RESUMO
Activity of adjuvanted vaccines is difficult to predict in vitro and in vivo. The wide compositional and conformational range of formulated adjuvants, from aluminum salts to oil-in-water emulsions, makes comparisons between physicochemical and immunological properties difficult. Even within a formulated adjuvant class, excipient selection and concentration can alter potency and physicochemical properties of the mixture. Complete characterization of physicochemical properties of adjuvanted vaccine formulations and relationship to biological response is necessary to move beyond a guess-and-check paradigm toward directed development. Here we present a careful physicochemical characterization of a two-component nanosuspension containing synthetic TLR-4 agonist glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at various molar ratios. Physicochemical properties were compared with potency, as measured by stimulation of cytokine production in human whole blood. We found a surprising, nonlinear relationship between physicochemical properties and GLA-DPPC ratios that corresponded well with changes in biological activity. We discuss these data in light of the current understanding of TLR4 activation and the conformation-potency relationship in development of adjuvanted vaccines.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Adjuvantes Imunológicos/química , Dissacarídeos/química , Lipídeo A/análogos & derivados , Miristatos/química , Nanoestruturas/química , Receptor 4 Toll-Like/agonistas , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Acilação , Adjuvantes Imunológicos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Fenômenos Químicos , Citocinas/agonistas , Citocinas/metabolismo , Dissacarídeos/farmacologia , Combinação de Medicamentos , Humanos , Testes de Liberação de Interferon-gama , Lipídeo A/química , Lipídeo A/farmacologia , Miristatos/farmacologia , Concentração Osmolar , Tamanho da Partícula , Fosforilação , Propriedades de Superfície , Suspensões , Temperatura de TransiçãoRESUMO
BACKGROUND: Adjuvant formulations are critical components of modern vaccines based on recombinant proteins, which are often poorly immunogenic without additional immune stimulants. Oil-in-water emulsions comprise an advanced class of vaccine adjuvants that are components of approved seasonal and pandemic influenza vaccines. However, few reports have been published that systematically evaluate the in vitro stability and in vivo adjuvant effects of different emulsion components. OBJECTIVES: To evaluate distinct classes of surfactants, oils, and excipients, for their effects on emulsion particle size stability, antigen structural interactions, and in vivo activity when formulated with a recombinant H5N1 antigen. METHODS: Emulsions were manufactured by high pressure homogenization and characterized alone or in the presence of vaccine antigen by dynamic light scattering, zeta potential, viscosity, pH, hemolytic activity, electron microscopy, fluorescence spectroscopy, and SDS-PAGE. In vivo vaccine activity in the murine model was characterized by measuring antibody titers, antibody-secreting plasma cells, hemagglutination inhibition titers, and cytokine production. RESULTS: We demonstrate that surfactant class and presence of additional excipients are not critical for biological activity, whereas oil structure is crucial. Moreover, we report that simplified two-component emulsions appear more stable by particle size than more complex formulations.Finally, differences in antigen structural interactions with the various emulsions do not appear to correlate with in vivo activity. CONCLUSIONS: Oil-in-water emulsions can significantly enhance antibody and cellular immune responses to a pandemic influenza antigen. The dramatic differences in adjuvant activity between squalene-based emulsion and medium chain triglyceride-based emulsion are due principally to the biological activity of the oil composition rather than physical interactions of the antigen with the emulsion.