Dissertations / Theses on the topic 'Vaccine engineering'
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1
Brune, Karl Dietrich. "Engineering modular platforms for rapid vaccine development." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:41d57165-6e7c-4ca7-8025-b5ec31794c8c.
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Vaccines have saved more lives than any other medical intervention. Recombinant vaccines provide unmatched safety profiles, but at the expense of reduced immunogenicity. Virus-like particles (VLPs) resemble viruses in size, shape and repetitive arrangement but are devoid of pathogenic genetic material and therefore safe. Poor immunogens can be rendered immunogenic by display on VLPs. Successfully decorating VLPs is still a major challenge. Genetic fusion or chemical modification is often time-consuming and can lead to misassembly or misfolding, which obstructs generation of the desired immune response. SpyCatcher is a genetically encodable protein, previously engineered to form a covalent isopeptide bond to its peptide-partner SpyTag. Presented in this thesis are SpyCatcher-VLPs, based on the fusion of SpyCatcher to the bacteriophage VLP AP205. SpyCatcher- VLPs can be conveniently conjugated with SpyTag fused antigens, simply by mixing. I demonstrate the modularity of this approach by covalently linking several complex, cysteine-rich malarial antigens to SpyCatcher-VLPs, such as the transmission-blocking antigen Pfs25 and the blood-stage antigen CIDR. A single administration of Pfs25-SpyTag conjugated to SpyCatcher-VLPs induced potent antibody generation against Pfs25, even in the absence of adjuvant. Anti-Pfs25 antibodies induced by this platform conveyed potent transmission-blocking activity in the mosquito vector. The thesis further demonstrates the feasibility of more complex Catcher-nanoparticle architectures. The previously engineered SnoopCatcher covalently reacts with SnoopTag peptide and is orthogonal to the SpyCatcher / SpyTag pair. IMX313 is an engineered chimera of the multimerization domain of chicken complement inhibitor C4-binding protein. This work describes fusion of SnoopCatcher and SpyCatcher to IMX313, which yields independently addressable Catcher-moieties on a single IMX313 nanoparticle. Display of two antigens on one particle may enable single-particle, multi-disease vaccines as well as multi-stage vaccines to tackle immune evasion of parasites. The platforms presented should accelerate and enhance vaccine development and may create opportunities for imaging and metabolic engineering.
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Hanes, Justin Scott. "Polymer microspheres for vaccine delivery." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/10153.
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Webster, Gina. "Engineering immunoglobulin genes for novel Tuberculosis vaccine production in plants." Thesis, St George's, University of London, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.753991.
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Tuberculosis is ranked in the top ten leading causes of death from an infectious disease. In 2015, an estimated 10.4 million people developed TB and 1.8 million died from the disease. There is a long-standing vaccine against TB, called BCG, but its ineffectiveness highlights the need for a new novel vaccine that can help prevent pulmonary TB. Ag85B, the most abundantly expressed protein in mycobacterial culture fluids, is a leading vaccine candidate currently used in various TB subunit vaccines in clinical trials. In this project Ag85B was used to form a novel polymeric immunoglobulin G scaffold (PIGS) vaccine candidate by fusion to truncated IgG y-chain. Moreover, IgM ptp was fused to the y-chain to facilitate polymeric structure formation. The predicted immune-complex-like structures should bind complement and FcRs with increased avidity. PIGS were cloned and expressed in Nicotiana benthamiana, purified using protein G affinity chromatography, and polymeric structures were observed by Native PAGE, dynamic light scattering and size exclusion chromatography. These PIGS were shown to be biologically active as they bind to C lq component of the complement cascade as well as FcyRs. More importantly, it was shown that PIGS had greater avidity to low affinity FcyRs, as would be expected for polymeric structures. PIGS were shown to bind to monocytic cells and be internalised. Immunisation and challenge studies in BALB/c and CD64 transgenic mice showed that PIGS are immunogenic but did not improve protection of mice from challenge with Mycobacterium tuberculosis compared to BCG. Adding polylC adjuvant to human PIGS increased immunogenicity, resulting in spleen CFU counts of M. tuberculosis that were statistically reduced compared to CFU counts from BCG immunised mice. One of the major drawbacks to plant molecular pharming is that often relatively low levels of recombinant protein expression are achieved. As plants were used to express the vaccine candidate, a codon optimization algorithm was tested using synonymous codon variants of the native human 2G12 heavy chain which could be applied to increase the expression levels of any plant-made antibody, including the tuberculosis vaccine candidate. However, preliminary results indicated no improvement in 2G12 monoclonal antibody yield using any of the codon optimized variants, compared to native sequence. In conclusion, this study demonstrated that plant-produced PIGS contained polymeric structures and were capable of eliciting an immune response in vivo. Addition of adjuvant, polyIC, resulted in increased immunogenicity of the human PIGS candidate and improved protection of mice from Mycobacterium tuberculosis, measured by the lower CFU counts, in the spleen, compared to BCG immunized mice.
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Kaczorowski, Kevin J. "Data-driven strategies for vaccine design." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/117327.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2018.Cataloged from PDF version of thesis.
Includes bibliographical references.
Vaccination is one of the greatest achievements in immunology and in general medicine, and has virtually eradicated many infectious diseases that plagued humans in the past. Vaccination involves injecting an individual with some version of the pathogen in order to allow the individual to develop a memory immune response that will protect them from future challenge with the same pathogen. Until recently, vaccine development has largely followed empirical paradigms that have proven successful against many diseases. However, many pathogens have now evolved that defy success using the traditional approaches. Rational design of vaccines against such pathogens will likely require interdisciplinary approaches spanning engineering, immunology, and the physical sciences. In this thesis, we combine theoretical approaches with protein sequence and clinical data to address two contemporary problems in vaccinology: 1. Developing an antibody vaccine against HIV, an example of a highly mutable pathogen; and 2. Understanding how the many immune components work collectively to effect a systemic immune response, such as to vaccines. In HIV-infected individuals, antibodies produced by the immune system bind to specific parts of an HIV protein called Envelope (Env). However, the virus evades the immune response due to its high mutability, thus making effective vaccine design a huge challenge. To predict the mutational vulnerabilities of the virus, we developed a model (a fitness landscape) to translate sequence data into knowledge of viral fitness, a measure of the ability of the virus to replicate and thrive. The landscape accounts explicitly for coupling interactions between mutations at different positions within the protein, which often dictate how the virus evades the immune response. We developed new computational approaches that enabled us to tackle the large size and mutational variability of Env, since previous approaches have been unsuccessful in this case. A small fraction of HIV-infected individuals produce a class of antibodies called broadly neutralizing antibodies (bnAbs), which neutralize a diverse number of HIV strains and can thus tolerate many mutations in Env. To investigate the mechanisms underlying breadth of these bnAbs, we combined our landscape with 3D protein structures to gain insight into the spatial distribution of binding interactions between bnAbs and Env. Based on this, we designed an optimal set of immunogens (i.e. Env sequences), with mutations at key residues, that are potentially likely to lead to the elicitation of bnAbs via vaccination. We hope that these antigens will soon be tested in animal models. Even when the right immunogens are included in a vaccine, a potent immune response is not always induced. For example, some individuals do not respond to protective influenza vaccines as desired. The human immune system consists of many different immune cells that coordinate their actions to fight infections and respond to vaccines. The balance between these cell populations is determined by direct interactions and soluble factors such as cytokines, which serve as messengers between cells. A mechanistic understanding of how the various immune components cooperate to bring about the immune response can guide strategies to improve vaccine efficacy. To investigate whether differences in immune response could be explained by variation in immune cell compositions across individuals, we analyzed experimental measurements of various immune cell population frequencies in a cohort of healthy humans. We demonstrated that human immune variation in these parameters is continuous rather than discrete. Furthermore, we showed that key combinations of these immune parameters can be used to predict immune response to diverse stimulations, namely cytokine stimulation and vaccination. Thus, we defined the concept of an individual's "immunotype" as their location within the space of these key combinations of parameters. This result highlights a previously unappreciated connection between immune cell composition and systemic immune responses, and can guide future development of therapies that aim to collectively, rather than independently, manipulate immune cell frequencies.
by Kevin J. Kaczorowski.
Ph. D.
5
DeMuth, Peter C. (Peter Charles). "Engineered microneedles for transcutaneous vaccine delivery." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81667.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 151-165).
Immunization is a powerful approach for the prevention and control of infectious disease, however despite the successes of modem vaccine development, there remain several notable obstacles for the advancement of vaccine-mediated improvements in global healthcare. Many of the current limitations in vaccine availability and administration are the result of obligate needle-based delivery, which in addition to contributing to reduced speed, ease, and compliance in administration, has been shown to contribute to reduced overall safety due to needle re-use and needle-based injuries. Needle-based vaccine delivery to immunologically passive tissues such as muscle may limit efficacy, thus motivating the targeting of more inherently potent immune-competent sites. These inherent limitations of needle-based vaccination on global health have led to a strong impetus to develop needle-free vaccination strategies which have the potential to improve vaccine efficacy and availability, enhance the ease, speed, and safety of vaccine administration, and reduce vaccination associated costs world-wide. Here we present the design and preclinical testing of several parallel materials strategies for the noninvasive delivery of subunit vaccines to the skin. We have utilized laser ablative micro-molding of poly(dimethylsiloxane) to generate bio resorbable poly(lactide-co-glycolide) micro-structured skin patches bearing -100 micron-scale needles arrayed across their surface. Upon topical application, these 'microneedle arrays' are able to safely, and painlessly insert into the immune-competent epidermal skin layers to generate microscopic conduits through which otherwise impermeant vaccines and therapeutics are able to passage into the body. We have leveraged this approach in combination with layer-by-layer (LbL) directed assembly to generate vaccine-loaded conformal coatings on the surface of these microneedle arrays, which are then delivered into the skin through topical patch application. The construction of coatings containing antigen-expressing plasmid DNA (pDNA), together with immune-stimulatory RNA, and degradable cationic polymers provided tunable control over vaccine dosage, rapid and effective vaccine delivery in murine and primate skin models, and potent immunogenicity against a model HIV antigen in mice. In this case, DNA vaccine delivery was able to elicit strong functional CD8' T cell and humoral responses matching or exceeding the potency of in vivo electroporation, currently the most promising approach for clinical DNA delivery in humans. Further efforts have explored the use of LbL for encapsulation and delivery of soluble and particulate protein subunit vaccines, giving enhanced generation of diverse and potent humoral responses in mice. In other work, we have developed an approach enabling rapid delivery of micron-scale degradable polymer matrices or hydrogel depots using dissolvable composite microneedle structures for the delivery of vaccines with programmable kinetics. These efforts have demonstrated the potential of persistent vaccine release on tuning immune potency following non-invasive microneedle delivery, including induction of potent effector and memory CD8* T cell responses and more powerful and diverse antigen-specific humoral responses. Finally, we have developed an approach for simple loading and delivery of clinically advanced recombinant adenoviral vaccine vectors from sugar-glass coatings on bioresorbable microneedles. Formulation in microneedle coatings improved vaccine stability at room temperature and preclinical testing of these vaccine patches in mice and nonhuman primates demonstrated equivalent immunogenicity compared to parenteral injection, eliciting strong systemic and disseminated mucosal CD8' and CD4* T cell responses to a model HIV antigen. These cellular responses were correlated with a similarly potent systemic and mucosal humoral response, together suggesting the utility of this approach for non-invasive adenoviral immunization in a model close to humans. Together these results strongly demonstrate the potential of materials engineering strategies for the effective formulation, delivery, and release of recombinant vaccines by microneedle patches targeting the skin. In addition to the significant practical advantages enabled by microneedle delivery including improved safety, convenience, and storage, we have shown that advanced formulation strategies paired with controlled release are able to initiate humoral and cellular adaptive immunity more potently than possible through parenteral injection. Comprehensive tests in both mice and primates have suggested that these principles may be broadly applied to enhance various recombinant vaccination strategies potentially targeting numerous disease targets. Finally, initial tests performed in nonhuman primates have indicated the promise of engineered microneedle approaches for successful translation to humans. Overall, these findings provide a strong basis for the continued development of similar vaccination strategies for the comprehensive transformation of conventional vaccination enabling significant vaccine-mediated improvements in global health.
by Peter C. DeMuth.
Ph.D.
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Wikman, Maria. "Rational and combinatorial protein engineering for vaccine delivery and drug targeting." Doctoral thesis, Stockholm : Department of Biotechnology, Royal Insitute of Technology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-231.
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Howland, Shanshan W. "Yeast-based vaccine approaches to cancer immunotherapy." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45949.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2008.Includes bibliographical references.
Saccharomyces cerevisiae stimulates dendritic cells and represents a promising candidate for cancer immunotherapy development. Effective cross-presentation of antigen delivered to dendritic cells is necessary for successful induction of cellular immunity. Using a yeast vaccine model, we investigated the phagosome-to-cytosol pathway of cross-presentation. We demonstrate that the rate of antigen release from phagocytosed yeast directly affects cross-presentation efficiency, with an apparent time limit of about 25 min post-phagocytosis for antigen release to be productive. Antigen expressed on the yeast surface is cross-presented much more efficiently than antigen trapped in the yeast cytosol by the cell wall. The cross-presentation efficiency of yeast surface-displayed antigen can be increased by the insertion of linkers susceptible to cleavage in the early phagosome. Antigens indirectly attached to yeast through antibody fragments are less efficiently cross-presented when the antibody dissociation rate is extremely slow. Next, we present a yeast-based cancer vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the dendritic cell phagosome and subsequent translocation for cross-presentation.
(cont.) Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast cell wall fragments instead of whole cells, and by conjugating multiple layers of antigen. The novel multi-layer conjugation scheme is site-specific and takes advantage of Sfp phosphopantetheinyl transferase, enabling the antigen dose to grow linearly. We show that whole yeast cells coated with one layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing three layers were able to cross-prime naive CD8+ T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after ten days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied towards the development of other particulate vaccines.
by Shanshan W. Howland.
Ph.D.
8
Chen, Hongming. "Polymerized liposomes as potential oral vaccine delivery vehicles." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/10343.
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Panas, Cynthia Dawn Walker. "Design and manufacture of low cost vaccine cooler." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40937.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.Includes bibliographical references (p. 58).
Vaccines are very sensitive to temperature, needing to be held between 2 and 80°C to maintain potency. In developing countries where electricity and fuel supplies are unreliable, many vaccines are ruined due to thermal exposure. These are also the locations where vaccines are needed the most, yet often many of the vaccines given are ineffective. Long holdover vaccine coolers are designed to maintain a proper internal temperature during long periods of power loss. The most prevalent technology is the ice-lined cooler, but in the field these often have problem with freezing the vaccines. A vaccine cooler was designed that modifies the ice-jacket idea by separating the ice compartment and the vaccine chamber, connecting them through a heat transfer regulating device. The objective of this research is to design and prototype the heat transfer regulating device. After several design iterations a cooling loop filled with R-134a made of 1/8 piping, a 0.055 in ID capillary, and a Clippard normally-closed valve was combined with a modified car thermostat, using peanut oil as its working fluid, to create a thermosyphon type heat transfer device with a safety shutoff to prevent freezing. The prototype was manufactured and tested. It was found that with the proper amount of working fluid, it is possible to run the cooling loop at 4°C and pull heat from the vaccine chamber side to the ice. The peanut oil thermostat was tested and was found to open at a slightly lower temperature than expected, 2.5°C, but still within range. These results indicate that the concept is viable and should be tested in the vaccine cooler.
by Cynthia D. Walker.
S.B.
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Kang, Myungsun(Myungsun Sunny). "Optimizing vaccine dosing kinetics for stronger antibody response." Thesis, Massachusetts Institute of Technology, 2018. https://hdl.handle.net/1721.1/124586.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019Cataloged from PDF version of thesis. "The pagination in this thesis reflects how it was delivered to the Institute Archives and Special Collections. The Table of Contents does not accurately represent the page numbering"--Disclaimer Notice page.
Includes bibliographical references (pages 95-102).
One of the barriers to rational vaccine design against evolving pathogens is our lack of mechanistic understanding of how innate and adaptive immune response systematically emerge and evolve. Immune response is comprised of dynamic events that require many components to cooperate collectively in a manner that spans a range of scales. These characteristics make it hard to predict mechanisms for immune response based solely on experimental observations. This thesis investigates various aspects of affinity maturation that are relevant to vaccination and therapeutic strategies but are not yet fully understood mechanistically, ranging from the evolution of the heterogeneity of the antibody population with respect to affinity to optimal design parameters for temporal dosing of vaccines. Our approach is to apply computational techniques to mathematically model the immune system, and being synergistic with complementary experiments. 1.
As affinity maturation ensues, average affinity of antibodies increase with time while resulting affinity distribution becomes increasingly heterogeneous. To shed light on how the extent of this heterogeneity evolves with time during affinity maturation, we have taken advantage of previously published data of antibodies isolated from individual serum samples. Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we find that after a single injection of small antigen doses, the ratio decreases progressively over time. This is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal. 2.
What are the aspects of affinity maturation being altered by various temporal patterns of antigen dosing? Certain extended-duration dosing profiles increase the strength of the humoral response, with exponentially-increasing(EI) dosage providing the greatest enhancement. While this is an exciting result, it is necessary to establish a mechanistic understanding of how immune response be enhanced to further engineer and optimize the temporal patterns. From our computational model, the effect is driven by enhanced capture of antigen in lymph nodes by evolving higher-affinity antibodies early in the GC response. We validate the prediction from independent experimental data, where EI dosage result in promoted capture and retention of the antigen in lymph nodes. To our knowledge, this work is the first to demonstrate a key mechanism for vaccine kinetics in the response of B cells to immunization, and may prove to be an effective method for increasing the efficacy of subunit vaccines. 3.
Are there optimal dosing profiles that maximize total protection? That is, lead to the evolution of the most antibodies of high affinity? In extension of mechanistic studies in 2, we propose a stochastic simulation method that can be used as a tool for optimizing dosage protocols for vaccine delivery. Using this tool, we analyze experimental conditions for EI dosage induce suboptimal immune response and investigate two approaches for the optimization. Specifically, reducing the total dosage optimizes affinity of resulting antibodies, while total protection is optimal neither at constant or EI dosage but that corresponding to a "linear-like" dosing profile. Our approach can be extended to broader applications in vaccine design.
by Myungsun (Sunny) Kang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering
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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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Hanson, Melissa C. (Melissa Catherine). "Enhancement of HIV vaccine efficacy via lipid nanoparticle-based adjuvants." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/97975.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, February 2015..Cataloged from PDF version of thesis. "December 2014."
Includes bibliographical references (pages 93-108).
Adjuvants are immunomodulators and/or formulations/delivery vehicles which enhance immune responses to vaccines. The lack of progress in the development of an HIV humoral vaccine is due, in part, to the absence of available adjuvants which can be sufficiently potent with minimal adverse side effects. The main goal of this thesis was to develop nanoparticles as HIV vaccine adjuvants. Building upon previous work in the Irvine lab, we determined the potency of lipid-coated microparticles was due in part to the in situ generation of antigen-displaying liposomes. Synthetic liposomes were nearly as potent as lipid-coated microparticles, but with a 10-fold greater antigen conjugation efficiency. We subsequently optimized unilamellar liposomes as delivery vehicles for surface-displayed HIV antigens. For vaccines with a recombinant gpl20 monomer (part of the HIV envelope trimer), immunization at 0 and 6 weeks with 65 nm or 150 nm diameter liposomes with 7.5 pmol gpl20 was found to induce strong anti-gp120 titers which competed with the broadly-neutralizing antibody VRC01. The second HIV antigen used was a peptide derived from the membrane proximal external region (MPER) of the gp41 protein. High-titer IgG responses to MPER required the presentation of MPER on liposomes and the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced optimizing the MPER density to a mean distance of -10-15 nm between peptides on the liposomes surfaces. Lastly, we explored the adjuvant potential of cyclic dinucleotides (CDNs) with MPER liposome vaccines. Encapsulation of CDN in PEGylated liposomes enhanced its accumulation in draining lymph nodes (dLNs) 15-fold compared to unformulated cyclic dinucleotide. Liposomal CDN robustly induced type I interferon in dLNs, and promoted durable antibody titers comparable to a 30-fold larger dose of unformulated CDN without the systemic toxicity of the latter. This work defines several key properties of liposome formulations that promote durable, high-titer antibody responses against HIV antigens and demonstrates the humoral immunity efficacy of nanoparticulate delivery of cyclic dinucleotides, which is an approach broadly applicable to small molecule immunomodulators of interest for vaccines and immunotherapy.
by Melissa C. Hanson.
Ph. D.
13
Werts, Kendall (Kendall Marie). "Synthesis of biodegradable hydrogel microparticles for vaccine protein delivery." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/44811.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007.Includes bibliographical references (p. 21).
Soluble protein antigens used in vaccines have shown lower immune responses when compared with certain particulate forms of these same antigens. For example, it has been shown that micro- and nano-particle mediated delivery of protein antigen can use up to 100 times less protein and still produce an effective immune response [1]. In order to use this phenomenon to make vaccines more efficient, we need a biodegradable delivery particle. This thesis modifies a particle created by Jain et al., which consists of a polymer network surrounding and trapping a protein, by removing the non-degradable crosslinker used in the original particle design and replacing it with a poly (ethylene glycol) acrylate molecule attached to ovalbumin protein. When a dendritic cell degrades the particle, the ovalbumin protein will be degraded, as will the connections between the polymer network that holds the particle together [2]. The particles degraded to 56% of their original size in 3 days, while the non-degradable particle degraded to only 80% of its original size.
by Kendall Werts.
S.B.
14
Lau, Ting Ting Alice. "Engineering a pro-apoptotic BCG strain to improve efficacy of the current tuberculosis vaccine." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60692.
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Bacillus Calmette-Guérin (BCG), introduced almost 100 years ago, is the only vaccine designed to prevent tuberculosis (TB). BCG effectively protects newborns from meningeal TB yet fails to prevent adult pulmonary TB. In fact, TB kills 1.3 million people annually in areas where BCG vaccination is widely practiced. Thus, more efficient TB vaccines are urgently needed.
We and others have shown that BCG possesses the same virulence traits of Mycobacterium tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the macrophage’s cysteine protease cathepsin S (CatS), which leads to prevention of MHC II molecule maturation and proper antigen presentation. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to i) contain and process ingested bacteria and ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells.
To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (rBCG-CatS). Since caspase-3 plays a central role in the execution of apoptosis, we also constructed a BCG strain that secrets an active form of caspase-3 (rBCG-C3).
Macrophages infected with either recombinant strain elicited a pro-apoptotic phenotype as indicated by increased levels of annexin V surface staining, PARP degradation, and caspase-3 cleavage compared to parental BCG. Furthermore, macrophage transcriptomic profiling revealed that rBCG-CatS up-regulates key pro-apoptotic genes and down-regulates anti-apoptotic genes, which were further confirmed by RT-qPCR analyses. Consistent with these findings, mice vaccinated with rBCG-CatS or rBCG-C3 showed increased antigen-specific CD4+ and CD8+ T-cell responses, as well as enhanced cytokine production and proliferation upon ex vivo re-stimulation. Of particular note, immunogenicity responses from mice vaccinated with rBCG-C3 exceeded the effects observed with rBCG-CatS, demonstrating that induction of apoptosis is key to achieving high immunogenicity of TB vaccines.
Collectively, we have shown that by modifying BCG we can promote key host traits that confer high potential in improving efficacy of the TB vaccine.Medicine, Faculty of
Experimental Medicine, Division of
Medicine, Department of
Graduate
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Smith, Mark T. "Engineering Cell-Free Systems for Vaccine Development, Self-Assembling Nanoparticles and Codon Reassignment Applications." BYU ScholarsArchive, 2014. https://scholarsarchive.byu.edu/etd/4449.
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This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness” of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.
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Sandford, Erika J. (Erika Jaye). "Experimental technique for optimizing aerosolized vaccine efficacy by Erika J. Sandford." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/59930.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2010.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 26).
Vaccination via aerosol has been proven to be as safe, as effective, and more appropriate for transportation when compared with vaccination via injection. These advantages make aerosolized vaccinations a realistic alternative to traditional injection vaccines for the developing world, where cold chain systems are impractical and the use of hypodermic needles can be unsafe. However, more research is needed to determine optimal parameters for vaccine aerosolization. This thesis presents an experimental setup to test Aerovax, a device designed to deliver aerosolized vaccinations in developing regions of the world. The experimental technique is the first effort to optimize vaccine aerosols across multiple variables, including input pressure, nebulizer geometry, and vaccine reconstitution. The setup provides a pressure input, sensors for ambient properties, and a device to measure particle size distribution.
S.B.
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Gallovic, Matthew D. "Acid-Sensitive Polymer Microparticles for Subunit Vaccine Delivery." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1468803443.
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Bershteyn, Anna. "Lipid-coated micro- and nanoparticles as a biomimetic vaccine delivery platform." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/101866.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2010.Vita. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 128-146).
Biomaterials provide a unique opportunity to control the display, release, and in vivo trafficking of vaccine components. We have designed and characterized a system for vaccine delivery that uses a "lipid surfactants" approach to combine a degradable polymer core with a two-dimensionally fluid lipid bilayer shell. Using optical and electron microscopy, we characterized the distribution, lamellarity, nanostructure, and mobility of the lipid shell. Single, tightly apposed bilayers could be formed around either microparticles or nanoparticles, mimicking bacteria or viruses in size. Alternative nanostructures could be formed at different lipid concentrations and compositions, such as multilamellar lipid "onions" stacked against a polymer core, or lipid "flowers" with petal-like projections emanating from a polymer core. The lipid nanostructure was characterized during the process of emulsion and solvent evaporation, and during degradation by hydrolysis. Design of this carrier was guided foremost by the goal of properly displaying minimal peptide epitopes from the Membrane-Proximal External Region of HIV gp41 (MPER) and enhancing their immunogenicity. Display within a lipid context was needed to provide the chemical environment appropriate for neutralizing antibodies, such as 4E10, to bind efficiently. Multifunctional vaccines were created through a combination of multivalent display, delivery of helper stimuli, and insertion of lipophilic adjuvant molecules in the lipid shell. We further explored the ability of this system to potentiate humoral immune responses against recombinant protein vaccines, using ovalbumin as a model antigen. In studies of both cellular and humoral immune responses, we found that lipid-coated microparticles co-displaying protein and lipophilic adjuvant molecules could potentiate immune responses in vivo. Notably, we found that the dose-sparing capabilities of the particles reached a potency that is seldom reported: a single injection of 2 ng antigen co-displayed on particles with c-galactosylceramide elicited measurable titers, and a prime-boost regimen of 2.5 ng ovaparticles adjuvanted with monophosphoryl lipid A elicited high titers that were sustained for >150 days. No studies to our knowledge have reported dose sparing to this degree with titers sustained over time. The mechanism of this dose sparing effect is of great interest, and will be a subject of future work.
by Anna Bershteyn.
Ph. D.
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Shi, Amy (Amy J. ). "Versatility of M13 bacteriophage in medicine : vaccine storage and cancer diagnostics." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38583.
Full textAbstract:
Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007.Includes bibliographical references (p. 88-94).
Two novel ways of engineering the filamentous bacteriophage, M13, for the prevention, diagnosis, and treatment of human disease are proposed. Both ways are founded on the unique structural properties of the M13 bacteriophage and the ability of its major and minor coat proteins, p3 and p8, to be manipulated to serve as virus-based multifunctional platforms. The first project addresses the problem of vaccine storage and the cold chain (requirement to store vaccines at 2-80C or lower). The need for refrigeration leads to high cost, difficult field delivery, and high potential for vaccine instability. By capitalizing on the liquid crystalline nature and unique diffraction patterns of phage films, we aim to encapsulate vaccines in a 3-D liquid crystalline matrix that would not only allow for stability at elevated temperatures but would also allow for easy detection of viability by using a laser light and noting the diffraction pattern. We chose luciferase as a model for a protein-based vaccine, and found several phage-borne peptide sequences with increased affinity to luciferase compared to controls. Two of these sequences, CKLHGTSRC and CTHKNQAC were chosen to form luciferase-encapsulated phage films. The second project addresses the need of more sensitive imaging techniques for early detection of cancer.
(cont.) M13 bacteriophage were used in combination with quantum dots and magnetic nanoparticles as bigger and brighter markers for cancer lesions. A 100% expressed p8 library was created for screening against potential cancer markers and work will soon proceed with screening against several cancer cell lines. In addition, a Type 83 phage was created that had a sequence directed against vascular cell adhesion molecule-1 (VCAM-1) expressed on the p3 and a tri-glutamate sequence (E3) on the p8 that could bind well to positively charge molecules like amines. Successful attachment of amine-terminated CdSe/ZnS quantum dots to p8E3 phage (E3 on p8 and wildtype on p3) was shown and III-V quantum dots (GaN and InN) were water solubilized for use in vivo. The goal is to combine all parts and start in vivo testing and screening, as well as to expand our cancer targeting repertoire.
by Amy Shi.
S.M.
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O'Keeffe, Roderic S. "The production, harvest and adsorptive recovery of an infectious herpes simplex virus vaccine." Thesis, Aston University, 1999. http://publications.aston.ac.uk/9630/.
Full textAbstract:
At present there is not a reliable vaccine against herpes virus. Viral protein vaccines as yet have proved unsuccessful to meet the challenge of raising an appropriate immune response. Cantab Pharmaceuticals has produced a virus vaccine that can undergo one round of replication in the recipient in order to produce a more specific immune reaction. This virus is called Disabled Infectious Single Cycle Herpes Simplex Virus (DISC HSV) which has been derived by deleting the essential gH gene from a type 2 herpes virus. This vaccine has been proven to be effective in animnl studies. Existing methods for the purification of viruses rely on laboratory techniques and for vaccine production would be on a far too small a scale. There is therefore a need for new virus purification methods to be developed in order to meet these large scale needs. An integrated process for the manufacture of a purified recombinant DISC J-ISV is described. The process involves culture of complementing Vero (CR2) cells, virus infection and manufacture, virus harvesting and subsequent downstremn processing. The identification of suitable growth parameters for the complementing cell line nnd optimal limes for both infection and harvest are addressed. Various traditional harvest methods were investigated and found not to be suitable for a scaled up process. A method of harvesting, that exploits the elution of cell associated viruses by the competitive binding of exogenous heparin to virus envelope gC proteins, is described and is shown to yield significantly less contaminated process streams than sonication or osmotic approaches that involve cell rupture (with> 1O-fold less complementing cell protein). High concentrations of salt (>0.8M NaCl) exhibit the same effect, although the high osmotic strength ruptures cells and increase the contamination of the process stream. This same heparin-gC protein affinity interaction is also shown to provide an efficient adsorptive purification procedure for herpes viruses which avoids the need to pre-treat the harvest material, apart from clarification, prior to chromatography. Subsequent column eluates provide product fractions with Cl 100- fold increase in virus titre and Iow levels of complementing cell protein and DNA (0.05 pg protein/pfu and 1.2 x 10-4 pg DNA/pfu respectively).
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Jain, Siddhartha Ph D. Massachusetts Institute of Technology. "Synthesis of a hydrogel-based vaccine to mimic dendritic cell responses to pathogens." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37957.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (p. 143-160).
Live or attenuated pathogens are the basis of many successful vaccines due in part to the orchestrated response of dendritic cells (DCs) triggered by these immunizations, which includes (1) DC and DC precursor attraction to the immunization site, (2) efficient antigen delivery to class I and class II MHC loading pathways coincident with maturation of DCs, and (3) emigration to draining lymph nodes for T cell activation. We have developed a model immunization system designed to allow these steps in the DC life cycle to be controlled in the context of a subunit vaccine. The system is comprised of microspheres encapsulating chemokines and hydrogel nanoparticles; each nanoparticle contains antigen and DC maturation signals (e.g., TLR ligands). The nanoparticles remain sequestered within the carrier microspheres but the chemokine is released at a controllable rate, creating a local chemoattractant gradient centered on each microsphere. DCs are attracted to individual microspheres where nanoparticles are concentrated; attracted DCs extract nanoparticles from the carrier microspheres, and receive maturation signals coincident with the delivery of antigen into both class I and class II MHC processing pathways.
(cont.) In addition, the nanoparticles may be labeled to allow subsequent tracking of particle-carrying DCs in vivo. These components allow the attraction (or if desired, emigration) of dendritic cells and their precursors to be selectively modulated at an immunization site, and the activation signals received by these cells when they encounter antigen to be tailored. In vitro experiments indicate that chemokine-releasing microspheres effectively attract DCs and monocytes over significant distances, and that the gel nanoparticles efficiently trigger DC maturation and lead to both CD4+ and CD8+ T cell activation in vitro and in vivo. This system provides both a platform for rational immunotherapy as well as a powerful set of tools by which the function of dendritic cells can be manipulated and dissected to improve our understanding of how DC trafficking and functional state impacts immune responses.
by Siddhartha Jain.
Ph.D.
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Nicholson, Laura A. (Laura Ann). "Design of experimental setup for identification of parameters for optimal aerosolization of measles vaccine." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/54520.
Full textAbstract:
Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 29).
Given the pressing worldwide need for measles vaccination coverage, measles vaccine administration via inhalation is a viable option which overcomes many obstacles currently facing vaccine distribution. Although aerosolization is well understood, studies have focused on pharmaceuticals and very little data is available regarding vaccines. The proposed study analyzes the relationships between various pre- and post-aerosolization parameters in order to calculate the "aerosol fingerprint," or combination of pre-aerosolization parameters optimized to produce the most effective aerosol particle size distribution for measles vaccination. Relevant pre-aerosolization parameters are identified as relative humidity, nebulizer temperature, vaccine reconstitution, solution pH, surface tension, viscosity, air pressure, and nebulizer geometry. Relevant post-aerosolization parameters are identified particle size distribution, aerosolization endurance and bioavailability, and drug delivery rate. Sensing, actuation, automation and special concerns for each variable are considered.
by Laura A. Nicholson.
S.B.
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Little, Steven (Steven Ronald). "Poly ([beta]-amino ester)s as pH sensitive biomaterials for microparticulate genetic vaccine delivery." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/34159.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.In title on t.p., "beta" appears as lower-case Greek letter.
Includes bibliographical references.
Genetic vaccination is the administration of nucleic acids to induce cellular expression of antigens, leading to an immune response. Unlike traditional vaccines, this technology has tremendous potential for treating or preventing diseases such as HIV, malaria, and cancer. However, this potential is currently unrealized because of the safety concerns which plague viral vaccine carriers and the inefficiency of nonviral delivery systems when compared to their viral counterparts. A promising and versatile nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect their payload and target it to phagocytic, antigen-presenting immune cells. However, the biomaterial conventionally used in these microparticle formulations, an FDA-approved polyester called poly lactic-co-glycolic acid (PLGA), was not designed specifically to deliver DNA, takes too long to release encapsulated payload, and therefore fails to induce high levels of target gene expression. A new class of novel biomaterials have been synthesized called poly([beta]-amino ester)s which are biodegradable and can have similar physical properties to PLGA, but are pH-sensitive and have gene delivery functionalities.
(cont.) Using these materials we can fabricate microparticle-based delivery systems which have relatively high DNA loadings and can significantly buffer the destructive acidic pH microenvironment created by ester bond degradation. These formulations generate an increase of up to 5 orders of magnitude in DNA delivery efficiency when compared to PLGA alone and can be potent stimulators of antigen presenting cells in vitro. We have also demonstrated that incorporating these new biomaterials into microparticulate genetic vaccines can lead to antigen-specific, immune-mediated rejection of a lethal tumor dosage in vivo, a significant advance over conventional formulations. Finally, with the synthesis of libraries containing thousands of structurally diverse PBAEs, it is warranted to develop new methods of fabrication which enable the high-throughput screening of such libraries. Herein, we describe, for the first time, such a rapid fabrication technique and demonstrate that plasmid encapsulated in these formulations is transcriptionally active.
by Steven Little.
Ph.D.
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El, Meleigy May Ahmed Nagib. "Schistosoma mansoni heat shock protein 60 : cloning, expression and preliminary vaccine testing." Thesis, University of London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272135.
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Fung, Peter W. (Peter Waitak). "Evaluation of polyelectrolyte multilayer thin-film coated microneedle arrays for transcutaneous vaccine delivery." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/69787.
Full textAbstract:
Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 43-46).
The skin is an ideal organ for the safe and convenient delivery of vaccines, small molecules, and other biologics. Members of the Irvine and Hammond groups have developed a polyelectrolyte multilayer thin film-coated microneedle platform that can achieve simultaneous DNA and nanoparticle delivery. This delivery platform has the advantage of direct delivery of DNA or polymer nanoparticles to immune-active cells at the interface between the dermis and epidermis, enhancing uptake of the delivered cargo by resident immune cells. Ideal for the delivery of DNA vaccines, this platform aims to bridge the gap in the lack of efficient delivery platforms hampering the effectiveness of DNA vaccines. The ability to co-deliver polymer nanoparticles can serve as a conduit for delivering immune stimulating adjuvants or other drugs for therapeutic applications. An overview of current vaccine and delivery system research is presented. Market factors for the commercialization of the polyelectrolyte multilayer thin film-coated microneedle delivery platform are considered along with the risk factors in bringing this invention to market. An assessment of the intellectual property surrounding the platform is performed and a preliminary market entry strategy is developed for minimizing the risks commercialization.
by Peter W. Fung.
M.Eng.
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Atukorale, Prabhani U. (Prabhani Upeka). "Amphiphilic gold nanoparticles: mechanisms for interaction with membranes and applications in drug and vaccine delivery." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/90145.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 70-82).
Materials that can interact with and transit membranes without toxic bilayer disruption or poration are of great interest in the drug delivery field. These materials can presumably bypass endocytosis to directly enter the cell cytosol through the plasma membrane, which is often the desired site of action for therapeutics, thereby avoiding potential and likely cargo degradation if trapped in endosomes. Alternatively, if these materials are endocytosed, they are often able to escape the endosome by interacting with and transiting the endosomal membrane, and moving into the cytosol. Here, we present a unique membrane-interacting, amphiphilic gold nanoparticle (amph-AuNP) system, which we show can interact with, embed within, and even penetrate through multiple adjacent lipid bilayers without evidence of membrane disruption or poration. By virtue of these key properties, we also present these amph-AuNPs as effective carriers for therapeutic molecules. Using a one-step reaction, we synthesized small -2-4 nm core size amph- AuNPs with an amphiphilic ligand shell comprised of one or two alkanethiols. Each amph-AuNP is coated by a mixture of long-chain mercaptoundecanesulfonate (MUS) terminated by a water-soluble sulfonate group, and in some cases, short-chain hydrophobic octanethiol (OT). First, we describe our efforts to adapt and develop a method to synthesize giant multilamellar model membranes to study amph-NP-membrane interaction in a well-defined setting. We show that giant membranes can be synthesized and fine-tuned by varying lipid composition and buffer salt concentration, can be fluorescently labeled with lipid tracers, and can be analyzed robustly with confocal microscopy and flow cytometry. Second, we describe our systematic analysis of amph- AuNP and membrane characteristics that influence mechanisms of NP association with bilayers. We study effects of general membrane properties such as electrostatics and phase that govern NP-membrane interactions, and found that NP penetration of bilayers was blocked under conditions where strong electrostatic repulsion or gel-phase lipids were employed. We further studied effects of AuNP core diameter, surface charge, and surface hydrophobicity on NP-membrane interactions at the nanoscale. We found that MUS particles with an optimal gold core size -2- 3nm in diameter and MUS:OT particles of a broader size range were capable of inducing hemifusion between liposomal membranes, while MUS:OT 2:1 particles of intermediate hydrophobicity were capable of spontaneously aggregating within the bilayer of vesicles to form Janus egg-like morphologies. Third, we built on these NP membrane-embedding properties to explore and characterize NP-embedding in erythrocyte membranes, with particular attention to the glycocalyx and membrane fluidity, for the future application of constructing therapeutic erythrocyte 'pharmacytes' in situ. Finally, we describe work in engineering amph-AuNPs to carry short antigenic peptide cargoes for in vivo vaccine applications, where immunization experiments have shown much promise for antigen-ferrying amph-AuNPs in eliciting robust and long-lasting CD8+ T cell responses.
by Prabhani U. Atukorale.
Ph. D.
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Deitche, Alexandra. "Refrigerated Vaccine Transport: A Heat Transfer Analysis of Periodic Heating Through a Multi-Layer Cylinder." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17417573.
Full textAbstract:
There are vaccines against many of the worst diseases, as well as money to pay for them, but one main issue with the supply chain is that while most vaccines must be kept cool, almost half of all health posts have minimal or no grid access. In 2007, 151 million vaccine doses were wasted due to improper refrigeration. Using heat transfer analysis, this self-powered vaccine refrigerator will keep its payload at the CDC regulated temperatures for the 3-5 days necessary to distribute vaccines in areas without power access but very high temperatures.Mechanical Engineering
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Rynda, Agnieszka. "Low dose tolerance vaccine platform, reovirus protein sigma 1 and treatment of autoimmunity." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/rynda/RyndaA0808.pdf.
Full textAbstract:
Effective treatments for multiple sclerosis (MS) are problematic due to its unknown etiology. Experimental autoimmune encephalomyelitis (EAE) in rodents mimics MS. Mucosal treatment of EAE with antigens to induce tolerance is effective, but requires large and/or multiple administrations, which introduces an allergy risk. We utilized reovirus adhesin, protein sigma 1 (p sigma1), to improve mucosal auto-antigen delivery and show that a single low-dose of pσ1-based vaccines induces tolerance and prevents autoimmunity when administered nasally. We engineered three pσ1-based vaccines carrying chicken ovalbumin (OVA-pσ1) and/or myelin antigens (PLP:OVA-pσ1, MOG-pσ1). When mice were nasally immunized with OVA-pσ1, tolerance to OVA was established. This tolerance resisted co-administration of mucosal adjuvants and peripheral challenge with OVA. Pσ1-mediated tolerance relied upon specific IL-10- producing regulatory T (Treg) cells, which inhibited OVA-specific CD4+ T cell proliferation. OVA-pσ1 did not generate tolerance in IL-10-deficient mice presumably by a failure to induce Treg cells. Mucosal, but not systemic pσ1 delivery, induced tolerance, while mice lacking mucosal inductive tissues were resistant to pσ1-mediated tolerance. Likewise, PLP:OVA-pσ1 and MOG-pσ1 protected mice against relapsing-remitting or acute EAE, respectively. Protection against PLP 139-151-induced EAE was accomplished by PLP:OVA-pσ1, but not OVA-pσ1, implicating antigen-specificity of pσ1-mediated tolerance. Moreover, MOG-pσ1, but not PLP:OVA-pσ1, ameliorated MOG 35-55-induced EAE via apoptosis of encephalitogenic CD4+ T cells. The PLP:OVA-pσ1- or MOG-pσ1-mediated protection against EAE depends on specific IL-10+ Treg cells and is supported by IL-4+ Th2-type cells. Adoptive transfer of PLP:OVA-pσ1-primed Treg cells entirely prevented EAE development in mice; however, transfer of PLP:OVA-pσ1-specific CD25-CD4 + Th2 cells significantly reduced and delayed clinical EAE. Aggressive EAE, due to the TGF-β which induced activation of Th17 cells, was observed in mice dosed with PLP:OVA-pσ1 and were functionally depleted of Treg cells. Concomitant inactivation of TGF-β and Treg cells induced Th2 cells bias and re-established PLP:OVA-pσ1-mediated protection against EAE. IL-10-producing B cells supported MOG-pσ1-mediated protection against EAE, as MOG-pσ1-dosed B cell-deficient mice developed attenuated disease. Adoptive transfer of Treg cells, but not Th2 or B cells from MOG-pσ1-dosed B6 mice to diseased IL-10-/- mice, significantly accelerated recovery from EAE. These data demonstrate the feasibility of using pσ1-based single-dose delivery system to prevent and/or treat autoimmunity.
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James, Emmanuel Robin. "Optimisation of a recombinant Hepatitis B vaccine through the cultivation and fermentation of Aspergillus Niger." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1058.
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Leong, Tein Foong. "Engineering novel antibody-based molecules to probe the role of the immunoglobulin hinge and generate a potential cancer vaccine." Thesis, University of Dundee, 2017. https://discovery.dundee.ac.uk/en/studentTheses/5fe856e6-ce7a-4ff8-92c3-40c555aafd14.
Full textAbstract:
In different immunoglobulin (Ig) isotypes, the hinge region, which separates the antigen-binding Fab region from the Fc region, varies considerably in length. The hinge region is believed to provide flexibility for the Ig to conduct better positioning for antigen binding via the Fab arms or better receptor association via the Fc region. However, the reason for the difference in hinge length among different Igs is not fully understood. In this project it is hypothesised that the longer the upper hinge length of an IgG the further the distance that the antigen binding sites at the tips of Fab arms are able to reach between neighbouring antigens. In order to investigate the relation between upper hinge region of IgG and the distance between antigen binding sites, human IgG1 mutants specific for the hapten NIP which featured different upper hinge lengths were generated. Rigid bivalent ligands or “molecular rulers” (dsNIP-DNA) comprising defined lengths of double stranded DNA with NIP covalently attached to both ends were produced. ELISA tests demonstrated that anti-NIP human IgG1 bound to dsNIP-DNA. Through employment of molecular rulers of different lengths, surface plasmon resonance experiments indicated that the maximum distance between antigen binding sites is influenced by the length of the upper part of the IgG hinge. In addition, the role of upper hinge length on the ability of IgG to bridge between antigen on a target cell surface and Fc receptors on the surface of a phagocyte was investigated. Rosette formation between antibody-coated erythrocytes and Fc receptor-positive effector cells was utilised as an assay for this bridging. When anti-NIP human IgG1 hinge mutants were compared to wildtype IgG1, differences in rosette formation efficiency were observed indicating that the upper hinge region has a role in facilitating the bridging between an antigenic target cell and effector cell. In a second IgG engineering approach, potential cancer vaccines that feature the Fc region of IgG fused to the tumour associated cancer-testis antigen, MAGE-A, were generated and tested. Such vaccines are designed to promote formation of immune complexes, which are preferentially taken up by dendritic cells via their FcRn receptors, with the aim of stimulating a strong anti-tumour immune response. Human MAGE-A2 fused to either human IgG1 Fc or mouse IgG2a Fc had been successfully produced and purified using Protein-G Sepharose. Results from Coomassie staining and western blotting analyses suggested that the Fc fusion proteins were suffering from protein aggregations and possibly protein degradations. To test if MAGE-A2 Fc fusion protein can be a potential cancer vaccine, an 8 amino acid peptide sequence, SIINFEKL, was incorporated into MAGE-A2 protein of the Fc fusion protein containing mouse IgG2a Fc. IgG-immune complexes of SIINFEKL incorporated Fc fusion protein were incubated with bone marrow derived dendritic cells (BMDC) and corresponding cytotoxic T cells, B3Z, to investigate the induction of antigen cross presentation. Unfortunately, SIINFEKL incorporated Fc fusion protein was unable to activate B3Z T cells. Further investigation showed that BMDC seems to have a low expression of FcRn receptor, which is crucial for inducing an effective antigen cross presentation, and might be the reason for the unsuccessful antigen cross presentation assay. As a result, further investigations are required to determine if MAGE-A2 Fc fusion protein can be a potential cancer vaccine. More studies are also required to understand the characteristics of MAGE-A2 Fc fusion protein to minimise protein aggregations or protein degradations.
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Patel, Bindi Patel. "Plant Viral Nanoparticle-based Vaccine Targeting NY-ESO-1+ Triple Negative Breast Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1523873757595623.
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Kotze, Lara. "Development of Pichia pastoris as a production system for HPV16 L1 virus-like particles as component to a subunit vaccine." Thesis, Stellenbosch : University of Stellenbosch, 2007. http://hdl.handle.net/10019.1/1946.
Full textAbstract:
Human papillomavirus (HPV) is a sexually transmitted virus and known precursor to
cervical cancer, the second most lethal cancer in females across the world. Two virus-like
particle (VLP) vaccines exist that provide immunity against the main serotypes of the
disease and are produced in Saccharomyces cerevisiae (S. cerevisiae) and baculovirus
infected insect cells. Pichia pastoris (P. pastoris) was chosen as an alternative expression
system for HPV VLP production based on its history as prolific heterologous protein
producer that circumvent many of the problems associated with aforementioned
expression systems. The strongly inducible AOX promoter allows three-phase
fermentations (1.3 L bioreactors) in which high cell densities (>100gCDW.L-1) are
obtained prior to induction with methanol. During the induction phase the dissolved
oxygen concentration may be used to control addition of methanol. It is also possible to
use predetermined methanol feed rates and to adjust the amount of additional oxygen
sparged to maintain a constant dissolved oxygen level. The effects of these control
strategies, different gene constructs and multiple gene integrations were quantified
through monomer-, VLP- and mRNA production levels.
Increased biomass concentrations in the 20% dissolved oxygen control strategy led to the
highest volumetric VLP concentration (68.53 mg.L-1). VLPs were located intracellularly
in both the cytoplasm and membranes of the yeast cells. Despite lower codon adaptation
of the h-L1 gene expressed in the X33[h-L1] strain it still had higher volumetric VLP
concentrations under 40% dissolved oxygen control than the X33[Syn-L1] and
X33[SA-L1] strain containing the SA-L1 and Syn-L1 genes. This was ascribed to the
possible presence of rare codons in the Syn-hL1 and SA-L1 genes and a lower A+T
content in the h-L1 gene. Multiple gene integrations of the h-L1 gene had a negative
effect on VLP production and this conclusion was supported by lower mRNA
concentrations indicating lower transcriptional efficiency. Increased methanol induction
efficiency in the DO control strategies was indicated by higher specific L1 monomer
levels. Decreased VLP to monomer ratios in the DO control strategies indicated that a
bottleneck existed in the assembly process due to increased L1 monomer concentrations. Due to the hydrophobic region on the L1 protein, these proteins associated with the
membranes within the yeast cells especially when efficient assembly to VLPs did not
occur. HPV16 L1 VLP concentrations obtained in P. pastoris in this study are
comparable to the study by Li et al., (2003), but much lower than expression levels
obtained in baculovirus infected insect cells. Based on the expression levels of HBsAg
VLPs obtained in P. pastoris, this system, with the necessary recommended optimisation,
has the capacity for increased HPV VLP production ability.
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Bhattacharjee, Partha Sarathi S. M. Massachusetts Institute of Technology. "VacSeen : semantically enriched automatic identification and data capture for improved vaccine logistics." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/107582.
Full textAbstract:
Thesis: S.M. in Technology and Policy, Massachusetts Institute of Technology, School of Engineering, Institute for Data, Systems, and Society, Technology and Policy Program, 2016.Thesis: S.M. in Engineering and Management, Massachusetts Institute of Technology, School of Engineering, Institute for Data, Systems, and Society, System Design and Management Program, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 79-82).
Vaccines are globally recognized as a critical public health intervention. Routine immunization coverage in large parts of the developing world is around 80%. Technology and policy initiatives are presently underway to improve vaccine access in such countries. Efforts to deploy AIDC technologies, such as barcodes, on vaccine packaging in developing countries are currently ongoing under the aegis of the 'Decade of Vaccines' initiative by key stakeholders. Such a scenario presents an opportunity to evaluate novel approaches for enhancing vaccine access. In this thesis I report the development of VacSeen, a Semantic Web technology-enabled platform for improving vaccine access in developing countries. Furthermore, I report results of evaluation of a suite of constituent software and hardware tools pertaining to facilitating equitable vaccine access in resource-constrained settings through data linkage and temperature sensing. I subsequently discuss the value of such linkage and approaches to implementation using concepts from technology, policy, and systems analysis.
by Partha Sarathi Bhattacharjee.
S.M. in Technology and Policy
S.M. in Engineering and Management
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Reid, Sandy M. "Nano-sized Polymeric Particles for Safe Delivery of Vaccine Adjuvants to Combat Fungal Pathogens." Ohio University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1543927420834628.
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Yang, Fan [Verfasser], and Frank [Akademischer Betreuer] Rösl. "Re-engineering a Nanoparticle Human Papillomavirus Prophylactic Vaccine Antigen Based on the Minor Capsid Protein L2 / Fan Yang ; Betreuer: Frank Rösl." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/121816798X/34.
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Ahmed, Kawther Khalid. "Assembly and characterization of a cell-particle hybrid system as a potential cancer vaccine." Thesis, University of Iowa, 2013. https://ir.uiowa.edu/etd/3034.
Full textAbstract:
Cancer vaccines represent a promising treatment modality for a world-wide health problem. Whether as an adjuvant or as a stand-alone therapy, cancer vaccines represent a tumor-specific and systemic treatment potentially capable of eliminating metastatic lesions without the severe side-effects often associated with chemotherapy. Specifically, whole cell tumor vaccines have shown promise in preclinical and clinical settings and the studies presented here represent the beginnings of an approach to improve the antitumor potency of these vaccines.
This project demonstrates as "proof of concept" the feasibility of manufacturing tumor cell-particle hybrids. The coupled use of these two components, whole tumor cells and cargo-carrying biodegradable particles, as one entity in a cancer vaccine system is a new line of research. Stable cell-particle hybrids were assembled using avidin-biotin chemistry where cargo-carrying PLGA particles (500 nm diameter) were coated with streptavidin and allowed to bind to tumor cells that had been indirectly labeled with biotin (using an integrin-specific biotinylated antibody). That successful cell-particle hybrids were assembled was determined by multiple means, including flow cytometry, laser scanning confocal microscopy and scanning electron microscopy. Two murine tumor cell lines (representing melanoma and prostate cancer) were investigated in this study and successfully demonstrated the general applicability of the assembly method. Particles appeared to be localized on the cell surface (rather than endocytosed) as determined by microscopic imaging. The cell-particle hybrid was shown to be stable to irradiation, an important consideration since whole tumor cells need to be treated with ionizing radiation prior to being used as vaccines in order to render them nonproliferative and immunogenic. We also characterized loading and release profiles of CpG, a prospective vaccine adjuvant, into PLGA particles.
We conclude that we have developed a method for manufacturing cell-particle hybrids comprising PLGA nanoparticles and irradiated tumor cells. The next step would be to use CpG-loaded particles in the assembled hybrid and test the anti-tumor immune efficiency of this cancer vaccine formulation in either a melanoma or prostate cancer model.
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Chebolu, Seethamahalakshmi. "EXPRESSION OF GAL/GALNAC LECTIN OF ENTAMOEBA HISTOLYTICA IN TRANSGENIC CHLOROPLASTS TO DEVELOP A VACCINE FOR AMEBIASIS." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3081.
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Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.M.S.
Department of Biology
Arts and Sciences
Biology
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Regele, Oliver Brian. "Applied discrete event simulation for root cause analysis and evaluation of corrective process change Efficacy within vaccine manufacturing." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/126896.
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Thesis: M.B.A., Massachusetts Institute of Technology, Sloan School of Management, May, 2020Thesis: S.M., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, May, 2020
Cataloged from the official PDF of thesis.
Includes bibliographical references (pages 137-141).
Digital Transformation of the Biopharmaceutical industry is enabling improved operations through smart manufacturing. One area of interest is the application of advanced data analytics techniques to supplement traditional workflows. The focus of this research was developing a process simulation model to address a defect observed at a manufacturing line at the Sanofi Pasteur Lyon site. This defect entailed a series of Out-of-Trend batches with abnormally low content of a certain attribute, at the end of a two-year process with complex product batch genealogy, which complicated the use of a traditional approaches to Root Cause Analysis. This study performed a statistical analysis of the defect batch attribute content through production stages to determine which contained a Root Cause. Once this analysis identified the Valence Assembly process as a stage of origin, a Discrete Event Simulator for this process was developed based on historical process data and specifications. This simulator was able to model the current process and replicate the defect in-silico. The simulator identified a specific Root Cause in the batch testing protocol as well as the expected incidence rate of the defect over future campaigns. Finally, the simulator evaluated the efficacy of two potential Corrective Process Changes. This work functions as a practical exploration of integrating novel data analysis and simulation techniques into traditional vaccine manufacturing activities.
by Oliver Brian Regele.
M.B.A.
S.M.
M.B.A. Massachusetts Institute of Technology, Sloan School of Management
S.M. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science
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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.
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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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Zuma, Medeiros Mauricio. "Industrial development in a high tech sector of a developing country : the unfinished technological transition in the Brazilian vaccine industry." Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/6322/.
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This thesis investigates the development of the Brazilian vaccine industry. This industry has experienced a sharp growth in the last decades supported by public policies and a protected and fast-growing public market. In addition, this development is apparently characterized by continuous processes of technology acquisition, rather than indigenous R&D, as the main source of its technological knowledge, and by other specificities in the vaccine context. The research draws on studies of the dynamics of technological capability building in catching-up industries of latecomer contexts, especially during the transition period when they are approaching the innovation frontier. It also draws on those studies focusing on new directions/paths as an alternative strategy adopted to overcome barriers and disadvantages to develop. It has been argued that the specificities of the Brazilian context and, of the vaccine sector, may be determining a particular pattern of technological accumulation to this industry, and that interpreting its pattern of development may be useful to understand how and if this industry has overcome its constraints to develop. A framework based on linear approaches of catching-up, and that integrates the innovation transition approach was built as a benchmark model for the search for similarities and differences in the pattern of development of this industry. The findings show similarities and new directions in the process of technological accumulation of the industry, suggesting that, more recently, it has actually developed through a distinct pattern. They also show the strong role of the government and its public market as one of the drivers of this new path. Distinct roles of the technology acquisition strategy and a high level of technological capabilities currently developed are also revealed. Finally, they show that the technology acquisition strategy has effectively contributed to the development of this industry and that the constraints to the completion of the transition phase is linked less to technical and scientific issues and more to managerial and policy ones.
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Richard, Emeline. "Synthèse par ingénierie métabolique d'oligosaccharides sialylés pour l'élaboration de glycoconjugués d'intérêt médical." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV007/document.
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Les structures sialylées sont présentes à la surface des cellules sous forme de glycoconjugués,couplés à des protéines ou des lipides. Ces structures jouent un rôle important dans divers processusbiologiques que ce soit à travers l’interaction avec des lectines, ou de par leurs propriétés physicochimiques.Ces structures sont également impliquées dans diverses pathologies et on constatenotamment une forte augmentation du taux d’acides sialiques chez les individus atteints de cancer,due à une surexpession de structures naturelles mais aussi à l’apparition de nouveaux motifs,naturellement absent chez l’individu sain. L’ensemble de ces structures sialylées présente un intérêtsoit par leur rôle biologique soit à cause de leur expression spécifique dans les cancers. Leurobtention est très difficile par voie chimique et la synthèse enzymatique in vitro est efficace mais trèscoûteuse en nucléotide-sucre et ne sont pas adaptées à une production à l'échelle préparative.Dans un premier temps, ces travaux de thèse s’intéressent à la synthèse bactérienne par ingénieriemétabolique d'acides polysialiques fonctionalisés. Ces polysaccharides présentent divers intérêts.Tout d’abord il est possible de les coupler à des protéines actives pour en augmenter le temps dedemi-vie in vivo. Mais ces polysaccharides peuvent également être utilisés dans le cadre de thérapievaccinale, soit contre des bactéries pathogènes de types Neisseria meningitidis qui le présententcomme polysaccharide capsulaire, soit contre les cellules cancéreuses surexprimant cette structure.Ensuite nous avons cherché à obtenir des oligosaccharides spécifiques des cancers, les motifssialylTn, et siallTF, toujours par ingénierie métabolique d’E. coli. Le sialylTn a été couplé à uneplateforme peptidique immunogène afin de construire un candidat vaccin qui a été testé in vitro et invivo sur la sourisSialylation is an important feature of glycolipids and glycoproteins of animal cell surfaces. Sialylatedmotifs are involved in many biological processes through lectin interactions or because of theirphysico-chemical properties. There is a great variety of sialylated structural motifs, and in manycases, there is a structure-relationship between the sialylated profile of and some pathologicprocesses. In cancer, there is an increase of sialylation including the apparition of newly andspecifically related sialylated structures belonging to the so-called tumor-associated carbohydrateantigens (TACA). Those structures present a particular interest, but their chemical or chemoenzymaticsynthesis is costly and quite unappropriated for preparative scale.This work addresses to the bacterial synthesis of sialylated motifs through the metabolic engineeringof Escherichia coli. The first part of the thesis deals with the biosynthesis of polysialylatedconjugatable motifs. Those motifs present various biological properties, such as an increase of thelife-time of therapeutic proteins; they also belong to the TACA family since over-expressed incancers. In addition, some of them are bacterial-specific motifs such as in pathogenic Neisseriameningitidis. Altogether, polysialylated conjugates can be useful for the synthesis of therapeuticdrugs and vaccines. The second part of the thesis describes a new way of producing sialylated Tn andTF carbohydrate antigens by metabolic engineering. The sialylTN motif was coupled to a peptidic andimmunogenic scaffold being a potential vaccine candidate, and its ability of raising specific antibodieswas assayed in mouse
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Kou, Peng Meng. "Elucidation of dendritic cell response-material property relationships using high-throughput methodologies." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/44911.
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Ongoing advances in tissue engineering with the goal to address the clinical shortage of donor organs have encouraged the design and development of biomaterials to be used in tissue-engineered scaffolds. Furthermore, biomaterials have been used as delivery vehicles for vaccines that aim to enhance the protective immunity against pathogenic agents. These tissue-engineered constructs or vaccines are usually combination products that combine biomaterial and biological (e.g. cells, proteins, and/or DNA) components. Upon introduction into the body, the host response towards these products will be a combination of both a non-specific inflammatory response towards the biomaterial and an antigen-specific immune response towards the biological component(s). Recently, the biomaterial component was shown to influence the immune response towards a co-delivered antigen. Specifically, poly(lactic-co-glycolic acid) (PLGA), but not agarose, scaffolds or microparticles (MPs) enhanced the humoral response to a model antigen, ovalbumin. This in vivo result echoed with the in vitro study that PLGA, but not agarose, supported a mature phenotype of dendritic cells (DCs), the most potent antigen-presenting cells. Therefore, it is hypothesized that the effect of biomaterials on DC phenotype may influence the adaptive immunity against a co-delivered antigen. Understanding how biomaterials affect DC response will facilitate the selection and design of biomaterials that direct a desired immune response for tissue engineering or vaccine delivery applications.
The objectives of this research were to elucidate the correlations between material properties and DC phenotype, develop predictive models for DC response based on material properties, and uncover the molecular basis for DC response to biomaterials. Well-defined biomaterial systems, including clinical titanium (Ti) substrates and two polymer libraries, were chosen to study induced DC phenotype.
Due to the time-consuming nature of conventional methods for assessing DC phenotype, a high-throughput (HTP) method was first developed to screen for DC maturation based on surface marker expression (CHAPTER 4). A 96-well filter plate-based HTP methodology was developed and validated for the assessment of DC response to biomaterials. A "maturation factor", defined as CD86/DC-SIGN and measured by immunostaining, was found to be a cell number-independent metric for DC maturation and could be adapted to screen for DC maturation in a microplate format. This methodology was shown to reproducibly yield similar results of DC maturation in response to biomaterial treatment as compared to the conventional flow cytometric method upon DC treatment in 6-well plates. In addition, the supernatants from each treatment could easily be collected for cytotoxicity assessment using glucose-6-phosphate dehydrogenase (G6PD)-based assay and cytokine profiling using multiplex technology. In other words, the 96-well filter plate-based methodology can generate three outcomes from one single cell culture: 1) maturation marker expression, 2) cytotoxicity, and 3) cytokine profile.
To examine which material properties were critical in determining DC phenotype, a set of three clinical titanium (Ti) substrates with well-defined surfaces was used to treat DCs (CHAPTER 5). These Ti substrates included pretreatment (PT), sand-blasted and acid-etched (SLA), and modified SLA (modSLA), with different roughness and surface energy. DCs responded differentially to these substrates. Specifically, PT and SLA induced a mature DC (mDC) phenotype, while modSLA-treated DCs remained immature based on surface marker expression, cytokine production profiles and cell morphology. Both PT and SLA induced higher CD86 expression as compared to iDC control, while modSLA maintained CD86 expression at a level similar to iDC. PT- or SLA-treated DCs exhibited dendritic processes associated with a mDC phenotype, while modSLA-treated DCs were rounded, a morphology associated with an iDC phenotype. Furthermore, PT induced increased secretion of MCP-1 by DCs compared to iDCs, indicating that PT promoted a pro-inflammatory environment. SLA induced higher IL-16 production, which is a pleiotropic cytokine, by DCs, most likely as a pro-inflammatory response due to the enhanced maturation of DCs induced by SLA. In contrast, modSLA did not induced enhanced production of any cytokines examined. Principal component analysis (PCA) were used to reduce the multi-dimensional data space and confirmed these experimental results, and it also indicated that the non-stimulating property of modSLA co-varied with certain surface properties, such as high surface hydrophilicity, % oxygen and % titanium of the substrates. In contrast, high surface % carbon and % nitrogen were more associated with a mDC phenotype. Furthermore, PCA also suggested that surface line roughness (Ra) did not contribute to the expression of CD86, an important maturation marker, suggesting that roughness had little impact on DC response (CHAPTER 5).
DC response-material property relationships were also derived using more complex materials from a combinatorial library of polymethacrylates (pMAs) (CHAPTER 6). Twelve pMAs were selected and were found to induce differential DC response using the HTP method described in CHAPTER 4. These pMAs resulted in a trend of increasing DC maturation represented by the metric CD86/DC-SIGN, which was consistent with the trends of the production of pro-inflammatory cytokine, TNF-α, and chemokine, IL-8. Interestingly, this set of pMAs induced an opposite trend of IL-16 production, which is most likely released as an anti-inflammatory cytokine in this situation. These polymers were characterized extensively for a number of material properties, including surface chemical composition, glass transition temperature (Tg), air-water contact angle, line roughness (Ra), surface roughness (Sa), and surface area. Similar to the results from the Ti study, PCA determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an iDC phenotype. In addition, Tg, Ra, and surface area were unimportant in determining DC response. Partial square linear regression (PLSR), a multivariate modeling approach, was implemented using the pMAs as the training set and a separate polymer library, which contained methacrylate- and acrylate-based terpolymers, as the prediction set. This model successfully predicted DC phenotype in terms of surface marker expression with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80 (CHAPTER 6). Nonetheless, one should note that a predictive model can be only as good as what it is trained on and cannot be used to predict the DC response induced by a type of materials different from the training set. Also, this model might not contain all the important material properties such as polymer swelling and cannot predict specific types of immune responses. However, these results demonstrated that a generalized immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection (CHAPTER 6).
From the pMA library, pMAs that induced the two extremes of DC phenotype (mature or immature) were identified for elucidating the mechanistic basis of biomaterial-induced DC responses (CHAPTER 7). Two pMAs, polyhydroxyethylmethacrylate (pHEMA) and poly(isobutyl-co-benzyl-co-terahydrofurfuryl)methacrylate (pIBTMA), were selected because they induced the least and the most mature DC phenotype, respectively. These pMAs were used to elucidate the activation profiles of transcription factors in DCs after biomaterial treatment and were compared to the iDC and mDC controls. In addition, a combined treatment of pHEMA and LPS was also included to determine if pHEMA could maintain an iDC phenotype in the presence of LPS. Interestingly, pIBTMA induced DC maturation primarily through the activation of NF-κB, while pHEMA mediated suppression of DC maturation through multiple TFs, including the activation of ISRE, E2F-1, GR-PR, NFAT, and HSF. GR-PR and E2F-1 have been shown to be associated with the suppression of DC maturation; ISRE, E2F-1, and NFAT are linked to apoptosis induction; HSF regulates the production of heat shock proteins (HSPs) that induce DC maturation and inhibit apoptosis. The activation of HSF by pHEMA was most likely a natural defensive mechanism against the other apoptotic signals. Therefore, pHEMA suppressed DC maturation through the induction of apoptosis. Surprisingly, in the presence of pHEMA, the effect of LPS was completely eliminated, suggesting that biomaterials can override the effect of soluble factors. The morphology and surface marker expression of DCs treated with these different biomaterials or controls were consistent with TF activation profiles (CHAPTER 7).
Overall, this research illustrates that biomaterial properties, within the chosen biomaterial space, can be correlated to DC phenotype and more importantly, can be used as predictors for relative levels of DC phenotype. Furthermore, the differential responses induced by different biomaterials were mediated through the distinct activation profiles of transcription factors. Together, these findings are expected to facilitate the design and selection of biomaterials that direct desired immune responses.
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Mahajan, Rutuja. "Analyzing Public View towards Vaccination using Twitter." Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright1578379698895464.
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Pelin, Adrian. "Bio-Engineering Vaccinia Viruses for Increased Oncolytic Potential." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39909.
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Vaccinia virus has a large and still incompletely understood genome although several strains of this virus are already in clinical development. For the most part, clinical candidates have been attenuated from their wild type vaccine strains through deletion of metabolic genes like the viral thymidine kinase gene. In the present work, we thoroughly examined the genetic elements of vaccinia which could be modulated to improve tailor the virus as a cancer therapeutic. Using a variety of cancer cell lines and primary tumor explants, we performed a fitness assay that directly compares multiple wild-type Vaccinia strains to identify the genetic elements that together create an optimal “oncolytic engine”. Using a transposon insertion strategy and deep sequencing of viral populations we systematically examined Vaccinia genes that do or do not play a role in the therapeutic activity of the virus. Our studies allowed us to identify a variety of genes in the vaccinia genome that when deleted, augment the oncolytic activity of a newly engineered Vaccinia virus. In the context of this thesis, I define enhanced oncolytic activity as superior therapeutic activity, increased immunogenicity and an improved safety profile, all aspects which we used to compare this novel virus to Vaccinia viruses currently in the clinic.
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Murphy, Jane Clare. "The engineering of viral fusion proteins in the baculovirus expression system." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240912.
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Chewachong, Godwill Mih. "Engineering Plant Virus " Vaccines" Using Pepino mosaic virus as a Model." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384203201.
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Mehta, Naveen K. Ph D. Massachusetts Institute of Technology. "Engineering more potent vaccines for the treatment of cancer and autoimmunity." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/123065.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019Cataloged from PDF version of thesis.
Includes bibliographical references (pages 171-181).
Vaccination against infectious diseases has long been heralded as one of the greatest advancements in public health, yet its application to other clinical indications has fallen short of expectations. In this thesis, we apply engineering principles to develop more potent vaccines in the treatment of cancer and autoimmunity. Both major components of molecular vaccines, antigen and adjuvant, are independently explored as a part of this work. Our antigen studies sought to improve the delivery of peptide epitopes to lymphoid organs by fusing epitopes to inert protein carriers with defined pharmacokinetic properties. To promote anti-tumor immunity, we found that antigen carriers should 1) protect peptide cargo from proteolytic degradation, 2) be appropriately bulky to drain into the lymphatics, and 3) be rapidly cleared once in the blood to prevent tolerization at distal poorly inflamed organs.
Applying these principles, we identified transthyretin as an optimal delivery protein, and demonstrated efficacy against a number of clinically relevant antigens. Because our protein-epitope fusion approach is fully recombinant in nature, we were able to convert our protein vaccines into nucleic acid modalities, including plasmid DNA and self-replicating RNA, which are significantly easier and cheaper to manufacture at scale. Finally, we applied our learnings to purposefully induce tolerization in the treatment of autoimmunity, and found that albumin is a particularly efficacious antigen carrier protein for this application due to its extended half-life. On the adjuvant front, we attempted to engineer novel Toll-like receptor 3 (TLR3) agonists via yeast surface display. Although we successfully developed high affinity TLR3 binders, all tested clones failed to agonize TLR3 despite the utilization of several multimerization strategies.
Separately, in an effort to better understand adjuvant biology, we conducted a detailed mechanistic study of lipo-CpG, a particularly potent amphiphilic CpG variant previously developed by the Irvine lab. We uncovered a cascade of inflammatory signals originating from monocytes that facilitates the induction of high magnitude T cell responses, largely by acting in trans rather than directly on the antigen-presenting cell. Overall, these studies have elucidated a number of design principles that should aid in the engineering of next generation vaccines to better treat cancer and autoimmunity.
by Naveen K. Mehta.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering
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Wang, Nick X. "Controlled Delivery of Protein Therapeutics for HIV Prevention." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1327614039.
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Van, der Westhuizen Maria Jacoba. "The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen." Thesis, North-West University, 2012. http://hdl.handle.net/10394/9852.
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Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa.Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.
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Moynihan, Kelly D. (Kelly Dare). "Engineering immunity : enhancing T Cell vaccines and combination immunotherapies for the treatment of cancer." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/113960.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 127-140).
Checkpoint blockade with antibodies against CTLA-4 or PD-1 has demonstrated that an endogenous adaptive immune response can be stimulated to elicit durable tumor regressions in metastatic cancer, but these dramatic responses are confined to a minority of patients¹-³. This outcome is likely due in part to the complex network of immunosuppressive pathways present in advanced tumors, which necessitates the development of novel therapeutics and combination immunotherapies to generate a counter-directed network of pro-immunity signals⁴-⁸. In Chapters 2 and 3 of this thesis, we describe methods for enhancing T cell priming against tumor antigens via covalent modification of molecular vaccines to enhance lymphatic drainage, serum stability, or cytosolic access to improve presentation on MHC class I. In Chapter 4, we demonstrate a combination immunotherapy that recruits a diverse set of innate and adaptive effector cells, enabling robust elimination of large tumor burdens that to my knowledge have not previously been curable by treatments relying on endogenous immunity. Maximal anti-tumor efficacy required four components: a tumor antigen targeting antibody, an extended half-life IL-2⁹, anti-ƯPD-1, and a powerful T-cell vaccine¹⁰. This combination elicited durable cures in a majority of animals, formed immunological memory in multiple transplanted tumor models, and induced sustained tumor regression in an autochthonous BRraf[superscript V600E]/Pten[superscript -/-] melanoma model. Finally, in Chapter 5, we show preliminary data on combination immunotherapies used to treat antigenically heterogeneous tumors. Taken together, these data define design criteria for enhancing the immunogenicity of molecular vaccines and elucidate essential characteristics of combination immunotherapies capable of curing a majority of tumors in experimental settings typically viewed as intractable.
"During my doctorate by the John and Fanny Hertz Foundation Fellowship (specifically the Wilson Talley Hertz Fellowship), the NSF Graduate Research Fellowship Program, and the Siebel Scholarship"--Page 141. "This thesis work was supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the US National Institutes of Health (NIH) grant CA174795, the Bridge Project partnership between the Koch Institute for Integrative Cancer Research and the Dana Farber-Harvard Cancer Center (DF-HCC), the V Foundation, the Ragon Institute, and the Howard Hughes Medical Institute"--Page 141.
by Kelly D. Moynihan.
Ph. D.