Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

A busca de adjuvantes que estimulem de forma eficiente à resposta imune celular representa uma importante contribuição para a pesquisa de vacinas. O principal objetivo deste trabalho foi a avaliação do efeito imunoestimulador de flagelinas expressas por linhagens de Salmonella sp., particularmente na ativação de respostas mediadas por células T CD8+, ao antígeno modelo ovalbumina. Em uma segunda etapa, foram investigados os efeitos adjuvantes da flagelina FliCd frente a um epítopo T CD8+, específico da proteína CS de P. yoelli. Animais foram imunizados com linhagens atenuadas de S. Dublin que expressam flagelinas geneticamente fusionadas ao epítopo CS ou com flagelina purificada co-administrada ou geneticamente fusionada ao antígeno alvo. A ativação de células T CD8+, foi monitorada por ELISPOT após estimulação com peptídeos específicos. Os resultados obtidos demonstram que a FliCd é capaz de modular o sistema imune, atuando particularmente na ativação de respostas mediadas por células T CD8+, sugerindo seu potencial para a composição de vacinas terapêuticas. / The search for adjuvants stimulating efficient cellular immune responses represents an important contribution in vaccine research. In the present work, we evaluated immunostimulatory effect of flagellins expressed by Salmonella sp. strains, particularly on activation of CD8+, T cells using ovalbumin as a model antigen. Secondly, we explored the adjuvant effect of the FliCd flagellin using an epitope from the CS protein of P. yoelli. Mice were immunized with S. Dublin strains that express flagellin genetically fused to CS epitope or purified flagellin co-administrated or genetically fused to the target antigen. The CD8+, T cells activation was monitored by ELISPOT after stimulation with specific peptides. Our results show that FliCd flagellin modulates the immune system, increasing CD8+, T cell activation, unveiling its potencial use in therapeutic vaccine approaches.

A busca de adjuvantes que estimulem de forma eficiente à resposta imune celular representa uma importante contribuição para a pesquisa de vacinas. O principal objetivo deste trabalho foi a avaliação do efeito imunoestimulador de flagelinas expressas por linhagens de Salmonella sp., particularmente na ativação de respostas mediadas por células T CD8+, ao antígeno modelo ovalbumina. Em uma segunda etapa, foram investigados os efeitos adjuvantes da flagelina FliCd frente a um epítopo T CD8+, específico da proteína CS de P. yoelli. Animais foram imunizados com linhagens atenuadas de S. Dublin que expressam flagelinas geneticamente fusionadas ao epítopo CS ou com flagelina purificada co-administrada ou geneticamente fusionada ao antígeno alvo. A ativação de células T CD8+, foi monitorada por ELISPOT após estimulação com peptídeos específicos. Os resultados obtidos demonstram que a FliCd é capaz de modular o sistema imune, atuando particularmente na ativação de respostas mediadas por células T CD8+, sugerindo seu potencial para a composição de vacinas terapêuticas. / The search for adjuvants stimulating efficient cellular immune responses represents an important contribution in vaccine research. In the present work, we evaluated immunostimulatory effect of flagellins expressed by Salmonella sp. strains, particularly on activation of CD8+, T cells using ovalbumin as a model antigen. Secondly, we explored the adjuvant effect of the FliCd flagellin using an epitope from the CS protein of P. yoelli. Mice were immunized with S. Dublin strains that express flagellin genetically fused to CS epitope or purified flagellin co-administrated or genetically fused to the target antigen. The CD8+, T cells activation was monitored by ELISPOT after stimulation with specific peptides. Our results show that FliCd flagellin modulates the immune system, increasing CD8+, T cell activation, unveiling its potencial use in therapeutic vaccine approaches.

Un des enjeux majeurs de la thérapie contre le cancer est le développement d’immunothérapies innovantes ciblées et efficaces sur le long terme. Dans ce but, nous avons tiré profit de la versatilité des liposomes pour concevoir une plateforme vaccinale modulable associant i) un épitope peptidique CD4 universel capable de stimuler les lymphocytes T CD4+, ii) un épitope peptidique CD8 dérivé de la tumeur, induisant la différenciation des lymphocytes T CD8+ en lymphocytes T cytotoxiques et iii) un ou des agonistes des récepteurs Toll-like ou NOD-like qui agissent comme des adjuvants en activant les cellules dendritiques. Après plusieurs étapes de criblage, trois vaccins ont été développés, spécifiques d’un modèle orthotopique de cancer du poumon (TC-1) et différant uniquement par leur adjuvant. Une étude comparative dans un modèle murin a été réalisée. Ces travaux ont permis de comparer leur limite temporelle d’efficacité, de mettre en évidence leur mécanismes immunitaires respectifs et de démontrer la supériorité thérapeutique des liposomes contenant un agoniste du TLR4 (MPLA). Ces travaux ont montré l’intérêt d’une plateforme liposomique modulable pour la conception de vaccins personnalisés. / Currently, a challenging goal in the area of cancer treatment is the development of innovative targeted antitumoral immunotherapies with a long-term efficiency. In this context, we took advantage of liposomal nanoparticles properties for the conception of a tunable vaccine platform allowing the strategical conception of vaccines containing: i) a CD4 epitope peptide able to stimulate CD4+ T helper cells ii) a tumor CD8 epitope peptide, which induces the differentiation of CD8+ T cells in cytotoxic T cells and iii) Toll or Nod-Like receptor agonist(s), which act as adjuvant for the activation of dendritic cell. After several screening steps, three vaccines, specific tardeting an orthotopic lung tumor model (TC-1) and differing only by their adjuvant composition, were successfully developed. Subsequently, a comparative study of their efficacy time limit and their immunologic mode of action was performed. This study showed the therapeutic supremacy of liposomal vaccines containing a TLR4 agonist (MPLA). In this work, we demonstrated the value of a customizable liposomal platform for the conception of personalized vaccines and we highlighted the necessity of immune monitoring for a better understanding of vaccines impact and the prediction of their efficacy.