RESUMEN
Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Tetrahymena thermophila/genética , Animales , Anticuerpos Antivirales/biosíntesis , Perros , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Macaca , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Proteína Adaptadora de Señalización NOD2/administración & dosificación , Poliésteres/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunologíaRESUMEN
Respiratory syncytial virus (RSV) infection is the most important viral cause of severe respiratory disease in infants and children worldwide and also forms a serious threat in the elderly. The development of RSV vaccine, however, has been hampered by the disastrous outcome of an earlier trial using an inactivated and parenterally administered RSV vaccine which did not confer protection but rather primed for enhanced disease upon natural infection. Mucosal administration does not seem to prime for enhanced disease, but non-replicating RSV antigen does not induce a strong mucosal immune response. We therefore investigated if mucosal immunization with inactivated RSV supplemented with innate receptor ligands, TLR9 (CpG ODN) and NOD2 (L18-MDP) through the upper or total respiratory tract is an effective and safe approach to induce RSV-specific immunity. Our data show that beta-propiolactone (BPL) inactivated RSV (BPL-RSV) supplemented with CpG ODN and L18-MDP potentiates activation of antigen-presenting cells (APC) in vitro, as demonstrated by NF-κB induction in a model APC cell line. In vivo, BPL-RSV supplemented with CpG ODN/L18-MDP ligands induces local IgA responses and augments Th1-signature IgG2a subtype responses after total respiratory tract (TRT), but less efficient after upper respiratory tract (intranasal, IN) immunization. Addition of TLR9/NOD2 ligands to the inactivated RSV also promoted affinity maturation of RSV-specific IgG antibodies and shifted T cell responses from mainly IL-5-secreting cells to predominantly IFN-γ-producing cells, indicating a Th1-skewed response. This effect was seen for both IN and TRT immunization. Finally, BPL-RSV supplemented with TLR9/NOD2 ligands significantly improved the protection efficacy against a challenge with infectious virus, without stimulating enhanced disease as evidenced by lack of eotaxin mRNA expression and eosinophil infiltration in the lung. We conclude that mucosal immunization with inactivated RSV antigen supplemented with TLR9/NOD2 ligands is a promising approach to induce effective RSV-specific immunity without priming for enhanced disease.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteína Adaptadora de Señalización NOD2/administración & dosificación , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Receptor Toll-Like 9/administración & dosificación , Administración a través de la Mucosa , Animales , Línea Celular , Femenino , Inmunidad Mucosa , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Células TH1/inmunología , Proteína Tumoral Controlada Traslacionalmente 1 , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
BACKGROUND: Airway tolerance is critical for protecting the lung from inflammatory disease driven by allergens. However, factors that disrupt tolerance processes and then lead to susceptibility to developing allergic asthma remain elusive. OBJECTIVE: To investigate whether recognition of bacterial microbial-associated molecular patterns in the lung may result in susceptibility to developing allergic reactions, and to understand the molecular mechanisms by which such triggers block natural tolerance. METHODS: Ligands of intracellular microbial-associated molecular pattern recognition receptors-the nucleotide-binding oligomerization domain (Nod)-like receptors, Nod1 and Nod2-were given intranasally with antigen, and their ability to modulate airway tolerance was analyzed. RESULTS: Intranasal Nod2 ligand rapidly induced lung expression of the innate cytokines thymic stromal lymphopoietin and IL-25, and thymic stromal lymphopoietin promoted expression of OX40 ligand, a T-cell-costimulatory ligand, on lung CD11c(+)CD11b(+) cells and B220(+) cells. Together these 3 molecules blocked the generation of antigen-specific CD4(+)forkhead box protein 3(+) adaptive regulatory T cells and concomitantly drove IL-4-producing CD4 T cells. By altering the regulatory T/T(H)2-cell balance, tolerance was blocked, and sensing of Nod2 ligand resulted in subsequent susceptibility to developing eosinophil-dominated airway inflammation. CONCLUSION: We show that a Nod-like receptor is a novel, previously unrecognized, pathway that adversely links innate and adaptive immunity and leads to allergic disease and asthmatic lung inflammation.