Development of adjuvants using cytokine and polymeric particles for efficient mucosal immunization

Abstract

Mucosal surfaces are a main portal of entry for not only nutrients but also many pathogens that are the cause of infectious disease in livestock animals. Immunization through mucosal routes may be more effective in terms of inducing protective immunity against mucosal pathogens. In addition, mucosal vaccines can offer needle-free delivery and improve accessibility, safety and cost-effectiveness in farm industry. However, challenge to successful mucosal vaccination include poor induction of mucosal immunity, the availability of safe and effective mucosal adjuvant and delivery systems. Recently, major efforts are made to develop new mucosal vaccines by selecting appropriate antigens with high immunogenicity, designing mucosal route of administration, selecting immune-stimulatory adjuvant molecules and developing appropriate delivery systems. The aim of the present study is to develop an effective mucosal adjuvant and delivery vehicles to improve efficacy of the livestock mucosal vaccine, which is divided into two parts

i) Development of an oral cytokine vaccine adjuvant which was designed to be produced in Lactococcus lactis IL1403, ii) Development of mucoadhesive and mannan decorated thiolated polymeric microspheres, designed to target mucosal antigen presenting cells (APCs) as nasal vaccine delivery vehicles. In study 1, a recombinant cytokine, IL-6-CKS9, was generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secreted the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreted IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting mice immunization assays. In addition, the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses. Collectively, the results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated that the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination. In study 2, mucoadhesive and mannan decorated microspheres, designed to target APC and were evaluated the efficiency of vaccine delivery carrier after intranasal immunization. The aim was to develop mannan-decorated mucoadhesive thiolated hydroxypropylmethyl cellulose phthalate (HPMCP) microspheres (Man-THM) that contain ApxIIA subunit vaccine – an exotoxin fragment, as a candidate for a subunit nasal vaccine against A. pleuropneumoniae. For adjuvant activity, mucoadhesive thiolated HPMCP microspheres decorated with mannan could be targeted to the PRRs (pathogen recognition receptors) and mannose receptors (MR) of APCs in the respiratory immune system. The potential adjuvant ability of Man-THM for intranasal immunization was confirmed by in vitro and in vivo experiments. As following in a mechanistic study using APCs in vitro, it was found that Man-THM enhanced receptor-mediated endocytosis by the MR of APCs. In vivo, the nasal vaccination of ApxIIA-loaded Man-THM in mice resulted in higher levels of mucosal sIgA and serum IgG than mice immunized with the ApxIIA or ApxIIA-loaded THM due to the specific recognition of the mannan by the MRs on the APCs. Moreover, ApxIIA-containing Man-THM protected immunized mice when challenged with strains of A. pleuropneumoniae serotype 5. These results suggest that mucoadhesive Man-THM could be considered as a promising candidate for a nasal vaccine delivery system to elicit systemic and mucosal immunity that can protect from pathogenic bacteria infection.