Дисертації з теми "HIV vaccine; adjuvants"
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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.
Повний текст джерелаCataloged 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.
Buglione-Corbett, Rachel. "Adjuvant-Specific Serum Cytokine Profiles in the Context of a DNA Prime-Protein Boost HIV-1 Vaccine: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/666.
Повний текст джерелаBuglione-Corbett, Rachel. "Adjuvant-Specific Serum Cytokine Profiles in the Context of a DNA Prime-Protein Boost HIV-1 Vaccine: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/666.
Повний текст джерелаBraga, Catarina Joelma Magalhães. "Pesquisa de novos adjuvantes para vacinas terapêuticas." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-13022012-080631/.
Повний текст джерелаThe search for adjuvants that are able to stimulate an efficient cellular immune response represents an important contribution in vaccine development. In this study, we evaluated the potential of Salmonella flagellin as adjuvant for activation of T lymphocytes, with emphasis on the activation of CD8+ T cells, on the mice immunized with different approaches such as vaccines based on attenuated Salmonella strains; acellular vaccines with purified flagellin co-administered or genetically fusioned to the target antigen; or even DNA vaccines. Our results demonstrate that the use of flagellin in the form of DNA vaccines induced greater therapeutic protection than the same formulation used in the form of subunit vaccines, suggesting that the adjuvant effects of flagellin to the activation of CTLs are related not only to link to TLR5. The results presented in this work contribute significantly to the understanding of the mechanisms of flagellin adjuvanticity, particularly in the context of activation of responses mediated by T cells.
Gutjahr, Alice. "Évaluation de combinaisons de ligands de PRR et de particules biodégradables pour la vaccination muqueuse." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1325.
Повний текст джерелаThere are many barriers to the development an effective HIV vaccine. The use of adjuvants is a promising option to overcome these obstacles. In this context, the objective of this PhD is the evaluation of combinations of PRR ligands and biodegradable particles for mucosal vaccination.The first part of this study aimed at assessing the added value of hybrid molecules composed of two PRR ligands compared to the co-administration of the two agonists. TLR7 and TLR2 stimulating molecules followed by TLR7 and NOD2 were evaluated. We demonstrated the interest of the association of PRR ligands within the same molecule for the induction of systemic and mucosal immune responses.Recent studies showed the interest of STING agonists as a vaccine adjuvant. We investigated the induction of immune responses by STING agonists administered parenterally or mucosally. We confirmed the strong potential of STING ligands for the induction of cellular and mucosal responses.In these studies, we demonstrated that the interest of vectorization of PRR agonists depends on the molecule. Indeed, although the encapsulation of a TLR7/TLR2 hybrid molecule has no impact on the induced immune response, the vectorization of STING agonists potentiates their immunostimulatory effect.Finally, we showed that the route of administration has an impact on the immune response induced. In order to better understand the mechanisms involved, a biodistribution study of PLA NP formulations after systemic or mucosal administration was performed
Xu, Lin. "HIV-1 mucosal immunity : from infection to prevention : HIV-1 envelope gp41 conserved region P1 modulates the mucosal innate immune response and acts as a potential mucosal adjuvant The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry By shaping the antigen binding site in IgA, the CH1α domain is crucial for HIV-1 protection in highly exposed sero-negative individuals The antigen HIV-1 envelope gp41 conserved region P1 can act as mucosal adjuvant by modulating the innate immune response". Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB071.
Повний текст джерелаMucosal vaccination, especially intranasal administrated ones, has been considered to be ideal for protection from pathogens invading through mucosal sites. However, the lack of specific adjuvant and insufficient acknowledgement of nasal immune system limits the development of vaccine. P1, a conserved region of gp41 envelope glycoprotein, was recently developed into a prophylactic HIV-1 vaccine immunized via both the intramuscular and intranasal routes. It showed high efficiency in pre-clinical and phase I clinical trial due to induction of P1 specific mucosal IgA with transcytosis blocking activity and IgG inducing antibody dependent cell cytotoxicity. In this study, we characterized the immunological mechanism underneath P1-vaccine in nasal mucosa. Firstly, we demonstrated that P1 initiate immune responses by inducing nasal epithelial cells to secret the Th2 cytokine Thymic Stromal LymphoPoietin (TSLP). TSLP has been reported to be a strong mucosal adjuvant, and its receptor TSLP-R plays a critical role in IgA response. We showed that P1 induce TSLP expression via the interaction with galactosyl ceramide, the receptor of HIV-1 mucosal entry. Furthermore, we identified Calcineurin/NFAT signaling pathway and microRNA-4485 as important players in the regulation of TSLP production induced by P1. Secondly, we showed that P1 modulates innate immune responses by activate dendritic cells (DCs). P1 stimulation results in enhanced expression of costimulatory molecules on DCs. Furthermore, the secretion of IL-6, IL-10 were increased, while IFN-γ was reduced, indicating that P1 activated DCs polarize into a Th2 and IgA prone phenotype. In addition, IL-8, CCL20, CCL22 were produced indicating a capacity at recruiting immune cells to mucosal surface for initiation of an adaptive immune response. MMP-9 was also produced allowing degradation of the extracellular matrix and facilitating the migration of immune cells out of the mucosa. Stricingly, a TSLP autocrine loop was observed as P1 induced DCs to secret TSLP and meanwhile, enhanced DC expression of TSLP-R. Finally, P1 activated DCs enhanced the proliferation of CD4+ T cells. In conclusion, we demonstrated that P1 is a multi-functional protein with a great interest for vaccine development, not only as an antigen for vaccine candidate, but also as a potential adjuvant that can be combined to other mucosal vaccines
Brinckmann, Sarah Anna. "Polyethyleneimine as a candidate vaccine adjuvant for Env-based HIV-1 infection." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559758.
Повний текст джерелаBridge, Simon Harwood. "HIV-Neutralising response to recombinant, cross-clade, adjuvanted, VLP-forming vaccine candidates." Thesis, University of Liverpool, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485898.
Повний текст джерелаKhodami, 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.
Повний текст джерела"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.
Smith, Jeffrey D. "Vaccination of BALB/c Mice with an Alhydrogel Adjuvanted Whole Cell Trichomonas vaginalis Formulation." Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30424.
Повний текст джерелаWang, 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.
Повний текст джерелаSantana, Vinicius Canato. "Desenvolvimento de estratégias para aumento da imunogenicidade da vacina de DNA HIVBr18 baseadas na fusão com a glicoproteína D do herpes vírus humano tipo 1 e na coadministração de citocinas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-23092014-091246/.
Повний текст джерелаThe formulation HIVBr18, previously developed and tested, is based on a DNA vaccine encoding 18 conserved and promiscuous HIV-1 CD4 epitopes and after immunization of transgenic mice for many human HLA class II molecules using this DNA vaccine, could be observed proliferation of CD4+ and CD8+ T cells and IFN-y production directed to multiple epitopes encoded by the vaccine. We intend to explore here, strategies based on fusion or combination of epitopes encoded by HIVBr18 vaccine with glycoprotein D (gD) of HSV- 1 and also the coadministration of cytokine-encoding plasmids (pIL-2, -12, -15 and pGM -CSF) aiming to enhance immunogenicity of HIVBr18. The DNA sequence of epitopes encoded by HIVBr18 vaccine was amplified by PCR and cloned into a plasmid that contained the sequence of gD, giving rise to plasmid pVAX-gDh-HIVBr18. After mice immunization, animals immunized with this construct showed similar immune response to the group that received HIVBr18, and also the group of animals that received gDh-HIVBr18 plasmid that had been modified by exchange in peptides order to assure to the molecule a better hydrophobic distribution and allow translocation to the extracellular face of cell membrane. We constructed and injected mice with a bicistronic plasmid expressing gDh and HIVBr18, simultaneously and isolated, but no increase in the magnitude of the immune response was observed. HIVBr18 coadministration with cytokine-encoding plasmids pIL-12, pIL-15 and pGM-CSF, provides an increase in the magnitude of immune response induced against the peptides encoded by the vaccine, and similar breadth. In addition, co-immunization with pGM-CSF induced greater number of polyfunctional CD4 + T cells. We also demonstrate that, even in a low dose approach coadministration of pGM-CSF induced a higher immune response than HIVBr18 alone in the same dose. However, we observed that this cytokine is not a good adjuvant when used in combination with an adenovirus that expresses the 18 HIV-1 epitopes.
Garrod, Tamsin Jade. "Evaluating the immuno-modulatory effects of a natural adjuvant as a novel vaccine strategy." Thesis, 2014. http://hdl.handle.net/2440/92055.
Повний текст джерелаThesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014
Xi, Yang. "Adjuvant activity of Interferon epsilon and toll-like receptor-9 to enhance HIV-specific mucosal immunity." Phd thesis, 2013. http://hdl.handle.net/1885/156393.
Повний текст джерелаDuarte, Henrique José Xavier. "INULIN PARTICLES AS A VACCINE ADJUVANT FOR HEPATITIS B. DEVELOPMENT AND IN VITRO EVALUATION OF THEIR IMMUNOTOXICITY." Master's thesis, 2021. http://hdl.handle.net/10316/99161.
Повний текст джерелаO vírus da Hepatite B (HBV) é um vírus de DNA com envelope associado à família Hepadnaviridae, o qual é altamente hepatotrófico e espécie-específico, ou seja, replica-se exclusivamente em hepatócitos humanos. A infeção por HBV pode ocorrer no início da vida, o que geralmente leva a uma infeção crónica, caso não seja tratada corretamente. Caso a infeção por HBV ocorra mais tarde, geralmente resulta numa infeção aguda que tem a capacidade de se auto-resolver. Atualmente, para prevenir a infeção pelo HBV existe uma vacina injectável profilática, que consiste em 3 ou 4 doses (dependendo da região geográfica do indivíduo) contendo o antigénio de superfície do HBV (HBsAg) combinado com sais de alumínio. Apesar da vacina disponível ser eficaz, a prevalência do HBV ainda é um sério problema de saúde pública, razão pela qual eliminar a hepatite é uma das principais metas inscrita na Agenda 2030 das Nações Unidas. Embora o futuro pareça promissor, existem dois problemas que constituem uma ameaça à erradicação do HBV, sendo o primeiro a necessidade de melhorar a vacinação nos países em desenvolvimento e o segundo a inexistência de uma vacina terapêutica para uso em doentes crónicos. Uma forma de tentar resolver estes problemas é o uso da nanotecnologia como ferramenta para o desenvolvimento de adjuvantes de vacinas, uma vez que esta permite “desenhar” as nanopartículas com diversas composições, tamanhos e propriedades de superfície. A inclusão do antigénio em nanopartículas ajuda a aumentar a estabilidade do antigénio, a imunogenicidade e permite a entrega direcionada deste mesmo antigénio a células apresentadoras de antigénio. A delta inulina (denominada Advax ™) um polissacarídeo composto por unidades de frutose, tem vindo a ser testada em ensaios clínicos para uso na vacina contra o HBV. Uma das qualidades mais importantes da inulina, observada em estudos com animais, foi o aumento da resposta imune adaptativa, confirmada numa ampla variedade de antigénios virais e bacterianos, como influenza, encefalite japonesa, HIV, entre outros. Por conseguinte, esta dissertação de mestrado tem como objetivo desenvolver e produzir partículas de inulina combinadas ou não, com um polímero previamente estudado, como a Policaprolactona (PCL) ou Quitosano, para ser usado como adjuvante em vacinas. O método de produção desenvolvido e otimizado foi baseado em técnicas de nano-precipitação e as partículas foram caracterizadas quanto às suas características físico-químicas e imunotoxicológicas. Neste contexto três sistemas de entrega foram desenvolvidos: partículas de inulina, partículas de PCL-inulina e partículas de PCL. Enquanto as partículas de PCL-inulina e PCL apresentaram tamanhos médios de 301,26 nm e 275,32 nm respetivamente, as partículas de inulina eram muito maiores, encontravam-se fora da nano-escala (1320,86 nm). Os resultados das determinações de tamanho permitiram-nos inferir que as partículas são estáveis por 42 dias, com exceção das partículas de inulina que apresentaram uma tendência para aumento de tamanho ao longo do tempo. Em termos de imunotoxicidade, as partículas de inulina e PCL-inulina não mostraram toxicidade em culturas de células primárias do baço de murganho, embora na linha celular RAW 264,7 os resultados tenham sido ligeiramente diferentes, uma vez que as partículas de PCL-inulina mostraram alguma toxicidade em concentrações mais elevadas, o que foi também observado nas partículas PCL. Considerando os estudos de avaliação de produção de óxido nítrico (NO) e citocinas, as partículas não induziram a produção de NO quando incubadas com a linha celular RAW 264,7, nem a produção de GM-CSF. O mesmo foi verificado para o TNF-α, IL-6, IL-10, e IL-12 após incubação das partículas de PCL-inulina a 2 μg/mL com as DCs, onde se observou que não foram induzidos níveis significativos de citocinas. No entanto, a 4 μg/mL pode ver-se uma pequena indução da IL-12 e IL-10. No geral, relatamos o desenvolvimento e produção de partículas de inulina e policaprolactona, cujos resultados preliminares de diversos ensaios in vitro sugerem que são adequados para uso, como sistemas de entrega de antigénios, sem toxicidade esperada associada. No entanto, são necessários mais estudos para confirmar os resultados obtidos para a produção de citocinas com DCs, uma vez que a variabilidade entre dadores foi significativa. Por fim, estudos in vivo em murganhos usando o antigénio do HBV com as partículas de inulina e PCL-Inulina desenvolvidas, deverão elucidar-nos em futuras experiências sobre a real capacidade das formulações como estratégia para desenvolver novas vacinas e melhorar a vacinação contra o HBV.
Hepatitis B virus (HBV) is an enveloped DNA virus associated with the Hepadnaviridae family, being highly hepatotrophic and species-specific, meaning that HBV replicates exclusively in human hepatocytes. HBV infection can occur early in life, generally leading to chronic infection if not treated correctly. If HBV infection occurs later, an acute self-resolving infection might take place. To prevent HBV infection, there is currently a prophylactic vaccine, that consists in 3 or 4 doses (depending on the individual geographic region) of the injectable HBV surface antigen (HBsAg) adjuvanted with aluminum salts. Despite the effective vaccine available, HBV prevalence is still a serious global health problem, thus, eliminating hepatitis is a major goal in the United Nations 2030 Agenda. Although the future looks prospective, two main problems constitute a threat to the eradication of HBV, with the first problem being the need to improve vaccination in developing countries, and the second the non-existence of a therapeutic vaccine to be used in chronic patients. A way of trying to solve these problems is the use of nanotechnology to design vaccine adjuvants, since it allows to obtain nanoparticles with different compositions, sizes, and surface properties, which help to increase antigen stability, immunogenicity, and allow targeted delivery. Delta inulin isoform (called Advax ™) is a polysaccharide comprised of fructose units, that has been tested in clinical trials with the HBV vaccine. One of the important qualities of inulin shown in animal studies, was the enhancement in the adaptive immune response, which was confirmed against a wide variety of viral and bacterial antigens, like influenza, Japanese encephalitis, HIV, and others. Therefore, this master dissertation aims to develop and produce Inulin particles blended or not with other polymers, previously studied for vaccine adjuvant purposes, such as Polycaprolactone (PCL) or Chitosan. The production method developed and optimized was based on nanoprecipitation techniques and the particles were characterized regarding their physicochemical and immune-toxicological characteristics. Three main delivery systems were developed: inulin particles, PCL-inulin particles and PCL particles. While PCL-inulin and PCL particles had sizes of 301.26 nm and 275.32 nm respectively, inulin particles, were much bigger, and out of the nanoscale (1320.86 nm). The size measurement results allowed us to infer that the particles were stable for 42 days, with the exception of Inulin based particles that present a tendency to increase their size. In terms of immunotoxicity, Inulin and PCL-Inulin particles have shown no toxicity in primary murine spleen cells, although in RAW 264.7 cell line, the results were a little different, as PCL-Inulin particles have shown some toxicity in higher concentrations, which was the same observed in PCL particles. Considering the nitric oxide (NO) and cytokine production studies, the particles did not induce NO production when incubated with RAW 264.7 cell line nor GM-CSF production. Considering the TNF-α, IL-6, IL-10, and IL-12 production by DCs incubated with different particles, the results revealed that the PCL-Inulin at 2 µg/mL does not induce the production of significant cytokine levels. However, at 4 µg/mL concentration, we observed a small induction of the production of the IL-12 and IL-10. Overall, we reported a successful development and production of Inulin particles blended with Polycaprolactone polymer, which preliminary results suggest that they are suitable for use as antigen delivery systems, with no expectable toxicity associated. However further studies are required to confirm the results obtained for cytokine production with DCs since donor variability was significant. Ultimately, in vivo studies in mice using HBV antigens with the developed Inulin and PCL-Inulin particles, should elucidate about the real ability of these formulations as a strategy to improve HBV vaccination.
FCT
Duarte, Henrique José Xavier. "INULIN PARTICLES AS A VACCINE ADJUVANT FOR HEPATITIS B. DEVELOPMENT AND IN VITRO EVALUATION OF THEIR IMMUNOTOXICITY." Master's thesis, 2021. http://hdl.handle.net/10316/99174.
Повний текст джерелаO vírus da Hepatite B (HBV) é um vírus de DNA com envelope associado à família Hepadnaviridae, o qual é altamente hepatotrófico e espécie-específico, ou seja, replica-se exclusivamente em hepatócitos humanos. A infeção por HBV pode ocorrer no início da vida, o que geralmente leva a uma infeção crónica, caso não seja tratada corretamente. Caso a infeção por HBV ocorra mais tarde, geralmente resulta numa infeção aguda que tem a capacidade de se auto-resolver. Atualmente, para prevenir a infeção pelo HBV existe uma vacina injectável profilática, que consiste em 3 ou 4 doses (dependendo da região geográfica do indivíduo) contendo o antigénio de superfície do HBV (HBsAg) combinado com sais de alumínio. Apesar da vacina disponível ser eficaz, a prevalência do HBV ainda é um sério problema de saúde pública, razão pela qual eliminar a hepatite é uma das principais metas inscrita na Agenda 2030 das Nações Unidas. Embora o futuro pareça promissor, existem dois problemas que constituem uma ameaça à erradicação do HBV, sendo o primeiro a necessidade de melhorar a vacinação nos países em desenvolvimento e o segundo a inexistência de uma vacina terapêutica para uso em doentes crónicos. Uma forma de tentar resolver estes problemas é o uso da nanotecnologia como ferramenta para o desenvolvimento de adjuvantes de vacinas, uma vez que esta permite “desenhar” as nanopartículas com diversas composições, tamanhos e propriedades de superfície. A inclusão do antigénio em nanopartículas ajuda a aumentar a estabilidade do antigénio, a imunogenicidade e permite a entrega direcionada deste mesmo antigénio a células apresentadoras de antigénio. A delta inulina (denominada Advax ™) um polissacarídeo composto por unidades de frutose, tem vindo a ser testada em ensaios clínicos para uso na vacina contra o HBV. Uma das qualidades mais importantes da inulina, observada em estudos com animais, foi o aumento da resposta imune adaptativa, confirmada numa ampla variedade de antigénios virais e bacterianos, como influenza, encefalite japonesa, HIV, entre outros. Por conseguinte, esta dissertação de mestrado tem como objetivo desenvolver e produzir partículas de inulina combinadas ou não, com um polímero previamente estudado, como a Policaprolactona (PCL) ou Quitosano, para ser usado como adjuvante em vacinas. O método de produção desenvolvido e otimizado foi baseado em técnicas de nano-precipitação e as partículas foram caracterizadas quanto às suas características físico-químicas e imunotoxicológicas. Neste contexto três sistemas de entrega foram desenvolvidos: partículas de inulina, partículas de PCL-inulina e partículas de PCL. Enquanto as partículas de PCL-inulina e PCL apresentaram tamanhos médios de 301,26 nm e 275,32 nm respetivamente, as partículas de inulina eram muito maiores, encontravam-se fora da nano-escala (1320,86 nm). Os resultados das determinações de tamanho permitiram-nos inferir que as partículas são estáveis por 42 dias, com exceção das partículas de inulina que apresentaram uma tendência para aumento de tamanho ao longo do tempo. Em termos de imunotoxicidade, as partículas de inulina e PCL-inulina não mostraram toxicidade em culturas de células primárias do baço de murganho, embora na linha celular RAW 264,7 os resultados tenham sido ligeiramente diferentes, uma vez que as partículas de PCL-inulina mostraram alguma toxicidade em concentrações mais elevadas, o que foi também observado nas partículas PCL. Considerando os estudos de avaliação de produção de óxido nítrico (NO) e citocinas, as partículas não induziram a produção de NO quando incubadas com a linha celular RAW 264,7, nem a produção de GM-CSF. O mesmo foi verificado para o TNF-α, IL-6, IL-10, e IL-12 após incubação das partículas de PCL-inulina a 2 μg/mL com as DCs, onde se observou que não foram induzidos níveis significativos de citocinas. No entanto, a 4 μg/mL pode ver-se uma pequena indução da IL-12 e IL-10. No geral, relatamos o desenvolvimento e produção de partículas de inulina e policaprolactona, cujos resultados preliminares de diversos ensaios in vitro sugerem que são adequados para uso, como sistemas de entrega de antigénios, sem toxicidade esperada associada. No entanto, são necessários mais estudos para confirmar os resultados obtidos para a produção de citocinas com DCs, uma vez que a variabilidade entre dadores foi significativa. Por fim, estudos in vivo em murganhos usando o antigénio do HBV com as partículas de inulina e PCL-Inulina desenvolvidas, deverão elucidar-nos em futuras experiências sobre a real capacidade das formulações como estratégia para desenvolver novas vacinas e melhorar a vacinação contra o HBV.
Hepatitis B virus (HBV) is an enveloped DNA virus associated with the Hepadnaviridae family, being highly hepatotrophic and species-specific, meaning that HBV replicates exclusively in human hepatocytes. HBV infection can occur early in life, generally leading to chronic infection if not treated correctly. If HBV infection occurs later, an acute self-resolving infection might take place. To prevent HBV infection, there is currently a prophylactic vaccine, that consists in 3 or 4 doses (depending on the individual geographic region) of the injectable HBV surface antigen (HBsAg) adjuvanted with aluminum salts. Despite the effective vaccine available, HBV prevalence is still a serious global health problem, thus, eliminating hepatitis is a major goal in the United Nations 2030 Agenda. Although the future looks prospective, two main problems constitute a threat to the eradication of HBV, with the first problem being the need to improve vaccination in developing countries, and the second the non-existence of a therapeutic vaccine to be used in chronic patients. A way of trying to solve these problems is the use of nanotechnology to design vaccine adjuvants, since it allows to obtain nanoparticles with different compositions, sizes, and surface properties, which help to increase antigen stability, immunogenicity, and allow targeted delivery. Delta inulin isoform (called Advax ™) is a polysaccharide comprised of fructose units, that has been tested in clinical trials with the HBV vaccine. One of the important qualities of inulin shown in animal studies, was the enhancement in the adaptive immune response, which was confirmed against a wide variety of viral and bacterial antigens, like influenza, Japanese encephalitis, HIV, and others. Therefore, this master dissertation aims to develop and produce Inulin particles blended or not with other polymers, previously studied for vaccine adjuvant purposes, such as Polycaprolactone (PCL) or Chitosan. The production method developed and optimized was based on nanoprecipitation techniques and the particles were characterized regarding their physicochemical and immune-toxicological characteristics. Three main delivery systems were developed: inulin particles, PCL-inulin particles and PCL particles. While PCL-inulin and PCL particles had sizes of 301.26 nm and 275.32 nm respectively, inulin particles, were much bigger, and out of the nanoscale (1320.86 nm). The size measurement results allowed us to infer that the particles were stable for 42 days, with the exception of Inulin based particles that present a tendency to increase their size. In terms of immunotoxicity, Inulin and PCL-Inulin particles have shown no toxicity in primary murine spleen cells, although in RAW 264.7 cell line, the results were a little different, as PCL-Inulin particles have shown some toxicity in higher concentrations, which was the same observed in PCL particles. Considering the nitric oxide (NO) and cytokine production studies, the particles did not induce NO production when incubated with RAW 264.7 cell line nor GM-CSF production. Considering the TNF-α, IL-6, IL-10, and IL-12 production by DCs incubated with different particles, the results revealed that the PCL-Inulin at 2 µg/mL does not induce the production of significant cytokine levels. However, at 4 µg/mL concentration, we observed a small induction of the production of the IL-12 and IL-10. Overall, we reported a successful development and production of Inulin particles blended with Polycaprolactone polymer, which preliminary results suggest that they are suitable for use as antigen delivery systems, with no expectable toxicity associated. However further studies are required to confirm the results obtained for cytokine production with DCs since donor variability was significant. Ultimately, in vivo studies in mice using HBV antigens with the developed Inulin and PCL-Inulin particles, should elucidate about the real ability of these formulations as a strategy to improve HBV vaccination.
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