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Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli , the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.

The available HBV vaccines based on the HBV surface protein are manufactured in yeasts and demonstrate excellent prophylactic but no therapeutic activity and are thus ineffective against chronic HBV infection. Five different HBV core proteins (HBc)-full length and C-terminally truncated-were used for the insertion of the short, preS1,aa 20-47 and long, preS1phil, aa 12-60 + 89-119 fragments. Modified virus-like particles (VLPs) were compared for their biotechnological and immunological properties. The expression level of HBc-preS1 proteins was high for all investigated proteins, allowing us to obtain 10-20 mg of purified VLPs from a gram of biomass with the combination of gel filtration and ion-exchange chromatography to reach approximately 90% purity of target proteins. The immunogenicity of chimeric VLPs was tested in BALB/c mice, showing a high anti-preS1 response and substantial T-cell proliferation after stimulation with HBc protein. Targeted incorporation of oligonucleotide ODN 1668 in modified HBc-preS1 VLPs was demonstrated., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The core proteins (HBc) of the hepatitis B virus (HBV) genotypes A, B, C, D, E, F, and G were cloned and expressed in Escherichia coli (E. coli ), and HBc-formed virus-like particles (VLPs) were purified with ammonium sulfate precipitation, gel filtration, and ion exchange chromatography (IEX). The best VLP yield was found for the HBc of the HBV genotypes D and G. For the HBc of the HBV genotypes D, F, and G, the possibility of dissociation and reassociation maintaining the native HBc structure was demonstrated. Single-stranded (ss) and double-stranded (ds) ribonucleic acid (RNA) was successfully packed into HBc VLPs for the HBV genotypes D and G.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. Through a complex enzootic cycle, the bacteria transfer between two different hosts: Ixodes ticks and mammalian organisms. At the start of the tick blood meal, the spirochetes located in the tick gut upregulate the expression of several genes, mainly coding for outer surface proteins. Outer surface proteins belonging to the paralogous gene family 54 (PFam54) have been shown to be the most upregulated among the other borrelial proteins and the results clearly point to the potential importance of these proteins in the pathogenesis of Lyme disease. The significance of PFam54 proteins is confirmed by the fact that of all ten PFam54 proteins, BBA64 and BBA66 are necessary for the transfer of B. burgdorferi from infected Ixodes ticks to mammalian hosts. To enhance the understanding of the pathogenesis of Lyme disease and to promote the development of novel therapies against Lyme disease, we solved the crystal structure of the PFam54 member BBA65. Additionally, we report the structure of the B. burgdorferi BBA64 orthologous protein from B. spielmanii. Together with the previously determined crystal structures of five PFam54 members and several related proteins, we performed a comprehensive structural analysis for this important group of proteins. In addition to revealing the molecular aspects of the proteins, the structural data analysis suggests that the gene families PFam54 and PFam60, which have long been referred to as separate paralogous families, should be merged into one and designated as PFam54_60., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determined the crystal structures of the key outer surface protein BBE31 from B. burgdorferi and its orthologous protein BSE31 (BSPA14S_RS05060 gene product) from B. spielmanii. BBE31 is known to be important for the transfer of B. burgdorferi from the gut to the hemolymph in the tick after a tick bite. While BBE31 exerts its function by interacting with the Ixodes scapularis tick gut protein TRE31, structural and mass spectrometry data revealed that BBE31 has a glutathione (GSH) covalently attached to Cys142 suggesting that the protein may have acquired some additional functions in contrast to its orthologous protein BSE31, which lacks any interactions with GSH. In the current study, in addition to analyzing the potential reasons for GSH binding, the three-dimensional structure of BBE31 provides new insights into the molecular details of the transmission process as the protein plays an important role in the initial phase before the spirochete is physically transferred to the new host. This knowledge will be potentially used for the development of new strategies to fight against Lyme disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The periplasmic lipoprotein BB0365 of the Lyme disease agent Borrelia burgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG subunits of the NADH:quinone and ferredoxin:NAD + sodium-translocating oxidoreductase complexes, which points to a potential role for BB0365 as a component of the sodium pump. Additionally, BB0365 coordinated Zn 2+ by the His51, His55, His140 residues, and the Zn 2+ -binding site indicates that BB0365 could act as a potential metalloenzyme; therefore, this structure narrows down the potential functions of BB0365, an essential protein for B. burgdorferi to cause Lyme disease., (© 2019 Federation of European Biochemical Societies.)

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis - the most common tick-borne disease in Europe and the United States. Spirochetes are transmitted from infected Ixodes ticks to the mammalian host when the ticks feed. In general, the transfer process of the borreliae is quite complicated, as the environments in the tick and the new mammalian host differs significantly. Therefore, Borrelia changes the expression profile of dozens of proteins, mainly outer surface proteins, to adapt to the new tasks and needs in the new organism. In the transfer process from the tick to the mammalian host, spirochetes that cause Lyme disease show the strongest upregulation of members of paralogous gene family 54 (PFam54). PFam54 members encode 10 proteins, and BBA69 is one of its members. Although several PFam54 members play an important role in the pathogenesis of Lyme disease, the exact function has been determined only for CspA, which binds complement regulator factor H (CFH) and factor H-like protein 1 (FHL-1); thus, CspA is essential to resist the vertebrate host's immune response. In the current study, we determined the crystal structure of BBA69 at a 2.25 Ǻ resolution. The BBA69 structure revealed a seven α-helical BbCRASP-1 fold previously found only in PFam54 member proteins. Among the PFam54 members, BBA69 shares the highest sequence identity (61%) and 3-D similarity with CspA. Although none of the PFam54 members besides CspA bind CFH and FHL-1, in the current study, we investigated the structural differences accounting for the divergence in the functions of these proteins. The results clearly indicated that the C-terminal α-helix is the main determinant of this functional divergence. The results provide better insight into the PFam54 proteins that play an important role in the pathogenesis of Lyme disease., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Borrelia (B.) bavariensis, B. burgdorferi, B. afzelii, B. garinii, B. spielmanii, and B. mayonii are the causative agents in Lyme disease. Lyme disease spirochetes reside in infected Ixodes ticks and are transferred to mammalian hosts during tick feeding. Once transmitted, spirochetes must overcome the first line of defense of the innate immune system either by binding complement regulators or by terminating the formation of the membrane attack complex (MAC). In B. bavariensis, the proteins BGA66 and BGA71 inhibit complement activation by interacting with the late complement components C7, C8, and C9, as well as with the formed MAC. In this study, we have determined the crystal structure of the potent MAC inhibitor BGA71 at 2.9 Ǻ resolution. The structure revealed a cysteine cross-linked homodimer. Based on the crystal structure of BGA71 and the structure-based sequence alignment with CspA from B. burgdorferi, we have proposed a potential binding site for C7 and C9, both of which are constituents of the formed MAC. Our results shed light on the molecular mechanism of immune evasion developed by the human pathogenic Borrelia species to overcome innate immunity. These results will aid in the understanding of Lyme disease pathogenesis and pave the way for the development of new strategies to prevent Lyme disease.

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1-173, 1-152, and 147-191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147-191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147-191 reached 10(5); core aa 1-152, 5 × 10(5); core aa 1-173 and F-protein, 10(6). Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature - Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the host's immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H-like protein 1 (CFHL-1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL-1 binding protein CspZ (also known as BbCRASP-2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N-terminal part of the CspZ protein forms a four-helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α-catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL-1., (© 2014 FEBS.)

Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host organisms by infected Ixodes ticks. Transfer of the spirochaetal bacteria from Ixodes ticks to the warm-blooded mammalian organism provides a challenge for the bacteria to adapt and survive in the different environmental conditions. B. burgdorferi has managed to differentially express genes in response to the encountered changes such as temperature and pH variance or metabolic rate to survive in both environments. In recent years, much interest has been turned on genes that are upregulated during the borrelial transfer to mammalian organisms as this could reveal the proteins important in the pathogenesis of Lyme disease. BBA66 is one of the upregulated outer surface proteins thought to be important in the pathogenesis of B. burgdorferi as it has been found out that BBA66 is necessary during the transmission and propagation phase to initiate Lyme disease. As there is still little known about the pathogenesis of B. burgdorferi, we have solved the crystal structure of the outer surface protein BBA66 at 2.25Å resolution. A monomer of BBA66 consists of 6 α-helices packed in a globular domain, and the overall folding is similar to the homologous proteins BBA64, BBA73, and CspA. Structure-based sequence alignment with the homologous protein BBA64 revealed that the conserved residues are mainly located inwards the core region of the protein and thus may be required to maintain the overall fold of the protein. Unlike the other homologous proteins, BBA66 has an atypically long disordered region at the N terminus thought to act as a "tether" between the structural domain and the cell surface., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

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.

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.

Lyme disease is a tick-borne infection caused by the transmission of Borrelia burgdorferi from infected Ixodes ticks to a mammalian host during the blood meal. Previous studies have shown that the expression of B. burgdorferi surface-localized lipoproteins, which include BBA64, is up-regulated during the process of tick feeding. Although the exact function of BBA64 is not known, this lipoprotein is critical for the transmission of the spirochete from the tick salivary glands to the mammalian organism after a tick bite. Since the mechanism of development of the disease and the functions of the surface lipoproteins associated with borreliosis are still poorly understood, the crystal structure of the B. burgdorferi outer surface lipoprotein BBA64 was solved at 2.4 Å resolution in order to obtain a better insight into the pathogenesis of B. burgdorferi and to promote the discovery of novel potential preventive drugs against Lyme disease. In this study, the crystal structure of BBA64 was also compared with that of the paralogous protein CspA (also referred to as BbCRASP-1, CRASP-1 or BBA68). CspA is the complement regulator-acquiring surface protein-1 of B. burgdorferi; its structure is known, but its function apparently differs from that of BBA64. It is demonstrated that unlike the homologous CspA, BBA64 does not form a homodimer. Their differences in function could be explained by divergence in their amino-acid sequences, electrostatic surface potentials and overall tertiary structures. The C-terminal part of BBA64 has a different conformation to that of CspA; the conformation of this region is essential for the proper function of CspA.

Borrelia burgdorferi, which is the causative agent of Lyme disease, is transmitted from infected Ixodes ticks to a mammalian host following a tick bite. Upon changing the host organism from an Ixodes tick to a warm-blooded mammal, the spirochete must adapt to very different conditions, which is achieved by altering the expression of several genes in response to a changing environment. Recently, considerable attention has been devoted to several outer surface proteins, including BBA73, that undergo dramatic upregulation during the transmission of B. burgdorferi from infected Ixodes ticks to mammals and that are thought to be important for the establishment and maintenance of the infection. These upregulated proteins could reveal the mechanism of pathogenesis and potentially serve as novel drug targets to prevent the transmission of the pathogenic bacteria. To promote effective treatments for Lyme disease and to gain better insight into B. burgdorferi pathogenesis, we have determined the crystal structure of the upregulated outer surface protein BBA73 at 2.0 Å resolution. We observed that the BBA73 protein exists as a homodimer both in the crystal and in solution. The monomers interact with their N-terminal α-helices and form a cleft that could potentially serve as a ligand or receptor binding site. To confirm that the protein dimerizes through the interaction of the N-terminal regions, we produced an N-terminal deletion mutant of BBA73 to disrupt dimerization, and we determined the crystal structure of the truncated BBA73 protein at 1.9 Å resolution. The truncated protein did not form a homodimer, and the crystal structure confirmed that the overall fold is identical to that of the native BBA73 protein. Notably, a paralogous protein CspA from B. burgdorferi with known crystal structure also forms a homodimer, albeit through an entirely different interaction between the monomers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., From the Clinical Editor: This paper discusses the nanotechnology-based application of self-assembling viral-like peptides (VLP-s) for targeted delivery using a hepatitis B core protein based system. Creating fusion VLPs may be an attractive model for cellular targeting applications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Plasmids pQE-60 and pQE-30 containing 6 x His-tag sequence were used for expression of fragments of HCV structural and non-structural proteins in Escherichia coli (E. coli). The following fragments were used: core (1-98 aa), NS3 (202-482 aa), and tetramer of hypervariable region 1 (HVR1) of E2 protein. The constructed plasmids directed high levels of expression of HCV proteins in E. coli JM109. After purification by the metal-affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose, the His-tagged HCV proteins were used for immunization of BALB/c mice. All three proteins were able to induce high levels of specific antibodies and, in the case of the NS3 and HVR1 tetramer, also to mount vigorous cell-proliferating responses. High immunogenicity of the tested fragments of HCV proteins shows them as good candidates for inclusion into the future HCV vaccine preparations.

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.

Hepatitis B virus (HBV) core particles carrying the amino-terminal 120 amino acids (aa) of the nucleocapsid (N) protein of the hantaviruses Dobrava, Hantaan or Puumala have been demonstrated to be highly immunogenic in mice when complexed with adjuvants. Here we demonstrate that even without adjuvant, these chimeric particles induced high-titered, and strongly cross-reactive N-specific antibody responses in BALB/c and C57BL/6 mice. The induced N-specific antibodies represented all IgG subclasses. Pre-existing core-specific antibodies did not abrogate the induction of an N-specific immune response by a hantavirus N insert presented on core particles. Therefore, chimeric core particles should represent promising vaccine candidates even for anti-core positive humans.

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 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 (HBc) 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 HBc 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' HBc 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 HBc 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 HBc Delta-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 HBc Delta-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., (Copyright 2002 S. Karger AG, Basel)

Spatial and immunochemical elucidation of hepatitis B core antigen suggested unique organization of its major immunodominant region (MIR) localized within the central part of molecule around amino acid residues 74-83. This superficial loop was recognized as the most prospective target for the insertion of foreign epitopes ensuring maximal antigenicity and immunogenicity of the latter. MIR allowed a substantial capacity of insertions up to about 40 amino acid residues without loss of the capsid-forming ability of core particles. Vector capacity as well as structural behavior and immunological fate of inserted epitopes were dependent on their primary structure. Special sets of display vectors with retained but cross-sectioned MIR as well as with uni- and bidirectionally shortened MIR have been investigated.