Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core 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 to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves 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.

Hepatitis B small surface antigen particles are octahedral.

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.

Differential induction of interleukin-1beta and tumour necrosis factor-alpha may account for specific patterns of leukocyte recruitment in the brain.

In peripheral tissue, IL-1beta has been shown to induce TNFalpha expression and vice versa, resulting in mixed neutrophil and mononuclear cell recruitment to the site of injury. This has led to the concept of crosstalk in peripheral cytokine signalling pathways. In the brain parenchyma, however, restricted patterns of leukocyte recruitment following the focal injection of pro-inflammatory agents into the brain are observed. This study investigates the expression of the principal pro-inflammatory cytokines--IL-1beta and TNFalpha--in the brain after IL-1beta, TNFalpha, NMDA or endotoxin injection into the brain parenchyma of rats. Each of these agents gives rise to a distinct pattern of acute leukocyte recruitment at 24 h. We found that IL-1beta induces de novo synthesis of additional IL-1beta but not TNFalpha, as determined by RT-PCR and ELISA, and TNFalpha does not induce either itself or IL-1beta. Injection of NMDA results in IL-1beta, but not TNFalpha up-regulation. Injection of IL-1beta or NMDA is associated with neutrophil recruitment whereas injection of TNFalpha is associated with mononuclear cell recruitment. Following injection of endotoxin, both TNFalpha and IL-1beta levels are elevated and neutrophils and mononuclear cells are recruited to the brain. These data suggest that the signalling pathways that are present in the periphery are modified in the brain and that differential induction of TNFalpha and IL-1beta may have a role in the atypical pattern of leukocyte recruitment observed in the brain.