An Engineered Non-Toxic Superantigen Increases Cross Presentation of Hepatitis B Virus Nucleocapsids by Human Dendritic Cells

et al. (2014) An Engineered Non-Toxic Superantigen Increases Cross Presentation of Hepatitis B Virus Nucleocapsids by Human Dendritic Cells. PLoS ONE 9(4): e93598. doi:10.1371/journal.pone.0093598 An Engineered Non-Toxic Superantigen Increases Cross Presentation of Hepatitis B Virus Nucleocapsids by Human Dendritic Cells Julie D. McIntosh 0 Kristy Manning 0 Shilpa Chokshi 0 Nikolai V. Naoumov 0 John D. Fraser 0 P. Rod Dunbar 0 John A. Taylor 0 Stephen J. Turner, University of Melbourne, Australia 0 1 School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland , Auckland , New Zealand , 2 School of Medical Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland , Auckland , New Zealand , 3 Institute of Hepatology, University College London , London , United Kingdom Virus like particles (VLPs) are potent immunogens capable of priming strong protective antibody responses due to their repetitive structural arrangement and affinity for specific B cell receptors. By contrast, T cell responses to VLPs can be weak due to inefficient uptake and processing by antigen presenting cells. We report here a novel strategy for increasing the T cell reactivity of a VLP, the nucleocapsid of hepatitis B virus, through covalent coupling of M1, an engineered form of the Streptococcal superantigen SMEZ2, that binds MHC II with high affinity but lacks its T cell mitogenic capability. M1:HBcAg conjugates bound to dendritic cells and were efficiently endocytosed into late endosomes. Human dendritic cells pulsed with M1:HBcAgs stimulated HBV-specific CD8+ T cells more effectively than cells pulsed with native capsids indicating that the modified VLP was more effectively cross presented by APCs. Coupling of M1 was also able to induce significantly greater reactivity of human CD4+ T cells specific for a common T-helper epitope. These studies indicate the potential of recombinant superantigens to act as flexible molecular adjuvants that can be incorporated into various subunit vaccine platforms leading to enhanced T cell reactivity in humans. - Funding: This research was funded by a Project Grant awarded by the Health Research Council of New Zealand (05/252). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Generation of cytotoxic T lymphocytes (CTL) by subunit vaccines requires cross-presentation of antigen to Class I Major Histocompatability Complex (MHC I) on the surface of antigen presenting cells (APCs) and cross-priming of antigen-specific CD8+ T cells. Dendritic cells, a specialized subset of APC represent critical targets of subunit vaccines due to their intrinsic ability to capture exogenous antigens and direct them into the cross presentation pathway. Cross presentation of subunit vaccines in mice can be enhanced by strategies designed to target antigens to specific receptors on the surface of DCs leading to receptormediated endocytosis of antigen and delivery to intracellular compartments where processing and cross presentation of key epitopes to MHC I occurs. For example, targeting antigen of the model antigen ovalbumin (OVA) to the lectin DEC-205 expressed on DCs through coupling of OVA to Dec-205-specific antibodies can significantly increase the efficiency of antigen presentation on MHC class I and II molecules leading to elevated numbers of CD4+ T and CD8+ T cells in vaccinated mice [13]. An alternative approach to targeting proteins to DCs utilises synthetic or natural ligands of DC surface proteins. For example, the family of calcium-dependent lectins (CLRs) has received significant attention as DC receptors to which antigens can be targeted after coupling to either antibody or a specific sugar moiety and several studies have reported significantly improved immune responses in both mouse models and human clinical trials (reviewed in [4]). A further extension of this approach is to utilise the specificity and affinity of natural protein ligands that are recognised by molecules present at the surface of the cell. Cell surface receptors that have been targeted with their natural ligands include heat-shock proteins [5,6], bacterial-derived toxins [7] and C-type lectins [8]. Increasing the efficiency of cross presentation requires not only targeting to the DC surface but the subsequent delivery of antigen to an endocytic pathway that precedes processing and loading of epitopes to class I MHC. Different mechanisms for crosspresentation have been proposed largely based on studies with murine DC subsets. The cytosolic pathway requires the transfer of internalized antigens to the cytosol where they are degraded by the proteasome. The resulting peptides are translocated into the endoplasmic reticulum (ER) by TAP transporters and loaded onto MHC class I molecules effectively entering the endogenous pathway. This model is supported by the observation that elements of the ER retrotranslocation machinery can be detected within specialised phagolysosomal compartments containing exogenous antigen [913]. Alternatively some antigens may be processed by endosomal proteases, MHC I loading being independent of TAP (reviewed in [14]). The precise mechanism(s) by which cross presentation occurs is likely to be dependant on the route of internalisation and the specific subset of DCs in which it occurs. In this study we have explored a novel method for targeting a VLP to human monocyte derived DCs with the aim of improving the T cell responses to prospective VLP-based vaccines in humans. Our approach utilises conjugation to a bacterial superantigen engineered to lack its T cell mitogenic activity while retaining high affinity binding to MHC II on the surface of APCs. Internalization of VLPs via MHC II directs the particles to a late endosomal/ lysosomal compartment where transfer of epitopes to MHC I can occur leading to cross priming of CD8+ T cells. Ethics Statement Blood was collected from healthy donors after obtaining written informed consent. All procedures related to blood collection were approved by The University of Auckland Human Subjects Ethics Committee. Expression and purification of HBcAg and SMEZ2.M1 cDNA encoding amino acids 1142 of HBcAg was amplified by PCR from the viral genome (subtype ayw) and cloned into the plasmid pET17xb (Novagen). To facilitate covalent coupling using the bifunctional crosslinker sulpho-MBS, the wild-type HBcAg sequence was mutated to replace the proline and alanine at residues 7980 respectively with the amino acid sequence GAKGG. In addition, the cysteine residues at positions 48 and 107 were mutated to alanine to ensure coupling of the M1 only occurred via the introduced cysteine as described previously [15]. Expression of HBcAg in E.coli was induced by 2 mM IPTG and the protein purified as described previously (...truncated)