Dissertations / Theses on the topic 'Mucolas vaccination'

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB.
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

Here I examine the properties of a live Salmonella-based vaccine and a mucosal adjuvant based on a bacterial protein. Initially I examined the immunogenicity of the M. tuberculosis fusion antigen Ag85B-ESAT6 using a number of different mucosal vaccination strategies. These strategies included (i) intranasal immunisation with Ag85B-ESAT6 protein with and without Heat Labile toxin as an adjuvant (ii) oral immunisation with Salmonella enterica Typhimurium expressing Ag85B-ESAT6 from in vivo inducible or constitutive promoters (with and without intranasal boosts). Mice immunised with the various vaccine candidates were found to have significant anti-Ag85B-ESAT6 serum and mucosal antibody titres as well as strong T_H1 type cytokine responses, with IFN-γ levels particularly high. Intranasal boosting served to further enhance these immune responses. Following vaccination with the constitutive Salmonella vector, mice challenged with M. tuberculosis were found to have significantly reduced CFU in the liver when compared to non-vaccinated animals. Mice primed with Salmonella and then boosted intranasally with Ag85B-ESAT6/LTK63 led to a significant increase in protection, equivalent to that observed in mice vaccinated with BCG. In a separate study, flow cytometry and confocal microscopy were used to examine the frequencies and localisation of innate immune cells, their activation status, as well as the expression of cell adhesion molecules following intranasal immunisation. I found striking differences between the cell surface phenotype of leukocytes and their pattern of distribution in the tissues examined at all time points tested after immunisation. Following on from these results one particular cell type was examined in more depth to determine its role in adaptive immune responses.

Influenza viruses have sporadically caused pandemics during the last century, with the most severe occurring in 1918 when the “Spanish flu”, an A/H1N1 influenza virus, passed around the globe killing about 20-100 million people. Today 250 000-500 000 deaths occur annually due to influenza virus or secondary infection after influenza, e.g. pneumonia. Influenza viruses cause severe infections in susceptible age groups like children and elderly and in individuals with impaired immune response due to other medical conditions. The best way to prevent an influenza epidemic is by vaccination. Since the 1950´s we have vaccines against seasonal flu, but vaccine efficacy is not 100 % and there is a need to develop better and more effective vaccines, especially for the risk groups. Since the virus enters the host through the nasal cavity, nasal vaccination is a good approach. By stimulating a mucosal immune response already in the nasal cavity, the goal with nasal vaccination is to stop the virus before it enters the host. Nasal vaccination also reduces the risk of transmission of blood-borne diseases, and is less painful and easier to administer, compared to injectable vaccines. In order to be able to use less immunogenic antigens, like split and subunit antigens, as nasal vaccine components, an adjuvant is needed to enhance the immune response. At the moment there is no licensed mucosal adjuvant for human use. Several studies are ongoing, but it is a complicated and long way to reach the market. In this thesis nasal vaccination with influenza antigen together with the mucosal adjuvant Endocine™ and other mucosal adjuvants has been evaluated. The Endocine™ adjuvant has been shown to be safe and well tolerated in clinical trials. Depending on the pathogen of interest, different approaches are necessary. For HIV, DNA-vaccination has been evaluated together with a plasmid encoding Salmonella typhimurium flagellin C and the mucosal adjuvant N3. The results found in paper I-IV show that by adding adjuvant to the antigen enhances the protective immune response towards the antigen. Enhanced systemic, mucosal and cell-mediated immunity were observed. Hopefully in the future these adjuvants evaluated in this thesis, will be used in vaccines for humans.

Malgré 30 ans de recherche aucun vaccin VIH n'a permis d'apporter une protection efficace et de manière stable. Au regard des échecs obtenus jusqu'à présent, de nouvelles formes vaccinales ont été développées. Parmi ces dernières les VLP présentent l'avantage notables d'être très immunogènes du fait de leur forme particulaire mimant les virus natifs, et sécuritaire puisque ne véhiculant pas de génome viral. Ce travail de thèse a pour objectif d’établir et d’évaluer une stratégie vaccinale utilisant ces VLP administrées par voies muqueuses dans le but d’initier une réponse humorale et cellulaire au niveau systémique et muqueux. Dans cette étude, nous avons montré que la voie muqueuse est indispensable pour l’induction d’une réponse locale forte. De plus, nos résultats révèlent que la forme particulaire de l’antigène est décisive dans la génération d’une immunité de qualité, générant une réponse TFH forte, une réponse cellulaire locale polyfonctionnelle ainsi qu’une réponse humorale forte et stable dans le temps, caractérisée par des anticorps de qualité.Cherchant à mieux caractériser la stratégie vaccinale établie, nous avons analysé les mécanismes de prise en charge des VLP et d’initiation de la réponse immunitaire après administration IN. Nous avons observé que de nombreuses cellules de l’immunité innée pulmonaire, notamment les macrophages alvéolaires et les neutrophiles, captaient massivement les VLP limitant alors la réponse TFH et potentiellement la réponse humorale qui en découle. Au final, ce travail de thèse aura permis de mettre en avant la voie d’immunisation muqueuse ainsi que la forme particulaire de l’antigène dans la mise en place d’un vaccin VIH
Currently no HIV vaccine elicit full and stable protection against viral acquisition. In view of the failures until now, new vaccines strategies were developed. Among these, VLP have the advantage to be highly immunogenic because of their particulate structure mimicking native pathogens and safe because of the lack of viral genome.This thesis work aims to develop and evaluate a VLP-based vaccine strategy by mucosal administration in order to initiate systemic and local humoral and cellular responses. In this study, we showed that the mucosal administration is mandatory to generate a strong local immunity. Moreover, our results show that particular form of the antigen is crucial in the generation of the quality of the immune response, generating strong TFH response, polyfunctional T-cell responses in the mucosa and a strong and stable humoral response characterized by high-quality antibodies.We also investigated mechanisms involved in the generation of immune responses following IN VLP injections. We determined which cells take in charge VLP and their role in the followed immune responses. Our preliminary results show many innate immune cells in the lungs, such as alveolar macrophages and neutrophils, have an important role in the particles uptake, limiting TFH response and potentially the followed humoral response.Finally, this thesis work will show the determining role of the mucosal route of immunization and the particulate form antigen for the development of an HIV vaccine

The objective of this investigation was to evaluate the effects of administering CpG-ODN to commercial strain chickens as a potential adjuvant to vaccination against Salmonella, Eimeria spp., and Newcastle disease virus, or immunization to bovine serum albumin (BSA). During Experiment 1, which evaluated the dual application of CpG-ODN and a Newcastle disease virus vaccine, in the first of three replicate trials, on day 28 of the experiment, animals in the Vaccine + CpG 1& 14 experimental group were observed to have the highest levels of (p<0.05) anti-NDV IgG in serum. These levels were elevated above levels in animals from all other experimental groups. This suggestion for an adjuvant effect associated with CpG-ODN administration was not supported in the remaining two trials of experiment 1. Experiment 2 evaluated the potential for CpG-ODN to adjuvant a commercial live oocyst coccidial vaccine when applied by an oral route to neonatal broiler chickens. Overall, when body weight gain during challenge, development of intestinal lesions, and anti-Eimeria IgG levels were evaluated, vaccine administration alone was demonstrated to provide the best measure of protection among animals in all experimental groups, including those receiving either CpG-ODN or Non CpG-ODN. Experiment 3 investigated the simultaneous administration of CpG-ODN or Non-CpG ODN and a commercially acquired Salmonella typhimurium vaccine to SCWL chickens. Similar to experiments 1 and 2, antigen specific IgG responses in serum and indices of protection against field strain Salmonella challenge were variable and inconsistent. Anti-BSA IgG levels were compared in broiler and SCWL chickens immunized against BSA by a drinking water route of administration alone, or in combination with two different concentrations of CpG-ODN or Non CpG-ODN in experiment 4. The only observation where CpG-ODN and BSA co-administration resulted in anti-BSA IgG levels that were elevated above BSA alone immunized chickens was measured in broilers at day 19 post-final immunization. Taken together, given the variable results reported in this investigation related to the co-administration of ODN and vaccine or protein antigen, these data are largely inconclusive for suggesting that CpG-ODN can effectively adjuvant humoral immune responses in commercial strain chickens.

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

L’avènement des multi-thérapies antirétrovirales a permis une réduction importante de la mortalité associée au VIH en induisant notamment la chute de la charge virale à moins de 50 copies/mL et une récupération progressive du nombre de lymphocytes T CD4+ (LT-CD4). Cependant, certains patients définis comme mauvais répondeurs immunologiques (MRI) ne parviennent pas à récupérer un taux de CD4 généralement considéré comme « protecteur » (>500cellules/µL). L’interleukine-7 (IL-7), cytokine essentielle à la thymopoïèse et à l’homéostasie lymphocytaire T, a été utilisée en étude clinique afin de restaurer et maintenir le taux de LT-CD4 chez les patients MRI. La première partie de mon travail de thèse visait à évaluer l’impact d’une telle thérapie sur le réservoir viral circulant. Dans l’essai clinique sur lequel nous avons travaillé (INSPIRE 3, Cytheris), des cycles d’administration d’IL-7 ont induit une augmentation significative du nombre de LT-CD4 et CD8 circulants, avec une expansion majoritaire des populations naïves et centrales mémoires. Nous avons montré qu’un cycle d’injections d’IL-7 induisait une augmentation significative de la quantité de cellules infectées circulantes 28 jours et 3 mois post-injection. Cependant, malgré l’accroissement de la fréquence de LT-CD4 infectés 28 jours post-injection, nous avons observé une diminution significative de la charge virale ADN par million de LT-CD4 chez la majorité des patients 3 mois après l’initiation de la thérapie, suggérant une élimination partielle de cellules infectées. Suite au second cycle d’injections, nous n’avons pas observé d’évolution de la quantité de cellules infectées circulantes ni de la fréquence de LT-CD4 infectés, suggérant un impact différent des 2 cycles d’injections sur la dynamique du réservoir viral périphérique. Enfin, certains patients ayant développé des anticorps neutralisants anti-IL-7 (Nab) suite au second cycle d’injections d’IL-7, nous avons cherché à identifier des facteurs prédictifs de l’apparition de ces anticorps ainsi que leurs conséquences physiologiques in vivo. Le seul paramètre caractérisant ces patients est l’amplitude de la reconstitution T-CD4 au cours du premier cycle d’injections d’IL-7. Il semble donc qu’une meilleure réponse à l’IL-7 ait pour conséquence de faciliter le développement de la réponse immune contre cette cytokine. Cependant, ces anticorps ne sont détectables que de façon transitoire chez les patients. De plus, nous avons observé une diminution significative, mais transitoire, de la prolifération des thymocytes chez les patients présentant des Nab, démontrant un impact fonctionnel de ces anticorps sur l’activité biologique de l’IL-7 endogène. L’injection systémique d’IL-7 induit la migration des cellules circulantes vers différents compartiments tissulaires lymphoïdes et non lymphoïdes. Dans une seconde partie de mon travail de thèse, j’ai étudié le pouvoir adjuvant de cette cytokine administrée localement par pulvérisation à la surface de la muqueuse vaginale. Dans le modèle macaque rhésus, nous avons mis en évidence une augmentation de la production d’un large spectre de chimiokines dans le chorion et l’épithélium vaginal des animaux 48 heures après l’administration vaginale d’IL-7. Cette surexpression de chimiokines s’accompagne d’une migration massive de LT-CD4, CD8, macrophages, cellules dendritiques et cellules NK dans cette muqueuse, suggérant l’augmentation de la vigilance immunologique. L’effet adjuvant de cette cytokine a été confirmé par l’analyse de la réponse humorale muqueuse de macaques vaccinés par pulvérisation vaginale d’antigènes 48h après l’administration du spray d’IL-7. Dans les lavages cervicovaginaux (CVL) des animaux traités à l’IL-7, nous avons mis en évidence des réponses spécifiques de type IgA et IgG plus rapides, plus fortes et plus durables que chez les animaux contrôles, démontrant la capacité de l’IL-7 à préparer la muqueuse vaginale à répondre à une stimulation antigénique locale
Highly Active Antiretroviral Therapy (HAART) has led to significant reduction of HIV-associated mortality by maintaining an undetectable viral load and inducing progressive CD4-T cell restoration. However, some patients, defined as poor immunological responders (PIR), fail to restore their CD4 counts to 500cells/µL during treatment, a threshold considered as the protective against AIDS related or non AIDS related malignancies, opportunistic infections and cardiovascular events. Interleukin-7 (IL-7), an essential cytokine for thymopoïesis and T cell homeostasis has been used in clinical trials aimed at restoring and maintaining CD4 counts in PIR patients. The first part of my thesis project aimed at assessing the impact of IL-7 therapy on circulating HIV reservoir. In the clinical study we worked on (INSPIRE 3, Cytheris), cycles of IL-7 injections led to a significant increase of the number of circulating CD4 and CD8 T-cells, with a predominance of naïve and central memory T cell expansion. We have shown that one cycle of IL-7 injections induced a significant increase in the number of circulating infected cells 28 days and 3 months post-injections. However, despite a significant increase in the frequency of infected CD4 T-cells 28 days post-injections, we observed a significant decrease of HIV-DNA load in CD4 T-cells in the majority of patients 3 months after the therapy initiation. These data suggest a partial elimination of HIV infected cells. After the second cycle of IL-7 injections, we did not observed any change in the number or frequency of circulating infected cells, suggesting a differential impact of the two IL-7 injection cycles on the dynamics of circulating HIV-reservoir. Finally, considering that some patients developed anti-IL-7 neutralizing antibodies (Nab) after the second cycle of IL-7 injections, we looked for predictive factors of this immunogenicity and analyzed its physiological consequences in vivo. The only parameter that distinguished Nab and non-Nab patients was the extent of CD4 T-cell reconstitution during the first cycle of therapy. This suggests that a better response to IL-7 also facilitates the development of auto-antibodies to the cytokine. However, these antibodies were only transiently detectable after the second cycle of therapy. Moreover, the appearance of Nab was associated with a significant but transient decrease of thymocyte proliferation, suggesting a functional impact of these antibodies on the endogenous IL-7 function. Systemic injection of IL-7 induces circulating T cells homing from the blood into lymphoid and non-lymphoid tissues. In the second part of my thesis project, I evaluated whether this cytokine could be used as an adjuvant when sprayed on the vaginal mucosa. Ten micrograms of IL-7 directly sprayed in the vaginal tract of rhesus monkeys induced, 48h after administration, the production of a large pattern of chemokines in the vaginal chorion and epithelium. This chemokine expression was accompanied by massive homing of CD4 and CD8-T cells, macrophages, dendritic cells and NK cells in the vaginal mucosa, suggesting an increased immunological vigilance. Finally, the adjuvant potential of this cytokine was confirmed by analyzing local humoral immune response after vaginal administration of antigens 48h following IL-7 spray. In cervicovaginal washes (CVL) of treated animals, we observed a faster, stronger and longer-lasting specific IgA and IgG response than in control animals, highlighting the capacity of IL-7 to prepare the vaginal mucosa response to local antigen stimulation

Le virus de la grippe infecte les muqueuses du tractus respiratoire. Un vaccin intranasal induit une réponse immunitaire proche de celle faisant suite à une infection naturelle en bloquant le virus directement sur le site de l'infection et permet une vaccination sans aiguille. Par ailleurs, les vaccins à base de pseudo-particules virales ou Virus-like Particles (VLP) produites sur cellules représentent une alternative intéressante au vaccin classique produit sur oeufs. Les VLP sont des particules non-réplicatives qui ressemblent au virus et qui peuvent être immunogènes même sans adjuvant, en particulier par voie intranasale. Au cours de ma thèse, une plateforme de production de VLP grippales composées d'hémagglutinine, de neuraminidase et de protéine de matrice M1 a été développée par transfection transitoire des cellules de mammifères. Des immunisations de souris BALB/c ont montré que les VLP de type A et B, purifiées et caractérisées, étaient immunogènes à de faibles doses par voie intramusculaire. L'administration par voie intranasale de VLP avec la sous-unité B de la toxine cholérique, comme adjuvant muqueux, a permis d'obtenir des taux d'anticorps sériques comparables à ceux obtenus par immunisation en intramusculaire mais également une forte réponse IgA au niveau des muqueuses. Par ailleurs, le rendement des VLP s'est révélé souche-dépendant et lié aux protéines HA et NA à la surface de la particule. Pour contourner ce problème, un vaccin quadrivalent composé de deux VLP bivalentes exprimant chacune deux HA et NA différentes à la surface a été produit montrant ainsi la flexibilité de cette plateforme
The influenza virus infects the mucous membranes of the respiratory tract. An intranasal vaccine induces an immune response close to the one induced by the natural infection by blocking the virus directly at the site of infection and allows needle-free vaccination. In addition, vaccines based on Virus-like Particles (VLP) produced in cells represent an interesting alternative to the traditional egg-based vaccine. VLPs are non-replicative particles that mimic the virus. Studies on influenza VLPs have shown protection by the intranasal route without adding an adjuvant. During my thesis, a platform for the production of influenza VLPs composed of the hemagglutinin, the neuraminidase and the M1 matrix proteins was developed by transient transfection of mammalian cells. Immunizations of BALB/c mice showed that the purified and characterized type A and B VLPs were immunogenic at low doses by the intramuscular route. The intranasal administration of VLPs with the B subunit of cholera toxin as a mucosal adjuvant resulted in serum antibody levels comparable to those obtained by intramuscular immunization but also a strong IgA response in the mucosal secretions. In addition, VLP yield was found to be strain-dependent and linked to the HA and NA proteins on the surface of the particle. To overcome this problem, a quadrivalent vaccine based on two bivalent VLPs each expressing two different HAs and NAs at the surface was produced, demonstrating the flexibility of this platform

Master of Science
Department of Clinical Science
Elizabeth Davis
Background: While there is a commercially-available vaccine for Streptococcus equi subsp. equi licensed for the intranasal route of administration, some equine practitioners are administering this vaccine orally despite a lack of evidence for its efficacy by this route of administration. Objectives: To compare systemic and local immune responses following intranasal or oral administration of the USDA-approved, live-attenuated Streptococcus equi subspecies equi vaccine (Pinnacle IN®, Zoetis, Florham Park, New Jersey). Study Design: Experimental, randomized clinical trial Methods: Eight healthy horses with low Streptococcus equi M protein (SeM) titers (<1:1600) were randomly assigned to an intranasal or oral two-vaccine series. SeM-specific serum immunoglobulins G (IgG) and A (IgA) and nasal secretion IgA were assessed using a commercially-available ELISA (Equine Diagnostic Solutions, LLC, Lexington, Kentucky) and a novel magnetic microsphere assay utilizing fluorescence. A general linear mixed models approach was used for statistical data analysis. Results: As expected, intranasal vaccinates showed substantial increases in both serum SeM-specific IgG and IgA levels post-vaccination (P=0.0006 and P=0.007, respectively). Oral vaccinates showed an increase in serum SeM-specific IgG post-vaccination (P=0.0150), though only one-third the magnitude of intranasal vaccinates. Oral vaccinates showed no evidence of change in SeM-specific IgA post-vaccination (P=0.15). Main Limitations: Changes in mucosal antibody responses were not identified in this study which may be related to small change in antibody response, timing of sample collection, or method of nasal secretion collection. Conclusions: Results indicate that intranasal or oral vaccine administration resulted in increased serum SeM-specific IgG, though the magnitude of response differed between routes.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

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

Les entérobactéries Salmonella sont divisées en plusieurs sérovars dont les quatre principaux Typhimurium, Enteritidis, Typhi et Paratyphi sont responsables soit de gastroentérites soit de fièvres typhoïdes, à raison de plus de 90 millions de cas et 400 000 décès par an. L’apparition de souches multi-résistantes nécessite la mise en place d’une vaccination prophylactique muqueuse. L’environnement intestinal est caractérisé par une balance entre tolérance immunitaire et réaction inflammatoire régulée par les immunoglobulines (Ig) A sécrétoires. Les IgA des sécrétions muqueuses sont dimériques, les IgA sériques sont monomérique et deux isotypes ont été décrits chez l’Homme: IgA1 et IgA2. Nous avons tout d’abord exploré les fonctions des différents isotypes et isoformes des IgA humaines. Nous avons pu noter un rôle anti-inflammatoire des IgA1 à l’inverse d’un rôle pro-inflammatoire des IgA2 et nous avons souligné un processus de régulation de l’expression des récepteurs aux IgA par les IgA elles-mêmes ainsi qu’un axe IgA/lymphocytes T CD8 cytotoxiques. Nous avons ensuite mis en place un vaccin multivalent composé des antigènes SseB et OmpC de Salmonella liés à des Ig sécrétoires. Cette étude a mis en évidence une solide réponse immunitaire humorale et cellulaire spécifique aux antigènes couplés à des IgA ou IgM après vaccination intra-nasale au niveau systémique et muqueux. Par ailleurs, de plus fortes réponses humorales et systémiques spécifiques ont été observées en couplant à la fois OmpC et SseB sur l’IgA. Ce travail de thèse ouvre de nouvelles perspectives pour la mise en place de vaccins muqueux multivalents et pourrait apporter des réponses quant au rôle des IgA
The enterobacteria Salmonella species are divided into several serovars such as Typhimurium, Enteritidis, Typhi and Paratyphi which are the major causative agents of either gastroenteritis or typhoid fever. They are responsible for more than 90 million cases and 400 000 deaths each year. The increase in multi-drug resistant strains requires the implementation of prophylactic mucosal vaccines. Besides, the intestinal environment is characterized by a balance between immune tolerance and inflammatory response tightly regulated by secretory immunoglobulins (Ig) A. Mucosal IgA are mainly dimeric, serum IgA monomeric and two IgA isotypes have been described in humans: IgA1 and IgA2. We firstly explored the functions of the different isotypes and isoforms of human IgA. We pointed out a pro-inflammatory role of IgA2 whereas IgA1 rather oriented the immunity towards an anti-inflammatory response. We have also highlighted both the regulation of IgA receptors expression by IgA and an IgA/CD8 cytotoxic T cells axis. We also designed a multivalent vaccine against Salmonella by coupling two antigens – SseB and OmpC – to secretory Ig. We pointed out solid specific humoral and cellular responses against both these antigens coupled to either IgA or IgM after intra-nasal immunization in mucosal but also systemic compartments. We have also demonstrated the possibility to preserve and increase the antigen immunogenicity with a multivalent vaccine. This thesis thus paves the way for new secretory Ig-vectorized mucosal vaccines. In addition, the immune response could be modulated through the chosen isotype or isoform and the differences in immune activation generated by structural changes in IgA could shed some light on their role in mucosal homeostasis

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

Vaccination is the most effective way to control a global disease such as tuberculosis (TB); however, conventional intradermal (ID) injection of the licensed TB vaccine, BCG, provides only limited protection. Mucosal delivery of vaccines to pulmonary surfaces is a loqical approach to generate a protective immune response at the primary site of infection. Non-human primate (NHP) models have been used to compare the safety and immunogenicity of mucosal and ID TB vaccination strategies using BCG and modified vaccinia virus Ankara expressing TB antigen 85A (MVA85A). Mucosal vaccination strategies were well tolerated and induced potent antigen-specific CD4 and CD8 T-cell responses, including polyfunctional T-cells in the lung and peripheral immune compartment. Systemic immune responses induced by mucosal BCG revaccination and MVA85A boost vaccination were relatively transient, whereas antigen-specific responses detected in mucosal tissues and bronchoalveolar lavage (BAL) samples were more persistent. Unlike ID vaccination, aerosol vaccination with MVA85A did not induce a detectable serum anti-vector antibody response. Polyfunctional CD4 T-cells were detected in BAL and peripheral blood mononuclear cells (PBMC) following aerosol and ID BCG vaccination. The immunogenicity of aerosol-delivered BCG was dose dependent and consisted of both Th 1 and Th 17 cytokine responses. The aerosol BCG-induced T -cell response measured in BAL and PBMC was significantly delayed relative to ID-vaccination, with a similar trend apparent in serum anti-PPD IgG levels. Mucosal CD4 and CD8 populations were polarised toward an effector memory phenotype, whereas frequencies of peripheral central memory T-cells increased significantly, and remained elevated, following aerosol vaccination. Expression of the 04131 integrin lung homing markers remained consistently high on C04 and C08 T-cells isolated from BAL, and varied on peripheral T -cells. These studies indicate, that delivery of TB vaccines to the pulmonary surfaces is practical and safe, with the potential to target the cell-mediated immune response to the lung. The comparison of aerosol and ID BCG vaccination highlighted differences in the mycobacteria-specific immune response that may contribute to the enhanced protection previously reported from aerosol BCG studies, and will inform future investigations of mucosal TB vaccination strategies.

The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.

Les SIgA possèdent la capacité de pouvoir adhérer spécifiquement à la membrane apicale des cellules M présentes au niveau des muqueuses monostratifiées. La capacité des cellules M à transporter les SIgA de la lumière intestinale jusqu'au GALT par un mécanisme de transcytose inverse a également été décrite. J'ai donc souhaité évaluer la capacité des SIgA à transporter efficacement un antigène vaccinal, à travers la barrière épithéliale par l'intermédiaire des cellules M vers les DCs présentes dans le MALT. Le mécanisme exact et notamment la structure moléculaire du récepteur permettant la transcytose inverse des IgA n'a pas été identifiée. Il m’a donc paru intéressant d'améliorer la compréhension des mécanismes physiologiques impliqués dans le transport d'une SIgA de la lumière intestinale jusqu’aux cellules immunitaires sous-muqueuses. Cette étude a permis de démontrer le rôle majeur de deux nouveaux récepteurs présents à la surface des cellules M, la dectine-1 et le siglec-5, dans l'activité physiologique rétrograde des SIgA. Cette étude a permis d'identifier les domaines des SIgA impliqués dans ce mécanisme. J'ai ensuite utilisé les SIgA comme vecteur vaccinal permettant le ciblage des cellules M. Les applications de cette approche à la vaccination par voie orale et nasale sont décrites dans la publication 4 en cours de rédaction. Durant ma thèse, j'ai pu démontrer que la transcytose inverse des SIgA est un mécanisme physiologique dépendant par exemple de récepteurs aux sucres. J'ai pu également démontrer que leur utilisation dans des approches de vaccination muqueuse peuvent être une voie très prometteuse notamment contre le VIH ou d'autres pathogènes muqueux
Secretory IgA (SIgA) are the main effectors of the mucosal immune response. More, SIgA have the capacity to adhere to the apical membrane of M cells present in the intestinal and nasal mucosa. After binding to M cells, SIgA are transported from the intestinal lumen to the GALT by a reverse transcytosis mechanism. In this work, I have assessed the capacity of SIgA to effectively deliver a vaccine antigen through the epithelial barrier via M cells to sub-mucosal dendritic cells (DCs). Precise mechanisms and the IgA-specific receptor(s) for reverse transcytosis have not yet been identified. In this work, I identified the receptors involved in SIgA reverse transcytosis. Both dectin-1 and siglec-5 allow the transport of the Cα1 domain of SIgA by murine an human M cells in vitro and also in vivo. This work is currently undergoing to immunity (publication 1) and should also be patented. Next, I tried to use the reverse transcytosis mechanism mediated by M cells to efficiently deliver an HIV-1 antigen by mucosal routes. We applied results obtained using SIgA as a vaccine vector for M cells targeting. This approach should help to protect antigen in the mucosal environment. Applications of this approach to oral and nasal immunisation are described in the incomplete publication 4. During any PhD, I was able to demonstrate that SIgA reverse transcytosis is a physiological mechanism depending on sugar receptors. I was also able to demonstrate that their use could be a very promising vaccine approach especially for mucosa] diseases or pathogens as HIV

L’administration de vaccins par voie muqueuse (orale et nasale) est une alternative efficace aux injections classiques. En effet, au-delà d’une plus grande compliance pour les patients et les soignants, ces voies ont l’avantage de déclencher une immunité dite muqueuse, grâce à la présence d’un système immunitaire propre (aussi appelé MALT pour Mucosal Associated Lymphoid Tissue). Ce MALT est situé à la surface des épithélia de revêtement. Il est différencié en îlots distinguables, les Follicular Associated Epithelium (FAE), et est constitué de cellules épithéliales spécialisées dans la surveillance et le prélèvement de pathogènes, les cellules M. Celles-ci surplombent une zone riche en cellules présentatrices d’antigènes (CPA) et lymphocytes, les Interfollicular Regions (IFR). Ainsi lors d’une infection, les cellules M sont capables de prélever et transloquer des fragments antigéniques vers les CPA, qui initieront la réponse immunitaire auprès des lymphocytes. Cette réponse se traduira par une immunité humorale et cellulaire au niveau de l’épithélium infecté, au niveau des muqueuses plus distantes, mais aussi au niveau systémique. Comme la majorité des infections se produisent au niveau des muqueuses, cette stratégie d’immunisation est de plus en plus étudiée.Si les antigènes sous-unitaires sont moins toxiques que les vaccins vivants, ils sont aussi moins immunogènes, et leur administration nécessite la présence d’un adjuvant pour stimuler efficacement les CPA et initier une réponse immunitaire forte. Or, par la voie des muqueuses, les molécules immunomostimulantes classiquement utilisées pour les voies injectables (par exemple les toxines bactériennes, le LPS ou encore certaines émulsions) sont souvent reportées comme étant toxiques. L’utilisation de systèmes de délivrance pour vectoriser les antigènes jusque dans les CPA semble donc être une alternative séduisante.On distingue deux types de vecteurs d’antigènes : les particules immunomodulatrices, et les systèmes de délivrance purs. Les premiers délivrent les antigènes dans les CPA, et en parallèle, stimulent fortement les voies pro-inflammatoires, pour orienter la balance immunitaire Th1/Th2. Parmi ces vecteurs, les plus utilisés en tests cliniques sont les virus-like particles (VLP), les émulsions à base de saponines (ISCOMs) ou les liposomes contenant des éléments bactériens (MPL, CpG…). En revanche, leur utilisation par voie muqueuse, et notamment nasale, est confrontée aux mêmes risques de toxicité que les adjuvants classiques. Les systèmes de délivrance purs, quant à eux, n’améliorent l’immunogénicité des antigènes qu’en les délivrant dans les cellules, mimant ainsi une infection naturelle. Et bien qu’ils soient mieux tolérés par les muqueuses, leur efficacité doit s’orienter vers une pénétration du mucus, ainsi qu’une association, protection et délivrance des antigènes dans les CPA bien plus performantes.Durant cette thèse, nous avons ainsi étudié les mécanismes permettant à des nanoparticules (NPs) de maltodextrine cationiques et lipidées (NPL) de délivrer des antigènes par voie nasale.Nous avons tout d’abord évalué la capacité des NPL à franchir le mucus des voies respiratoires, en comparaison avec des nanoparticules mucopénétrantes (des PLGA recouvertes de PEG, ou PLGA-PEG) et des nanoparticules mucoadhérentes (des PLGA recouvertes de chitosan, ou PLGA-CS). En mesurant le déplacement des différentes NPs dans du mucus respiratoire reconstitué, nous avons observé que, grâce à la présence du coeur de phospholipides anioniques, la NPL était capable de se déplacer dans le mucus, contrairement aux PLGA-CS qui restaient immobiles [...]
The mucosal routes of immunization present several advantages compared to classical injection routes. Indeed, besides a better compliance towards patients, these routes possess their own immune system, also known as the Mucosal Associated Lymphoid Tissue (MALT), able to trigger a local mucosal response after immunization. This tissue is mainly located in the nasal and intestinal mucosa, where it is spread in small extents called Follicular Associated Epithelium (FAE). On their apical surface, the FAE contain specialized epithelial microfold cells (or M cells), whose role is to survey potential infections by sampling pathogenic fragments, and which overlay a lymphocyte and antigen presenting cells (APC) zone. Then, when an infection occurs, M cells sample and translocate antigenic fragments to CPA, which could therefore trigger lymphocyte maturation and the initiation of the subsequent immune response. This activation will lead to both humoral and cellular immunity in the infected epithelium and could also spread to distant mucosa. As many pathogens infect the body through mucosa, this way of immunization is often considered.Adjuvants are frequently added to subunit vaccines to enhance their immunogenicity toward APC. Indeed, despite their lower toxicity, they are also less immunogenic than live-attenuated vaccines. However, the administration of classical adjuvanting molecules, such as toxins or immunostimulating emulsions, via mucosal routes, has often led to serious adverse effects. Therefore, the alternative use of delivery systems to deliver antigen in APC after mucosal administration is more and more studied.Antigen delivery systems include immunomodulating particles, and inert delivery systems. The first ones can enhance the mucosal antigen bioavailability by vectorizing antigens to APC, and at the same time trigger intracellular pro-inflammatory pathways, to drive the Th1/Th2 immune balance. Among them, virus-like particles (VLP), saponin-based emulsions (ISCOMs) or MPL-containing liposomes are the most represented in clinical trials. However, their mucosal administration can lead to the same adverse effects than classical immunostimulating molecules. In parallel, true delivery systems can enhance the antigens immunogenicity by increasing their intracellular delivery, thus mimicking a natural infection. They are therefore far less toxic for the mucosa than immunomodulating particles but need to be more efficient in the mucus penetration, in the antigen association and in the APC intracellular delivery.During this thesis, we deciphered the mechanisms allowing cationic and lipidated maltodextrine nanoparticles (NPL) to deliver antigens after nasal administration.We first evaluated the ability of NPL to cross the airway mucus barrier, compared to mucopenetrant particles (PEG-coated PLGA or PLGA-PEG) and mucoadherent particles (chitosan-coated PLGA or PLGA-CS), by measuring their displacement in reconstituted mucus. We observed that in presence of the phospholipid core, the NPL were able to move in the mucus, while PLGA-CS NPs remained stuck in the gel. Moreover, we observed that the NPL uptake and the protein delivery in airway epithelial cells were not impaired by the presence of mucins, contrary to PLGA-CS that were hindered by the mucins, and to PLGA-PEG which were not taken up by the cells, due to their neutral surface charge. We finally demonstrated that the NPL mucopenetration was allowed thanks to steric and repulsive electrostatic forces between the anionic phospholipid core and the mucins.In parallel, we studied the mechanisms allowing the NPL to enhance the immunogenicity of subunit antigens after nasal administration, with a highlight on the importance of the NP’s density [...]

La vaccination muqueuse est un moyen efficace de lutter contre les pathogènes qui utilisent les muqueuses comme porte d'entrée. Cependant, la vaccination muqueuse avec des antigènes non réplicatifs nécessite l'utilisation d'adjuvants. Les molécules de la famille de la toxine du choléra, l'entérotoxine thermolabile d'E.coli (LT), la toxine du choléra (CT) ainsi que le mutant LT-R192G et les sous-unités B non toxiques de ces toxines (LTB et CTB) ont été montrées augmenter les réponses immunitaires contre des antigènes coadministrés par voie muqueuse. Cependant leur mécanisme d'action est complexe et reste encore mal connu et des différences entre molécules entières et sous-unités B ont été rapportées ainsi que, pour une même molécule, des différences selon le modèle utilisé. Dans ce travail, nous avons étudié les effets de ces cinq molécules sur les réponses anticorps ainsi que sur les lymphocytes T CD4 dans un modèle murin d'immunisation intrarectale avec des pseudoparticules virales de rotavirus (VLP-2/6). Chez les souris non immunisées, nous avons montré que ces molécules, à l'exception de la CTB, diminuent in vitro les lymphocytes T régulateurs naturels CD4+CD25+Foxp3+, probablement par un mécanisme d'apoptose. Chez les souris immunisées, toutes les molécules étudiées induisent une même réponse anticorps sérique et fécale spécifique des VLP-2/6, qu'il s'agisse des molécules entières connues pour leur fort pouvoir adjuvant ou des sous-unités B qui, elles, ont été rapportées avoir un plus faible effet adjuvant voire un effet tolérogène dans certaines études. Concernant la réponse T CD4, les réponses spécifiques de l'antigène et de l'adjuvant ont été analysées. Des différences importantes ont été mises en évidence entre ces molécules. Notamment, seules les molécules entières (LT, LT-R192G et CT) induisent la production d'IL-2 et l'activation de lymphocytes T CD4+CD25+Foxp3- mémoires spécifiques de l'antigène tout en permettant la mise en place d'une régulation médiée par des lymphocytes T régulateurs CD4+CD25+Foxp3+ (boucle d'autorégulation), qui pourraient jouer un rôle majeur lors d'une réponse secondaire, afin d'éviter les réactions inflammatoires délétères. Malgré ces différences, toutes les molécules étudiées induisent la production d'IL-17, suggérant le rôle majeur de cette cytokine dans l'effet adjuvant.L'influence de la voie d'administration sur ces effets est en cours d'étude grâce à la comparaison avec la voie intranasale

Das humorale Immungedächtnis wird von reifen Plasmazellen des Knochenmarks vermittelt, welche bei Immunreaktionen aus aktivierten B-Lymphozyten gebildet werden. Dabei sind im Blut Plasmablasten als unmittelbare Vorläufer der Plasmazellen nachweisbar, die von dort aus in das Knochenmark einwandern. Anhand der durchflusszytometrischen Detektion spezifischer Plasmablasten gelang es hier, das simultane Auftauchen von Wellen neu generierter, migratorischer Plasmablasten und reifer, nicht-migratorischer Plasmazellen im Blut eine Woche nach einer Tetanusimpfung nachzuweisen. Plasmablasten und Plasmazellen lagen stets im Gleichgewicht vor, wodurch auf die stöchiometrische Mobilisierung reifer Plasmazellen des Knochenmarks durch systemisch induzierte Plasmablasten geschlossen wurde. Ein solcher Verdrängungsmechanismus wird hier erstmalig als Anpassungsmechanismus des humoralen Immungedächtnisses dargestellt, der die Aufnahme neuer Spezifitäten in das Gedächtnis unter Wahrung der Stabilität präexistierender Spezifitäten erlaubt. Anders als systemisch induzierte Plasmablasten, weisen Plasmablasten, die im immunologischen Ruhephase zirkulieren, Kennzeichen mukosaler Immunreaktionen auf: sie exprimieren IgA sowie die mukosalen Zellmigrationsrezeptoren alpha4beta7-Integrin und CCR10. Wahrscheinlich wandern sie in mukosale Plasmazelldepots ein und interferieren nicht mit den Plasmazellen des Knochenmarks, sodass die Stabilität des humoralen Gedächtnisses in der Ruhephase gewahrt bleibt. Eine Anpassung des humoralen Gedächtnisses findet somit nur im Rahmen systemischer Immunreaktionen statt. Bei splenektomierten Patienten und unter der B-Zell-Depletionstherapie bei Rheumapatienten bleiben mukosale Plasmablasten im Blut nachweisbar. Dies belegt deren autonome Bildung aus mukosalen, therapie-refraktären B-Zellen. Insgesamt wird hier eine bisher unbeachtete Komplexität menschlicher peripherer Plasmablasten und Plasmazellen und ihren Beziehungen zum humoralen Immungedächtnis dargestellt.
Humoral memory, i.e. persistence of specific antibody titers, is provided by plasma cells in the bone marrow, which are generated from activated B cells during immune responses. At this, immediate plasma cell precursors, the plasmablasts, migrate via the blood to the bone marrow. Using cytometric detection of antigen-specific plasmablasts, synchronous circulation of waves of recently generated, migratory plasmablasts and non migratory plasma cells with a mature phenotype was demonstrated one week after tetanus vaccination. Circulating plasmablast and plasma cell numbers were always in homeostasis, so that the stoichiometric mobilization of old bone marrow plasma cells by recently generated plasmablasts was hypothesized. This plasma cell replacement mechanism is herein described for the first time as an adaption mechanism of the humoral memory that allows incorporation of new antibody specificities while maintaining pre-existing ones. In immunological steady state, very low numbers of plasmablasts are detectable in any donor. These express IgA and receptors for mucosal homing, alpha4beta7 integrin and CCR10, and therefore most likely migrate into mucosal plasma cell depots and do not interfere with plasma cells of the bone marrow, preserving the stability of humoral memory during steady state. Hence, adaption of humoral memory is only possible during systemic immune reactions. Circulating mucosal plasmablasts produced during steady state remain detectable in patients with rheumatoid arthritis during B cell depletion therapy as well as in asplenic patients. Hence, this type of plasmablasts is self-sufficiently generated from mucosal B cells that are refractory to B cell depletion therapy. This work demonstrates a hitherto disregarded complexity of peripheral plasmablast and plasma cell subsets in healthy humans, with implications for the regulation of induction and maintenance of humoral memory.

Influenza A virus (IAV) and Streptococcus pneumoniae are two of the most prominent respiratory pathogens affecting humans worldwide. While IAV and S. pneumoniae cause considerable morbidity and mortality individually, their synergistic pathogenicity poses the greatest threat to human health. Co-infection is associated with dramatically increased disease severity, particularly during IAV pandemics. The 1918 pandemic remains the most lethal on record, with an estimated 50 million deaths. However, a large portion of fatalities have since been attributed to secondary bacterial infection, with S. pneumoniae being heavily implicated. Given the ongoing risk of a future pandemic with highly pathogenic avian IAV, protective strategies against both IAV and S. pneumoniae represent an urgent and unmet need. Current vaccines against each of the individual pathogens are restricted to the induction of strain- and serotype-specific responses. Thus, our group has been developing novel vaccines that confer broad-spectrum protection after mucosal administration. These vaccines consist of whole IAV and whole un-encapsulated pneumococci that have been sterilised using gamma (γ)-irradiation, to generate γ-Flu and γ-PN. Irradiation effectively sterilises each pathogen by damaging the genomic material, whilst pathogen structure and antigenic proteins are maintained. This study describes the enhancement of safety and immunogenicity of both vaccines to facilitate future clinical advancement. Treatment of γ-Flu with a high radiation dose of 50 kGy was shown to have minimal impact on vaccine efficacy whilst exceeding a Sterility Assurance Level of 10-6. Establishing the efficacy of 50 kGy-treated preparations will aid in the inclusion of highly pathogenic strains in future vaccine formulations, such as avian H5N1 or H7N9. Such strains must be irradiated with a very high dose for sterilisation, and generation of g-Flu based on 50 kGy-treated avian strains would be immensely beneficial in the event of a future pandemic. Utilisation of high radiation dose may also aid in the transfer of this inactivation approach to other highly pathogenic agents for vaccine purposes, particularly when CD8⁺ T-cell responses are needed. The safety profile of our g-PN vaccine was also heightened in the current study. A growth attenuating mutation was introduced (generating γ-PN(∆PsaA)), which is an additional safety parameter to facilitate future clinical use of our vaccine. Interestingly, the supplementation of media with manganese required to restore normal growth in vitro was found to have immunomodulatory effects. Specifically, manganese supplementation was associated with enhanced TLR2 signalling by both live and irradiated samples of the pneumococcal vaccine. This phenomenon was unique to the further attenuated strain and is expected to enhance the magnitude of immune responses induced in vivo. In addition, antibody responses induced by g- PN(DPsaA) were found to react against a wider range of pneumococcal antigens compared to those induced by the original γ-PN. While the adjuvant activity of γ-Flu to co-administered γ-PN has been reported previously, the subsequent combination of the two optimised vaccines revealed direct interaction of γ-Flu and γ-PN(∆PsaA) in suspension, suggesting bi-directional adjuvant activities. Mixing the two vaccines resulted in enhanced uptake of γ-Flu virions by epithelial cells and macrophages in vitro, and co-vaccination with γ-Flu + γ-PN(∆PsaA) was associated with significant enhancement of IAV-specific Tissue Resident Memory cell populations in the lung. Furthermore, co-vaccination enhanced the protection in mice against lethal challenge with both drifted and heterosubtypic IAV strains. My data indicate that our novel approach of mixing whole inactivated viral and bacterial vaccine components could enhance pathogen-specific immunity, and may revolutionise vaccine design to combat infectious diseases.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018

Tese de doutoramento em Farmácia (Tecnologia Farmacêutica) apresentada à Fac. de Farmácia de Coimbra
A nova geração de vacinas são, na maioria dos casos, resultado dos rápidos avanços que se têm verificado nos últimos anos na área da biologia molecular e da imunologia, permitindo o uso da biotecnologia na produção de proteínas recombinantes com acção imunológica protectora. A vacina contra o vírus da hepatite B é disso o melhor exemplo. Esta vacina foi licenciada pela primeira vez em 1981, sendo nesta altura um produto derivado do plasma de portadores crónicos da hepatite B. Só mais tarde, em 1986, o mesmo antigénio, uma proteína de superfície do vírus da hepatite B, começou a ser produzido em sistemas de cultura celular. A vacina da hepatite B foi a primeira vacina recombinante a ser licenciada e a sua concepção tem servido de modelo ao desenvolvimento de novas vacinas, nomeadamente em doenças provocadas por vírus. Por outro lado, tem sido constatado pela comunidade científica que estas vacinas recombinantes são em geral antigénios fracos e por isso necessitam de ser administrados conjuntamente com substâncias que ampliem o seu efeito, os adjuvantes. Assim, paralelamente ao crescente desenvolvimento, que se tem verificado nos últimos anos, da tecnologia para o desenvolvimento e produção de novas vacinas, assiste-se também a uma crescente investigação na descoberta de adjuvantes mais seguros e potentes que tornem essas vacinas mais eficazes e estáveis. O termo “adjuvante” vem do termo latim adjuvare que significa ajuda. Recentemente, num livro editado por Virgil Schijns e Derek O’Hagan, dois de entre os maiores especialistas na área das vacinas e adjuvantes, o termo adjuvante é definido tendo em conta o seu efeito biológico. Estes investigadores classificam-nos em duas categorias principais: os adjuvantes cuja função principal é o controlo da biodisponibilidade da vacina nos tecidos linfoides (“delivery mechanism”) e os imunopotenciadores que têm uma acção directa sobre componentes da resposta imune inata. O presente trabalho teve por objectivo desenvolver um sistema nanoparticular (“delivery mechanism”), com potencial para a encapsulação de vacinas resultantes da tecnologia recombinante, com a finalidade de transportar e controlar a apresentação do antigénio nos tecidos linfóides. Tem sido referido por alguns investigadores que o sistema de libertação de vacinas ideal deve ter em conta o fenómeno de maturação da afinidade, que ocorre durante uma resposta imune. Quando a concentração de um antigénio é reduzida, as células com elevada afinidade para os receptores são estimuladas selectivamente. De acordo com este modelo, o padrão do sistema ideal deve imitar os perfis de concentração de antigénios que são observados no decurso de uma infecção natural: elevadas doses de antigénios, nos primeiros dias da administração, seguidos por um período em que há decréscimo da quantidade de antigénios. A biodisponibilidade inicial dos antigénios irá influenciar a extensão da formação da memória das células T, enquanto a subsequente diminuição dos antigénios irá beneficiar o desenvolvimento da maturação da afinidade dos anticorpos. Por outro lado, será útil fazer uma reflexão sobre qual a via de administração mais indicada para a administração de vacinas. Visto que as mucosas, nomeadamente a oral, a nasal, a pulmonar e genitourinária, são os locais por onde entram a maior parte dos microorganismos patogénicos, então a protecção contra esses microorganismos será mais eficiente pela presença de anticorpos nas secreções locais do que no soro. Alguns autores referem mesmo que a imunoglobulina A secretória (sIgA) local previne de forma mais eficiente, não só a colonização dos tecidos das mucosas, mas também a entrada de microorganismos na corrente sanguínea, quando comparada com a acção dos anticorpos sistémicos. Por outro lado, a indução de anticorpos nas mucosas não parece ser possível através da administração subcutânea ou intramuscular, usadas vulgarmente nos esquemas de vacinação. As evidências científicas, até hoje reunidas, indicam que, para haver indução de anticorpos (sIgA) nas mucosas, a administração do antigénio deve ser feita através das mucosas que se encontram revestidas por tecido linfoíde. Por esta razão, parece desejável que a próxima geração de vacinas, particularmente para os microorganismos patogénicos que invadem o organismo através das superfícies das mucosas, deve ser desenvolvida ou optimizada tendo em atenção a potencial indução de uma resposta imune nas mucosas. Para além da importante vantagem apresentada anteriormente, a administração através das mucosas, particularmente a administração oral, tem sido apontada como a via mais natural para introdução de fármacos no organismo, a de mais fácil acesso e sem os inconvenientes de outras vias de administração, como sejam os riscos de infecção por utilização de agulhas contaminadas, riscos de efeitos hemolíticos ou possível dor durante a administração. Para além disso, a via oral é sem dúvida a melhor aceite, nomeadamente pelas crianças que são a população alvo da maioria das vacinas. Acresce ainda referir que a vacinação em larga escala, num curto espaço de tempo, seria muito facilitada se tivéssemos vacinas orais em que, para a sua administração, não fossem necessários técnicos especializados. É o caso de situações de bioterrorismo, de ameaças de surtos infecciosos ou, simplesmente, o caso de países não industrializados que, por escassez de recursos humanos e também financeiros, continuam a ter taxas elevadas de prevalência de doenças para as quais já existem vacinas eficazes, como por exemplo de hepatite B. A administração oral de macromoléculas como o DNA, as proteínas e os peptídeos tem-se mostrado ineficaz, conduzindo a que, em alternativa, esta classe de fármacos seja administrado por uma das vias parentéricas. A limitada biodisponibilidade oral deve-se fundamentalmente a três ordens de razões. Por um lado ao extensivo metabolismo pré-sistémico a que estão sujeitos, devido à degradação enzimática que sofrem antes e durante os processos de absorção, não só ao nível do lúmen, como da mucosa gastrointestinal. A segunda barreira encontrada, prende-se com as fracas características de absorção destes fármacos e, por último, a terceira razão terá a ver com o efeito da primeira passagem pelo fígado (hepatic first pass effect). Consciente destas dificuldades, a comunidade Científica tem procurado novos sistemas terapêuticos, que permitam contornar ou minimizar as condições adversas do meio que impedem a administração oral destas moléculas. Neste contexto, os sistemas de transporte mais estudados, para a encapsulação destas macromoléculas, são as nanopartículas poliméricas e dentro destas são preferidos os sistemas biodegradáveis. A encapsulação irá proteger o antigénio do meio ácido e rico em enzimas proteolíticas como é o tubo digestivo. Para além das vantagens apontadas, acresce ainda referir que um sistema de transporte polimérico permite uma cedência gradual das moléculas activas, prolongando o seu efeito no organismo e contribuindo assim para a simplificação dos esquemas posológicos. Por outro lado, estes sistemas contribuem igualmente para a simplificação da logística de produção, armazenamento e distribuição de vacinas. Um dos sistemas que tem sido estudado é o de bioadesivos de libertação de fármacos (BDDS- bioadhesive drug delivery system). Os Tecnologistas estão confiantes que este novo conceito poderá contornar as dificuldades relacionadas com a libertação oral de peptídeos e análogos de peptídeos. Os BDDS são produzidos com a finalidade de se fixarem ao revestimento mucoso do tracto gastrointestinal. Desta forma, este sistema deverá exercer uma influência positiva na absorção dos fármacos que transportam. Essa influência deverá fazer-se por vários mecanismos: - prolongamento do tempo de residência no local de absorção do fármaco, com vista à redução da frequência de administração. - Intensificação do contacto com a barreira epitelial da mucosa subjacente, com vista ao incremento do transporte através do epitélio. - Pensa-se que alguns polímeros mucoadesivos têm a capacidade de modular a permeabilidade dos tecidos epiteliais, actuando ao nível das junções das células do epitélio (tight junctions). - Pensa-se igualmente, que alguns polímeros mucoadesivos podem actuar como inibidores de enzimas proteolíticas. Considerações finais: neste trabalho foi apresentado um novo sistema de libertação constituído por nanopartículas poliméricas. As nanopartículas são constituídas por um núcleo de quitosano onde foi posteriormente adsorvido o antigénio e finalmente revestido com alginato de sódio. O método desenvolvido permite encapsular, com elevada eficiência, antigénios do tipo proteico em condições reconhecidamente não agressivas, minimizando assim a possibilidade de perda de acção biológica por parte da vacina. As nanopartículas revestidas mostraram ter um efeito adjuvante relativamente à vacina da hepatite B quando administradas pela via subcutânea. A adição do imunopotenciador CpG ODN à suspensão das nanopartículas, contendo o antigénio da hepatite, permitiu modificar o tipo de resposta imune de Th2 para uma resposta mista Th1/Th2, mais adequada no caso do vírus da hepatite B. A administração, através das mucosas oral e nasal, da vacina da hepatite B encapsulada nas nanopartículas de quitosano revestidas permitiu confirmar a opinião de muitos especialistas na área da vacinologia, que defendem que não será suficiente a inclusão do antigénio em sistemas de libertação de nanopartículas, principalmente quando se trata de um antigénio fraco. Para se obter uma resposta imunológica adequada, para além da encapsulação do antigénio, vai ser ainda necessário adicionar um imunopotenciador. De facto, os melhores resultados foram obtidos com formulações que continham na sua composição o CpG ODN. Para finalizar, o sistema nanoparticular desenvolvido mostrou ser igualmente útil para a encapsulação do imunopotenciador em formulações orais. Em contraste, na mucosa nasal, o imunopotenciador produziu melhores resultados quando foi administrado em solução, pelo que a futura utilização destas nanopartículas para a encapsulação de moléculas que demonstrem uma elevada afinidade para o quitosano, como o caso do CpGODN, estará condicionada a uma optimização conveniente destas nanopartículas. As nanopartículas de quitosano, revestidas com alginato provaram ter um efeito adjuvante com o antigénio da hepatite B pela via subcutânea. Por conseguinte, os próximos estudos serão feitos com o objectivo de avaliar se o sistema nanopartícular poderá substituir, com vantagem, o actual adjuvante (compostos de alumínio), da vacina da hepatite B. Para finalizar, o efeito adjuvante, observado para a vacina da hepatite B, deverá ser avaliado com outras vacinas, de preferência mais fortes, não só pela via subcutânea mas também através das mucosas oral e nasal.
Este trabalho foi parcialmente realizado na qualidade de bolseiro da Fundação para a Ciência e Tecnologia no âmbito do POCTI – Formar e Qualificar – Medida 1.1 (referência SFRH/BD/5327/2001).

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
A vacinação é considerada como uma das medidas de saúde publica de controlo de doenças infetocontagiosas mais eficazes na civilização moderna. Os programas de imunização em larga escala diminuíram significativamente a morbilidade e mortalidade atribuídas às doenças infeciosas. A melhoria das vacinas atuais, e o desenvolvimento de vacinas para doenças contra as quais ainda não existem vacinas eficazes, são linhas de investigação de grande importância e com grande impacto na saúde pública e na sociedade. Particularmente, o desenvolvimento de vacinas para administração pelas mucosas será altamente desejável dado que, para além de permitir uma mais fácil administração, permitirá conferir proteção imunológica específica no local de entrada dos patogéneos. A colocação no mercado deste tipo de vacinas tem sido adiada devido à falta de adjuvantes seguros e eficazes para estas vias de administração. O objetivo deste projeto foi desenvolver um novo sistema de entrega de antigénios, com função adjuvante, para vacinação nasal. Este sistema de entrega é composto por dois adjuvantes para as mucosas, os quais, individualmente, em ensaios pré-clínicos, já mostraram ser muito promissores: nanopartículas de quitosano e um ativador de mastócitos, o composto 48/80 (C48/80). Esta é a primeira vez que é descrita a combinação do C48/80 com nanopartículas com o objetivo de desenvolver uma melhor formulação adjuvante para vacinas. Foram desenvolvidos dois sistemas de entrega, tendo por base o quitosano para a formação de nanopartículas, e nas quais se encapsulou o C48/80: Quitosano-C48/80 NP (Chi-C48/80 NP) e Quitosano/Alginato-C48/80 NP (Chi/Alg-C48/80 NP). As duas formulações foram caracterizadas e avaliadas in vitro. À posteriori, estudos in vivo avaliaram o seu potencial como adjuvantes para vacinas nasais. Para auxiliar no desenvolvimento das nanopartículas carregadas com C48/80, foi desenvolvido um método colorimétrico com deteção por espetrofotometria UV-Vis para a quantificação do C48/80. Este método foi validado de acordo com as recomendações das normas ICH para os seguintes parâmetros: especificidade, gama de trabalho e linearidade, exatidão, precisão e limites de deteção e de quantificação. As nanopartículas desenvolvidas no decurso deste projeto, Chi-C48/80 NP e Chi/Alg-C48/80 NP apresentaram um tamanho médio de 501 nm e 564 nm, respetivamente, e ambas apresentaram um potencial zeta positivo. A eficácia de carregamento do C48/80 foi 19 % para as Chi-C48/80 NP e 30 % para as Chi/Alg-C48/80 NP. Estudos de citotoxicidade demonstraram que a incorporação do C48/80 nos sistemas de entrega diminui a toxicidade do imunopotenciador. Estudos in vitro de internalização revelaram que as Chi-C48/80 NP foram internalizadas por células apresentadoras de antigénio mais eficientemente que as Chi/Alg-C48/80 NP. A capacidade das nanopartículas desenvolvidas para ativarem mastócitos foi também testada in vitro usando o ensaio de libertação de β-hexosaminidase. Os resultados demonstraram que a associação do C48/80 com as Chi NP, mas não com as Chi/Alg NP, resultou numa maior ativação de mastócitos quando comparado com o C48/80 em solução. Para avaliar se as particulas mucoadesivas, à base de quitosano, conseguiam aumentar o tempo de residência do antigénio na cavidade nasal, foi realizado um estudo in vivo de clearance nasal. Com esse objetivo foi usada ovalbumina marcada com fluorescência, adsorvida às nanopartículas. Neste estudo as Chi-C48/80 NP superaram novamente as Chi/Alg-C48/80 NP aumentando significativamente o tempo de residência do antigénio na cavidade nasal. A aptidão das nanopartículas desenvolvidas para potenciarem uma resposta imune, foi avaliada em murganhos C57BL/6 usando a imunização nasal com o antigénio protetor (PA) do B. anthracis como sistema modelo. A imunização usando as Chi-C48/80 NP como adjuvante induziu títulos elevados de anticorpos neutralizantes anti-PA no soro e títulos de anticorpos IgG2c anti-PA mais elevados do que as Chi/Alg-C48/80 NP. A incorporação do C48/80 nas Chi NP também promoveu uma imunidade nas mucosas melhor que a induzida por todos os outros grupos testados. Estes estudos de vacinação demonstraram que as Chi-C48/80 NP exibiram um desempenho melhor como adjuvante para a mucosa nasal que as Chi/Alg-C48/80 NP. Além disso, um estudo de dose-resposta revelou que as Chi-C48/80 NP permitiram diminuir seis vezes a dose de antigénio usada, sem afetar os títulos de anticorpos IgG específicos e neutralizantes. Estes resultados sugerem que esta inovadora combinação de adjuvantes, Chi NP com C48/80, tem potencial para diminuir a dose de antigénio necessário a incluir numa formulação comercial da vacina. De um modo geral, os resultados obtidos com este projeto demonstram que a combinação de nanopartículas de quitosano com um ativador de mastócitos é uma estratégia promissora para a imunização nasal, induzindo uma resposta imune potente, tanto sistemicamente como nas mucosas.
Vaccination is considered one of the greatest medical achievements of modern civilization and large scale immunization programs greatly reduced the global burden of infectious diseases. Therefore, improvement of current vaccines and development of vaccines against diseases for which successful vaccines are not currently available would bring huge benefits for public health and for the society. The development of mucosal vaccines would be highly desirable since it would provide protection at the local of entry of pathogens. However, the development of mucosal vaccination strategies has been delayed for the lack of effective and safe mucosal adjuvants. The objective of this project was to develop a novel prototypic delivery system for nasal vaccination composed by two highly promising mucosal adjuvant candidates: chitosan nanoparticles and the mast cell activator compound 48/80 (C48/80). This is the first time that the combination of C48/80 with nanoparticles in order to develop an improved adjuvant formulation is described. Two different C48/80 loaded chitosan-based delivery systems were developed: Chitosan-C48/80 NP (Chi-C48/80 NP) and Chitosan/Alginate-C48/80 NP (Chi/Alg-C48/80 NP). The two C48/80 loaded delivery systems were characterized and evaluated in vitro. Subsequently, in vivo studies assessed their potential as nasal adjuvants. To support the development of C48/80 loaded nanoparticles, a UV-Vis spectrophotometric method for quantification of C48/80 was developed. This method was validated according to the recommendations of ICH Guidelines for specificity, linearity, range, accuracy, precision and detection and quantification limits. Chi-C48/80 NP and Chi/Alg-C48/80 NP had a mean size of 501 nm and 564 nm, respectively, and were both positively charged. Loading efficacy of C48/80 was 19 % for Chi-C48/80 NP and 30 % for Chi/Alg-C48/80 NP. Cytotoxicity studies performed in two different cell types showed that incorporation of C48/80 in both formulations resulted in a decreased toxicity of the immunopotentiator compared with C48/80 in solution. In vitro uptake studies showed that Chi-C48/80 NP were more efficiently internalized by antigen presenting cells than Chi/Alg-C48/80 NP. The ability of the developed nanoparticles to activate mast cells was tested in vitro using the β-hexosaminidase release assay. Results demonstrated that association of C48/80 with Chi NP but not with Chi/Alg NP enhanced mast cell activation when compared with C48/80 in solution. To see if the mucoadhesive chitosan-based nanoparticles could increase the residence time of an antigen, an in vivo nasal clearance study of fluorescently labelled ovalbumin loaded on nanoparticles was performed. Once again, Chi-C48/80 NP outperformed Chi/Alg-C48/80 NP significantly increasing ovalbumin residence time in the nasal cavity. Both delivery systems, Chi-C48/80 NP and Chi/Alg NP were then compared for their ability to induce antigen-specific serum IgG, mucosal IgA and serum lethal toxin-neutralizing antibody responses in C57BL/6 mice using nasal immunization with anthrax recombinant protective antigen (PA) as a model system. Nasal immunization with Chi-C48/80 NP as adjuvant elicited high levels of serum anti-PA neutralizing antibodies and higher antigen-specific IgG2c than Chi/Alg-C48/80 NP. The incorporation of C48/80 within Chi NP also promoted a mucosal immunity greater than all the other adjuvanted groups tested. These vaccination studies showed that Chi-C48/80 NP displayed a better performance as nasal adjuvant than Chi/Alg-C48/80 NP. Additionally, an antigen dose-response study showed that Chi-C48/80 NPs allowed a 6-fold decrease of the antigen dose without affecting the levels of specific IgG titers and its neutralizing ability. These results suggest the potential of this novel adjuvant combination to decrease the antigen dose required for vaccination. Overall, the findings of this project show that the combination of a mast cell activator with chitosan nanoparticles is a promising strategy for nasal immunization inducing potent systemic and mucosal immune responses.

博士
國防醫學院
醫學科學研究所
96
Current clinical vaccination has included different pathways, including intravenous, intradermal, subcutaneous and intramuscular routes. The efficacy of subcutaneous immunotherapy for allergic diseases is well documented, but the possibility of fatal anaphylaxis can not be excluded. The efficacy and safety of sublingual immunotherapy has been also confirmed in recent years. In fact, mucosal immunity plays a crucial role to counteract aeroallergens and mucosal pathogens at their first encounter. SIgA is the first line of mucosal defense. Its effects to minimize mucosal permeability of allergens and invasion of bacterial pathogens have been termed as “immune exclusion”. Therefore, induction of mucosal immunity is important to prevent mucosal infections and allergic reactions. Vaccination with protein vaccines and adjuvants such as cholera toxin and CpG oligodeoxynucleotides (CpG) can effectively trigger systemic immunity. Denaturation of proteins can abrogate allergenicity and oral tolerogenicity, but there is so far no study of the effect of sublingual vaccination with a denatured protein. Adult BALB/c mice were thus sublingually vaccinated with native OVA or its denatured form CM-OVA. Sublingual vaccination together with adjuvant CpG enhanced saliva specific SIgA antibody responses and systemic Th1 responses, including higher IFN-r production by spleen and cervical lymph node (CLN) cells, higher serum specific IgG2a and lower serum specific IgE antibody responses. In contrast, sublingual vaccination together with adjuvant CT increased saliva specific SIgA antibody responses and systemic Th2 responses, including higher IL-4, IL-5 and IL-6 production by spleen and CLN cells, and enhanced serum specific IgG1 antibody responses. However, the effect of sublingual vaccination on previously sensitized mice had not been studied. Those sublingually treated with OVA or CM-OVA, plus either CT or CpG, stimulated saliva SIgA antibody responses. The mice sublingually vaccinated together with adjuvant CpG had much enhanced serum specific IgG2a but markedly suppressed IgE antibody responses. Vaccination with strong adjuvant may be more helpful for newborns to overcome their immune immaturity. However, there is no study of the effect of sublingual vaccination on newborns. Salmonella enteritidis is a common intestinal pathogen. Symptomatic Salmonella infections range from mild enteritis to severe life-threatening sepsis. Most experimental vaccines against Salmonella have been applied by injection. It will be better to try other routes to have no pains. Newborn mice were sublingually vaccinated for the first time. Neonatal sublingual vaccination with sonicated Salmonella proteins (SSP) and adjuvant CpG enhanced saliva specific SIgA antibody responses and systemic Th1 responses, including higher IFN-r production by spleen and CLN cells, and increased serum specific IgG2a antibody responses. In contrast, neonatal sublingual vaccination with SSP and CT increased saliva specific SIgA antibody responses and systemic Th2 responses, including enhanced IL-4, IL-5 and IL-6 production by spleen and CLN cells, and higher serum specific IgG1 antibody responses. After further challenge with live Salmonella enteritidis, only the adult mice receiving neonatal sublingual vaccination with SSP and adjuvant CpG or CT had effective prophylaxis against necrotic enteritis, and the best survival rate. Sublingual vaccination with antigens and mucosal adjuvants can induce systemic and mucosal immunity and further prevent allergic and infectious reactions in adult and newborn mice. It is a simple and easy way to develop mucosal vaccines for prophylaxis and immunotherapy.

Coccidiosis vaccine associated side effects, oocyst shedding patterns, intestinal lesions, and mucosal gene expression in the turkey were studied. The first study examined vaccine associated side effects and oocyst shedding patterns under experimental conditions. Peak oocyst shedding occurred on days 5-6, 13-17, and 19-20 days post vaccination. Throughout the course of the study, several poults exhibited clinical coccidiosis. Based on body weights, growth was correlated with vaccine cycling. The second study examined coccidiosis vaccine induced lesions and changes in mucosal gene expression on day 5, 10, 13, 17, and 20 days post vaccination. Poults were gavaged the equivalent of 0x, 1/2x, 1x, and 2x the available vaccine dose. Intestinal sections adjacent to the Meckel's diverticulum, ileocecal junction, and middle of the ceca were collected for histological analysis and gene expression. Measurements from the tip of the villus to the base of the lamina propria, villus width, and the muscularis mucosae thickness were acquired from the histological sections. Interleukin-10, IL-1beta, and GAPDH gene expression were measured by extracting mRNA in the tissues and quantified using real-time RT-qPCR. Starting on day five post vaccination, the control group weighed significantly more than the group that received the 2x dose. Body weight and oocyst dose were inversely related through day 17. Intestinal measurements did not necessarily correlate with the vaccine dose, although there appears to be some correlation on day five. There were no significant changes in the mucosal gene expression of IL-10 and IL-1beta in the intestinal tissue adjacent to the Meckel's diverticulum throughout the course of the study. On day five post vaccination, IL-10 and IL-1beta were significantly upregulted in the ileocecal junction. Interleukin-10 was significantly upregulated on day 17 and IL-1beta was significanlty down regulated on day 20 in the ileocecal junction. Both IL-10 and IL-1beta were significantly upregulated in the ceca days 5, 10, and 13 post vaccination. Interleukin-10 was significnalty upregulated in the ceca on day 17 and significantly down regulated on day 20. Individual variation among poults in the same group merits further attention.

Most pathogens gain access to their host through mucosal surfaces. It is therefore desirable to develop mucosal vaccines that elicit an immune response to prevent this crucial first step in infection. Current mucosal vaccines are live attenuated strains of pathogens. More recent efforts have focused on the use of recombinant non-pathogenic gram-positive bacteria as live vaccine delivery vectors. Here I have tested the potential of Lactococcus lactis to be used as a vaccine vector. A recombinant strain of L. lactis has been constructed which expresses and displays on its surface the C repeat region (CRR) of the M6 protein of Streptococcus pyogenes. I show that nasal vaccination of mice with this strain elicited strong salivary IgA and serum lgG response. These responses protected mice against a nasal challenge with S. pyogenes. Subcutaneous vaccination with the same strain of L. lactis produced a strong serum lgG response, but no salivary lgA response. Subcutaneous vaccination did not protect the mice against nasal infections when the mice were challenged with S. pyogenes. The immune response and protection afforded by concomitant vaccination by both nasal and subcutaneous routes were better that that seen in nasal vaccination alone. This study shows that an effective vaccine against S. pyogenes is possible using L. lactis as a vaccine vector. It also opens up the potential of L. lactis to be used in the development of vaccines to other mucosal infections.
Graduation date: 2004

Tese de doutoramento em Ciências Farmacêuticas, na especialidade de Tecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Vaccines are one of medicine greatest achievements, reducing the incidence of infectious diseases and eradicating otherwise fatal diseases worldwide. However, hepatitis B virus (HBV) infection is still a major global health concern and the most common cause of chronic liver disease and mortality from hepatocellular carcinoma. New generation vaccines are needed in order to overcome the limitations of the current HBV vaccines in the market. In this regard, mucosal immunization constitutes an attractive alternative to the available parenteral vaccine, especially in developing countries, since it would be best suited for mass immunization and would provide protection at the pathogen entry site. The main objective of this thesis was to develop the next generation of HBV vaccines exploiting the immunomodulatory and mucoadhesive properties of chitosan-based delivery nanoparticles. This strategy would improve not only mucosal- and cell-mediated immunity, but would also allow the vaccine to be efficiently administered through the nasal mucosa. To achieve this goal, two different approaches were developed and tested. First, a novel prototypic system combining two well-established immunopotentiators, chitosan and aluminium salts, was produced to deliver hepatitis B surface antigen (HBsAg). Adjuvant combination has been considered a promising strategy to boost immunogenicity. The second approach involved the generation of a gene delivery system consisting of complexes of human serum albumin (HSA)-loaded chitosan nanoparticles with DNA (HSA-CH NP/DNA) encoding HBsAg. The two delivery systems were characterized and evaluated, both in vitro and in vivo. In order to fulfill the main goal, we established a detailed methodology to easily obtain large quantities of endotoxin-free chitosan without modifying its immunomodulatory properties. Bacterial endotoxins content was assessed according to the recommendations of the International Council for Harmonization (ICH) guideline and validated with in vitro data. Chitosan-aluminium nanoparticles (CH-Al NPs) were prepared using a nanoprecipitation technique; the optimal formulation exhibited a mean diameter of 280 nm and a positive surface charge, showing no cytotoxic effects in two different cell lines and in a primary culture of splenocytes, in the dose used for in vivo studies. In vitro uptake studies showed that CH-Al NPs were efficiently internalized by epithelial cells, demonstrating potential as a delivery system for a wide range of model antigens. Immunization studies showed that mice subcutaneously immunized with HBsAg adjuvanted with CH NPs displayed enhanced humoral and cellular immune responses. To understand the underlying mechanisms of adjuvanticity of CH-Al NPs, the ability of CH-Al NPs to promote dendritic cell (DC) activation and their potential to stimulate innate and adaptive immune responses was assessed. Results were correlated to those obtained with chitosan in solution (CH sol.) and conventional chitosan particles (CH-Na NPs). All the formulations were capable of modulating Toll-like receptor (TLR)-9 agonist, CpG, induced cytokine secretion in bone-marrow derived dendritic cells (BMDCs) and induced DC maturation in the absence of cytokine production. After intraperitoneal (I.P.) injection, CH-Al NPs were capable of generating a local immune response comparable to that elicited by the vaccine adjuvant alum, with recruitment of neutrophils and eosinophils and concomitant disappearance of resident macrophages and mast cells. After vaccination with CH-Al NPs in combination with HBsAg, mice developed high antigen-specific immunoglobulin G (IgG) titers in the serum, as well as in nasal and vaginal washes, generating an overall improved immune profile in comparison to the commercially available vaccine Engerix-B. In the second approach, a DNA vaccine was developed in which a plasmid coding for the HBsAg was adsorbed on the surface of the HSA-loaded CH NPs. The presence of HSA enhanced transfection activity and facilitated DNA release from the complex by weakening the interaction between positively charged nanoparticles and negatively charged plasmid DNA (pDNA). To assess in vivo the value of the developed formulation, immunization studies were conducted. Nasal immunization with HSA-CH NP/DNA complexes elicited high levels of serum anti-HBsAg IgG and antigen-specific IgA in nasal and vaginal secretions, while no systemic or mucosal responses were detected after immunization with DNA alone. These results confirm the ability of this novel delivery system to generate a mucosal immune response, making it a valuable adjuvant for nasal vaccination against HBV. Overall, our findings add to our knowledge of the mechanism of action of chitosan-based formulations and illustrate that proper design is vital in order to generate an effective adjuvant for HBV vaccines, capable of driving mucosal immune responses in addition to potent humoral and cell-mediated immunity.
As vacinas são uma das maiores conquistas da medicina, reduzindo a incidência de doenças infeciosas e erradicando mundialmente doenças que outrora seriam fatais. Apesar de existir no mercado uma vacina profilática contra a hepatite B, a infeção pelo vírus da hepatite B (VHB) continua a ser um dos principais problemas de saúde pública e a causa mais comum de doença hepática crónica e mortalidade por carcinoma hepatocelular. É por isso necessário uma nova geração de vacinas que ultrapassem as limitações das vacinas contra o VHB atualmente no mercado. Nesse sentido, a imunização pelas mucosas constitui uma alternativa apelativa à vacina parentérica disponível, especialmente nos países em desenvolvimento, uma vez que seria mais adequada para imunização em massa e proporcionaria proteção no local de entrada do patogéneo, nomeadamente no caso de uma doença sexualmente transmitida. Neste sentido, o principal objetivo desta tese consiste em desenvolver a próxima geração de vacinas contra o VHB explorando as propriedades imunomoduladoras e mucoadesivas de nanopartículas à base de quitosano. Esta nova vacina melhoraria não só a imunidade ao nível das mucosas e a mediada por células, como permitiria que a vacina fosse administrada de forma eficiente através da mucosa nasal. Para atingir este objetivo, duas estratégias diferentes foram desenvolvidas e testadas. Primeiro, foi otimizado um novo sistema protótipico de liberação do antigénio de superfície do vírus da hepatite B (HBsAg), combinando dois imunopotenciadores bem estabelecidos, o quitosano e os sais de alumínio, uma vez que a combinação de adjuvantes tem sido considerada uma estratégia promissora para potenciar a resposta imunológica; segundo, foram também desenvolvidos complexos de nanopartículas de quitosano carregados com albumina de soro humano (HSA) e complexados com ADN (HSACH NP/ADN), usados para estimular a resposta imune sistémica e nas mucosas após administração intranasal. Os dois sistemas de libertação de moléculas ativas foram caracterizados e avaliados quer in vitro, quer in vivo. Para cumprir o objetivo principal, estabelecemos uma metodologia detalhada de modo a obter facilmente quitosano livre de endotoxinas, sem comprometer as suas propriedades bioquímicas. O conteúdo das endotoxinas bacterianas foi avaliado em conformidade com as recomendações da diretiva do Conselho Internacional de Harmonização (ICH) e validado com dados obtidos em estudos in vitro. As nanopartículas de quitosano alumínio (CH-Al NPs) foram preparadas utilizando uma técnica de nanoprecipitação; a formulação otimizada exibiu um diâmetro médio de 280 nm e uma carga de superfície positiva, não apresentando efeitos citotóxicos em duas linhas celulares diferentes e numa cultura primária de esplenócitos, para a dose utilizada posteriormente em estudos de imunização. Estudos de internalização in vitro demonstraram que as CH-Al NPs foram eficientemente internalizadas por células epiteliais exibindo potencial como sistema de libertação para uma ampla gama de antigénios modelo. Estudos in vivo mostraram que murganhos imunizados pela via subcutânea com HBsAg coadjuvado com CH NP apresentaram uma melhor resposta imune humoral e celular quando comparado com o grupo de murganhos vacinados apenas com o antigénio. Para entender os mecanismos subjacentes de adjuvanticidade das CH-Al NPs, avaliou-se a sua capacidade para promover a ativação de células dendríticas (DC), e o seu potencial para estimular respostas imunes inatas e adaptativas. Os resultados foram correlacionados com os obtidos com quitosano em solução (CH sol.) e partículas de quitosano convencionais (CH-Na NP). Todas as formulações foram capazes de modular a secreção de citoquinas induzida por agonistas dos recetores tipo-Toll (TLR)-9, CpG, em células dendríticas derivadas da medula óssea de murganho (BMDCs) e induziram a maturação de DC na ausência de produção de citoquinas. Após a injeção intraperitoneal (I.P.), as CH-Al NPs foram capazes de gerar uma resposta imunitária local comparável à evocada por sais de alumínio usado com adjuvantes em vacina que se caracterizou por recrutar neutrófilos e eosinófilos e por um desaparecimento de macrófagos e mastócitos. Após a vacinação com CH-Al NP pela via subcutânea em combinação com HBsAg, os murganhos desenvolveram títulos elevados de anticorpos IgG anti-HBsAg no soro, bem como nas lavagens nasais e vaginais, gerando um perfil imunológico melhorado em comparação com a vacina comercialmente disponível, Engerix-B. Na segunda abordagem foi desenvolvida uma vacina de ADN na qual um plasmídeo que codifica o HBsAg foi adsorvido na superfície das CH-NPs carregadas com HSA. A presença de HSA aumentou a atividade de transfecção e facilitou a libertação de ADN a partir do complexo, por enfraquecer a interação entre nanopartículas positivamente carregadas e o ADN carregado negativamente. Para avaliar o potencial da formulação desenvolvida foram conduzidos estudos in vivo. A imunização com complexos HSA-CH NP/ADN originou níveis elevados de anticorpos IgG anti-HBsAg e anticorpos IgA específicos contra o HBsAg nas secreções nasais e vaginais, enquanto não foram detetadas respostas sistémicas ou nas mucosas após imunização com ADN sozinho. Estes resultados demonstram o potencial deste novo sistema de administração em gerar uma resposta imune nas mucosas, tornando-o num adjuvante promissor para a vacinação nasal contra o HBV. De modo geral, os resultados obtidos melhoram o nosso conhecimento de formulações à base de quitosano e demonstram que o design apropriado é vital para a formular um adjuvante eficaz para vacinas contra o HBV, capaz de produzir respostas imune ao nível das mucosas além de potenciar imunidade humoral e celular.