Adjuvancy enhancement of muramyl dipeptide by modulating its release from a physicochemically modified matrix of ovalbumin microspheres

摘要

The weak immunogenicity of subunit vaccines has necessitated research into the development of novel adjuvants and methods to enhance the adjuvancy associated with vaccine delivery systems. A combination approach of the sustained release of vaccine components (antigen and adjuvant) and the use of novel adjuvants has been investigated. Besides enhancing immunogenicity, such an approach also holds the potential of reducing the dose and adverse effects associated with the bolus administration of adjuvants. Among novel adjuvants, N-acetylmuramyl-L-alanyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP) is an important immunostimulating compound, because of its potential to be used in human vaccines. The adverse effects associated with the required but high doses have led research efforts into the development of a depot system for MDP. However, studies systematically characterizing a sustained release adjuvant system in vitro and demonstrating its efficacy in the animal models are lacking.;The purpose of the present research project was to enhance the adjuvancy of MDP in vivo, by modulating its release from a physicochemically modified matrix of ovalbumin microspheres (OVA-MSs). These MSs utilized OVA as the model antigen (Ag) and polymer and MDP as the adjuvant. The OVA-MSs were prepared by a w/o emulsion method, followed by suspension cross-linking using glutaraldehyde. The MS matrix was modified with respect to the degree of cross-linking (by varying the concentration of glutaraldehyde) and density, a function of disulfide bond formation (by changing the pH of the OVA solution). The modifications in the MS matrix were characterized using SDS-PGE, SEM, DSC, and thin layer wicking (TLW). The structural modifications in the OVA-MSs modulated the release of MDP through controlled surface erosion and bulk degradation of the MSs.;In an effort to choose the optimum route of immunization in mice, the potential of the intradermal (id) route was investigated using OVA and fluorescent latex MSs. As compared to the traditionally used subcutaneous (sc) route, the id administration of OVA-MSs induced a significantly (p < 0.05) higher antibody (Ab) immune response in mice. This was explained by more efficient phagocytosis and lymph node targeting of the id administered fluorescent latex MSs.;Mice were immunized id using various preparations of OVA-MSs in order to investigate the physicochemical and physiological factors affecting the induction of the OVA-specific IgG Ab immune response. These factors included the degree of crosslinking and the matrix density of MSs, the NIS surface adsorbed MDP, the use of threonyl-MDP, and the MS dose---Ab response relationship. An inverse relationship was observed between the in vitro release rate of MDP from OVA-MSs and the in vivo Ab response in mice. This shows that a modulated release of MDP led to its enhanced adjuvancy.