k and anti-H-2d CTL in mixed lymphocyte cultures, strongly suggesting that the generation of allospecific CTL during acute LCMV-infection is antigen specific. During the LCMV infection there was blastogenesis of the CDB+ T cell population, but the HY-specific T cells remained small in size, and did not alter their expression of the activation molecules CD44 and MEL-14. In order to examine the potential for bystander stimulation under conditions of a very strong CTL response, T cell chimeras were made between normal and HY-transgenic mice. Even in the context of a normal vicus-induced CTL response, no stimulation of HY -specific T cells was observed, and HY-specific cells were diluted in number by day 9 post-infection. In LCMV-carrier mice in which donor and host T cells could be distinguished by Thy 1 allotypic markers, adoptive transfer of LCMV-immune T cells into LCMV-carrier mice, whose T cells were tolerant to LCMV, resulted in activation and proliferation of donor CDB cells but little or no activation of host CDB+ T cells. These results show that TcR-independent bystander activation of non virus-specific T cells is not a significant component of an anti-viral T cell response and support the hypothesis that the massive polyclonal CTL response to LCMV infection is virus-specific.
T cells activated during potent anti-viral immune responses are sensitized to undergo apoptosis after strong TcR-stimulation in a process known as activation-induced cell death (AICD). To determine if T cells, not participating in the immune response were also subject to AICD, LCMV-carrier mice were used. Using TUNEL flow cytometry, it was shown that after reconstitution of Thy 1.2+ LCMV-carrier mice with spleen cells from Thy 1.1+ LCMV-immune mice, the Thy 1.2+ host T cells which were not specific for the virus and did not proliferate in a bystander fashion, were rendered sensitive to TcR-induced apoptosis in vitro. This bystander sensitization to AICD was shown not to be dependent on the continued presence of activated proliferating donor cells during the in vitro culture period. Bystander sensitization to AICD was not the result of an antigen presenting cell defect, but rather was the result of an in vivo conditioning of the T cells themselves. The mechanism of this sensitization was, at least, partially dependent on the ability of host T cells to respond to IFNγ, and on the expression of Fas ligand on the activated, proliferating donor cells. This bystander sensitization to AICD may explain why memory T cell responses are so poor during acute viral infection and can serve as a potential mechanism for virus-induced immunosuppression.
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Carlsson, Björn. "Adoptive T Cell Therapy of Viral Infection and Cancer : Ex vivo Expansion of Cytomegalovirus- and Prostate Antigen-specific T Cells." Doctoral thesis, Uppsala University, Clinical Immunology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4821.
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The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system.
To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer.
I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.
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Abeywickrama, Samarakoon Natali. "Small hepatitis Delta antigen mimics a histone H3 epitope to facilitate the remodeling of the Hepatitis D virus (HDV) viral ribonucleoprotein." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1193.
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Le virus de l'hépatite Delta (HDV) est un agent infectieux transmissible satellite du virus de l'hépatite B (HBV), induisant des maladies du foie plus sévères que la mono–infection par le HBV. Aucun traitement totalement efficace n'est disponible contre l'HDV et les 15 millions de personnes infectées par le HDV dans le monde sont exposées a un risque élevé de cirrhose et de carcinome hépatocellulaire. HDV est un virus unique qui ne code pas pour une polymérase virale contrairement aux autres virus a ARN. La réplication de l'ARN HDV s'effectue par un double mécanisme de cercle roulant générant des brins d'ARN de longueur génomique ou antigénomiques unitaires. La synthèse de l'ARN génomique est sensible à de faibles concentrations d'alpha–amanitine, ce qui suggère qu'elle soit médiée par l'ARN polymérase II (ARN Pol II) classiquement ADN dépendante. Ce processus repose sur la petite protéine du HDV (S–HDAg), qui doit être acétylée sur l'acide amine K72 pour activer la synthèse de l'ARN génomique. Nous avons récemment identifié la protéine BAZ2B (Bromodomain Associated Zinc finger protein 2B) comme un interactant majeur de S–HDAg par capture par affinité, couplée à la spectrométrie de masse à partir de l'expression de S– HDAg étiqueté par un double motif Strep–TagR dans les cellules HepaRG différentiées. La fonction biologique de BAZ2B est inconnue. Cependant, en comparant avec des protéines apparentées BAZ (BAZ–1A/1B/2A), on postule que BAZ2B représente la sous–unité accessoire d'un nouveau complexe de remodelage de chromatine de type ISWI, qui régule le positionnement des nucléosomes par hydrolyse de l'ATP. Des études récentes ont révélé que le bromodomaine de BAZ2B (BRD) reconnait la signature épigénétique spécifique K14ac–X–X–R sur l'histone H3. Cela pourrait impliquer le mode d'action du complexe de remodelage de la chromatine dont BAZ2B représente l'unité régulatrice reconnaissant des marques spécifiques d'acétylation des histones propagées séquentiellement modifiant la dynamique de la chromatine et favorisant le recrutement de l'ARN Pol II pour activer la transcription. Nous émettons l'hypothèse que l'acétylation, médiée par p300, du motif K72–X–X–R conserve dans les S–HDAg interagissant avec l'ARN antigénomique pseudo double brin, mimerait l'acétylation des histone H3 en K14 permettant de recruter le complexe de remodelage de la chromatine BAZ2B associée et de lancer la réplication HDV. Brièvement, pour confirmer la pertinence fonctionnelle du recrutement BAZ2B pour la réplication HDV, nous avons transfecté des lignées cellulaire Huh–7 exprimant de façon stable, soit la protéine sauvage S–HDAg ou le mutant R75A pour étudier la réplication HDV à partir plasmide pSVLD2m défectif pour l'expression de S–HDAg. Nos résultats indiquent que la synthèse de l'ARN génomique est fortement réduite dans les cellules exprimant le mutant R75A S–HDAg par rapport aux cellules exprimant le type sauvage S–HDAg, alors que la quantité d'ARN antigénomique est restée le même dans les deux cas. Des expériences de co–cristallisation et de siRNA sont actuellement menées afin de mieux caractériser au niveau moléculaire l'association entre BAZ2B BRD et des peptides dérivés de la séquence de S–HDAg et d'étudier les conséquences de l'inhibition par siRNA de BAZ2B. L'implication des BAZ2B dans la réplication de HDV pourra ouvrir des possibilités de développement de médicaments anti–HDV, basées sur l'optimisation des inhibiteurs émergents de BAZ2B–BRDHepatitis Delta Virus (HDV) is a satellite of Hepatitis B Virus (HBV), leading to more severe life threatening liver diseases than HBV mono–infection. No efficient therapy is available against HDV and the estimated 15 million HDV infected individuals worldwide are at a high risk of cirrhosis and hepatocellular carcinoma. HDV is a unique RNA virus as it does not encode a viral polymerase. HDV RNA replication occurs via a double rolling circle mechanism generating unit–length genomic or antigenomic RNA strands. The synthesis of the genomic RNA is sensitive to low concentrations of α–amanitin, suggesting that the RNA–dependent RNA synthesis is mediated by DNA–dependent RNA polymerase II (RNA Pol II). This process relies on the HDV encoded Small Hepatitis Delta antigen (S–HDAg), which must be acetylated at K72 to activate the synthesis of the genomic RNA. We recently identified BAZ2B (Bromodomain Associated to Zinc finger protein 2B) as a major interactant of S–HDAg by affinity capture coupled to mass spectrometry in differentiated HepaRG cells. The biological function of BAZ2B is however unknown. In comparison with related BAZ proteins (BAZ–1A/1B/2A), it is postulated that BAZ2B is the accessory subunit of a new chromatin remodeling complex of ISWI–type, which regulates nucleosome positioning through ATP hydrolysis. Recent studies revealed that the BAZ2B bromodomain (BRD) recognizes the distinct epigenetic signature K14ac–X–X–R on histone H3. This suggests that the mode of action of BAZ2B associated chromatin remodeling complex involves recognizing propagated specific histone acetylation marks to subsequently alter the chromatin dynamic and recruit the RNA Pol II for transcriptional activation. We hypothesized that the p300–mediated acetylation of the conserved K72–X–X–R motif in S–HDAg mimics acetylated histones on the pseudo–double stranded antigenomic RNA, to recruit the BAZ2B associated chromatin remodeling complex to initiate RNA Pol II mediated synthesis of HDV genome. To confirm the functional relevance of BAZ2B recruitment for HDV replication, we transfected Huh 7 cells stably expressing either wild–type S–HDAg or R75A mutant S–HDAg with the HDV replication defective plasmid pSVLD2m. Our results indicate that the synthesis of genomic RNA was greatly reduced in cells expressing the R75A mutant S–HDAg in comparison to cells expressing wild–type S–HDAg, whereas the amount of antigenomic RNA remained the same in both cases. Co–crystallization experiments are currently being carried out to better characterize at the molecular level the association between BAZ2B BRD and S–HDAg derived peptides. Furthermore, siRNA experiments directed against the BAZ2B gene are expected to reveal the consequences of BAZ2B inhibition on HDV viral replication. The involvement of BAZ2B in HDV replication may open anti–HDV drug development opportunities, based on the optimization of emerging BAZ2B–BRD inhibitors
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Moetlhoa, Boitumelo. "The preparation of antigen for the generation of polyclonal antibodies against the capsid subunit, VP1, and the viral protease, 3Cpro, of Theiler's murine encephalomyelitis virus (TMEV)." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1013225.
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The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.
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Catamo, Eulalia. "Genetic variation in hla-g: its influence in autoinflammatory autoimmune and viral diseases." Doctoral thesis, Università degli studi di Trieste, 2014. http://hdl.handle.net/10077/9982.
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2012/2013L'antigene leucocitario umano (HLA)-G presenta, in condizioni fisiologiche, una ristretta espressione tessuto-specifica ed ha funzione immuno-tollerogenica. Però, la presenza della molecola HLA-G è stata associata a diverse patologie autoimmuni e virali. In questo progetto di dottorato di ricerca abbiamo analizzato la possibile associazione tra la varianti genetiche nel gene HLA-G, che si suppone regolino l'espressione di HLA-G, e la suscettibilità allo sviluppo e al decorso della malattia celiaca, del lupus eritematoso sistemico, dell'artrite reumatoide e dell'infezione dal virus dell'epatite C. Inoltre, abbiamo analizzato se variazioni all'interno del promotore di HLA-G possano alterare la sua trascrizione genica, a questo scopo è stato condotto il saggio della luciferasi. Per gli studi di associazione sono stati analizzati 800 bp del promotore, l’intero 3’UTR e la delezione di una citosina all’esone 3 (ΔC, allele HLA-G*0105N) in 402 pazienti celiaci e 509 controlli italiani; 114 pazienti con lupus eritematoso sistemico e 128 controlli sani provenienti dal Nord -Est del Brasile, 127 pazienti con artrite reumatoide e 128 controlli dal Nord-Est del Brasile, e 286 pazienti caucasici HCV positivi e 285 controlli provenienti dalla stessa area geografica. Il saggio della luciferasi è stato condotto su 9 diversi aplotipi al promotore del gene HLA-G: CCTAGGACCG, CGTAGGACCG, CTTAGGACCG, TCGGTACGAA, TGGGTACGAA, TTGGTACGAA, CCTAGGAGCG, CGTAGGAGCG, e CTTAGGAGCG. Numerosi SNPs e aplotipi del gene HLA-G sono stati associati con le malattie analizzate. Inoltre, il saggio della luciferasi ha permesso di constatare che la presenza di polimorfismi, nel promotore del gene HLA-G, altera la trascrizione genica; nello specifico, in condizioni di stress, l’allele -725 C era significativamente associato ad un aumento della trascrizione del gene rispetto agli alleli -725 G e T. I nostri i risultati indicano un'associazione tra i polimorfismi del gene HLA-G e la suscettibilità allo sviluppo delle malattie studiate, suggerendo che molecola HLA-G è coinvolta nella patogenesi di queste malattie. Inoltre, possiamo ipotizzare che il gene HLA-G sia un gene stress-inducibile e che la presenza di SNPs al promotore alterati i livelli di trascrizione del gene
The Human Leukocyte Antigen (HLA)-G present a physiological restricted tissue-specific expression and an immuno-tolerogenic functions. The HLA-G expression has been associated with various autoimmune and viral diseases. In this PhD project we analyzed the possible association between genetic variant in the HLA-G gene, supposed to regulate HLA-G expression, and the susceptibility to develop celiac disease, systemic lupus erythematosus, rheumatoid arthritis and hepatitis C virus infection. Furthermore, we analyzed if variations within the HLA-G promoter could alter its transcription, and for this reason luciferase reporter gene assays was conducted. The HLA-G 5’ upstream regulatory region (URR), 3’ untranslated region (UTR) and a cytosine deletion at exon 3 (ΔC, HLA-G*0105N allele) were analyzed in 402 celiac patients and 509 controls from Italy; 114 systemic lupus erythematosus patients and 128 healthy controls from North East Brazil; 127 rheumatoid arthritis patients and 128 controls from North East Brazil; and 286 Hepatitis C virus Caucasian patients and 285 controls from the same geographical area. The luciferase reporter gene assay was used for the HLA-G promoter CCTAGGACCG, CGTAGGACCG, CTTAGGACCG, TCGGTACGAA, TGGGTACGAA, TTGGTACGAA, CCTAGGAGCG, CGTAGGAGCG, and CTTAGGAGCG haplotypes. Several HLA-G SNPs and haplotypes were associated with the diseases analyzed. By luciferase reporter gene assay, we found that the presence of polymorphisms in the HLA-G promoter altered gene transcription, specifically -725 C allele was significantly associated with an increased of the HLA-G transcription with respect to -725 G and T alleles in stress condition. Our findings indicate an association between HLA-G gene polymorphisms and susceptibility to diseases development, suggesting that HLA-G molecule is involved in the pathogenesis of the diseases. Also, we can hypothesize that HLA-G gene is a stress-inducible gene and that the presence of SNPs to the promoter alter levels of transcription of the gene
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Almqvist, Jenny. "Epstein-Barr virus nuclear antigen 1, Oct & Groucho/TLE in control of promoter regulation /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-523-2/.
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Warnatsch, Annika. "Impact of proteasomal immune adaptation on the early immune response to viral infection." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16775.
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Im Kampf gegen eine Virusinfektion spielen CD8+ T Zellen des adaptiven Immunsystems eine besondere Rolle. Sie patroullieren im Körper und entdecken spezifische Virusepitope, welche mittels MHC Klasse I Molekülen auf der Oberfläche infizierter Zellen präsentiert werden. Wird eine virus-infizierte Zelle erkannt, kann diese schnell und effizient eliminiert. Für die Generierung viraler Peptide, welche auf MHC Klasse I Komplexe geladen werden, ist das Ubiquitin-Proteasom-System von essentieller Bedeutung. Kürzlich wurden weitere Funktionen des Immunoproteasoms aufgedeckt wie zum Beispiel der Schutz gegen oxidativen Stress. Innerhalb der vorliegenden Arbeit konnte die Fähigkeit des Immunoproteasoms gegen eine Akkumulation oxidativ geschädigter Proteine zu schützen mit der Generierung von MHC Klasse I Liganden kombiniert und neu interpretiert werden. Es konnte gezeigt werden, dass während einer Virusinfektion in Nicht-Immunzellen die Produktion reaktiver Sauerstoffspezies durch die alternative NADPH Oxidase Nox4 eine bedeutende Rolle spielt. Die Aktivierung von Nox4 resultiert in der Akkumulation oxidativ geschädigter Proteine. Innerhalb von zwei Stunden nach dem Eintreten von Viruspartikeln in die Zellen wurden strukturelle Virusproteine oxidiert und anschließend ubiquityliert. Die gleichzeitige, virus-induzierte Expression von Immunoproteasomen führte zu einem schnellen und effizienten Abbau ubiquitylierter Virusantigene. Infolgedessen konnten immundominante Virusepitope vermehrt freigesetzt werden. Folglich wurde ein soweit unbekannter Mechanismus gefunden, welcher Substrate für das Proteasom zur Generierung von MHC Klasse I Liganden bereitstellt. Zusammenfassend konnte innerhalb dieser Arbeit gezeigt werden, dass das Immunoproteasom den Schutz vor oxidativen Stress mit der Generierung antigener Peptide verbindet, wodurch eine effektive adaptive Immunantwort etabliert werden kann.An efficient immune control of virus infection is predominantly mediated by CD8+ T cells which patrol through the body and eliminate infected cells. Infected cells are recognized when they present viral antigenic peptides on their surface via MHC class I molecules. To make antigenic peptides available for loading on MHC class I complexes, the ubiquitin proteasome system plays a crucial role. Moreover, the induction of the i-proteasome is known to support the generation of MHC class I ligands. Recently, new functions of the i-proteasome have been discovered. Evidence is increasing that the i-proteasome is involved in the protection of cells against oxidative stress. Within this thesis the characteristic of the i-proteasome to protect cells against the accumulation of oxidant-damaged proteins could be linked to its role in improving the generation of MHC class I ligands. It could be demonstrated that during a virus infection in non-immune cells the production of reactive oxygen species by the alternative NADPH oxidase Nox4 is of critical importance resulting in the accumulation of potentially toxic oxidant-damaged proteins. Indeed, within two hours of infection structural virus proteins were oxidized and subsequently poly-ubiquitylated. The concomitant formation of i-proteasomes led to a rapid and efficient degradation of ubiquitylated virus antigens thereby improving the liberation of immunodominant viral epitopes. In conclusion, a so far unknown mechanism to fuel proteasomal substrates into the MHC class I antigen presentation pathway has been revealed. A new protein pool consisting of exogenously delivered viral proteins provides proteasomal substrates in the very early phase of a virus infection. Within the scope of this thesis the i-proteasome has been shown to link the protection against oxidative stress, initiated directly by pathogen recognition, with the generation of antigenic peptides. Together, an effective adaptive immune response is triggered.
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Söderholm, Jonas. "The hepatitis C virus and immune escape : relation between sequence variations and the in vitro and in vivo functionality of the non-structural 3/4A complex /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7357-042-7/.
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Trujillo, Jonathan Anthony. "T cell responses to S-glutathionylated And heteroclitic viral epitopes and CCl2-mediated immune dysregulation in mice infected with a neurotropic coronavirus." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4775.
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Mice infected with neurotropic variants of the murine coronavirus, mouse hepatitis virus, (strains JHMV or J2.2–V–1) develop acute and chronic CNS infections, and provide a model system to study the pathogenesis of virus–induced neuroinflammation, mechanisms of virus persistence, and anti–viral immune responses in the CNS.
Using the J2.2–V–1 model of CNS infection, we addressed the role of sustained CCL2 production during viral infection using mice in which CCL2 was expressed transgenically in oligodendrocytes. Tonic CCL2 expression in the CNS resulted in delayed kinetics of virus clearance, and converted what is typically a mild, nonlethal disease to acutely lethal encephalitis, with the majority of mice succumbing to the infection. CCL2 induced a rapid and dysregulated inflammatory response that was no longer protective and was unable to efficiently clear virus from the CNS. Infected CCL2 Tg mice had increased numbers of Foxp3–expressing CD4 T cells (Tregs) and of macrophages and microglia expressing elevated levels of YM–1, a marker for alternatively activated macrophages, and nitric oxide. Our results showed that CCL2 has effects beyond serving as a chemoattractant for leukocytes, and has effects on the composition and function of inflammatory cells at sites of infection.
In a separate set of experiments, I identified and characterized two additional heteroclitic variants of the JHMV epitope S598 that induced CD8 T cells with greater antigen sensitivity to the native S598 determinant relative to the cells primed by the native epitope. One of these heteroclitic epitopes elicited a T cell response with nearly complete cross–reactivity towards the native peptide. The structural data show that these heteroclitic epitopes induced modest conformational changes in the local environment of the peptide–MHCI complex. I also provide data to support the notion that heteroclitic determinants augment functional avidity by increasing surface epitope density. Collectively, these data will help guide the design of heteroclitic epitopes in the setting of vaccine development.
Lastly, I examined the consequences of oxidative stress induced by viral infection on antigen presentation. The brains of JHMV–infected mice were found to have signs of oxidative stress, with significantly decreased ratios of reduced (GSH) to oxidized (GSSG) glutathione, suggesting that there is an environment that is conducive for cysteine modification with oxidized glutathione. We found that virus–induced oxidative stress resulted in the presentation of both native and S–glutathionylated forms of the JHMV epitope S510 by infected cells. A subset of the S510–specific CD8 T cells failed to recognize the modified form of the epitope, suggesting that GSH–modification of a cysteine–containing viral epitope might interfere with T cell recognition. Further, GSH-modified peptides were identified in stressed human cells, including herpes virus–transformed B cells, suggesting that the modification is not limited to mouse cells. Collectively these findings have implications for both anti–viral immunity and anti–tumor immunity, where oxidative stress has been shown to play a role during infection and tumorgenesis.
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Monjezi, Razieh [Verfasser], and Michael [Gutachter] Hudecek. "Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing / Razieh Monjezi ; Gutachter: Michael Hudecek." Würzburg : Universität Würzburg, 2018. http://d-nb.info/1162062231/34.
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Lazaro, Estibaliz. "Impact du sous-type viral et de l’apprêtement antigénique sur la présentation des épitopes du VIH-1 par les molécules HLA de classe I." Thesis, Bordeaux 2, 2010. http://www.theses.fr/2010BOR21776/document.
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Les lymphocytes T cytotoxiques (CTLs) dirigés contre le VIH jouent un rôle essentiel dans la défense anti-virale. L’identification des facteurs impliqués dans la variabilité de ces réponses est indispensable à la mise au point de vaccins efficaces.Nous avons focalisé notre travail sur deux facteurs potentiellement impliqués dans la reconnaissance du virus par le HLA : le sous-type viral et la qualité de l’apprêtement antigénique. L’extrême variabilité du virus avec à ce jour 11 sous-types et 48 formes recombinantes (CRFs) circulant au sein de populations au typage HLA hétérogène implique un polymorphisme important avec des mutations d’échappement multiples.Nos résultats montrent que dans la population Vietnamienne infectée par le VIH, le CRF01_AE prédomine largement et que l’affinité pour la molécule HLA des épitopes CTL classiquement décrits dans les sous-types B est drastiquement diminuée, ce qui favorise l’échappement de ce sous-type viral au système immunitaire.Par ailleurs, nous avons montré que l’apprêtement des épitopes CTL dépend du type de cellule impliquée, les monocytes se caractérisant par une capacité de présentation significativement plus forte à l’origine d’une réponse CTL plus efficiente comparativement aux lymphocytes T CD4 . Des tests de dégradation in vitro ont démontré que la stabilité intracellulaire des épitopes est hautement variable, dépendante de la séquence en acides aminés et contribue à l’optimisation de la réponse CTL.L’ensemble de ces résultats indiquent que, au delà de l’affinité pour le HLA ou le TCR et des facteurs d’épuisement cellulaire, la réponse CTL peut aussi être modulée par le sous-type viral et l’apprêtement antigéniqueHIV-specific cytotoxic T lymphocytes (CTLs) play a critical role for clearance of virus-infected cells and induction of these cells is a necessary component of any successful vaccine strategy against AIDS. Therefore, identification of the factors defining and modulating the efficiency of these protective responses are urgently needed. We focused our study on two factors potentially involved in HLA recognition: HIV-1 sub-type and antigen processing.The extreme variability of the virus with to date 11 HIV-1 subtypes and 48 circulant recombinant forms (CRFs) circulating worlwide among heterogeneous populations imply high polymorphism and different mutational escape patterns.We demonstrate that among the HIV-1 infected Vietnamese population where the CRF01_AE is largely predominant, the HLA binding of known CTL epitopes is strongly reduced compared to the subtype B due to intraepitopic mutations, facilitating immune evasion of these viral strains.Moreover, we show that the presentation of adequate amounts of epitopes leading to CTL recognition depends on the subset cells involved in the antigen processing, monocytes having a significantly higher and more efficient proteolytic activity. Using in vitro degradation assays, we measured the intracellular HIV-1 epitope stability and demonstrated that this factor is highly variable, sequence dependent and also contributes to a more efficient presentation.Together, these data indicate that, besides HLA and TCR binding and exhaustion factors, HIV-1 CTL recognition can also be modulated by the viral sub-type and the antigen processing machinery
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Chernyukh, O. G. "Qualitative detection of Ig G antibodies to SARS-CoV-2 coronavirus nucleocapsid antigen in the blood serum of patients who had viral respiratory infection, caused by SARS-CoV-2." Thesis, БДМУ, 2022. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/19534.
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Xu, Dan. "Cellular Immunity in Recombinant Adeno-Associated Virus Vector Mediated Gene Therapy." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1313504203.
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Mukherjee, Siddhartha. "The processing and presentation of viral antigens." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390538.
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McMahon, Christopher Wolcott. "Structure-function analysis of mouse mammary tumor virus superantigens /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/8342.
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Lafon, Monique. "La nucleocapside du virus rabique : une nouvelle cible pour la reponse immunitaire et pour la therapie." Paris 7, 1987. http://www.theses.fr/1987PA077219.
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Jowett, Jeremy Bryan Mark. "Properties of recombinant HIV and SIV antigens." Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:5de3119b-4aa1-469a-80b6-f1f28dff4f5c.
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When expressed in Spodoptera frugiperda cells using recombinant baculoviruses, the HIV - 1 gag gene product, p55, self assembles to form immature HIV core like particles that are secreted into the culture medium. Using this system, the gag open reading frame was progressively truncated from the carboxy terminus, and each deleted Gag protein examined for its ability to produce core like particles. Deletions that removed the distal region of the Gag nucleocapsid domain, including both Cys - His motifs thought to function as RNA capture signals, did not disrupt particle formation. Analysis of the truncation mutants by north - western blot with a radiolabelled HIV - 1 RNA encapsidation signal, confirmed that only precursors with the Cys - His motif present were able to bind the probe. It was concluded that HIV - 1 Gag particle formation per se does not require RNA encapsidation. This finding clarifies uncertainties proposed previously regarding the role of RNA encapsidation in particle assembly. Further deletion mutants led to the delineation of the carboxy terminal boundary of a Gag assembly domain. Properties of the SIV env encoded TM glycoprotein were also explored. Previous reports have found that the cytoplasmic tail of this membrane spanning protein was preferentially deleted in vivo when SIV was grown in a variety of cultured human cells. However, when propagated in cells of simian origin, the TM glycoprotein cytoplasmic tail was retained. Analysis of the function of this domain was addressed by expression of both the truncated and the full length proteins in insect cells using recombinant baculoviruses, coupled with a study of their biochemical characteristics. A role for the cytoplasmic tail was identified in the post - translational modification and localization of the glycoprotein. The use of the Gag particles in designing vaccines was investigated. Acting as non - infectious carriers of immunogenic antigens, the particles represent a safe and efficient means for presentation of epitopes for eliciting a protective immune response. Two possible approaches for loading the particles with foreign antigen were examined. In the first, the deleted portion of Gag was replaced with a sequence encoding the foreign antigen. The second method involved co - expression of Gag with another recombinant virus that expressed a foreign antigen on the surface of the cell. Both methods were found to be feasible, although limitations were identified when addition of the fusion protein promoted excess degradation of the Gag precursor.
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Perry, Sara Jane St John. "Novel delivery systems for SIV antigens." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321085.
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Stevenson, Philip G. "The immune response to viral antigens in the mouse brain." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364023.
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MAZZOLENI, ELISA. "Exploration of new techniques for purification and chemo-selective conjugation of bioreagents for immunodiagnostic applications." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/68468.
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Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici.
La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale.
La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.
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