Galina Borisova

Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy

Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transcription to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.

Hepatitis B Virus Core Particles as Epitope Carriers

HBV core (HBc) particle is one of the most intensively studied particulate carriers for the insertion of foreign peptide sequences. Recombinant HBc protein expressed from the cloned gene undergoes the correct folding in a large variety of bacterial, yeast, insect and mammalian cells. Unique assembly properties and shape of 30/34-nm HBc particles allow substantial insertions into their primary structure without loss of their capsid-forming ability. N- and C-terminal regions, as well as the immunodominant loop in the middle of the molecule are widely accepted as targets for the introduction of foreign epitopes, ensuring retention and even enhancement of the original immunological activity of inserted sequences. Special sets of display vectors have been constructed on the basis of the cloned HBc gene. Epitope sequences of viral (BLV, FeLV, FMDV, HBV, HCV, HIV-1, HRV2, MCMV, PV-1, SIV) and nonviral (human chorionic gonadotropin) origin have been studied as model display moieties.

Chimaeric HBV core particles carrying a defined segment of Puumala hantavirus nucleocapsid protein evoke protective immunity in an animal model.

Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carriers of foreign protective epitopes.

Evaluation of HBs, HBc, and frCP Virus-Like Particles for Expression of Human Papillomavirus 16 E7 Oncoprotein Epitopes

Objectives: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35–98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). Methods: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. Results: The HBs*-E7(35–54) protein expressing E7 residues 35–54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae. The HBcΔ-E7(35–98), but not the frCP-E7(35–98), ensured VLP formation in Escherichia coli. In Balb/C mice, the HBs*-E7(35–54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBcΔ-E7(35–98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response. The frCP-E7(35–98) protein elicited equally high antibody responses to both E7 and frCP carrier. Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset. Conclusions: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes. All chimeric proteins may be regarded as potential vaccine candidates.

Spatial Structure and Insertion Capacity of Immunodominant Region of Hepatitis B Core Antigen

Spatial and immunochemical elucidation of hepatitis B core antigen suggested unique organization of its major immunodominant region (MIR) localized within the central part of molecule around amino acid residues 74-83. This superficial loop was recognized as the most prospective target for the insertion of foreign epitopes ensuring maximal antigenicity and immunogenicity of the latter. MIR allowed a substantial capacity of insertions up to about 40 amino acid residues without loss of the capsid-forming ability of core particles. Vector capacity as well as structural behavior and immunological fate of inserted epitopes were dependent on their primary structure. Special sets of display vectors with retained but cross-sectioned MIR as well as with uni- and bidirectionally shortened MIR have been investigated.

Fine Mapping and Functional Characterization of Two Immuno-Dominant Regions from the preS2 Sequence of Hepatitis B Virus

A set of monoclonal antibodies (mAbs) directed against the preS2 region of hepatitis B virus (HBV) surface antigen (HBsAg) was generated by immunization of mice with native HBsAg isolated from the blood of HBV carriers. According to (1) mutual competition binding of mAb to natural HBsAg, (2) recognition of full-length preS2 displayed on hepatitis B core particles, (3) recognition of synthetic partial preS2 peptides, and (4) Western blotting using a fusion protein library of truncated preS2 fragments of different legths, mAbs were assigned to two groups which coincided with groups I and III described by Mimms et al. [Virology 1990; 176:604-619]. All mAbs recognized linear epitopes and were glycosylation independent. Six out of eight fine-mapped mAbs recognized common epitopes located in the amino-terminal part of the preS sequence between amino acids 131 and 144 (group I), and inhibited binding of HBsAg to polymerized human serum albumin. Only two mAbs recognized a carboxy-terminal HBV-genotype-specific epitope covering amino acid residues 162 to 168 (group III). These mAbs bound to the highly variable proteolysis-sensitive hinge of preS2. Although four out of six mAbs targeted to immunodominant region I require the full-length sequence 131-L[Q/L]DPRVRGLY[F/L]PAG-144, two mAbs recognize the shorter and slightly carboxy-terminal-shifted sequences 133-DPRVRGLY[F/L]-141 or 135-PVRGLY[F/L]PAG-144. Together with previously identified preS2 epitopes 133-DPRVRGL-139, 137-RGLYFPA-143, and 132-QDPR-135, these data indicate diversity of the immune response against epitopes within the same immunodominant region. This diversity may be generated by a labile secondary structure. Sequence analysis suggests the transition from an alpha-helix to a loop structure at this site.

Behavior of a short preS1 epitope on the surface of hepatitis B core particles.

Abstract The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79–81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79–88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79–93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79–81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune polyclonal or hyperimmune anti-HBc antibodies

Expression, assembly competence and antigenic properties of hepatitis B virus core gene deletion variants from infected liver cells

Previous studies have shown that the progression of hepatitis B virus-related liver disease in long-term immunosuppressed kidney transplant recipients is associated with the accumulation of virus variants carrying in-frame deletions in the central part of the core gene. A set of naturally occurring core protein variants was expressed in Escherichia coli in order to investigate their stability and assembly competence and to characterize their antigenic and immunogenic properties. In addition, a library of core gene variants generated in vitro with deletions including the major immunodominant region (MIR) of the core protein was investigated. The position and length of deletions determined the behaviour of mutant core proteins in E. coli and their assignment to one of the three groups: (i) assembly-competent, (ii) stable but assembly-incompetent and (iii) unstable proteins. In vivo core variants with MIR deletions between amino acids 77 and 93 belong to the first group. Only proteins with the shortest deletion (amino acids 86-93) showed stability and self-assembly at the same level as wild-type cores, and they showed reduced antigenicity and immunogenicity. Mutants with deletions extending N-terminally beyond residue G73 or C-terminally beyond G94 were found to be assembly-incompetent. We suggest that G73 and G94 are involved in the folding and the native assembly of core molecules, whereas the intervening sequence determines the antibody response. Depending on their ability to form stable proteins or to assemble into particles, core mutants could contribute to liver cell pathogenesis in different ways.

Characterization of Potential Insertion Sites in the Core Antigen of Hepatitis B Virus by the Use of a Short-Sized Model Epitope1

Core particles of hepatitis B virus (HBV) are able to improve the immunogenicity of foreign sequences exposed on the particle surface. The insertion site in the core antigen of HBV (HBcAg) determines the surface presentation and thus the immunogenicity of the foreign sequence. For direct comparison of the value of potential insertion sites in the core antigen, we constructed vectors allowing insertions of a model marker epitope DPAFR. This epitope was inserted at the N-terminus, the c/e1 loop, behind amino acid (aa) 144 and behind aa 183 (DPAF only). In addition, we generated a mosaic construct allowing the co-expression of HBcAg and a HBcAg/DPAFR fusion protein due to a suppressor tRNA-mediated readthrough mechanism. All 6 constructs allowed the formation of chimaeric or mosaic core-like particles. Western blot analyses and a direct ELISA demonstrated the presence of the DPAFR sequence in the chimaeric and mosaic particles. Competitive ELISA and immune electron-microscopic data suggested the c/e1 loop as the insertion site of choice for presenting foreign sequences on the surface of chimaeric HBV core particles. However, the N-terminal fusion also allowed partial surface exposure of the DPAFR motif. In contrast, in particles of constructs carrying the DPAFR insert at aa position 144 or 183, respectively, the epitope seemed not to be surface accessible.

Stop Codon Insertion Restores the Particle Formation Ability of Hepatitis B Virus Core-Hantavirus Nucleocapsid Protein Fusions

In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBcΔ) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBcΔ mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain [J Gen Virol 1997;78:2049–2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of ‘non-mosaic’ HBcΔ fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBcΔ particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1–114, aa 1–213 and aa 340–433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1–114 or aa 1–213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and ‘problematic’, e.g. hydrophobic, hantavirus sequences.