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Virus-like particles (VLPs) are multisubunit self-assembly competent protein structures with identical or highly related overall structure to their corresponding native viruses. To construct a new filamentous VLP carrier, the coat protein (CP) gene from potato virus M (PVM) was amplified from infected potato plants, cloned, and expressed in Escherichia coli cells. As demonstrated by electron microscopy analysis, the PVM CP self-assembles into filamentous PVM-like particles, which are mostly 100-300 nm in length. Adding short Gly-Ser peptide at the C-terminus of the PVM, CP formed short VLPs, whereas peptide and protein A Z-domain fusions at the CP N-terminus retained its ability to form typical PVM VLPs. The PVM-derived VLP carrier accommodates up to 78 amino acid-long foreign sequences on its surface and can be produced in technologically significant amounts. PVM-like particles are stable at physiological conditions and also, apparently do not become disassembled in high salt and high pH solutions as well as in the presence of EDTA or reducing agents. Despite partial proteolytic processing of doubled Z-domain fused to PVM VLPs, the rabbit IgGs specifically bind to the particles, which demonstrates the functional activity and surface location of the Z-domain in the PVM VLP structure. Therefore, PVM VLPs may be recognized as powerful structural blocks for new human-made nanomaterials.

This work demonstrates studies of ferromagnetic nanoparticle functionalization by a lipid-like compound, cationic pyridine amphiphile derived from 1,4-dihydropyridine, 1,1'-{[3,5-bis(dodecyloxycarbonyl)-4-phenyl-1,4-dihydropyridine-2,6-diyl]dimethanediyl}dipyridinium dibromide, and determination of optimal conditions for the production of magnetoliposomes. The following methodologies, such as sonication, spontaneous swelling, and reverse-phase evaporation, were used for the preparation of liposomes. Ferrofluid containing γ-Fe2O3 nanoparticles with a positively charged surface was used for the preparation of magnetoliposomes. The obtained soft matter objects were studied by optical microscopy, transmission electron microscopy, and dynamic light scattering techniques. The optimal conditions for the production of magnetoliposomes were found by spontaneous swelling and reverse-phase evaporation methods using ferrofluid with positively charged γ-Fe2O3 nanoparticles and cationic 1,4-dihydropyridine derivative.

The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine.

Three variants of the major rubella virus (RV) E1 protein virus-neutralizing epitope from position 214 to 285 were exposed on the hepatitis B virus (HBV) C-terminally truncated core (HBcΔ) in a virus-like particle (VLP) vector and were produced in Escherichia coli . All three chimeras demonstrated VLPs in bacterial cell lysates, but only HBcΔ-E1(245-285) demonstrated the correct VLP structure after purification. The other chimeras, HBcΔ-E1(214-285) and HBcΔ-E1(214-240), appeared after purification as non-VLP aggregates of 100 to 900 nm in diameter according to dynamic light scattering data. All three variants possessed the intrinsic antigenic activity of RV E1, since they were recognized by natural human anti-RV E1 antibodies and induced an anti-RV E1 response in mice. HBcΔ-E1(214-240) and HBcΔ-E1(245-285) can be regarded as prototypes for a putative RV vaccine because they were able to induce antibodies recognizing natural RV E1 protein in RV diagnostic kits.

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8 mg from 1 g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

The preS1phil, a hydrophilic component of the hepatitis B virus (HBV) preS1 sequence, was exposed on the surface of three widely used virus-like particle (VLP) carriers by (i) insertion into the HI loop of the murine polyomavirus (MPyV) VP1, (ii) N-terminal addition to the hepatitis B surface (HBs) protein, and (iii) insertion into the major immunodominant region (MIR) of three hepatitis B core (HBc) vectors with different structure of their C-termini. Adjuvant-free immunisation of Balb/c mice demonstrated high preS1-specific antibody responses, but strong Th1 cell activation with efficient induction of IgG2a isotype antibodies was observed only in those VLPs, and namely in two of three HBc derivatives, which contained packaged RNA.

RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.

The dynamics of the motile magnetotactic bacterium Magnetospirillum gryphiswaldense in a rotating magnetic field is investigated experimentally and analyzed by a theoretical model. These elongated bacteria are propelled by single flagella at each bacterial end and contain a magnetic filament formed by a linear assembly of approximately 40 ferromagnetic nanoparticles. The movements of the bacteria in suspension are analyzed by consideration of the orientation of their magnetic dipoles in the field, the hydrodynamic resistance of the bacteria, and the propulsive force of the flagella. Several novel features found in experiments include a velocity reversal during motion in the rotating field and an interesting diffusive wandering of the trajectory curvature centers. A new method to measure the magnetic moment of an individual bacterium is proposed based on the theory developed.

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.

The major immunodominant region (MIR) and N-terminus of the hepatitis B virus (HBV) core (HBc) protein were used to expose foreign insertions on the outer surface of HBc virus-like particles (VLPs). The additions to the HBc positively charged arginine-rich C-terminal (CT) domain are usually not exposed on the VLP surface. Here, we constructed a set of recombinant HBcG vectors in which CT arginine stretches were substituted by glycine residues. In contrast to natural HBc VLPs and recombinant HBc VLP variants carrying native CT domain, the HBcG VLPs demonstrated a lowered capability to pack bacterial RNA during expression in Escherichia coli cells. The C-terminal addition of a model foreign epitope from the HBV preS1 sequence to the HBcG vectors resulted in the exposure of the inserted epitope on the VLP surface, whereas the same preS1 sequences added to the native CT of the natural HBc protein remained buried within the HBc VLPs. Based on the immunisation of mice, the preS1 epitope added to the HBcG vectors as a part of preS1(20–47) and preS1phil sequences demonstrated remarkable immunogenicity. The same epitope added to the original C-terminus of the HBc protein did not induce a notable level of anti-preS1 antibodies. HBcG vectors may contribute to the further development of versatile HBc VLP-based vaccine and gene therapy applications.

The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3' terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete "mature" VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants of hybrid particles induced high humoral and cellular responses to HBcAg. Immunization with the HBc/HCV core particles led to relatively low antibody and T-cell proliferative responses to HCV core epitopes. The HBc/HCV NS3 particles were able to induce high levels of anti-NS3 antibodies in the absence of proliferative responses to HCV epitopes. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCV-specific immunogen.

Objective: Pulmonary valve autografts have been reported as clinically effective for replacement of diseased aortic valve (Ross procedure). Published data about pulmonary valve mechanical and structural suitability as a long-term substitute for aortic valve are limited. The aim of this study was to compare aortic and pulmonary valve properties. Methods: Experimental studies of biomechanical properties and structure of aortic and pulmonary valves were carried out on pathologically unchanged human heart valves, collected from 11 cadaveric hearts. Biomechanical properties of 84 specimens (all valve elements: cusps, fibrous ring, commissures, sinotubular junction, sinuses) were investigated using uniaxial tensile tests. Ultrastructure was studied using transmission and scanning electron microscopy. Results: Ultimate stress in circumferential direction for pulmonary valve cusps is higher than for aortic valve (2.78 ± 1.05 and 1.74 ± 0.29 MPa, respectively). Ultimate stress in radial direction for pulmonary and aortic cusps is practically the same (0.29 ± 0.06 and 0.32 ± 0.04 MPa, respectively). In ultrastructural study, different layout and density in each construction element are determined. The aortic and pulmonary valves have common ultrastructural properties. Conclusions: Mechanical differences between aortic and pulmonary valve are minimal. Ultrastructural studies show that the aortic and pulmonary valves have similar structural elements and architecture. This investigation suggests that the pulmonary valve can be considered mechanically and structurally suitable for use as an aortic valve replacement.

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.

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.

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 organ's 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.

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.

Icosahedral virus-like particles (VLPs) of RNA phage Qbeta are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between beta-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 Qbeta 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 Qbeta VLPs, along with normal icosahedral capsids. The length of the rod-like particles exceeded 4-30 times the diameter of icosahedral Qbeta VLPs.

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

The Q beta 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 phage Q beta-based vectors suggested application of the 195-amino acid extension of coat protein (CP) within the so-called A1 protein for insertion of the appropriate immunological epitopes. 'Mosaic' capsids presenting model hepatitis B virus preS1 and HIV-1 gp120 epitopes and formed by Q beta CP together with A1-derived proteins were obtained as a result of (1) suppression of leaky UGA stop codon of the CP gene and (2) simultaneous expression of 'pure' CP and full-length A1-derived genes obtained after the changing of CP-terminating UGA to strong UAA stop codon or sense GGA codon, respectively.

An efficient pBR327- and Ptrp-based E. coli expression system was used to generate a large-scale library of virus like particles (VLP) formed by recombinant hepatitis B virus (HBV) core (HBc) protein derivatives. To construct the library, the gene of HBc protein of the genotype D/subtype ayw2 virus was gradually truncated from the 3`-end and twenty-two HBc variants (with truncation up to 139 aa) were expressed at high levels. The proteins were purified by salt precipitation and gel filtration. Background RNA binding was observed for VLPs formed by HBc1-149, which lacked all C-terminal Arg blocks, and the addition of three Arg residues (HBc1-152) only slightly increased RNA binding. The presence of two Arg blocks (proteins HBc1-162 and HBc1-163) resulted in approximately half of the typical level of RNA binding, and the presence of three blocks (protein HBc1-171) led to approximately 85% of the typical level of binding. Only a small increase in the level of RNA binding was found for the HBc1-175 VLPs, which contained all four Arg blocks but lacked the last 8 aa of the full-length HBc protein. VLPs containing high levels of RNA had higher antigenicity according to an ELISA with anti-HBc mAbs than the VLPs formed by HBc variants without C-terminal Arg blocks and lacking RNA. The results indicate that the VLPs were stabilised by nucleic acids. The immunogenicity in BALB/c mice was comparable for VLPs formed by different HBc proteins, but a clear switch from a Th1 response to a Th2 response occurred after the loss of encapsidated RNA. We did not observe significant differences in lymphocyte proliferation in vitro for the tested VLP variants; however, the loss of RNA encapsidation correlated with a decreased level of IFN-γ induction, which is a measure of the potential CTL activity of immunogens.

Background Subviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution. Findings The findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization. Conclusions The large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.

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.

Virus-like particles (VLPs) are created by the self-assembly of multiple copies of envelope and/or capsid proteins from many viruses, mimicking the conformation of a native virus. Such noninfectious nanostructures are mainly used as antigen-presenting platforms, especially in vaccine research; however, some of them recently were used as scaffolds in biotechnology to produce targeted nanoparticles for intracellular delivery. This study demonstrates the creation of fusion VLPs using hepatitis B core protein-based system maintaining a fibronectin-binding property from B. burgdorferi BBK32 protein, including the evidence of particles’ transmission to BHK-21 target cells via caveolae/rafts endocythosis. These results make this construct to be an attractive model in development of HBc-based nanoparticles for cellular targeting applications and highlights the fragment of B. burgdorferi BBK32 as a novel cellular uptake-promoting peptide., This work was supported by grant of Latvian Council of Science, Nr. 10.0029.3 and by ESF Project

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.

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

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.

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.

Vaccination of melanoma patients with tumor-specific antigens recognized by cytotoxic T lymphocytes (CTLs) may produce significant tumor regressions. Here, we suggest a novel type of tumor vaccines, with well-studied CTL epitopes presented on highly immunogenic virus-like particle (VLP) carriers. Cancer-germline gene MAGE-3 encodes for an antigenic nonapeptide (MAGE-3(168-176) peptide) that is recognized by CTLs on human leukocyte antigen (HLA)-A1 and HLA-B35 molecules. A set of recombinant genes encoding hepatitis B virus core protein carrying MAGE-3 epitope was constructed and expressed in Escherichia coli cells. Variants that led to formation of chimeric VLPs in vivo were purified and analyzed for their DNA binding properties in vitro. VLPs exhibiting the most pronounced nucleic acid binding affinity were selected and loaded either with single-stranded DNA oligodeoxynucleotides rich in nonmethylated CG motifs, or with longer double-stranded DNA fragments. Packaged DNA was protected, at least partially, against the action of bacterial DNase. Such highly purified chimeric VLPs with entrapped immunomodulatory sequences could possibly be used as antitumor vaccines.

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.

Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well.

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.

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.

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 anti-HBc antibodies.

The construction of a mouse hybridoma FRS2 secreting neutralizing monoclonal antibody specific for RNA bacteriophages fr, MS2 and GA is reported. The genes encoding the variable domains of the monoclonal antibody FRS2 heavy and light chains were cloned and sequenced and the corresponding complementarity determining region (CDR) peptides were chemically synthesized. The CDR-peptides were tested for their ability to neutralize the activity of RNA phage fr and related RNA phages MS2 and GA. The CDR-derived peptides H2, L2 and L3 interacted with the fr phage particles and neutralized fr phage activity. Two of these peptides-H2 and L3 also had the ability to neutralize partly the activity of related bacteriophage MS2; butLJande-speciilll'y L3 neutralize the activity of the RNA phage GA. These results provide an excellent system for further antibody­ antigen interaction studies and raise the possibility that simple CDR-peptides may serve as a new class of anti­ viral molecules. [Hum Antibod Hybridomas 1996; 7: 106-112]

The Escherichia coli RNA phage Q beta coat protein-encoding gene (C) was amplified from native Q beta RNA using a reverse transcription-PCR technique. Gene C contains sequences coding for both the 133-amino acid (aa) Q beta coat protein (CP) and the 329-aa read-through protein (A1) consisting of CP and an additional 196-aa C-terminal sequence, separated from CP within the C gene by an opal (UGA) stop codon. Primers ensuring the natural environment for gene C, especially within the ribosome-binding site, and supplying C with unique restriction sites at both ends have been prepared. An amplified 1062-bp PCR fragment was positioned under the control of the strong E. coli trp promoter (Ptrp) within a pGEM-derived plasmid. The synthesis of gene C products was confirmed electrophoretically and immunologically. An immunodiffusion test with anti-Q beta phage antibodies and electron microscopy evaluation of the purified recombinant products showed that when expressed, the Q beta C gene was responsible for high-level synthesis and correct self-assembly of Q beta CP monomers into capsids indistinguishable morphologically and immunologically from Q beta phage particles, which we plan to use as surface display vectors.