Hepatitis B Virus Capsid-like Particles Can Display the Complete, Dimeric Outer Surface Protein C and Stimulate Production of Protective Antibody Responses against Borrelia burgdorferi Infection

Abstract

Hepatitis B virus capsid-like particles (CLPs), icosahedral assemblies formed by 90 or 120 core protein dimers, hold promise as immune-enhancing vaccine carriers for heterologous antigens. Insertions into the immunodominant c/e1 B cell epitope, a surfaceexposed loop, are especially immunogenic. However, display of whole proteins, desirable to induce multispecific and possibly neutralizing antibody responses, can be restrained by an unsuitable structure of the foreign protein and by its propensity to undergo homomeric interactions. Here we analyzed CLP formation by core fusions with two distinct variants of the dimeric outer surface lipoprotein C (OspC) of the Lyme disease agent Borrelia burgdorferi. Although the topology of the termini in the OspC dimer does not match that of the insertion sites in the carrier dimer, both fusions, coreOspC a and coreOspC b , efficiently formed stable CLPs. Electron cryomicroscopy clearly revealed the surface disposition of the OspC domains, possibly with OspC dimerization occurring across different core protein dimers. In mice, both CLP preparations induced high-titered antibody responses against the homologous OspC variant, but with substantial cross-reactivity against the other variant. Importantly, both conferred protection to mice challenged with B. burgdorferi. These data show the principal applicability of hepatitis B virus CLPs for the display of dimeric proteins, demonstrate the presence in OspC of hitherto uncharacterized epitopes, and suggest that OspC, despite its genetic variability, may be a valid vaccine candidate.

A Structural Model for Duck Hepatitis B Virus Core Protein Derived by Extensive Mutagenesis

“…The interdimer contacts are mainly provided by the "hand region" (6) consisting of ␣5 (residues 112 to 127) onto which downstream residues to about position 140 fold back. Although the individual interdimer contacts are weak (58), the intact particles are so stable that even complete foreign proteins can be inserted into the c/e1 epitope (28,35,44); this is achieved by an inherent flexibility within the subunits, as well as in their arrangement on the icosahedral lattice (5,7). Such structural plasticity may be crucial for the active role of the capsid in reverse transcription, although only subtle differences between HBV CLPs and genome-containing nucleocapsids were detected in a recent cryo-electron microscopic (cryo-EM) study (40).…”

Internal Core Protein Cleavage Leaves the Hepatitis B Virus Capsid Intact and Enhances Its Capacity for Surface Display of Heterologous Whole Chain Proteins

“…Negative staining EM revealed a strongly enhanced proportion of spherical particles compared with the uncleaved sample (Fig. 5C, panel ϩTEV); their average diameters, though not accurately measurable due to imperfections in shape, the rough surface appearance, plus potential structural impacts of the staining procedure per se, were clearly larger (around 40 -45 nm, similar to those observed for CLPs carrying GFP or OspC (28,41,29)) than for the CLPs from the two other proteins (around 31 and 34 nm, likely corresponding to T ϭ 3 versus T ϭ 4 architectures). Finally, the bulk of the cleaved protein migrated as a distinct band in NAGE, particularly well visible after succinylation (Fig.…”

“…Native presentation of the whole protein antigen overcomes these restrictions, yet properly accommodating a large, structured protein into the carrier structure is much more demanding. Except for the recent successful insertion of anthrax toxin receptor 2 into surface-exposed loops of the Flock House insect virus (30), HBcAg is the only system for which native display of complete foreign proteins has been clearly demonstrated (28,29). However, all successfully inserted proteins had closely juxtaposed termini with a natural fit to the carrier acceptor sites.…”

“…While well established for short peptides (6, 7), we have demonstrated that even some complete proteins can be inserted into this site, without compromising folding of the insert or the carrier part (27). Insertion of the green fluorescent protein (GFP) yielded, in Escherichia coli, highly fluorescent CLPs that evoked a strong anti-GFP antibody response (28); efficient CLP formation and strong, including neutralizing, antibody responses were also achieved with the outer surface protein C (OspC) of the Lyme disease agent Borrelia burgdorferi (29). With the recent exception of the Flock House virus capsid (30), HBcAg is the only carrier for which such native whole chain protein display has been accomplished.…”

The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

“…Cryomicroscopy shows that the flexibly linked hantavirus fragment forms an extra shell of partially ordered material on the outside of the core protein (figure 10). Modified hepatitis B core particles may thus provide the basis for vaccines against hantavirus and other infections, such as malaria (Oliveira et al 2005) or lyme disease (Skamel et al 2006). For hepatitis B itself, there is already a very effective vaccine based on expressed surface protein particles.…”