Dissertations / Theses on the topic 'HIV vaccine; adjuvants'

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.

In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches. My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine. Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.

In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches. My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine. Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.

A busca por adjuvantes capazes de estimular eficiente resposta imunológica celular representa uma importante contribuição para a pesquisa de vacinas. No presente trabalho avaliamos o potencial adjuvante de flagelinas de Salmonella para a ativação de linfócitos T, com ênfase na ativação de linfócitos T CD8+, em animais imunizados com diferentes estratégias vacinais, desde vacinas baseadas em linhagens atenuadas de Salmonella; vacinas acelulares que empregaram flagelinas purificadas co-administradas, ou em fusão, a antígenos alvo e, ainda, vacinas de DNA. Nossos resultados demonstram que a utilização de flagelina na forma de vacina de DNA induziu maior proteção terapêutica que a mesma formulação utilizada na forma de vacinas de subunidades, sugerindo que os efeitos adjuvantes da flagelina para ativação de CTLs não estão relacionados apenas a ligação ao TLR5. Os resultados apresentados na presente tese contribuem para a compreensão dos mecanismos de adjuvanticidade da flagelina de Salmonella, particularmente no contexto da ativação de células T.
The 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.

De nombreux obstacles freinent le développement d'un vaccin efficace contre le VIH. Pour franchir ces barrières, le recours aux adjuvants est une option prometteuse. Dans ce contexte, l'objectif de ce doctorat est l'évaluation de combinaisons de ligands de PRR et de particules biodégradables pour la vaccination muqueuse. La première partie de l'étude vise à analyser la valeur ajoutée de molécules hybrides composée de deux ligands de PRR comparée à la co-administration des deux agonistes. Des molécules stimulant TLR7 et TLR2 puis TLR7 et NOD2 ont été évaluées. Nous avons démontré l'intérêt de l'association de ligands de PRR au sein d'une même molécule, pour l'induction de réponses immunitaires systémiques et muqueuses. Des études récentes ont montré l'intérêt d'agonistes de STING comme adjuvant vaccinaux. Nous avons étudié l'induction de réponses immunitaires par les agonistes de STING, administrés par voie parentérale ou muqueuse. Nous avons démontré le fort potentiel de ligands de STING pour l'induction de réponses cellulaires et muqueuses. Lors de ces études, nous avons démontré que l'intérêt de la vectorisation d'agonistes de PRR dépend de la molécule. En effet, bien que la vectorisation d'une molécule hybride TLR7/TLR2 n'ait pas d'impact sur la réponse immunitaire induite, la vectorisation d'agonistes de STING potentialise leur effet immunostimulant. Pour finir, nous avons montré que la voie d'administration a un impact sur la réponse immunitaire induite. Afin de mieux comprendre les mécanismes mis en jeu, une étude de biodistribution des formulations de NP de PLA après administration par voie systémique ou muqueuse a été réalisée
There 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

La vaccination muqueuse, notamment celle administrée par voie nasale, est considérée comme la méthode optimale permettant de protéger les sites muqueux de l'infection par des pathogènes. Cependant, le manque d'adjuvants muqueux spécifiques ainsi qu'une prise en compte insuffisante du système immun nasal limite le développement des vaccins muqueux. P1, un domaine conservé de gp41, la glycoprotéine d'enveloppe du VIH-1, a été récemment utilisé par le laboratoire d'acceuil pour développer un vaccin prophylactique muqueux après immunisation combinée par voie nasale et intra-musculaire. Ce vaccin s'est montré efficace lors études pré-cliniques et clinique de Phase I chez l'Homme, grâce à sa capacité d'induire des IgA muqueux contre P1 bloquant la pénétration muqueuse du VIH-1 par transcytose et de stimuler la production d'IgG spécifiques de gp41 induisant la lyse des cellules infectées par le VIH-1 par cytotoxicité cellulaire médiée par anticorps (ADCC). Dans le travail présenté ici, nous avons caractérisé les mécanismes immuns induits par ce vaccin basé sur P1 au niveau de la muqueuse nasale humaine. Tout d'abord, nous avons démontré que P1 initie une réponse immune en induisant la sécrétion de la cytokine TH2, la Thymic Stromal LymphoPoietin (TSLP), par les cellules épithéliales nasales. TSLP est connyu pour ses propriétés d'adjuvant muqueux puissant et son récepteur, le TSLP-R, joue un rôle déterminant dans la réponse muqueuse à IgA. Nous avons montré que P1 induit l'expression de TSLP via l'interaction de P1 avec le galactosyl céramide, un récepteur du VIH-1 permettant l'entrée muqueuse du virus. De plus, nous avons identifié la voie de signalisation Calcineurin/NFATet le microRNA- comme partenaire décisifs dans la régulation de la production de TSLP induite par P1. Dans un second temps, nous avons montré que le peptide P1 module la réponse innée en activant les cellules dendritiques (DCs). Cette stimulation par P1 induit l'augmentation de l'expression des molécules de co-stimulation par les DCs. La sécrétion de l'IL-6 et de l'IL-10 par les DCs est aussi augmentée par P1 alors que celle de l'interféron gamma, IFN-', est réduite, démontrant ainsi que les DCs activée par P1 se polarisent en une phénotype facilitant une réponse Th2 et IgA. De plus, l'IL-8 et les chimiokines CCL20 et CCL22 sont secrétées indiquant que les DCs ont acquis la capacité de recruter des cellules immunes dans la muqueuse pour initier une réponse muqueuse adaptative. La métalloprotéinase MMP-9 permettant la dégradation de la matrice extracellulaire et facilitant la migration des cellules hors de la muqueuse, est aussi produite. Une boucle positive autocrine de TSLP est observée, puisque P1 induit la sécrétion par les DCs de TSLP en conséquence l'augmentation de l'expression du TSLP-R par les DCs induite par P1. Finalement, P1 active la prolifération des lymphocytes TCD4+ médiée par les DCs. En conclusion, nous avons démontré que P1 est un peptide multifonctionnel avec un grand potentiel dans le développement de vaccin, non seulement comme antigène vaccinal candidat mais aussi comme potentiel adjuvant qui pourrait être combinés à d'autres vaccins muqueux
Mucosal 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

Almost 30 years after the identification of HIV-I as the causative agent of AIDS, an effective prophylactic HIV-I vaccine has yet to be developed. Despite the design of a vast array of therapeutic agents and prevention strategies, which have helped to reduce AIDS mortality and the growth of the pandemic, HIV-I is still the fourth biggest killer worldwide and an effective vaccine will be needed to halt the pandemic. Over the past three decades, various HIV-I vaccination strategies have been explored leading to the key understanding that sterile protection against HIV-I infection will require a combination of cellular and humoral responses, particularly at the mucosal compartment, the common route of infection. Consequently, it has been postulated that this can only be achieved through a potent and effective immunisation strategy involving mucosal immunisation and the application of a heterologous prime-boost regimen. However, the mucosal route of immunisation, in particular for subunit vaccines, has not been well established as a mainstream mode of vaccination in human and results with experimental mucosal immunisation have generally been impeded by a lack of safe and effective mucosal adjuvants. In the work presented herein, I investigated whether the highly cationic polyethyleneimine (PEI), a gold standard DNA delivery agent, has adjuvant activity applied parenterally and mucosally in formulation with glycoprotein antigens, in particular HIV-I Env gp140, the sole target of neutralising antibodies (nAbs) and therefore the most attractive HIV-I sub unit vaccine candidate. I demonstrated that PEI is a potent inducer of Env-specific Ab responses in a mixed Ti.l and Th2 context. PEI is particularly potent via the mucosal route and superior to gold standard mucosal adjuvant Cholera Toxin Beta subunit (CTB) as measured by HIV-I specific IgA responses at the mucosal compartment. PEI showed direct immune stimulatory effects, marked by a significant recruitment of local leukocytes, including inflammatory monocytes and dendritic cells (DCs). Further it associated with antigen to form gpI40-PEI particles, which enhanced antigen targeting to, and uptake by, immune cells, in particular monocytes, macro phages, and DCs. The observed adjuvant activity was maintained with the use of various forms of PEI, including linear or branched, and durable or biodegradable, and under a range in molecular weight (MW), suggesting that immune potentiation is a shared attribute of this group of PEI polymers. This is the first demonstration of PEI as a mucosal adjuvant for protein immunisation. These results merit further exploration of PEI in protection models with the implication for clinical trials.

The development of a safe, effective and affordable HIV-1 vaccine is urgently needed especially in resource poor settings. It is widely accepted that for a vaccine to confer sterilising immunity to HIV-1 it will have to elicit broadly neutralising antibodies (nAbs) to primary isolates of HIV but this goal has remained elusive. There are two key problems to be addressed in this regard: vaccine candidates have to express the correct epitopes capable of eliciting broadly cross-reactive neutralising antibodies to HIV, and they have to provide potent stimulatory signals to the relevant B cells so that high titres ofneutralising antibodies are obtained. Plasmid DNA and recombinant poxviruses such as modified vaccinia virus Ankara (MYA) and fowlpox virus (FPV) have been safely administered to humans and offer the advantage of accommodating large amounts of additional recombinant DNA. In addition, they have provided promising cytotoxic T-Iymphocyte inducing HIV-l vaccine candidates, especially in heterologous prime-boost combinations. The hypothesis investigated here is that sequentially immunising with envelopes of different clades of HIV might lead to the antibody response being focussed on neutralising epitopes held in common. To overcome the poor immunogenicity of cross-clade neutralising epitopes, and the generally poor antibody responses elicited to priming with DNA, followed by boosting with recombinant poxviruses, B cells were stimulated by the coexpression of cholera toxin B and human complement component C3d. To further stimulate antibody production the candidate vaccines coencoded envelope and capsid components so that there would be in vivo virus-like particle formation. The candidate HIV vaccines proceeded directly to a non-human primate study of immunogenicity because this model most closely resembles the immune response in humans, and the benefits of virus-like particle formation and the adjuvant effect ofhuman C3d were most likely to be demonstrated. The work in this thesis describes the construction and characterisation of complex, adjuvanted poxviral recombinants for use as HIV vaccine candidates in a DNA primeFPV boost-MVA boost in macaques and the subsequent efficacy of macaque sera to neutralise primary isolates ofHIV-l in a validated HIV-l neutralisation assay.

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.

A human safe, Alhydrogel adjuvanted whole cell Trichomonas vaginalis vaccine was tested for efficacy in a BALB/c mouse model of vaginal infection. Additionally, the systemic and local immune response were measured. Vaccination reduced incidence and increased clearance of infection, and induced both systemic and local humoral immune responses. CD4+ cells were detected in vaginal tissues following intravaginal challenge with T. vaginalis, but were not seen in uninfected mice. CD4+ cells were detected more often, earlier, and in greater numbers in vaccinated vaginal tissues compared to unvaccinated controls. Presence of CD4+ T cells following infection can have significant implications of increasing HIV susceptibility and transmission. These data suggest that the vaccine induces local and systemic immune responses, and confers significantly greater protection against vaginal challenge than unvaccinated vaginal challenge. These data support the potential for a human vaccine against T. vaginalis infection that could also impact the incidence of HIV infections.

A formulação HIVBr18, previamente desenvolvida e testada, é uma vacina de DNA que codifica 18 epítopos CD4, promíscuos e conservados do HIV-1, e que após imunização de camundongos transgênicos para diversas moléculas de HLA de classe II humanas, observou-se proliferação de linfócitos T CD4+ e CD8+ e produção de IFN-? direcionadas a múltiplos epítopos codificados pela vacina. Abordamos aqui estratégias baseadas na fusão ou combinação dos epítopos codificados pela vacina HIVBr18 à glicoproteína D (gD) do HSV-1, e também na coadministração de plasmídeos que codificam citocinas (IL-2, -12, -15 e GM-CSF) visando aumentar a imunogenicidade de HIVBr18. A sequencia de DNA que codifica os 18 peptídeos da vacina HIVBr18 foi amplificada por PCR e clonada em um plasmídeo que abrigava a sequencia da gD do HSV-1. dando origem ao plasmídeo pVAX-gDh-HIVBr18. Animais imunizados com gDh-HIVBr18 apresentaram resposta imunológica similar ao grupo que recebeu somente HIVBr18, não sendo diferente também daqueles que receberam plasmídeos gDh-HIVBr18 que sofreram alterações nas sequências para melhorar o padrão de distribuição hidrofóbica e permitir a migração da proteína de fusão para o meio extracelular. Construímos e testamos um plasmídeo bicistrônico que expressa gDh e HIVBr18 isoladamente, mas também não observamos aumento na resposta imune induzida. A coadministração com o plasmídeo HIVBr18 e plasmídeos que codificam as citocinas IL-12, IL-15 e GM-CSF, proporciona um aumento na magnitude da resposta imunológica induzida contra o pool de peptídeos codificados pela vacina, entretanto sem alteração da amplitude da resposta. Além disso, o plasmídeo de GM-CSF induziu maior número de células T CD4+ polifuncionais. Demonstramos também que a coadministração do plasmídeo que codifica GM-CSF, induz uma resposta imune celular de maior magnitude mesmo em uma condição de dose reduzida. Entretanto, observamos que esta citocina não é um bom adjuvante quando utilizamos como vetor de imunização um adenovírus que expressa os 18 epítopos
The 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.

Introduction: Traditional vaccine strategies are inefficient at protecting against more complex pathogens including HIV, therefore novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but reduced immunogenicity in humans is a limitation. The key to improving DNA vaccine immunogenicity is enhanced DNA uptake and localised inflammatory responses, which may be achieved with the use of an appropriate adjuvant. HSP70 is a damage associated molecular pattern (DAMP) that binds to and activates dendritic cells, making HSP70 a potential natural adjuvant. The immunogenicity of a DNA vaccine encoding HIV gag and HSP70 was examined in which the latter was genetically modified to encode cytoplasmic, fusion, secreted or membrane-bound forms of HSP70, controlled by a separate promoter within the same construct, ensuring delivery of the antigen and adjuvant to the same cell. Furthermore, immune responses induced by DNA vaccines encoding multiple antigens compared with a single antigen were measured. Methodology: C57Bl/6 mice were vaccinated three times at two weekly intervals with 10 μg doses of DNA via the intradermal route. Gag-specific T cell responses were determined by EliSpot, intracellular cytokine staining (ICS) and proliferation assays. Short term and long term protection was evaluated by challenge with EcoHIV, a chimeric HIV that can infect mouse, but not human, leukocytes. Results: A bicistronic vector containing the CMV and SV40 promoters was produced. The CMV promoter was shown to be approximately 10-fold stronger than the SV40 promoter using the eGFP reporter gene. Therefore, initially, the HIV gag antigen was cloned downstream of the CMV promoter and the wild type and modified HSP70 genes were inserted downstream of the SV40 promoter, to ensure that the adjuvant properties of HSP70 targeted antigen-positive cells. Membrane-bound or secreted HSP70 significantly enhanced gag-specific T cell responses in vaccinated mice and increased the breadth of T cell responses as determined by the recognition of otherwise subdominant gag epitopes. The adjuvant effect of membrane-bound or secreted HSP70 also significantly improved the ability of HIV-specific T cells to secrete multiple cytokines, a characteristic thought to be important for vaccine efficacy. Finally, inclusion of genes encoding membrane-bound HSP70 resulted in increased protection as shown by a significant reduction in the titre of EcoHIV in peritoneal exudate cells after virus challenge, compared with mice vaccinated with the gag-only DNA. To increase the expression of membrane-bound or secreted HSP70, these genes were also inserted downstream of the CMV promoter, while expression of gag was controlled by the SV40 promoter. However, this significantly decreased gag-specific T cell responses as shown by EliSpot and ICS analysis. Furthermore, DNA vaccines which included the HIV pol gene with gag compared with gag alone reduced the gag-specific T cell responses in vaccinated mice and reduced the protection in mice challenged with EcoHIV. Conclusion: This study demonstrated that expression of membrane-bound or the secreted form of HSP70 from a DNA vaccine encoding HIV gag significantly improved gag-specific T cell function, multifunctionality and proliferation. Most importantly, the adjuvant effect of membrane-bound HSP70 increased protection in mice challenged with EcoHIV.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014

Effective mucosal vaccines that elicit strong sustained CD8 T cell immunity at mucosal surfaces are thought to be critical against mucosal transmitted infection like HIV. A novel type I Interferon epsilon (IFN-e) and toll-like receptor member 9 (TLR9) were co-expressed together with HIV antigens and were used in a homologous prime-boost setting to assess mucosal immunity against HIV-1. The immuno-biology studies of IFN-e indicated that intranasal (IN) infection of VV co-expressing IFN-e (VV-HIV-IFN-e) can induce rapid VV clearance in lung that correlated with (i) elevated lung VV-specific CD8+CD107a+IFNg+ population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) heightened cytotoxic CD8+CD4+ T cell subset (CD3high/CCR7high/CD62Llow) in lung lymph nodes. These responses were different to that observed with VV-HA-IFN-alpha or VV-HA-IFN-beta infection. When IFN-e was used in an IN/intramuscular(IM) HIV prime-boost immunization, elevated HIV-specific effector (but not memory) CD8 T cell response was observed in spleen, genito-rectal nodes, and peyer's patches. Homing markers (alpha4beta7 & CCR9) analysis indicated that unlike other type I IFNs, IFN-e could promote migration of antigen-specific CD8 T cells to the gut mucosae. The induction of gut-specific immunity was independent of the route of delivery. This suggested that IFN-e at the vaccination site may have the ability to activate unique APC subsets that promoted CD8 T cell homing to gut mucosae. These results for the first time have established that IFN-e plays an important and unique role in the mucosae. Following vaccination the short-lived CD8 T cell responses observed, indicate that rather than a vaccine adjuvant IFN-e could be used as a therapeutic to control local lung and/or gut infections. Specifically, IFN-e may have great potential to be used as microbicide to prevent mucosal disease (e.g TB, Chlamydia or HIV-1). When the immuno-biological properties of TLR9 were evaluated using the VV co-expression system, the IN VV-HIV-TLR9 infection showed a rapid viral clearance in lung compared to the control VV-HIV infection. These results also correlated with enhanced lung DC and lung alveolar macrophage subset that was specific to TLR9 infection. Next, in an IN/IM immunization modality, the TLR9 adjuvanted vaccination showed promising HIV-specific memory CD8 T cell responses at both systemic and mucosal compartments following 8-week post booster immunization. Compared to control vaccination, TLR9 adjuvanted vaccine generated higher HIV-tetramer reactive memory CD8 T cells and IFNg+TNFa+CD8+ T cells in spleen and lung, but unlike at the effector stage of immunity no difference in the IL-2+CD8+ T cells were detected. Interestingly, elevated IL-4 producing T cells were detected following TLR9 adjuvanted vaccination (expression of IL-4/IL-13 by CD8+ T cells is associated with low avidity T cells). The latter correlated with TLR9 vaccination strategy not inducing elevated numbers of high avidity CD8+ T cells and suboptimal protective immunity. In summary, data in this thesis demonstrated that IFN-e is uniquely involved in mucosal immunity among other type I IFNs. Both IFN-e and TLR9 have good potential to be used as mucosal therapeutics to enhance or modulate tissue-specific mucosal immunity at the first line of defense.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
O 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

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
O 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