Development of polymeric carriers for enhanced immune response of foot-and-mouth disease virus subunit vaccine

Abstract

Foot-and-mouth disease (FMD) is a highly contagious disease susceptible to cloven-hoofed animals such as cattle, pigs, goats, etc. affecting livestock industry. In this aspect, FMDV subunit vaccines have been developed to prevent the spread of this fatal animal epidemic, because they provide several advantages such as no need for attenuation and serological tests that can differentiate infected animals from vaccinated ones and they can be produced with epitopes and have less side effects than live attenuated/inactivated vaccine although FMDV is continuously evolving and mutating, making it difficult to develop FMDV vaccine (mainly live attenuated or inactivated vaccines) to protect animals from disease. However, there are several limitations such as for practical application of subunit vaccines the low stability of subunit vaccine, easy degradation by enzyme and physiological environment. In addition, their low immunogenicity compared to live attenuated vaccines limits the efficacy of subunit vaccine. Therefore, enhancement of stability and immunogenicity of subunit vaccines is main bottleneck in vaccine development. To overcome these limitations, polymeric adjuvants have been introduced to enhance the immunogenicity of subunit vaccines because they introduce immunomodulatory properties and provide the flexibility in the route of vaccine delivery depending on the vaccination strategies. Furthermore, immune response can be greatly regulated by single factor or combination of multiple factors by modification of the polymeric adjuvants such as size of polymeric particle, surface charge, hydrophilicity, molecular weight and chemical properties. In chapter I, pH-sensitive and mucoadhesive thiolated CAP (T-CAP) as a polymeric carrier was developed for efficient delivery of mucosal subunit vaccine M5BT through oral route. In this study, cellulose acetate phthalate (CAP), the pH-sensitive polymer that dissolve at > pH 6.2 was modified by thiolation to introduce mucoadhesive property and to dissolve at ileum pH. FMDV recombinant antigen M5BT was encapsulated into thiolated CAP microparticles (T-CAP MPs) using double emulsion solvent evaporation method. As a result, T-CAP MPs showed sustained release of encapsulated M5BT from the MPs at intestinal pH (pH 7.4), while releasing less M5BT at gastric pH (pH 2) due to its pH-sensitive property. Also, porcine mucosa assay showed 1.4-fold enhanced mucoadhesiveness of T-CAP MPs than non-modified CAP MPs in vitro due to the formation of disulfide bond between thiol group in T-CAP and mucin glycoproteins in mucus layer by thiol/disulfide exchange reactions. Finally, M5BT delivered by T-CAP MPs elicited higher IgA production than only M5BT in in vivo mouse experiment. Therefore, this study represents an effective mucosal subunit vaccine delivery through oral route. In chapter II, mannan-decorated inulin acetate (M-INAC) MPs as an immunostimulatory polymeric carrier were developed for efficient delivery of subunit vaccine M5BT. In this study, inulin was modified by acetylation to introduce hydrophobic moiety. And vaccine FMDV recombinant antigen M5BT was encapsulated into INAC MPs and decorated with mannan using double emulsion solvent evaporation method. As a result, M-INAC MPs showed released more than 90% of loaded antigen for 6 days, while less than 50% of M5BT was released from INAC MPs. As a result of in vivo immunization in murine model, after 4 weeks of immunization, M5BT/M-INAC MPs and M5BT/INAC MPs showed similar level of FMDV serotype O specific antibody with the M5BT group coinjected with conventional adjuvant CFA. And M5BT encapsulated in M-INAC MPs elicited higher IgG titer than M5BT/INAC MPs groups exhibiting similar level of IgG titer with M5BT group coinjected with CFA. It indicates that antigen-loaded INAC MPs can enhance antigen specific immune response comparable to the conventional group implying the potential polymeric adjuvant system for subunit vaccine. Therefore, this study represents an effective subunit vaccine for the better enhancement of adaptive immune response.