Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins

Abstract

In order to design an optimized liposome immunoadjuvant for inducing cell-mediated immune response against soluble proteinaceous antigens, we investigated the effect of liposomal surface charge on the immunoadjuvant action. Positively charged liposomes containing soluble antigens functioned as a more potent inducer of antigen-specific cytotoxic T lymphocyte responses and delayed type hypersensitivity response than negatively charged and neutral liposomes containing the same concentrations of antigens. To clarify the reason of the differential immune response, we examined the delivery of soluble proteins by the liposomes into the cytoplasm of macrophages, using fragment A of diphtheria toxin (DTA) as a marker. We found that positively charged liposomes encapsulating DTA are cytotoxic to macrophages, while empty positively charged liposomes, DTA in negatively charged and neutral liposomes are not. Consistent with this, only macrophages pulsed with OVA in positively charged liposomes could significantly stimulate OVA-specific, class I MHC-restricted T cell hybridoma. These results suggest that the positively charged liposomes can deliver proteinaceous antigens efficiently into the cytoplasm of the macrophages/antigen-presenting cells, where the antigens are processed to be presented by class I MHC molecules to induce the cell-mediated immune response. Possible development of the safe and effective vaccine is discussed.

Introduction

Much evidence suggests that effective protection against such pathogens as human immunodeficiency virus, Leishmania major and Mycobacterium leprae requires cell-mediated immunity, including DTH and CTL responses, rather than antibody-mediated immunity [1], [2], [3]. Therefore, the candidate vaccine against these pathogens must be able to induce cell-mediated immunity quite efficiently. Although live vaccines can satisfy this criterion, development of attenuated vaccines against these microorganism are not practical because of their strong pathogenicity.

Non-live vaccines, including subunit vaccines that consist of the recombinant proteins or synthetic peptide, cannot induce strong cell-mediated immunity in general, although they are potentially much safer than the live vaccine. So non-live vaccines may require potent immunoadjuvants for effective antigen-specific immunization. A key point in inducing effective cell-mediated immune responses by using an immunoadjuvant is how to efficiently stimulate the processing and presentation of antigens to the immune system. Thus, a critical target of vaccines for enhancing cell-mediated immune responses is the antigen-presenting cell (APC). In support of this idea, it was reported recently that APC primed with various antigens in vitro can induce potent antigen-specific cell-mediated immune response after administration in naive mice [4], [5], [6], [7]. Therefore, targeting and delivery of antigenic molecules to APCs in vivo with an appropriate adjuvant will be necessary to success in inducing cell-mediated immunity.

Positively charged carriers are suitable for this purpose, because they are taken up preferentially by phagocytotic cells such as macrophages which are one of the APC populations [8], [9], [10]. In this article, we considered liposome to be the candidate safe adjuvant and demonstrated that the soluble antigens can induce cell-mediated immunity quite efficiently in vivo with the help of positively charged liposomes. We also found that the ability of various liposomes to support cell-mediated immunity against soluble antigens is correlated with the ability of these liposomes to deliver the soluble protein to APCs. These results indicate that the positive charge on the surface of liposomes represents an important factor for enhancing their immunoadjuvancy in the induction of antigen-specific cell-mediated immune responses.