A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

Abstract

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.

Introduction

The recent understanding of the molecular mechanisms by which the immune system is triggered to fight infection is opening new perspectives for effective induction of specific immunity through strategies targeting the innate immune system (Akira, 2009, Palm and Medzhitov, 2009). It is now well established that the innate immune system is able to sense microorganisms through a variety of germ-line encoded receptors which recognise pathogen-associated molecular patterns (PAMPs). These pattern recognition receptors (PRRs) include, among others, the membrane associated Toll-like receptors (TLRs) and the cytosolic nucleotide-binding oligomerisation domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) (Kawai and Akira, 2009). Together, PRRs can detect a broad range of microorganisms, discriminating the self from the non-self at an early stage of the immune response, by the recognition of conserved, structural and essential components of bacteria, viruses, fungi and parasites. Those components are in most cases cell wall elements or nucleic acids with microbe specific features. The stimulation of antigen presenting cells through PRRs results on up-regulation of major histocompatibility complex (MHC) and co-stimulatory molecules, transitory enhancement of antigen internalisation, enhanced direct and cross presentation and induction of cytokines that shape the specific immune response (van Duin et al., 2006, Watts et al., 2010). Consequently, the direct or indirect conjugation of antigens with PRR ligands may profoundly alter their immunogenicity, both in terms of magnitude and type of immune response elicited, and this can be explored for the development of improved subunit vaccines (Ishii and Akira, 2007, Warshakoon et al., 2009, Coffman et al., 2010).

Being relevant for the design of effective immunogens, several studies show that formulations with a direct linkage between the antigens and the adjuvant molecules have a superior capacity to elicit immunity, including cytotoxic T lymphocyte (CTL) responses (Wille-Reece et al., 2005, Huleatt et al., 2007, Khan et al., 2007, Schlosser et al., 2008). Studies suggesting TLR-dependent autonomous phagosome maturation may contribute to explain the molecular basis of these observations (Blander and Medzhitov, 2004, Blander and Medzhitov, 2006). Moreover, recent research reveals that activation of different signalling pathways and cooperation between them lead to antigen presenting cell phenotypes with different stimulatory and T cell-polarising characteristics (reviewed by Trinchieri and Sher, 2007, Manicassamy and Pulendran, 2009). These observations open the possibility to modulate the immune response through the careful selection of the PRR ligands associated with the antigen.

Based on the adjuvant properties of bacterial lipoproteins, now known to be TLR2 ligands, an Escherichia coli expression system for the production of antigens in fusion with the small OprI lipoprotein from the outer membrane (OM) of Pseudomonas aeruginosa was developed in the past (Cornelis et al., 1996). The cloning vectors were based on the oprI gene modified by the insertion of a polylinker near its end for the cloning of heterologous sequences, conferring to the fusion proteins built-in adjuvant properties. Using African swine fever virus (ASFV) fusion antigens we have previously demonstrated their capacity to enter the class I pathway of antigen presentation. This made possible the identification of ASFV epitopes specifically recognised by porcine CTL (Leitão et al., 1998) and enabled the stimulation of CTL activity in vitro (Leitão et al., 2000). Later, in a murine model, an OprI-based immunogenic formulation against Leishmania major allowed to direct the immune response towards a protective Th1 profile. In this study it was demonstrated that the fusion of the antigen with non-lipidated OprI enhanced the specific IgG1 response but the lipid moiety was found essential to bias the immune response profile and achieve protection (Cote-Sierra et al., 2002). In an asthma murine model, the intranasal administration of OprI led to the suppression of allergen-specific Th2 effector cells (Revets et al., 2005). Other studies, on HIV (Piedade, 2003), classical swine fever (Rau et al., 2006) and tuberculosis (Gartner et al., 2007), corroborate the adjuvant properties of OprI and the possibility to modulate adaptive immune responses with this lipoprotein. More recently, it was shown that OprI can bind in vitro and in vivo to epithelial cells of the trachea and the small intestine of chickens, and might consequently act as a carrier for antigen delivery at mucosal surfaces (Loots et al., 2008).

The impact of PRR activation on the development of specific immunity is now an emerging field of research, for which the OprI-based system may represent a tool with particularly interesting characteristics. However, plasmid instability due to basal leakage of expression and the laborious purification methods required to obtain purified fusion antigens posed limitations to its utilisation. Here we develop and characterise a new expression system based on OprI, which solves these major difficulties and further enables the formulation of novel recombinant bacterial cell wall-derived immunogens with distinct potential for PRR activation. The diversity of immunogenic formulations in which recombinant antigens can be obtained may be used for the development of experimental vaccines and can be explored for studies on the modulation of acquired immunity.