Elsevier

Vaccine

Volume 20, Issues 17–18, 22 May 2002, Pages 2287-2295
Vaccine

Virosome-mediated delivery of protein antigens to dendritic cells

https://doi.org/10.1016/S0264-410X(02)00103-2Get rights and content

Abstract

Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.

Introduction

Effective vaccination against a protein antigen requires dendritic cells (DCs), which are essential antigen-presenting cells (APCs) in the induction of primary immune responses [1]. Immature DCs can effectively internalize and process antigens whereas mature DCs are very efficient in presentation of these antigens. Inflammatory signals, such as viral infection, double stranded RNA, bacterial products or cytokines, can induce DC maturation and upregulation of co-stimulatory molecules [2]. The final maturation of DCs is mediated by T cell–DC contact [3]. Uptake of antigen by DCs and its partial proteolysis in endosomes will result in MHC class II presentation of antigenic peptides to CD4+ helper T cells [1]. Stimulation of CD8+ T cells by class I MHC-associated peptides from exogenous antigen requires transport of the antigen to the cytosol of the APCs prior to its translocation to the endoplasmic reticulum for association with nascent MHC class I molecules [4]. Consequently, agents which augment delivery of exogenous antigen into the cytoplasm of APCs and thereby into the classical MHC class I route could be effective in induction of cytotoxic T lymphocyte (CTL) responses [5], [6].

We are using virosomes to deliver antigen into the cytosol of APCs. Virosomes are reconstituted viral envelopes, which contain the cell binding and fusion proteins of the native virus but do not contain the genetic material of the virus. Therefore, virosomes made from influenza virus retain the cell entry and membrane fusion capacity of this virus [7], [8]. Functionally reconstituted influenza virosomes will bind to sialic acid residues on the surface of cells and enter the cell via receptor-mediated endocytosis [9], [10]. Upon endocytosis, the low pH in the endosomes induces fusion of the virosomal membrane with the endosomal membrane, causing the release of the contents of the virosome into the cytoplasm of the cell. The fusion process is mediated by hemagglutinin, the major envelope glycoprotein of influenza virus [11], [12], [13].

Previously we have shown that influenza virosomes can deliver whole proteins to the cytoplasm of cells [14], [15]. Gelonin or subunit A of diphtheria toxin (DTA) were encapsulated in virosomes and upon incubation of cells with these virosomes cellular protein synthesis was inhibited. We have also shown that virosomes containing the cationic lipid DODAC in their membrane can bind plasmid DNA and deliver this DNA to transfect cells [16]. Both of these effects were dependent on the fusion activity of the virosomes, as they could be inhibited by pre-exposing the virosomes to low pH, resulting in irreversible inactivation of the hemagglutinin. These experiments demonstrate that virosome-encapsulated substances enter the cytosol of target cells, and indicate that fusion of the virosomal membrane with the endosomal membrane is needed for delivery.

Likewise, it is to be expected that protein antigens encapsulated in virosomes can be delivered into the cytosol of an APC and therefore into the classical MHC class I presentation pathway. Since not all of the virosomes are likely to fuse with the endosomal membrane, some of the virosomes will continue into the late endosomal/lysosomal route. These virosomes and their contents are expected to be degraded in these compartments and their peptides will thus become available for loading onto MHC class II molecules. Antigen delivered to an APC by a fusogenic virosome is therefore expected to be presented in association with both MHC class I and II molecules, resulting in stimulation of both CD4+ and CD8+ T cells. This property makes virosomes an excellent antigen delivery system for stimulation of both helper and cytotoxic responses.

Liposomes are vesicles composed of lipids, which, unlike virosomes, do not contain viral glycoproteins [17]. “Classical” or conventional liposomes, composed of phospholipids and cholesterol are not able to fuse with the endosomal membrane when taken up by APCs. Thus, in the absence of cellular mechanisms in APCs that may exist for the purpose of mediating cytoplasmic delivery of exogenous protein antigens, liposome contents are not expected to be delivered to the cytoplasm. Liposomes can be associated with ligands, such as antibody, which will increase their binding to and uptake by APCs, at least in vitro. Previously, we have described targeting of antigen-containing liposomes to the FcγR of DCs by opsonizing the liposomes with IgG. This targeting results in uptake of the liposomes and presentation of peptides of the antigen in the context of MHC class II [18]. Uptake of FcγR-targeted liposomes also resulted in the presentation of antigenic peptides in the context of MHC class I, but only when DCs were maintained in culture for longer than about 12 days or at higher antigen concentrations [19].

To investigate whether arrival in the cytoplasm of short-term cultured DCs is sufficient for the presentation of an exogenous antigen in the context of MHC class I, we compare the efficiency of MHC class I and II presentation of a whole protein antigen ovalbumin (OVA) by DCs when delivered by fusion-competent virosomes, fusion-incompetent virosomes or FcγR-targeted liposomes. Only fusion-competent virosomes were capable of inducing potent MHC class I presentation of OVA peptide by these cells. Fusion activity was not required for MHC class II presentation of OVA peptide.

Section snippets

Mice

OT-1 mice (a kind gift from Matthias Merkenschlager, MRC, London, UK) are transgenic for an αβ TCR specific for the chicken OVA peptide 257–264 (SIINFEKL) in the context of H-2Kb [20]. They were maintained on the C57BL/6 background and identified by FACS analysis as those mice in which a majority of peripheral blood CD8+ cells express Vα2. T cells obtained from the spleens of 6–12 weeks old transgenic mice were purified by passage over nylon wool columns.

DCs

DCs were derived from bone marrow of

Characterization of the virosomes

The morphology of influenza virosomes was similar to that of native virus as determined by transmission electron microscopy (Fig. 1). The images clearly demonstrate the hemagglutinin and neuraminidase spikes on the virosomes. No morphological difference could be seen between the empty virosomes and the OVA virosomes. The mean diameter of the virosomes was about 200 nm, comparable to that of the liposomes we used.

The pH-dependent fusion activity of virosomes was determined using a lipid mixing

Discussion

The experiments described in this paper show that fusion-active virosomes are highly effective in their delivery of encapsulated protein antigen for MHC class I presentation by bone marrow-derived DCs. DCs cultured in the presence of picomolar concentrations of OVA encapsulated in fusion-active virosomes are able to stimulate specific CD8+ T cells. Moreover, these CD8+ T cells are primed to become CTLs, as shown by killing of SIINFEKL-loaded target cells. MHC class I presentation depends on the

Acknowledgements

This work was supported by a travel grant from The Netherlands Organization for Scientific Research (NWO) under the auspices of the Foundation for Chemical Research (CW), and from the Institut National de la Santé et de la Recherche Médicale (INSERM). The authors thank the Laboratory of Cellbiology and Electron microscopy for preparing the electron microscopic images.

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