Velta Ose

Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy

Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transcription to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

Background Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. Methodology/Principal Findings Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. Conclusions/Significance AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

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

Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods

Icosahedral virus‐like particles (VLPs) of RNA phage Qβ are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between β‐strands F and G (FG loop) of the monomeric subunits, which determine the five‐fold and quasi‐six‐fold symmetry contacts of the VLPs. In order to reduce the stability of Qβ VLPs, we mutationally converted the amino acid stretch 76‐ANGSCD‐81 within the FG loop into the 76‐VGGVEL‐81 sequence. It led to production in Escherichia coli cells of aberrant rod‐like Qβ VLPs, along with normal icosahedral capsids. The length of the rod‐like particles exceeded 4–30 times the diameter of icosahedral Qβ VLPs.

Evaluation of HBs, HBc, and frCP Virus-Like Particles for Expression of Human Papillomavirus 16 E7 Oncoprotein Epitopes

Objectives: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35–98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP). Methods: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied. Results: The HBs*-E7(35–54) protein expressing E7 residues 35–54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae. The HBcΔ-E7(35–98), but not the frCP-E7(35–98), ensured VLP formation in Escherichia coli. In Balb/C mice, the HBs*-E7(35–54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBcΔ-E7(35–98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response. The frCP-E7(35–98) protein elicited equally high antibody responses to both E7 and frCP carrier. Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset. Conclusions: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes. All chimeric proteins may be regarded as potential vaccine candidates.

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.

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

Potato Virus Y-Like Particles as a New Carrier for the Presentation of Foreign Protein Stretches

Virus-like particle (VLP) technology represents a promising approach for the creation of efficient vaccines and materials for use in nanotechnological applications. For construction of a new carrier for foreign protein sequences, the coat protein (CP) gene from potato virus Y (PVY) was cloned and expressed in Escherichia coli cells. The PVY CP self-assembles into PVY-like particles, as demonstrated by electron microscopy analysis of purified VLP preparations. The PVY CP with an N-terminal insertion of a foreign epitope (preS1) or of a whole protein (rubredoxin) retains its ability to form filamentous particles, whereas adding a foreign sequence to the C-terminus of the PVY CP generates mostly unstructured protein aggregates. This new filamentous plant virus-derived VLP carrier accommodates a foreign protein sequence that is up to 71 amino acids in length on the VLP surface and can be produced in E. coli in preparative amounts. The PVY CP VLPs are stable in physiological conditions, but they are sensitive to EDTA, high salt, and extreme pH. The presence of the preS1 epitope decreases the stability of the chimeric PVY CP particles at elevated temperatures. Mice that are immunized with chimeric PVY CP particles carrying preS1 epitopes exhibit a strong anti-preS1 immune response, even in the absence of adjuvants.

Biomechanical properties of oesophagus wall under loading

In this investigation, firstly, the biomechanical properties of different parts of oesophagus were determined. Oesophagus stress and strain are the greatest in the cervical part for all age groups. The human oesophagus deforms unevenly, depending on the direction of load in relation to the organs axis, it exhibits anisotropical behaviour. With the age the values of mechanical parameters of the oesophagus wall reduce, in particular beginning from 45 years of age, but the modulus of elasticity increases. Biomechanical properties of the oesophagus depend on the architecture of its structure. By loading the organ in the circumferential direction, microfibrilae rupture and deformation of the muscular fibres occurs. With increase of load, collagenous fibres straighten and microruptures in collagenous fibrilae occur. With stretching of oesophagus longitudinally, collagenous fibres partially preserve their wavy and helical configuration. Therefore, higher resistance of the oesophageal wall occurs in the longitudinal direction.