[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Structures of hepatitis B virus cores presenting a model epitope and their complexes with antibodies.

The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T=3 and T=4 icosahedral shells, and atomic coordinates are available for the T=4 shell. Since the modified protein assembles predominantly into T=3 shells, a quasi-atomic model of the native T=3 shell was made. The spikes in this T=3 structure resemble those in T=4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.

HBV core particles as a carrier for B cell/T cell epitopes.

In the middle 80s, recombinant hepatitis B virus cores (HBc) gave onset to icosahedral virus-like particles (VLPs) as a basic class of non-infectious carriers of foreign immunological epitopes. The recombinant HBc particles were used to display immunodominant epitopes of hepatitis B, C, and E virus, human rhinovirus, papillomavirus, hantavirus, and influenza virus, human and simian immunodeficiency virus, bovine and feline leukemia virus, foot-and-mouth disease virus, murine cytomegalovirus and poliovirus, and other virus proteins, as well as of some bacterial and protozoan protein epitopes. Practical applicability of the HBc particles as carriers was enabled by their ability to high level synthesis and correct self-assembly in heterologous expression systems. The interest in the HBc VLPs was reinforced by the resolution of their fine structure by electron cryomicroscopy and X-ray crystallography, which revealed an unusual alpha-helical organization of dimeric units of HBc shells, alternative packing into icosahedrons with T = 3 and T = 4 symmetry, and the existence of long protruding spikes. The tips of the latter seem to be the optimal targets for the display of foreign sequences up to 238 amino acid residues in length. Combination of numerous experimental data on epitope display with the precise structural information enables a knowledge-based design of diagnostic, and vaccine and gene therapy tools on the basis of the HBc particles.

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

Mutilation of RNA phage Qbeta virus-like particles: from icosahedrons to rods.

Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-strands F and G (FG loop) of the monomeric subunits, which determine the five-fold and quasi-six-fold symmetry contacts of the VLPs. In order to reduce the stability of Qbeta VLPs, we mutationally converted the amino acid stretch 76-ANGSCD-81 within the FG loop into the 76-VGGVEL-81 sequence. It led to production in Escherichia coli cells of aberrant rod-like Qbeta VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs.

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles.

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

Characterization of potential insertion sites in the core antigen of hepatitis B virus by the use of a short-sized model epitope.

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.

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.

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

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 anti-HBc antibodies.

Hepatitis B core particles as a universal display model: a structure-function basis for development.

Because it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self-assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers. Structurally, the unusual alpha-helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes. Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc-based structures has brought closer the knowledge-based design of future vaccines, gene therapy tools and other artificial particulate objects.

T cell-independent type I antibody response against B cell epitopes expressed repetitively on recombinant virus particles.

Recombinant viral or virus-like particles offer new tools for vaccine development. This study investigated hepatitis B core antigen (HBcAg) capsids and RNA phage Qbeta coats as carriers of a foreign epitope to induce antibody responses in mice. HBcAg capsids were shown to induce T cell-independent (TI) antibodies. We found that these particles behave as antigen-specific TI type 1 (TI-1) Ag comparable to other rigidly structured viruses. When a 5-aa long epitope of the pre-S1 domain of hepatitis B surface antigen (HBsAg) was introduced into the optimal position of the HBc molecule, it also behaved as a TI-1 Ag. Best efficiency of the antibody response to the foreign epitope was achieved by a compensatory deletion after the epitope to retain the regular structure of the HBcAg capsid with a highly repetitive superficial exposition of the foreign epitope. For recombinant Qbeta phage coats, a much more efficient antibody response to the foreign epitope was achieved when the foreign epitope was expressed repetitively on a particulate derivate of Qbeta phage coats. Thus, recombinant virus particles are suitable vaccine carriers for the introduction of foreign B cell epitopes, if precise structural requirements are fulfilled.

Mosaic Qbeta coats as a new presentation model.

The new protein carrier was developed on the basis of recombinant RNA phage Qbeta capsid. C-terminal UGA extension of the short form of Qbeta coat, so-called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qbeta particles. In conditions of enhanced UGA suppression, the proportion of A1-extended to short coats in mosaic particles dropped from 48% to 14%, with an increase of the length of A1 extension. A model insertion, short preS1 epitope 31-DPAFR-35 of hepatitis B surface antigen, demonstrated superficial location on the mosaic Qbeta particles and ensured specific antigenicity and immunogenicity.

Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments.

Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.

Relationship between antigenicity and immunogenicity of chimeric hepatitis B virus core particles carrying HIV type 1 epitopes.

We have developed a comparative study of antigenic and immunogenic properties of selected immunodominant HIV-1 epitopes from p24 and gp120 proteins added to C-terminally truncated hepatitis B virus (HBV) core protein and exposed on the surface of chimeric core particles. Inserted p24 (121-210) and gp120/MN (306-328) epitopes induced the appropriate humoral and cellular immune responses against HIV-1. Superficially exposed region 160-192 of p24 also showed maximal B cell immunogenicity whereas buried region 148-162 induced maximal T cell response. Both recombinant proteins were also able to be recognized in vitro by T lymphocytes of HIV-1 asymptomatic carriers.

RNA phage Q beta coat protein as a carrier for foreign epitopes.

The Q beta gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known 'display vectors' on the basis of coat proteins of RNA phage group I, group III phage Q beta-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called A1 protein for insertion of the appropriate immunological epitopes. 'Mosaic' capsids presenting model hepatitis B virus preS1 and HIV-1 gp120 epitopes and formed by Q beta CP together with A1-derived proteins were obtained as a result of (1) suppression of leaky UGA stop codon of the CP gene and (2) simultaneous expression of 'pure' CP and full-length A1-derived genes obtained after the changing of CP-terminating UGA to strong UAA stop codon or sense GGA codon, respectively.

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.

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.

Crystal structure of bacteriophage fr capsids at 3.5 A resolution.

The structure of recombinant capsids of the bacterial virus fr has been determined by X-ray crystallography at 3.5 A resolution. The capsids were produced by expressing the fr coat protein in Escherichia coli, the natural host of the virus, and are probably essentially identical to the protein shell of the native virus. The structure was determined using molecular replacement with the protein shell of the related MS2 virus, and refined to a crystallographic R-factor of 0.228. A comparison of the protein shells of the viruses shows that they are very similar, and indicates that they may have a similar regulation of the assembly of the quasi-symmetrical protein shell.