ENHANCING IMMUNE RESPONSE IN THE ORAL CAVITY BY ANTIGEN-TARGETING

Abstract

Mucosal vaccines that induce both local mucosal and systemic immune responses are ideal for preventing infections at the site of pathogen entry. Delivering antigen via molecules targeted specifically for receptors on the surface of antigen-presenting cells is potentially an excellent strategy to improve the immune response against the antigens delivered to mucosal sites. Many studies have reported that targeting antigen to CD40 via CD40 ligand or anti-CD40 antibody was able to strongly enhance antigen specific immune response; however, this approach has not been tested with protein-based mucosal immunization in the oral cavity. In this study, an antigen-targeting fusion protein OVA-CD40LS consisting of the C-terminal fragment of ovalbumin fused to the extracellular domain of mouse CD40 ligand and C-terminal fragment of ovalbumin alone (rOVA) as control protein were constructed and cloned into Escherichia coli. The two recombinant proteins were successfully purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography coupled with different protein-refolding approaches. Biologically active OVA-CD40LS was generated via a ‘two-round’ protein-refolding strategy that was developed in the present study. The rOVA was successfully refolded via dialysis. Buccal intramucosal injection of 1 μg of OVA-CD40LS in the absence of adjuvant induced a rapid and strong Th1-skewed systemic antibody response while rOVA did not. Buccal topical immunization of 1 μg OVA-CD40LS did not induce a systemic or mucosal immune response. The most possible reasons for this lack immune response is the amount of fusion protein used was inadequate. This study has demonstrated that antigen targeting to CD40 via CD40LS could induce a strong systemic antibody response in the mouse oral cavity when the protein antigen was delivered into the mucosal layer.