Diana Koletzki

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.

HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein.

Abstract Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles (‘mosaic particles’); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid- readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.

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.

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/Hällnäs into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.

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.