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1.
Eur J Pharm Biopharm ; 87(2): 310-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24434202

RESUMO

Next generation vaccine adjuvants include Toll like receptor agonists, which are mostly extracted from microorganisms, but synthetic small molecule TLR agonists have also been identified. However, their delivery systems have not been optimized for effective administration in conjunction with antigens. Here, we describe a novel approach in which a small molecule TLR agonist was directly conjugated to antigen to ensure effective co-delivery. We describe the conjugation of a recombinant protective antigen from Streptococcus pneumoniae linked to a TLR7 agonist. Following thorough characterization to ensure no aggregation, the conjugate was evaluated in a murine infection model. Results showed that the conjugate extended the animals' survival after lethal challenge with S. pneumoniae. Comparable results were obtained with a dose 10-fold lower than that of the native unconjugated antigen. Notably, the animals immunized with the same dose of unconjugated TLR7 agonist and antigen showed no adjuvant effect. The increased immunogenicity was likely a consequence of the co-localization of TLR7 agonist and antigen by chemical binding and was more effective than simple co-administration. This approach can be adopted to increase potency of a broad variety of antigens and reduce the dose of antigen required to induce protective immunity.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/farmacologia , Imunoconjugados/farmacologia , Glicoproteínas de Membrana/agonistas , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/farmacologia , Streptococcus pneumoniae/imunologia , Receptor 7 Toll-Like/agonistas , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Carga Bacteriana , Modelos Animais de Doenças , Feminino , Células HL-60 , Humanos , Imunização , Imunoconjugados/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , Fagocitose/efeitos dos fármacos , Infecções Pneumocócicas/sangue , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/genética , Fatores de Tempo , Receptor 7 Toll-Like/metabolismo
2.
Eur J Biochem ; 268(24): 6491-7, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11737204

RESUMO

alpha-Chymotrypsin activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide (GPNA) in aqueous media in the presence of synthetic surfactants, which differ in the flexibility of their bulky head groups. Superactivity can be ascribed to the presence of the tributylammonium residue on the surfactant head group, as in p-octyloxybenzyltributylammonium bromide (pOOTBABr), while in the presence of a more rigid moiety, i.e. a cyclic one, no activation was found. A nonmicellizable quaternary ammonium salt, the tetrabutylammonium bromide (TBABr), which has a head group structure very similar to pOOTBABr, not only induces a remarkable superactivation, at a concentration 80-fold higher than pOOTBABr, but also allows the enzyme to retain a high residual activity for long periods of time. The presence of a lipophilic chain, which by interacting with apolar residues on the enzyme surface, probably penetrates into hydrophobic pockets of the protein and causes a rapid inactivation. In 0.4 m TBABr, a 20-fold increase both in kcat and Km values, with respect to buffer alone, was found. The increase of Km could be attributed either to a true decrease in affinity between enzyme and substrate or alternatively to the presence of TBA+ ions near the catalytic region. They could interact with protein residues around the active site and bind to negatively charged GPNA molecules, lowering the local substrate concentration. Spectroscopic experiments (CD and fluorescence) show minor changes of protein conformation in 0.4 m TBABr, while at 1 m a strong modification of both spectra was observed.


Assuntos
Quimotripsina/metabolismo , Animais , Cátions , Bovinos , Dicroísmo Circular , Estabilidade Enzimática , Cinética , Espectrometria de Fluorescência , Tensoativos/farmacologia
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