Stop Codon Insertion Restores the Particle Formation Ability of Hepatitis B Virus Core-Hantavirus Nucleocapsid Protein Fusions (original) (raw)
Related papers
Journal of Biotechnology, 1999
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.
Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments
Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured ' virus-like particles ' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg∆) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg∆ and a readthrough fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.
Virology, 2004
Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.
Journal of General Virology, 2004
The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium-and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.
The sequence of the preS domain of the hepatitis B virus (HBV, genotype D) envelope was inserted into the major immunodominant region (MIR) of the C-terminally truncated HBV core (HBc) protein. In Escherichia coli, the HBc-preS fusion protein was partially soluble and did not produce particles. Co-expression of the wild-type HBc as a helper protein along with the fusion protein led to the formation of mosaic HBc particles that exhibited HBc, preS1 and preS2 antigenicity. Two alternative combinations of medium-and high-copy plasmids were used for co-expression of fusion and helper proteins, in an attempt to improve mosaic particle production. However, the preS fusion content of the particles remained the same in both expression combinations. In a third co-expression in which the modified HBc helper lacked aa 76-85 in the MIR, the incorporation level of HBc-preS fusion into the particles was noticeably lower. Purified chimeric particles were immunogenic in mice.
Archives of Virology, 1994
The properties of three different recombinant hepatitis B virus core proteins expressed in Escherichia eoli were compared: an N-terminal fusion protein, a C-terminally truncated protein and a sequence-authentic protein. All three proteins assembled into capsid-like particles with typical HBcantigenicity, sedimentation behavior and distinctive electron microscopical images. Apart from this, however, variant HBc proteins displayed properties different from sequence-authentic HBc protein p21.4. Unlike p21.4, the particles of the N-terminal fusion protein p22.2 were sensitive to proteolytic attack by trypsin at variable sites within its arginine-rich C-terminus but not in its extended N-terminus. We therefore conclude that the C-terminal region is located on the surface of the p22.2 particle. These particles also showed increased HBe-antigenicity, as did the C-terminally truncated core particles p17.6, and to an even greater extent p18* particles which were derived from p22.2 by tryptic digestion. This might be interpreted as evidence for an-albeit .minor-structural change. All variant core particles were less stable and contained less RNA. Electron microscopic indication for DNA binding of C-terminal deleted p17.6 particles was obtained using an aqueous spreading technique.
Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs)
Methods in molecular biology (Clifton, N.J.), 2018
The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferre...
FEBS Letters, 1989
Insertion of foreign oligopeptide sequences (4G50 amino acids in length) into the Pro144 position of hepatitis B core antigen (HBcAg) leads to the formation of chimeric capsids in Escherichia co/i cells. These capsids are morphologically and immunogically similar to native HBcAg, but expose the inserted oligopeptides on their outer surface and exhibit antigenic and immunogenic characteristics of the latter. As a source of model antigenic determinants, the appropriate DNA copies excised from cloned viral genes such as the pm-S region of hepatitis B virus, the transmembrane protein gp41 of human immunodeficiency virus 1 and the envelope protein gp51 of bovine leukemia virus have been used. The localization of the inserted antigenic determinants on the surface of chimeric capsids does not depend on the presence or absence of the arginine-rich, 39 amino acidlong C terminus of HBcAg. Hepatitis B core antigen; PreS region; Human immunodeficiency virus-l transmembrane protein gp41; Bovine leukemia virus envelope protein gp51; Antigenic determinant
Core particles of hepatitis B virus as carrier for foreign epitopes
Advances in virus research, 1998
To be effective as vaccines, most monomeric proteins and peptides either require chemical coupling to high molecular weight carriers or application together with adjuvants. More recently, recombinant DNA techniques have been used to insert foreign epitopes into proteins with inherent multimerization capacity, such as particle-forming viral capsid or envelope proteins. The core protein of hepatitis B virus (HBcAg), because of its unique structural and immunological properties, has gained widespread interest as a potential antigen carrier. Foreign sequences of up to approximately 40 amino acid residues at the N terminus, 50 or 100 amino acids in the central immunodominant c/e 1 epitope region of HBcAg, and up to 100 or even more residues at the C terminus, did not interfere with particle formation. The humoral immunogenicity of inserted epitopes is determined by the immunogenicity of the peptide itself and its surface exposure, and is influenced by the route of application. The probab...