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.

Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR

“…For further purification, the virus band between the sucrose cushions was pipetted on a CsCl solution (1.39 kg/L) and centrifuged for 45 h at 100 000g. The CsCl gradient was unloaded from the bottom and fractionated into 0.5-mL portions (16 ). The CsCl density of the fractions was determined by refractometry.…”

HBV Core Particles as a Carrier for B Cell/T Cell Epitopes

“…However, addition of 120 N-terminal aa of the hantavirus nucleocapsid to the N-terminus of HBc¢ prevented self-assembly, in contrast to their insertion into position 78 [110] (see below).…”

“…In spite of its internal location, the MIR allows for a surprisingly high capacity of insertions. For example, the entire 120 aa long immunoprotective region of the hantavirus nucleocapsid was inserted into the MIR of the C-terminally truncated HBc¢ particles, whereas N-and C-termini failed to accept this fragment for self-assembly [110]. It is necessary to emphasize that the shorter, aa 1-45 segment of hantavirus nucleocapsid within the MIR also ensured protection of bank voles against virus challenge after immunization with chimeric particles [113,114].…”

“…A strategy to construct mosaic particles was based on the introduction of a linker containing translational stop codons (UGA, or UAG) between sequences encoding a C-terminally truncated HBc¢ and a foreign protein sequence [103,110,114,152,162]. Expression of such recombinant gene in an E. coli suppressor strain leads to the simultaneous synthesis of both HBc¢ as a helper moiety and a read-through fusion protein containing a foreign sequence.…”

“…Expression of such recombinant gene in an E. coli suppressor strain leads to the simultaneous synthesis of both HBc¢ as a helper moiety and a read-through fusion protein containing a foreign sequence. This technology allowed incorporation into, and presentation onto mosaic particles of 45 [109], 114 [114], 120 [110], and even 213 [Kazaks et al, in preparation] aa long segments of hantavirus nucleocapsid, although nonmosaic HBc¢ carrying the hantavirus segment at the C-terminus were unable to self-assemble. However, in the animal model mosaic particles carrying 45 and 114 aa of the hantavirus nucleocapsid protein failed to induce or induced only a marginal protective response [109,114].…”