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• Partial virus-like particle models were constructed for chimeric vaccine simulations. • MIR-inserted sequence properties have a greater impact on HBc self-assembly than its length. • MD simulation well predicts the surface hydrophobicity and overall stability of HBc-based vaccines. • MD simulation minimizes design failures and provides guidance for the downstream processing of chimeric VLP vaccines. Self-assembling virus-like particles (VLPs) are promising platforms for vaccine development. However, the unpredictability of the physical properties, such as self-assembly capability, hydrophobicity, and overall stability in engineered protein particles fused with antigens, presents substantial challenges in their downstream processing. We envision that these challenges can be addressed by combining more precise computer-aided molecular dynamics (MD) simulations with experimental studies on the modified products, with more to-date forcefield descriptions and larger models closely resembling real assemblies, realized by rapid advancement in computing technology. In this study, three chimeric designs based on the hepatitis B core (HBc) protein as model vaccine candidates were constructed to study and compare the influence of inserted epitopes as well as insertion strategy on HBc modifications. Large partial VLP models containing 17 chains for the HBc chimeric model vaccines were constructed based on the wild-type (wt) HBc assembly template. The findings from our simulation analysis have demonstrated good consistency with experimental results, pertaining to the surface hydrophobicity and overall stability of the chimeric vaccine candidates. Furthermore, the different impact of foreign antigen insertions on the HBc scaffold was investigated through simulations. It was found that separately inserting two epitopes into the HBc platform at the N-terminal and the major immunogenic regions (MIR) yields better results compared to a serial insertion at MIR in terms of protein structural stability. This study substantiates that an MD-guided design approach can facilitate vaccine development and improve its manufacturing efficiency by predicting products with extreme surface hydrophobicity or structural instability. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1–6 suppressed the production of cccDNA. These results suggest that KPNA1–6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1–6 suppressed the production of cccDNA. These results suggest that KPNA1–6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.

YongLi Hou,1 Ting Yan,2 Hui Cao,1 Peng Liu,1 Kang Zheng,1 Zhenyu Li,1 Qing Deng,1 SiHai Hu11Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Institution of Pathogenic Biology, Hengyang Medical College, University of South China, Hengyang 421001, People’s Republic of China; 2Department of Health Services, Air Force Medical University, Xi’an, Shaanxi 710032, People’s Republic of ChinaCorrespondence: SiHai HuInstitution of Pathogenic Biology, Hengyang Medical College, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, People’s Republic of ChinaTel +86 1 397 340 7567Fax +86 734 828 2907Email hhsshh_518@163.comPurpose: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB).Methods: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1–144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer.Results: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue.Conclusion: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.Keywords: hepatitis B core protein, Neisserial surface protein A, virus-like particles, recombinant protein vaccine, Neisseria meningitidis serogroup B

Abstract Background Truncated mis-disordered tau protein plays an important role in the pathogenesis of Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Tau294–305, an epitope in the truncated tau, is essential for pathological tau-tau interaction and aggregation. A tau294–305-targeted approach may have beneficial effects in the treatment of AD and FTD. Methods In this study, we genetically fused tau294–305 epitope to the hepatitis B virus core protein (HBc) major immunodominant region (MIR) (with the resultant protein termed T294-HBc), and we subcutaneously immunized a Tau.P301S transgenic mouse model of FTD and AD with T294-HBc four times. The levels and characteristics of antibodies induced by T294-HBc were determined by enzyme-linked immunosorbent assay. The effect of T294-HBc on the cognitive deficits of Tau.P301S mice was tested using the Morris water maze test, novel object recognition, and a Y-maze test. Western blot analysis and IHC were applied to measure the effect of T294-HBc on tau pathologies and neuroinflammation in the mouse brains. Results The results showed that T294-HBc self-assembled into HBc chimeric virus-like particles (VLPs) with tau294–305 displayed on the surface and that it induced high antibody titers specifically against the mis-disordered truncated tau. Further investigation showed that these antibodies simultaneously bound to microtubule-binding regions 1–4 (MTBR1–4) [tau263–274, tau294–305, tau325–336, tau357–368 and tau294–305(P301S)]. Moreover, T294-HBc VLP vaccination significantly ameliorated memory and cognitive decline; reduced the levels of AT8-positive tau, truncated tau monomer, and oligomer; attenuated microgliosis and astrogliosis; and rescued synaptic deficits in Tau.P301S transgenic mice. Conclusions T294-HBc VLP vaccine elicited strong immune response and alleviated cognitive deficits and neuropathology progression in Tau.P301S mice, indicating that the T294-HBc VLP vaccine has promising therapeutic potential for the treatment of AD and FTD.

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.

Highlights • The HBc particles are heat stable and can withstand temperature up to 80℃ for 30 min. • Host cell protein in cell lysate supernatant can enter and stay in HBc particles above 70℃ in real separation. • Entering of HCP lead to increase in HBc particle size as observed by DLS and MALLS. • A two-step heat treatment strategy achieved 85.8% HBc particle recovery and 74.7% purity. • Further one-step HIC increase the HBc particle purity to 99.0% with overall recovery of 77.7%. Abstract The thermal stability of HBc particles was systematically investigated for efficient removal of host cell proteins (HCP) by heat treatment before chromatographic step. The HBc particles were found stable up to 80°C for 30 min without any noticeable change in circular dichroism spectra, fluorescence spectra and transmission electron microscope observation. When heating was applied to precipitate the HCP in the cell disruption supernatant of HBc fermentation, the HCP removal effect was more obvious as the temperature went higher. However, a phenomenon was found beyond 70°C where the recovered HBc particles had larger than normal size and molecular weight as observed by dynamic light scattering and multi-angle laser light scattering. Analysis found that the HBc particles possess nanopores which expand with temperature. When the temperature was above 70℃, the pores were large enough for some HCP to penetrate in, but not being able to get out after cooling down. To fully utilize the thermal stability and avoid the interference of HCP entering, a two-step heat treatment strategy was designed. The supernatant was firstly heated up to 60°C for 30 min to precipitate most HCP, then another 30 min at 70°C was used to remove the rest impurities. The two-step heat treatment effectively avoided the HCP entering problem, achieving 85.8% particle recovery and 74.7% purity. With further one-step hydrophobic interaction chromatography, the purity was increased to 99.0% with overall process recovery of 77.7%, considerably higher than those reported in the literature. The same process design was applied to purify three HBc-related products, including OVA-HBc, M2e-HBc and NP-HBc. All recoveries were higher than 50% with purity greater than 97%. [ABSTRACT FROM AUTHOR]

Self-assembling protein nanoparticles showed promise for vaccine design due to efficient antigen presentations and safety. However, the unpredictable formations of epitopes-fused protein assemblies remain challenging in the upstream design. This study suggests employing molecular dynamic (MD) simulations to investigate the assembly properties of Hepatitis B core protein (HBc) from thermodynamic perspectives. Eight HBc derivatives were expressed in E. coli , with their self-assembly properties characterised by high-performance liquid chromatography and transmission electron microscopy. MD simulations on the dimers, based on AlphaFold-predicted 3D structures, analysed the derivative at the atomic level. Results revealed that HBc derivatives can form dissociative polymers or large multi-subunit structures due to assembly failures. The instability of the dimer in aqueous solvents or inappropriate intradimer distances could cause major assembly failures. Polar solvation energies played a vital role too in forming assemble-incompetent dimers. Importantly, our study demonstrated that MD simulations on dimers can provide preliminary predictions on the assembly properties of HBc derivatives, thus aiding vaccine design by lowering the risk of self-assembling failures in engineered proteins.Communicated by Ramaswamy H. Sarma.

Virus-mimicking particles have made great contribution to the development of nanomedicine. Herein, several modularized peptides (lipophilic NS5A peptide, 6xHis tag, and tumor-targeting peptide RGD) were genetically inserted into the C-terminus and the major immunodominant loop region (MIR) of hepatitis B core protein (HBc), respectively. This study demonstrated that the recombinant HBc-based VLPs could participate in self-assembly of monodisperse nanoparticles (33.6 ± 3.5 nm) with well-defined morphology, and DOX can be packaged into VLNPs without any chemical modification. Moreover, the HBc-based VLPs could specifically target to cancer cells via the interaction with overexpressed integrin α v β 3 . The treatment with DOX-loaded HBc-based VLPs showed a significant inhibition of tumor growth (90.7% TGI) and less cardiotoxicity in B16F10 tumor-bearing mice models than that with the free DOX. Importantly, the results may offer an easy way to give a variety of ideal functional modulations for VLPs, thereby extending its potential biomedicine applications. [ABSTRACT FROM AUTHOR]

The hepatitis B core protein (HBc) has been suggested to interact with covalently closed circular DNA (cccDNA) and regulate hepatitis B virus (HBV) transcription. However, direct evidence is lacking. We aimed to identify the specific HBc region(s) responsible for transcription regulation and its interaction with cccDNA. Seventeen mutants with mutations at the four arginine-rich clusters of the HBc carboxyl-terminal domain (CTD) were created. The effect of HBc mutations on the levels of HBV DNA, RNA, and hepatitis B surface antigen (HBsAg) were measured. The association of cccDNA with mutant HBc and histone acetyltransferases (HATs) was assessed by chromatin immunoprecipitation (ChIP). Compared with wild-type HBc, HBc mutants with mutations in clusters III and IV resulted in a significant reduction in HBV RNA levels (all P < 0.05). HBc arginine clusters III and IV mutants also had a significantly lower levels of intracellular HBV DNA (<5% of wild-type; P < 0.001) and HBsAg (<10% of wild-type; P < 0.0001). cccDNA-ChIP assay demonstrated that HBc clusters III and IV mutants had a smaller degree of association with cccDNA ( P < 0.001). In the HBc mutants, the association between HATs with cccDNA were reduced. In conclusion, HBc-CTD arginine residues at clusters III and IV play an important role in the regulation of HBV transcription as well as subsequent replication steps, likely through the reduced interaction of HBc with cccDNA and reduced acetylation of cccDNA-bound histones. These findings may provide clues to the identification of novel therapeutic targets against HBV. [ABSTRACT FROM AUTHOR]

Occult hepatitis B virus (HBV) infection (OBI) is characterized by presence of HBVDNA in blood or liver tissue without detectable HBV surface antigen (HBsAg), with or without antibodies to hepatitis B core antigen (anti-HBc) or antibodies against HBsAg (anti-HBs). A molecular and serological characterization was done of OBI in blood donors from Yucatan, Mexico. HBV DNA was found in 24 (6.4%) of the 372 evaluated samples. Anti-HBs was present in 15/24 samples (62.5%), and no significant difference was observed between HBV DNA positivity and anti-HBs levels. HBV genotype H was detected in 66.7% of samples, followed by genotypes D (20.8%) and F (8.3%). Amino acid substitutions were identified in the core region of nine samples, and most of these changes were located in immunodominant epitopes. No precore stop codon 28 mutant (W28Stop) was identified among the analyzed HBV isolates. In conclusion, genotype H is the main circulating HBV strain among OBI blood donors from Yucatan, Mexico. Mutations in the core region may contribute to viral persistence.

Viral nanoparticles (VNPs) are a fascinating class of natural nanoparticles which provide excellent architectural templates that possess the ideal properties of monodisperse size, shape and composition and therefore offer great promise for developing innovative bioinspired nanomaterials. The use of VNPs as templates, carriers, containers and scaffolds for nanotechnology is a new field that has received increasing interest in the last decade. Hepatitis B virus core protein can form VNPs during self-assembly in vivo (in planta) and in vitro. HBV capsid protein forms highly stable icosahedral VNPs with diameter ≈ 30-33 nm. In this study we investigate the potential of modified HB core protein to self-assemble into VNPs during the expression in Nicotiana benthamiana. Interestingly, the stability of HBV VNPs can be controlled by alterations in the length of the capsid protein. In order to optimize chemical modification of HBV VNPs additional Lys residues have been added by site directed mutagenesis to surface exposed c/1 loop. A part of c/1 epitope has been replaced by peptide Gly-Lys-Gly. The introduced additional Lys residues that have reactive ε amino group side chains will facilitate interaction with chemical reagents such as activated NHS esters. This opens up a new opportunity to produce chemically conjugated HBV VNP for biomedical purpose. [ABSTRACT FROM AUTHOR]

Background: The core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes' expression. Results: Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS). Conclusion: HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection. [ABSTRACT FROM AUTHOR]

Abstract: The nucleocapsids formation is a pivotal step of hepatitis B virus (HBV) life cycle. The inhibition of HBV nucleocapsids assembly is a promising strategy for the anti-HBV treatment. HBc78–117 is an internal fragment of hepatitis B core protein (HBc). In this study, we used lentiviral vector to deliver HBc78–117 cDNA sequence into HepG2.2.15 cells and examined the effect of HBc78–117 on HBV replication. We confirmed by immunoprecipitation analysis that HBc78–117 interacted with full-length HBc in HepG2.2.15 cells. The nucleocapsids and HBV DNA replication intermediates were markedly reduced in the cells expressing HBc78–117, although HBV pregenome RNA was not affected. The level of HBV DNA was also significantly reduced in culture supernatant. These suggest that HBc78–117 can inhibit HBV DNA replication by interfering with nucleocapsids assembly. [Copyright &y& Elsevier]

Hepatitis B virus (HBV) replicates through reverse transcription inside its icosahedral nucleocapsid. The internal genome status is signaled to the capsid surface, predicting regulated conformational changes in the capsid structure. To probe their nature and extent, we imposed local conformational stress on the outer surface of HBV capsid-like particles, and monitored its consequences by electron cryomicroscopy and image reconstruction. The capsid structure had an enormous flexibility and robustness as a whole, as well as within the subunits, whose spikes were able to rotate by as much as 40° against the distal interdimer contact sites. The likely hinge for the swiveling movement was the conserved Gly111 residue at the inner surface of the capsid. The stress imposed from the outside also affected the internal capsid organization, implying a specific route for the flow of conformational information between capsid interior and exterior as required for signaling of the genome status. [Copyright &y& Elsevier]

Abstract Background The core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression. Results Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS). Conclusion HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection.

Personalized cancer vaccine which targets neoepitopes shows great promise for cancer treatment. However, rapid preparation is a critical challenge for clinical application of personalized cancer vaccine. Genetic recombination and chemical modification are a time-consuming "trial and error" pattern for making vaccines. Here we first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of hepatitis B core protein (HBc) (1-183). The resulted recombinant protein HBc (1-183) -SpyCatcher (HBc (1-183) -S) assembled to virus-like particles (VLPs) and readily bound to SpyTag conjugated with OVA epitope peptides by just mixing, forming HBc (1-183) -S-OVA. HBc (1-183) -S-OVA VLPs effectively induced dendritic cell maturation. Our further results indicated that HBc (1-183) -S-OVA VLPs vaccination inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice by generating significant OVA-specific cytotoxic T lymphocyte responses. Our study provides a simple, rapid, efficient and universal HBc-based platform for the preparation of personalized cancer vaccine. HBc (1-183) -S VLPs, which are universal vaccine carriers, were constructed by inserting the gene of ΔN1SpyCatcher into the immunodominant region (MIR). OVA peptide synthesized with SpyTag can be glued onto HBc (1-183) -S by just mixing to form HBc (1-183) -S-OVA vaccine which inhibits tumor growth by eliciting Th1 type OVA specific immune responses. Unlabelled Image • SpyTag conjugated OVA MHC I-restricted epitope peptides can be glued onto HBc (1-183) -S VLPs by simple mixing to form stable HBc (1-183) -S-OVA. • HBc (1-183) -S-OVA VLPs remarkably inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice. • HBc (1-183) -S-OVA VLPs effectively entered drainage lymph node, induced dendritic cell maturation and markedly simulated OVA-specific cytotoxic T lymphocyte (CTL) responses. [ABSTRACT FROM AUTHOR]

Personalized cancer vaccine which targets neoepitopes shows great promise for cancer treatment. However, rapid preparation is a critical challenge for clinical application of personalized cancer vaccine. Genetic recombination and chemical modification are a time-consuming "trial and error" pattern for making vaccines. Here we first constructed a platform for peptide vaccine preparation by inserting SpyCatcher into the major immunodominant region (MIR) of hepatitis B core protein (HBc) (1-183). The resulted recombinant protein HBc (1-183) -SpyCatcher (HBc (1-183) -S) assembled to virus-like particles (VLPs) and readily bound to SpyTag conjugated with OVA epitope peptides by just mixing, forming HBc (1-183) -S-OVA. HBc (1-183) -S-OVA VLPs effectively induced dendritic cell maturation. Our further results indicated that HBc (1-183) -S-OVA VLPs vaccination inhibited tumor growth in both prophylactic and treatment ways in E.G7-OVA tumor bearing mice by generating significant OVA-specific cytotoxic T lymphocyte responses. Our study provides a simple, rapid, efficient and universal HBc-based platform for the preparation of personalized cancer vaccine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein nanocages are promising multifunctional platforms for nanomedicine owing to the ability to decorate their surfaces with multiple functionalities through genetic and/or chemical modification to achieve desired properties for therapeutic and diagnostic purposes. Here, we describe a model antigen (OVA peptide) that was conjugated to the surface of a naturally occurring hepatitis B core protein nanocage (HBc NC) by genetic modification. The engineered OVA-HBc nanocages (OVA-HBc NCs), displaying high density repetitive array of epitopes in a limited space by self-assembling into symmetrical structure, not only can induce bone marrow derived dendritic cells (BMDC) maturation effectively but also can be enriched in the draining lymph nodes. Naïve C57BL/6 mice immunized with OVA-HBc NCs are able to generate significant and specific cytotoxic T lymphocyte (CTL) responses. Moreover, OVA-HBc NCs as a robust nanovaccine can trigger preventive antitumor immunity and significantly delay tumor growth. When combined with a low-dose chemotherapy drug (paclitaxel), OVA-HBc NCs could specifically inhibit progression of an established tumor. Our findings support HBc-based nanocages with modularity and scalability as an attractive nanoplatform for combination cancer immunotherapy.

Virus-mimicking particles have made great contribution to the development of nanomedicine. Herein, several modularized peptides (lipophilic NS5A peptide, 6xHis tag, and tumor-targeting peptide RGD) were genetically inserted into the C-terminus and the major immunodominant loop region (MIR) of hepatitis B core protein (HBc), respectively. This study demonstrated that the recombinant HBc-based VLPs could participate in self-assembly of monodisperse nanoparticles (33.6±3.5nm) with well-defined morphology, and DOX can be packaged into VLNPs without any chemical modification. Moreover, the HBc-based VLPs could specifically target to cancer cells via the interaction with overexpressed integrin α v β 3 . The treatment with DOX-loaded HBc-based VLPs showed a significant inhibition of tumor growth (90.7% TGI) and less cardiotoxicity in B16F10 tumor-bearing mice models than that with the free DOX. Importantly, the results may offer an easy way to give a variety of ideal functional modulations for VLPs, thereby extending its potential biomedicine applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)