RESUMO
As an alternative strategy to classical inactivated viral vaccine against FMDV, naked DNA vaccine is attractive because of safety, flexibility and low cost. However DNA vaccination is usually poorly efficient in target species. Indeed we found that naked DNA plasmids encoding for P1-2A3C3D and GM-CSF proteins did not induce any detectable immunity against FMDV in sheep. Interestingly, we demonstrate herein that formulations of DNA on poly(D,L-lactide-co-glycolide) (PLG) or in lipofectin triggered divergent types of immune responses: PLG stimulated a T cell response and could elicit significant neutralising antibody titers, whereas lipofectin generated even higher antibody titers but no significant T cell response. The DNA/PLG regimen used in five sheep protected against clinical symptoms and viraemia and prevented the carrier state in four of them. Thus formulated DNA can be remarkably efficient against FMDV in a ruminant species that is usually refractory to DNA vaccination.
Assuntos
Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Doenças dos Ovinos/prevenção & controle , Vacinas Virais/imunologia , Animais , Formação de Anticorpos/imunologia , Portador Sadio , Ensaio de Imunoadsorção Enzimática , Excipientes , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Hipersensibilidade Tardia/imunologia , Imunidade Celular/imunologia , Interferon gama/biossíntese , Ácido Láctico , Linfonodos/citologia , Linfonodos/imunologia , Fosfatidiletanolaminas , Plasmídeos/genética , Plasmídeos/imunologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Ovinos , Linfócitos T/imunologia , Transfecção , Vacinas de DNA/imunologia , Viremia/sangue , Viremia/imunologia , Viremia/virologiaRESUMO
We investigated the ability of pig ileal Peyer's patch segments to transport intestinal poly (D,L-lactide-co-glycolide) microspheres (PLGA MS) from intestinal lumen across the mucosae using in situ and ex vivo segments with confocal laser scanning microscopy (CLSM) and transmission electronic microscopy (TEM). From a global aspect, CLSM suggested that PLGA MS were translocated by M cells labelled with a FITC-conjugated anti-cytokeratin peptide 18, and transported through the follicle-associated epithelium (FAE) in the dome area in both types of experiments. At the ultrastructural level, TEM showed the traffic of PLGA MS throughout M cells, their transport into the basolateral invaginations of the M cells and their subsequent migration into the dome area and the follicular area in contact with macrophages and lymphatic vessels. Although in situ experiments allowed following the migration of PLGA MS until mesenteric lymph nodes, an ex vivo model could be used as a useful tool to study the targeting ability of PLGA MS formulations to the gut-associated lymphoid tissue (GALT).
Assuntos
Íleo/metabolismo , Ácido Láctico , Nódulos Linfáticos Agregados/metabolismo , Ácido Poliglicólico , Polímeros , Animais , Fenômenos Químicos , Físico-Química , Portadores de Fármacos , Fluorescência , Íleo/anatomia & histologia , Imuno-Histoquímica , Técnicas In Vitro , Absorção Intestinal , Mucosa Intestinal/metabolismo , Linfonodos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Microesferas , Nódulos Linfáticos Agregados/anatomia & histologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , SuínosRESUMO
The study aimed to investigate on a pig alveolar macrophage culture model the influence of: (1) poly(D,L-lactide-co-glycolide) characteristics, (2) the residual poly(vinyl alcohol) (PVA) and (3) the nature of encapsulated proteins, immunoglobulin Y (IgY) or bovine serum albumin, on the microspheres phagocytosis efficiency. The phagocytosis evaluation was performed by flow cytometry and has allowed a screening of microspheres formulations. The hydrophilicity of microspheres resulting from the nature of the polymer and/ or from the residual hydrophilic surface agent (PVA) led to a decrease of phagocytosis intensity. The phagocytosis results of IgY-loaded microspheres strongly suggested that the phagocytosis was increased when the phagocytic cell possessed a receptor for this protein on its surface.
Assuntos
Ácido Láctico/metabolismo , Macrófagos Alveolares/metabolismo , Fagocitose , Ácido Poliglicólico/metabolismo , Polímeros/metabolismo , Álcool de Polivinil/farmacologia , Animais , Citometria de Fluxo , Imunoglobulinas/administração & dosagem , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Soroalbumina Bovina/administração & dosagem , SuínosRESUMO
The phagocytosis of fluorescent poly(D,L-lactide-co-glycolide) microspheres by fresh and frozen pig alveolar macrophages was investigated by optical microscopy on adherent cell culture and by flow cytometry with cell suspension. The kinetic of phagocytosis was studied on a 360 min period as a function of the ratio between microspheres and macrophages (MS:AM ratio from 1:1 to 10:1). No difference of phagocytosis between fresh and frozen macrophages was observed whatever the MS:AM ratio following flow cytometric evaluation while a significant phagocytosis pattern was noticed following optical microscopic evaluation for the highest ratio. The intensity of phagocytosis was dependent on the duration of incubation and dependent, but not proportionally, to the MS:AM ratio showing that the highest efficiency was obtained with the MS:AM ratio of 1:1. Flow cytometry analysis has shown a correlation between cell population and fluorescent events suggesting that phagocytosis of nonfluorescent antigen-loaded particles with different characteristics could be investigated.
