A imunização transcutânea (IT) é uma técnica promissora de vacinação, na qual a formulação contendo o antígeno é aplicada sobre a pele para induzir resposta imune. O sucesso desse tipo de imunização ocorre devido à presença de células apresentadoras de antígenos (APCs) na epiderme viável, as quais são potentes estimuladoras de linfócitos T. Porém, é necessário que o antígeno transponha a barreira imposta pelo estrato córneo e atinja a epiderme viável em concentrações adequadas para que uma resposta imune efetiva seja induzida. Neste trabalho, a influência da iontoforese, método físico que utiliza uma corrente elétrica fraca para aumentar a penetração cutânea de fármacos, foi investigada pela primeira vez na IT. Sua associação com lipossomas, para direcionar a liberação do antígeno para a epiderme viável, e com nanopartículas de prata (NPAg), para potencializar a resposta imune, também foi avaliada pela primeira vez. A ovalbumina (OVA) foi utilizada como antígeno modelo e a injeção subcutânea de OVA como controle positivo da imunização. Foram obtidos lipossomas aniônicos contendo 5 mg/mL de OVA e catiônicos contendo 0,25 mg/mL de OVA, adicionados ou não de NPAg. Os lipossomas aniônicos apresentaram tamanho médio de aproximadamente 120 nm e eficiência de encapsulação da OVA de 78,4%. Os lipossomas catiônicos apresentaram tamanho médio de aproximadamente 260 nm e eficiência de encapsulação da OVA de 83,08%. A adição das NPAg não alterou significativamente o tamanho dos lipossomas, mas alterou o potencial zeta (de -7,5 mV para -14 mV para os lipossomas aniônicos e de +41 mV para +36 mV para os lipossomas catiônicos). As microscopias de força atômica e eletrônica de transmissão (MET) confirmaram a presença de vesículas lipossomais, sendo que a TEM mostrou as NPAgs dispersas no meio aquoso externo dos lipossomas aniônicos e no meio aquoso interno dos lipossomas catiônicos. A OVA e os lipossomas se mostraram estáveis ao processo de obtenção e frente a corrente elétrica. Nos estudos de penetração cutânea in vitro observou-se que a encapsulação da OVA nos lipossomas direcionou sua liberação para a epiderme viável. A associação com a iontoforese aumentou 108 vezes a liberação da OVA na epiderme viável quando lipossomas aniônicos contendo NPAg foram administrados e 92 vezes quando lipossomas catiônicos contendo NPAg foram administrados. A presença das NPAg nas formulações aumentou a quantidade de OVA na epiderme viável quando a iontoforese foi utilizada, mas diminuiu sua penetração passiva. As quantidades de OVA penetradas por iontoforese anódica e catódica a partir dos lipossomas aniônicos não foram significativamente diferentes. O coeficiente de penetração da OVA na epiderme viável a partir da iontoforese dos lipossomas aniônicos foi 1,6 vezes (na ausência de NPAg) e 39 vezes (na presença de NPAg) maior do que o dos lipossomas catiônicos. Nos experimentos de imunização transcutânea in vivo observou-se que as quantidades de OVA que penetraram a pele por iontoforese foram suficientes para induzir resposta imune humoral semelhante a induzida pela injeção subcutânea da OVA para os lipossomas aniônicos, na presença e ausência de NPAg, e para o lipossoma catiônico apenas na presença da NPAg, sugerindo que as NPAg, na presença de baixas concentrações de OVA, funcionam como adjuvantes imunológicos. A iontoforese anódica das formulações, assim como a injeção subcutânea da OVA, não estimularam resposta imune celular significativa. A iontoforese catódica, no entanto, induziu tanto resposta humoral como celular, além de estimular a produção de INF-? e o recrutamento de células apresentadoras de antígeno, tanto no baço como nos linfonodos inguinais. Sendo assim, a iontoforese de lipossomas contendo OVA e NPAg foi capaz de direcionar a liberação da OVA para a epiderme, induzir altos títulos de anticorpos IgG1 e ainda ativar a resposta imune celular, sendo uma estratégia promissora para a IT. / Transcutaneous immunization (TI) is a promising strategy for vaccine in which the antigen-containing formulation is applied to the skin to induce immune response. The success of this type of immunization occurs due to the presence of antigen presenting cells (APCs) in the viable epidermis, which are potent stimulator of T lymphocytes. However, the antigen should transpose the barrier imposed by the stratum corneum to reach the viable epidermis in appropriate concentrations so that an effective immune response is induced. In this work, the influence of iontophoresis, a physical method that uses a weak electrical current to increase skin penetration of drugs, was investigated for the first time in TI. Its association with liposomes, to target the antigen release to the viable epidermis, and with silver nanoparticles (NPAg), to enhance the immune response, was also evaluated for the first time. Ovalbumin (OVA) was used as a model antigen and OVA subcutaneous injection as a positive control of immunization. Anionic and cationic liposomes containing 5 mg/ml and 0.25 mg/mL of OVA, respectively, were obtained and were added or not by NPAg. Anionic liposomes had an average size of approximately 120 nm and 78,4% OVA encapsulation efficiency. Cationic liposomes had an average size of approximately 260 nm and 83,08% OVA encapsulation efficiency. The addition of NPAg did not significantly alter the size of the liposomes, but changed the zeta potential (-7.5 mV to - 14 mV for anionic liposomes and +41 mV to +36 mV for cationic liposomes). The atomic force microscopy and transmission electron microscopy (TEM) confirmed the presence of liposomal vesicles; TEM showed NPAgs dispersed in the external aqueous medium of the anionic liposomes and in the internal aqueous medium of the cationic liposomes. The OVA and the liposomes were stable during the preparation process and in front of the electric current. In the in vitro skin penetration studies it was observed that the encapsulation of OVA into liposomes directed their release to the viable epidermis. The association with iontophoresis increased 108-fold the release of OVA in the viable epidermis when anionic liposomes containing NPAg were administered and 92-fold when cationic liposomes containing NPAg were administered. The presence of NPAg in the formulations increased the amount of OVA released in the viable epidermis when iontophoresis was applied, but decreased OVA passive penetration. The amount of OVA penetrated by anodic and cathodic iontophoresis of anionic liposomes was similar. The OVA penetration coefficient in the viable epidermis from the iontophoresis of anionic liposomes was 1.6-fold (in the absence of NPAg) and 39- fold (in the presence of NPAg) greater than the iontophoresis of cationic liposomes. In transcutaneous immunization in vivo experiments, it was observed that the amount of OVA that penetrated the skin by iontophoresis was sufficient to induce similar humoral immune response than that induced by subcutaneous injection of OVA when anionic liposomes, in the presence and absence of NPAg, and cationic liposome, only in the presence of NPAg, were administered. These results suggest that NPAg in the presence of low concentrations of OVA acted as immunological adjuvants. Altough induced humoral immune response; anodal iontophoresis of the formulations as well as subcutaneous injection of OVA did not stimulate significant cellular immune response. Cathodic iontophoresis, on the other hand, induced both humoral and cellular immune response, as well as stimulating the production of IFN-? and the recruitment of antigen presenting cells, both in spleen and in inguinal lymph nodes. Therefore, iontophoresis of liposomes containing OVA and NPAg was able to target the release of OVA to the epidermis, induce high titers of IgG1 and activate the cellular immune response, being a promising strategy for TI.

Το δέρμα λειτουργεί σαν μηχανικός φραγμός ενάντια σε ένα εχθρικό περιβάλλον. Παράλληλα λειτουργεί ως ένα ανοσολογικό εμπόδιο, το οποίο είναι πλούσιο σε αντιγονοπαρουσιαστικά κύτταρα, όπως τα κύτταρα Langerhans. Αν και είναι γενικά παραδεκτό ότι το δέρμα δεν είναι περατό από μεγαλομοριακές ουσίες, και συνεπώς δεν μπορεί να χρησιμοποιηθεί ως οδός χορήγησης αντιγόνων, πρόσφατες μελέτες έδειξαν ότι το δέρμα μπορεί να αποτελέσει οδό για τη χορήγηση αντιγόνων. Συγκεκριμένα έδειξαν ότι η διαδερμική χορήγηση ενός αντιγόνου μαζί με την τοξίνη της χολέρας ως ανοσοενισχυτικό επάγει ικανοποιητική ανοσοαπόκριση έναντι του αντιγόνου. Επιπρόσθετα, η διαδερμική χορήγηση της τοξίνης της χολέρας δεν εμφανίζει τοξικότητα όπως με άλλες οδούς χορήγησης. Η υποδόρια χορήγηση αντιγόνων εγκλεισμένων σε PLA και PLGA μικροσφαίρες και νανοσφαίρες έχει ευρεθεί ότι επάγει ισχυρή και μακράς διάρκειας ανοσοαπόκριση. Μέχρι σήμερα δεν έχει μελετηθεί η δυνατότητα διαδερμικής χορήγησης αντιγόνων εγκλεισμένων σε πολυμερικά νανοσωματίδια. Έτσι, στην παρούσα μελέτη μελετήθηκε η ανοσοαπόκριση που λαμβάνεται μετά την διαδερμική χορήγηση οβαλβουμίνης (OVA) εγκλεισμένης σε νανοσφαίρες πολύ (γαλακτικού οξέως) (PLA) σε BALB/c μύες με ή χωρίς την συγχορήγηση ανοσοενισχυτικού, της τοξίνης της χολέρας (CT). Επίσης διερευνήθηκε ο πιθανός μηχανισμός εισόδου των νανοσφαιρών στο άθικτο δέρμα των μυών. Για τη παρούσα μελέτη πραγματοποιήθηκε σε πρώτο στάδιο σύνθεση πολύ (γαλακτικού οξέως) (PLA) το οποίο χαρακτηρίστηκε ως προς το μοριακό του βάρος και ως προς την καθαρότητα του. Επίσης νανοσφαίρες PLA με ενκαψακιωμένη οβαλβουμίνη παρασκευάστηκαν με την μέθοδο του διπλού γαλακτώματος και χαρακτηρίσθηκαν. Στην συνέχεια μελετήθηκε η ικανότητα των νανοσφαιρών να εισχωρούν στο δέρμα μυών μετά από εφαρμογή τους σε περιοχή της πλάτης των μυών από την οποία είχαν απομακρυνθεί οι τρίχες με ξύρισμα. Για αυτή την μελέτη χρησιμοποιήθηκαν αρχικά κενές νανοσφαίρες φθορίζοντος πολυμερούς PLA-pyren-butanol (μονο-επισημασμένες) και νανοσφαίρες φθορίζοντος πολυμερούς PLA-pyren-butanol με ενκαψακιωμένη ροδαμίνη (διπλά-επισημασμένες). Με την βοήθεια φθορίζοντος μικροσκοπίου παρατηρήθηκε ότι οι νανοσφαίρες έχουν εισχωρήσει στις εσωτερικές στοιβάδες του ιστού, σε στοιβάδες αρκετά πιο βαθιά από την κερατίνη στοιβάδα. Στην συνέχεια διερευνήθηκε η οδός εισόδου νανοσφαιρών επισημασμένων με φθορίζουσα αλβουμίνη (FITC-albumin) στο δέρμα χρησιμοποιώντας συνεστιακό μικροσκόπιο σάρωσης (confocal laser microscopy). Παρατηρήθηκε ότι οι νανοσφαίρες εμφανίστηκαν ικανές να διεισδύουν στο εσωτερικό του δέρματος μέσω των θυλάκων των τριχών του ιστού ενώ δεν παρατηρήθηκε άλλη οδός εισόδου των νανοσφαιρών στο εσωτερικό του δέρματος Για την ανίχνευση της ειδικής ανοσοαπόκρισης μελετήθηκαν αντιοροί προς την ολική ΙgG και των ισοτύπων IgG1 και IgG2a έναντι της οβαλβουμίνης. Επίσης πραγματοποιήθηκαν πειράματα σε κυτταρικό επίπεδο στα σπληνοκύτταρα που ελήφθησαν από τους μύες οι οποίοι ανοσοποιήθηκαν διαδερμικά, με την μέτρηση του πολλαπλασιασμού των σπληνοκυττάρων και μετρήθηκαν τα επίπεδα των κυτταροκινών (IL-4, IL-10, IFN-γ και IL-2) στα υπερκείμενα καλλιεργειών των σπληνοκυττάρων κατόπιν in vitro διέγερσης με το αντιγόνο. Στο πρώτο μέρος της μελέτης έγινε διαδερμική χορήγηση (σε ξυρισμένη περιοχή της πλάτης) διαφορετικών μορφών αντιγόνου (ενκαψακιωμένη ή ελεύθερη οβαλβουμίνη και παρουσία ή απουσία ανοσοενισχυτικού). Οι νανοσφαίρες με το αντιγόνο παρουσίασαν παρόμοια επίπεδα ολικών αντισωμάτων IgG με την ελεύθερη οβαλβουμίνη (οβαλβουμίνη σε υδατικό διάλυμα). Επιπρόσθετα, και με τις δύο μορφές χορήγησης του αντιγόνου (διάλυμα και νανοσφαίρες) η συγχορήγηση τοξίνης της χολέρας προκάλεσε αύξηση της παραγωγής αντισωμάτων IgG. Μεγαλύτερη αύξηση παρατηρήθηκε στην περίπτωση των νανοσφαιρών. Τα αποτελέσματα του πειράματος σε κυτταρικό επίπεδο δείχνουν ότι οι νανοσφαίρες με το ενκαψακιωμένο αντιγόνο συν το ανοσοενισχυτικό προκάλεσαν αρκετά υψηλότερες αποκρίσεις IFN-γ σε σύγκριση με όλες τις υπόλοιπες μορφές χορήγησης. Στο δεύτερο μέρος της μελέτης διερευνήθηκε πρωταρχικά η ικανότητα της διαδερμικής χορήγησης ενκαψακιωμένου και μη-ενκαψακιωμένου αντιγόνου (OVA) να επάγει αυξημένη ανοσοαπόκριση μετά από μετέπειτα «πρόκληση» με το αντιγόνο (priming efficiency) και δευτερευόντως η ανοσοαπόκριση που λαμβάνεται με την συνχορήγηση μικρότερης δόσης ανοσοενισχυτικού (50μg τοξίνης της χολέρας ανά μυ). Διαπιστώθηκε ότι η συνχορήγηση έστω και μικρότερης δόσης ανοσοενισχυτικού προκαλεί βελτίωση της ανοσοαπόκρισης σε αντισώματα, ιδιαίτερα στην περίπτωση του ελεύθερου αντιγόνου. Πιο σημαντικό όμως ίσως είναι ότι τα επίπεδα ολικής IgG μετά από υποδόρια χορήγηση 50 μg OVA ανά μυ (δόση «πρόκλησης») σε μύες που είχαν ήδη ανοσοποιηθεί με δύο διαδερμικές δόσεις ενκαψακιωμένης και μη-ενκαψακιωμένης OVA ήταν υψηλότερα (περίπου διπλάσια με όλες τις μορφές χορήγησης) από τα επίπεδα ολικής IgG που λήφθηκαν από μύες που δέχτηκαν μονάχα την υποδόρια δόση με το αντιγόνο. Σε κυτταρικό επίπεδο, οι νανοσφαίρες παρουσίασαν ελαφρώς υψηλότερα επίπεδα παραγωγής IFN-γ και IL-2 από το διάλυμα του αντιγόνου ενώ επέδειξαν παρόμοια επίπεδα IL-4 και IL-10 με το διάλυμα του αντιγόνου. Τα επίπεδα των IFN-γ και IL-2 που μετρήθηκαν για τις νανοσφαίρες με το αντιγόνο συν το ανοσοενισχυτικό ήταν σημαντικά υψηλότερα από όλες τις άλλες μορφές χορήγησης του αντιγόνου. Με βάση τα αποτελέσματα αυτά φαίνεται ότι σε κυτταρικό επίπεδο η διαδερμική χορήγηση του αντιγόνου ενκαψακιωμένου σε PLA νανοσφαίρες πλεονεκτεί της χορήγησης του ελεύθερου αντιγόνου. Τα αυξημένα επίπεδα IFN-γ και IL-2 με την ενκαψακιωμένη μορφή του αντιγόνου είναι πιθανόν να σχετίζονται με αλλαγή της ισορροπίας της ανοσοαπόκρισης προς μία περισσότερο Th1 κατεύθυνση. Οι διαφορές που προκύπτουν στην ανοσογονική συμπεριφορά μεταξύ της ενκαψακιωμένης οβαλβουμίνης στις νανοσφαίρες και της ελεύθερης οβαλβουμίνης μπορεί να οφείλονται στον διαφορετικό τρόπο πρόσληψης και παρουσίασης του αντιγόνου από τα ανοσοπαρουσιαστικά κύτταρα. Η διαδερμική χορήγηση του αντιγόνου ενκαψακιωμένου σε νανοσφαίρες PLA δεν παρείχε σημαντικό πλεονέκτημα όσον αφορά την χυμική ανοσοαπόκριση (παραγωγή αντισωμάτων) σε σύγκριση με την διαδερμική χορήγηση ελεύθερου αντιγόνου. Τα αποτελέσματα της παρούσας μελέτης δείχνουν ότι το σύστημα διαδερμικής χορήγησης του αντιγόνου (η σύνθεση του διαδερμικού «εμβολίου») έχει σημαντική επίδραση στην λαμβανόμενη ανοσοαπόκριση και δικαιολογούν την περαιτέρω μελέτη της χρησιμότητας των PLA νανοσφαιρών στην διαδερμική χορήγηση αντιγόνων. / The skin is part of the epithelial system of the body, which serves as an effective barrier against a potentially hostile environment. As a structural barrier, the skin keeps water and other vital substances in and foreign material out. As an immunological barrier the skin is rich of immunocompetent cells, such as Langerhans cells. Recent studies have demonstrated the potential of skin as a non-invasive route for administering antigens. In the case of protein antigens, the skin barrier limits the penetration of high molecular weight molecules, preventing their use for therapeutic purposes. However, co-administration of proteins with cholera toxin (CT) has been shown to enhance protein-specific antibody responses. Also, CT was not toxic when applied onto bare skin. Using non-invasive routes such as the skin for vaccine delivery could be advantageous for vaccination for several reasons. Subcutaneous delivery of antigen-loaded PLA- and PLGA-microspheres and nanospheres has been found capable of inducing efficient and long-lasting immune responses. In the present study we investigated the immune responses obtained after transcutaneous administration of a model antigen (ovalbumin, OVA) encapsulated in PLA nanospheres. OVA-loaded PLA nanospheres were applied onto bare skin of Balb/c mice in the presence or the absence of CT and the immune responses obtained were compared to those obtained with free OVA (OVA aqueous solution). Also, we investigated the possible route of entry of the nanospheres in the skin. PLA polymer was synthesized by melt polymerization. OVA-loaded nanospheres were prepared by a double emulsion technique. We investigated the ability of nanospheres labeled with one fluorescent dye (1- pyrene-butanol coupled to PLA) and nanospheres labeled with two fluorescent dyes (1-pyrene-butanol/PLA and dextran-rhodamine) to penetrate into the mouse skin using fluorescent microscopy. The results indicated that the nanospheres were capable of entering into the inner layers of the skin. Then, we investigated the possible route of nanospheres entrance into the skin using confocal laser microscopy. The nanopsheres appeared capable to enter the skin only through the duct of the hair follicles. We did not observe other modes of nanospheres entry into the skin in any of the skin samples examined. We proceeded in the evaluation of immune responses elicited after transcutaneous immunization with OVA aqueous solution and OVA-loaded PLA nanospheres. For the evaluation of the immune responses, total IgG, IgG1 and IgG2a levels were measured in anti-serum samples. We also measured the proliferative responses of splenocytes retrieved from the immunized mice and the IFN-γ, IL-2, IL-4 and IL-10 responses in the supernatant of cultured splenocytes after in vitro stimulation with OVA. On the first stage of the study, we transcutaneously immunized mice onto their bare back with OVA in solution or OVA-loaded nanospheres (200 μg OVA per mouse) in the presence or absence of CT (100 μg CT per mouse). The OVA-loaded nanospheres elicited similar total IgG responses with the OVA solution. With both modes of antigen delivery (aqueous solution and nanospheres-entrapped), the coadministration of CT adjuvant increased IgG response, especially that obtained with the OVA-loaded nanospheres. Also, the OVA-loaded nanospheres plus CT exhibited higher IFN-γ responses than the other formulations tested but similar IL-4 and IL-10 responses. On the second stage of the study, we investigated mainly the ability of transcutaneous delivery of OVA-loaded nanospheres and OVA solution to induce an increased immune response after a subcutaneous booster (“challenge”) with the antigen. Also, we investigated the immune responses obtained by transcutaneous immunization with a lower dose of CT. We observed that even a relatively small amount of CT (50 μg per mouse) could augment antibody responses, especially in the case of the free antigen. It is important to note that the IgG responses obtained after subcutaneous booster with OVA (50 μg per mouse) of mice previously primed with 2 transcutaneous doses of the different OVA formulations were significantly higher than the IgG responses obtained by mice which received only the subcutaneous dose of OVA. This would indicate that transcutaneous administration of antigens in the form of aqueous solution or antigen-loaded nanospheres can prime antibody responses (can induce “memory” response). All formulations elicited both IgG1 and IgG2a responses, indicating a balanced type of immune response. The OVA-loaded nanospheres exhibited a little higher IFN-γ and IL-2 responses than the OVA-solution and similar IL-4 and IL-10 responses with the OVA solution. On the other hand, the OVA-loaded nanospheres plus CT induced much higher IFN-γ and IL-2 responses than all other formulations tested. These results indicate that as far as the cellular responses induced by transcutaneous antigen administration is concerned it may be advantageous to deliver the antigen in nanosphere-encapsulated form rather than in free (aqueous solution) form. The increased IFN-γ and IL-2 levels obtained with the encapsulated forms of OVA compared to the soluble forms of OVA may indicate a possibility of altering the balance of immune response towards a Th1-type of response using nanosphere-encapsulated antigens. The differences in the immunogenic behavior between the encapsulated antigen (OVA entrapped in nanospheres) and free antigen (OVA solution) may arise from the facilitated uptake and presentation of the encapsulated antigen by antigen presenting cells. Taking into account that the encapsulation of OVA in the nanospheres was accompanied by a significant (around 30%) reduction of OVA antigenicity, it might be expected that optimized OVA-loaded nanospheres, in which the protein would retain its full antigenic potential, could have resulted to more potent IgG responses than those obtained in this study. The results of the present study reveal that the type of antigen formulation could have a pronounced effect on the immune response obtained after transcutaneous administration of the antigen and justify the further investigation of the possible advantages of using PLA nanospheres as the antigen delivery system in transcutaneous immunization.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Skin is the largest immune organ and an ideal site to administer vaccines. However, by nature, skin is not permeable to antigens, which are macromolecules. The major hurdle in skin permeation is the outermost stratum corneum layer. Microneedles have proven feasible to create micron-sized channels in the epidermis of the skin, through which protein and plasmid DNA antigens can penetrate into the viable skin epidermis and dermis. However, the immune responses induced by microneedle-mediated transcutaneous immunization with protein or plasmid DNA alone are generally weak, and a vaccine adjuvant is often required to induce strong immune responses. Data from numerous previous studies have shown that nanoparticles as a vaccine carrier can significantly enhance the immunogenicity of antigens, but the feasibility of utilizing nanoparticles as a vaccine carrier to enhance the immune responses induced by microneedle-mediated transcutaneous immunization has rarely been studied. In this dissertation, using protein antigen (OVA) chemically conjugated onto the surface of solid-lipid nanoparticles and plasmid DNA (pCMV-beta, pVax/opt-BoNT/C-Hc50, and pCI-neo-sOVA) physically coated on the surface of cationic polymeric nanoparticles, we showed that the immune responses induced by microneedle-mediated transcutaneous immunization with protein antigens or plasmid DNA vaccines are significantly enhanced by delivering the proteins and plasmid DNA with nanoparticles. Importantly, microneedle-mediated transcutaneous immunization with proteins or plasmid DNA induces not only systemic immune responses, but also mucosal immune responses. In addition, it is generally believed that microneedles are safe. However, it remained unclear whether the micropores created by microneedles on the skin will also facilitate the permeation of microbes such as bacteria into the skin. In this dissertation, we also designed an unique ex vivo model to evaluate the permeation of live bacteria through mouse skin pretreated with microneedles. The results demonstrated that the risk of potential bacterial infection associated with microneedle treatment is not greater than that associated with a hypodermic needle injection. / text