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1.
BMC Immunol ; 14 Suppl 1: S4, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23458578

RESUMO

The use of new adjuvants in vaccine formulations is a subject of current research. Only few parenteral adjuvants have been licensed. We have developed a mucosal and parenteral adjuvant known as AFCo1 (Adjuvant Finlay Cochleate 1, derived from proteoliposomes of N. meningitidis B) using a dialysis procedure to produce them on lab scale. The immunogenicity of the AFCo1 produced by dialysis has been already evaluated, but it was necessary to demonstrate the feasibility of a larger-scale manufacturing process. Therefore, we used a crossflow diafiltration system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The aim of this work was to produce AFCo1 on pilot scale, while conserving the adjuvant properties. The proteoliposomes (raw material) were resuspended in a buffer containing sodium deoxycholate and were transformed into AFCo1 under the action of a calcium forming buffer. The detergent was removed from the protein solution by diafiltration to a constant volume. In this CFS, we used a hollow fiber cartridge from Amicon (polysulfona cartridge of 10 kDa porosity, 1mm channel diameter of fiber and 0.45 m² area of filtration), allowing production of a batch of up to 20 L. AFCo1 were successfully produced by tangential filtration to pilot scale. The batch passed preliminary stability tests. Nasal immunization of BALB/c mice, induced specific saliva IgA and serum IgG. The induction of Th1 responses were demonstrated by the induction of IgG2a, IFNγ and not IL-5. The adjuvant action over Neisseria (self) antigens and with co-administered (heterologous) antigens such as ovalbumin and a synthetic peptide from haemolytic Streptococcus B was also demonstrated.


Assuntos
Adjuvantes Imunológicos , Vacinas Meningocócicas/biossíntese , Neisseria meningitidis Sorogrupo B/imunologia , Proteolipídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Imunização , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Interleucina-5/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Projetos Piloto , Células Th1/imunologia
2.
PLoS One ; 7(10): e46461, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077508

RESUMO

Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.


Assuntos
Cálcio/química , Imunidade nas Mucosas , Fosfolipídeos/química , Proteolipídeos/química , Vibrio cholerae/química , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica de Fratura por Congelamento , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Vibrio cholerae/ultraestrutura
3.
Methods ; 49(4): 309-15, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19545630

RESUMO

Conservative estimates place the death toll from cholera at more than 100,000 persons each year. A particulate mucosal vaccine strategy combining antigens and immune stimulator molecules from Vibrio cholerae to overcome this problem is described. Proteoliposomes extracted from V. cholerae O1 were transformed into cochleates (AFCo2, Adjuvant Finlay cochleate 2) through a calcium inducible rotary dialysis method. Light microscopy was carried out and tubules of 16.25+/-4.57 microm in length were observed. Western blots were performed to verify the immunochemical properties of the main AFCo2 incorporated antigens, revealing full recognition of the outer membrane protein U (OmpU), lipopolysaccharide (LPS), and mannose-sensitive hemagglutinin (MSHA) antigens. AFCo2 were administered by the intranasal route using a two or three dose schedule and the immune response against V. cholerae antigens was assessed. Three AFCo2 doses were required to induce significant (p<0.05), antigen specific IgA in saliva (1.34+/-0.135) and feces (0.60+/-0.089). While, two or three doses of AFCo2 or proteoliposomes induce similar specific IgG and vibriocidal activity responses in sera. These results show for the first time that AFCo2 can be obtained from V. cholerae O1 proteoliposomes and have the potential to protect against the pathogen when administered intranasally.


Assuntos
Mucosa/imunologia , Proteolipídeos/administração & dosagem , Proteolipídeos/imunologia , Vibrio cholerae O1/imunologia , Administração Intranasal , Animais , Ácido Desoxicólico/administração & dosagem , Ácido Desoxicólico/imunologia , Ácido Edético/administração & dosagem , Ácido Edético/imunologia , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/efeitos dos fármacos
4.
Methods ; 49(4): 301-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19410000

RESUMO

Most pathogens either invade the body or establish infection in mucosal tissues and represent an enormous challenge for vaccine development by the absence of good mucosal adjuvants. A proteoliposome-derived adjuvant from Neisseria meningitidis serogroup B (AFPL1, Adjuvant Finlay Proteoliposome 1) and its derived cochleate form (Co, AFCo1) contain multiple pathogen-associated molecular patterns as immunopotentiators, and can also serve as delivery systems to elicit a Th1-type immune response. The present studies demonstrate the ability of AFPL1and AFCo1 to induce mucosal and systemic immune responses by different mucosal immunizations routes and significant adjuvant activity for antibody responses of both structures: a microparticle and a nanoparticle with a heterologous antigen. Therefore, we used female mice immunized by intragastric, intravaginal, intranasal or intramuscular routes with both structures alone or incorporated with ovalbumin (OVA). High levels of specific IgG antibody were detected in all sera and in vaginal washes, but specific IgA antibody in external secretions was only detected in mucosally immunized mice. Furthermore, antigen specific IgG1 and IgG2a isotypes were all induced. AFPL1 and AFCo1 are capable of inducing IFN-gamma responses, and chemokine secretions, like MIP-1alpha and MIP-1beta. However, AFCo1 is a better alternative to induce immune responses at mucosal level. Even when we use a heterologous antigen, the AFCo1 response was better than with AFPL1 in inducing mucosal and systemic immune responses. These results support the use of AFCo1 as a potent Th1 inducing adjuvant particularly suitable for mucosal immunization.


Assuntos
Imunização/métodos , Mucosa/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Proteolipídeos/administração & dosagem , Proteolipídeos/imunologia , Administração Intranasal , Administração Intravaginal , Animais , Células Cultivadas , Ácido Desoxicólico/administração & dosagem , Ácido Desoxicólico/imunologia , Ácido Edético/administração & dosagem , Ácido Edético/imunologia , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/efeitos dos fármacos
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