Dissertations / Theses on the topic 'Antigen delivery'
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Lu, Zeyu (Zeyu Mike). "Protective antigen-mediated delivery of biomolecules." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/120906.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.
Includes bibliographical references.
The intracellular delivery of therapeutic biomolecules such as oligonucleotides and proteins remains a key challenge today. Protective antigen, a naturally evolved protein translocase derived from Bacillus anthracis, has shown promise as a platform of protein delivery due to its ability to form a transmembrane pore that allows the cargo to have cytosolic access. We and others have used the LFN/PA system to deliver a wide variety of natural and non-natural peptides and proteins. Despite the significant progress made with the LFN/PA delivery platform, some aspects including cargo selection and targeting still remain limited. In the first part of the thesis, we greatly expand the application of the platform by demonstration of efficient delivery of peptide nucleic acids (PNAs), an oligonucleotide analog. Using this technology, we successfully exploited a cancer- specific gene dependency by the intracellular delivery of an anti-sense PNA in a receptor-dependent manner. In addition to exploiting new types of cargo for delivery, we developed a new strategy to target the LFN/PA system to specific cell types. In the second part of the thesis, we chemically conjugated a full-length immunoglobulin G (IgG) to a mutant PA (mPA). Significantly, we took advantage of the fact that PA activation is protease-dependent and created highly specific delivery vehicles that can only be activated by the concurrent presence of two entities on the cell surface. We showed a protein toxin delivered by these IgG-mPA variants effectively inhibited cell growth in different cancer cell lines and exhibited a significantly increased therapeutic window over previously reported PA variants both in vitro and in vivo. In the last part of the thesis, we explored the possibility of simplifying the LFN/PA system by directly ligating protein cargos to PA. In the absence of LFN, the chemically created single-component system significantly increased the amount of delivered cargo. Moreover, the single-component system combined with a short N-terminal polylysine tag further improved the delivery efficiency by more than 100-fold. Our findings raise the prospect of a simpler PA-mediated delivery platform..
by Zeyu (Mike) Lu.
Ph. D.
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Rodgers, Emily Sarah. "Polymeric nanoparticles as immunopotentiating antigen delivery systems." Thesis, Queen's University Belfast, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337114.
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Al-Mamari, Ahmed. "Biocontainment system for bacterial antigen delivery carriers." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28793.
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Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.
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McNeil, Sarah E. "Liposome-mediated antigen delivery: formulation and optimisation." Thesis, Aston University, 2005. http://publications.aston.ac.uk/11037/.
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Live conventional vaccines are generally effective at provoking protective immunity against the infectious agent. However, there are many disadvantages regarding their adverse side effects and overall safety profile. Alternative vaccine strategies such as subunit and plasmid-based vaccines, using recombinant technology, are much safer, yet less effective. Therefore, the immunogenicity of such vaccines could be enhanced by utilising delivery and/or adjuvant systems, to provoke the appropriate immune responses. The role of liposomal systems within plasmid-based delivery was examined, looking at the effects of varying liposomal composition and method of preparation on the physical characteristics, transfection efficiency in vitro and immunogenicity in vivo. Compared to naked DNA alone, entrapment of plasmid DNA within liposomal vesicles results in complete protection from degradation by intracellular enzymes, with the DNA maintaining full structure and function. For liposome-mediated gene delivery small cationic lipids have been shown to be potent candidates, acquiring a net positive charge, which effectively interact with the anionic charges of the DNA to generate high incorporation values. In contrast, neutral liposomes are much larger aggregated structures with lower incorporation of DNA. The method of preparation was shown to effect the spatial location of plasmid DNA to liposomal systems. The dehydration-rehydration procedure (DRV) carried out in the presence of DNA effectively entraps the plasmid with little effect on liposome size and surface charge. Alternatively, upon addition of plasmid DNA the measured vesicle size of small unilamellar vesicles (SUV) or 'empty' (water containing) DRV increases due to the formation of SUV-DNA or DRV-DNA complexes, with the majority of the DNA localised on the surface of the liposomes. When applied in vitro, transfection efficiency of SUV-DNA complexes was greater than DRV(DNA). Transfection efficiency of SUVDNA complexes varied depending on the cationic lipid present within the lipid bilayer, with DC-Chol showing most efficiency. Furthermore, these DC-Chol cationic liposomes were formulated in combination with two different 'helper' lipids, the fusogenic lipid dioleoyl phosphatidylethanolamine (DOPE) or the stabilising lipid Cho!. The manner in which complexes form, the resultant structure and their transfection efficiency in vitro varied depending on the combining effects of both the type of 'helper' lipid incorporated within the lipid bilayer and total lipid to DNA charge ratio, with the overall structural size playing a significant role in promoting transfection. Transfection efficiency in vitro was significantly reduced when complexes were stabilised by the inclusion of phosphatidylcholines, with both the phospholipid head group and the alkyl-chain length influencing transfection efficiency. The production of DRV vesicles incorporating DNA were also produced in the range of IOO-200nm by the addition of a disaccharide (i.e. sucrose), prior to freeze-drying during the dehydration-rehydration procedure. In this instance, with an increase in sucrose/lipid mass ratio, the z-average diameter of liposomes decreased, while the percentage plasmid DNA, pRc/CMV HBS, entrapment remained relatively high (92%). Despite this, these small DRV(DNA) were found to be poor transfecting agents in vitro. After an initial screening process in vitro, a select few liposomal systems were subcutaneously administered in vivo. For all the liposomal formulations tested there was no induction of a humoral immune response, as no antibody titres were detected against the encoded antigen. However, SUV-DNA complexes composed of PC:Choi:DC-Chol (16:8:4 Ilmole/ml) and the production of small modified DRV(DNA) by the addition of sucrose generated sufficiently high levels of cell-mediated immunity. With regard to protein antigen delivery and adjuvanticity, the association with liposomal systems significantly enhances the immunogenicity of the fusion protein, Ag8SB-ESAT -6, a promising tuberculosis vaccine antigen. Several factors were shown to influence adjuvanticity of these liposomal systems. For example, the inclusion of the immunomodulator, TDB, effectively enhanced immunity against tuberculosis by increasing the adjuvanticity of these liposomal systems. Such immune responses were prolonged and most effective when these liposomal systems were either neutral or possessed a net positive charge rather than a negative charge and when the protein antigen was entrapped within these vesicles rather than surface complexed. Therefore, the overall protection against infection by tuberculosis was enhanced, presumably as a result of these liposomes forming depots, whereby the protein antigen is released slowly and at a controlled rate, maintaining therapeutic levels of the antigen in vivo to exert its therapeutic effect.
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Myschik, Julia, and n/a. "Immunostimulatory lipid implants as delivery systems for model antigen." University of Otago. School of Pharmacy, 2008. http://adt.otago.ac.nz./public/adt-NZDU20080806.114447.
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Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated.
Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies.
Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity.
Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.
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Cahill, Edward Sean. "Antigen delivery systems for nasal immunisation against B. pertussis." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321455.
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Neil, Stuart John Douglas. "Lentiviral mediated gene delivery to human antigen presenting cells." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251820.
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Saxena, Manvendra, and s3031657@student rmit edu au. "Utilising salmonella to deliver heterologous vaccine antigen." RMIT University. Applied Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080522.095907.
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Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.
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Guan, Holly H. "Development of liposomal antigen delivery system for synthetic MUC1 peptides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0005/NQ29044.pdf.
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Garmory, Helen Susan. "Vaccine vector-based delivery of the Yersinia pestis V antigen." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407227.
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Jeffery, Hayley. "The preparation and characterization of biodegradable microparticles in antigen delivery." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335608.
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Arikat, Farah. "Microneedle delivery of antigen-specific immunotherapy for Type 1 diabetes." Thesis, Cardiff University, 2019. http://orca.cf.ac.uk/118826/.
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Antigen-specific immunotherapy (ASI) involves induction of tolerance to autoantigens. An important protein in the development of type 1 diabetes (T1D) is the autoantigen, proinsulin (PI), the precursor of insulin. Microneedles (MNs) are micron-sized needles that penetrate into the upper skin layers. MNs provide advantages for autoantigen delivery including targeted delivery to the skin's dendritic cells (DCs), with minimal inflammation. The aim of this Thesis was to develop a PI-coated solid MN system and investigate the potential of this system to induce peripheral tolerance in the non-obese diabetic (NOD) mouse model of T1D. A highly concentrated PI MN coating formulation was developed containing the PI, diluent and a surfactant. The formulation enabled uniform and reproducible coating of the PI on to MNs. Delivery of PI from the MN system was investigated in mouse skin. MN application method and duration were optimised and resulted in skin puncture and reproducible delivery of PI to the skin. In vitro studies identified the insulin-reactive G9 CD8+ T cell as an appropriate biological readout for PI delivery. In vivo delivery studies indicated that MNdelivered PI was delivered to the skin and subsequently processed by DCs into PI peptides, which were cross-presented in the skin draining lymph nodes to adoptively transferred G9 CD8+ T cells. This demonstrated that the PI-coated MN system has potential for inducing peripheral tolerance in the NOD mouse. T1D development was significantly delayed in NOD SCID mice that received cells from PI-treated NOD mice and cells from diabetic NOD mice (experimental group). However, no statistically significant difference in time to T1D development was observed between the experimental group and the control NOD SCID mice that received cells from untreated NOD mice and diabetic NOD mice. Further investigation of the dosage and dosing frequency of PI using the coated MN system is, therefore, warranted.
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Isaac, Samine Jessica. "Investigating antigen delivery and presentation of OMV-based cancer vaccines." Doctoral thesis, Università degli studi di Trento, 2020. http://hdl.handle.net/11572/277831.
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Immunisation is the single most successful medical intervention to date. With the improved understanding of cancer immunity, cancer vaccines have quickly become weapons to be honed in the fight against cancer. The low immunogenicity of malignant cells hampers cancer immunotherapies. Cancer vaccines promise to overcome this problem.Outer membrane vesicles (OMVs) are spherical structures shed from all gram-negative bacterial outer membranes. Currently, OMV-based vaccines focus on infectious disease. There are gaps in their use as cancer antigen delivery vehicles for cancer vaccines. Due to their adjuvanticity and simplicity for genetic engineering, OMVs have great potential as a cancer vaccine platform. Here the aim is to show that it is possible to express an antibody derived single-chain variable fragment (scFv), specific for dendritic cell receptor DEC205, on the surface of OMVs using a bacterial protein as a carrier. Then demonstrate OMVs can be used to elicit good CD8+ T cell responses. And finally show that their adjuvant and delivery vehicle properties lend them for use in in situ cancer vaccination.I have shown we can engineer OMVs to express a functional scFv specific for the dendritic cell receptor DEC205 using a carrier. And that our proteome-minimised OMVs improved surface expression of the scFv fusion protein. The expression of the dendritic cell specific scFv increases internalisation of OMVs in dendritic cells. Importantly, this demonstrates an effective method for modifying OMV interaction with antigen presenting cells. Moreover, our OMVs, without additional dendritic cell targeting, are efficient vehicles for antigen delivery and can be used to elicit strong CD8+ T cell responses. Finally, we show that our proteome-minimised OMVs alone induce anti-tumour immunity when injected intratumorally. Additionally, in the presence of tumour-specific neoepitopes we elicit an improved anti-tumour response.The flexibility of OMVs both as adjuvants and vehicles for antigen delivery means they make a versatile vaccine platform, which can be fine-tuned using the techniques demonstrated.
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Alzahrani, Sharifah Yahya. "Dissolving microneedle arrays for enhanced transcutaneous delivery of a model antigen." Thesis, Queen's University Belfast, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602410.
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Vaccination remains the most important approach to offering protection from infectious diseases. However, using needles and syringes for vaccine administration continues to be a matter of concern, especially in developing countries. Re-use of needles, needle stick injuries and improper disposal of needles in these regions of the world increase the risk of spreading blood-borne pathogens among health care workers, patients and the wider community. The concerns about the safe vaccination practice have led to an intensive effort to develop safe delivery methods for vaccines and replace hypodermic injections. The opportunity to develop a safe and effective method of vaccination using a minimally invasive method is becoming real. The most promising approaches is microneedle (MN) array which has proven to be a safe and cost effective method for vaccination. The current thesis was focus on dissolvable MNs fabricated from 20%w/w poly(methyl vinyl ether/maleic acid) loaded with a model antigen, ovalbumin (OVA). Various experiments were carried out during this thesis to investigate the feasibility and efficacy of using MNs for vaccine delivery. The in vitro studies showed that MNs loaded with OVA were strong enough to avoid breaking under high compression force. The integrity of the primary and secondary structure of OVA loaded into MN arrays successfully ensured. Further, MNs enhanced the release of OVA into the skin compared to passive permeation. In in vivo studies, the OVA released from the MNs' matrix upon insertion into mouse skin targeted dendrite cells (DCs). This thesis showed that av A was engulfed by DCs, processed and migrated to the lymph nodes. Consequently, the processed antigen encountered naive T cells, which led to initiation of robust humoral and cellular immune responses indicated by production of IgG, IgG 1, IgG2a, IFN-γ and IL-4. Interestingly, the PMVE/MA copolymer used to fabricate MNs seems to have adjuvant effects, indicated by the higher IgG level of mice immunized using MNs fabricated from PMVE/MA loaded with OVA compared with MNs fabricated from PMVE/MA and loaded with OVA plus adjuvant imiquimod.
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Amores, Da Silva Pedro M. "The utilization of biodegradable PLPG microparticles as controlled antigen delivery systems." Doctoral thesis, Universite Libre de Bruxelles, 1999. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211917.
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Schumacher, Dominik. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18547.
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Antikörper und Antigen-bindende Proteine, die an Fluorophore, Tracer und Wirkstoffe konjugiert sind, sind einzigartige Moleküle, welche die Entwicklung wertvoller diagnostischer und therapeutischer Werkzeuge ermöglichen. Allerdings ist der Konjugationsschritt sehr anspruchsvoll und trotz intensiver Forschung noch immer ein bedeutender Engpass. Zusätzlich sind Antigen-bindende Proteine oftmals nicht dazu in der Lage, die Zellmembran zu durchdringen und im Zellinneren nicht funktionsfähig. Daher ist ihre Verwendung auf extrazelluläre Targets beschränkt, was eine bedeutende Anzahl wichtiger Antigene vernachlässigt. Beide Limitierungen bilden Kernaspekte dieser Arbeit. Mit Tub-tag labeling wurde ein neuartiges und vielseitiges Verfahren für die ortsspezifische Funktionalisierung von Biomolekülen und Antigen-bindenden Proteinen entwickelt, und so die Palette der Proteinfunktionalisierungen bedeutend erweitert. Tub-tag wurde erfolgreich für die ortsspezifische Funktionalisierung verschiedener Proteine und Antigen-bindender Nanobodies angewendet, die für konfokale Mikroskopie, Proteinanreicherung und hochauflösende Mikroskopie eingesetzt wurden. In einem weiteren Projekt wurden zellpermeable Antigen-bindende Nanobodies hergestellt und somit das schon lange Zeit bestehende Ziel, intrazelluläre Targets durch in vitro funktionalisierte Antigen-bindende Proteine zu visualisieren und manipulieren, erreicht. Hierzu wurden zwei verschiedene Nanobodies an ihrem C-Terminus cyclischen zellpenetrierenden Peptiden unter Verwendung von Expressed Protein Ligation funktionalisiert. Diese Peptide ermöglichten die Endozytose-unabhängige Aufnahme der Nanobodies mit sofortiger Bioverfügbarkeit. Mit Tub-tag labeling und der Synthese von zellpermeablen Nanobodies konnten wichtige Bottlenecks im Bereich der Proteinfunktionalisierung und Antikörperforschung adressiert werden und neue Tools für die biochemische und zellbiologische Forschung entwickelt werden.Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.
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Roberts, Mark J. J. "The production and characterization of a model microparticulate oral antigen delivery system." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291890.
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Francis, Donny [Verfasser]. "Development of protein loaded microparticles and nanoparticles for antigen delivery / Donny Francis." Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1080561382/34.
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Bowen, Joanne C. "Influence of microbial antigen formulation and delivery route on the immune response." Thesis, Aston University, 1990. http://publications.aston.ac.uk/12545/.
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Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccha-ride vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH arok mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.
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de, Castro Fernanda V. V. "Antibody-based vaccines for delivery of antigen to dendritic cells in situ." Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484853.
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Numerous studies have documented the crucial role of dendritic cells (DC) in the cross-priming of cytotoxic T-Iymphocyte responses against exogenous antigens. However, these responses, particularly to tumour specific antigens, are often suboptimal. Aiming to investigate the effectiveness of vaccines targeting anti~en to DC in situ and their ability to potentiate immunity, we generated a panel of conjugates consisting of ovalbumin (Ova) protein linked to monoclonal antibodies (mAb) that target molecules expressed mainly on DC. The effectiveness of the various [FabxOva] conjugates was investigated in in vitro co-culture assays and in an in vivo system where Ova-specific CDS and CD4 T· cells were adoptively transferred into naive mice. The results revealed that targeting of the integrin CDllc, induced the strongest CD4 and CDS T cell responses, followed by DEC205 and MHC-II targeting. Co-administration with the adjuvant a-CD40 mAb, 'prevented the induction of tolerance and generated functional memory effector T cells. In addition, immunization with . a single lo. w dose of [a-CD11cxOva] had the unique ability to rapidly generate high titres of a-Ova IgG. Together with data from biodistribution studies, we demonstrate here that delivery of antigen to DC in situ, in particular via CD11c, can efficiently potentiate T and B cell immunity and propose that this rpolecule plays an important and as yet poprly characterized role in the transfer of intact antigen to B cells and possibly CDS+DC. Immunization with [a-CDllcxOva] was observed to enhance the resistance to tumour development and these encouraging results highlight the potential clinical benefit of this strategy in the treatment of cancer.
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Reddin, Karen Margaret. "Purification, immunogenicity and protective potency of the F1 antigen from Yersinia pestis." Thesis, Open University, 1998. http://oro.open.ac.uk/54548/.
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The area of self-esteem in people with learning disabilities has been largely neglected, and previous researchers have employed a variety of approaches. It is important to further our understanding in the context of providing appropriate clinical interventions and in monitoring the effect of social policy developments on the individuals at the receiving end of service provision. The study aimed to assess the reliability and validity of a set of measures devised specifically for use with learning disabled people, by Szivos-Bach (1993). The measures assess social comparisons, perception of stigma and aspirations and expectations. The study was carried out with 30 adults with mild and moderate learning disabilities between the ages of 18 and 65. The results provide initial support for the social comparisons test as a measure of self-esteem. Less evidence was found for the stigma questionnaire and the aspirations-expectations test. The results are discussed in the light of comparable research into self-esteem measures with non-learning disabled populations. Further research is required, and the most profitable way forward seems to be development of multi-dimensional measures of self-esteem.
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Du, Roure Camille. "The regulated long-term delivery of therapeutic proteins using antigen-specific B lymphocytes." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424715.
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Chu, Hin Lun. "Intracellular delivery of radioimmunoconjugates that target the cancer testis antigen, NY-ESO-1." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:84c830c4-c216-4b2c-8383-e1119d77c295.
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Cancer testis antigens (CTA) represent attractive targets for targeted radiotherapy and imaging as their expression is restricted to cancer and germ cells. NY-ESO-1, a member of the CTA family, is highly immunogenic and expressed in multiple tumor types including carcinoma of bladder, liver lung. The aim of this study was to develop radioimmunoconjugates (RIC) to target NY-ESO-1 protein in cancer cells. Anti-NY-ESO-1 antibodies were modified by addition of DTPA for 111In-labelling or, in the presence of Iodogen, were 123I-labelled. Delivery of radiolabeled immunoconjugates across the cell membrane was achieved using a protein transfection (PT) reagent (SAINT-PhD) and by chemical linkage with the cell-penetrating and nuclear-localizing peptide, TAT (YGRKKRRQRRR). Cellular internalization, distribution and efflux of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT were investigated in cell fractionation and retention assays. It was shown that protein transfection reagent has promoted the cellular uptake of RICs into SK-MEL-37 and both of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT was retained longer in SK-MEL-37 cells in comparison to their isotope control RIC. In clonogenic assays, 111In-DTPA-anti-NY-ESO-1-TAT-PT significantly reduced surviving fraction of SK-MEL-37 cells. Cytotoxicity was inversely proportional to specific activity and the concentration of cells exposed to 111In-DTPA-anti-NY-ESO-1-TAT-PT. siRNA knock down of NY-ESO-1 resulted in partial reversal of 111In-DTPA-anti-NY-ESO-1-TAT-PT associated cytotoxicity. These promising results obtained from the in vitro study has brought the probe further into in vivo study. In preliminary biodistribution studies in SK-MEL-37 xenograft-bearing mice, tumour:muscle ratio for 111In-DTPA-anti-NY-ESO-1-TAT-PT was statistically significant compared to the control RIC 48 h post injection. This clearly indicated that the probe can be delivered into tumour in in vivo model and the successful uptake of radioactivity increased the chance of causing cytotoxicity to tumour cells through DNA damage. All of these findings have suggested that intracellular cancer associated antigen NY-ESO-1 can be reached by protein transfection reagent and cell penetrating peptide and initiates DNA damage through radio-isotope mediated cytotoxicity. Therefore, it represents a novel approach to the treatment of CTA-expressing cancers.
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Roos, Anna-Karin. "Delivery of DNA vaccines against cancer /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-895-9/.
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Maxwell, Tammy Joy. "Dendritic cell mRNA delivery strategies for ovarian cancer immunotherapy." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16495/.
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Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.
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Lu, Dongmei Hickey Anthony J. "Aerosol delivery of recombinant antigen 85B in microparticle vaccine systems for protection against tuberculosis." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1388.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmaceutical Sciences." Discipline: Pharmaceutical Sciences; Department/School: Pharmacy.
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Walters, Adam Alexander. "The development and evaluation of a nanoparticulate antigen delivery system for vaccination of cattle." Thesis, University of Surrey, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.600038.
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Conventional subunit vaccine regimes can be modified in order to stimulate strong immune responses resulting in memory cell formation. An adjuvant system that has gained increased attention in recent years is the use of particulate antigen delivery. Particulate systems have a number of advantages over conventional approaches since they are believed to be taken up preferentially by dendritic cells (DC) where they prolong the release of antigen resulting in enhanced T cell stimulation. Furthermore, they offer a versatile system that allows for targeted delivery of antigen and adjuvant to the same DC. A wide range of particle types have been used to enhance vaccine potency. Poly (Iactic-co-glycolic) acid (PLGA), in particular, has been used successfully by many groups. However, there are a great variety of means to synthesise and characterise the desired particles. This study, firstly, set out to develop and optimise a protocol to generate nanoparticles with defined properties. A number of parameters were evaluated including, particle size, protein loading, protein coating and surface charge. In addition to conventional methods, such as electron microscopy and dynamic light scattering, particles were characterised by novel flow cytometric methods. While particles have been shown to adjuvant candidate vaccine proteins, this property should be enhanced when the particle is targeted to dendritic cells by increasing specific uptake. Specific targeting has previously been performed through either targeting with natural receptor ligands or monoclonal antibodies. However, there is currently conflicting data in other studies as to whether this can be achieved. It was thus the second objective of this study to devise methods to implement this technology and to determine whether targeting can be achieved through either approach. It was 1 Abstract found that there was potential in targeting DC populations with monoclonal antibodies, while targeting with natural ligands yielded more mixed results. As a final component, the adjuvant properties of the particles rationally loaded with antigens and molecular adjuvant was tested in vivo in cattle using a viral challenge model. The experiment had a promising outcome with the vaccine particles inducing both T cell and antibody responses resulting in a degree of protection against virus challenge. Furthermore, it highlighted areas where the system, both the particles and the model, may be improved, which could form the basis of future work.
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Grimaldi, Elizabeth Anne. "The induction of cytotoxic T-lymphocytes against respiratory syncytial virus using toxin-mediated antigen delivery." Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422140.
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Foged, Camilla. "Human dendritic cells : cell culture, models for studies of particulate antigen, formulation in vitro /." Cph., Stockholm : Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Division of Hematology, Center for Molecular Medicine, Karolinska Hospital and Institute, 2003. http://www.dfh.dk/phd/defences/Camillafoged.htm.
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Doty, Raymond Thomas. "The role of the cytoplasmic tail of antigen-presenting cell surface molecule CD80 in delivery of T cell costimulation /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/8327.
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Ibrahim, Nesma Elsayed Ahmed [Verfasser]. "Theranostic Gelatin Nanoparticles for Antigen Delivery and Combined Strategies for Transcutaneous Application / Nesma Elsayed Ahmed Ibrahim." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1222973731/34.
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Rohrbach, Florian. "Induction of anti-tumor immunity by targeted delivery of ErbB2 cancer vaccines to antigen-presenting cells." Université Louis Pasteur (Strasbourg) (1971-2008), 2004. http://www.theses.fr/2004STR13026.
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Lavelle, Edward Charles. "Gastrointestinal antigen processing and its relevance to enteric vaccine delivery in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)." Thesis, University of Plymouth, 1994. http://hdl.handle.net/10026.1/2330.
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An investigation of antigen processing in the rainbow trout gastrointestine was carried out to provide a rational basis for the design of oral delivery systems for protein antigens. Using in vitro systems involving isolated lumenal enzymes and gut cell suspensions the degradation of human gamma globulin (HGG) and bovine serum albumin (BSA) was analysed by Western blotting and laser densitometry. Proteolysis by lumenal enzymes was dependant on pH and temperature and serine proteases were found to be partly responsible for antigen degradation in the intestine. The extent of intracellular proteolysis depended on the antigen used and on the gut region from which the cells were isolated. To test the predictive value of results obtained from the in vitro studies, the processing of HGG in the digestive tract after oral administration was investigated. The findings indicated that different regions of the gut perform distinct bur complementary roles in proteolysis. Measurement of the uptake of HGG into the bloodstream of these fish by enzyme linked immunosorbent assay (ELISA) and Western blotting indicated that the nature of proteins. absorbed from the gut could be influenced by altering the conditions in the gastrointestine. After parenteral and oral immunisation of HGG the antibody response was investigated in plasma and in mucosal and biliary secretions and found that a fragment of HGG produced by partial digestion with intestinal enzymes was highly antigenic in trout. The methods developed to study antigen processing in the gut were applied to assess the potential value of modern enteric delivery systems in teleosts. Encapsulation of HGG in poly lactide-co-glycolide (PLG) microparticles partially protected HGG from degradation in the gut and increased its absorption into the bloodstream. A live attenuated strain of Aeromonas salmonicida was shown to adhere to and invade isolated trout enterocytes and Atlantic Salmon tissue culture cells using a range of light - and electron microscopical techniques. These results indicate that an investigation into antigen processing by the gut is a valuable preliminary step in the formulation of oral delivery systems for teleosts.
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Afraz, Zahra [Verfasser]. "Investigation of Virus-Like-Particles and Antigen-Loaded Poly-Lactic-Acid Particles for Transcutaneous Vaccine Delivery / Zahra Afraz." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1074871049/34.
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Kaye, P. M. J. "Particle mediated co-delivery of IL-10 and antigen inhibits T cell activation but fails to induce tolerance." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302067/.
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Immune disorders such as allergy and autoimmunity are becoming increasingly common in developed countries. Self-reactive T cells exist in both healthy and autoimmune individuals. It is generally understood that hyperimmune disorders are caused by insufficient regulation, namely loss of activity of regulatory T cells. Whilst regulatory T cells exist naturally it is also possible to induce them both in vitro and in vivo. Immunotherapeutic techniques aim to provide noninflammatory exposure of antigen to the immune system with the aim of inducing antigen-specific regulatory T cells. Interleukin-10 (IL-10) is a cytokine with well known immunosuppressive qualities. It inhibits both the migration and the antigen-presenting ability of dendritic cells. It also has direct effects on T cells. Indeed, IL-10-secreting TR1 regulatory T cells were identified almost 15 years ago; their in vitro generation being dependent on exposure to IL-10. Particle-mediated DNA delivery (PMDD) is a promising method of immunisation and is especially suited to vaccines intended to have greater control over the response they induce. One of the main reasons for this is the possibility of including genes encoding immunomodulatory molecules alongside the antigen gene. This study utilises a mouse model involving the adoptive transfer of TCR-transgenic CD4+ T cells and establishes the response of these cells to PMDD immunisation. The model was then used to examine the effect of coadministration of the IL-10 gene. Its inclusion in the vaccine suppressed the response to antigen. This effect was maximal when the IL-10 gene was expressed in the same cell as the antigen gene. Using sequential immunisations the model was extended in order to study long-term effects, namely tolerance and the induction of regulatory T cells. Finally a mouse model of allergic asthma was used to examine any tolerogenic/therapeutic effects of the antigen-IL-10 vaccine. No significant longterm tolerance to antigen was identified. These results demonstrate that whilst the presence of IL-10 clearly inhibits the T cell response to antigen it does not necessarily confer tolerogenic properties on these cells. This brings into question whether IL-10 in the periphery, supplied, for example, by TR1 cells, generates fresh regulatory T cells or merely inhibits the response to a particular antigenic challenge.
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Hayavi, Sima. "Novel formulations for antigen delivery using biodegradable polymers: new approaches for the use of new and established adjuvants." Thesis, Aston University, 2002. http://publications.aston.ac.uk/12621/.
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The use of immunological adjuvants has been established since 1924 and ever since many candidates have been extensively researched in vaccine development. The controlled release of vaccine is another area of biotechnology research, which is advancing rapidly with great potential and success. Encapsulation of peptide and protein drugs within biodegradable microspheres has been amongst the most successful of approaches within the past decade. The present studies have focused on combining the advantages of microsphere delivery systems composed of biodegradable polylactide (PLLA) and polylactide-co-glycolide (PLGA) polymers with that of safe and effective adjuvants. The research efforts were directed to the development of single-dose delivery vehicles which, can be manufactured easily, safely, under mild and favourable conditions to the encapsulated antigens. In pursuing this objective non ionic block copolymers (NIBCs) (Pluronics@ LI01 and L121) were incorporated within poly-dl-lactide (PDLA) micorospheres prepared with emulsification-diffusion method. LI0I and L121 served both as adjuvants and stabilising agents within these vaccine delivery vehicles. These formulations encapsulating the model antigens lysozyme, ovalbumin (OVA) and diphtheria toxoid (DT) resulted in high entrapment efficiency (99%), yield (96.7%) and elicited high and sustained immune response (IgG titres up to 9427) after one single administration over nine months. The structural integrity of the antigens was preserved within these formulations. In evaluating new approaches for the use of well-established adjuvants such as alum, these particles were incorporated within PLLA and PLGA microspheres at much lesser quantities (5-10 times lower) than those contained within conventional alum-adsorbed vaccines. These studies focused on the incorporation of the clinically relevant tetanus toxoid (TT) antigen within biodegradable microspheres. The encapsulation of both alum particles and TT antigen within these micropheres resulted in preparations with high encapsulation efficiency (95%) and yield (91.2%). The immune response to these particles was also investigated to evaluate the secretion of serum IgG, IgG1, IgG2a and IgG2b after a single administration of these vaccines. The Splenic cells proliferation was also investigated as an indication for the induction of cell mediated immunity. These particles resulted in high and sustained immune response over a period of 14 months. The stability of TT within particles was also investigated under dry storage over a period of several months. NIBC microspheres were also investigated as potential DNA vaccine delivery systems using hepatitis B plasmid. These particles resulted in micro spheres of 3-5 μm diameter and were shown to preserve the integrity of the encapsulated (27.7% entrapment efficiency) hepatitis B plasmid.
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Gungor, Hatice. "Antigen specific delivery of chemokines by activated T cells : potential strategy for inducing inflammation at the tumour site." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9492.
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We have developed a new strategy to induce inflammation and other tumour destructive responses in a tumour specific manner. Inflammation can play an important role in tumour supression by stimulating an anti-tumour response. In this project we have harnessed the action of the angiostatic members of non-ELR CXC family chemokines (CXCL9 and CXCL10) with a view to directing lymphocyte chemotaxis to the tumour site. CXCL9 and CXCL10 recruit selective subsets of inflammatory cells by driving various cell types into the site of inflammation. They can also reduce angiogenesis in tumours. These chemokines are highly pleotropic, and in addition there are splice variants of the non-ELR CXC family receptor CXCR3 which may have contrasting effects. CXCR3A promotes cell survival and endothelial cell angiogenesis, while CXCR3B acts as a cellular inhibitor and is involved in angiostasis. Therefore, expression of these receptors is as important as expression of their ligands within the cancer in controlling the microenvironment and determining tumour behaviour. In order to target expression of CXCL9 and CXCL10, various constructs were created, with chemokines expressed under the control of the inducible promoters derived from the promoter of cytokine IL-2 gene, that is only expressed when T cells are activated. These constructs were transfected into primary T cells, and we showed that the promoter is activated when the T cell receptor is engaged and that production and activity of chemokine was restricted to activated T cells. The delivery method, by transfection of the constructs into tumour specific T cells, is designed to limit the production of the chemokine to the active tumour sites. The function and fidelity of the constructs were compared and optimized using T cell lines. We have determined the function and fidelity of the promoter and also confirmed the biological action of CXCL9 and CXCL10 in the construct. Quantitative real-time PCR experiments confirmed that expression of the construct results in production of the chemokine only when the T cell receptor is engaged. ELISAs were used to quantify, the amount of chemokine production. Chemotaxis assays showed the migration of cytotoxic lymphocyte populations and NK cells towards the supernatants of cells transfected with constructs. In preliminary experiments, tumour infiltrating lymphocytes from liver metastasis of colorectal adenocarcinomas were isolated and grown in culture. Transduction of the constructs into tumour specific cells will result in production and activity of the chemokine only in the presence of tumour antigen. Expression of CXCL9, CXCL10 and CXCR3A and CXCR3B in colorectal adenocarcinoma was validated by quantitative real time PCR in 50 fresh frozen liver metastasis samples from colorectal adenocarcinomas which showed strong correlation between the expression of two chemokines and also correlation between each chemokine and CXCR3B variant expression. This project forms a proof of concept, and would be a prelude for further work to demonstrate the in vivo efficacy of this approach.
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Kirby, Daniel J. "Formulation and characterisation of an effective particulate delivery vehicle for the novel sub-unit vaccine antigen, Ag85B-ESAT-6." Thesis, Aston University, 2007. http://publications.aston.ac.uk/11065/.
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This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-?), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.
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Bachmann, Michael, Holger Bartsch, Biji T. Kurien, Robert Hal Scofield, Achim Temme, Knut Schäkel, Senming Zhao, et al. "A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)." PLOS, 2011. https://tud.qucosa.de/id/qucosa%3A29020.
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Background
Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells.
Methodology/Principal Findings
Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells.
Conclusions/Significance
In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.
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Bachmann, Michael, Holger Bartsch, Biji T. Kurien, Robert Hal Scofield, Achim Temme, Knut Schäkel, Senming Zhao, et al. "A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-186316.
Full textAbstract:
Background
Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells.
Methodology/Principal Findings
Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells.
Conclusions/Significance
In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.
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Hauptmann, Nicole. "Ni(II)-NTA-modifizierte dendritische Glycopolymere als Trägersysteme für Antigen-Peptide in Zell-basierter Immuntherapie." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-129273.
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Dendritische Polymere werden im zunehmenden Maße als nicht-virale Vektoren für virus- oder tumor-assoziierte Antigen-Peptide zur Entwicklung neuer immuntherapeutischer Strategien eingesetzt. Diese beruhen auf der Verwendung von dendritischen Zellen (DCs), welche Schlüsselzellen bei der Induktion und Aufrechterhaltung einer T-Zell-basierten Immunantwort darstellen. Im Rahmen dieser Arbeit wurden Nitrilotriessigsäure-funktionalisierte dendritische Glycopolymere (NTA-DG) für den Transport von Antigen-Peptiden in DCs etabliert. Die Ni(II)-NTA-DGs waren durch definierte Komplexierungs- und Freisetzungseigenschaften charakterisiert. So wurde das Antigen-Peptid bei einem pH-Wert unter 6 vom polymeren Träger freigesetzt. Die gebildeten Polyplexe, zwischen Ni(II)-NTA-DG und dem Antigen-Peptid, bewirkten eine Erhöhung der Antigen-Peptid-Aufnahme in immaturen DCs (iDCs). Dieses war nach der Endozytose im frühen endosomalen und lysosomalen Kompartiment von iDCs lokalisiert. Somit kann das Antigen-Peptid am MHC Klasse II-Molekül im lysosomalen Kompartiment ohne sterische Hinderungen durch die Polymeroberfläche binden. Die Polyplexe bewirkten eine Aktivierung der iDCs durch Aufregulation der kostimulatorischen Moleküle CD86 und CD80 sowie der pro-inflammatorischen Zytokine IL-6 und IL-8. Weiterhin wurde die Migrationsfähigkeit und das pro-inflammatorische Potential der Antigen-Peptid enthaltenen maturen DCs (mDCs) aufrechterhalten. Somit stellen Ni(II)-NTA-DGs ein vielversprechendes polymeres Trägersystem für Antigen-Peptide dar.
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Bayon, Emilie. "Nouveau système de délivrance d'antigènes à base de nanoparticules lipidiques (Lipidots) pour formulation vaccinale." Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAV003/document.
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Les vaccins représentent l’un des progrès majeurs de l’Histoire pour la santé publique, concrétisant notamment l’éradication de la variole en 1980. Les vaccins historiques, à base de pathogènes entiers atténués ou inactivés et donc très immunogènes ont été progressivement remplacés par des vaccins à sous-unités, beaucoup plus sûrs mais en contrepartie moins immunogènes. Des adjuvants tels que des vecteurs et des molécules immunostimulantes ont donc été incorporés dans les formulations vaccinales dans le but de générer des réponses immunitaires de grande amplitude. Cependant, les principaux adjuvants actuellement autorisés chez l’homme induisent exclusivement une réponse immunitaire humorale, à savoir la production d’anticorps permettant de neutraliser les pathogènes extracellulaires. Or, certains pathogènes comme le VIH requièrent une immunité cellulaire, indispensable à l’élimination du virus persistant dans les cellules infectées. Dans ce contexte, les adjuvants de vaccin sont en plein essor dans le but d’identifier de nouveaux candidats plus performants et sûrs. Nous décrivons ici la démarche suivie afin de proposer un vecteur lipidique nanoparticulaire (LNP), dont la stabilité, l’innocuité et la versatilité en font un outil idéal pour la délivrance d’antigènes. Nous avons dans un premier temps réalisé la preuve de concept sur la base de l’antigène modèle ovalbumine, dont la délivrance aux cellules immunitaires a permis d’augmenter significativement la réponse humorale in vivo chez la souris. D’autre part, l’induction d’une réponse cellulaire a été observée par la double délivrance de l’antigène et d’un immunostimulant. Plusieurs combinaisons et stratégies de vectorisations ont été évaluées, dans le but d’identifier la formulation la plus performante en vue d’une étude de protection anti-tumorale. Finalement, nous avons appliqué ces technologies au cas concret du VIH avec l’antigène de capside p24, ce qui s’est conclu par une étude d’immunogénicité chez le primate non-humain. L’ensemble de ces résultats met en lumière la versatilité des LNP et leur capacité à induire des réponses immunitaires de grande magnitude, à médiation humorale et cellulaireThe development of vaccines was one of the major health advances of the last century, with the success of smallpox eradication in 1980. Historical vaccines, based on attenuated or killed pathogens thus strongly immunogenic were finally replaced by subunit candidates, much safer but also poorly immunogenic. Therefore, adjuvants such as vectors and immunostimulants were incorporated in vaccine formulations in order to generate immune responses of high magnitude. However, actual adjuvants authorized in human vaccines only trigger humoral immune responses, with the production of antibodies which neutralize extracellular pathogens. Yet, some pathogens such as HIV require the induction of a cell-mediated immunity, necessary to eliminate viral reservoirs in infected cells. In this context, new adjuvant systems are being developed in order to identify the most efficient and safe candidates. Here we describe the approach followed to prepare a stable, safe and versatile vector consisting in lipid nanoparticles (LNP), for the delivery of antigens. We first report the proof of concept of antigen delivery based on the model ovalbumin, leading to the significant enhancement of humoral responses in vivo in mice. Thereafter, we focused on the induction of cell-mediated immune responses through the vectorization of both antigens and immunostimulants. Several combinations and vectorization strategies were assessed in the aim to identify the best prototype for a study of protection against tumor challenge. Finally, we applied these systems to HIV and its capsid antigen p24, which allowed us to conduct an immunogenicity study on a non-human primate model. Altogether, these results highlight the versatility of LNP and their ability to induce potent humoral and cell-mediated immune responses
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Yap, Jonathan Woon Teck. "Dendritic cell maturation and activation via RNA/DNA danger signals : co-delivery of protein antigen with siRNA or CpG DNA." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/38449.
Full textAbstract:
Thesis (M. Eng.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005."June 2005."
Includes bibliographical references (p. 40-43).
Traditional vaccines consisting of live attenuated pathogens or inactivated toxins cannot be readily applied to the more challenging diseases of the present e.g. hepatitis C and the human immunodeficiency virus. As such, there is a need to develop new methods of priming the immune system against such foreign invaders. Recombinant protein subunits and peptides are relatively safe alternatives to live attenuated pathogens. However, these antigens are poorly immunogenic when administered alone in solution form and thus require the use of an adjuvant. To this end, we have developed a hydrogel-based nanoparticulate system to encapsulate protein antigen and to co-deliver it with DNA/RNA-based adjuvants to dendritic cells, the key antigen presenting cells in primary immune responses. Using CpG oligonucleotides or siRNA as adjuvants, we observed via enzyme-linked immunosorbent assays for interleukin 12 and interferon-[alpha], respectively, that DCs were activated by CpG oligonucleotide- and siRNA-functionalized nanoparticles [approx.]10-fold more potently than by soluble CpG or siRNA ligands.
by Jonathan Woon Teck Yap.
M.Eng.
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Khodami, Pantea. "An evaluation of novel lipid-enveloped nanoparticles for adjuvant and antigen delivery for an HIV vaccine : stepping from laboratory into potential markets." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/62742.
Full textAbstract:
Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, February 2011."February 2011." Cataloged from PDF version of thesis.
Includes bibliographical references (p. 69-80).
Enormous effort has been devoted to the development of a vaccine against human immunodeficiency virus (HIV). The purpose of this paper is to evaluate the technological and economical aspects of a potential vaccine designed by Professor Irvine's group. Lipid-enveloped virion-sized nano-particles with a biodegradable polymer core are used as synthetic pathogens to deliver HIV specific antigens and adjuvants. The nano-particles are designed to display multiple copies of the antigen on their surfaces and to elicit humoral immunity response. Topics such as patent ability, obtaining an FDA licensure, storage, cost of manufacturing, and supply of the vaccine are explored. A business model for commercialization of the vaccine is outlined, and some possible future business opportunities for the nano-particles are discussed.
by Pantea Khodami.
M.Eng.
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Boschi, Bazan Silvia [Verfasser], and Manfred J. [Akademischer Betreuer] Schmitt. "Comparative study of using different yeast genera as vehicles for protein delivery to antigen-presenting cells / Silvia Boschi Bazan. Betreuer: Manfred J. Schmitt." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2011. http://d-nb.info/1051434270/34.
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Schmitt, Saskia Maria [Verfasser], and Karl-Peter [Akademischer Betreuer] Hopfner. "Novel multifunctional antibody constructs combining antigen and adjuvant delivery to dendritic cells as a therapeutic vaccine / Saskia Maria Schmitt ; Betreuer: Karl-Peter Hopfner." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1221524437/34.
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Schumacher, Dominik [Verfasser], Christian P. R. [Gutachter] Hackenberger, Dorothea [Gutachter] Fiedler, and Heinreich [Gutachter] Leonhardt. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation / Dominik Schumacher ; Gutachter: Christian P. R. Hackenberger, Dorothea Fiedler, Heinreich Leonhardt." Berlin : Humboldt-Universität zu Berlin, 2017. http://d-nb.info/1185578390/34.
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Volckmar, Julia [Verfasser], and Stefan [Akademischer Betreuer] Dübel. "Characterizing the potential of DEC-205-mediated antigen delivery to dendritic cells as a tool to induce adaptive immunity against hepatitis C virus infection / Julia Volckmar ; Betreuer: Stefan Dübel." Braunschweig : Technische Universität Braunschweig, 2011. http://d-nb.info/1175824933/34.
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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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Anderson, Richard John. "Novel delivery systems for vaccination with bacterial antigens." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362376.
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