Dissertations / Theses on the topic 'Vaccine schedule and formulation'

Many people may have become increasingly concerned about the risks associated with vaccines. At the same time, there is a lack of qualitative research on the impact of various vaccinations schedules on individuals' physio-psychological health. In addition, "mandatory" versus "nonmandatory, but recommended" vaccines are still under debate in some Western countries. The purpose of this ethnographic study was to provide an in-depth understanding of the beliefs, experiences, and perceptions of adolescents, parents, and health care providers regarding different vaccination schedules. The health belief model was used as the theoretical framework. The sample consisted of adolescents and parents from different vaccination backgrounds, as well as of healthcare providers who were involved with vaccination schedules (N=72). Purposeful sampling strategy was applied and individual interviews were conducted. All interviews were recorded and transcribed verbatim, and the obtained data were analyzed thematically. According to the results of the study, participants' perceptions on vaccination were generally positive, and a mandatory vaccination schedule was mostly recommended. Adolescents who received mandatory vaccination reported that this scheme was appropriate against several diseases. Further, health care members indicated that vaccination side effects were mainly emotional, and they suggested that public health agencies should disseminate more scientifically-sound information on the benefits and risks of vaccination. The findings of this study may be used as the basis for the formulation of an effective public health policy to adopt a nationally-and internationally-accepted vaccination schedule.

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Though vaccine uptake and public support have risen since the release of the first HPV vaccines, the United States has far lower initiation and completion rates for the HPV vaccine series in comparison to other vaccines indicated for youth. Disparities are even greater in the Appalachian regions. Understanding factors contributing to these discrepancies is vital to improving raise vaccine rates in Appalachia. A comprehensive literature search identified all articles pertaining to HPV vaccination in children and adolescents living in Appalachia. The final 15 articles were included in a systematic review of the topic. Findings: HPV disease and HPV vaccine-related knowledge and communication were low in Appalachian communities, and vaccine uptake was lower in all areas of Appalachia as compared to non-Appalachian U.S. Moreover, large variations in uptake existed among Appalachian subregions. Many variables appear to contribute to this variation, including vaccine acceptance for younger adolescents, local and press-driven critical reports of the vaccine, physician communication, and views of the family matriarchs. Targeting the Appalachian subregions, specific campaigns or intervention may be more impactful than viewing the region as a homogenous whole.

Healthy People 2020 was launched in December 2010 with a target human papilloma virus (HPV) vaccination rate of 80%. As of 2014, we were well short of this goal, especially among males, for whom the HPV vaccine became recommended in 2011. An estimated 14-20% of adolescent-aged males had completed the vaccine schedule as of 2015. This is particularly problematic in northeast Tennessee, as multiple risk factors for lower vaccination rates are characteristic of the population, including being white and living in the South. Reasons to decline vaccination vary, and usually involve concerns about safety, efficacy, and necessity. Worries about sexual disinhibition from being vaccinated are often cited by opponents. Parents also perceive a lack of benefit from getting the vaccination starting at age 11 before their sons are sexually active. The media and internet are also barriers to appropriate vaccination in males, as previous research has demonstrated that media coverage is more likely to focus on political controversies instead of benefits, and is more likely to emphasize the benefits to females. Research on improving vaccine uptake on males is currently limited. Doctors who appear knowledgeable and are willing to spend time talking about the HPV vaccine for male patients may increase vaccination rates. Other interventions that may also be effective include vaccinating as part of nurse visits or through school programs. Early studies have been mixed on the effect of patient and parent education on vaccine uptake, although a 2015 review demonstrated that most practice- and community-based educational interventions have some positive effect on uptake. The purpose of this project was to improve HPV vaccine uptake among male patients in a family practice residency program through patient and parent education. After informed consent was obtained, the patients and/or their parents were given a handout produced by the CDC highlighting the benefits of vaccination for males. A chart review was done to determine vaccine coverage among males before the intervention instituted in November of 2016. The intervention was completed in August 2017, and a repeat chart review is currently ongoing to determine vaccine coverage in the post-intervention period. Data collection and analysis is ongoing at the time of abstract submission. We expect a statistically significant increase in the number of male patients who have received any doses of vaccine, and in the number who have completed the vaccine series. Future research should involve broadening the intervention to include local family medicine and pediatrician’s offices to increase vaccine uptake in these populations as well.

Currently, biopharmaceuticals including vaccines, proteins, and DNA are delivered almost exclusively through the parenteral route using hypodermic needles. However, injection by hypodermic needles generates pain and causes bleeding. Disposal of these needles also produces biohazardous sharp waste. An alternative delivery tool called microneedles may solve these issues. Microneedles are micron-size needles that deliver drugs or biopharmaceuticals into skin by creating tiny channels in the skin. This thesis focuses on dissolving microneedles in which the needle tips dissolve and release the encapsulated drug or vaccine upon insertion. The project aimed to (i) design and optimize dissolving microneedles for efficient drug and vaccine delivery to the skin, (ii) maintain vaccine stability over long-term storage, and (iii) immunize animals using vaccine encapsulated microneedles. The results showed that influenza vaccine encapsulated in microneedles was more thermally stable than unprocessed vaccine solution over prolonged periods of storage time. In addition, mice immunized with microneedles containing influenza vaccine offered full protection against lethal influenza virus infection. As a result, we envision the newly developed dissolving microneedle system can be a safe, patient compliant, easy to-use and self-administered method for rapid drug and vaccine delivery to the skin.

This research focused on the formation of particulate delivery systems for the sub-unit fusion protein, Ag85B-ESAT-6, a promising tuberculosis (TB) vaccine candidate. Initial work concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyl dioctadecyl ammonium (DDA). These studies demonstrated that addition of the immunomodulatory trehalose dibehenate (TDB) enhanced the physical stability of the system whilst also adding further adjuvanticity. Indeed, this formulation was effective in stimulating both a cell mediated and humoural immune response. In order to investigate an alternative to the DDA-TDB system, microspheres based on poly(DL-lactide-co-glycolide) (PLGA) incorporating the adjuvants DDA and TDB, either alone or in combination, were first optimised in terms of physico-chemical characteristics, followed by immunological analysis. The formulation incorporating PLGA and DDA emerged as the lead candidate, with promising protection data against TB. Subsequent optimisation of the lead microsphere formulation investigated the effect of several variables involved in the formulation process on physico-chemical and immunological characteristics of the particles produced. Results revealed that the DDA-TDB liposome system proved to be the most immunologically efficient delivery vehicle studied, with high levels of antibody and cytokine production, particularly gamma-interferon (IFN-?), considered the key cytokine marker for anti-mycobacterial immunity. Of the microsphere systems investigated, PLGA in combination with DDA showed the most promise, with an ability to initiate a broad spectrum of cytokine production, as well as antigen specific spleen cell proliferation comparable to that of the DDA-TDB formulation.

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.

Avec pour problématique une importante variabilité des lots de production vaccinale contre la diphtérie, une analyse intégrative du métabolisme central, du fer et du transcriptome de Corynebacterium diphtheriae, cultivé en conditions les plus proches de la culture industrielle, ont permis d’élargir les connaissances sur le comportement de la bactérie en conditions de production industrielle, et plus particulièrement sur l’existence de corrélations entre activité métabolique et virulence. L'analyse du métabolisme central et la comparaison de l'expression des gènes de C. diphtheriae, ont permis de mettre en évidence l'importance du maltose dans l'établissement de la virulence. Ce sucre, largement présent dans notre alimentation, est ainsi rencontré par la bactérie lors de son processus de colonisation de l'hôte. La consommation du maltose lors de la phase stationnaire, coïncident avec la production de la toxine, semble ainsi liée aux réactions de maintenance et à la production de métabolites secondaires plutôt qu'à la croissance. Plusieurs gènes liés à la capture et au catabolisme du maltose sont liés à la production de la toxine diphtérique. Cependant, les mécanismes de régulation sont complexes, impliquant plusieurs régulateurs. Une étude sur les besoins en fer de la bactérie et les liens avec la pathogénicité a été réalisée. En effet, le gène de la toxine diphtérique est régulé par DtxR, répresseur activé par Fe2+. Elle a permis d'évaluer la capacité de stockage en fer de la bactérie et la limite de concentration intracellulaire pour obtenir une production de toxine. Un effort a été apporté afin de visualiser les ferritines synthétisées par la bactérie par la technique NanoSIMS. Enfin, l'analyse de l'expression des gènes de C.diphtheriae cultivé en condition de limitation en fer et supplémentée en fer a apporté une quantité d'informations sur les liens entre métabolisme central et du fer, virulence et stress oxydatif. Cette démarche a été mise à profit afin de proposer à l'industriel des pistes d'optimisations du procédé et d’améliorations de la productivité en toxine diphtérique. Via l’ajustement de certains paramètres physico-chimiques (visant l'oxygénation et une meilleure métabolisation du maltose), il est possible d'obtenir un gain de production significatif (titre final en toxine multiplié par 2,5). La productivité spécifique en toxine peut être également multipliée par 2,2 par une astucieuse étape de dilution et recyclage de la biomasse en fin de culture
Faced with an important variability in production yields of vaccines against diphtheria, a dynamic systemic approach, including central and iron metabolism and transcriptome analysis, led to an improved knowledge of Corynebacterium diphtheriae physiology, notably as regards the connection of central metabolism and virulence. Gene expression analysis coupled to metabolic characterization enabled a correlation between maltose consumption and virulence to be established. Because of the typical human diet, maltose is present in the human oropharynx where it may serve as a key nutrient source for C. diphtheriae. Maltose consumption during stationary phase, coupled with toxin production, seems to be linked to maintenance and secondary metabolites rather than growth. Several genes, including uptake and catabolism of maltose, are related to diphtheria toxin production. However, mechanisms of regulation are complex and may involve several transcriptional regulators. Bacterial iron requirements and its relation to pathogenicity were considered. Indeed, diphtheria toxin gene is regulated by Fe2+ activated DtxR. These studies revealed that C. diphtheriae is able to store an important quantity of intracellular iron within ferritin-like proteins visualized by NanoSIMS microscopy and the definition of an intracellular threshold concentration provoking expression of toxin production. Finally, genome-wide gene expression analysis of C.diphtheriae in iron starvation and iron excess conditions provided information on relations between central and iron metabolism, virulence establishment and oxidative stress. The resulting knowledge was exploited to suggest process optimization strategies to enhance toxin production, currently being assessed by the industrial partner. Adjusting some key physico-chemical parameters (targeting oxygenation and better maltose metabolization) enabled significant gains in toxin production (2.5 fold increase). Specific productivity could be increased by 2.2 thanks to a novel biomass dilution and recycling step at the end of the culture

Le développement de vaccins à ARNm est en plein essor dans le domaine de la vaccinologie. Un des défis majeurs de ces approches est de parvenir à transporter et délivrer les ARNm dans le cytoplasme des cellules dendritiques (DC) pour permettre la production d’antigène et l’activation des réponses immunitaires.L’objectif de ce travail a porté sur la conception et l’évaluation de nouvelles stratégies de vectorisation des ARNm par des nanoparticules de poly(acide lactique) (NP-PLA). Une stratégie basée sur l’adsorption des ARNm à la surface des NP-PLA par l’intermédiaire du LAH4-L1, un peptide cationique amphipathique, a été développée. Des polyplexes formés suite à la condensation de l’ARNm par le LAH4-L1 ont été adsorbés sur les NP-PLA pour former des nanocomplexes. L’intérêt des polyplexes et nanocomplexes pour le ciblage des DC et l’activation des réponses immunitaires in vitro a été évalué. Dans des tests de transfection, ces formulations ont induit une expression efficace d’ARNm modèles dans des DC. La présence des NP-PLA dans les formulations semble favoriser l’expression des ARNm. Le traitement avec des inhibiteurs a révélé que les polyplexes et nanocomplexes semblent internalisés par phagocytose et endocytose clathrine-dépendante, et s’échappent des endosomes par un mécanisme dépendant de la pompe à protons v-ATPase. Suite à la transfection de DC dérivées de monocytes (moDC), nous avons montré que nos formulations stimulent les récepteurs de l’immunité innée et induisent une réponse pro-inflammatoire. Cette activation est associée à la maturation des moDC, à la présentation de peptides antigéniques sur le CMH-I et -II et à la sécrétion de cytokines et chémokines impliquées dans l’immunité adaptative. Ces données soulignent l’intérêt des NP-PLA associées au LAH4-L1 pour vectoriser des ARNm, cibler des DC et activer les réponses immunitaires. Dans le contexte du VIH-1, ce type de réponse pourrait aider le système immunitaire à contrôler la charge virale
MRNA-based vaccines currently raise a growing interest in vaccinology. However, the transport and delivery of mRNAs to DC cytoplasm in order to induce antigen production and immune responses remains challenging. The objective of this thesis concerns the design and evaluation of novel strategies to vectorize vaccine mRNAs by poly(lactic acid) nanoparticles (PLA-NPs). We developed a strategy based on mRNA adsorption onto PLA-NPs using, as intermediate, LAH4-L1, an amphipathic cationic peptide. To do this, mRNAs were condensed by LAH4-L1 to form polyplexes which was then adsorbed onto PLA-NPs in a second step to form nanocomplexes. The LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes ability to target DCs and induce immune responses in vitro was evaluated. We showed that formulations induce an efficient transfection of mRNA in DCs in vitro. The addition of PLA-NPs in formulations seems to increase sustained expression of mRNAs. DC treatment by inhibitors revealed that polyplexes and nanocomplexes are taken up by phagocytosis and clathrin-dependent endocytosis, and escape endosomes by a v-ATPase-dependent mechanism. Transfection of monocyte-derived DCs (moDCs) showed that LAH4-L1/mRNA polyplexes and PLA-NP/LAH4-L1/mRNA nanocomplexes trigger innate-sensing activation with pro-inflammatory responses. This activation is associated with moDCs maturation, MHC-I and MHC-II presentation, and the secretion cytokines and chemokines involved in adaptive immunity.These data highlight the interest of these new platform formulations to vectorize mRNAs, target DCs and induce immune responses, which in the context of HIV-1, could help the immune system to control the viral load

Avec l’identification des antigènes tumoraux et la compréhension de la réponse immunitaire mucosale, la vaccination par voie respiratoire est devenue un champ d’investigation prometteur pour le traitement du cancer. L’objectif de cette thèse était de concevoir des vaccins peptidiques nanoparticulaires à base de liposomes, destinés à la vaccination antitumorale par voie respiratoire ou nasale. Ainsi, nous avons formulé des liposomes vectorisant un épitope peptidique TCD8+ dérivé de l’oncogène ErbB2, un épitope TCD4+ et une molécule adjuvante. Cette construction a ensuite été optimisée en faisant varier les caractéristiques physicochimiques du vecteur liposomique (taille, structure, composition) ou de la formulation (viscosité). L’efficacité antitumorale des différents vaccins ainsi obtenus a été évaluée après administration respiratoire ou nasale, prophylactique ou thérapeutique, dans un modèle de tumeurs pulmonaires ou de tumeurs sous-cutanées chez la souris. L’ensemble de ces travaux a montré un intérêt indéniable des vaccins peptidiques à base de liposomes dans la vaccination antitumorale par voie respiratoire et nasale, ouvrant de nouvelles perspectives pour le traitement du cancer
With the identification of tumor antigens and the better understanding of the mucosal immune response, the vaccination by the respiratory route has become a promising field of investigation for cancer treatment. The purpose of this study was to develop nanoparticulate peptide-based liposomal vaccines for antitumor vaccination by respiratory or nasal route. Thus, we have prepared liposomes associating ErbB2 TCD8+ and HA TCD4+ peptide epitopes with an adjuvant molecule. This construct was then optimized by varying its physicochemical characteristics (size, structure, composition) or its formulation (viscosity). The antitumor efficacy of the various vaccines obtained thereby was evaluated in a model of pulmonary or subcutaneous tumors in mice after prophylactic or therapeutic, nasal or respiratory immunization. All our data showed an undeniable interest of peptide vaccines based on liposomes in the antitumor vaccination by the respiratory and nasal routes, opening new perspectives for cancer treatment

Mathematical models provide unique insights to real-world problems. Within the context of infectious diseases, models are used to explore the dynamics of infections and control mechanisms. Human papillomavirus (HPV) globally infects about 630 million people, many of these infections develop into cancers and genital warts. Vaccines are available to protect against the most prevalent and devastating strains of HPV. The introduction of this vaccine as part of a national immunization program in Canada is a complex decision for policy-makers in which mathematical models can play a key role. We use the current recommendations provided by the World Health Organization to explore the integral role mathematical models have in the decision to incorporate the HPV vaccine within a national immunization program. We then provide a review of the literature discussing the role of mathematical models in the decision to include a vaccine in a national immunization program within the context of the HPV vaccine. Next, we evaluate the current standing of mathematical models used within the context of HPV immunization, to highlight the types of models used, underlying assumptions and general recommendations made about these immunization programs. Then, we create and analyze a model to explore the possibility of bettering the current HPV vaccine strategy in Canada. We focus on the effects of the grade of vaccination and the number of doses required to eradicate the targeted strains of HPV.

Deux vaccins sont actuellement disponibles pour la prévention des infections invasives à pneumocoques (IIP) : un vaccin polysaccharidique Pneumovax® (PPV23) et un vaccin conjugué Prevenar13® (PCV13), induisant respectivement une protection contre 23 et 13 sérotypes. Le PPV23 est considéré comme faiblement immunogène, en particulier chez les personnes âgées et les patients immunodéprimés. Le PCV13, en revanche, en raison de la conjugaison à une protéine porteuse, présente l'avantage d'induire une réponse immunitaire T-dépendante, non observée avec le vaccin PPV23. Dans notre travail nous avons donc évalué l'impact des stratégies vaccinales utilisant le PCV13 et le PPV23 sur différentes populations de patients à risque. Dans une première étude, nos résultats sur la vaccination anti-pneumococcique chez des patients atteints de myélome indolent (SMM) ont montré qu'une dose de PCV13 seul, induisait une réponse immune transitoire et de faible persistance. Ces résultats suggéraient l'utilisation d'un schéma vaccinal incluant plusieurs doses de PCV13 ou une association avec le PPV23. Depuis 2013, ce schéma combiné du PCV13 et du PPV23 est le schéma recommandé par la Haute Autorité de Santé en France chez les patients à risque, avec les délais suivants : une dose de PCV13 suivie d'une dose de PPV23, 8 semaines après. Nous avons par la suite étudié cette stratégie vaccinale combinée chez des patients à risque d'IIP : patients atteints de lupus érythémateux systémique (SLE) et patients atteints de polyarthrite rhumatoïde (PR). Nos résultats montrent une immunogénicité à court terme de la stratégie combinée, mais une protection qui ne persiste pas au-delà de deux ans. De façon surprenante, les taux d'anticorps 2 ans après la vaccination, sont inférieurs aux taux pré-vaccinaux pour les patients PR. Cet effet délétère du PPV23 sur la réponse vaccinale induite par le PCV13 est appelé hyporéponse. Ce phénomène, observé chez les patients PR, ne se retrouve pas chez les patients SLE dont la vaccination PPV23 a été effectuée plus à distance du PCV13. Ces résultats suggèrent que le schéma vaccinal plus tardif (c'est-à-dire une vaccination par le PPV23 six mois après le PCV13 au lieu de deux mois) inhiberait le phénomène d'hyporéponse. Dans une troisième partie, nous avons comparé différents schéma vaccinaux modulant les doses des vaccins et les délais d'injection chez des volontaires sains mais également dans un modèle murin d'hyporéponse développé au sein du laboratoire. Notre hypothèse était que la modulation du schéma vaccinal utilisant les 2 vaccins pouvait à la fois induire une protection à long terme et prévenir l'hyporéponse. Nos résultats ont montré que l'utilisation d'une dose diminuée de PPV23 ou l'injection concomitante des deux vaccins n'empêchaient pas l'hyporéponse. En revanche, en allongeant le délai entre le PCV13 et le PPV23, le phénomène d'hyporéponse est limité. Des études cliniques chez les patients à risque d'IIP sont nécessaires afin d'évaluer une stratégie combinée tardive, où le PPV23 serait reçu au moins 6 à 12 mois après le PCV13
Two vaccines are currently available for the prevention of invasive pneumococcal diseases (IPD): a polysaccharide vaccine, Pneumovax® (PPV23) and a conjugate vaccine, Prevenar13® (PCV13), inducing protection against 23 and 13 serotypes, respectively. PPV23 is considered to be weakly immunogenic, particularly in the elderly and immunocompromised patients. PCV13, however, due to the conjugation to a carrier protein, has the advantage of inducing a T-dependent immune response, not observed with PPV23 vaccine. In our work, we therefore evaluated the impact of vaccine strategies using PCV13 and PPV23 on different populations of patients at risk of IPD. In a first study, our results on anti-pneumococcal vaccination in patients with smoldering myeloma (SMM) showed that a single dose of PCV13 induces a transient immune response and long term persistence. These results suggested the use of a vaccination schedule including several doses of PCV13 or association with the PPV23. Since 2013, this combined strategy of PCV13 and PPV23 is recommended by la Haute Autorité de Santé (HAS) for patients at risk, with the following delays: a dose of PCV13 followed by a dose of PPV23, 8 weeks later. We then studied this combined vaccine strategy in patients at risk of IPD: patients with systemic lupus erythematosus (SLE) and patients with rheumatoid arthritis (RA). Our results show a short-term immunogenicity of the combined strategy, but a protection that does not persist beyond two years. Surprisingly, antibody levels 2 years after vaccination are lower than pre-vaccine levels for RA patients. This negative effect of PPV23 on PCV13-induced immune response is called hyporesponsiveness. This phenomenon, observed in RA patients, is not found in SLE patients who received PPV23 vaccination at distance from PCV13. These results suggest that the delayed vaccination schedule (ie, PPV23 vaccination six months after PCV13 instead of two months) could inhibit the hyporesponsiveness phenomenon. In a third study, we compared different vaccine strategies modulating vaccine doses and injection times in healthy volunteers but also in a mouse model of hyporesponsiveness developed in our laboratory. Our hypothesis was that modulation of the vaccine schedule using both vaccines could both induce long-term protection and prevent hyporesponsiveness. Our results showed that decreased doses of PPV23 or concomitant injection of both vaccines did not prevent hyporesponsiveness. However, by increasing the delay between PCV13 and PPV23, the phenomenon of hyporesponsiveness is limited. Clinical studies in patients at risk of IPD are needed to evaluate a delayed combined strategy, where PPV23 would be received at least 6 to 12 months after PCV13

Thesis (Ph.D.)--Bioengineering program, Georgia Institute of Technology, 2008.
Committee Chair: Dr. Mark R. Prausnitz; Committee Co-Chair: Dr. Mark Feinberg; Committee Member: Dr. Mark Allen; Committee Member: Dr. Niren Murthy; Committee Member: Dr. Peter Hesketh; Committee Member: Dr. Robert Swerlick

BACKGROUND: The widespread use of vaccines led to significant decline in multiple potentially fatal infectious diseases. Recently, there has been an increase in vaccine hesitancy. Measles and pertussis outbreaks throughout the United States have put a spotlight on this urgent healthcare issue. Motivational interviewing is a counseling tactic that is gaining popularity and is being studied for its efficacy in preventative medicine and psychological disorders. It aims to inspire people to make behavioral changes through collaborative relationships with their provider by understanding how current actions do not translate into their health goals. LITERATURE REVIEW FINDINGS: Vaccine hesitancy is growing. Communities with decreased immunization rates are associated with a higher risk of disease outbreak. Increasing rates of undervaccinated children are likely due to increases in non-medical exemptions. Many parents, regardless of their vaccine hesitancy status, are concerned about vaccine safety. Vaccine hesitant parents refuse vaccines due to philosophical and religious beliefs, conspiracy theories, and safety concerns. Parents feel that providers do not adequately address their concern. Providers report not having the training to discredit parental concerns. The majority of parents describe their child’s pediatrician as their most trusted source of vaccine information. Parents who receive vaccine information from a provider are more likely to comply with the recommended childhood vaccine schedule. The most efficient way to discuss vaccines with parents has yet to be determined. PROPOSED PROJECT: This is a proposed QI research project for the Pediatric Clinic at Boston Medical Center. Providers would be trained in motivational interviewing during several sessions that included lectures and small group practice sessions with systematic feedback. During the intervention, parents who refuse vaccines for their child, aged 0-6 years old, will receive motivational interviewing from the provider. The proportion of the vaccine hesitant parents who accept the offered vaccine after will be analyzed. The pre and post intervention vaccination rates for the entire clinic will also be assessed. Data collection will be preformed through retrospective chart review. The project aims to increase provider confidence on vaccine counseling, educate providers on reasons for hesitancy, and improve compliance with the CDC recommended vaccine schedule. CONCLUSION: While most Americans continue to vaccinate their children according to the CDC’s recommended schedule, constant vigilance is required to maintain high immunization rates to protect our communities. Motivational interviewing is goal-oriented to alter a specific behavior and would allow providers to engage in an open, persuasive dialogue about parental vaccine concerns.

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
A vacinação tem um papel essencial na área da saúde pública e um enorme impacto na sociedade moderna. O desenvolvimento de formulações mais seguras tem levado a que a investigação de vacinas baseadas em microorganismos vivos inativados ou mortos seja preterida em prol da utilização de pequenas proteínas ou de outras moléculas contendo epítopos antigénicos, cuja imunogenicidade é reduzida. Contudo, o sucesso dessas vacinas depende da presença de adjuvantes imunológicos, por forma a aumentar a resposta imune gerada. Ao longo dos últimos anos diversas nanoparticulas (NPs) políméricas têm sido estudadas como sistemas adjuvantes de vacinas, funcionando como sistemas de entrega, protegendo os antigénios e aumentando o reconhecimento e internalização destes por parte de células especializadas do sistema imunológico. Face ao exposto, o objetivo do trabalho subjacente à presente dissertação consistiu na preparação e avaliação de NPs poliméricas de poli-caprolactona (PCL) e quitosano como adjuvantes de uma vacina para a hepatite B. As NPs preparadas através do método de nanoprecipitação foram pormenorizadamente caracterizadas e as suas capacidades como adjuvantes avaliadas através de ensaios in vitro e in vivo. Quando suspensas em água, as NPs apresentaram um tamanho aproximado de 200 nm e um potencial zeta positivo (+ 25 mV). Estas NPs, constituídas pela mistura física dos dois polímeros, revelaram citocompatibilidade com diversas linhas celulares e células primárias, boa interação e internalização celular. A presença de quitosano na superfície das NPs maioritariamente constituídas por PCL, permitiu obter partículas com uma elevada capacidade de carregamento de proteínas modelo e de plasmídeos através de adsorção à superfície. Usando células mononucleares isoladas a partir de sangue periférico humano, demonstrou-se que estas NPs não induzem a secreção do fator de necrose tumoral alfa (TNF-), uma citocina ligada a reações inflamatórias, que, por vezes, está também na base do mecanismo de ação de alguns adjuvantes. Por outro lado, descobriu-se que as NPs de PCL/quitosano são ativadoras de mastócitos. A ativação destas células leva à sua desgranulação, favorecendo a libertação de mediadores imunológicos que tem um papel activo na geração da resposta imune. Ambas as descobertas foram importantes contribuições sobre o mecanismo adjuvante das NPs de PCL/quitosano. Neste trabalho, estudos de vacinação de murganhos C57BL/6 permitiram validar, pela primeira vez, a aplicação das NPs de PCL/quitosano como um sistema adjuvante na vacinação contra a infeção pelo vírus da hepatite B. Inicialmente, testou-se uma vacina de ADN, na qual o plasmídeo codificado para a expressão do antigénio de superfície do vírus da hepatite B (HBsAg) foi adsorvido à superfície das NPs. No entanto, a resposta imune obtida com esta abordagem foi negligível. Por sua vez, asformulações desenvolvidas com a proteína recombinante HBsAg adsorvida à superfície das NPs levaram a uma forte resposta humoral quando administradas pela via subcutânea, superior à resposta gerada pela vacinação com a vacina comercial Engerix-B®, na mesma dose. A reposta gerada foi dependente da dose de NPs administrada, sendo que doses mais elevadas deste adjuvante nanoparticulado geraram um aumento estatísticamente significativo na produção de citocinas secretadas por linfócitos do tipo Th1 e Th17. Numa outra abordagem, foram ainda usados exosomas, adjuvantes imunológicos ainda pouco explorados para vacinas baseadas em proteínas recombinantes, mas cujas propriedades têm revelado vantagens em estratégias de vacinação contra tumores. Os resultados da vacinação subcutânea com exosomas como adjuvantes da formulação de NPs adsorvida com o HBsAg, demonstraram o aumento da resposta imune inata in vivo, ilustrada pelos níveis basais elevados de interferão gama (IFN-) secretado por esplenócitos. No entanto, a utilização desta formulação não se mostrou vantajosa na resposta imune específica. Por fim, realizaram-se estudos de vacinação pela via nasal encorajados pelos bons resultados da vacinação subcutânea e pela capacidade que as NPs demonstraram de adsorverem mucina, uma glicoproteína abundante no muco e de ficarem retidas sobre uma camada artificial de muco produzida in vitro. Estes estudos mostraram a possibilidade da utilização das NPs de PCL/quitosano para vacinação nasal contra a hepatite B, utilizando quantidades muito reduzidas de antigénio. Em suma, ao longo dos trabalhos realizados no âmbito da presente tese de doutoramento, foi possível caracterizar e testar com sucesso as NPs de PCL/quitosano como adjuvantes da vacinação, quer pelo efeito de transporte, quer pelo efeito imunoestimulante. Apesar da extrapolação dos resultados entre antigénios recombinantes apresentar limitações, as conclusões inferidas ao longo dos estudos são de grande importância para futuras aplicações deste sistema polimérico.
Vaccination is a major achievement in global public health, with a huge impact on modern society. The development of safer formulations has instigated the use of small proteins and other molecules containing antigenic epitopes instead of the inactivated or killed microorganisms. Those antigens have reduced immunogenicity and their efficacy is dependent on the presence of immunological adjuvants. Over the last years, different polymeric nanoparticles (NPs) have been tested as vaccine adjuvants acting as delivery systems, which protect and enhance the recognition of the antigen and its internalization by the specialized immune system cells. Given the aforementioned considerations, the studies underlying the present dissertation aimed at preparing and evaluating polymeric NPs, based on poly-caprolactone (PCL) and chitosan, as adjuvants for hepatitis B vaccine. PCL/chitosan NPs, prepared by a nanoprecipitation technique, were characterized and their capabilities as a vaccine adjuvant tested through in vitro and in vivo assays. When suspended in water, NPs measured approximately 200 nm and presented a positive zeta potential (+ 25 mV). These NPs, constituted by the physical blend of two polymers, demonstrated to be cytocompatible with various cell lines and primary cells and revealed a good cellular interaction and internalization. The presence of chitosan on the surface of the NPs mainly constituted by PCL, yielded particles with a high loading capacity for model proteins and DNA plasmids through surface adsorption. Using mononuclear cells isolated from human peripheral blood, it was possible to demonstrate that PCL/chitosan NPs did not induce TNF- secretion, a cytokine linked to inflammatory response and sometimes related to adjuvants mechanism of action. On the other hand, it was observed that PCL/chitosan NPs are mast cell activators. By inducing the activation, NPs contribute to mast cell degranulation, favoring the release of immune mediators with an active role in the generation of a protective immune response. These discoveries are important contributions to the knowledge of the PCL/chitosan NPs adjuvant mechanism. Vaccination studies were performed in C57BL/6 mice, allowing for the first time, the validation of PCL/chitosan NPs as an adjuvant for a vaccine against hepatitis B virus. One of the approaches considered was the development of a DNA vaccine, in which a plasmid encoding for the hepatitis B surface antigen (HBsAg) recombinant protein was adsorbed on the surface of the PCL/chitosan NPs. However, this approach conducted to a negligible immune response. On the other hand, a formulation developed with the recombinant HBsAg protein adsorbed on NPs surface led to a strong humoral immune response when administered subcutaneously, superior to the response generated by vaccination with the commercial vaccine Engerix-B®, at the same dose. It was also found that the response generated was dependent on the NPs dose since highest doses of the adjuvant caused a significant increase in the production of cytokines secreted by Th1 and Th17 lymphocytes. In another strategy, exosomes were used as co-adjuvants of the nanoparticulate formulation vaccine. In the literature, exosomes are described as effective immunological adjuvants for tumor vaccination strategies but they are not yet widely explored as vaccine adjuvants for recombinant antigens. The study developed in this thesis demonstrated an increase in mice innate immune response after subcutaneous vaccination with the formulation co-adjuvanted with exosomes, as illustrated by the high production of basal IFN-from splenocytes. However, this formulation showed no advantage in the specific immune response. Finally, vaccination studies through the nasal route were developed, encouraged by the good results of the subcutaneous immunization, together with the results demonstrating the ability of this NPs to adsorb mucin, an abundant glycoprotein in the mucus, and to be retained in an artificial mucus layer produced in vitro. The results showed the possibility of using PCL/chitosan NPs for nasal vaccination against hepatitis B using very small amounts of antigen. Overall, with the work herein developed and described, it was possible to characterize PCL/chitosan NPs and test their ability as a delivery system and immunostimulatory adjuvant for vaccination purposes. Despite the extrapolation of these results to other recombinant antigens presents some limitations, the conclusions drawn here are of great importance for future applications of PCL/chitosan NPs.
FCT - SFRH/BD/81350/2011

Many vaccines require an adjuvant to induce a strong immune response. Aluminum–containing adjuvants have been approved by the United States Food and Drug Administration for human use for many years. There are two main aluminum-containing adjuvants, aluminum hydroxide and aluminum phosphate. Due to their favorable safety profile, aluminum-containing adjuvants have been widely used in human vaccines for decades. Many currently licensed and commercially available vaccines contain aluminum-containing adjuvants. However, aluminum-containing vaccine adjuvants suffer from two major limiting factors: (1) aluminum-containing adjuvants can only weakly or moderately potentiate antigen-specific antibody responses and are generally considered incapable of inducing cellular immune responses; (2) vaccines that contain aluminum-containing adjuvants require cold-chain refrigeration for storage and distribution, and may not be frozen, because freezing of the vaccine in dispersion causes irreversible coagulation that damages vaccines (e.g., loss in potency and stability). In this dissertation, the first limitation was addressed by reducing the size of the aluminum hydroxide from micrometers (3-10 micrometer) to nanometers of less than 200 nm, and the second limitation mentioned above was addressed by freeze-drying vaccines that contain aluminum salts as adjuvants into a dry powder using thin-film freeze-drying. In addition, using an improved experimental design, the vaccine adjuvant activities of nanoparticles of around 200 nm was compared to that of the nanoparticles of around 700 nm. The smaller 200 nm nanoparticles showed a more potent adjuvant activity than the larger nanoparticles. When dispersed in an aqueous medium, both aluminum hydroxide and aluminum phosphate are physically 1–20 micrometer particulates. There are data showing that particulate vaccine carriers of around 200 nm (or less) may be optimal in potentiating the immunogenicity of vaccines. Based on this finding, aluminum hydroxide nanoparticles of 112 nm were synthesized, and its adjuvant activity was compared to that of the traditional aluminum hydroxide adjuvant, which have particulates of 3-20 micrometer. Using ovalbumin and Bacillus anthracis protective antigen protein as model antigens, it was found that protein antigens adsorbed on the aluminum hydroxide nanoparticles induced stronger antigen-specific antibody responses than the same protein antigens adsorbed on the traditional aluminum hydroxide microparticles of around 9.3 µm. Importantly, the inflammation reactions induced by aluminum hydroxide nanoparticles in the injection sites were milder than that induced by microparticles. Simply reducing the particle size of the traditional aluminum hydroxide adjuvant in suspension from micrometers into nanometers represents a novel and effective approach to improve its potency. The second limitation was addressed by converting vaccines that contain an aluminum salt as an adjuvant from an aqueous dispersion into a dried powder using thin-film freeze-drying. There is evidence that aluminum-containing vaccines can be lyophilized to dry powders using high speed freezing methods. Thin-film freezing is a high speed freezing method with a freezing rate between 100 to 10,000 K/s, but the feasibility of using thin-film freeze-drying to freeze-dry vaccines that contain aluminum salts as adjuvants has not been tested before. In this dissertation, Using ovalbumin as a model protein antigen and aluminum hydroxide or aluminum phosphate as an adjuvant, it was confirmed that vaccines that are adjuvanted with aluminum hydroxide or aluminum phosphate can be freeze-dried with as low as 2% (w/v) of trehalose as a cryoprotectant by thin-film freeze-drying without causing vaccine aggregation while preserving the immunogenicity of the vaccine. Finally, the feasibility of using the thin-film freeze-drying method to freeze-drying vaccines that contain aluminum salts as adjuvants was further confirmed by drying a commercial aluminum salt-adjuvanted tetanus toxoid vaccine. Vaccines that contain aluminum salts as adjuvants may be converted to a dry powder using the thin-film freeze-drying method to avoid loss of potency due exposure to freezing conditions during transport and storage.
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Dissertação de mestrado em Biophysics of Bionanosystems
The main goal of this work was to characterize and explore the potential of Dioctadecyldimethylammonium Chloride (DODAC) / Monoolein (MO) liposomes in a 1:2 proportion and identify the formulations that could be used in the development of an immunoprotective protocol for Chronic Myeloid Leukemia (CML). CML has long been recognized as one of the most responsive leukemic disorder to immunotherapy. CML is potent model for immune therapy in humans because there is a specific gene rearrangement, BCR/ABL, which product, P210bcr/abl, can be the target antigen. The loading of drugs into particles at the nanometer size range is a recognized technique for the optimization of controlled drug delivery. In its use in vaccines, liposomes have the advantage of being able to maintain antigens present in the organism for long enough to obtain an immune response. Different methods of preparation and distinct peptide/lipid molar ratios were used to prepare P210bcr/abl / DODAC:MO (1:2) nanoparticles. This thesis describes results for biophysical characterization of the peptide/lipid system, encapsulation efficiency and exposure of THP-1 cells to the nanoparticles. The lipid content was essential to achieve the desired nanoparticles. The highest lipid concentration showed higher encapsulation, however, a lower lipid content induced a more efficient cell response. The peptide/lipid system was capable of inducing a stronger cell response than the peptide by itself, emphasizing the potential of this system in vaccine development for the treatment of CML.
O objetivo principal deste trabalho foi caracterizar e explorar o potencial dos lipossomas de cloreto de Dioctadecildimetilamónio (DODAC) / Monooleina (MO) numa proporção de 1:2 e identificar as formulações que poderão ser usadas no desenvolvimento de um tratamento imunoprotetor para a Leucemia Mieloide Crónica (LMC). A LMC é desde há muito tempo conhecida como uma das desordens imunológicas mais responsivas à imunoterapia. A LMC é um poderoso modelo para imunoterapia em humanos devido à existência de um gene específico BCR/ABL, cujo produto, P210bcr/abl, pode ser usado como antigene-alvo. A incorporação de fármacos em partículas a uma escala nanométrica é uma técnica reconhecida para a optimização da entrega controlada de fármacos. No seu uso em vacinas, os lipossomas possuem a vantagem de ser capazes de manter os antigenes presentes no organismo o tempo suficiente para se obter uma resposta imune. Diferentes métodos de preparação e várias razões molares de péptido/lipido foram usadas para preparar nanoparticulas de P210bcr/abl / DODAC:MO(1:2). Esta tese descreve os resultados obtidos da caracterização biofísica do sistema péptido/lípido, eficiência de encapsulação e exposição das células THP-1 às nanopartículas. O conteúdo lipídico foi essencial para obter nanopartículas desejáveis. A concentração mais alta de lípido demonstrou maior eficiência de encapsulação, no entanto, uma concentração de lipído mais baixa mostrou-se mais eficiente em induzir uma resposta por parte das células. O sistema péptido/lipido foi capaz de induzir uma resposta mais forte do que o pétido por si só, enfatizando o seu potencial no desenvolvimento de uma vacina para o tratamento da LMC.

Mucosal delivery of recombinant adenovirus serotype 5 (rAd5)-based vaccine preparations are appealing for vaccine development in terms of lowering toxicity induced by high viral loads and substantial liver accumulation following systemic injection of the vaccine. However, this mode of delivery is currently under-developed due to the relatively low T-cell mediated immune responses generated against the encoded transgene. The first study described in this thesis demonstrated that sublingual immunization induces rapid migration of MHCII+, CD11C+ antigen presenting cells to the delivery site and elicit antigen-specific T and B cell-mediated immune responses in naïve mice and those with pre-existing immunity (PEI) to Ad5 at a level higher than that achieved after oral immunization. More importantly, this strategy improved protection of animals with PEI to Ad in contrast to poor protection after IM injection. The second study was designed to establish a method for inducing PEI that most accurately reflects natural infection in rodents and identifies the immunologic parameters elicited by rAd5-based Ebola vaccine necessary for protection against lethal infection. When immunization occurred by the same route in which PEI was induced, the antigen-specific multifunctional CD8+ T cell and antibody responses were significantly reduced. This correlated with poor survival after challenge with a lethal dose of Ebola Zaire in rodents. The data suggests that 1) establishment of PEI by the same route used for immunization is the most stringent test for a novel formulation designed to be effective in those with PEI to Ad5, and 2) for a formulation to be effective in those with PEI, it must be capable of restoring antigen-specific multifunctional CD8+ T cell and antibody responses, compromised by PEI. The third study screened novel formulations for their ability to improve in vitro transduction efficiency and immunogenicity and efficacy in vivo in the presence of anti-Ad5 neutralizing antibodies. Formulations consisting of pharmaceutically acceptable, non-immunogenic excipients that can prime the arms of immune response compromised by PEI improved survival after lethal challenge with Ebola Zaire challenge for rAd5-based Ebola vaccine in rodents with PEI. Taken together, these studies provide insight on how to reconstitute necessary immune responses in vaccine protocols by establishing a reliable PEI model in rodents, testing routes of administration, and formulations of the rAd5-based Ebola vaccine.
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This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane.
Graduation date: 2003