Instead, and presumably due to flexibility in the linkers between the MIR and the GFP, an icosahedrally-averaged pattern of density is seen with the greatest concentration being over the 2-fold axes where apparent spikes arise from the capsid surface, and at around the 3-fold and 5-fold axes. particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused Indolelactic acid to the surface of the tandem core particle Indolelactic acid (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves Rabbit Polyclonal to MRPS16 the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody. Introduction The core protein (HBcAg) of hepatitis B virus (HBV) readily assembles into highly immunogenic virus-like particles (VLPs) when expressed in either prokaryotic or eukaryotic expression systems [1C3]. These icosahedral hepatitis B core (HBc) particles are assembled from dimers of the 183C5 amino acid HBcAg protein and occur in two size classes with T = 3 or T = 4 symmetry, constructed from 90 and 120 dimeric subunits, respectively. The HBcAg protein has a high -helical content and the dimer contacts form four-helix bundles which appear as prominent spikes at the surface of assembled particles. The structure of HBc particles has been determined by cryo-EM and X-ray crystallography [4C6] (Fig. 1A). Open in a separate window Fig 1 Tandem core technology.a) The structure of a monomeric HBc VLP with one HBcAg dimer shown in a surface representation coloured yellow and green. b) Two HBcAg sequences fused together via a flexible linker makes a tandem core construct, with either full-length (hetero-tandem) or truncated (homo-tandem) C-terminus, and two modifiable major insertion regions (MIRs). c) Structure of a tandem core protein: N-terminal core 1 (in green) is fused via a Indolelactic acid flexible linker (red) to C-terminal core 2 (yellow). The two views are related by a 90 rotation. HBc particles have immunologically interesting properties: they induce very high titres of antibody, they can function as both T-cell independent and T-cell dependent antigens, and are potent activators of macrophages [7C9]. The HBcAg protein contains strong T cell epitopes and it has been proposed that the nucleic acid encapsidated within HBc VLPs further contributes to the immunogenicity of the particles via stimulation of Toll-like receptors [10,11]. Furthermore, third party antigens can be introduced at several positions in the HBcAg protein without compromising particle assembly. The most favourable of these sites is the major insertion region (MIR, or c/e1 loop) located at the tip of the -helical spike [12,13]. Recombinant HBc particles bearing foreign antigenic sequences at this site induce strong immune responses to both HBc and the insert [14]. Early studies showed that HBc particles containing short foreign peptide sequences induced high serological responses to the inserted epitopes and, in the case of foot-and-mouth disease, solid protection of laboratory animals against virulent virus challenge was achieved [15]. More recently, HBc particles modified to display a malaria antigen were used in a phase I clinical trial [16,17]. Despite its apparent advantages, HBc has not been fully developed as a vaccine vector, in part because of Indolelactic acid limitations on the types of antigen that could be inserted. The preferred insertion site (the MIR), located at the tips of the surface-oriented spikes, inevitably presents two copies of the inserted sequence closely located in space, and so subject to potential steric clashes [12]. It has Indolelactic acid been demonstrated that the.