Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation

Inserting foreign epitopes to hepatitis B core (HBc) virus‐like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost‐effective purification process was developed for two chimeric HBc VLPs displaying Epstein–Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core‐HBc was found to be less stable in water environment compared with EBNA1‐HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α‐helix of HCV core‐HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost‐effective purification approach.     

Melanoma vaccine candidates from chimeric hepatitis B core virus-like particles carrying a tumor-associated MAGE-3 epitope

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.     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。     

HBc-based virus-like particle assembly from inclusion bodies using 2-methyl-2, 4-pentanediol

Hepatitis B virus core antigen (HBc) has recently been used as carriers to develop recombinant vaccines. However, not virus-like particles (VLPs) but inactive inclusion bodies are often formed for the chimeric proteins when expressed in Escherichia coli. A novel method for in vitro assembly of chimeric HBc-MAGE3 II from inclusion bodies to VLPs was established in this study. The method utilized 2-methyl-2, 4-pentanediol (MPD), an amphipathic di-alcohol, to dissociate sodium dodecyl sulfate (SDS) from the solubilized chimeric protein to initiate VLP assembly. The HBc-MAGE3 II could assemble into VLPs only when the molar ratio of SDS/protein subunit was less than 0.14. After removing SDS/MPD by desalting and further purification, VLPs with similar morphology to the natural virus were obtained. This method could be used for preparation of other VLPs expressed as inclusion bodies.     

Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies.     

Human dendritic cells are activated by chimeric human papillomavirus type-16 virus-like particles and induce epitope-specific human T cell responses in vitro

Human papillomavirus (HPV)-derived chimeric virus-like particles (VLPs) are the leading candidate vaccine for the treatment or prevention of cervical cancer in humans. Dendritic cells (DCs) are the most potent inducers of immune responses and here we show for the first time evidence for binding of chimeric HPV-16 VLPs to human peripheral blood-derived DCS: Incubation of immature human DCs with VLPs for 48 h induced a significant up-regulation of the CD80 and CD83 molecules as well as secretion of IL-12. Confocal microscopy analysis revealed that cell surface-bound chimeric VLPs were taken up by DCS: Moreover, DCs loaded with chimeric HPV-16 L1L2-E7 VLPs induced an HLA-*0201-restricted human T cell response in vitro specific for E7-derived peptides. These results clearly demonstrate that immature human DCs are fully activated by chimeric HPV-16 VLPs and subsequently are capable of inducing endogenously processed epitope-specific human T cell responses in vitro. Overall, these findings could explain the high immunogenicity and efficiency of VLPs as vaccines.     

Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies

Antibody to the capsid (PORF2) protein of hepatitis E virus (HEV) is sufficient to confer immunity, but knowledge of B-cell epitopes in the intact capsid is limited. A panel of murine monoclonal antibodies (MAbs) was generated following immunization with recombinant ORF2.1 protein, representing the C-terminal 267 amino acids (aa) of the 660-aa capsid protein. Two MAbs reacted exclusively with the conformational ORF2.1 epitope (F. Li, J. Torresi, S. A. Locarnini, H. Zhuang, W. Zhu, X. Guo, and D. A. Anderson, J. Med. Virol. 52:289-300, 1997), while the remaining five demonstrated reactivity with epitopes in the regions aa 394 to 414, 414 to 434, and 434 to 457. The antigenic structures of both the ORF2.1 protein expressed in Escherichia coli and the virus-like particles (VLPs) expressed using the baculovirus system were examined by competitive enzyme-linked immunosorbent assays (ELISAs) using five of these MAbs and HEV patient sera. Despite the wide separation of epitopes within the primary sequence, all the MAbs demonstrated some degree of cross-inhibition with each other in ORF2. 1 and/or VLP ELISAs, suggesting a complex antigenic structure. MAbs specific for the conformational ORF2.1 epitope and a linear epitope within aa 434 to 457 blocked convalescent patient antibody reactivity against VLPs by approximately 60 and 35%, respectively, while MAbs against epitopes within aa 394 to 414 and 414 to 434 were unable to block patient serum reactivity. These results suggest that sequences spanning aa 394 to 457 of the capsid protein participate in the formation of strongly immunodominant epitopes on the surface of HEV particles which may be important in immunity to HEV infection.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

嵌合型口蹄疫病毒样颗粒构建、表达及鉴定

为提高口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)病毒样颗粒(Virus-likeparticles,VLPs)的特异性识别和递呈,为靶向疫苗研究奠定基础,利用反向PCR技术,将卵清蛋白(Ovalbumin,OVA)第257–264位氨基酸(Amino acids,aa)的短肽嵌入FMDV结构蛋白VP3第171–172位aa或第173–174位aa,通过大肠杆菌表达FMDV结构蛋白VP0、VP1和嵌合型VP3,体外组装得到嵌合OVA257-264肽的病毒样颗粒(VLPOVA)。用动态光散射、透射电镜检测VLPOVA大小和形态,免疫印迹、酶联免疫吸附试验和激光共聚焦显微镜检测短肽的嵌入情况。结果显示在VP3的第173–174位aa嵌入OVA,不影响蛋白表达和VLPs的组装且OVA位于VLPOVA的表面,VLPOVA粒径比VLPs稍大。     

Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris

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.     

Development of a vaccine marker technology: display of B cell epitopes on the surface of recombinant polyomavirus-like pentamers and capsoids induces peptide-specific antibodies in piglets after vaccination

Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage. Here, a comparative analysis revealed that antibody responses raised against the carrier entities, KLH or VP1 pentamers, did not significantly differ up to 18 weeks, demonstrating the highly immunogenic nature of VP1-based particulate structures. The carrier-specific antibody response was reproducibly detected in the meat juice after processing. More importantly, chimeric VP1 pentamers and VLPs carrying peptides of 12 and 14 amino acids in length, inserted into the BC2 loop, induced a strong and long-lasting humoral immune response against VP1 and the inserted foreign epitope. Remarkably, the epitope-specific antibody response was only moderately decreased when VP1 pentamers were used instead of VLPs. In conclusion, we identified polyomavirus VP1-based structures displaying surface-exposed immunodominant B cell epitopes as being an efficient carrier system for the induction of potent peptide-specific antibodies. The application of this approach in vaccine marker technology in livestock holding and the meat production chain is discussed.     

Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion

The major capsid protein L1 of human papillomavirus (HPV) contains the immunodominant neutralization epitopes of the virus and can auto-assembles to form virus-like particles (VLPs). Therefore, HPV L1 capsid proteins have been well investigated as potential vaccine candidates. To express large quantities of human papillomavirus type 16 (HPV-16) L1 in Escherichia coli (E. coli), The HPV-16 L1 gene was cloned into pGEX-4T-1, resulting in only low expression levels of HPV-16 L1 in E. coli. The first 129 nucleotides of the 5' end of the L1 gene, which contains the major inhibitory RNA element, were then deleted. The deletion RNA was efficiently translated, resulting in about 2-fold higher L1 accumulation in E. coli. The N-terminal amino-acid deletion did not affect the ability of L1 to auto-assemble in E. coli and form small VLPs.     

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris

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.     

乙型肝炎病毒核心蛋白作为表位疫苗载体的应用

乙型肝炎病毒核心蛋白(Hepatitis B viruscore protein,HBc)可以形成二十面体对称的颗粒样结构,由于其N端、C端和主要免疫显性区域(Major immunodominant region,MIR)允许一定程度的缺失和外源插入,并且能够将外源序列重复且高密度地暴露在颗粒的表面,诱发强烈的外源序列特异的体液和细胞免疫反应,从上世纪80年代中期就开始被运用于表位疫苗的研究。以下主要从影响HBc作为表位疫苗载体的因素,包括HBc长度、外源插入位点和表位序列的性质等来介绍HBc作为表位疫苗载体的应用。     

SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定

为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据。     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.     

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.