Andris Dislers

RNA Phage Qβ Coat Protein as a Carrier for Foreign Epitopes

The Qβ 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 p

Construction and Immunological Evaluation of Multivalent Hepatitis B Virus (HBV) Core Virus-Like Particles Carrying HBV and HCV Epitopes

ABSTRACT A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.

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.

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

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

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.

Protein-prime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice.

BACKGROUND Therapeutic vaccination is a novel treatment approach for chronic hepatitis B, but only had limited success so far. We hypothesized that optimized vaccination schemes have increased immunogenicity, and aimed at increasing therapeutic hepatitis B vaccine efficacy. METHODS Modified Vaccinia virus Ankara (MVA) expressing hepatitis B virus (HBV) antigens was used to boost protein-prime vaccinations in wildtype and HBV-transgenic (HBVtg) mice. RESULTS Protein-prime/MVA-boost vaccination was able to overcome HBV-specific tolerance in HBVtg mice with low and medium but not with high antigenemia. HBV-specific antibody titers, CD8+ T-cell frequencies and polyfunctionality inversely correlated with HBV antigen levels. However, optimization of the adjuvant formulation, increasing the level of antigen expression and utilization of HBsAg of heterologous subtype induced HBV-specific CD8+ and CD4+ T-cells and neutralizing antibodies even in high-antigenemic HBVtg mice. CONCLUSIONS Our results indicate that high HBV antigen levels limit the immunological responsiveness to therapeutic vaccination but optimization of the vaccine formulation can overcome tolerance even in the presence of high antigenemia. These findings have important implications for the development of future therapeutic hepatitis B vaccination strategies and potentially also for the stratification of chronic hepatitis B patients for therapeutic vaccination.

Complete Genome Sequence of the Enterobacter cancerogenus Bacteriophage Enc34

ABSTRACT Enterobacter cancerogenus is widely distributed in nature and is generally recovered from environmental or vegetal sources. In some cases, it has also been associated with human infections. In this study, the complete genomic sequence of virulent E. cancerogenus bacteriophage Enc34 was determined. The Enc34 genome is 60,364 bp in length and contains 80 open reading frames. To our knowledge, this is the first report of a bacteriophage infecting E. cancerogenus.

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.

Assembly of mixed rod-like and spherical particles from group I and II RNA bacteriophage coat proteins

The capsids of single-stranded RNA bacteriophages show remarkable structural similarity. In an attempt to test whether the coat protein (CP) from one bacteriophage could substitute for the CP of another and form mixed particles, we reassembled capsids in vitro from a mixture of different RNA phage CP dimers together with E. coli ribosomal RNA. Surprisingly, mixing CPs from phages belonging to groups I and II led to appearance of rod-like particles along with icosahedral spherical capsids, both containing a mixture of the two CPs. Rods and mixed spherical capsids containing host RNA were also obtained in vivo in bacteria expressing simultaneously fr and GA CPs. In a co-infection of the two phages, however, only authentic fr and GA virions were formed. Coat protein mutants in the FG loop were unable to assemble into rods, suggesting that these loops are involved in the formation of the aberrant particles.