Elmars Grens

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 α-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.

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 α‐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.

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.

Development of Virus-Like Particle Technology from Small Highly Symmetric to Large Complex Virus-Like Particle Structures

Virus-like particle (VLP) technology is a promising approach for the construction of novel vaccines, diagnostic tools, and gene therapy vectors. Initially, VLPs were primarily derived from non-enveloped icosahedral or helical viruses and proved to be viable vaccine candidates due to their effective presentation of epitopes in a native conformation. VLP technology has also been used to prepare chimeric VLPs decorated with genetically fused or chemically coupled epitope stretches selected from immunologically defined target proteins. However, structural constraints associated with the rigid geometrical architecture of icosahedral or helical VLPs pose challenges for the expression and presentation of large epitopes. Complex VLPs derived from non-symmetric enveloped viruses are increasingly being used to incorporate large epitopes and even full-length foreign proteins. Pleomorphic VLPs derived from influenza or other enveloped viruses can accommodate multiple full-length and/or chimeric proteins that can be rationally designed for multifunctional purposes, including multivalent vaccines. Therefore, a second generation of VLP carriers is represented by complex particles reconstructed from natural or chimeric structural proteins derived from complex enveloped viruses. Further development of safe and efficient VLP nanotechnology may require a rational combination of both approaches.

Molecular Epidemiology and Immunology of Hepatitis B Virus Infection – An Update

Hepatitis B virus (HBV) continues to be one of the most important viral pathogens in humans. This review provides an update on the molecular epidemiology and immunology of HBV infection. DNA sequencing has allowed replacement of the initial serotypic classification of HBV strains by a more systematic genotype system that currently consists of 7 members (genotypes A–G). More recently, sequence analysis of virus isolates from many individual patients has revealed the occurrence of certain mutational hot spots in the genome, some of which appear to correlate with the patient’s immunological and/or disease status; however, cause and effect are not always easily discernible. This holds particularly for the issue of whether virus variants exist that have, per se, an increased pathogenic potential; due to the scarcity of appropriate experimental in vivo models, such hypotheses are difficult to prove. Similarly, because of the compact organization of the HBV genome, almost every single mutation may have pleiotropic phenotypic effects. Nonetheless, there is accumulating evidence that at least some frequently observed mutations are causally related to viral escape from selective pressures, such as the presence of antibodies against dominant B cell epitopes, or drugs that inhibit the viral reverse transcriptase; possibly, this is also true for the cellular immune response. Therefore, despite the availability of an effective prophylactic vaccine, further extensive efforts are required to monitor the emergence of vaccination- and therapy-resistant HBV variants and to prevent their spread in the general population.

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.

Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen : identification of internal and surface-exposed sequences

The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.

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.