RESUMO
Adjuvants are essential components of subunit, recombinant, nonreplicating and killed vaccines, as they are substances that boost, shape, and/or enhance the immune response triggered by vaccination. Saponins obtained from the Chilean Q. saponaria tree are used as vaccine adjuvants in commercial vaccines, although they are scarce and difficult to obtain. In addition, tree felling is needed during its extraction, which has ecological impact. Q. brasiliensis leaf-extracted saponins arise as a more sustainable alternative, although its use is still limited to preclinical studies. Despite the remarkable immunostimulating properties of saponins, they are toxic to mammalian cells, due to their intrinsic characteristics. For these reasons they are mostly used in veterinary vaccines, although recently the Q. saponaria purified saponin QS-21 has been included in adjuvant systems for human vaccines, such as Mosquirix and Shingrix (GSK). In order to abrogate the toxicity of the saponins fractions, they can be formulated as immunostimulating complexes (ISCOMs). ISCOM-matrices are cage-like nanoparticles of approximately 40 nm, formulated combining saponins and lipids, without antigen, and are great adjuvants able to promote Th1-biased immune responses in a safe manner. Herein we describe how to formulate ISCOM-matrices nanoparticles using Q. brasiliensis purified saponin fractions (IMXQB) by the dialysis method. In addition, we indicate how to verify the appropriate size and homogeneity of the formulated nanoparticles.
Assuntos
ISCOMs , Nanopartículas , Saponinas , Adjuvantes Imunológicos/farmacologia , Adjuvantes de Vacinas , Animais , Humanos , ISCOMs/farmacologia , Vacinas Antimaláricas , Mamíferos , Quillaja , Saponinas de Quilaia , Saponinas/farmacologiaRESUMO
In the last decades, significant efforts have been dedicated to the search for novel vaccine adjuvants. In this regard, saponins and its formulations as "immunostimulating complexes" (ISCOMs) have shown to be capable of stimulating potent humoral and cellular immune responses, enhanced cytokine production and activation of cytotoxic T cells. The immunological activity of ISCOMs formulated with a saponin fraction extracted from Quillaja brasiliensis (QB-90 fraction) as an alternative to classical ISCOMs based on Quil A(®) (IQA) is presented here. The ISCOMs prepared with QB-90, named IQB-90, typically consist of 40-50 nm, spherical, cage-like particles, built up by QB-90, cholesterol, phospholipids and antigen (ovalbumin, OVA). These nanoparticles were efficiently uptaken in vitro by murine bone marrow-derived dendritic cells. Subcutaneously inoculated IQB-90 induced strong serum antibody responses encompassing specific IgG1 and IgG2a, robust DTH reactions, significant T cell proliferation and increases in Th1 (IFN-γ and IL-2) cytokine responses. Intranasally delivered IQB-90 elicited serum IgG and IgG1, and mucosal IgA responses at distal systemic sites (nasal passages, large intestine and vaginal lumen). These results indicate that IQB-90 is a promising alternative to classic ISCOMs as vaccine adjuvants, capable of enhancing humoral and cellular immunity to levels comparable to those induced by ISCOMs manufactured with Quillaja saponaria saponins.
Assuntos
Adjuvantes Imunológicos/farmacologia , ISCOMs/farmacologia , Imunidade Celular , Imunidade Humoral , Imunidade nas Mucosas , Saponinas de Quilaia/farmacologia , Adjuvantes Imunológicos/química , Administração Intranasal , Animais , Células da Medula Óssea/imunologia , Chlorocebus aethiops , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , ISCOMs/química , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Injeções Subcutâneas , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Quillaja/química , Saponinas de Quilaia/química , Coelhos , Saponinas/química , Saponinas/farmacologia , Linfócitos T Citotóxicos/imunologia , Células VeroRESUMO
Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk, with improved opsonic capacity, compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A, fibronectin binding protein A and ß-toxin formulated with Iscom Matrix, characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate+recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells, in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition, antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin, and neutralizing ß-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis.
Assuntos
Imunização/veterinária , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Vacinas Sintéticas/imunologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Bovinos , Linhagem Celular , Coagulase/genética , Coagulase/imunologia , Citocinas/sangue , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , ISCOMs/farmacologia , Imunização/métodos , Mastite Bovina/imunologia , Mastite Bovina/prevenção & controle , Leite/microbiologia , Gravidez , Distribuição Aleatória , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/administração & dosagem , Estatísticas não Paramétricas , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/normasRESUMO
Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)3. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas/imunologia , ISCOMs/farmacologia , Mastite Bovina/imunologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Contagem de Colônia Microbiana/veterinária , Citocinas/sangue , Citocinas/genética , Feminino , Imunização/normas , Imunização/veterinária , Mastite Bovina/microbiologia , Mastite Bovina/prevenção & controle , Leite/microbiologia , Gravidez , RNA/química , RNA/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/genética , Estatísticas não ParamétricasRESUMO
A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n=10) received two doses (100 microg/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n=10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n=10) and G4 (unvaccinated unchallenged, n=8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T. gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine.