Andris Kazaks

Mosaic Qβ coats as a new presentation model

The new protein carrier was developed on the basis of recombinant RNA phage Qβ capsid. C‐terminal UGA extension of the short form of Qβ coat, so‐called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qβ 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 Qβ particles and ensured specific antigenicity and immunogenicity.

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

New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge

Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV mosaic particles in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.

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.

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.

Mosaic particles formed by wild‐type hepatitis B virus core protein and its deletion variants consist of both homo‐ and heterodimers

Co‐expression in Escherichia coli of wild‐type (wt) hepatitis B virus core protein (HBc) and its naturally occurring variants with deletions at amino acid positions 77–93 or 86–93 leads to formation of mosaic particles, which consist of three dimer subunit compositions. These compositions are wt/variant HBc heterodimers and two types of homodimers, formed by wt HBc or the variant HBc themselves. Mosaic particles were found also when both HBc deletion variants 77–93 and 86–93 were co‐expressed in E. coli. These findings are discussed in terms of their significance for hepatitis B virus pathogenesis and prospective use of mosaic particles in vaccine development.